Heterogeneity of glycosylphosphatidylinositol‐anchored alkaline phosphatase of calf intestine

Abstract

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 acidmol 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 K,,, V,,,,,, 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 phosphatidylinositolspecific 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 11-V differ in their susccptibility to phospholipases. Fractions TI and IV are completcly split by PtdIns-PLC or PLD action, almost 50% of fraction I11 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 11, 111, 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 11-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 I1 and IV are tetramers and octamers with two molecules fatty acid/subunit. Fraction 111 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 glycosylPtdInsanchored alkaline phosphatase of the mucosa is released into the chyme without changing the anchor molecule constituents.A favoured line of investigation of proteins which are anchored to glycosylphosphatidylinositol (glycosylPtdIns) concerns research on the mechanism of their release by specific anchor-cleaving activities e.g. phospholipases C and D. This process is interesting regarding not only the effects evoked by the proteins released but also regarding the fact that the anchor molecules liberated can potentially act as sig- nals for a broad spectrum of metabolic pathways [l-61. Considerably different susceptibilities to phospholipases have been found for diffe...

Characterizing the interactions between GPI-anchored alkaline phosphatases and membrane domains by AFM

“…The minimal size of bright dots was compatible with structural data [56] and the time course of their visualization at the membrane surface agreed with previous work done on direct incorporation of AP-GPI into liposomes [15]. The presence of larger spots at the membrane surface also gave direct support for BIAP having a marked tendency to form clusters, a hypothesis formulated from indirect biochemical [13] and physicochemical [78] evidences. It is worth noting that pretreatment of BIAP with phosphatidylinositol-specific phospholipase C to remove the GPI anchor prevented the imaging of the protein at the SLBs surface.…”

“…BIAP contains only fatty acid esters, with a remarkably high content of octadecanoate and hexadecanoate saturated fatty acids. Moreover, a significant fraction of the GPI anchor contains an additional long-chain fatty acid, possibly inositol-linked [13]. On the other hand, the GPI membrane anchor of human PLAP is made of 1-O-alkyl-2-O-acylglycerol that contains a significant amount of unsaturated C 18:1 [77], i.e.…”

Microheterogeneity of the Hydrophobic and Hydrophilic Part of the Glycosylphosphatidylinositol Anchor of Alkaline Phosphatase from Calf Intestine

“…Two of the eight species observed contained a fatty acid residue presumably attached to the inositol moiety and, consistent with the previous findings (Bublitz et al, 1993), these species were restricted to the alkaline phosphatase fractions F2 and F4.…”

“…The purified enzyme from both sources, mucosa and intestine, proved to be a mixture of four species differing in aggregation behavior, hydrophobicity and susceptibility to PtdIns-specific phospholipase C (Bublitz et al, 1993). On the basis of these properties a model was proposed suggesting two tetrameric forms (Fl,F2) and two octameric forms (F3, F4), with the tetramer and octamer of lower hydrophobicity (F1 , F3) containing two fatty aciddsubunit, those of higher hydrophobicity (F2, F4) containing 2.5 and 3 fatty acids, respectively.…”

“…that large amounts of this enzyme are liberated into chyme as glycosyl-PtdIns species (Bublitz et al, 1993) contained in large particles called chymosomes (Halbhuber et al, 1994). The purified enzyme from both sources, mucosa and intestine, proved to be a mixture of four species differing in aggregation behavior, hydrophobicity and susceptibility to PtdIns-specific phospholipase C (Bublitz et al, 1993).…”