{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/19577"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/19577","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Studies on the properties of two intracellular serine proteases from Bacillus subtilis","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.","abstract_html":"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.","abstract_has_math":true,"creators":["Sheehan, Shannon Mark"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Biochemistry","degree_department":null,"school":null,"contributors":["Switzer, Robert L."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2011,"date_issued":"2011-05-07T12:11:58Z","date_published":"2011-05-07T12:11:58Z","updated_at":"2026-07-22T22:25:14Z","subjects":["Biology, Microbiology","Chemistry, Biochemistry"],"languages":["eng"],"rights":["Copyright 1990 Sheehan, Shannon Mark"],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["AAI9114409","(UMI)AAI9114409"],"render_values":[{"text":"AAI9114409","href":null,"code":true},{"text":"(UMI)AAI9114409","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/2142/19577","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Switzer, Robert L."]},{"key":"dc:creator","label":"Author","values":["Sheehan, Shannon Mark"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2011-05-07T12:11:58Z","10000-01-01","1990"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biochemistry"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Biology, Microbiology","Chemistry, Biochemistry"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 1990 Sheehan, Shannon Mark"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["AAI9114409","(UMI)AAI9114409","http://hdl.handle.net/2142/19577"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["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.","Made available in DSpace on 2011-05-07T12:11:58Z (GMT). No. of bitstreams: 2 license.txt: 4922 bytes, checksum: 910b249b4beec47e7ab768910c8f966f (MD5) 9114409.pdf: 4966933 bytes, checksum: 3b83d6b6f6200f1cc4b9c5e79427632d (MD5) Previous issue date: 1990","Item marked as restricted to the 'UIUC Users [automated]' Group (id=2) by Howard Ding (hding2@illinois.edu) on 2011-05-07T14:37:59Z Item is restricted indefinitely.","Restriction data tranferred 2014-07-01T11:15:43-05:00 Original Data Group with Access UIUC Users [automated] Release Date: none Reason: ETDs are only available to UIUC Users without author permission","ETDs are only available to UIUC Users without author permission","U of I Only"]},{"key":"dc:title","label":"Title","values":["Studies on the properties of two intracellular serine proteases from Bacillus subtilis"]}]}],"canonical_facts":{"dc:contributor":["Switzer, Robert L."],"dc:creator":["Sheehan, Shannon Mark"],"dc:date":["2011-05-07T12:11:58Z","10000-01-01","1990"],"dc:description":["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.","Made available in DSpace on 2011-05-07T12:11:58Z (GMT). No. of bitstreams: 2 license.txt: 4922 bytes, checksum: 910b249b4beec47e7ab768910c8f966f (MD5) 9114409.pdf: 4966933 bytes, checksum: 3b83d6b6f6200f1cc4b9c5e79427632d (MD5) Previous issue date: 1990","Item marked as restricted to the 'UIUC Users [automated]' Group (id=2) by Howard Ding (hding2@illinois.edu) on 2011-05-07T14:37:59Z Item is restricted indefinitely.","Restriction data tranferred 2014-07-01T11:15:43-05:00 Original Data Group with Access UIUC Users [automated] Release Date: none Reason: ETDs are only available to UIUC Users without author permission","ETDs are only available to UIUC Users without author permission","U of I Only"],"dc:identifier":["AAI9114409","(UMI)AAI9114409","http://hdl.handle.net/2142/19577"],"dc:language":["eng"],"dc:rights":["Copyright 1990 Sheehan, Shannon Mark"],"dc:subject":["Biology, Microbiology","Chemistry, Biochemistry"],"dc:title":["Studies on the properties of two intracellular serine proteases from Bacillus subtilis"],"dc:type":["text"],"thesis:degree_discipline":["Biochemistry"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."],"thesis:institution_name":["University of Illinois at Urbana-Champaign"]},"updated_at":"2026-07-22T22:25:14Z"}