The synthesis, characterization, and use of a protein-cysteine proteinase inhibitor complex for the study of endosome/lysosome fusion

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dc.contributor.authorMountz, Adele K.en
dc.contributor.committeechairMason, R.W.en
dc.contributor.committeememberNewton, William E.en
dc.contributor.committeememberCastagnoli, Neal Jr.en
dc.contributor.committeememberGregory, Eugene "Mick"en
dc.contributor.committeememberGregory, D.R.en
dc.contributor.departmentBiochemistry and Anaerobic Microbiologyen
dc.date.accessioned2014-03-14T21:14:51Zen
dc.date.adate2006-06-07en
dc.date.available2014-03-14T21:14:51Zen
dc.date.issued1994en
dc.date.rdate2006-06-07en
dc.date.sdate2006-06-07en
dc.description.abstractThe cysteine proteinases cathepsins B, L, and S are lysosomal enzymes responsible for the degradation of endocytosed proteins. Their presence in human cell monocytic lines THP1 and U937 was detected by the use of the membrane-permeable, irreversible, active-site directed inhibitor Fmoc-(¹²⁵I)Tyr- Ala-CHN₂ followed by immunoprecipitation of the enzymes, SDSPAGE, and autoradiography. All three enzymes were detected in THP1 cells; only after differentiation of U937 cells to macrophage-like cells were the enzymes detectable. Both cell lines show multiple forms of cathepsin S, at 35 kDa, 28 kDa, and 26 kDa, suggesting the presence of an active pro-form of cathepsin S as well as the processing of cathepsin S into single- and two-chain forms. This is the first evidence for an active pro-form of a cysteine proteinase and for the processing of cathepsin S to a two-chain enzyme form. Multiple forms of cathepsin L were analyzed by isoelectric focusing followed by denaturing polyacrylamide gel electrophoresis. The multiple forms are not due to the presence of carbohydrate chains on the protein. The inhibitor Fmoc-Tyr-Ala-CHN₂ synthesized and its inhibitory properties against cathepsins B, L, and S were determined. Both in vitro and in vivo studies show that this inhibitor is an effective reagent for studying lysosomal cysteine proteinases. In order to be useful in the study of the delivery of lysosomal enzymes to vesicles containing recently internalized compounds, the deblocked peptidyl diazomethane inhibitor NH₂-Tyr-Ala-CHN₂ was cross-linked to bovine serum albumin (BSA) using the heterobifunctional crosslinking agent sulfo-SANPAH. This non-reducible cross-linked complex was used to characterize the inhibitory properties of the protein-inhibitor complex against cathepsins B, L, S and papain in vitro and in vivo.en
dc.description.degreePh. D.en
dc.format.extentxiv, 188 leavesen
dc.format.mediumBTDen
dc.format.mimetypeapplication/pdfen
dc.identifier.otheretd-06072006-124205en
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-06072006-124205/en
dc.identifier.urihttp://hdl.handle.net/10919/38555en
dc.language.isoenen
dc.publisherVirginia Techen
dc.relation.haspartLD5655.V856_1994.M686.pdfen
dc.relation.isformatofOCLC# 30924531en
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subject.lccLD5655.V856 1994.M686en
dc.subject.lcshCysteine proteinasesen
dc.subject.lcshEndocytosisen
dc.subject.lcshLysosomesen
dc.titleThe synthesis, characterization, and use of a protein-cysteine proteinase inhibitor complex for the study of endosome/lysosome fusionen
dc.typeDissertationen
dc.type.dcmitypeTexten
thesis.degree.disciplineBiochemistry and Anaerobic Microbiologyen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.leveldoctoralen
thesis.degree.namePh. D.en

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