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Title:Studies on the properties of two intracellular serine proteases from Bacillus subtilis
Author(s):Sheehan, Shannon Mark
Doctoral Committee Chair(s):Switzer, Robert L.
Department / Program:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Biology, Microbiology
Chemistry, Biochemistry
Abstract:Intracellular serine protease-1 (ISP-1) from Bacillus subtilis had been previously purified to homogeneity. The purified protease (Mr = 31,000) had undergone processing to remove 17 to 20 amino-terminal amino acid residues. Then observations and a number of other studies led to the proposal that ISP-1 is synthesized in B. subtilis cells as an inactive precursor, which may undergo activation by amino-terminal processing. To examine these questions, monospecific polyclonal antibodies against ISP-1 were raised. A variety of procedures for extracting ISP-1 from cells under conditions that prevent proteolysis in vitro were evaluated by immunobloting and ISP-1 activity assays. ISP-1 was found to be always produced in a form (Mr = 34,000) that was larger than the purified form. This larger form was readily converted to the smaller form in vitro in crude extracts at pH 8.5 in the presence of Ca$\sp{2+}$ ions. It was also found that the appearance of ISP-1 activity and immunologically cross-reactive protein were temporally coincident. Thus, amino-terminal processing of ISP-1 is an artifact of in vitro proteolysis, and no evidence for an inactive ISP-1 precursor was found.
ISP-1 could be stabilized in vitro in the unprocessed form at low pH(5.0 to 6.5) and by inclusion of high concentrations of chelators, such as ethylene diamine tetraacetate. A procedure was developed for purifying the unprocessed form of ISP-1 about 50 fold, but further purification steps resulted in degradative ions or processing of the protein. Unprocessed ISP-1 was tightly associated with a high molecular weight complex (10 to 20 MDa), from which it could not be dissociated in an active or unprocessed form.
During attempts to purify ISP-1, a previously undiscovered B. subtilis intracellular serine protease was found, which was named ISP-4. This protease hydrolyzed azocasein and the same chromogenic substrate (Cbz-Ala-Ala-Leu-pNA) as ISP-1, but did not cross-react with antiserum against ISP-1. Surprisingly, ISP-4 was absent from a mutant from which the gene encoding ISP-1 had been deleted. ISP-4 was also associated with a high molecular weight complex (8 to 10 MDa), but could be largely separated from ISP-1 by chromatography on Sepharose S-500 or by chromatography on omega-amino propyl-Agarose. ISP-4 was purified by about 250-fold, but further purification was prevented by an extreme tendency toward autodigestion. ISP-4 had a pH and temperature optima of 9.0 and 37$\sp\circ$C, respectively. The enzyme was strongly inhibited by phenylmethyl sulfonyl fluoride, antipain, and chymostatin.
Immunoinhibition studies indicated that ISP-1 and ISP-4 represent about 35%, and 65%, respectively, of the Cbz-Ala-Ala-Leu-pNA hydrolyzing activity present in extracts of stationary B. subtilis cells. ISP-1 was essentially inactive in the absence of added Ca$\sp{2+}$ ions; activity found in the presence of chelators is probably due to ISP-4.
Issue Date:1990
Rights Information:Copyright 1990 Sheehan, Shannon Mark
Date Available in IDEALS:2011-05-07
Identifier in Online Catalog:AAI9114409
OCLC Identifier:(UMI)AAI9114409

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