Purification and properties of bovine synovial fluid alkaline phosphatase.

Alkaline phosphatase from bovine synovial fluid was purified 2300-fold. A molecular weight of 72,300 was determined from sucrose density gradient studies. The following monoesters were hydrolyzed by the enzyme: P-glycerophosphate, galactosamine 6-phosphate, glucosamice 6-phosphate, glucose 6-phosphate, o-phospho-L-serine, o-carboxyphenyl phosphate, phenyl phosphate, and p-nitrophenyl phosphate. Kinetic studies of the enzyme were made with p-nitrophenyl phosphate as substrate. Adenosine triphosphate and pyrophosphate were not hydrolyzed by the enzyme. Activation of the enzyme was observed with Sr2+, Ca2+, and Mg2+ ions. Cyanide and fluoride, as well as ethylenediaminetetraacetate, inhibited the enzyme activated by MgZf while the chelating agent, 1,2-bis(-2-dicarboxymethylaminoethoxy)ethane (EGTA), inhibited the enzyme activated by Ca2f. Diisopropylfluorophosphate and p-chloromercuribenzoate were virtually without effect on the activity. The pH optimum increased with increasing substrate concentration. The pH profiles were obtained for pK,, log V IIIBX, and log V,,,/K,. These data show the existence of two groups at the active site having pK values of 8.6 and 9.6.