Porcine germinal angiotensin I-converting enzyme: isolation, characterization and molecular cloning.
Germinal angiotensin I-converting enzyme (gACE) was purified to homogeneity from porcine seminal plasma. The molecular weight of the purified enzyme was calculated to be 182,000 on non-denaturing PAGE and 94,000 and 93,000 on SDS-PAGE in the absence and presence of beta-ME, respectively. These findings suggest that the enzyme is composed of two identical subunits in seminal plasma. The K(m), V(max), K(cat) and K(cat)/K(m) values of gACE at optimal pH (pH 7.2) were 680 microM, 1.0 micromol/mg/min, 33.1 s(-1) and 4.87 x 10(4) s(-1) M(-1) for Z-Val-Lys-Met-MCA, respectively. gACE was potently inhibited by EDTA, 1,10-phenanthroline, captopril and lisinopril, and it promptly released the dipeptides His-Leu and Phe-Arg from angiotensin I and bradykinin. Met- and Leu-enkephalins, neuromedine B and beta-neo-endorphin were also good natural substrates for gACE. We determined the structure of gACE cDNA from the porcine testis, and deduced the amino acid sequence of gACE. The cDNA is composed of 2508 bp of nucleotides in length and encodes 745 amino acids in the coding region. The overall homology of amino acid sequences between porcine, human, sheep and rat gACEs is 72.6 to 84.7%. Zinc-binding motif, chloride-binding site and positions of cysteine residues were well conserved.
Takeuchi
Keisuke
K
Department of Medical Biochemistry, Shiga University of Medical Science, Seta, Otsu, Japan.
Araki
Hisazumi
H
Sakaue
Tomohisa
T
Yamamoto
Yoshio
Y
Fujiwara
Manabu
M
Nishi
Katsuji
K
Ohkubo
Iwao
I
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002216:Captopril / Q000494:pharmacology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D007700:Kinetics; D017706:Lisinopril / Q000494:pharmacology; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D007703:Peptidyl-Dipeptidase A / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D012661:Semen / Q000201:enzymology / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D034421:Sus scrofa / Q000235:genetics*; D013696:Temperature; D013737:Testis / Q000166:cytology / Q000201:enzymology / Q000378:metabolism
D018076:DNA, Complementary; D002216:Captopril; D017706:Lisinopril; D007703:Peptidyl-Dipeptidase A