Potassium ion-activated hydrolysis of p-nitrophenyl phosphate in pancreatic islet-cell membranes

Abstract

Hydrolysis of p-nitrophenyl phosphate was measured in a fraction enriched in plasma membranes from pancreatic islets of non-inbred ob/ob mice. Hydrolysis was stimulated by K+ (10mM) in the pH range 5--10; a small peak of K+-induced activation was observed between pH7.5 and 8. Both the K+-induced activation and the hydrolysis in the absence of K+ were Mg2+-dependent; maximum activation was obtained with 10mM-K+ plus 5 mM-Mg2+. Rb+ was as effective an activator as K+. Ouabain was inhibitory, the effect being inversely related to the K+ concentration; 0.1--0.2mM-ouabain caused about 50% inhibition in the presence of 1 mM-K+, but had no demonstrable effect in the presence of 4--5mM-K+. The K+-stimulated activity was markedly inhibited by 0.1mM-ATP, 35--140 MM-Na+, or 0.01 mM-p-chloromercuribenzenesulphonic acid. Similarities to Rb+ accumulation suggest that catalysis of univalent cation flow in pancreatic beta-cells may be coupled to a phosphoryl-transfer reaction with ATP as natural substrate or regulator.

86Rb+ fluxes and K+-stimulated nitrophenyl phosphatase acitvity in the pancreatic islets of genetically diabetic mice (C57BL/KsJ-db/db)

“…B-cells of diabetic KsJ-db/db-mice have an abnormal regulation of their electric activity with persistant depolarization of the plasma membrane [6]. The genesis of the membrane potential in B-cells ; is incoriapletely known, but a fundamental role ~s generally attributed to univalent cation pumping [9,14,15]. Previous experiments indicate that accumulation of S6Rb+ reflects operation of the B-cell's univalent cation pump [7].…”

“…The assay of K+-activated p-nitrophenyl phosphatase in islets has been described in detail [9]. In the present study, 20-60 islets were washed twice in 300 ~tl of Tris-aeetate buffer (0.1 tool/1 Tris base and acetic acid to give pH 7.8).…”

“…In a few experiments, 10 mmol/l boric acid/NaOH (pH 7.8) was used instead of the Tris-aeetate buffer; the choice of buffer had no noticeable effect on enzyme activity, so results obtained with the two buffers have been pooled. Proteins were assayed with fluorescamine [9].…”

“…Accumulation of 86Rb+ is a useful measure of univalent cation pumping in well-functioning B-cells, and is markedly inhibited by the diabetogenic compound, alloxan [7,8]. The univalent cation pump may be closely associated with a K+-activated p-nitrophenyl phosphatase that resides in B-cell plasma membranes [9]. Studies on 86Rb+ efflux from preloaded islets led to the proposal that glucose-induced depolarization of the B-cells is at least in part due to a decrease of K + permeability [10].…”

Stimulus-Secretion Coupling of Glucose-Induced Insulin Release

“…Most published studies on ATPase activ ity in crude homogenates or subcellular frac tions derived from pancreatic islets concern the Na, K-ATPase [13,[17][18][19]. The present results on Ca-ATPase activity in islet homog enates are, in several respects, in good agree ment with the findings of Formby et al [4], The Ca-ATPase displayed two Km values for Ca2+, namely 0.13 and 4-5 \iM in the present study as compared with 0.07 and 4.2 pM according to Formby et al [4].…”