Dipeptidyl peptidase I from goat brain: purification, characterization and its action on Leu-enkephalin.
Brain dipeptidyl peptidase (DPP) I has been purified 2990-fold to apparent homogeneity shown by a single protein band in electrophoreses at pH 4.5, 8.4 and in SDS-PAGE at pH 7.2. The purification techniques included homogenization of brain acetone powder, autolysis at pH 4.2, 30-80% (NH4)2SO4 fractionation. Sephadex G-100 column chromatography, heat treatment at 65 C. organomercurial affinity chromatography. CM-Sephadex cation-exchange chromatographies at pH 5.6 and 5.0 and anion-exchange chromatography on DEAE-Sephadex at pH 6.8. The enzyme hydrolysed synthetic substrate Gly-Arg-4-methoxy-beta-naphthyl-amide maximally at pH 6.0. The Km values for Gly-Arg-beta-naphthylamide and Gly-Arg-4-methoxy-beta-naphthylamide substrates were 0.10 mM and 0.14 mM respectively. The enzyme was inhibited by thiol inhibitors like p-chloromercuribenzoic acid, iodoacetic acid, iodoacetamide and microbial inhibitors leupeptin and antipain. Molecular weight estimations on a calibrated Sephadex G-200 column afforded a value of 180,000 Da while in denaturing conditions on sodium dodecyl sulphate polyacrylamide gel electrophoresis, the subunit molecular weight was 22,000 Da. The subunit structure of the native enzyme was unfolded in presence of different concentrations of urea. In 8 M urea, the enzyme dissociated completely into monomers of 25,000 Da but 6, 5 and 4 M urea concentrations revealed the existence of dimers, tetramers and hexamers. Leu-enkephalin. Tyr-Gly-Gly-Phe-Leu was degraded by DPPI into Tyr-Gly and Gly-Phe-Leu with no further degradation of the newly generated tripeptide.
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Department of Chemistry, Kurukshetra University, Haryana, India.
Raghav
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Kamboj
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Singh
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H
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D020789:Cathepsin C; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004743:Enkephalin, Leucine / Q000378:metabolism*; D004795:Enzyme Stability; D006041:Goats; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight
D046911:Macromolecular Substances; D004743:Enkephalin, Leucine; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D020789:Cathepsin C