Selective preparation and characterization of membranous and soluble forms of alkaline phosphatase from rat tissues. A comparison with the serum enzyme.
We developed a method for selective preparation of two forms of alkaline phosphatase from rat tissues. The enzyme was extracted by n-butanol treatment at pH 5.5 and pH 8.5 as soluble and aggregated (membranous) forms, respectively. The soluble form prepared from liver was found to be identical with the serum enzyme. Complete solubilization of the membrane-bound enzyme without detergents had a great advantage in its purification. Rat hepatoma AH-130 cells enriched in alkaline phosphatase were first used for purification of the liver-type enzyme. The hepatoma enzyme, purified by chromatographies on concanavalin-A-Sepharose, Sephacryl S-300 and hydroxyapatite was used for production of antibodies specific for the liver-type isozyme. An immunoaffinity column, prepared with anti-(hepatoma-enzyme) IgG was utilized for the enzyme purification from other tissues including the membranous form. Analyses of amino acid composition of the purified enzymes revealed that all the liver-type enzymes from hepatoma, liver, kidney and serum had the same composition, whereas the intestinal type consisted of the composition distinctly different from that in the liver type. In addition, there was no significant difference in amino acid composition between the soluble and membranous forms, suggesting a possible involvement in the membranous form of a hydrophobic component other than its polypeptide domain. The present method for selective preparation of the soluble and membranous forms of alkaline phosphatase will be useful for a further investigation on the interaction of the enzyme with membranes.
D000469:Alkaline Phosphatase / Q000097:blood / Q000302:isolation & purification*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D007120:Immunochemistry; D007422:Intestines / Q000201:enzymology; D007668:Kidney / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D008114:Liver Neoplasms, Experimental / Q000201:enzymology; D008566:Membranes / Q000201:enzymology; D017830:Octoxynol; D011092:Polyethylene Glycols; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility
D000596:Amino Acids; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D000469:Alkaline Phosphatase