Heterogeneity of glycosylphosphatidylinositol-anchored alkaline phosphatase of calf intestine.

A method is described for large-scale purification of glycosylphosphatidylinositol-anchored alkaline phosphatase from intestinal mucosa and chyme to homogeneity. Both enzyme preparations contain approximately 2 mol fatty acid/mol subunit and exhibit a very similar fatty acid composition with octadecanoate and hexadecanoate as prevalent components. No significant differences between native glycosylPtdIns-anchored and hydrophilic alkaline phosphatases from both sources were found regarding Km, Vmax, the type of inhibition and inhibition constants of the amino acids L-leucine, L-phenylalanine, and L-tryptophan. The purified enzymes of both sources yield diacylglycerol and phosphatidic acid, after treatment with phosphatidylinositol-specific phospholipase C (PtdIns-PLC) and glycosylphosphatidylinositol phospholipase D (PLD), respectively. Enzyme preparations of both sources appear as heterogeneous mixtures of five fractions separable by octyl-Sepharose chromatography. Fraction I corresponds to the anchorless enzyme, fractions II-V differ in their susceptibility to phospholipases. Fractions II and IV are completely split by PtdIns-PLC or PLD action, almost 50% of fraction III is split by PtdIns-PLC, while fraction V is resistant. The susceptibility of these two fractions toward the action of PLD is considerably higher. Fatty acid analysis yields molar ratios of fatty acids/alkaline phosphatase subunit of 1.78, 2.58, 2.24, and 3.37 for fractions II, III, IV, and V, respectively. Aggregates of glycosylPtdIns-anchored alkaline phosphatase of all fractions are seen in native PAGE in the presence of Triton X-100. By gel chromatography in the presence of Brij 35, fractions II-V form stable multiple aggregates of dimers and may bind different amounts of the detergent. These data, together with fatty acid analysis, can be interpreted by the following model. Fractions II and IV are tetramers and octamers with two molecules fatty acid/subunit. Fraction III is a tetramer, bearing one additional fatty acid molecule, localized on the dimer. Fraction V is an octamer, containing glycosylPtdIns-anchor molecules with three molecules fatty acids/anchor molecule. The additional fatty acid residue is possibly located on inositol and responsible for the reduced susceptibility to PtdIns-PLC. The similarity of all measured parameters of both enzymes suggests that the glycosylPtdIns-anchored alkaline phosphatase of the mucosa is released into the chyme without changing the anchor molecule constituents.