Immunoaffinity purification and characterization of native placental leucine aminopeptidase/oxytocinase from human placenta.
cDNA cloning of placental leucine aminopeptidase (P-LAP)/cystinyl aminopeptidase (CAP)/oxytocinase demonstrated that this enzyme is a type II integral membrane protein, which means that native P-LAP, found in placenta, is membrane-bound and that the soluble form of this enzyme, found in maternal sera, is most likely derived from the native form. The presence of the different forms of the protein makes it difficult to purify homogeneously. In the current study we prepared antibody specific to native P-LAP and used it to purify native P-LAP from microsomal fractions of human placenta to homogeneity, 5039-fold within 4 h, by immunoaffinity chromatography. Zn(2+)and Cu(2+)strongly inhibited the enzyme but Ca(2+)did not. Amastatin was a more potent inhibitor than bestatin and leupeptin. Using antibodies against native P-LAP, protein having 83 per cent of l -methionine insensitive Leu-p-nitroanilide cleaving activity, was immunoprecipitated from the microsomal fraction of human placenta. The availability of a specific antibody against native P-LAP permits the rapid purification and the preliminary immunoassay of the enzyme. Establishment of simple purification and assay methods for the native, membrane bound form of P-LAP pave the way to elucidating the roles and processing systems of this enzyme.
Nakanishi
Y
Y
Department of Obstetrics and Gynecology, Nagoya University School of Medicine, Nagoya, 466-8550, Japan.  
Nomura
S
S
Okada
M
M
Ito
T
T
Katsumata
Y
Y
Kikkawa
F
F
Hattori
A
A
Tsujimoto
M
M
Mizutani
S
S
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002846:Chromatography, Affinity; D003300:Copper / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006801:Humans; D007158:Immunologic Techniques; D007931:Leucyl Aminopeptidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D010455:Peptides; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D015032:Zinc / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D010455:Peptides; C016977:amastatin; D003300:Copper; D007931:Leucyl Aminopeptidase; D015032:Zinc; D002118:Calcium