Purification and fluorometric assay of proteinase A from yeast

A kinetic assay system which provides reliable measurements of Na-K-ATPase activity on 0.2 to 0.5-mm segments of renal proximal convoluted tubules isolated from collagenase-digested renal cortical slices is described. The use of collagenase digestion provides higher values for Na-K-ATPase, possibly by making the enzyme more accessible to the reaction system. The advantages of a kinetic vs an endpoint assay include the ability to use the same tubule as its own reference for the determination of total, ouabain-sensitive, and ouabain-insensitive ATPase activity. In addition, it allows dose-response studies on the effect of inhibitors on ATPase activity in the same tubule segment.