Comparative Studies of K⊕-p-Nitrophenylphosphatase, K⊕-Acylphosphatase and (Na⊕+ K⊕)Adenosinetriphosphatase in Synaptosomes of Rat Brain

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Abstract
Synaptosomes prepared from whole rat brains by gradient ultracentrifugation were assayed for K[image]-nitrophenylphosphatase and K[image]-acylphosphatase activities and compared with the activity of (Na[image] + K[image])adenosinetri-phosphatase (ATPase). p-Nitrophenylphosphate and acetylphosphate each competitively inhibits the hydrolysis of the other. Both phosphatases are competitively inhibited by ATP. The effects of the inhibitors: N-ethylmaleimide, diisopropylfluorophosphate (DFP), N-chloroacetyl-chlorothiazid and fluoride were studied. N-ethylmaleimide progressively inhibited K[image]-p-nitrophenylphosphatase, K[image]-acylphosphatase and (Na[image] + K[image])ATPase, when preincubated with synaptosomes at 0[degree]C The presence of ATP in the preincubation mixture had a protective effect. DFP noncompetitively inhibited K[image]-p-nitrophenylphosphatase and (Na[image] + K[image][image])ATPase vhenpreincubated with synaptosomes at 20[degree]C. The inhibition of K[image]-p-nitrophenylphosphatase was markedly potentiated by K[image], the effect of which was antagonized by Na[image] and ATP. Fluoride inhibited K[image]-p-nitrophenylphosphatase, K[image]-acylphosphatase and (Na[image] + K[image])ATPase noncompetitively. Noradrenaline abolished the fluoride inhibition of K[image]-p-nitrophenylphosphatase. N-chloroacetyl-chlorothiazid caused complete inactivation of K[image]-p-nitrophenylphosphatase, K[image]-acylphosphatase and (Na[image] + K[image])ATPase after a preincubation period of 80 h. The activating effect of polar phospholipids and deoxycholate was studied. The partition of polar phospholipids in synaptosomes and their suspension medium before and after treatment with deoxycholate is described. The results are discussed and the occurrence of serine, histidine and Mg2[image] at or near the active sites of K[image]-p-nitrophenylphosphatase, K[image]-acylphosphatase and (Na[image]+ K[image])ATPase, the activities of which may be due to the same enzyme system, is suggested.