Identification of placental leucine aminopeptidase and triton-slowed aminopeptidase N in serum of pregnant women.
BACKGROUND
Previously, we found characteristic triton-slowed bands of aminopeptidase N (APN) in cholestatic serum by triton-polyacrylamide gel electrophoresis (triton-PAGE) [Makoto Kawai, Yukichi Hara, Triton-polyacrylamide gel electrophoresis and leucine aminopeptidase activity staining detect Triton-slowed bands including high-molecular-mass aminopeptidase N (CD13) isoform in cholestatic patient sera. Clin Chim Acta 2006; 364:188-195].


METHODS
Sera from 14 normal pregnant women were electrophoresed on polyacrylamide gel containing 0.02 l/l triton (triton-PAGE) or a 0-0.02 l/l horizontal gradient of triton (gradient-triton-PAGE), and stained with leucine-beta-naphthylamide. Some samples were pretreated with a monoclonal APN antibody or rabbit anti-placental leucine aminopeptidase (PLAP) serum. The stained bands were eluted from the gel, treated with N- and O-glycosidase, and analyzed by Western blotting with rabbit anti-APN or anti-PLAP serum.


RESULTS
Triton-PAGE clearly differentiated 5 LAP activity bands (1-5 from the front). Gradient-triton-PAGE revealed that bands 4-5 were slowed by triton (triton-slowed bands) much more than bands 1-3. Triton-PAGE of antibody-treated serum showed that bands 1, 2, 4, and 5 are mainly APN and that band 3 is PLAP. The molecular mass of PLAP was about 130-140 kDa before treatment with glycosidases but 100 kDa after. Triton-PAGE detected PLAP in 13 and triton-slowed APN in 4 of the 14 women.


CONCLUSIONS
Triton-PAGE differentiates PLAP from APN. Triton-slowed APN as well as PLAP is present in the serum of pregnant women.
Kawai
Makoto
M
Department of Gastroenterology, Tokyo Kyosai Hospital, Tokyo, Japan. mkawai@tkh.meguro.tokyo.jp
Araragi
Keiko
K
Shimizu
Yasufumi
Y
Hara
Yukichi
Y
D000818:Animals; D000906:Antibodies / Q000378:metabolism; D001774:Blood Chemical Analysis / Q000379:methods*; D015153:Blotting, Western; D018826:CD13 Antigens / Q000097:blood*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005260:Female; D006801:Humans; D007931:Leucyl Aminopeptidase / Q000097:blood* / Q000378:metabolism; D010920:Placenta / Q000201:enzymology*; D011092:Polyethylene Glycols; D011247:Pregnancy; D012680:Sensitivity and Specificity
D000906:Antibodies; D011092:Polyethylene Glycols; D007931:Leucyl Aminopeptidase; D018826:CD13 Antigens