Alanyl Aminopeptidase from Human Seminal Plasma: Purification, Characterization, and Immunohistochemical Localization in the Male Genital Tract

Abstract

Alanyl aminopeptidase (AAP) was purified to homogeneity from human seminal plasma. The calculated molecular weight of the purified enzyme was approximately 137,000+/-5,000 from light scattering, 140,000 (main) and 137,000 (minor) from non-denatured PAGE and 153,000 from SDS-PAGE in the absence or presence of 2-mercaptoethanol (2-ME). These findings suggest that the enzyme is monomeric in form in human seminal plasma. The enzyme hydrolyzed several amino acid 4-methyl-coumaryl-7-amide (MCA) substrates. The order of Kcat/Km values of AAP at optimal pH (pH 7.5) was Ala- > Lys-Ala- > or = Met- > Leu- > Phe- > Arg- > or = Arg-Arg- > Lys- > Gly-MCAs. AAP was potently inhibited by bestatin, leuhistin, actinonin, amastatin, and 1,10-phenanthroline. These findings suggest that AAP is an aminopeptidase. We determined that the amino acid sequence of the first 22 residues of the enzyme was Ser1-Thr-Thr-Pro-Ser5-Ala-Ser-Ala-Thr-Thr10-Asn-Pro-Al a-Ser-Ala15-Thr-Thr-Leu-Asp-Gln20-Ser-Lys-. This sequence was completely coincident with that downstream of the transmembrane site of human intestinal alanyl aminopeptidase N (CD13). We also isolated cDNA encoding AAP from human prostate cDNA library, sequenced its structure, and confirmed human seminal plasma AAP to be identical with alanyl aminopeptidase N. We postulated that native human seminal plasma alanyl aminopeptidase is released into the seminal plasma after the specific site is cleaved by elastase or an elastase-like enzyme. The enzyme level in human seminal plasma determined by single radial immunodiffusion was 5.2+/-3.2 mg/100 ml (mean+/-SD, n=20) in individuals 20-47 years of age. AAP was immunohistochemically stained in the luminal site-cell membrane of epithelial cells in the prostatic gland and ductuli efferentes of the testis.

Characterization of Prostate Cell Types by CD Cell Surface Molecules

“…Our findings agree, for the most part, with previous studies of CD expression in the prostate, ie, CD10, 6 CD13, 7,8 CD26, 7,9 CD38, 10 CD82,…”

Substrate Specificity of Aminopeptidase from the Mid-gut Gland of the Scallop (Patinopecten yessoensis)

“…Compared with the actions for Ala-Ala, (Ala) 3 , and (Ala) 4 , the hydrolysis rate of Ala-Ala was faster and that of (Ala) 3 slower. (Ala) 4 was not hydrolyzed by the enzyme. The enzyme strongly cleaved Ala-Phe, whereas Ala-Phe-Ser-Ser-Trp-Gly were scarcely attacked.…”

“…1) There are substantial reports concerning substrate specificity, biological function, and structure of aminopeptidases in prokaryotes and mammals. 2,3) Among various aminopeptidases, the alanyl aminopeptidases (EC 3.4.11.2, 3.4.11.14), which preferentially liberate amino-terminal amino acids, such as Ala, Met, Leu, and Tyr of peptides, are widely distributed in mammalian tissues and body fluids, [4][5][6][7][8][9] and in plant tissues 10) and cyanobacteria, 11) as a membrane or cytosolic type. These enzymes from mammals are believed to participate in the metabolism of hormones and neurotransmitters.…”

“…Asp-MCA, Ser-MCA, Tyr-MCA, His-Ala, Met-Tyr-Phe, Ala-Gl-Gly, (Ala) 4 , and Ala-Phe-Ser-Ser-Trp-Gly were purchased from Bachem Inc. (Bubendorf, Switzerland), and all other amino acid-pNAs and peptides from Sigma. The aminopeptidase was purified from the mid-gut gland of the scallop and assayed using Ala-pNA as a substrate as in our previous experiment.…”

“…The k cat =K m values for various pNA and MCA substrates were of the same levels as those for MCA substrates in alanine aminopeptidase from guinea-pig brain, 9) for peptide substrates in ratliver cytosol, 8) and for pNA substrates and dipeptides in Aeromonas caviae aminopeptidase. 16) The results of the action for various peptides and the kinetic study for pNA and MCA substrates showed that scallop aminopeptidase is specific for alanine in the amino-termini of substrates and resembles mammal cytosol alanyl aminopeptidases [4][5][6]8,9) with respect to substrate specificity. Our previous paper 15) demonstrated, however, that the enzyme differs from mammal cytosol alanyl aminopeptidase in molecular weight and sensitivity to puromycin, SH-blocking reagents, and divalent metal ions.…”