Amylase from human serous ovarian tumors: purification and characterization.

Abstract

Abstract Human serous-type ovarian tumors contain an acidic isoenzyme of amylase. Previous attempts at purification of tumor amylases have yielded preparations contaminated with other proteins. The purification scheme presented here incorporates an affinity-chromatography procedure, with use of cycloheptaamylose linked to epoxy-activated Sepharose, that is specific for alpha-amylase (EC 3.2.1.1). Purified amylase isoenzyme from a human serous ovarian tumor was characterized and compared with the purified salivary and pancreatic isoenzymes. All three were similar in amino acid composition, pH optimum, substrate specificity, calcium requirement, heat inactivation, and Km for maltotetraose substrate. The ovarian tumor amylase was similar to the salivary and distinct from the pancreatic enzyme by apparent molecular mass and doublet formation on sodium dodecyl sulfate--polyacrylamide electrophoresis, specific activity of pure enzyme, and sensitivity to specific alpha-amylase inhibitors. All three isoenzymes differed in net electrical charge as evidenced by diethylaminoethyl-Sephadex ion-exchange chromatography and isoelectric focusing. The tumor amylase is clearly distinct from the pancreatic and differs from the salivary enzyme in net electrical charge. Evidence is presented that this charge difference may reflect, at least in part, deamidation of an amylase that is similar to or identical with salivary amylase.