Inhibition by Equine α₂-Macroglobulin of an N-Succinyl-L-Trialanine p-Nitroanilide-Hydrolyzing Protease Purified from Pronase

Abstract

The binding of an N-succinyl-L-trialanine p-nitroanilide-hydrolyzing protease (STA-protease) purified from pronase to equine α₂-macroglobulin (α₂M) was investigated in comparison with that of trypsin. The α₂M subunits (about 90000 daltons), which were electrophoretically detected in the reaction mixture of α₂M and trypsin, were undetectable in that of α₂M and STA-protease. The binding molar ratios of enzyme to α₂M were estimated from the inhibition curves of caseinolytic activity be 1.5 : 1 for native and acetylated STA-protease and 2 : 1 for native and acetylated trypsin. The finding of greater incorporation of monodansylcadaverine into α₂M reacted with acetylated enzymes than into that reacted with the native enzymes suggests that free amino groups in the enzymes are involved at least partly in the formation of the α₂M-proteinase complexes. The numbers of thiol groups generated in α₂M bound to STA-protease and in α₂M bound to trypsin were both estimated to be approximately 4 mol per mol of α₂M by the use of thiol-directed fluorescent probes, though there were slight differences in the microenvironments of thiol groups generated in the two α₂M-proteinase complexes. The values of K_cat/K_m were one-half (α₂M-STA-protease complex) and one-sixth (α₂M-trypsin complex) of those of the uninhibited enzymes. These results suggest that STA-protease binds to α₂M both covalently and noncovalently, as does trypsin, and its hydrolytic activities towards casein and low-molecular-weight substrates are inhibited to various extents.