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...

A versatile, multidimensional, and non-denaturing proteome separation procedure using microplate technology is presented, yielding a digitized image of proteome composition. In the first dimension, the sample under study is separated into 96 fractions by size exclusion chromatography (SEC). In the second dimension, the fractions of the first dimension are transferred by the liquid-handling device CyBi-Well (CyBio AG, Jena, Germany) to 96 parallel anion exchange chromatography columns. In this way the proteins are conserved in their native states and are distributed in 2400 liquid fractions with high recovery rates and sufficient reproducibility. The resulting fractions are subjected to protein quantitation and identification. Spectrophotometrical and immunological methods and enzyme activity measurements are used for quantitation. To identify proteins, the fractions are subjected to MALDI-MS, and their tryptic digests to both MALDI- and LC-ESI-MS/MS. All preparation steps except the first are applied in parallel to sets of multiples of 96 samples. The procedure may be refined by adding more separation steps and may be adapted to various protein amounts and to various proteomes. Moreover, the method offers the opportunity to investigate functional protein complexes. The method was applied to separate the normal human serum proteome. Within 255 fractions exhibiting the highest protein concentrations, 742 proteins were identified by LC-ESI-MS/MS peptide sequence tags.

A method is introduced to evaluate protein concentrations using the height sum of all MALDI-MS peaks that unambiguously match theoretic tryptic peptide masses of the protein sought after. The method uses native chromatographic protein fractionation prior to digestion but does not require any depletion, labeling, derivatization, or preparation of a compound similar to the analyte. All peak heights of tryptic peptides are normalized with the peak height of a unique standard peptide added to the MALDI-MS samples. The sum of normalized peak heights, S(n), or the normalized mean peak height, M(n), reflects the concentration of the respective protein. For fractions containing various proteins, S(n) and M(n) can be used to compare concentrations of a protein between different fractions. For fractions with one predominating protein, they can be used to estimate concentration ratios between fractions, or to quantify the fractional protein concentration after calibration with pure protein solutions. Initial native fractionation retains the possibility to apply all conventional analytic procedures. Moreover, it renders the method relatively robust to MS mass accuracy. The method was validated with albumin, transferrin, alpha1-antitrypsin, and immunoglobulin G within highly complex chromatographic fractions of pathological and normal sera, which contained the respective intact native protein in dominating as well as minor concentrations. The correlation found between S(n) and the protein concentration as determined with ELISA showed that the method can be applied to select markers for distinguishing between normal and pathological serum samples.

Surprisingly alkaline phosphatase (AP) (EC 3.1.3.1) of calf intestine is found in large amounts, e.g. 80%, within chyme. Most of the enzyme is present as a mixture of four differently hydrophobic anchor-bearing forms and only the minor part is present as an anchorless enzyme. To investigate whether changes in the N-glycosylation pattern are signals responsible for large-scale liberation from mucosa into chyme, the glycans of the two potential glycosylation sites predicted from cDNA were investigated by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry in combination with exoglycosidase treatment after tryptic digestion and reversed-phase chromatography. The glycans linked to Asn249 are at least eight different, mainly non-fucosylated, biantennary or triantennary structures with a bisecting N-acetylglucosamine. For the most abundant glycopeptide (40%) the following glycan structure is proposed: [carbostructure: see text]. The glycans linked to Asn410 are a mixture of at least nine, mainly tetraantennary, fucosylated structures with a bisecting N-acetylglucosamine. For the most abundant glycopeptide (35%) the following glycan structure is proposed: [carbostructure: see text]. For the structures the linkage data were deduced from the reported specificities of the exoglycosidases used and the specificities of the transglycosidases active in biosynthesis. The majority of glycans are capped by alpha-galactose residues at their non-reducing termini. In contrast to the glycans linked to other AP isoenzymes, no sialylation was observed. Glycopeptide 'mass fingerprints' of both glycosylation sites and glycan contents do not differ between AP from mucosa and chyme. These results suggest that the observed large-scale liberation of vesicle-bound glycosylphosphatidylinositol (GPI)-anchored AP from mucosa into chyme is unlikely to be mediated by alteration of glycan structures of the AP investigated. Rather, the exocytotic vesicle formation seems to be mediated by the controlled organization of the raft structures embedding GPI-AP. (c) 2001 John Wiley & Sons, Ltd.

Digestion of calf intestine alkaline phosphatase with pronase and subsequent dephosphorylation of the released peptidyl-(Etn-P),-glycosyl-PtdIns with HF generated 8 glycosyl-Ins species the largest of which (G1 and G2) have the following proposed structures:G1 aManand G5 are lower homologues of G1 and G2, respectively, being one a l -2 linked mannopyranosyl residue shorter. G4 is analogous to G2 lacking the N-acetylgalactosaminyl residue and G6 is the next lower homologue of G4. Most of G4 and G6 occur substituted with a palmitoyl (G4, G6) or a myristoyl residue (G6) probably attached to the inositol moiety. Thus, the basic Man,Glc-Ins species are either substituted with an N-acetylgalactosaminyl residue or a fatty acid ester. The structures were deduced from compositional analysis, molecular-mass determination by matrix-assisted laser desorption MS, sequential hydrolysis with appropriate exoglycosidases and treatment with CrO,. Purification of the glycosylinositol species was achieved by a novel reverse-phase HPLC technique using fluorescent fluoren-9-yl-methoxycarbonyl (Fmoc) derivatives. These stable derivatives were susceptible to hydrolysis with exoglycosidases which allowed sequential cleavages to be carried out and kinetics to be followed at the picomole level.We observed recently that native alkaline phosphatase separates on octyl-Sepharose into four distinct fractions of increasing hydrophobicity (F1 -F4). Here we show that all four fractions contain G1 -G6. The acylated species G4 and G6 were restricted to F2 and F4 which had been shown earlier to contain, on average, 2.5 and 3 fatty acid residueshbunit, respectively. In all four fractions the diradylglycerol moiety was predominantly diacylglycerol, alkylacylglycerol being less than 10 % which is in contrast to most glycosyl-PtdIns-anchored proteins of mammalian origin.