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Purification and Properties of Alkaline Phosphatase from Rat Chloroma¹

Department of Medicine and Biochemistry and The Howard Hughes Medical Institute, University of Miami School of Medicine, Miami, Florida 33152

Alkaline phosphatase from rat chloroma has been purified by butanol extraction, acetone and ammonium sulfate precipitation, column chromatography on diethylaminoethyl cellulose, and filtration on Sephadex G-200. Upon electrophoresis on polyacrylamide gel, the enzyme travels in at least three activity bands. Molecular weight determinations by sucrose density gradient sedimentation yield a value of 140,000. Like alkaline phosphatase from other tissues, chloroma alkaline phosphatase is a nonspecific phosphomonoesterase capable of hydrolyzing a wide variety of phosphate esters. It requires magnesium for maximal activity and is relatively stable at pH 7.5. The enzyme is inactivated by ethylenediaminetetraacetate and virtually full reactivation can be accomplished only by the addition of Zn⁺⁺. Antibody prepared in rabbit against purified chloroma alkaline phosphatase reacts with the enzyme from rat chloroma and rat leukocytes but fails to cross-react with human placental or leukocyte alkaline phosphatase.

¹ This work was supported by USPHS Grants AM 09001-06, AM 05472-05, and AM 13087-02 and American Cancer Society Grant P-391.

Received 6/22/70. Accepted 9/18/70.