Chemical identification of lipid components in the membranous form of rat liver alkaline phosphatase.
Membranous and soluble forms of rat liver alkaline phosphatase were selectively prepared by extracting microsomes with n-butanol at pH 8.5 and 5.5, respectively, and purified in homogeneous forms by the method previously established (Miki et al. (1986) Eur. J. Biochem. 160, 41-48). When subjected to polyacrylamide gel electrophoresis, the two forms migrated to the same position in the presence of sodium dodecyl sulfate, while the membranous form remained at the top of gels in the absence of the detergent. Treatment of the membranous form with phosphatidylinositol-specific phospholipase C resulted in its conversion to a soluble form with the same electrophoretic mobility even in the absence of the detergent as that of the soluble form extracted at pH 5.5. Automated Edman degradation analysis showed that the two forms have the same N-terminal amino acid sequence up to the 30th residue determined. Chemical analyses of hydrolysates of the two forms by gas-liquid chromatography demonstrated that the membranous form contains palmitic acid, stearic acid, and inositol, while the soluble form contains inositol but is devoid of the fatty acids. Taken together, these results suggest that rat liver alkaline phosphatase is covalently attached to phosphatidylinositol acylated with palmitic acid and stearic acid, which functions as the membrane-anchoring domain of the enzyme molecule.
Ogata
S
S
Department of Biochemistry, Fukuoka University School of Medicine.
Hayashi
Y
Y
Yasutake
K
K
Ikehara
Y
Y
D000469:Alkaline Phosphatase / Q000032:analysis*; D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology*; D002849:Chromatography, Gas; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008055:Lipids / Q000032:analysis*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000032:analysis; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010716:Phosphatidylinositols / Q000032:analysis; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013229:Stearic Acids / Q000032:analysis
D008055:Lipids; D010169:Palmitic Acids; D010716:Phosphatidylinositols; D013229:Stearic Acids; D019308:Palmitic Acid; C031183:stearic acid; D000469:Alkaline Phosphatase; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase