Free lipid acyl residues

Takayama and coworkers (60) introduced the h.p.l.c. separation technique for such amphiphilic molecules as lipid A, and in earlier experiments they applied paired-ion reverse-phase h.p.l.c. for the preparation of homogeneous fractions deriving from 4,-monophosphated lipid A of S. typhimur-ium. The purified preparations obtained were suitable for f.a.b. - m.s. analysis. However, monophosphated lipid A isolated in this way expressed a considerable heterogeneity with respect to the number and location of 0-acyl residues (60). In order to further improve the purification procedure, as well as to obtain lipid A derivatives suitable for n.m.r. spectroscopy, Qureshi et al. (174) prepared the dimethyl phosphate derivative of S. minnesota (R595) lipid A, which, after purification by reverse-phase h.p.l.c. (C18), could be analyzed by1 H-n.m.r. The n.m.r. spectrum of, for example, the heptaacyl lipid A dimethyl monophosphate fraction, unequivocally revealed 0-acyl substitution [14 0(3-OH)J at position 3 and a free hydroxyl group at position 4 of GlcN(I). 

In order to conduct research studies on this novel lipid mediator, it is necessary to have available a sufficient quantity of purified, well-defined material. This has not been an easy task, but an approach to preparation of sphingosine-l-P in milligram amounts was outlined by Van Veldhoven et al. (1989). In this procedure, a commercial preparation of sphingosylphospho-choline (which contains a free amine group due to removal of a fatty acyl residue from initial substrate, sphingomyelin) is incubated with phospholipase D in an ammonium acetate buffer at pH 8.0 for 1 hr. An insoluble reaction product is collected and subjected to purification by dissolution in water at room temperature followed by cooling to 4°C. A precipitate forms again and is collected and treated in a similar manner as above except that acetone is used as the solvent. 

Although fatty acyl residues are present in all lipids, the analysis of free fatty acids by MALDI-TOF MS is rather difficult as they show-due to their relatively low molecular weights-a significant overlap with the matrix. This particularly holds if fatty acids at low concentrations have to be analyzed. One possibility to overcome this problem is the use of mesotetrakis(pentafiuorophe-nyl)porphyrin (Ayorinde et al. 1999) as matrix It could be shown that the analysis of free fatty acids obtained by alkaline saponification of different plant oils is possible by this matrix (Ayorinde et al. 2000). As an excess of sodium acetate was added, exclusively the Na adducts of the sodium salts of the fatty acids were detected by positive ion MALDI-TOF MS. Therefore, problems with peak assignments did not occur. Unfortunately, this approach... 

Hydroxylaminolysis, treatment with stronger alkali (0.5 m NaOH, 2 h, 100°C) and alkaline methanolysis (0.25 m NaOMe, 1 h, 50°C) lead to complete O-deacylation of LPS and lipid A (176). Particularly in the case of alkaline methanolysis, ester-linked 3-acyloxyacyl residues undergo, in addition to transmethylation, a -elimination reaction, whereby the (R)-3-hy-droxy fatty acid ester is first transformed into the a,/ -unsaturated and then into the (S.-R -methoxy fatty acid methyl ester. The acyl substituent, on the other hand, is eliminated in the form of the free fatty acid (176). In fact, the presence of a 3-methoxyacyl derivative in the fatty acid spectrum of a given LPS is a strong indication for the presence of an ester-bound 3-acyloxyacyl... 

Inhibitor studies indicate that serine, histidine and, perhaps tyrosine, residues are involved in the active site of the enzyme (Hirayama et al., 1975). Activity toward membrane-bound phospholipids (or phospholipid liposomes) is considerably enhanced by detergents. Of a range of surface-active agents studied, unsaturated free fatty acids were the most effective (Gal-liard, 1971b). For example, although a crude extract of potato hydrolyzes mitochondrial or liposome-bound lipids, the purified enzyme is inactive unless a detergent is added the factor responsible for enhancement in crude extracts was identified as a mixture of free fatty acids. Thus, the reaction on membrane lipids is autocatalytic free fatty acids released by the enzyme disrupt membrane structures to allow access of the enzyme to the substrate. The activity of acyl hydrolase from potato on mitochondrial lipids has been confirmed by Hasson and Laties (1976b). 

Of course, many characteristic dyes available allow the specific staining of selected GPL classes [11]. However, the majority of these dyes are specific for the headgroup (for instance, free amino residues can be stained by ninhydrin) and, thus, information about the lipid class but neither about the fatty acyl composition nor the linkage type is available. 

The RBP molecule is a single polypeptide chain of about 180-185 amino acid residues containing three intramolecular disulfide bonds (as cystine residues) (Rask et al., 1981). No fatty acid or fatty acyl chains were detected in purified RBP (Kanai et al., 1968). Analyses for carbohydrate showed that RBP was free of neutral sugar and hexosamine (Peterson, 1971) and had a maximum of 0.9 mol of hexose per mol of protein (Raz et al., 1970). It is likely that RBP contains no bound lipid (other than retinol) and no carbohydrate. 

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