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Purification and Properties of Bovine Synovial Fluid Alkaline Phosphatase

From the ¹ From the Department of Anatomy and the Department of Physiological Chemistry, Wayne State University School of Medicine, Detroit, Michigan 48207

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: ß-glycerophosphate, galactosamine 6-phosphate, glucosamine 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 Mg²⁺ while the chelating agent, 1,2-bis(-2-dicarboxymethylaminoethoxy)ethane (EGTA), inhibited the enzyme activated by Ca²⁺. 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_m, log V_max, and log V_max/K_m. These data show the existence of two groups at the active site having pK values of 8.6 and 9.6.

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