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Aminopeptidase M from human liver. I. Solubilization, purification, and some properties of the enzyme
M Nakanishi1, A Moriyama, Y Narita
1Department of Biochemistry, Nagoya City University Medical School, Aichi.
Abstract:
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.
Insights
Human liver aminopeptidase M was purified and found to be inhibited by bile acids. This enzyme, present on the cell membrane and in bile, plays a role in bile acid metabolism.
Area of Science:
- Biochemistry
- Enzymology
- Membrane Biology
Background:
- Aminopeptidase M (APM) is an enzyme found in various tissues, including the liver.
- Its role in liver function and bile acid metabolism is not fully understood.
- Microsomal fractions are a key source for purifying membrane-bound enzymes.
Purpose of the Study:
- To purify and characterize human liver aminopeptidase M.
- To investigate the effects of bile acids on purified aminopeptidase M.
- To determine the localization and potential function of liver APM in bile.
Main Methods:
- Enzyme purification using Triton X-100, urea, and multiple chromatography techniques (DEAE-cellulose, hydroxylapatite, Butyl-Toyopearl, Sephacryl S-300).
- Molecular weight determination via SDS-PAGE and gel filtration.
- Proteoliposome reconstitution to assess amphiphilic nature.
- Enzyme inhibition assays using various conjugated and unconjugated bile acids.
- Immunochemical analysis to detect enzyme presence in bile.
Main Results:
- Aminopeptidase M was purified 772-fold with 18.9% yield.
- Purified APM exhibited molecular weights of 140,000 (SDS-PAGE) and 280,000 (gel filtration), indicating an amphiphilic nature.
- Bile acids, particularly chenodeoxycholic acid and cholic acid, potently and reversibly inhibited APM activity.
- Inhibition potency followed the order: unconjugated > glycoconjugated > tauroconjugated bile acids.
- Immunochemistry confirmed significant release of liver APM into the bile duct.
Conclusions:
- Human liver aminopeptidase M is an amphiphilic enzyme that can be purified using standard biochemical methods.
- Bile acids are potent inhibitors of liver APM, suggesting a regulatory role in bile acid metabolism.
- The presence of APM in both the bile canalicular membrane and bile indicates its involvement in hepatic bile formation and transport.