Protein Lecture 3,4 PDF

Reasons for Protein Digestion: A. To get through the membrane (i.e., uptake/absorption). B. To resynthesize necessary proteins. C. For the immune process/purpose. Digestion: I- In the mouth: - No protein digestion. II- In stomach: (A) HCl: - Gastric juice in the stomach (HC1) is important for...

Reasons for Protein Digestion: A. To get through the membrane (i.e., uptake/absorption). B. To resynthesize necessary proteins. C. For the immune process/purpose. Digestion: I- In the mouth: - No protein digestion. II- In stomach: (A) HCl: - Gastric juice in the stomach (HC1) is important for conversion of native protein to acid-metaprotein. This phase causes denaturation of protein as a result of disruption of its secondary, tertiary and quaternary structures. HCl Native Protein Acid Meta protein (B) Pepsin: - Source: it is secreted by the chief cells of gastric mucosa. -pH: 1-2. - Mode of secretion: it is secreted as a zymogen called pepsinogen (inactive form). - Mode of activation: HCL Pepsinogen Pepsin - Pepsinogen is activated by HCl or by pepsin (autoactivation). Action: it is endopeptidases which act on the amino acids in the middle of the polypeptide chain hydrolyzing the bonds between phenyl alanine , tyrosine and tryptophan. - End product: the products of pepsin digestion are a mixture of Protein , Proteoses, Peptones , Polypeptides (C) Rennin: - Source: parietal cells. - pH: 4. It is present in infants' stomach only because its pH is suitable for the enzyme action. - Mode of secretion: it is secreted as a zymogen called pro-rennin (inactive form). - Mode of activation: the pro-rennin is activated by calcium ions to the active rennin Ca++ Pro-rennin Rennin - Action: it acts on casein of milk (main protein of milk) converting it in presence of calcium ions into insoluble calcium caseinate (milk clot). (Rennin Ca ++ ) Casein Para-casein Calcium Caseinate (Insoluble) - The digestion of insoluble calcium caseinate is completed by pepsin enzyme. Formation of milk clot in infants' stomach give the sense of fullness. Rennin is absent in stomach of adults because its pH is unsuitable for the enzyme action. - End product: insoluble calcium caseinate (milk clot). III-In Duodenum: Pancreatic juice reaches the duodenum via the common bile duct and it contains the following enzymes: (A) Trypsin and chymotrypsin: - Source: pancreatic acini. - pH : 8. - Mode of secretion: Trypsin and Chymotrypsin are secreted as zymogens which are called trypsinogen and chymotrypsinogen respectively. - Mode of activation: Enterokinase Trypsinogen Trypsin. Trypsin Trypsinogen Trypsin Trypsin Chymotrypsinogen Chymotrypsin - Action of trypsin: it is endo-peptidase hydrolyzing the bonds between basic amino acids as arginine and lysine. - Action of chymotrypsin: it is an endo-peptidase act on peptide bonds between uncharged amino acids as aromatic amino acids. - End products: products of trypsin and chymotrypsin digestion are mixtures of proteoses, peptone and polypeptides. (B) Carboxypeptidase: - Source: pancreatic acini. - pH: 8. - Mode of secretion: inactive zymogen called pro-carboxypeptidase. - Mode of activation: by trypsin. - Action: It is an exo-peptidase which acts on the periphery of polypeptide Chains produced by the action of endo-peptidases. It acts on the peptide bond at the free- COOH of the polypeptide chain. - End products: single amino acids. (C) Elastase(s): (1) Proelastase is also activated by trypsin. (2) Only enzyme active against elastin. (3) Elastase is an endopeptidase, and acts especially on nonpolar AA (Val, Leu, Ser & Ala) & produces peptides. pH : 8. III - In Small Intestines: (A) Amino-peptidase: - Source: from glands of_Bruner and Lieberkuhn. - pH : 5 - 7. - Mode of secretion: active. - Action: it is exo-peptidase acting on the peptide bond at the free NH2 of the polypeptide chain. - End products: free amino acids. (B) Tri-peptidases and di-peptidases: - Source: from glands of Bruner and Lieberkuhn. - pH: 5-7. - Mode of secretion: active. - Action:.they is exo-peptidases acting on tri-peptides and di- peptides respectively. - End products: free amino acids.  Absorption  - Under normal conditions the dietary proteins are almost completely digested into amino acids which are then rapidly absorbed from the intestines into the portal blood (as carbohydrates).  - Site of absorption: jejunum and ileum.  - It is active processes that need energy which gain from the hydrolysis of ATP molecules. Mechanism of absorption:  Intestinal epithelial cells absorb protein by endocytosis. Enterocytes can take up by endocytosis a small amount of intact protein, most of which is degraded in lysosomes. A small amount of intact protein appears in the interstitial space. (A) Active transport: - L-amino acid (naturally occurring) are actively absorbed where energy is required which is derived from sodium pump. - They are absorbed by specific carrier protein present in small intestines by a mechanism similar to that of glucose absorption. - The carrier has a site for an amino acid and another site for sodium. - There are 5 or more different amino acid carrier systems each of which can transport a group of closely related amino acids: 1- Small neutral amino acids. 2- Large neutral amino acids. 3- Basic amino acids. 4- Acidic amino acids. 5- L- amino acids. -There is a competition of absorption between the amino acids of each group which means that amino acid fed in excess can retard the absorption of other amino acids of the same group. - Pyridoxal (vitamin B6) & Mn++ play a role in amino acid absorption. (B) Passive transport: - D-amino acids are absorbed by simple diffusion. - Absorption of proteins without digestion occurs in the following conditions: (1) Normally in infants: absorption of immunoglobulins present in colostrum which is the secretion of mammary gland in the first few days after labour, provide immunity to the baby against infections during the first 6 months of life. (2) Abnormally in adults: a small pan of protein escapes digestion and is absorbed as such. The body will react against this foreign material which acts as an antigen leading to formation of antibodies against this specific protein. If the person eats the same type of protein once more, this antigen will react with its specific antibody already formed leading to what is called antigen-antibody reaction producing allergic manifestations. Mechanism of amino acids absorption: There are two mechanisms of amino acids absorption as following: 1- Carrier proteins transport system - It is the main system for amino acid absorption. - It is an active process that needs energy. - The energy needed is derived from ATP, as the absorption of amino acid needs one molecule. - There are 7 carrier proteins, one for each group of amino acid. - Each carrier protein has two sites one for amino acid and one for Na+. - It co-transports amino acid and Na+ from intestinal lumen to cytosol of intestinal mucosa cells. - The absorbed amino acid passes to the portal circulation, while Na+ is extruded out of the cell in exchange with K+ by sodium pump. 2- Glutathione transport system (ɤ-Glutamyle cycle): - Glutathione is used to transport amino acid from intestinal lumen to cytosol of intestinal mucosa cells. - It is an active process that needs energy. - The energy needed is derived from ATP where the absorption of amino acid needs 3 molecules of ATP. -Glutathione reacts with amino acid in the presence of ɤ-Glutamyle trans-peptidase to form ɤ-Glutamyle amino acid. - ɤ-Glutamyle amino acid releases amino acid in the cytosol of the intestinal mucosa with formation of 5-oxoproline that is used for the generation of glutathione to begin another turn of the cycle. Dynamic state of body protein: - All the body proteins with the exception of collagen are in constant state of degradation and re-synthesis. The total amount of protein synthesized in the body is much greater than that degraded which proves the body needs of protein for growth replacement of cells after their death and formation of milk protein during lactation. Protein requirement: - Adult man needs about 30-60 gm/day (0.8 gm/kg body weight/day). -Exact daily protein requirement depends on many factors: (1) Age, sex and lactation: children, pregnant and lactating females need more protein than adult males do. (2) Quality of protein: according to the concentration of essential amino acids. - Nitrogen intake: Proteins and nucleoproteins of diet are the main sources of nitrogen to the body. Every 100 g proteins contain 16g nitrogen. - Nitrogen output: nitrogen is excreted from the body in: 1 - Urine (the main rout): in the form of non-protein nitrogenous compounds (NPN) which is: (1) Urea (10 - 50 gm/day) (2) Creatinine (0.7 - 1.7 gm/day). (3) Uric acid (0.7 gm/day) (4) Creatine (0 - 0.2 gm/day). (5) Ammonia (0.7 gm/day) (6) Amino acids (small amount). 2- Stools: in the form of digestive juices, shed off epithelial cells and non- digested fat e.g. phospholipids (about 1 gm/day). 3- Sweat: in the form of urea. 4 - Milk and menstrual fluids: in females. 5- Hairs and nails: normally 5-7 gm nitrogen is lost per day. - Definition: it is the quantitative difference between the nitrogen intake and nitrogen output in g per day. There are 3 states of nitrogen balance: (1) Nitrogen equilibrium: exists when nitrogen intake equals nitrogen output. It occurs in healthy adults on an adequate diet. (2) Positive nitrogen balance: exists when nitrogen intake is more than output as occurs in pregnancy, lactation, recovery from wasting diseases, babies and children during their growth period and muscular exercise. (3) Negative Nitrogen balance: exists when nitrogen output exceeds nitrogen intake. This excess nitrogen is derived from catabolism of tissue proteins as occurs in wasting diseases (e.g. cancer. T.B.. typhoid …etc) loss of proteins from the body (e.g. chronic hemorrhage, albuminuria and lactation with an inadequate diet, increased protein catabolism (e.g. in diabetes mellitus, Cushing syndrome. hyperthyroidism and infectious diseases), in low protein diet and in deficiency of essential amino acids.  - The plasma level of amino acids don't remain constant throughout the day, it varies from 4-8 mg/dl, depending upon the nutritional state whether it is post-absorption or fed state.  This is called the circadian changes of amino acids. (1) Exogenous sources which are absorbed amino acids of diet proteins. (2) Catabolism of tissue proteins. (3) Endogenous sources by the synthesis of non-essential amino acids. The absorbed amino acid may undergo one of the following fates: (1) Protein synthesis "anabolism”: the amino acids are incorporated into proteins as plasma proteins, tissue proteins, enzymes and some hormones. (2) Synthesis of important nitrogenous compounds: as glutathione, purine and pyrimidine bases. (3) Ketogenic amino acids give ketone bodies. (4) Glucogenic amino acids give glucose by transamination. (5) Synthesis of other specialized products: each amino acid can synthesize certain substances in the body e.g. phenylalanine gives adrenaline, tryptophan gives serotonin. (6) Catabolism of amino acids producing α -ketoacids and urea. Definition: - It is the removal of amino group (NH2) from amino acid in the form of ammonia (NH3). Site: - Liver and kidney. Types: 1- Transamination. 2- Oxidative deamination. 3- Trans-deamination. I- Transamination: - It is the transfer of an amino group from α-amino acid to form a new α -amino acid and a new α -keto acid. Site: - Transaminases are present in the mitochondria and cytosol of most tissues especially the liver. Mechanism: 1- Transamination is catalyzed by enzymes termed transaminases or amino transferases. 2- Pyridoxal phosphate (vitamin B6) is the coenzyme of transaminases. 3- All amino acids make transamination except lysine, threonine, proline and hydroxyproline. 4- Transamination reactions are reversible. 5- Transaminases of clinical importance are; ALT (GPT) and AST (GOT). Glutamate pyruvic transaminase (GPT) or Alanine transaminase (ALT): Glutamic oxaloacetic transaminase (GOT) or Aspartic transaminase (AST): Clinical importance of AST (SGOT) and ALT (SGPT): - AST and ALT are intracellular enzymes. - Their blood level increases if there is destruction to the cells as in: - Myocardial infarction (coronary thrombosis). - Acute hepatitis (liver infection). II- Oxidative deamination: - The removal of the amino group is accompanied by oxidation. Site: liver and kidney. Oxidative deamination occurs by the help of the following enzymes: L- Amino acid dehydrogenase: - It is an enzyme which works on L-amino acids (except glutamate). - This enzyme is not widely distributed in the body and has low activity. -It present in liver and kidney and needs FMN as a coenzyme. - All the reactions catalyzed are reversible. L-A.A. dehydrogenase is an aerobic dehydrogenase enzyme since FMNH2 gives its hydrogen directly to oxygen of atmosphere giving H2O2. - The hydrogen peroxide produced will be acted upon by catalase giving water plus oxygen. - Imino acid produced is hydrolyzed to give α-keto acid (glyoxalic acid) and NH3 In presence of water. D-amino acid dehydrogenase: - It is an enzyme working on D-amino acids (and glycine probably) - Its coenzyme is FAD. - D-amino acids are not present in the human body but are present in some animals and bacteria. - This enzyme is present in human liver and kidney but its function is not known. - Mode of action is similar to L-A.A. dehydrogenase. L-Glutamic acid dehydrogenase: - It acts on L-glutamic acid. - It is highly active and widely distributed in different tissues. Its coenzyme is NAD + or NADP +. - It is an anaerobic dehydrogenase because NADH+H+ can't give its hydrogen directly to the oxygen but is carried through respiratory chain. - The imino acid produced is hydrolyzed to NH3 and α-ketoglutaric acid. - All the reactions are reversible. III – Trans-deamination: - It is a combination of transamination and oxidative deamination. - The amino group is first removed from a given amino acid by transamination with α - ketoglutaric acid. - The resulting glutamic acid is then de-aminated by glutamic acid dehydrogenase giving free ammonia and α-ketoglutaric acid as previously explained. - This system is very active, reversible and widely distributed in animal tissues. Sources of ammonia: 1- Deamination of amino acids by different tissues. 2- Ammonia produced by the action of intestinal bacteria on: (a) Dietary protein. (b) Urea present in fluids secreted into the GIT tract. 3- Kidneys produce ammonia by renal tubular cells in cases of acidosis, a mechanism for regulation of acid-base balance. Blood ammonia: -Ammonia is highly toxic and its blood level is only 10- 20 ug/dl. When ammonia is formed by the tissues it is rapidly removed from the circulation by the liver. Fate of ammonia: A- Anabolic pathway: 1 -Formation of glutamate and other non-essential amino acids. 2 - Synthesis of purines and pyrimidines. B-Catabolic pathway: 1- Glutamine formation: - Glutamine synthetase enzyme is a mitochondrial enzyme present in the kidneys and the brain. Fate of ammonia: (a) In the brain: - Glutamine formation is the major mechanism for removal of ammonia from the brain. - Glutamine passes to the kidney then excreted in the urine. In liver cell failure, large amounts of ammonia appear in the blood, ammonia passes the blood brain barrier, the brain gets rid of this ammonia by conversion of glutamic acid to glutamine.  This will lead to depletion of α - ketoglutaric acid with subsequent inhibition of Kreb's cycle. This will deprive the brain from its energy leading to ammonia intoxication and coma. (b)- In the kidney: - Glutamine is stored in tubular cells. - Deamination of glutamine is the main source of NH3 in kidney. - NH3 formed from deamination of glutamine in kidneys represents 60 % of excreted ammonia. - This deamination reaction is catalyzed by glutaminase enzyme. Excretion in urine: - Ammonia resulting from the deamination of amino acids in the kidney is directly excreted in the urine. - This accounts for 40% of urinary ammonia. Ammonia enters in the formation of urea. - Urea is the main end product of amino acid catabolism. Site: Liver is the only site (in mitochondria). Steps: It is a 5 steps pathway called urea cycle.  - It is the main urinary non-protein nitrogenous compound (NPN).  - It is derived from amino acids of protein diet formed in the liver and passes to blood to be excreted by kidney.  - Blood urea level is 10-50 mg/dl;  - Urine urea level is 10-50 mg/day.  - Urea of urine is totally of exogenous origin. Regulation of urea cycle: - Carbamoyl phosphate synthetase acts with mitochondrial glutamate dehydrogenase. - The ammonia produced from glutamic acid and hence from all amino acids by glutamate dehydrogenase are used by carbamoyl phosphate synthetase to form carbamoyl phosphate,then urea. Carbamoyl phosphate synthetase I: - Present in the mitochondria of the liver cells. -Use ammonia for formation of carbamoyl phosphate. - It is activated by N-acetyl glutamate and Mg++ ions. - 2 molecules of ATP are used in this reaction. UREA CYCLE Factors affecting the level of urea in blood: (1) Protein diet: urea formation increase in case of increased protein intake and vice versa. (2) Liver: diseased liver is not able to synthesize urea so urea in blood and in urine is decreased, but there is high blood ammonia. (3) Kidney: - A diseased kidney is not able to excrete urea in urine. So urea is blood in increased and a fatal disease characterized called (uraemia). Metabolic disorders of urea cycle: - Metabolic disorders associated with a deficiency of each of the 5 enzymes of hepatic urea synthesis are known. (1) Hyperammonaemia type I: - This is due to deficiency of carbamoyl phosphate synthetase. - This probably is a familial disorder. (2) Hyperammonaemia type II: - It is due to deficiency of ornithine trans-carbamylase. - This disease is X-chromosome linked. (3) Citrullinaemia: - It is due to deficiency of argininosuccinate synthetase enzyme. - This rare disorder probably is recessively inherited. - Large quantities of citrulline are excreted in the urine and both plasma. (4) Argininosuccinic aciduria: - This is a rare recessive inherited disease. - It is due to absence of argininosuccinase enzyme. - It is characterized by elevated levels of argininosuccinate in blood, CSF and urine. (5) Hyperargininaemia: - It is due to deficiency of argininase enzyme. - This defect in urea synthesis is characterized by elevated blood and C.S.F. arginine levels.