Abstract
A highly active form of clostripain, composed of two polypeptide chains (Mr = 43,000 and 12,500), was isolated by hydrophobic chromatography from the culture medium of Clostridium histolyticum. It differs in amino acid composition, namely in the value for cyst(e)ine, from that previously reported. The analyses of the separated chains are given. Activity is related to the number of free cysteine residues and full activity is obtained only after complete reduction of the disulfide bonds. Specific modifications by sulfhydryl reagents and tosyl lysine chloromethyl ketone of one thiol group, the one implicated in the activity, are reported. High specificity of alpha-clostripain is restricted to arginyl peptide bonds as tested on parvalbumin.
Highlights
A highly active form of clostripain, composed of two polypeptide chains (Mr = 43,000 and 12,500), was isolated by hydrophobic chromatography from the culture medium of Clostridium histolyticum
Clostripain (EC 3.4.22.8) is a sulfhydryl protease isolated from the culture filtrate of an anaerobic bacterium Clostridium histolyticum
In the most active preparations, the direct titration after denaturation but without reduction showed that practically all the half-cystine residues present in clostripain are in the sulfhydryl form
Summary
The separation of clostripain into chromatographically homogeneous conformers, some without activity and one with a specific activity of 500 units/mg by means of chromatography on a w-aminoalkylagarose column is described. Thiol Number and Activation of Clostripain-As mentioned before, Porter and Mitchell (1973) assumed that the appearance of two distinct components of clostripain, after reduction by dithiothreitol and separation on an hydroxylapatite column, would indicate that the active site -SH groups were formed from at least two separate and stable a- Clostripain disulfide pairings, giving rise to two chromatographic conformers.
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