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+.

Purification and initial characterization of intrinsic membrane-bound alkaline phosphatase from chicken epiphyseal cartilage.

Alkaline phosphatase has been purified from microsomes of chicken epiphyseal cartilage by first selectively extracting certain adventitious proteins with 0.25 M trichloroacetate. The membrane-bound enzyme was then solubilized by 1% cholate in buffered 33% saturated ammonium sulfate and purified by column chromatography on Bio-Gel A-5m, extraction with 1-butanol, and ion exchange chromatography on DEAE-Bio-Gel A. The purified alkaline phosphatase from the cartilage membrane had a subunit molecular weight of 53,000 and a holoenzyme weight of 207,000-220,000, indicating a tetramer. The pH optima for p-nitrophenylphosphate, ATP, and pyrophosphate hydrolysis were 10.3, 9.0, and 8.5, respectively. Values of Vmax (in micromoles/min/mg) were 220, 3.1, and 0.8, respectively. Substrate inhibition was pronounced at values of pH below 8.5. Inhibition of p-nitrophenylphosphate hydrolysis at pH 10.3 showed that phosphate and arsenate were competitive inhibitors (KI = 1.88 and 0.15 mM, respectively) and levamisole was an uncompetitive inhibitor (KI = 0.32 mM), while L-phenylalanine and ZnCl2 were mixed inhibitors (KI = 15.8 and 0.02 mM, respectively). Inhibition by preincubation in 1 mM EDTA was reversible by readdition of 0.25 mM MgCl2 nd 20 microM ZnCl2. The data indicate that this membrane-bound alkaline phosphatase from chicken epiphyseal cartilage is a Zn2+ and possibly Mg2+-containing enzyme. While the subunit molecular weight and kinetic properties of the enzyme are quite typical of vertebrate alkaline phosphatases, the tightness of binding to the membrane lipids, the extreme sensitivity to substrate inhibition, and the tetrameric conformation of the holoenzyme are unusual.