A high activity divalent cation ATPase from the distal, caudal epididymis of bulls.

Abstract

A membrane fraction rich in divalent cation ATPase is sedimented at high centrifugal force from medium flushed through distal, caudal bovine epididymides after sperm and cytoplasmic droplets are removed. This membrane fraction rapidly liberates equimolar amounts of inorganic phosphate from ATP, CTP, ITP, CTP, UTP, and TTP. Inorganic phosphate is slowly hydrolyzed from nucleoside diphosphates by these membranes, but nucleoside monophosphates, pyrophosphate, phosphoenolpyruvate, glucose-6-phosphate, dihydroxyacetone phosphate, or p-glycerophosphate are not substrates. ADP and AMP are weak inhibitors of Mg’-ATPase activity. ATPase is optimal at pH 7, and the effectiveness of divalent cations to activate the ATPase is Mg2*> Ca2*> Mn’�> Co’�> Cd’. High concentrations of K�, Cs, Rb, Na, and choline produce a ouabain-insensitive stimulation of Mg’�-ATPase. Potassium also increases the maximal velocity of ATPase activated by Mn’ and Co’, but not Ca’�. Ouabain, ethacrynic acid, and dihydroethacrynic acid, in concentrations up to 1 mM, have no appreciable effect on Mg�- or Ca’�-ATPase. Mersalyl (0.1 mM) effectively inhibits both ATPase activities, whereas N-ethylmaleimide only weakly inhibits the enzyme. Very high concentrations of rutamycin and aurovertin have no effect on epididymal ATPase, while venturicidin and A23187 produce a slight inhibition. Mg�-ATPase is effectively inhibited by 50 mM sodium fluoride and sodium azide, whereas Ca’�-ATPase is less affected by these inhibitors. Electron micrographs of thin sections of the ATPase-rich fraction show numerous vesicles and long profiles, which appear to be microvilli detached from epididymal principal cells. Negatively stained preparations show numerous 3-4 nm particles covering the surface of these membranes. Histochemical studies indicate that both microvilli of distal, caudal epididymis and the membrane fraction from epididymal flushing medium contain an active magnesium-stimulated ATPase. The origin of the ATPase-rich fraction and possible roles of this enzyme in epididymal function are discussed.