Potassium dependent phosphatase in bovine parotid gland

Abstract

Naf, K+-dependent adenosine triphosphatase (Na+, Kf-dependent ATPase) [ATP phosphohydrolase, EC 3.6.1.31 may play an important role in active transport of monovalent cations. Hydrolysis of ATP by the Na+, K+-dependent ATPase is thought to be a two-staged reaction. The first step is the Na+-dependent phosphorylation of an intermediate, and second step is the K+-dependent dephosphorylation of the intermediate. It was reported that hydrolysis of p-nitrophenyl phosphate is catalyaed by an enzyme in the membrane fraction of various tissues and it is stimulated by the presence of potassium (JUDAH, AHMED and MCLEAN, 1962; AHMED and JUDAH, 1964). It was also suggested that, since p-nitrophenyl phosphate is known to serve as a substrate for phosphoprotein phosphatase [EC 3.1.3.161, it may be assumed that the action of K+-dependent phosphatase on p-nitrophenyl phosphate is representative of the dephosphorylation process in the Na+, K+-dependent ATPase reaction (NAGAI, IZUMI and YOSHIDA, 1965). Many investigators have demonstrated the presence and discussed the biological meaning of Na +, K+-dependent ATPase in salivary glands (SCHWARZ and MATSUI, 1967). However, K+-dependent phosphatase in salivary glands has not been reported. Therefore, in the present work the presence and some properties of K+-dependent phosphatase in bovine parotid gland were investigated. Female bovine parotid gland was homogenized in 4 volumes of 0 * 32 M sucrose. After removal of debris by centrifugation at 800 g for 10 min, the supernatant was centrifuged at 15,OOOg for 20 min to obtain the mitochondrial fraction. Then, microsomal and soluble fraction were separated by centrifugation of the supernatant at 105,000 g for 60 min. Each fraction was washed once, suspended in 0.32 M sucrose and used for enzyme assay. K+-dependent phosphatase activity was determined as follows. Two ml of the reaction mixture, containing SOpmoles of Tris buffer (pH 7*8), 10 pmoles of p-nitrophenyl phosphate disodium salt, 10 pmoles of MgC12, 20 pmoles of KC1 and a certain amount of the enzymes were incubated at 25°C for 20 min. The reaction was stopped by addition of 4 ml of O-5 M NaOH and, after centrifugation, an absorbance of the supernatant was estimated at 410 rnp in a spectrophotometer. The activity of the K+-dependent phosphatase was expressed as the difference between the activity in the presence and absence of K+ in the reaction mixture. The amount of protein was determined with Folin-Ciocalteu’s reagent (LOWRY et al., 1951).