Multiplicity of Metal-Independent Protein Phosphatases of Germinated Alfalfa Seeds

Abstract

Summary Nine enzyme activities that dephosphorylate casein in the presence of EDTA were separated out of an extract of alfalfa ( Medicago sativa ) seeds that had been germinated for 5 days in the dark at 21.5 °C. The separation was accomplished by means of a series of chromatographic steps through columns of DEAE-cellulose, Cibacron blue-Sepharose, Heparin-Sepharose and Concanavalin A-Sepharose. Except for one enzyme that acts optimally at pH 7.0, the enzymes function more efficiently at pH 5.0. None of the enzymes is metal-dependent although some are activated to varying degrees by divalent metal ions. In addition to casein, all enzymes dephosphorylate pNPP . For each enzyme, as well as for the non-specific calf intestinal phosphatase, the ratios of activities towards 32 32 P-casein and pNPP were determined. It is proposed that any enzyme that shows a higher activity ratio than the general non-specific phosphatase has a high probability of being a specific protein phosphatase. On the basis of this criterion (referred to as specificity index), eight of the nine enzymes appear to be specific protein phosphatases.