Characterization of the Complex between α₂-Macroglobulin and a Serine Proteinase from Bacillus natto

Abstract

The stoichiometry of the reaction of bovine α₂-macroglobulin (α₂M) with a serine proteinase from Bacillus natto (BSP) showed that BSP bound to α₂M in a molar ratio of about 2:1. The α₂M-BSP complex hydrolyzed casein and succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosyl-4-methylcoumarin-7-amide (Suc-LLVY-MCA) 65 and 55% less, respectively, while the activity of fibrin hydrolysis was completely lost. The purified complex from DEAE-Toyopearl 650 S and Toyopearl HW-60 F chromatographies was homogeneous on polyacrylamide gel electrophoresis at pH 9.4. Immunoelectrophoresis of the α₂M-BSP complex showed weak cross-reactivity with an antiserum to BSP (anti-BSP). The specificity of α₂M-BSP complex to the acyl-LSTR-amide substrates Boc-LSTR-MCA and Boc-LSTR-pNA was partially changed. Enzymatic activity of the complex was completely abolished by chymostatin. Inactivation was observed when α₂M-BSP complex was incubated with a 5-molar excess of Streptomyces subtilisin inhibitor (SSI) at pH 7.5 and 20°C. Interaction of α₂M-BSP with ¹⁴C-succinyl-SSI was done at 20°C and at 4°C. Incorporation of ¹⁴C-succinyl-SSI with α₂M-BSP was faster at 20°C than 4°C