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Analysis of a Bacillus subtilis Proteinase Mutant¹

^a Department of Molecular Biology and Biophysics, Yale University, New Haven, Connecticut 06520
Department of Biology, Temple University, Philadelphia, Pennsylvania 19122

ABSTRACT

A Bacillus subtilis mutant having a phenotype manifesting reduced extracellular proteolytic activity was investigated. An extracellular protein was isolated and shown by fingerprint analysis to be a fragment of the wild-type enzyme. By using previously established molecular weights for the wild-type enzyme (2.9 x 10⁴) and the two polypeptide chains derived from it (1.4 x 10⁴ each), with the amino acid analysis and fingerprints of both wild-type and mutant proteins, a molecular weight of 1.57 x 10⁴ was assigned to the mutant protein. ³²P-diisopropylphosphate labeling of the mutant protein showed only 1 in 53 molecules to be functional. Thin-layer chromatography on Sephadex G-75 demonstrated that the active molecules were separable from the bulk of the isolated protein and had the same mobility as the wild-type enzyme. Fingerprints of tryptic digests of ³²P-diisopropylphosphate-labeled wild-type and mutant proteins showed that the labeled peptides had identical characteristics.

² Present address: Wyeth Laboratory, Radnor, Pa.

¹ Taken in part from a dissertation submitted by R.S. to the faculty of the Graduate School of Yale University for the Ph.D. degree.