title: Dipeptidyl peptidase III from rat liver cytosol: purification, molecular cloning and immunohistochemical localization.
abstract: Dipeptidyl peptidase III (DPP III) was purified to homogeneity from rat liver cytosol. The calculated molecular weight of the purified enzyme was 82845.6 according to TOF-MS and 82000 on non-denaturing PAGE, and 82000 on SDS-PAGE in the absence or presence of beta-mercaptoethanol. These findings suggest that the enzyme exists in a monomeric form in rat liver cytosol. The enzyme rapidly hydrolyzed the substrate Arg-Arg-MCA and moderately hydrolyzed Gly-Arg-MCA in the pH range of 7.5 to 9.5. The Km, k(cat) and k(cat)/Km values of DPP III at optimal pH (pH 8.5) were 290 microM, 18.0 s(-1) and 62.1 s(-1) x nM(-1) for Arg-Arg-MCA and 125 microM, 4.53 s(-1) and 36.2 s(-1) x nM(-1) for Ala-Arg-MCA, respectively. DPP III was potently inhibited by EDTA, 1,10-phenanthroline, DFP, PCMBS and NEM. These findings suggest that DPP III is an exo-type peptidase with characteristics of a metallo- and serine peptidase. For further information on the molecular structure, we screened a rat liver cDNA library using affinity-purified anti-rat DPP III rabbit IgG antibodies, determined the cDNA structure and deduced the amino acid sequence. The cDNA, designated as lambdaRDIII-11, is composed of 2640 bp and encodes 738 amino acids in the coding region. Although the enzyme has a novel zinc-binding motif, HEXXXH, DPP III is thought to belong to family 1 in clan MA in the metalloprotease kingdom. The DPP III antigen was detected in significant amounts in the cytosol of various rat tissues by immunohistochemical examination.
authors.affiliation: Department of Medical Biochemistry, Shiga University of Medical Science, Seta, Otsu, Japan.
authors.forename: I
authors.initials: I
authors.lastname: Ohkubo
authors.forename: Y H
authors.initials: YH
authors.lastname: Li
authors.forename: T
authors.initials: T
authors.lastname: Maeda
authors.forename: Y
authors.initials: Y
authors.lastname: Yamamoto
authors.forename: T
authors.initials: T
authors.lastname: Yamane
authors.forename: P G
authors.initials: PG
authors.lastname: Du
authors.forename: K
authors.initials: K
authors.lastname: Nishi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology*; D018076:DNA, Complementary; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C100152:dipeptidyl peptidase III