Textbook of Biochemistry for Medical Students ( PDFDrive ).pdf

 Textbook of BIOCHEMISTRY  Textbook of BIOCHEMISTRY for Medical Students (Seventh Edition) Free online access to Additional Clinical Cases, Key Concepts & Image Bank DM Vasudevan MBBS MD FAMS FRCPath...

 Textbook of BIOCHEMISTRY  Textbook of BIOCHEMISTRY for Medical Students (Seventh Edition) Free online access to Additional Clinical Cases, Key Concepts & Image Bank DM Vasudevan MBBS MD FAMS FRCPath Distinguished Professor Department of Biochemistry College of Medicine, Amrita Institute of Medical Sciences Kochi, Kerala, India Formerly Principal, College of Medicine Amrita Institute of Medical Sciences, Kerala, India Dean, Sikkim Manipal Institute of Medical Sciences Gangtok, Sikkim, India Sreekumari S MBBS MD Professor and Head Department of Biochemistry Sree Gokulam Medical College and Research Foundation Thiruvananthapuram, Kerala, India Kannan Vaidyanathan MBBS MD Professor and Head Department of Biochemistry Pushpagiri Institute of Medical Sciences and Research Center Thiruvalla, Kerala, India ® JAYPEE BROTHERS MEDICAL PUBLISHERS (P) LTD New Delhi London Philadelphia Panama  ® Jaypee Brothers Medical Publishers (P) Ltd Headquarters Jaypee Brothers Medical Publishers (P) Ltd 4838/24, Ansari Road, Daryaganj New Delhi 110 002, India Phone: +91-11-43574357 Fax: +91-11-43574314 Email: [email protected] Overseas Offices J.P. Medical Ltd Jaypee-Highlights Medical Publishers Inc Jaypee Medical Inc 83, Victoria Street, London City of Knowledge, Bld. 237, Clayton The Bourse SW1H 0HW (UK) Panama City, Panama 111 South Independence Mall East Phone: +44-2031708910 Phone: +507-301-0496 Suite 835, Philadelphia, PA 19106, USA Fax: +02-03-0086180 Fax: +507-301-0499 Phone: + 267-519-9789 Email: [email protected] Email: [email protected] Email: [email protected] Jaypee Brothers Medical Publishers (P) Ltd Jaypee Brothers Medical Publishers (P) Ltd 17/1-B Babar Road, Block-B, Shaymali Shorakhute, Kathmandu Mohammadpur, Dhaka-1207 Nepal Bangladesh Phone: +00977-9841528578 Mobile: +08801912003485 Email: [email protected] Email: [email protected] Website: www.jaypeebrothers.com Website: www.jaypeedigital.com © 2013, DM Vasudevan, Sreekumari S, Kannan Vaidyanathan All rights reserved. No part of this book may be reproduced in any form or by any means without the prior permission of the publisher. Inquiries for bulk sales may be solicited at: [email protected] This book has been published in good faith that the contents provided by the authors contained herein are original, and is intended for educational purposes only. While every effort is made to ensure accuracy of information, the publisher and the authors specifically disclaim any damage, liability, or loss incurred, directly or indirectly, from the use or application of any of the contents of this work. If not specifically stated, all figures and tables are courtesy of the authors. Where appropriate, the readers should consult with a specialist or contact the manufacturer of the drug or device. Textbook of Biochemistry for Medical Students First Edition: 1995 Second Edition: 1998 Third Edition: 2001 Fourth Edition: 2004 Fifth Edition: 2007 Sixth Edition: 2010 Seventh Edition: 2013 ISBN 978-93-5090-530-2 Printed at  Dedicated to With humility and reverence, this book is dedicated at the lotus feet of the Holy Mother, Sri Mata Amritanandamayi Devi "Today's world needs people who express goodness in their words and deeds. If such noble role models set the example for their fellow beings, the darkness prevailing in today's society will be dispelled, and the light of peace and non- violence will once again illumine this earth. Let us work together towards this goal". —Mata Amritanandamayi Devi  Preface to the Seventh Edition We are glad to present the Seventh edition of the Textbook of Biochemistry for Medical Students. Now, this textbook is entering the 19th year of existence. With humility, we may state that the medical community of India has warmly received the previous editions of this book. The Medical Council of India has accepted it as one of the standard textbooks. We are happy to note that this book has also reached in the hands of medical students of neighboring countries of Nepal, Pakistan, Bangladesh, Sri Lanka, etc. and also to distant countries in Africa and Europe. We are very proud to report that the Textbook has a Spanish edition, with wide circulation in the Central and South America. Apart from the medical community, this book has also become popular to other biological group of students in India. In retrospect, it gives immense satisfaction to note that this book served the students and faculty for the past two decades. We are bringing out the new edition of the textbook every 3 years. A major addition of this edition is the incorporation of clinical case studies in almost all chapters. We hope that this feature will help the students to identify the clinical relevance of the biochemistry. Further, chapters on clinical chemistry have been extensively updated and clinically relevant points were further added. Rapid progress has been made in the area of molecular biology during past few years, and these advances are to be reflected in this book also. The major change in this Seventh edition is that advanced knowledge has been added in almost all chapters, clinical case studies have been added in relevant chapters; and a few new chapters were added. The print fonts and font size have also been changed for better readability. From the First edition onwards, our policy was to provide not only basic essentials but also some of the advanced knowledge. About 30% contents of the previous editions were not required for a student aiming for a minimum pass. A lot of students have appreciated this approach, as it helped them to pass the postgraduate (PG) entrance examinations at a later stage. However, this asset has paved the way for a general criticism that the extra details are a burden to the average students. Especially, when read for the first time, the student may find it difficult to sort out the essential minimum from the desirable bulk. In this Seventh edition, advanced topics are given in small prints. In essence, this book is composed of three complementary books. The bold printed areas will be useful for the student at the time of revision just before the examinations; regular printed pages are meant for an average first year MBBS student and the fine printed paragraphs are targeted to the advanced students preparing for the PG entrance. Essay questions, short notes, multiple choice questions and viva voce type questions are given as a separate book, but free of cost. These questions are compiled from the question papers of various universities during the last decade. These questions will be ideal for students for last-minute preparation for examinations. We are introducing the online study material, which provides concepts of major topic as well as clinical case studies. This shall be updated through the year. Hence, students are advised to check the web page at regular intervals. A textbook will be matured only by successive revisions. In the preface for the First edition, we expressed our desire to revise the textbook every 3 years. We were fortunate to keep that promise. This book has undergone metamorphosis during each edition. Chemical structures with computer technology were introduced in the Second edition. Color printing has been launched in the Third edition. The Fourth edition came out with multicolor printing. In the Fifth edition, the facts were presented in small paragraphs, so as to aid memory. In the Sixth edition, figures were drastically increased. In this Seventh edition, about 100 case studies are added. In this book, there are about viii Textbook of Biochemistry 1100 figures, 230 tables and 200 boxes (perhaps we could call it as illustrated textbook of biochemistry), altogether making the book more student-friendly. The quality of paper is also improved during successive editions. We were pleasantly surprised to receive many letters giving constructive criticisms and positive suggestions to improve the textbook. These responses were from all parts of the country (we got a few such letters from African and European students also). Such contributors include Heads of Departments, very senior professors, middle level teachers and mostly postgraduate students. We have tried to incorporate most of those suggestions, within the constraints of page limitations. In a way, this book thus became multi-authored, and truly national in character. This is to place on record, our deep gratitude for all those “pen-friends” who have helped us to improve this book. The first author desires more interaction with faculty and students who are using this textbook. All are welcome to communicate at his e-mail address As indicated in the last edition, the first author is in the process of retirement, and would like to reduce the burden in due course. A successful textbook is something like a growing institution; individuals may come and go, but the institution will march ahead. Therefore, we felt the need to induce younger blood into the editorial board. Thus, a third author has been added in the Sixth edition, so that the torch can been handed over smoothly at an appropriate time later on. In this Seventh edition, the first author has taken less responsibility in editing the book, while the third author has taken more effort. The help and assistance rendered by our postgraduate students in preparing this book are enormous. The official website of Nobel Academy has been used for pictures and biographies of Nobel laureates. Web pictures, without copyright protection, were also used in some figures. The remarkable success of the book was due to the active support of the publishers. This is to record our appreciation for the cooperation extended by Shri Jitendar P Vij (Group Chairman), Mr Ankit Vij (Managing Director) and Mr Tarun Duneja (Director-Publishing) of M/s Jaypee Brothers Medical Publishers (P) Ltd, New Delhi, India. We hope that this Seventh edition will be friendlier to the students and be more attractive to the teachers. Now this is in your hands to judge. “End of all knowledge must be building up of character” —Mahatma Gandhi DM Vasudevan Sreekumari S Kannan Vaidyanathan  Preface to the First Edition There are many textbooks of biochemistry written by Western and Indian authors. Then what is the need for yet another textbook? Putting this question to ourselves, we have waited for many years before embarking on this project. Most Western textbooks do not emphasize nutrition and such other topics, which are very vital to an Indian student. While Indian authors do cover these portions, they sometimes neglect the expanding fields, such as molecular biology and immunochemistry. Thus, during our experience of more than 25 years in teaching, the students have been seen compelled to depend on different textbooks during their study of biochemistry. We have tried to keep a balance between the basic essentials and the advanced knowledge. This book is mainly based on the MBBS curriculum. However, some advanced portions have also been given in almost all chapters. These areas will be very beneficial to the readers preparing for their postgraduate entrance examinations. Chapters on diabetes, cancer and AIDS are included in this book. During their clinical years, the students are going to see such cases quite more often, hence knowledge of applied biochemistry of these diseases will be very helpful. The authors, themselves medical graduates, have tried to emphasize medical applications of the theoretical knowledge in biochemistry in almost all the chapters. A few questions have been given at the end of most of the chapters. These are not comprehensive to cover all the topics, but have been included only to give emphasis to certain points, which may otherwise be left unnoticed by some students. We are indebted to many persons in compiling this textbook. We are highly obliged to Dr ANP Ummerkutty, Vice-Chancellor, University of Calicut, for his kind gesture of providing an introduction. Dr M Krishnan Nair, Research Director, Veterinary College, Trichur, has provided his unpublished electron micrographs for this book. Dr MV Muraleedharan, Professor of Medicine, and Dr TS Hariharan, Professor of Pharmacology, Medical College, Thrissur, have gone through the contents of this book. Their valuable suggestions on the applied aspects of biochemistry have been incorporated. Two of our respected teachers in biochemistry, Professor R Raghunandana Rao and Professor GYN lyer (both retired) have encouraged this venture. Professor PNK Menon, Dr S Gopinathan Nair, Assistant Professor, Dr Shyam Sundar, Dr PS Vasudevan and Mr K Ramesh Kumar, postgraduate students of this department, have helped in collecting the literature and compiling the materials. Mr Joby Abraham, student of this college has contributed the sketch for some of the figures. Professor CPK Tharakan, retired professor of English, has taken great pains to go through the entire text and correct the usage of English. The secretarial work has been excellently performed by Mrs Lizy Joseph. Many of our innumerable graduate and postgraduate students have indirectly contributed by compelling us to read more widely and thoroughly. Our expectation is to bring out the new edition every 3 years. Suggestions to improve the contents are welcome from the teachers. “A lamp that does not glow itself cannot light another lamp” —Rabindranath Tagore DM Vasudevan Sreekumari S  Contents SECTION A: Chemical Basis of Life 1. Biochemical Perspective to Medicine 3 Biomolecules 4; Study of metabolic processes 5; Stabilizing forces in molecules 5; Water: the universal solvent 6; Principles of thermodynamics 7; Donnan membrane equilibrium 8 2. Subcellular Organelles and Cell Membranes 10 Subcellular organelles 10; Nucleus 10; Endoplasmic reticulum 11; Golgi apparatus 12; Lysosomes 12; Peroxisomes 13; Mitochondria 13; Plasma membrane 14; Specialized membrane structures 16; Transport mechanisms 17 3. Amino Acids: Structure and Properties 24 Classification of amino acids 24; Properties of amino acids 27; General reactions of amino acids 29; Peptide bond formation 31 4. Proteins: Structure and Function 33 Structure of proteins 34; Study of protein structure 39; Physical properties of proteins 41; Precipitation reactions of proteins 41; Classification of proteins 42; Quantitative estimation 44 5. Enzymology: General Concepts and Enzyme Kinetics 47 Classification of enzymes 48; Co-enzymes 49; Mode of action of enzymes 51; Michaelis-Menten theory 53; Fischer's template theory 53; Koshland's induced fit theory 53; Active site or active center of enzyme 54; Thermodynamic considerations 54; Enzyme kinetics 55; Factors influencing enzyme activity 56; Specificity of enzymes 65; Iso-enzymes 66 6. Chemistry of Carbohydrates 69 Nomenclature 69; Stereoisomers 70; Reactions of monosaccharides 73; Disaccharides 76; Polysaccharides 78; Heteroglycans 79; Mucopolysaccharides 80; Glycoproteins and mucoproteins 81 7. Chemistry of Lipids 83 Classification of lipids 83; Fatty acids 84; Saturated fatty acids 85; Unsaturated fatty acids 85; Trans fatty acids 86; Neutral fats 87; Phospholipids 89 SECTION B: General Metabolism 8. Overview of Metabolism 97 Experimental study of metabolism 97; Metabolism 98; Metabolic profile of organs 99 9. Major Metabolic Pathways of Glucose 105 Digestion of carbohydrates 105; Absorption of carbohydrates 106; Glucose metabolism 107; Glycolysis 108; Metabolic fate of pyruvate 115; Gluconeogenesis 117 10. Other Metabolic Pathways of Glucose 123 Glycogen metabolism 123; Degradation of glycogen (glycogenolysis) 124; Glycogen synthesis (glycogenesis) 125; Glycogen storage diseases 128; Hexose monophosphate shunt pathway 129; Oxidative phase 130; Non-oxidative phase 130; Glucuronic acid pathway of glucose 134; Polyol pathway of glucose 135 xii Textbook of Biochemistry 11. Metabolic Pathways of Other Carbohydrates 137 Fructose metabolism 137; Galactose metabolism 138; Metabolism of alcohol 140; Metabolism of amino sugars 142; Glycoproteins 142 12. Metabolism of Fatty Acids 147 Digestion of lipids 147; Absorption of lipids 148; Beta oxidation of fatty acids 151; Oxidation of odd chain fatty acids 154; Alpha oxidation 155; Omega oxidation 155; De novo synthesis of fatty acids 156; Synthesis of triacylglycerols 160; Metabolism of adipose tissue 161; Fatty liver and lipotropic factors 162; Metabolism of ketone bodies 163; Ketosis 164 13. Cholesterol and Lipoproteins 169 Biosynthesis of cholesterol 170; Plasma lipids 173; Chylomicrons 175; Very low density lipoproteins 176; Low density lipoproteins 177; High density lipoprotein 179; Free fatty acid 181; Formation of bile acids 182 14. MCFA, PUFA, Prostaglandins and Compound Lipids 184 Monounsaturated fatty acids 185; Polyunsaturated fatty acids 186; Eicosanoids 188; Prostaglandins 188; Synthesis of compound lipids 191 15. General Amino Acid Metabolism (Urea Cycle, One Carbon Metabolism) 196 Digestion of proteins 196; Formation of ammonia 200; Disposal/detoxification of ammonia 203; Urea cycle 203; One-carbon metabolism 207 16. Simple, Hydroxy and Sulfur-containing Amino Acids (Glycine, Serine, Methionine, Cysteine) 210 Glycine 210; Creatine and creatine phosphate 211; Serine 213; Alanine 215; Threonine 215; Methionine 216; Cysteine 217; Cystinuria 219; Homocystinurias 220 17. Acidic, Basic and Branched Chain Amino Acids (Glutamic Acid, Aspartic Acid, Glutamine, Asparagine, Lysine, Arginine, Nitric Oxide, Valine, Leucine, Isoleucine) 223 Glutamic acid 223; Glutamine 224; Glutamate transporters 225; Aspartic acid 226; Asparagine 226; Arginine 226; Nitric oxide 227; Polyamines 229; Branched chain amino acids 230 18. Aromatic Amino Acids (Phenylalanine, Tyrosine, Tryptophan, Histidine, Proline) and Amino Acidurias 232 Phenylalanine 232; Tyrosine 233; Phenylketonuria 236; Alkaptonuria 237; Albinism 238; Hypertyrosinemias 239; Tryptophan 239; Histidine 243; Proline and hydroxyproline 244; Aminoacidurias 245 19. Citric Acid Cycle 247 Regulation of citric acid cycle 253 20. Biological Oxidation and Electron Transport Chain 255 Redox potentials 256; Biological oxidation 256; Enzymes and co-enzymes 257; High energy compounds 258; Organization of electron transport chain 260; Chemiosmotic theory 263 21. Heme Synthesis and Breakdown 270 Structure of heme 270; Biosynthesis of heme 271; Catabolism of heme 276; Hyperbilirubinemias 279 22. Hemoglobin (Structure, Oxygen and Carbon Dioxide Transport, Abnormal Hemoglobins) 283 Structure of hemoglobin 283; Transport of oxygen by hemoglobin 284; Transport of carbon dioxide 287; Hemoglobin derivatives 289; Hemoglobin (globin chain) variants 290; Thalassemias 293; Myoglobin 294; Anemias 295; Hemolytic anemia 295 SECTION C: Clinical and Applied Biochemistry 23. Clinical Enzymology and Biomarkers 301 Clinical enzymology 301; Creatine kinase 302; Cardiac troponins 303; Lactate dehydrogenase 303; Alanine amino transferase 305; Aspartate amino transferase 305; Alkaline phosphatase 305; Prostate specific antigen 306; Glucose-6-phosphate dehydrogenase 307; Amylase 307; Lipase 308; Enolase 308 24. Regulation of Blood Glucose; Insulin and Diabetes Mellitus 311 Regulation of blood glucose 311; Reducing substances in urine 316; Hyperglycemic hormones 322; Glucagon 322; Diabetes mellitus 323; Acute metabolic complications 326  Contents xiii 25. Hyperlipidemias and Cardiovascular Diseases 334 Atherosclerosis 334; Plasma lipid profile 336; Risk factors for atherosclerosis 336; Prevention of atherosclerosis 339; Hypolipoproteinemias 341; Hyperlipidemias 342 26. Liver and Gastric Function Tests 346 Functions of liver 346; Clinical manifestations of liver dysfunction 348; Studies on malabsorption 359 27. Kidney Function Tests 361 Renal function tests 361; Abnormal constituents of urine 364; Markers of glomerular filtration rate 366; Markers of glomerular permeability 371; Tests for tubular function 373 28. Plasma Proteins 378 Electrophoresis 378; Albumin 380; Transport proteins 382; Acute phase proteins 383; Clotting factors 385; Abnormalities in coagulation 386 29. Acid-Base Balance and pH 390 Acids and bases 390; Buffers 392; Acid-base balance 393; Buffers of the body fluids 393; Respiratory regulation of pH 395; Renal regulation of pH 395; Cellular buffers 397; Disturbances in acid-base balance 397 30. Electrolyte and Water Balance 407 Intake and output of water 407; Osmolality of extracellular fluid 408; Sodium 411; Potassium 413; Chloride 416 31. Body Fluids (Milk, CSF, Amniotic Fluid, Ascitic Fluid) 420 Milk 420; Cerebrospinal fluid 421; Amniotic fluid 422; Ascitic fluid 423 32. Metabolic Diseases 424 Prenatal diagnosis 424; Newborn screening 427; Laboratory investigations to diagnose metabolic disorders 427 33. Free Radicals and Antioxidants 433 Clinical significance 436 34. Clinical Laboratory; Quality Control 439 Reference values 439; Preanalytical variables 440; Specimen collection 441; Quality control 443 35. General Techniques for Separation, Purification and Quantitation 446 Electrophoresis 446; Chromatography 448; Radioimmunoassay 452; ELISA test 453; Colorimeter 455; Autoanalyzer 457; Mass spectrometry 458 SECTION D: Nutrition 36. Fat Soluble Vitamins (A, D, E, K) 463 Vitamin A 464; Vitamin D (cholecalciferol) 469; Vitamin E 473; Vitamin K 474 37. Water Soluble Vitamins - 1 (Thiamine, Riboflavin, Niacin, Pyridoxine, Pantothenic Acid, Biotin) 477 Thiamine (vitamin B1 ) 477; Riboflavin (vitamin B2 ) 479; Niacin 480; Vitamin B6 482; Pantothenic acid 484; Biotin 485 38. Water Soluble Vitamins - 2 (Folic Acid, Vitamin B12 and Ascorbic Acid) 488 Folic acid 488; Vitamin B12 491; Choline 494; Inositol 495; Ascorbic acid (vitamin C) 495; Rutin 499; Flavonoids 499 39. Mineral Metabolism and Abnormalities 502 Calcium 502; Phosphorus 511; Magnesium 512; Sulfur 513; Iron 514; Copper 520; Iodine 521; Zinc 522; Fluoride 522; Selenium 522; Manganese 523; Molybdenum 523; Cobalt 523; Nickel 523; Chromium 523; Lithium 524 xiv Textbook of Biochemistry 40. Energy Metabolism and Nutrition 527 Importance of carbohydrates 530; Nutritional importance of lipids 531; Importance of proteins 532; Protein-energy malnutrition 534; Obesity 536; Prescription of diet 538 41. Detoxification and Biotransformation of Xenobiotics 544 Phase one reactions 545; Phase two reactions; conjugations 546; Phase three reactions 548 42. Environmental Pollution and Heavy Metal Poisons 550 Corrosives 550; Irritants 551; Heavy metal poisons 551; Pesticides and insecticides 553; Occupational and industrial hazards 553; Air pollutants 553 SECTION E: Molecular Biology 43. Nucleotides: Chemistry and Metabolism 559 Biosynthesis of purine nucleotides 563; Uric acid 566; Gout 566; De novo synthesis of pyrimidine 569 44. Deoxyribonucleic Acid: Structure and Replication 574 Structure of DNA 574; Replication of DNA 578; DNA repair mechanisms 582 45. Transcription 587 Ribonucleic acid 587; Transcription process 589 46. Genetic Code and Translation 596 Protein biosynthesis 596; Translation process 599 47. Control of Gene Expression 608 Mutations 612; Classification of mutations 612; Cell cycle 614; Regulation of gene expression 616; Viruses 620 48. Recombinant DNA Technology and Gene Therapy 624 Recombinant DNA technology 624; Vectors 626; Gene therapy 629; Stem cells 631 49. Molecular Diagnostics and Genetic Techniques 633 Hybridization and blot techniques 633; Polymerase chain reaction 638; Mutation detection techniques 641 SECTION F: Hormones 50. Mechanisms of Action of Hormones and Signaling Molecules 649 51. Hypothalamic and Pituitary Hormones 659 Hypothalamic neuropeptides 659; Hormones of anterior pituitary 660 52. Steroid Hormones 664 Adrenal cortical hormones 664; Sex hormones 669 53. Thyroid Hormones 672 54. Gut Hormones 678 SECTION G: Advanced Biochemistry 55. Immunochemistry 685 Structure of immunoglobulins 687; Paraproteinemias 690; Complement system 691; Immunodeficiency states 692  Contents xv 56. Biochemistry of AIDS and HIV 699 The human immunodeficiency virus 701; Anti-HIV drugs 703 57. Biochemistry of Cancer 705 Oncogenic viruses 707; Oncogenes 709; Tumor markers 713; Anticancer drugs 716 58. Tissue Proteins in Health and Disease 720 Collagen 720; Elastin 723; Muscle proteins 724; Lens proteins 727; Prions 727; Biochemistry of aging 730 59. Applications of Isotopes in Medicine 732 Isotopes 733; Radioactivity 733; Biological effects of radiation 738 60. Signal Molecules and Growth Factors 740 Appendices 747 Index 763  SECTION A Chemical Basis of Life Chapter 1 Biochemical Perspective to Medicine Chapter 2 Subcellular Organelles and Cell Membranes Chapter 3 Amino Acids: Structure and Properties Chapter 4 Proteins: Structure and Function Chapter 5 Enzymology: General Concepts and Enzyme Kinetics Chapter 6 Chemistry of Carbohydrates Chapter 7 Chemistry of Lipids  CHAPTER 1 Biochemical Perspective to Medicine Chapter at a Glance The reader will be able to answer questions on the following topics: ¾¾History of biochemistry ¾¾Hydrophobic interactions ¾¾Ionic bonds ¾¾Principles of thermodynamics ¾¾Hydrogen bonding ¾¾Donnan membrane equilibrium Biochemistry is the language of biology. The tools for The word chemistry is derived from the Greek word "chemi" (the research in all the branches of medical science are mainly black land), the ancient name of Egypt. Indian medical science, even from ancient times, had identified the metabolic and genetic basis of diseases. biochemical in nature. The study of biochemistry is Charaka, the great master of Indian Medicine, in his treatise (circa 400 essential to understand basic functions of the body. This BC) observed that madhumeha (diabetes mellitus) is produced by the study will give information regarding the functioning of alterations in the metabolism of carbohydrates and fats; the statement cells at the molecular level. How the food that we eat is still holds good. Biochemistry has developed as an offshoot of organic chemistry, digested, absorbed, and used to make ingredients of the and this branch was often referred as "physiological chemistry". The body? How does the body derive energy for the normal term "Biochemistry" was coined by Neuberg in 1903 from Greek day to day work? How are the various metabolic processes words, bios (= life) and chymos (= juice). One of the earliest treatises in interrelated? What is the function of genes? What is the biochemistry was the "Book of Organic Chemistry and its Applications molecular basis for immunological resistance against to Physiology and Pathology", published in 1842 by Justus von Liebig invading organisms? Answer for such basic questions can only be derived by a systematic study of biochemistry. Modern day medical practice is highly dependent on the laboratory analysis of body fluids, especially the blood. The disease manifestations are reflected in the composition of blood and other tissues. Hence, the demarcation of abnormal from normal constituents of the body is another Hippocrates Charaka Sushrutha aim of the study of biochemistry. 460–377 BC 400 BC 500 BC  4 Textbook of Biochemistry (1803–73), who introduced the concept of metabolism. The "Textbook The large amount of data, especially with regard to single nucleotide of Physiological Chemistry" was published in 1877 by Felix Hoppe- polymorphisms (SNPs) that are available, could be harnessed by Seyler (1825–95), who was Professor of Physiological chemistry at "Bioinformatics". Computers are already helping in drug designing Strausbourge University, France. Some of the milestones in the develop­ process. Studies on oncogenes have identified molecular mechanisms of ment of the science of biochemistry are given in Table 1.1. control of normal and abnormal cells. Medical practice is now depending The practice of medicine is both an art and a science. The word more on the science of Medical Biochemistry. With the help of Human “doctor” is derived from the Latin root, "docere", which means “to genome project (HGP) the sequences of whole human genes are now teach”. Knowledge devoid of ethical back­ground may sometimes be available; it has already made great impact on medicine and related disastrous! Hippocrates (460 BC to 377 BC), the father of modern health sciences. medicine articulated "the Oath”. About one century earlier, Sushrutha (?500 BC), the great Indian surgeon, enunciated a code of conduct for the medical practitioners, which is still valid. He proclaims: “You must BIOMOLECULES speak only truth; care for the good of all living beings; devote yourself to More than 99% of the human body is composed of 6 the healing of the sick even if your life be lost by your work; be simply clothed and drink no intoxicant; always seek to grow in knowledge; in elements, i.e. oxygen, carbon, hydrogen, nitrogen, calcium face of God, you can take upon yourself these vows.” and phos­phorus. Human body is composed of about 60% Biochemistry is perhaps the most rapidly developing discipline water, 15% proteins, 15% lipids, 2% carbohydrates and in medicine. No wonder, the major share of Nobel prizes in medicine 8% minerals. Molecular structures in organisms are built has gone to research workers engaged in biochemistry. Thanks to the advent of DNA recombinant tech­no­logy, genes can now be transferred from 30 small precursors, sometimes called the alphabets from one person to another, so that many of the genetically determined of biochemistry. These are 20 amino acids, 2 purines, diseases are now amenable to gene therapy. Many genes, (e.g. human 3 pyrimidines, sugars (glucose and ribose), palmitate, insulin gene) have already been transferred to microorganisms for large glycerol and choline. scale production of human insulin. Advances in genomics like RNA In living organisms, biomolecules are ordered into interference for silencing of genes and creation of transgenic animals by gene targeting of embryonic stem cells are opening up new vistas a hierarchy of increasing molecular complexity. These in therapy of diseases like cancer and AIDS. It is hoped that in future, biomolecules are covalently linked to each other to form the physician will be able to treat the patient, understanding his genetic macromolecules of the cell, e.g. glucose to glyco­ gen, basis, so that very efficient "designer medicine" could cure the diseases. amino acids to proteins, etc. Major complex biomolecules are proteins, polysaccharides, lipids and nucleic acids. The TABLE 1.1: Milestones in history of Biochemistry macromole­cules associate with each other by noncovalent Scientists Year Landmark discoveries forces to form supramolecular systems, e.g. ribosomes, Rouelle 1773 Isolated urea from urine lipoproteins. Lavoisier 1785 Oxidation of food stuffs Wohler 1828 Synthesis of urea Berzelius 1835 Enzyme catalysis theory Louis Pasteur 1860 Fermentation process Edward Buchner 1897 Extracted enzymes Fiske and Subbarao 1926 Isolated ATP from muscle Lohmann 1932 Creatine phosphate Hans Krebs 1937 Citric acid cycle Lavoisier Berzelius Friedrich Justus von Liebig Avery and Macleod 1944 DNA is genetic material 1743–1794 1779–1848 Wohler 1803–1873 Lehninger 1950 TCA cycle in mitochondria 1800–1882 Watson and Crick 1953 Structure of DNA Nirenberg 1961 Genetic code in mRNA Holley 1963 Sequenced gene for tRNA Khorana 1965 Synthesized the gene Paul Berg 1972 Recombinant DNA technology Kary Mullis 1985 Polymerase chain reaction 1990 Human genome project started 2000 Draft human genome Frederick Louis Pasteur Johannes van Albert Lehninger Donnan 1822–1895 der Waals 1917–1986 2003 Human genome project completed 1870–1956 NP 1910, ENCODE 2012 ENCyclopedia Of DNA Elements 1837–1923  Chapter 1: Biochemical Perspective to Medicine 5 Finally at the highest level of organization in the electrons from the outer most orbit of an electropositive hierarchy of cell structure, various supramolecular atom to the outermost orbit of an electronegative atom. This comple­xes are further assembled into cell organelle. In transfer results in the formation of a ‘cation’ and an ‘anion’, prokaryotes (e.g. bacteria; Greek word "pro" = before; which get consequently bound by an ionic bond. Common karyon = nucleus), these macromolecules are seen in a examples of such compounds include NaCl, KBr and NaF. homogeneous matrix; but in eukaryotic cells (e.g. higher With regard to protein chemistry, positive charges are organisms; Greek word "eu" = true), the cytoplasm produced by epsilon amino group of lysine, guanidium contains various subcellular organelles. Comparison of group of arginine and imidazolium group of histidine. prokaryotes and eukaryotes are shown in Table 1.2. Negative charges are provided by beta and gamma carboxyl groups of aspartic acid and glutamic acid (Fig.1.3). STUDY OF METABOLIC PROCESSES Hydrogen Bonds Our food contains carbohydrates, fats and proteins as principal ingredients. These macromolecules are to be These are formed by sharing of a hydro­gen between two first broken down to small units; carbohydrates to mono- electron donors. Hydrogen bonds result from electrostatic saccharides and proteins to amino acids. This process is taking place in the gastrointestinal tract and is called digestion or primary metabolism. After absorption, the small molecules are further broken down and oxidized to carbon dioxide. In this process, NADH or FADH2 are generated. This is named as secondary or intermediary metabolism. Finally, these reducing equi­valents enter the electron transport chain in the mitochondria, where they are oxidized to water; in this process energy is trapped as ATP. This is termed tertiary metabolism. Metabolism is the sum of all chemical changes of a compound inside the body, which includes synthesis (anabolism) and breakdown Fig. 1.1: Covalent bond (catabolism). (Greek word, kata = down; ballein = change). STABILIZING FORCES IN MOLECULES Covalent Bonds Molecules are formed by sharing of electrons between atoms (Fig. 1.1). Ionic Bonds or Electrostatic Bonds Ionic bonds result from the electrostatic attraction Fig. 1.2: Ionic bond between two ionized groups of opposite charges (Fig.1.2). They are formed by transfer of one or more TABLE 1.2: Bacterial and mammalian cells Prokaryotic cell Eukaryotic cell Size Small Large; 1000 to 10,000 times Cell wall Rigid Membrane of lipid bilayer Nucleus Not defined Well defined Organelles Nil Several; including mitochondria and lysosomes Fig. 1.3: Ionic bonds used in protein interactions  6 Textbook of Biochemistry attraction between an electronegative atom and a hydrogen Johannes van der Waals (1837–1923). He was awarded atom that is bonded covalently to a second electronegative Nobel prize in 1910. These are short range attractive atom. Normally, a hydrogen atom forms a covalent bond forces between chemical groups in contact. Van der Waals with only one other atom. However, a hydrogen atom co- interactions occur in all types of molecules, both polar and valently bonded to a donor atom, may form an additional non-polar. The energy of the van der Waals interaction weak association, the hydrogen bond with an acceptor atom. is about 1 kcal/mol and are unaffected by changes in In biological systems, both donors and acceptors are usually pH. This force will drastically reduce, when the distance nitrogen or oxygen atoms, especially those atoms in amino between atoms is increased. Although very weak, van der (NH2) and hydroxyl (OH) groups. Waals forces collectively contribute maximum towards the With regard to protein chemistry, hydrogen releasing stability of protein structure, especially in preserving the groups are –NH (imidazole, in dole, peptide); –OH (serine, non-polar interior structure of proteins. threonine) and –NH2 (arginine, lysine). Hydrogen accep­ting groups are COO— (aspartic, glutamic) C=O (peptide); and S–S WATER: THE UNIVERSAL SOLVENT (disulphide). The DNA structure is maintained by hydrogen bonding between the purine and pyrimidine residues. Water constitutes about 70 to 80 percent of the weight of most cells. The hydrogen atom in one water molecule is Hydrophobic Interactions attracted to a pair of electrons in the outer shell of an oxygen atom in an adjacent molecule. The structure of liquid water Non-polar groups have a tendency to associate with each other contains hydrogen-bonded networks (Fig. 1.5). in an aqueous environment; this is referred to as hydrophobic The crystal structure of ice depicts a tetrahedral interaction. These are formed by interactions between arrangement of water molecules. On melting, the molecules nonpolar hydrophobic side chains by eliminating water get much closer and this results in the increase in density molecules. The force that causes hydrophobic molecules of water. Hence, liquid water is denser than solid ice. This or nonpolar portions of molecules to aggregate together also explains why ice floats on water. rather than to dissolve in water is called the ‘hydrophobic Water molecules are in rapid motion, constantly making bond’ (Fig.1.4). This serves to hold lipophilic side chains of and breaking hydrogen bonds with adjacent molecules. amino acids together. Thus non-polar molecules will have As the temperature of water increases toward 100°C, the minimum exposure to water molecules. kinetic energy of its molecules becomes greater than the energy of the hydrogen bonds connecting them, and the Van Der Waals Forces gaseous form of water appears. The unique properties of These are very weak forces of attraction between all atoms, water make it the most preferred medium for all cellular due to oscillating dipoles, described by the Dutch physicist reactions and interactions. Fig. 1.4: Hydrophobic interaction Fig. 1.5: Water molecules hydrogen bonded  Chapter 1: Biochemical Perspective to Medicine 7 a. Water is a polar molecule. Molecules with polar bonds A closed system approaches a state of equilibrium. that can easily form hydrogen bonds with water can Any system can spontaneously proceed from a state of low dissolve in water and are termed “hydrophilic”. probability (ordered state) to a state of high probability b. It has immense hydrogen bonding capacity both with (disordered state). The entropy of a system may decrease other molecules and also the adjacent water molecules. with an increase in that of the surroundings. The second This contributes to cohesiveness of water. law may be expressed in simple terms as Q = T × ∆S, c. Water favors hydrophobic interactions and provides a where Q is the heat absorbed, T is the absolute temperature basis for metabolism of insoluble substances. and ∆S is the change in entropy. Water expands when it is cooled from 4° C to 0° C, while normally liquids are expected to contract due to Gibb's Free Energy Concept cooling. As water is heated from 0° C to 4° C, the hydrogen The term free energy is used to get an equation combining bonds begin to break. This results in a decrease in volume the first and second laws of thermodynamics. Thus, ∆G = or in other words, an increase in density. Hence, water ∆H – T∆S, where ∆G is the change in free energy, ∆H is attains high density at 4° C. However, above 4° C the effect the change in enthalpy or heat content of the system and ∆S of temperature predominates. is the change in entropy. The term free energy denotes a portion of the total energy change in a system that is PRINCIPLES OF THERMODYNAMICS available for doing work. For most biochemical reactions, it is seen that ∆H is Thermodynamics is concerned with the flow of heat and nearly equal to ∆E. So, ∆G = ∆E – T∆S. Hence, ∆G or it deals with the relationship between heat and work. free energy of a system depends on the change in internal Bioenergetics, or biochemical thermodynamics, is the energy and change in entropy of a system. study of the energy changes accompanying biochemical reactions. Biological systems use chemical energy to Standard Free Energy Change power living processes. It is the free energy change under standard conditions. It is First Law of Thermodynamics designated as ∆G0. The standard conditions are defined for The total energy of a system, including its surroundings, biochemical reactions at a pH of 7 and 1 M concen­tration, remains constant. Or, ∆E = Q – W, where Q is the heat and differentiated by a priming sign ∆G0´. It is directly absorbed by the system and W is the work done. This is related to the equilibrium constant. Actual free energy also called the law of conservation of energy. If heat is changes depend on reactant and product. transformed into work, there is proportionality between Most of the reversible metabolic reactions are near the work obtained and the heat dissipated. A system is an equilibrium reactions and therefore their ∆G is nearly zero. object or a quantity of matter, chosen for observation. All The net rate of near equilibrium reactions are effectively other parts of the universe, outside the boundary of the regulated by the relative concentration of substrates system, are called the surrounding. and products. The metabolic reactions that function far from equilibrium are irreversible. The velocities of these Second Law of Thermodynamics reactions are altered by changes in enzyme activity. A The total entropy of a system must increase if a highly exergonic reaction is irreversible and goes to process is to occur spontaneously. A reaction occurs completion. Such a reaction that is part of a metabolic spontaneously if ∆E is negative, or if the entropy of the pathway, confers direction to the pathway and makes the system increases. Entropy (S) is a measure of the degree entire pathway irreversible. of randomness or disorder of a system. Entropy becomes Laws of thermodynamics have many applications in maximum in a system as it approaches true equilibrium. biology and biochemistry, such as study of ATP hydrolysis, Enthalpy is the heat content of a system and entropy membrane diffusion, enzyme catalysis as well as DNA is that fraction of enthalpy which is not available to do binding and protein stability. These laws have been used to useful work. explain hypothesis of origin of life.  8 Textbook of Biochemistry Three Types of Reactions In Figure 1.6, the left compartment contains NaR, which will dissociate into Na+ and R¯. Then Na+ can diffuse A. A reaction can occur spontaneously when ∆G is freely, but R¯ having high molecular weight cannot diffuse. negative. Then the reaction is exergonic. If ∆G is of The right compartment contains NaCl, which dissociates great magnitude, the reaction goes to completion and into Na+ and Cl¯, in which case, both ions can diffuse freely. is essentially irreversible. Thus, if a salt of NaR is placed in one side of a B. When ∆G is zero, the system is at equilibrium. membrane, at equilibrium C. For reactions where ∆G is positive, an input of energy Na+ × R¯ × H+ × OH¯ = Na+ × OH¯ × H+ is required to drive the reaction. The reaction is termed as endergonic. (Examples are given in Chapter 5). To convey the meaning of the mathematical values, a Similarly a reaction may be exothermic (∆H is negative), hypothetical quantity of each of the ion is also incorporated isothermic (∆H is zero) or endothermic (∆H is positive). in brackets. Initially 5 molecules of NaR are added to the Energetically unfavourable reaction may be driven left compartment and 10 molecules of NaCl in the right forward by coupling it with a favourable reaction. compartment and both of them are ionized (Fig.1.6A). Glucose + Pi → Glucose-6-phosphate (reaction1) When equilibrium is reached, the distributions of ions are ATP + H2O → ADP + Pi (reaction 2) shown in Figure 1.6B. According to Donnan's equilibrium, Glucose + ATP→ Glucose-6-phosphate+ADP (3) the products of diffusible electrolytes in both the Reaction 1 cannot proceed spontaneously. But the compartments will be equal, so that 2nd reaction is coupled in the body, so that the reaction [Na+] L × [Cl¯ ] L = [Na+] R × [Cl¯ ] R becomes possible. For the first reaction, ∆G0 is +13.8 kJ/ If we substitute the actual numbers of ions, the formula mole; for the second reaction, ∆G0 is –30.5 kJ/mole. When becomes the two reactions are coupled in the reaction 3, the ∆G0 9 × 4 in left = 6 × 6 in right becomes –16.7 kJ/mole, and hence the reaction becomes Donnan's equation also states that the electrical possible. Details on ATP and other high-energy phosphate neutrality in each compartment should be maintained. In bonds are described in Chapter 20. other words the number of cations should be equal to the Reactions of catabolic pathways (degradation or number of anions, such that oxidation of fuel molecules) are usually exergonic. On the In left : Na+= R¯+ Cl¯; substituting: 9 = 5 + 4 other hand, anabolic pathways (synthetic reactions or building In right : Na+ = Cl¯; substituting: 6 = 6 up of compounds) are endergonic. Metabolism constitutes The equation should also satisfy that the number anabolic and catabolic processes that are well co-ordinated. of sodium ions before and after the equilibrium are the same; in our example, initial Na+ in the two compartments DONNAN MEMBRANE EQUILIBRIUM together is 5 + 10 = 15; after equilibrium also, the value is When two solutions are separated by a membrane 9 + 6 = 15. In the case of chloride ions, initial value is 10 permeable to both water and small ions, but when one of and final value is also 4 + 6 = 10. the compartments contains impermeable ions like proteins, In summary, Donnan's equations satisfy the following distribution of permeable ions occurs according to the results: calculations of Donnan. 1. The products of diffusible electrolytes in both compartments are equal. 2. The electrical neutrality of each compartment is maintained. 3. The total number of a particular type of ions before and after the equilibrium is the same. 4. As a result, when there is non-diffusible anion on A B one side of a membrane, the diffusible cations are Fig. 1.6: Donnan membrane equilibrium more, and diffusible anions are less, on that side.  Chapter 1: Biochemical Perspective to Medicine 9 Clinical Applications of the Equation concentration of negative non-diffusible hemoglobin 1. The total concentration of solutes in plasma will be ions. This will cause unequal distribution of H+ ions more than that of a solution of same ionic strength with a higher concentration within the cell. containing only diffusible ions; this provides the net 4. The chloride shift in erythrocytes as well as higher osmotic gradient (see under Albumin, in Chapter 28). concentration of chloride in CSF are also due to 2. The lower pH values within tissue cells than in the Donnan's effect. surrounding fluids are partly due to the concentrations 5. Osmolarity of body fluid compartments and sodium of negative protein ions within the cells being higher concentration will follow Donnan equation. than in surrounding fluids. 6. Different steps of water purification employ the 3. The pH within red cells is lower than that of the same principle and may be cited as an example of surrounding plasma is due, in part, to the very high industrial application of the equation.  CHAPTER 2 Subcellular Organelles and Cell Membranes Chapter at a Glance The reader will be able to answer questions on the following topics: ¾¾Nucleus ¾¾Transport mechanisms ¾¾Endoplasmic reticulum ¾¾Simple and facilitated diffusion ¾¾Golgi apparatus ¾¾Ion channels ¾¾Lysosomes ¾¾Active transport ¾¾Mitochondria ¾¾Uniport, symport and antiport ¾¾Plasma membrane SUBCELLULAR ORGANELLES NUCLEUS Cells contain various organized structures, collectively 1. It is the most prominent organelle of the cell. All cells called as cell organelles (Fig.2.1). When the cell membrane in the body contain nucleus, except mature RBCs in is disrupted, either by mechanical means or by lysing the circulation. The uppermost layer of skin also may not membrane by Tween-20 (a lipid solvent), the organized possess a readily identifiable nucleus. In some cells, particles inside the cell are homogenized. This is usually nucleus occupies most of the available space, e.g. carried out in 0.25M sucrose at pH 7.4. The organelles small lymphocytes and spermatozoa. could then be separated by applying differential centrifugal forces (Table 2.1). Albert Claude got Nobel prize in 1974 for fractionating subcellular organelles. Marker Enzymes Some enzymes are present in certain organelles only; such specific enzymes are called as marker enzymes (Table 2.1). Albert Camillo Christian George Claude Golgi de Duve Palade After centrifugation, the separated organelles are identified NP 1974 NP 1906 NP 1974 NP 1974 by detection of marker enzymes in the sample. 1899–1983 1843–1926 b.1917 1912–2008  Chapter 2: Subcellular Organelles and Cell Membranes 11 2. Nucleus is surrounded by two membranes—the inner This is the area for RNA processing and ribosome one is called perinuclear membrane with numerous synthesis. The nucleolus is very prominent in cells pores (Fig. 2.2) and the outer membrane is continuous actively synthesizing proteins. Gabriel Valentine in with membrane of endoplasmic reticulum. 1836 described the nucleolus. 3. Nucleus contains the DNA, the chemical basis of 6. Vesicular transport across membrane is by endocytosis genes, which governs all the functions of the cell. and exocytosis. Importin and exportin proteins are The very long DNA molecules are complexed with involved, and it is helped by RanGAP proteins. proteins to form chromatin and are further organized into chromosomes. 4. DNA replication and RNA synthesis (transcription) ENDOPLASMIC RETICULUM (ER) are taking place inside the nucleus. 1. It is a network of interconnecting membranes enclos- 5. In some cells, a portion of the nucleus may be seen as ing channels or cisternae, that are continuous from lighter shaded area; this is called nucleolus (Fig. 2.2). outer nuclear envelope to outer plasma membrane. TABLE 2.1: Separation of subcellular organelles Subcellular Pellet formed at the Marker enzyme organelle centrifugal force of Nucleus 600–750 x g, 10 min Mitochondria 10,000–15,000 x g, Inner membrane: 10 min ATP Synthase Lysosome 18,000–25,000 x g, Cathepsin 10 min Golgi 35,000–40,000 x g, Galactosyl complex 30 min transferase Microsomes 75,000–100,000 x g, Glucose-6- 100 min phosphatase Cytoplasm Supernatant Lactate dehydrogenase Fig. 2.2: Nucleus Fig. 2.1: A typical cell 12 Textbook of Biochemistry Under electron microscope, the reticular arrange- secretion, e.g. secretion of immunoglobulins by ments will have railway track appearance (Fig. 2.1). plasma cells. George Palade was awarded Nobel prize in 1974, who b. They reach plasma membrane and form an integral identified the ER. part of it, but not secreted. 2. This will be very prominent in cells actively c. They form a secretory vesicle, where these products synthesizing proteins, e.g. immunoglobulin secreting are stored for a longer time. Under appropriate plasma cells. The proteins, glycoproteins and stimuli, the contents are secreted. Release of lipoproteins are synthesised in the ER. trypsinogen by pancreatic acinar cells and release 3. Detoxification of various drugs is an important of insulin by beta cells of Langerhans are cited as function of ER. Microsomal cytochrome P-450 examples. hydroxylates drugs, such as benzpyrine, amino- d. The synthesized materials may also reach pyrine, aniline, morphine, phenobarbitone, etc. lysosome packets. 4. According to the electron microscopic appearance, e. Golgi bodies are fragmented during mitosis, but the ER is generally classified into rough and smooth get reorganized by interaction with microtubules. varieties. The rough appearance is due to ribosomes Connective tissue disorders like Sjogren’s attached to cytoplasmic side of membrane where the syndrome are found to be associated with anti- proteins are being synthesized. golgi antibodies. 5. When cells are fractionated, the complex ER is disrupted in many places. They are automatically LYSOSOMES reassembled to form microsomes. 6. ERGIC (Endoplasmic reticulum - Golgi intermediate compartment): 1. Discovered in 1950 by Christian de Duve (Nobel prize The synthesized protein pass through this compartment before going 1974), lysosomes are tiny organelles. Solid wastes to the cis Golgi. Box 2.1: Clinical applications of lysosomes GOLGI APPARATUS 1. In gout, urate crystals are deposited around knee joints (see Chapter 39). These crystals when phagocyto­sed, cause 1. Camillo Golgi described the structure in 1898 (Nobel physical damage to lysosomes and release of enzymes. prize 1906). The Golgi organelle is a network of Inflammation and arthritis result. flattened smooth membranes and vesicles. It may be 2. Following cell death, the lysosomes rupture releasing the considered as the converging area of endoplasmic hydrolytic enzymes which bring about postmortem autolysis. reticulum (Fig. 2.1). 3. Lysosomal proteases, cathepsins are implicated in tumor metastasis. Cathepsins are normally restricted to the interior 2. While moving through ER, carbohydrate groups are of lysosomes, but certain cancer cells liberate the cathepsins successively added to the nascent proteins. These out of the cells. Then cathepsins degrade the basal lamina by glycoproteins reach the Golgi area. hydrolyzing collagen and elastin, so that other tumor cells can 3. Golgi apparatus is composed of cis, medial and trans cisternae. travel out to form distant metastasis. Glycoproteins are generally transported from ER to cis Golgi 4. There are a few genetic diseases, where lysosomal enzymes (proximal cisterna), then to medial Golgi (intermediate cisterna) are deficient or absent. This leads to accumulation of lipids or and finally to trans Golgi (distal cisterna) for temporary storage. polysaccharides (see Chapters 10 and 14). Trans Golgi is particularly abundant with vesicles containing 5. Silicosis results from inhalation of silica particles into the lungs glycoproteins. Newly synthesized proteins are sorted first which are taken up by phagocytes. Lysosomal membrane according to the sorting signals available in the proteins. Then they ruptures, releasing the enzymes. This stimulates fibroblast are packaged into transport vesicles having different types of coat to proliferate and deposit collagen fibers, resulting in fibrosis proteins. Finally they are transported into various destinations; this and decreased lungs elasticity. is an energy dependent process. 6. Inclusion cell (I-cell) disease is a rare condition in which 4. Main function of Golgi apparatus is protein sorting, lysosomes lack in enzymes, but they are seen in blood. This means that the enzymes are synthesized, but are not able to packaging and secretion. reach the correct site. It is shown that mannose-6-phosphate 5. The finished products may have any one of the is the marker to target the nascent enzymes to lysosomes. In following destinations: these persons, the carbohydrate units are not added to the a. They may pass through plasma membrane to enzyme. Mannose-6-phosphate deficient enzymes cannot the surrounding medium. This forms continuous reach their destination (protein targeting defect).  Chapter 2: Subcellular Organelles and Cell Membranes 13 of a township are usually decomposed in incinerators. MITOCHONDRIA Inside a cell, such a process is taking place within the lysosomes. They are bags of enzymes. Clinical 1. They are spherical, oval or rod-like bodies, about applications of lysosomes are shown in Box 2.2. 0.5–1 mm in diameter and up to 7 mm in length 2. Endocytic vesicles and phagosomes are fused with (Fig. 2.1). Erythrocytes do not contain mitochondria. lysosome (primary) to form the secondary lysosome The tail of sper­matozoa is fully packed with or digestive vacuole. Foreign particles are pro­ mitochondria. gressively digested inside these vacuoles. Completely 2. Mitochondria are the powerhouse of the cell, where energy released from oxidation of food stuffs is hydrolyzed products are utilized by the cell. As long trapped as chemical energy in the form of ATP (see as the lysosomal membrane is intact, the encapsulated Chapter 20). Metabolic functions of mitochondria are enzymes can act only locally. But when the membrane shown in Table 2.2. is disrupted, the released enzymes can hydrolyze 3. Mitochondria have two membranes. The inner mem­ external substrates, leading to tissue damage. brane convolutes into folds or cristae (Fig. 2.3). The 3. The lysosomal enzymes have an optimum pH around 5. These enzymes are: inner mitochon­drial membrane contains the enzymes a. Polysaccharide hydrolyzing enzymes (alpha-gluco­ sidase, of electron transport chain (see Chapter 20). The alpha-fucosidase, beta-galactosidase, alpha-mannosidase, beta- fluid matrix contains the enzymes of citric acid cycle, glucuronidase, hyaluronidase, aryl sulfatase, lysozyme) urea cycle and heme synthesis. b. Protein hydrolyzing enzymes (cathepsins, collagenase, elastase, 4. Cytochrome P-450 system present in mitochondrial peptidases) inner membrane is involved in steroido­ge­nesis (see c. Nucleic acid hydrolyzing enzymes (ribonuclease, deoxyribo- Chapter 52). Superoxide dismutase is present in nuclease) cytosol and mitochondria (see Chapter 33). d. Lipid hydrolyzing enzymes (fatty acyl esterase, phospholipases). 5. Mitochondria also contain specific DNA. The integral inner membrane proteins, are made by mitochondrial PEROXISOMES protein synthesizing machinery. However, the 1. The peroxisomes have a granular matrix. They are of majority of proteins, especially of outer membrane 0.3–1.5 mm in diameter. They contain peroxidases and are synthesized under the control of cellular DNA. catalase. They are prominent in leukocytes and platelets. The division of mitochondria is under the command 2. Peroxidation of polyunsaturated fatty acids in vivo of mitochondrial DNA. Mitochondrial ribosomes may lead to hydroperoxide formation, R-OOH → are different from cellular ribosomes. Antibiotics R-OO. The free radicals damage molecules, cell inhibiting bacterial protein synthesis do not affect membranes, tissues and genes. (see Chapter 33). cellular processes, but do inhibit mitochondrial 3. Catalase and peroxidase are the enzymes present in protein biosynthesis (see Chapter 45). peroxisomes, which will destroy the unwanted peroxides and other free radicals. Clinical applications of peroxisomes are shown in Box 2.2. Box 2.2: Peroxisomal deficiency diseases 1. Deficiency of peroxisomal matrix proteins can lead to adrenoleukodystrophy (ALD) (Brown-Schilder’s disease) characterized by progressive degeneration of liver, kidney and brain. It is a rare autosomal recessive condition. The defect is due to insufficient oxidation of very long chain fatty acids (VLCFA) by peroxi­somes (see Chapter 14). 2. In Zellweger syndrome, proteins are not transported into the peroxisomes. This leads to formation of empty peroxisomes or peroxisomal ghosts inside the cells. Protein targeting defects are described in Chapter 46. 3. Primary hyperoxaluria is due to the defective peroxisomal metabolism of glyoxalate derived from glycine (see Chapter 16). Fig. 2.3: Mitochondria 14 Textbook of Biochemistry 6. Mitochondria play a role in triggering apoptosis (see 2. The enzyme, nucleotide phosphatase (5' nucleotidase) Chapter 47). and alkaline phosphatase are seen on the outer 7. Taking into consideration such evidences, it is assumed that part of cell membrane; they are therefore called mitochondria are parasites, which entered into cells at a time ecto-enzymes. when multicellular organisms were being evolved. These parasites provided energy in large quanti­ ties giving an evolutionary 3. Membranes are mainly made up of lipids, proteins advantage to the cell; the cell gave protection to these parasites. and small amount of carbohydrates. The contents of This perfect symbiosis, in turn, evolved into a cellular organelle these compounds vary according to the nature of the of mitochondria. membrane. The carbohydrates are present as glyco- 8. Mitochondria are continuously undergoing fission and fusion, proteins and glycolipids. Phospholipids are the most resulting in mixing of contents of mitochondrial particles. Specific fission and fusion proteins have been identified and abnormalities common lipids present and they are amphipathic in in some of these proteins are implicated in diseases like Charcot- nature. Cell membranes also contain cholesterol. Marie-Tooth disease. 9. New evidence suggests a role for mitochondria in the genesis of systemic inflammatory response. The mitochondrial particles Fluid Mosaic Model released from damaged tissue may evoke an antigenic response The lipid bilayer was originally proposed by Davson and from the immune system. Danielle in 1935. Later, the structure of the biomembranes 10. A summary of functions of organelles is given in was described as a fluid mosaic model (Singer and Table 2.2 and Box 2.3. Nicolson, 1972). A. The phospholipids are arranged in bilayers with the polar head PLASMA MEMBRANE groups oriented towards the extracellular side and the cytoplasmic side with a hydrophobic core (Fig. 2.4A). The distribution of 1. The plasma membrane separates the cell from the the phospholipids is such that choline containing phospholipids external environment. It has highly selective permea­­ are mainly in the external layer and ethanolamine and serine containing phospholipids in the inner layer. Gerd Binnig and bility properties so that the entry and exit of compounds Heinrich Rohrer introduced the scanning electron microscopy in are regulated. The cellular metabolism is in turn influ­ 1981 by which the outer and inner layers of membranes could be enced and probably regulated by the membrane. The visualized separately. They were awarded Nobel prize in 1986. membrane is metabolically very active. B. Each leaflet is 25 Å thick, with the head portion 10 Å and tail 15 Å thick. The total thickness is about 50 to 80 Å. C. The lipid bilayer shows free lateral movement of its components, TABLE 2.2: Metabolic functions of subcellular organelles hence the membrane is said to be fluid in nature. Fluidity enables Nucleus DNA replication, transcription the membrane to perform endocytosis and exocytosis. D. However, the components do not freely move from inner to outer Endoplasmic Biosynthesis of proteins, glycoproteins, reticulum lipoproteins, drug metabolism, ethanol oxidation, layer, or outer to inner layer (flip-flop movement is restricted). During synthesis of cholesterol (partial) apoptosis (programed cell death), flip-flop movement occurs. This flip-flop movement is catalyzed by enzymes. Flippases Golgi body Maturation of synthesized proteins catalyze the transfer of amino phospholipids across the membrane. Lysosome Degradation of proteins, carbohydrates, lipids and Floppases catalyze the outward directed movement, which is nucleotides Mitochondria Electron transport chain, ATP generation, TCA cycle, beta oxidation of fatty acids, ketone body Box 2.3: Comparison of cell with a factory production, urea synthesis (part), heme synthesis Plasma membrane : F ence with gates; gates open (part), gluconeogenesis (part), pyrimidine when message is received synthesis (part) Nucleus : Manager’s office Endoplasmic reticulum : Conveyer belt of production units Cytosol Protein synthesis, glycolysis, glycogen metabolism, Golgi apparatus : Packing units HMP shunt pathway, transaminations, fatty acid synthesis, cholesterol synthesis, heme synthesis Lysosomes : Incinerators (part), urea synthesis (part), pyrimidine synthesis Vacuoles : Lorries carrying finished products (part), purine synthesis Mitochondria : Power generating units  Chapter 2: Subcellular Organelles and Cell Membranes 15 ATP dependent. This is mainly seen in the role of ABC proteins The nature of fatty acids and cholesterol content varies mediating the efflux of cholesterol and the extrusion of drugs from depending on diet. A higher proportion of PUFA, which increases cells. The MDR associated p-glycoprotein is a floppase. the fluidity favors the binding of insulin to its receptor, a trans- E. The cholesterol content of the membrane alters the membrane protein. The lipids making up components of membranes are of three fluidity of the membrane. When cholesterol concentra­ major classes that includes glycerophospholipids, sphingolipids, tion increases, the membrane becomes less fluid on and cholesterol. Sphingolipids and glycerophospholipids the outer surface, but more fluid in the hydrophobic constitute the largest percentage of the lipid weight of biological core. The effect of cholesterol on membrane fluidity membranes. Proteins that are found associated with membranes can also be modified by lipid attachment (lipoproteins). The lipid is different at different temperatures. At temperature portion of a lipoprotein anchors the protein to the membrane below the Tm, cholesterol increases fluidity and either through interaction with the lipid bilayer directly or through there-by permeability. At temperatures above the Tm, interactions with integral membrane proteins. Lipoproteins cholesterol decreases fluidity. associated with membranes contain one of three types of covalent In spur cell anemia and alcoholic cirrhosis, membrane lipid attachment. The lipids are isoprenoids such as farnesyl and studies have revealed the role of excess cholesterol. The decrease geranyl residues, fatty acids such as myristic and palmitic acid, and in membrane fluidity may affect the activities of receptors and glycosylphosphatidyl inositol (GPI). ion channels. This has been implicated in conditions like LCAT deficiency, Alzheimer’s disease and hypertension. Membrane Proteins Fluidity of cellular membranes responds to variations in diet and physiological states. Increased release of reactive oxygen A. The peripheral proteins exist on the surfaces of the species (ROS), increase in cytosolic calcium and lipid peroxidation bilayer (Fig. 2.4B). They are attached by ionic and have been found to adversely affect membrane fluidity. Anesthetics polar bonds to polar heads of the lipids. may act changing membrane fluidity. B. Anchoring of proteins to lipid bilayers: Several peripheral F. The nature of the fatty acids also affects the fluidity of membrane proteins are tethered to the membranes by covalent the membrane, the more unsaturated cis fatty acids linkage with the membrane lipids. Since the lipids are inserted increase the fluidity. into the hydrophobic core, the proteins are firmly anchored. A The fluidity of the membrane is maintained by the length of typical form of linkage is the one involving phosphatidyl inositol the hydrocarbon chain, degree of unsaturation and nature of the which is attached to a glycan. This glycan unit has ethanolamine, polar head groups. Trans fatty acids (TFA) decrease the fluidity phosphate and several carbohydrate residues. This glycan chain of membranes due to close packing of hydrocarbon chains. Cis includes a glucose covalently attached to the C terminus of a double bonds create a kink in the hydrocarbon chain and have protein by ethanolamine and to the phosphatidyl inositol by a marked effect on fluidity. Second OH group of glycerol in glucosamine. The fatty acyl groups of the phosphatidyl inositol membrane phospholipids is often esterified to an unsaturated fatty diphosphate (PIP2) are firmly inserted into the lipid membrane acid, monounsaturated oleic or polyunsaturated linoleic, linolenic thus anchoring the protein. This is referred to as glycosyl or arachidonic. phosphatidyl inositol (GPI) anchor. Fig. 2.4A: The fluid mosaic model of membrane Fig. 2.4B: Proteins are anchored in membrane by different mechanisms  16 Textbook of Biochemistry C. Microdomains on membranes: GPI anchored proteins are units of N-acetyl muramic acid (NAM) and N-acetyl glucosamine often attached to the external surface of plasma membrane at (NAG). This polysaccharide provides mechanical strength to the microdomains called lipid rafts. They are areas on the membrane plasma membrane. Synthesis of this complex polysaccharide is blocked having predominantly glycosphingolipids and cholesterol. by penicillin. This inhibition is responsible for the bactericidal action The localization and activity of the protein can be regulated by of penicillin. anchoring and release. Defective GPI anchors are implicated in Paroxysmal nocturnal hemoglobinuria (PNH). These lipid rafts SPECIALIZED MEMBRANE STRUCTURES are implicated in endocytosis, G protein signaling and binding of viral pathogens. Lipid rafts are areas on the membrane having Tight Junction predominantly glycosphingolipids and cholesterol. The GPI anchors that tether proteins to the membrane are also seen at When two cells are in close approximation, in certain areas, instead the lipid rafts. Membrane proteins may be anchored by covalent of 4 layers, only 3 layers of plasma membranes are seen. This tight bonding, palmitoylation and myristoylation. junction permits calcium and other small molecules to pass through D. Caveolae are flask shaped indentations on the areas of lipid rafts that from one cell to another through narrow hydrophilic pores. Some are involved in membrane transport and signal transduction. Caveolae sort of communication between cells thus results. Absence of tight contain the protein caveolin, along with other receptor proteins. junction is implicated in loss of contact inhibition in cancer cells Transport of macromolecules (IgA) from the luminal side occurs (see Chapter 57). Tight junctions also seal off subepithelial spaces by caveolae mediated transcytosis. The endocytosis of cholesterol of organs from the lumen. They contain specialized proteins, such as containing lipoproteins may be caveolae mediated. Similarly the occludin, claudins and other adhesion molecules. Most eukaryotic cells are in contact with their neighboring cells and fusion and budding of viral particles are also mediated by caveolae. these interactions are the basis of formation of organs. Cells that abut one E. The integral membrane proteins are deeply embed­ another are in metabolic contact, which is brought about by specialized ded in the bilayer and are attached by hydrophobic particles called gap junctions. Gap junctions are intercellular channels bonds or van der Waals forces. and their presence allows whole organs to be continuous from within. F. Some of the integral membrane proteins span the One major function of gap junctions is to ensure a supply of nutrients to cells of an organ that are not in direct contact with the blood supply. Gap whole bilayer and they are called transmembrane junctions are formed from a type of protein called connexin. proteins (Fig. 2.4). The hydrophobic side chains of the amino acids are embedded in the hydrophobic central Myelin Sheath core of the membrane. The transmembrane proteins It is made up of the membrane of Schwann's cells, (Theodor Schwann, can serve as receptors (for hormones, growth factors, 1858) condensed and spiralled many times around the central axon. The neurotransmitters), tissue specific antigens, ion channels, cytoplasm of Schwann cells is squeezed to one side of the cell. Myelin is membrane-based enzymes, etc. composed of sphingomyelin, cholesterol and cerebroside. Myelin sheaths thin out in certain regions (Node of Ranvier) (Anotoine Ranvier, 1878). Due to this arrangement, the propagation of nerve impulse is wave-like; Bacterial Cell Wall and the speed of propagation is also increased. Upon stimulation, there is Prokaryotic (bacterial) cells as well as plant cells have a cell wall rapid influx of sodium and calcium, so that depolarization occurs. Voltage surrounding the plasma membrane; this cell wall provides mechanical gradient is quickly regained by ion pumps. The ions flow in and out of strength to withstand high osmotic pressure. Animal cells are devoid membrane only where membrane is free of insulation; hence the wave- of the cell wall; they have only plasma membrane. Major constituent like propagation of impulse. In multiple sclerosis, demyelination occurs of bacterial cell wall is a heteropolysacc­haride, consisting of repeating at discrete areas, velocity of nerve impulse is reduced, leading to motor and sensory deficits. Microvilli Microvilli of intestinal epithelial cells and pseudopodia of macrophages are produced by membrane evagination. This is due to the fluid nature of membranes. Membranes of Organelle Membranes of endop