Purification and characterization of human placental aminopeptidase A.
Human placental aminopeptidase A (AAP) was purified 3,900-fold from human placenta and characterized. The enzyme was solubilized from membrane fractions with Triton X-100, then subjected to trypsin digestion, zinc sulfate fractionation, chromatographies with DE-52, Sephacryl S-300, and hydroxylapatite, affinity chromatography with Bestatin-Sepharose 4B, and finally immunoaffinity chromatography with the antibody against microsomal leucine aminopeptidase (LAP). Aminopeptidase A was completely separated from leucine aminopeptidase by the immunoaffinity chromatography. The apparent relative molecular mass (Mr) of the enzyme was estimated to be 280,000 by gel filtration. The purified enzyme was most active at pH 7.1 with L-aspartyl-beta-naphthylamide (L-Asp-NA) as substrate; the Km value for this substrate was 4.0 mmol/l in the presence of Ca2+. Human placental aminopeptidase A was markedly activated by alkaline earth metals (Ca2+, Sr2+, Ba2+), but strongly inhibited by metal chelating agents such as EDTA and o-phenanthroline. The highest activity was observed with L-glutamyl-beta-naphthylamide, while only minimal hydrolysis was found with some neutral and basic amino acid beta-naphthylamides.
Yamada
R
R
Department of Obstetrics and Gynaecology, Nagoya University School of Medicine, Japan.
Mizutani
S
S
Kurauchi
O
O
Okano
K
K
Imaizumi
H
H
Narita
O
O
Tomoda
Y
Y
D000626:Aminopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D002845:Chromatography / Q000379:methods; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005260:Female; D043384:Glutamyl Aminopeptidase; D006801:Humans; D006882:Hydroxyapatites; D007202:Indicators and Reagents; D007525:Isoelectric Focusing / Q000379:methods; D007700:Kinetics; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D013379:Substrate Specificity
D006882:Hydroxyapatites; D007202:Indicators and Reagents; D017886:Durapatite; D000626:Aminopeptidases; D043384:Glutamyl Aminopeptidase