Aminopeptidase M from human liver. I. Solubilization, purification, and some properties of the enzyme.
Aminopeptidase M [EC 3.4.11.2] was purified 772-fold to homogeneity from the microsomal fraction of human liver, with a yield of 18.9%, by a combination of solubilization with 0.5% Triton X-100 and then 1 M urea and chromatography on columns of DEAE-cellulose, hydroxylapatite, Butyl-Toyopearl, and Sephacryl S-300. The purified enzyme had a molecular weight of 140,000 by SDS-polyacrylamide gel electrophoresis and of 280,000 by gel filtration on a column of TSK gel 2000 SW. It was reconstituted into proteoliposomes with asolectin, showing its amphiphilic nature. The aminopeptidase M from liver was found to be efficiently inhibited by bile acids. The enzyme was almost completely inhibited by chenodeoxycholic acid and 70-90% inhibited by cholic acid at a concentration of 6 mM. The extent of inhibition by conjugated and unconjugated bile acids was in the order: unconjugated greater than glycoconjugated greater than tauroconjugated bile acid, independent of the nature of the substrates used. The inhibition by the various bile acids was totally reversible. Further, it was immunochemically revealed that a considerable amount of liver aminopeptidase M was released into the bile duct. The role of the aminopeptidase M on the bile canalicular membrane and of the enzyme released in the bile duct is discussed in relation to the effects of bile acids.
Nakanishi
M
M
Department of Biochemistry, Nagoya City University Medical School, Aichi.
Moriyama
A
A
Narita
Y
Y
Sasaki
M
M
D000596:Amino Acids / Q000032:analysis; D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D000813:Anilides / Q000378:metabolism; D000917:Antibody Formation; D001647:Bile Acids and Salts / Q000502:physiology*; D015153:Blotting, Western; D018826:CD13 Antigens; D002241:Carbohydrates / Q000032:analysis; D002614:Chelating Agents; D002853:Chromatography, Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008862:Microsomes, Liver / Q000201:enzymology*; D008970:Molecular Weight; D011506:Proteins / Q000032:analysis; D012995:Solubility; D013379:Substrate Specificity; D014357:Trypsin
D000596:Amino Acids; D000813:Anilides; D001647:Bile Acids and Salts; D002241:Carbohydrates; D002614:Chelating Agents; D011506:Proteins; C001536:1-leucine-4-nitroanilide; D000626:Aminopeptidases; D018826:CD13 Antigens; D014357:Trypsin