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dc.titlePurification and characterization of human brain prolyl endopeptidase
dc.contributor.authorKalwant, S.
dc.contributor.authorPorter, A.G.
dc.identifier.citationKalwant, S.,Porter, A.G. (1991). Purification and characterization of human brain prolyl endopeptidase. Biochemical Journal 276 (1) : 237-244. ScholarBank@NUS Repository.
dc.description.abstractProlyl endopeptidase (EC was purified from human brain by a series of column-chromatographic steps using DEAE-cellulose DE-52, hydroxyapatite, phenyl-Sepharose, Sephacryl S-200 and f.p.l.c. (Mono Q). The enzyme was purified by a factor of 943 and was homogeneous in a SDS/polyacrylamide gel as judged by Coomassie Blue staining. The M(r) estimated by SDS/PAGE is 79600, and under native conditions on Sephacryl S-200 it is 85600. Therefore the enzyme exists as a monomer. With benzyloxycarbonylglycylproline p-nitroanilide as substrate, the optimum pH of the enzyme is 6.8, and with the substrate concentration between 0.069 mM and 0.37 mM the K(m) is 9.0 x 10-4 M. The pI of the enzyme is 4.75. The enzyme is classified as a serine proteinase, as it is strongly inhibited by di-isopropyl fluorophosphate. However, other serine proteinase inhibitors do not inhibit the enzyme significantly, suggesting that the active site of prolyl endopeptidase differs from that of classical serine proteinases such as trypsin. Polyclonal antibodies were raised against purified human brain prolyl endopeptidase in rabbits. Western-blot analysis, enzyme-inhibition assays, antibody binding and immunoprecipitation experiments indicated that the polyclonal antibodies are both specific and inhibitory to the enzyme activity.
dc.contributor.departmentINSTITUTE OF MOLECULAR & CELL BIOLOGY
dc.description.sourcetitleBiochemical Journal
Appears in Collections:Staff Publications

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