Purification of a Ca2+-activated protease from rat erythrocytes and its possible effect on pyruvate kinase in vivo.

A Ca2+-activated protease with [32P]phosphopyruvate kinase as substrate was purified to about 50% from rat erythrocytes. The purification involved chromatography on Sepharose/Sephadex gels, DEAE-cellulose and (NH4)2SO4 precipitation. The protease required 3.3 mM Ca2+ for full activity. When pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) was purified from erythrocytes incubated with [32P]phosphate it contained 0.5 mol [32P]phosphate/mol enzyme subunit. When 3.3 mM Ca2+ were added at hemolysis this incorporation decreased. The possible importance of this Ca2+-activated protease for the regulation of pyruvate kinase in erythrocytes is discussed.