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.
BACKGROUND
Western blotting of aminopeptidase N (APN) detects a high-molecular-mass isoform (260 kDa) [M. Kawai, Y. Otake, Y. Hara High-molecular-mass isoform of aminopeptidase N/CD13 in serum from cholestatic patients. Clin Chim Acta 330 (2003) 141-149] in cholestatic patient serum but is time-consuming.


METHODS
Human sera were electrophoresed on polyacrylamide gel containing Triton-X100 (Triton-PAGE) and stained with leucine-B-naphthylamide (LAP-staining). The stained bands were eluted from the gel, treated with N- and O-glycosidase if necessary, and analyzed by Western blotting [M. Kawai, Y. Otake, Y. Hara High-molecular-mass isoform of aminopeptidase N/CD13 in serum from cholestatic patients. Clin Chim Acta 330 (2003) 141-149].


RESULTS
Triton-PAGE and LAP-staining clearly detected fast bands in all the sera examined. Almost parallel with leucine aminopeptidase activity, slow bands were strongly stained in all 11 cholestatic patients but clearly stained in 3 out of 14 patients with hepatobiliary diseases other than cholestasis. PAGE with various concentrations of Triton showed that Triton slows down slow bands but not fast bands. Western blotting showed that Triton-PAGE-slow bands of cholestasis contained 140 and 260-kDa APN and that fast bands were slightly smaller than monomer-size slow bands after glycosidase treatment.


CONCLUSIONS
Less time-consuming than Western blotting, Triton-PAGE and LAP-staining detect novel APN bands slowed by Triton and partly composed of the high-molecular-mass isoform in cholestasis. The slow bands seem to be homodimers of APN with transmembrane anchors. The polypeptide of the fast band seems to be processed differently from that of the slow band.
Kawai
Makoto
M
Department of Biochemistry and Biophysics, Graduate School of Health Sciences, Tokyo Medical and Dental University, 5-45, Yushima 1-chome, Bunkyo-ku, Tokyo 113-8519, Japan. m.kawai.bb@tmd.ac.jp
Hara
Yukichi
Y
D015153:Blotting, Western; D018826:CD13 Antigens / Q000097:blood* / Q000737:chemistry; D002779:Cholestasis / Q000097:blood* / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000097:blood / Q000737:chemistry; D007931:Leucyl Aminopeptidase / Q000378:metabolism*; D008099:Liver / Q000201:enzymology / Q000473:pathology; D008970:Molecular Weight; D017830:Octoxynol; D015203:Reproducibility of Results
D007527:Isoenzymes; D017830:Octoxynol; D006026:Glycoside Hydrolases; D007931:Leucyl Aminopeptidase; D018826:CD13 Antigens