Interaction of α²-Macroglobulin with Serine Proteinase from Aspergillus sojae

Abstract

The interaction of α₂-macroglobulin (α₂M) with a major fraction of serine proteinase (SepII) from Aspergillus sojae was investigated. The stoichiometry of the reaction of α₂M with SepII showed that SepII bound to α₂M in a molar ratio of about 2:1. The complex (α₂M-SepII) was separated by PAGE and chromatograph. About 80% inhibition of the proteinase activity as to the hydrolysis of casein and succinyl-l-leucyl-l-leucyl-l-valyl-l-tyrosyl-7-amino-4-methylcoumarin (Suc-LLVY-MCA) was observed. The residual activity of the complex was completely abolished by chymostatin and antipain, which were powerful inhibitors with Ki values of 3 × 10⁻⁸ and 5 × 10⁻⁸, respectively. Immunoelectrophoresis of the complex showed its weak cross-reactivity with antiserum to SepII (anti-SepII). The activity of the complex was partially abolished by the anti-SepII. Examination of electron micrographs left little doubt that a conformational change in shape took place. The entrapment reaction with α₂M was completed within 30 sec. When the α₂M-SepII complex was incubated at pH 3.6, a triphasic course was observed. Activation of the complex was observed within 10∼17min, while inactivation was observed after 10∼17 min incubation. And then the activity remained at several % of the initial level. The complex was found to exhibit enhanced pH stability at pH 3.6, while alkali and heat denaturation were observed not to have drastic effects on its stability.