HETEROGENEITY OF GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED ALKALINE-PHOSPHATASE OF CALF INTESTINE

A method is described for large-scale purification of glycosylphosphat idylinositol-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 comp osition with octadecanoate and hexadecanoate as prevalent components. No significant differences between native glycosylPtdIns-anchored and hydrophilic alkaline phosphatases from both sources were found regardi ng K(m), V(max), the type of inhibition and inhibition constants of th e amino acids L-leucine, L-phenylalanine, and L-tryptophan. The purifi ed enzymes of both sources yield diacylglycerol and phosphatidic acid, after treatment with phosphatidylinositol-specific phospholipase C (P tdIns-PLC) and glycosylphosphatidylinositol phospholipase D (PLD), res pectively. Enzyme preparations of both sources appear as heterogeneous mixtures of five fractions separable by octyl-Sepharose chromatograph y. Fraction I corresponds to the anchorless enzyme, fractions II-V dif fer 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 suscept ibility of these two fractions toward the action of PLD is considerabl y higher. Fatty acid analysis yields molar ratios of fatty acids/alkal ine 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 p resence 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 fat ty acid analysis, can be interpreted by the following model. Fractions II and IV are tetramers and octamers with two molecules fatty acid/su bunit. Fraction III is a tetramer, bearing one additional fatty acid m olecule, localized on the dimer. Fraction V is an octamer, containing glycosylPtdIns-anchor molecules with three molecules fatty acids/ancho r molecule. The additional fatty acid residue is possibly located on i nositol and responsible for the reduced susceptibility to PtdIns-PLC. The similarity of all measured parameters of both enzymes suggests tha t the glycosylPtdIns-anchored alkaline phosphatase of the mucosa is re leased into the chyme without changing the anchor molecule constituent s.