Porcine germinal angiotensin I-converting enzyme: Isolation, characterization and molecular cloning

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• 作者：Keisuke Takeuchi ; Hisazumi Araki ; Tomohisa Sakaue ; Yoshio Yamamoto ; Manabu Fujiwara ; Katsuji Nishi ; Iwao Ohkubo
• 关键词：Angiotensin I-converting enzyme ; Porcine seminal plasma ; Purification ; Characterization ; cDNA structure
• 刊名：Comparative Biochemistry and Physiology ; Part B
• 出版年：2007
• 期刊代码：50_10964959
• 类别：bio
• 出版时间：February 2007
• 卷：146
• 期：2
• 页码：215-226
• 文件大小：2179 K

摘要

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 β-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 μM, 1.0 μmol/mg/min, 33.1 s^{− 1} and 4.87 × 10⁴ 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 β-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.