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.