Studies on the properties of two intracellular serine proteases from Bacillus subtilis

Sheehan, Shannon Mark

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https://hdl.handle.net/2142/19577 Copy

Description

Title

Studies on the properties of two intracellular serine proteases from Bacillus subtilis

Author(s)

Sheehan, Shannon Mark

Issue Date

1990

Doctoral Committee Chair(s)

Switzer, Robert L.

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Biology, Microbiology
• Chemistry, Biochemistry

Language

eng

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.

Type of Resource

text

Permalink

http://hdl.handle.net/2142/19577

Copyright and License Information

Copyright 1990 Sheehan, Shannon Mark

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