INORGANIC PYROPHOSPHATE-PHOSPHOHYDROLYTIC ACTIVITY ASSOCIATED WITH RAT OSSEOUS PLATE ALKALINE-PHOSPHATASE

Purified membrane-bound alkaline phosphatase from rat osseous plate hy drolyzed pyrophosphate in the presence of magnesium ions, with a speci fic activity of 92.7 U/mg. Optimal apparent pH for pyrophosphatase act ivity was 8.0 and it remained unchanged on increasing the pyrophosphat e concentration. In the absence of magnesium ions the enzyme had a K-m = 88 mu M and V = 36.7 U/mg for pyrophosphate and no inhibition by ex cess substrate was observed. Pyrophosphatase activity was rapidly dest royed at temperatures above 40 degrees C, but magnesium ions apparentl y protected the enzyme against danaturation. Sodium metavanadate (Ki = 1.0 mM) was a competitive inhibitor of pyrophosphatase activity, whil e levamisole (Ki = 8.2 mM) and theophylline (Ki = 7.4 mM) were uncompe titive inhibitors. Magnesium ions (K-0.5 = 1.7 mu M) stimulated pyroph osphatase activity, while cobalt (Ki = 48.5 mu M) and zinc (Ki = 22.0 mu M) ions were non-competitive inhibitors. Manganese and calcium ions had no effect on pyrophosphatase activity. The M-w of the pyrophospha tase: protein was 130 kDa by gel filtration, but a value of 65 kDa was obtained by dissociative gel electrophoresis, suggesting that it was a dimer of apparently identical subunits. These results suggested that pyrophosphatase activity stems from the membrane-bound osseous plate alkaline phosphatase and not from a different protein.