You have two membrane vesicle preparations both of which contain a P-type Na+/K+

Question

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 directed 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 as well (as any other reagents we discussed in class related to transport).

How would you do this? In other words, design experiments to estimate the relative amounts of P-type ATPase in each vesicle preparation. 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 and you will have to account for these.

Explanation / Answer

Ouabian is know to inhibit P type Na+/K+ ATPase.

experiment -

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