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NAME_________________ 1 13th December, 2002 BIO 451 FINAL EXAM KEY This exam will be taken apart for grading. Please PRINT your name on each page. If you do not have sufficient room for your answer in the space provided, please continue on the back of the page on which the question appears. Question I. Maximum Points Earned Points 13 --------------- II. 8 --------------- III. 8 --------------- IV. 4 --------------- V. 12 --------------- VI. 8 --------------- VII. 10 --------------- VIII. 12 --------------- IX. 7 --------------- X. 16 --------------- XI. 10 --------------- XII. 10 --------------- XIII. 8 --------------- XIV 7 --------------- ================================================================== TOTAL 133 Normalized I II III Score on : Total* = Sum of two best scores + points earned on the Final [COURSE GRADE ON REVERSE SIDE] FINAL NOTE: FULL CREDIT WILL BE GIVEN FOR THOSE ANSWERS WHICH CLEARLY ADDRESS ALL RELEVANT ASPECTS OF THE QUESTION S IN THE CLEAREST AND MOST CONCISE MANNER. NAME_________________ I. [13 Points:1 point for each correct response] Match the metabolic pathways below with the cellular compartments in which they occur. A Cytosol B. Inner mitochondrial membrane C. Mitochondrial matrix D. Mitochondrial intermembrane space E. Endoplasmic reticulum ____A____Glycolysis _____A___Lactic fermentation ______C__Pyruvate dehydrogenation _______A,C______ Amino acid degradation ________A,C_____Urea cycle _________A,C____Gluconogenesis _______A___ Cholesterol synthesis II. _____C_______Palmitoyl-coA Oxidation ______A_______ Palmitate synthesis _______C______ Citric acid cycle ____B____Oxidative phosphorylation of ADP ____A____Pentose phosphate pathway ___F_____ Fatty acid elongation and desaturation [8 Points] Answer Either A or B not both. ONLY THE FIRST ANSWER WILL BE GRADED A. How does an increase in blood glucose affect triacylglycerol metabolism in adipocytes? Increasing levels of blood glucose favor TG synthesis as the rate of glucose uptake by fat cells increases. The 3-carbon backbone of TG’s is derived from DHAP, an intermediate of glycolysis. DHAP is converted to Glycerol-3-Phosphate to favor FA esterification. B. You obtain a liver homogenate from a fasting mouse and "spike" it with a high concentration of glucose. In what form would you expect glyogen phosphorylase? The addition of a large amount of glucose shifts the T/R equalibrium for phosphorylase towad the T state. Stabilization of the T state by glucose binding promotes dephosphorylation of Ser 14, which converts the enzyme to the less active phophorylase b. 2 NAME_________________ III. [8Points] A. During G- protein activation, which subunit exchanges GDP for GTP? What is the intermediate consequence of this exchange? (4 pts) 3 When G-protein binds to a hormone-receptor complex, the Gα subunit exchanges GDP for GTP. Binding of GTP induces a conformational change in Gα that causes it to dissociate from Gβδ . GTP binding also increases the affinity of Gα for adenylate cyclase, which is thereby activated. B. Describe the intracellular signaling cascade mediated by phospholipase C activity. How is phospholipase C activated? (4pts) SEE ANSWER TO CHAPTER 21 OF COMPANION PAGES 276 & 277. IV. [4 points] List the order in which the following energy sources are used by skeletal muscle to, produce ATP: protein, phosphocreatine, fatty acids, glycogen. Phosphocreatine, Glycogen, FA’s, Protein. NAME_________________ V. 4 [12 Points, 1.5 pts each] This question is designed to test your familiarity with heme biosynthesis and catabolism as well as inter tissue cooperation by writing the appropriate item beside each description. A. Principal site of red blood cell production in adult humans Bone marrow B. In mature red blood cell, aerobic; makes globin, heme and purine nucleotides C. Involved in transport of billirubin from the spleen to the liver reticulocyte Serum albumin D.Degrades worn out red blood cells, part of the reticuloendothelial system. Spleen E. Required for removal of glucuronic acid residues from billirubin diglucuronide. Enyzmes of intestinal bacteria F. Light-sensitive heme precursor that accumulates when urogen III cosynthase is defective. Uroporphirinogen I or Urogen I G. Storage form of iron in the bone marrow tissue. ferritin H. Principal site of feedback regulation of heme synthesis. ALA synthase VI. [8 points] 1. Malonic acid is a structural analog of succinic acid. Addition of malonate to a suspension of respiring, intact mitochondria is followed by a rapid cessation of O2 consumption. Explain; the correct answer is short but you must be specific. (4pts) O2 consumption depends on a functioning TCA cycle; malonate is a competitive inhibitor of succinic dehydrogenase and will therefore inhibit the cycle. 2. Short of removing the malonate, how might you restore respiration and coupled phosphorylation? Your assumptions and rationale must be explicitly stated. [Note: Conversion of the malonate to malonyl-SCoA is not an acceptable option.] Explain. (4pts) Add succinate in excess of malonate NAME_________________ 5 VII. [10 points] Answer Either A or B not both. ONLY THE FIRST ANSWER WILL BE GRADED A. A sample of liver tissue was obtained post mortem from the body of a person believed to be genetically deficient in one of the enzymes of carbohydrate metabolism. A homogenate of the liver sample had the following characteristics. 1) It degraded glycogen to produce G-6-P. 2) It did not synthesize glycogen from any sugar, nor did it utilize galactose as an energy source. 3) It synthesized G-6-P from lactate. Which of the following enzymes was deficient? Give specific reasons for your answer. You must also state specifically the basis for elimination of two choices. 1) glycogen phosphorylase #1 says this enzyme is OK 2) fructose-1,6-phosphatase #2 says that gluconeogenesis works, so FBPase-1 is OK 3) UDP-glucose pyrophosphorylase Required for glucogen synthesis and Glu to Gal, and both are non functional, the lesion is here. B. A weight-reducing diet heavily promoted several years ago required the daily intake of "liquid protein" (soup of hydrolyzed gelatin; a collagen product), water and an assortment of vitamins. All other food and drink were to be avoided. People on this diet typically lost 10 - 14 lb. in the first week. A number of people on this diet died. What are some of the dangers inherent in the diet and how can they lead to death? [Hint: It is not acceptable to invoke contamination or experimental artifacts. Consider ammonia toxicity and why this is likely in this case, as well as the composition of collagen.] The individuals are deficient in essential amino acids; collagen is 1/3 glycine and very poor in Lys, Trp, etc. There would also be extensive breakdown of body protein and a huge ammonia load to deal with, perhap more than can be handeled by te urea cycle NAME_________________ VIII. [ 12 points; 1 pt each] For each description (below) select the ONE most appropriate vitamin; there are two parts for which there are two possibilities – you are to provide either of them. 1. Required for oxidative decarboxylation of "-keto acids __D or K_____ 2. Its synthesis is inhibited by sulfanilamide ___F___ 3. Participates in the conversions involving free radicals ____B___ 4. Participates in 1C transfers at the level of a formyl group __F____ 5. Participates in 1C transfers at the level of a carboxyl group __A____ 6. Participates in hydride ion transfers ___K___ 7. Participates in the transfer of two hydrogen atom transfers, each with one electron __E or C_____ 8. Participates in postranslational hydroxylation __C____ 9. A derivative of cholesterol ___H___ 10. The precursor for FMN ___E___ 11. Contains a thiazoline ring ___D___ 12. Contains a redox active disulfide __L____ A. Biotin B. Vitamin B12 C. Ascorbic acid D. Thiamine E. Riboflavin F. Folic acid G. Vitamin A H. Vitamin D I. Vitamin K J. Vitamin E K. Niacin L. Lipoic acid 6 NAME_________________ 7 IX. [7 points] For each of the following, choose the ONE best answer A. Hormonal stimulation of the second messenger inositol 1,4,5-triphosphate [IP3] quickly leads to the release of which intracellular second messenger? __d___ a. cGMP b. cAMP c. prostaglandin d. calcium e. leukotriene B. A strong cup of coffee would be expected to ___c___ a. interfere with the synthesis of prostaglandins b. decrease the effects of glucagon c. enhance the effects of epinephrine d. provide the vitamin niacin e. none of the above C. During starvation, ketone bodies are synthesized in ____c__ a. adipose tissues b. brain c. liver d. muscles e. thymus D. After an injection of insulin in a diabetic patient with hyperglycemia, there is a prompt normalization of plasma glucose, due to the recruitment of which of the following transport proteins to the plasma membrane? ___d___ a. GLUT 1 b. GLUT 2 c. GLUT 3 d. GLUT 4 e. GLUT 5 E. Glycolysis is the only ATP-producing pathway in ___a__ a. erythrocytes b. lymphocytes c. reticulocytes d. hepatocytes e. neurons f. adipocytes F. In the normal resting state of humans, most of the blood glucose burned as fuel is consumed by___b__ a. the liver b. the brain c. the kidneys d. adipose tissue e. muscle NAME_________________ G. Elevation of cytosolic calcium from intracellular stores can be caused by ___d___ a. cAMP b. cGMP c. cCDP d. inositol 1,4,5-triphosphate [IP3] e. diacylglycerol 8 NAME_________________ 9 X. [16 points] This question is presented in the form of a crossword puzzle. It is designed to test your familiarity with terms and concepts associated purine nucleotide metabolism. As with most crossword puzzles, following the clues is a parenthetical expression which indicates the number of letters/numbers required for a particular answer; where answers are to be given in terms of abbreviations this is also indicated. You cannot possibly solve the entire puzzle in the time allotted; therefore, choose only 8 items. It is important, as with most crossword puzzles, that the answers are spelled properly. If you enter an answer and later decide that the answer is wrong cross it out or erase it. Only your first 8 answers will be graded. It is essential that you write legibly. Full credit will be awarded for correct, complete, legible, correctly-spelled answers. 2 points will be awarded for each correct answer therefore total credit for this question is 16 points. NAME_________________ 10 NAME_________________ 11 NAME_________________ XI. [10 Points] Identify ONE specific example of a genetic defect in metabolism that have been considered in class or in the text, and for which the biochemical basis is clear. You must describe BOTH the biochemical basis of the lesion as well as the metabolic/physiological consequences of the defect in as much detail as you are able. SEE CLASS NOTES/TEXT 12 NAME_________________ XII. 13 [10 points] A. Describe the biochemical and cellular processes that are involved in glucose stimulated insulin secretion (GSIS). (5pts) Insulin is secreted by the $-cells of the pancreas. Glucose is taken up via GLUT 2, a low affinity glucose transporter. The first stem in its metabolism is conversion to G6P via glucokinase (GK); subsequent catabolic processes, especially OxPhos produces ATP. ATP inhibits the ATP-dependent potassium channel. This depolarizes the membrane and allows calcium ions to enter the cell via the calcium channel. The influx of calcium ions enhances insulin secretion. B. kCAT Km Normal 8.4mM 54.4moles.mole!1.sec!1 Mutant (Val455Met) 2.9mM 50.2 " " " " The table above provides values for relevant kinetic parameters for glucokinase from $cells from normal individuals and patients having a mutation at residue 455 of the polypeptide. Keeping the normal processes involved in GSIS in mind, what would the effect of this mutation be on levels of insulin and glucose in the blood? Explain your rationale in as much detail as you are able. (5pts) The mutant GK has a significantly higher affinity for glucose, this means that for a given concentration of glucose from the blood, the GK activity is higher in the patients carrying the mutation, thus producing more ATP and increasing insulin secretion more than in normal individuals. These individuals will show hyperinsulinemia and hypoglycemia (insulin stimulates glucose uptake). NAME_________________ 1) Consider the situation where you want to check if two proteins A and B have a direct interaction using fluorescence resonnance energy transfer (FRET) technique. You have the options to use any of the fluorophores provided in the table. Which pair would you use and why? (6 points) The right pair to use if ECFP/ EYFP. However, Alexa488/ECFP, Bodipy/ FITC, Bodipy/EYFP, ECFP/Cy3 deserve 2 points. The selected pair is used because the emission wavelength of one fluorophore is close enough to excite the second fluorophore. 2) a- Why is X-ray crystallography important? (2 points) Insight into 3D structure of protein Better way to design drugs Understand Protein- Protein interaction b- What steps do you experimentally take to generate a 3D strucure? (4 points) Purify protein (higher than 98%) Grow crystal using the hanging drop method Collect diffraction data Generate electron density map Solve structure c- Which step is the most limitting and why? (2 points) Protein purification and/ or crystal growing step. XIII. [8 points] Consider the situation where you want to check if two proteins A and B have a direct interaction using fluorescence resonance energy transfer (FRET) technique. You have options to use any of the fluorophores provided in the table. Which pair would you use and why? Florophores Alexa 488 Bodipy 564/570 ECFP Cy3 FITC EYFP GFP Absorption wavelength (nm) 492 564 436 514 490 517 475 Emission wavelength (nm) 520 570 488 566 520 528 509 14 NAME_________________ XIV. [7 Points] a) Why is X-ray crystallography important? (2 pts) b) What steps do you experimentally take to generate a 3D structure? ( 3pts) c) Which step is the most limiting and why? (2 pts) 15