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Iwao Ohkubo, Yao-Hua Li, Toshinaga Maeda, Yoshio Yamamoto, Takuya Yamane, Pei-Ge Du, Katsuji Nishi

Dipeptidyl Peptidase III from Rat Liver Cytosol: Purification, Molecular Cloning and Immunohistochemical Localization

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 ?-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 K_m, k_cat and k_cat/K_m values of DPP III at optimal pH (pH 8.5) were 290?M, 18.0 s^?1 and 62.1 s^?1 .nm^?1 for Arg-Arg-MCA and 125?M, 4.53 s^?1 and 36.2 s^?1 .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 ?RDIII-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.

Biological Chemistry, Walter de Gruyter

Print ISSN: 1431-6730
Volume: 380, 12/1999
Pages: 1421 - 1430

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