Activity of epiphyseal cartilage membrane alkaline phosphatase and the effects of its inhibitors at physiological pH.
The kinetics of epiphyseal cartilage membrane-bound alkaline phosphatase activity was studied at physiological pH using p-nitrophenylphosphate and pyrophosphate (PPi) as substrates. The effect of three general types of alkaline phosphatase inhibitors was studied on both purified and membrane-bound forms of the enzyme: 1) uncompetitive inhibitors (L-tetramisole and theophylline), 2) competitive inhibitors (phosphate, arsenate, and vanadate), and 3) metal ions (Mg2+, Ca2+, and Zn2+). These studies were aimed at elucidating the physiological role of epiphyseal cartilage membrane alkaline phosphatase. Hydrolytic activity of enzyme at pH 7.5 toward both PPi and p-nitrophenylphosphate was only one-tenth and one-hundredth, respectively, of that observed at optimal pH. Arsenate (Ki - 1.0-2.7 microM) and vanadate (Ki = 0.7-1.3 microM) were powerful inhibitors of alkaline phosphatase; phosphate (Ki = 20-50 microM) was inhibitory at levels two orders of magnitude below the concentration in cartilage extracellular fluid. Neither Zn2+, Ca2+, or Mg2+ was inhibitory toward p-nitrophenylphosphate hydrolysis, whereas all three competitively inhibited PPi hydrolysis. The data suggest that formation of poorly hydrolyzable Ca.PPi, Mg2.PPi, and Zn2.PPi complexes was responsible. Inhibition of PPi hydrolysis by Ca2+ occurred at levels within the physiological range. The close similarity in inhibition of both the purified and membrane-bound forms of alkaline phosphatase at pH 7.5 indicates that interaction with the membrane does not significantly alter conformation at the active site. The data obtained suggest that under physiological conditions, cartilage membrane alkaline phosphatase would be essentially inactive as a phosphohydrolase due to its intrinsically weak activity at pH 7.5 and the strong inhibitory effect of physiological levels of phosphate and Ca2+.
D000469:Alkaline Phosphatase / Q000378:metabolism*; D000818:Animals; D001149:Arsenates / Q000494:pharmacology; D002356:Cartilage / Q000201:enzymology*; D002413:Cations, Divalent; D002462:Cell Membrane / Q000201:enzymology; D004838:Epiphyses / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007978:Levamisole / Q000494:pharmacology; D013379:Substrate Specificity; D014638:Vanadates; D014639:Vanadium / Q000494:pharmacology
D001149:Arsenates; D002413:Cations, Divalent; D014639:Vanadium; D007978:Levamisole; D014638:Vanadates; D000469:Alkaline Phosphatase