University of Pennsylvania - BIOL 203Fall2017_HW3_Key.

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Fall2017_BIOL203 Homework 3 1. You have two membrane vesicle preparations both of which contain a P-type Na+/K+-ATPase. Although you do not have the resources to purify the enzyme, and antibodies d... irected against this particular type of ATPase are not available, you would like to estimate the relative amounts of this enzyme in the two enzyme preparations. Assume that both membrane vesicle preparations have a random orientation (consist of a 1:1 mixture of rightside-out and inside-out vesicles), and you have a ready supply of Mg.[-32P]ATP, NaCl, KCl and ouabain. How would you do this? Explain the principles underlying your reasoning. Note that membrane preparations are often heavily ‘contaminated’ with non-specific phosphatases and non-P-type ATPases competent in the hydrolysis of ATP. 2. To study the mechanism a newly discovered ATP-energized K+ pump (an ATP-energized K+ uniporter), you solubilize, purify and reconstitute the integral membrane protein concerned to generate proteoliposomes containing the pump in a relatively pure state. The orientation of the reconstituted pump is such that the ATP-binding and hydrolysis sites of the K+-ATPase are accessible from the outer surface of the proteoliposomes. In the first series of experiments you attempt to measure the activity of the reconstituted pump as K+-stimulated ATP hydrolysis. However, when K+, as K2SO4, and MgATP are added to the assay medium, you obtain only a short burst of ATP hydrolytic activity, measured as the formation of Pi, after which the reaction ceases. Only after the addition of K2SO4 in combination with valinomycin do you see sustained Pi production after the addition of MgATP. Explain these results. 3. If in the system described in question 2 you determined that nigericin, unlike valinomycin, did not promote sustained Pi production from MgATP when K+ was provided as K2SO4 unless the nigericin was added in combination with 2,4-dinitrophenol (2,4-DNP), how would you explain your results? 4. A feature typical of coupled transport into membrane vesicles is what is referred to as an ‘uptake overshoot’, in which uptake transitorily exceeds that expected if solute were simply to equilibrate between the vesicle interior and external medium. In a study of [14C]glucose uptake by isolated membrane vesicles the results shown on the next page were obtained. In experiment A, vesicles loaded with 100 mM NaCl were rapidly transferred into a volume excess of solution containing 1 mM NaCl and 2.5 mM [14C]glucose, and [14C]glucose uptake into the vesicles was measured against time. 5. Mucilage is the term given to the gelatinous substance that is derived from the seed coat of various plants. Some of the major components of mucilage are very recognizable to even the young student of biochemistry: pectin, cellulose, and protein. Common forms of mucilage encountered in the laboratory are agar and agarose (both derived from different species of sea weed), and these are ground to a fine powder and then hydrated and boiled. Because of the abundance of carbohydrate and the ratio of protein, agar and agarose effectively swell and produce a Jell-o like substance that can be used for gel electrophoresis, plating of microorganisms, and more. In plants, the seed coat aids the seed in absorbing and retaining water which is required for germination. Another less commonly recognized mucilage is that of the mustard seed coat. Ground mustard seed is the primary component of Dijon and other mustards, used widely as a condiment and flavorful substance. A secret well known to chefs is that mustard is an excellent emulsifier – it prevents separation of oily and watery substances in food. Vinaigrettes often contain a bit of Dijon mustard which allows proper mixing of the dressing and application to the salad or dish of choice. The following experiments were performed by the culinary director of serious eats. Explain each result using your knowledge of biochemistry. Note that very little botany is required here – namely leaves are covered in a waxy coating that makes them waterproof. 6. A. Assuming that the free-energy change for ATP hydrolysis under cellular conditions is about —58 kJ/mol, determine the overall G for the action of a Na+/K+ ATPase pump at a -60mV (inside negative) transmembrane potential, given the concentrations listed in the figure below. 7. The currently accepted model of the architecture of the mammalian membrane is the fluid mosaic model, depicted in Figure 11-3 of your book. This model was presented in detail originally in a review article by SJ Singer in 1971. In the article, Singer presented three possible model of membrane structure that had been proposed at that time: 8. A. Explain why oleic acid (18:1, with 1 cis bond) has a lower melting point than stearic acid which has the same number of carbons but is saturated. How would you expected the melting point of trans-oleic acid to compare with cis-oleic acid? Why might most unsaturated fatty acids in phospholipids be in the cis rather than trans conformation? [Show More]

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