MICROHETEROGENEITY OF THE HYDROPHOBIC AND HYDROPHILIC PART OF THE GLYCOSYLPHOSPHATIDYLINOSITOL ANCHOR OF ALKALINE-PHOSPHATASE FROM CALF INTESTINE

Digestion of calf intestine alkaline phosphatase with pronase and subs equent dephosphorylation of the released peptidyl-(Etn-P)(2)-glycosyl- PtdIns with HF generated 8 glycosyl-Ins species the largest of which ( G1 and G2) have the following proposed structures: [GRAPHICS] G3 and G 5 are lower homologues of G1 and G2, respectively, being one alpha 1-2 linked mannopyranosyl residue shorter. G4 is analogous to G2 lacking the N-acetylgalaclosaminyl residue and G6 is the next lower homologue of G4. Most of G4 and G6 occur substituted with a palmitoyl (G4, G6) o r a myristoyl residue (G6) probably attached to the inositol moiety. T hus, the basic Man,Glc-Ins species are either subsituted with an N-ace tylgalactosaminyl residue or a fatty acid ester. The structures were d educed from compositional analysis, molecular-mass determination by ma trix-assisted laser desorption MS? sequential hydrolysis with appropri ate exoglycosidases and treatment with CrO3. Purification of the glyco sylinositol species was achieved by a novel reverse-phase HPLC techniq ue using fluorescent fluoren-9-yl-methoxycarbonyl (Fmoc) derivatives. These stable derivatives were susceptible to hydrolysis with exoglycos idases which allowed sequential cleavages to be carried out and kineti cs to be followed at the picomole level. We observed recently that nat ive alkaline phosphatase separates on octyl-Sepharose into four distin ct fractions of increasing hydrophobicity (F1-F4). Here we show that a ll four fractions contain G1-G6. The acylated species G4 and G6 were r estricted to F2 and F4 which had been shown earlier to contain, on ave rage, 2.5 and 3 fatty acid residues/subunit, respectively. In all four fractions the diradylglycerol moiety was predominantly diacylglycerol , alkylacylglycerol being less than 10% which is in contrast to most g lycosyl-PtdIns - anchored proteins of mammalian origin.