Molecular cloning and immunohistochemical localization of rat dipeptidyl peptidase III.
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.
Ohkubo
I
I
Department of Medical Biochemistry, Shiga University of Medical Science, Shiga, Japan.
Li
Y
Y
Maeda
T
T
Yamamoto
Y
Y
Yamane
T
T
Du
P G
PG
Nishi
K
K
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
D018076:DNA, Complementary; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C100152:dipeptidyl peptidase III