Student Number: THE UNIVERSITY OF MANITOBA April 11, 2011, 1:00 PM - 4:00 PM Page 1 (of 3)

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1 Name: Student Number: THE UNIVERSITY OF MANITOBA April 11, 2011, 1:00 PM - 4:00 PM Page 1 (of 3) Biochemistry II Laboratory Section Examiners: Drs. J. Galka 1. Answer ALL questions in the space provided. 2. The back side of each page may be used for your answer or for preliminary work. 3. Questions to invigilators about the exam will NOT be answered You are fiven a sample of 10% (w/v) beef liver homogenate to determine lactate dehydrogenase activity. First you must partially purify proteins and nucleic acids from the homogenate. Using a flow chart describe the procedure used during the term to separate RNA, DNA and protein from the original tissue homogenate. The names and purpose of the reagents must be indicated in the chart, however volumes and concentrations are NOT required.

2 April 11, 2011, 1:00 PM - 4:00 PM Page 2 (of 3) Biochemistry II Laboratory Section Examiners: Drs. J. Galka 2. Two methods of column chromatography were used to isolate lactate dehydrogenase from the above partially purified liver homogenate: Column A (Sephadex G-100, MW range ,000, ph 8.8) and Column B (DEAE-Sephadex, ph 8.8). A 5 ml portion of the partially purified liver homogenate was applied to Column A and another 5 ml applied to Column B. Several 10 ml fractions were collected from each column, with most of the enzyme activity being found in fraction 1 for both columns. Enzyme activity and protein assays for these fractions yielded the following results: Enzyme activity (µmoles pyruvate/min) Dilution Assay Volume (ml) Partially purified liver in homogenate Column A, Fraction in Column B, Fraction n/a 0.1 Protein content (mg) Dilution Assay Volume (ml) Partially purified liver in homogenate Column A, Fraction in Column B, Fraction n/a (a) Which of the two columns gave the best purification of the enzyme? Show all calculations and cite values to support your answer. 2 (b) If the extract was treated with urea (a protein denaturant) and then applied to column A (Sephadex G- 100, MW range 4000 to 120,000, ph 8.8), the enzyme activity appeared in fraction 9 (of 12) instead of fraction 1. Explain this observation.. 2 (c) With column B (DEAE-Sephadex, ph 8.8) most of the enzyme was eluted with 0.1 M TRIS, ph 8.8, in fraction 1. Explain this observation.

3 April 11, 2011, 1:00 PM - 4:00 PM Page 3 (of 3) Biochemistry II Laboratory Section Examiners: Drs. J. Galka 3 (d) The above enzyme fraction was subjected to electrophoresis at ph 8.8. Using a diagram, show an approximate migration pattern on the cellulose acetate strip. Be sure to label your diagram. Explain the observed pattern Draw the expected results of the anhydrous petroleum ether icing sugar column run this term. Clearly indicate the four main pigments extracted from the spinach and their relative positions on the column. One of these pigments is found in both an oxidized and a reduced form; indicate on your diagram both the oxidized and reduced form of this pigment. Beside your diagram, indicate the moving phase, the stationary phase, and the relative polarities of the pigments.

4 Name: Student Number: THE UNIVERSITY OF MANITOBA April 11, 2011, 1:00 PM - 4:00 PM Page 1 (of 4) 1. Answer ALL questions in the space provided. 2. The back side of each page may be used for your answer or for preliminary work. 3. Questions to invigilators about the exam will NOT be answered Using structural formulae and the names of enzymes, describe the mechanism by which changes in adrenalin levels in the blood affect the accumulation of glycogen Using diagrams, describe how equilibrium centrifugation involving a CsCl solution was used to analyze the mechanism of DNA replication and the conclusions that arose from the work.

5 April 11, 2011, 1:00 PM - 4:00 PM Page 2 (of 4) Using either structures and names of compounds, enzymes and coenzymes or a diagram, describe: (a) the role of rho factor in transcription; (b) the reaction catalyzed by the nitrogenase complex and its role; (c) the structures of a G-C base pair and a G-U base pair and the role of the G-U base pair; (d) the reaction converting CDP to dcdp utilizing NADPH as electron source; (e) the biochemical basis of phenylketonuria; (f) the reactions of the glyoxalate shunt and the role of the shunt.

6 April 11, 2011, 1:00 PM - 4:00 PM Page 3 (of 4) 8 4. Using diagrams with structures and coenzymes, describe the mechanism of reaction of transketolase starting with xylulose-5-phosphate and erythrose-4-phosphate in the Calvin Cycle Outline the process that produces the ATP and NADPH required for CO 2 fixation in chloroplasts. Your diagrams should include the names of all intermediates, an explanation for what the light energy is doing and a description of the quantum yield.

7 April 11, 2011, 1:00 PM - 4:00 PM Page 4 (of 4) Starting with methionine and lysine, use diagrams to describe the process by which the first peptide bond in a protein between methionine and a lysine is formed leaving the peptide ready for the addition of a third amino acid. Calculate the energy required in ATP equivalents The addition of glucose to a culture of E. coli growing on lactose medium changes expression of the lac operon, as does subsequent replacement of lactose with succinate. Draw a graph that describes these changes and using diagrams describe the molecular mechanisms that mediate these changes.