Primary Structure and Reactive Site of Streptoverticillium Anticoagulant (SAC), a Novel Protein Inhibitor of Blood Coagulation Produced by Streptoverticillium cinnamoneum subsp. cinnamoneum

Abstract

The complete amino acid sequence of SAC I, a novel protein inhibitor of blood coagulation produced by Streptoverticillium cinnamoneum subsp. cinnamoneum IFO 12852, was determined. Automated Edman degradation was employed for its fragment peptides, which were obtained by specific cleavage procedures including tryptic, chymotryptic, and peptic digestions, and cyanogen bromide treatment. SACI is composed of 110 amino acid residues with a molecular weight of 11, 642. It has two intramolecular disulfide bonds, Cys³¹-Cys⁴⁶ and Cys⁶⁸-Cys⁹⁸, but no cysteine residues. The overall sequence homology of SACI is 58% to plasminostreptin, a protein protease inhibitor produced by Streptomyces antifibrinolyticus, and 52% to S-SI, Streptomyces subtilisin inhibitor, produced by Streptomyces albogriseolus. Subtilisin [EC 3. 4. 21. 14]-modified SACI, which was prepared by incubating SACI with subtilisin, had a newly-generated amino terminal sequence, Glu⁷¹-Trp⁷²-Asn⁷³-, in addition to the original amino terminal sequence, and a newly-generated carboxyl terminal arginine in addition to the original phenylalanine. These results clearly show that the Arg⁷⁰-Glu⁷¹ bond was specifically cleaved on the limited proteolysis with subtilisin and that the bond is the reactive site for subtilisin.