Dipeptidyl peptidase III from rat liver cytosol: Purification, molecular cloning and immunohistochemical localization

Dipeptidyl peptidase III (DPP III) was purified to homogeneity from rat liv er cytosol, The calculated molecular weight of the purified enzyme was 8284 5.6 according to TOF-MS and 82000 on non-denaturing PAGE, and 82000 on SDS- PAGE in the absence or presence P-mercaptoethanol, These findings suggest t hat 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 value s of DPP III at optimal pH (pH 8.5) were 290 mu M, 18.0 s(-1) and 62.1 s(-1 ).nM(-1) for Arg-Arg-MCA and 125 mu 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-ph enanthroline, 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 liv er cDNA library using affinity-purified anti-rat DPP III rabbit IgG antibod ies, determined the cDNA structure and deduced the amino acid sequence. The cDNA, designated as lambda RDIII-11, is composed of 2640 bp and encodes 73 8 amino acids in the coding region. Although the enzyme has a novel zinc-bi nding motif, HEXXXH, DPP III is thought to belong to family 1 in dan MA in the metalloprotease kingdom. The DPP III antigen was detected in significant amounts in the cytosol of v arious rat tissues by immunohistochemical examination.