title: Molecular cloning and immunohistochemical localization of rat dipeptidyl peptidase III.
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-denatured PAGE and 82000 on SDS-PAGE in the absence or presence of beta-ME. These findings suggest that the enzyme assumes a monomeric form in rat liver cytosol. The enzyme rapidly hydrolyzed the substrate Arg-Arg-MCA and moderately hydrolyzed Ala-Arg-MCA in a pH range of 7. 5 to 9.5. The K(in), K(cat) and K(cat)/K(m) values of DPP III at optimal pH (pH 8.5) were 290 microM, 18.0 s(-1) and 6.21x10(4) s(-1)M(-1) for Arg-Arg-MCA and 125 microM, 4.53 s(-1) and 3.62x10(4) s(-1)M(-1) for Ala-Arg-MCA, respectively. DPP III was potently inhibited by EDTA, 1,10-phenanthroline, DFP, PCMBS, NEM, beta-ME and iodoacetamide. Furthermore, we screened a rat liver cDNA library using affinity-purified anti-rat DPP III rabbit IgG, and we determined the cDNA structure and deduced the amino acid sequence. The cDNA designated as lambdaRDIII-11 is composed of 2640 bp of nucleotides in length and encodes 738 amino acids in the coding region. Although the enzyme has a novel zinc-binding motif, HEXXXH in structure, DPP III is thought to belong to family 1 in clan MA in the metalloprotease kingdom. These findings suggest that DPP III is a metalloprotease that is probably regulated by SH modification. The DPP III antigen was extensively detected in the cytosol of various rat tissues by the immunohistochemical examination of the protein.
authors.affiliation: Department of Medical Biochemistry, Shiga University of Medical Science, Shiga, Japan.
authors.forename: I
authors.initials: I
authors.lastname: Ohkubo
authors.forename: Y
authors.initials: Y
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 / Q000379:methods*; D003600:Cytosol / Q000201:enzymology; D018076:DNA, Complementary / Q000235:genetics*; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000235:genetics* / Q000302:isolation & purification* / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007150:Immunohistochemistry / Q000379:methods*; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011817:Rabbits; D051381:Rats
chemical_list: D018076:DNA, Complementary; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C100152:dipeptidyl peptidase III