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 Β-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 290μM, 18.0 s⁻¹ and 62.1 s⁻¹ .nm⁻¹ for Arg-Arg-MCA and 125μM, 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 λRDIII-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.