Biochemistry- Paper II Feb 2007 I. Essay 1. What is the active form of methionine? How it is formed? What are its functions? Enumerate the steps of methionine metabolism and write the disorders associated with its metabolism.(20) Ans. Active form of methionine is S-Adenosyl Methionine(SAM). SAM is obtained by accepting adenosyl group from ATP by methionine by methionine adenosyl transferase. The function of SAM is transmethylation reactions. Transmethylation reaction is acceptance of a methyl group from a donor like S-adenosyl methionine(SAM) by a compound resulting in another compound. The transmethylation reactions are Methyl acceptor Methylated product Guanidoacetic acid Creatine Serine Choline Epinephrine Metanephrine Nor epinephrine Epinephrine tRNA Methylated tRNA Steps of methionine metabolism: 1. methionine converts to SAM 2. SAM donates methyl group to methyl acceptors by methyl transferases to form S-adenosyl homocysteine(SAH) 3. SAH loses adenosine using adenosine homocysteinase to form homocysteine. 4. Homocysteine forms methionine by homocysteine methyl transferase. This step uses methyl tetra hydrofolate which becomes THFA using B12. 5. Homocysteine combines with serine to form Cystathionine using Cystathionine synthase and Cystathionine is hydrolyzed to cysteine and homoserine by cystathioninase. Diseases in methionine metabolism: Homocystinurias are autosomal recessive disorders of about 1:200000 child births. Hyperhomocysteinemia is a risk factor for coronary heart disease. It is seen in smokers, alcoholics, and hypothyroidism. Homocystinurias are seen in the following: 1. Cystathionine beta synthase deficiency: - methionine and homocysteine level increase and seen in urine. - Charlie Chaplin gait, mental retardation, ectopia lentis are seen - homocysteine activates Hageman’s factor leading to thrombosis Cyanide Nitroprusside test will be positive. Urinary homocysteine levels are elevated. - Treatment is a diet low in methionine and rich in cysteine. 2. Cobalamin deficiency: -N5 methyl THFA homocysteine methyl transferase is dependent on B12. Hyperhomocysteinemia occurs 3. Deficient N5, N10 methylene THFA reductase 4. Cystathioninuria - it is due to cystathioninase deficiency. - mental retardation, anemia, thrombocytopenia and endocrinopathies. - acquired is seen in pyridoxine deficiency 5. Acquired hyper homocysteinemias - Nutritional deficiency of vitamins like Cobalamin, folic acid and pyridoxine. - metabolic: chronic renal diseases, hypothyroidism - folate agonists, Vit B12 antagonists, pyridoxine agonists, estrogen antagonists 2. What is the normal pH of blood. Discuss the mechanism involved in its regulation.(15) Ans. Normal blood pH is btween7.38-7.42. it is maintained by: I. Role of buffers in body fluids: Buffers resist changes in pH when small quantities of an acid or an alkali are added. Various buffers in body are: 1. Bicarbonate buffer system it is the most important buffer in plasma and is formed by (NaHCO3/H2CO3) - the base HCO3 is the metabolic component as it is regulated by kidney and the acid H2CO3 is called respiratory component since it is regulated by the lungs. The normal bicarbonate level in plasma is 24mmol/L. It has a pKa of 6.1-so it is a poor buffer. But the high blood concentration and the ratio of base to salt is high(20:1), which makes it an effective buffer. When acid(H+) is added + - o H + HCO3 H2CO3 H20 + CO2 excreted by lungs and kidney. When alkali is (HCO3-) added + - o H + HCO3 H2CO3 H20 + CO2 excreted by lungs and kidney. 2. Phosphate buffer system It is made of NaHPO4/NaH2PO4. It has a pKa of 6.8. + In acidosis NaHPO4 + H NaH2PO4 - excreted by the kidneys + In alkalosis NaH2PO4 - NaHPO4 + H 3. Protein buffer system The Histidine molecules in albumin acts as a buffer. In acidosis H+ + Pr- - HPr In alkalosis HPr H+ + Pr- II. Kidneys regulate acid base balance by: + 1. Excretion of H -In PCT cells CO2 combines with water to form carbonic acid using carbonic + - + anhydrase. Then it becomes H and HCO3 . this H is then excreted into lumen in exchange for Na+ . - 2. Reabsorption of HCO3 - sodium bicarbonate in the lumen becomes sodium and bicarbonate. + - Sodium is taken up by PCT cell in exchange of hydrogen ions. H combines with HCO3 to form carbonic acid, which forms CO2 and water and both are reclaimed into the cell and converted + - - back to carbonic acid and again to H and HCO3 . HCO3 is taken into blood with sodium. + - Fig. Excretion of H fig. Reabsorption of HCO3 + - 3. Excretion of titrable acid- The Na2 HPO4 becomes Na and NaHPO4 . sodium is exchanged + + – with H ions and H combines with NaHPO4 to become Na H2PO4 and gets excreted. Fig. Excretion of titrable acid and ammonium ions + 4. Excretion of NH4 - Glutamine in DCT becomes glutamate and ammonia. This ammonia is secreted into the lumen which combines with hydrogen ions to become ammonium ions and gets excreted. III. Role of lungs in Acid base balance When there is fall in pH the respiratory rate is stimulated resulting in hyperventilation. This would eliminate more CO2 thereby lowering H2CO3 . In tissues pCO2 is high and pH is low to the formation of acids by the cells like lactate and production of CO2 by cells. CO2 diffuses into RBC. It combines with water to form carbonic + - + acid by carbonic anhydrase. And dissociates into H and HCO3 . So RBC traps H from the tissues. - Some of the HCO3 diffuses out of the cell in exchange for chloride. + - In the lungs H combines with HCO3 to form H2CO3 which becomes H2O and CO2. This CO2 is released into the lungs. So lungs reduce the acid load of H2CO3 by excretion of CO2. In metabolic acidosis lungs hyperventilate to excrete more acid. In Metabolic alkalosis the reverse happens. Fig. Reactions in tissues fig. Reaction in lungs 3. Mention the sources, Daily requirement, functions and deficiency symptoms of Calcium. Explain how serum level of calcium is regulated.(15) Ans. Parathyroid hormone(PTH) is secreted by four parathyroid glands in the thyroid tissue. Decreased serum calcium leads to release of PTH from parathyroids. PTH activates adenylyl cyclase in target cells and increases intracellular calcium concentration. A protein kinase is activated which activates enzyme systems. PTH acts on 1. PTH and bones- PTH causes demineralization in bones. It activates pyrophosphatase in osteoclasts leading to bone resorption and solubilising calcium. Calcium is released into the blood stream and increases blood calcium level. This leads to loss of bone matrix. 2. PTH and kidneys: PTH causes decreased renal excretion of calcium and increased excretion of phosphates and increased reabsorption of calcium leading to increased blood calcium level. 3. PTH and intestines: PTH stimulates increased production of VIT D3 which acts on intestine to absorb more calcium leading to increased calcium level in blood. II. a. Oncogenes Ans. Functions of Oncogenes: Oncogenes are normal genes, whose products perform various functions in the cell. Products of many Oncogenes are polypeptide growth factors. Some of the products act as receptors for growth factors. Eg. Erb-B Some oncogene products act on key intracellular pathways involved in growth control. Eg. Src product The c-Oncogenes are under the control of regulatory genes and are expressed only when required. Activation of Oncogenes: Viruses, chemical carcinogens, chromosome translocations, gamma rays, spontaneous mutations and all such factors may converge into one biochemical abnormality, the activation of Oncogenes which leads to malignancy. Examples for Oncogenes causing cancer: erb-B1- lung cancer erb-B2-gastric tumors erb-B3-breast cancer sis- osteosarcoma by activating PDGF abl- leukemia ras- leukemias, lung cancer, pancreatic and colon cancer b. Electrophoresis Ans. The term electrophoresis refers to the movement of charged particles through an electrolyte when subjected to an electric field. Cations(positively charged ions) move towards cathode and anions(negative) to anode. When a biological mixture is subjected to electrophoresis, the compounds in the mixture move in relation to their net charge, size, molecular weight and mass and gets separated according to these characteristics, so that the desired compound can be identified and isolated. Factors affecting electrophoresis: rate of migration will depend on: Net charge of the particles (eg. more negative particles move faster than less negative) Mass and shape of particles(larger sized particles move slowly) pH of medium(particles move better in a pH in which they are more ionized) strength of electrical field(eg. Higher the voltage-faster the movement) properties of supporting medium temperature-increased temperature solidifies the support and impairs migration) The electrophoresis apparatus consists if a tank which contains electrodes connected to a power supply and buffer. The pH of buffer is selected so that it imparts maximum charge to the electrophoresed substances(eg. Proteins get separated well in a buffer pH of 8.6 Supporting medium is the surface on which electrophoresis is carried out. It may be agar gel, agarose gel, cellulose acetate, paper, etc., After the run the bands are visualized using naked eye or if needed to be quantified a densitometer can be used. Clinical applications: 1. serum protein electrophoresis: - in nephrotic syndrome – globulin is produced more by liver in compensation of renal loss of albumin. So alpha 2 band is prominent - cirrhosis- albumin band is less prominent - multiple myeloma- light chain immunoglobulins are produced more so there will be a prominence in gamma globulin region(M band) Fig. serum protein electrophoresis in health and disease 2. hemoglobin electrophoresis - S band is seen in sickle cell anemia - various hemoglobinopathies and thalasemias can be diagnosed c. Genetic code Ans. The letters A, G, T, and C correspond to the nucleotides found in DNA. Within the protein coding genes these nucleotides are organized into three-letter code words called codons, and the collection of these codons makes up the genetic code. The code provides a foundation for explaining the way in which protein defects may cause genetic disease and for the diagnosis and perhaps the treatment of these disorders.