Alanyl aminopeptidase from human seminal plasma: purification, characterization, and immunohistochemical localization in the male genital tract.
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.
Huang
K
K
Department of Medical Biochemistry, Shiga University of Medical Science, Otsu.
Takahara
S
S
Kinouchi
T
T
Takeyama
M
M
Ishida
T
T
Ueyama
H
H
Nishi
K
K
Ohkubo
I
I
D000328:Adult; D000595:Amino Acid Sequence; D018826:CD13 Antigens / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005837:Genitalia, Male / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D007202:Indicators and Reagents; D007526:Isoelectric Point; D007700:Kinetics; D008297:Male; D008670:Metals; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012661:Semen / Q000201:enzymology*; D013379:Substrate Specificity
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D008670:Metals; D018826:CD13 Antigens