Enzymatic changes of the bovine pituitary multicatalytic proteinase complex, induced by magnesium ions

Abstract

The effect of magnesium ions on the catalytic activities of the bovine pituitary multicatalytic proteinase complex (MPC) was studied. Mg 2+ markedly stimulated the breakdown of dephosphorylated β-casein (caseinolytic activity) and the hydrolysis of CbzLeuLeuGlu-2-naphthylamide (peptidylglutamyl peptide bond hydrolyzing activity) by a 1700-fold purified preparation of MPC. Cleavage of CbzD-AlaLeuArg-2-naphthylamide (trypsin-like activity) was strongly inhibited and cleavage of CbzGlyGlyLeu p-nitroanilide (chymotrypsin-like activity) was weakly inhibited. Similar results were produced when enzymatic activities in the absence of Mg 2+ were measured at 52 °C rather than at 37 °C. Trace protein impurities were removed by phenyl-Sepharose chromatography. This additional chromatographic step, while not changing the specific activities of hydrolysis of the three synthetic chromogenic substrates, led to a marked activation of the breakdown of dephosphorylated β-casein. Mg 2+ was not able to further stimulate the caseinolytic activities of either the phenyl-Sepharose-treated preparation or the preparation measured at 52 °C. Mg 2+ therefore converts a “repressed” form of MPC to an “activated” form, possibly by promoting dissociation of a protein inhibitor, and may serve as a physiological regulator of this enzyme complex.