Inhibition by equine .ALPHA.2-macroglobulin of an N-succinyl-L-trialanine p-nitroanilide-hydrolyzing protease purified from pronase.

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Abstract
The binding of an N-succinyl-L-trialanine p-nitroanilide-hydrolyzing protease (STA-protease) purified from pronase to equine .alpha.2-macroglobulin (.alpha.2M) was investigated in comparison with that of trypsin. The .alpha.2M subunits (about 9000 daltons), which were electrophoretically detected in the reaction mixture of .alpha.2M and trypsin, were undetectable in that of .alpha.2M and STA-protease. The binding molar ratios of enzyme to .alpha.2M were estimated from the inhibition curves of caseinolytic activity to 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 .alpha.2M 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 .alpha.2M-proteinase complexes. The numbers of thiol groups generated in .alpha.2M bound to STA-protease and in .alpha.2M bound to trypsin were both estimated to be approximately 4 mol per mol of .alpha.2M by the use of thiol-directed fluorescent probes, though there were slight differences in the microenvironments of thiol groups generated in the two .alpha.2M-proteinase complexes. The values of Kcat/Km were one-half (.alpha.2M-STA-protease complex) and one-sixth (.alpha.2M-trypsin complex) of those of the uninhibited enzymes. These results suggest that STA-protease bids to .alpha.2M both covalently and noncovalently, as does trypsin, and its hydrolytic activities towards casein and low-molecular-weight substrates are inhibited to various extents.