ABSTRACT
An endogenous inhibitor of calcium activated neutral proteinase has been purified from human placenta. The procedure included chromatography on DEAE cellulose, Ultrogel AcA 22 and milli calcium activated neutral proteinase-sepharose in succession. Endogenous calcium activated neutral proteinase inhibitor was a tetramer with identical subunits of molecular weight 68 kDa. It was specific for milli calcium activated neutral proteinase (Calpain II) which is inhibited by the formation of an inactive enzyme-inhibitor complex and not by sequestering Ca 2+ from the medium. Although micro calcium activated neutral proteinase (Calpain I) was not inhibited by endogenous calcium activated neutral proteinase inhibitor, it was protected from autolysis in the presence of the inhibitor. The placental endogenous calcium activated neutral proteinase inhibitor thus regulates Ca2+ activated proteolysis by ensuring micro calcium activated neutral proteinase activity, while inhibiting milli calcium activated neutral proteinase.
ABSTRACT
The subunits of human placental milli calcium activated neutral proteinase and micro calcium activated neutral proteinase have been separated by partial denaturation with urea followed by molecular sieving, with a recovery of 82–91% of activity. The separated subunits were homogeneous, as judged by sodium dodecyl sulphatepolyacrylamide gel electrophoresis. Their molecular sizes, catalytic activities and sulphydryl contents suggest that both the subunits of these two calcium activated neutral proteinases are distinct. The subunits were highly specific and could not be interchanged. Both the subunits of micro calcium activated neutral proteinase were catalytically active whereas only the 80 k subunit of milli calcium activated neutral proteinase was active. 30 k subunit of milli calcium activated neutral proteinase has a regulatory role since maximum activity of the 80 k subunit was elicited only in its presence. Activity of the reassociated subunits indicated that interaction is essential for the expression of optimum activity. Interaction of subunits rendered the enzymes less susceptible to inhibition by endogenous calcium activated neutral proteinase inhibitor.