Synthetic lipid A part structures corresponding structurally to a biosynthetic lipid A disaccharide precursor have been analyzed for endotoxic activity in several systems in vivo and in vitro.It was found that a synthetic P-1,6-linked D-glucosamine disaccharide, which carries four molar equivalents of (R)-3-hydroxytetradecanoyl residues in positions 2, 3, 2' and 3' and phosphoryl groups in positions 1 and 4' (preparation 406), exhibited lethal toxicity, B lymphocyte mitogenicity, the capacity to engender prostaglandin formation in macrophages and to induce endotoxic tolerance, as well as serological lipid A antigenicity. On a weight basis, preparation 406 was of comparable activity to lipid A precursor and bacterial free lipid A. Preparation 406, like lipid A precursor, lacked, however, the ability to induce the local Shwartzman phenomenon and both preparations were of moderate pyrogenicity. Two further synthetic analogues which contained only one phosphoryl group (preparation 404 at C-4', preparation 405 at C-1) showed comparable or diminished activity depending on the test system employed, except in the capacity to inactivate complement where they exhibited, in contrast to preparation 406, significant activity.The results show that the endotoxic principle of lipopolysaccharides, as postulated previously is embedded in the lipid A component. Our results also suggest initial conclusions on the structural requirements for the expression of endotoxin activities.
Cambial sap of spruce (Picea abies) proved to be a good source for isolation of cinnamoyl-CoA reductasc and cinnamyl alcohol :NADP+ dehydrogenase. Apparently homogeneous enzymes were obtained by a multistep procedure including dye-ligand chromatography and for the reductase also affinity chromatography on (coenzymc A)-agarose. An improved purification procedure for the reductase from soybean cell cultures is also reported.Molecular weights and subunit composition of reductase and dehydrogenase from spruce are very similar to those of the corresponding enzymes from soybean.Reduction of feruloyl-CoA to coniferaldehyde catalysed by the reductase is a freely reversible reaction with an equilibrium constant of 5.6 x M at pH 6.25. A strong dependence of the Michaelis constants on the type of buffer was found. For reductase the K,-value of feruloyl-CoA in phosphate buffer (5.2 pM) is about 14-times smaller than in citrate buffer (73 pM).Pronounced differences in substrate specificities between the enzymes from spruce and soybean were found, which reflect the different lignin composition of gymnosperms and dicotyledenous angiosperms. From the kinetic constants of the enzymes it can be concluded that under physiological conditions feruloyl-CoA is the preferred substrate for the reductase from both sources whereas sinapoyl-CoA is a substrate only for the soybean reductase and sinapyldehyde a substrate only for the soybean dehydrogenase.4-Coumaroyl-CoA is a poor substrate for the reductase from both spruce and soybean.This result is consistent with the low content of 4-coumaryl alcohol units in gymnosperm and angiosperm lignin.
Lipopolysaccharides of different wild-type and mutant gram-negative bacteria, as well as synthetic and bacterial free lipid A, were studied for their ability to activate arachidonic acid metabolism in mouse peritoneal macrophages in vitro. It was found that lipopolysaccharides of deep-rough mutants of Saln~onellu minnesotu and Escherirhia coli (Re to Rc chemotypes) stimulated macrophages to release significant amounts of leukotriene C4 (LTC,) and prostaglandin E2 (PGE,). Lipopolysaccharides of wild-type strains (S. abortus equi, S.friedenau) only induced PGE2 and not LTC, formation. Unexpectedly, free bacterial and synthetic E. coli lipid A were only weak inducers of LTC, and PGE2 production. Deacylated Re-mutant lipopolysaccharide preparations were inactive. However, co-incubation of macrophages with both deacylated lipopolysaccharide and lipid A lead to the release of significant amounts of LTC, and PGE2, similar to those obtained with Re-mutant lipopolysaccharide. The significance of the lipid A portion of lipopolysaccharide for the induction of LTC4 was indicated by demonstrating that peritoneal macrophages of endotoxin-low-responder mice or of mice rendered tolerant to endotoxin did not respond with the release of arachidonic acid metabolites on stimulation with Re-mutant lipopolysaccharide and that polymyxin B prevented the Re-lipopolysaccharide-induced LTC, and PGE2 release. Physical measurements showed that the phase-transition temperatures of both free lipid A and S-form lipopolysaccharide were above 37 "C while those of R-mutant lipopolysaccharides were significantly lower (30 -35 "C). Thus, with the materials investigated, an inverse relationship between the phase-transition temperature and the capacity to elicit LTC4 production was revealed.In a previous paper we demonstrated that, under specified conditions, lipopolysaccharides of a Salmonella minnesota Re mutant (strain R595) are capable of inducing, in cultured mouse peritoneal macrophages, leukotriene C4 (LTC,) formation and release in a dose-and time-dependent manner [l]. Chemically, this lipopolysaccharide consists of lipid A carrying an a(2,4)-linked 3-deoxy-~-manno-octu~osonic acid (dOclA) disaccharide [2-41. It has recently been proven that the lipid A component represents the endotoxic principle of lipopolysaccharides and that it elicits many of the biological effects seen with endotoxins [5]. On the other hand, a number of studies indicate that also the polysaccharide portion possesses intrinsic biological activity or that it may modulate lipid A activity [6, 71. I t was, therefore, of interest to investigate the significance of the lipid A and the polysaccharide region in the lipopolysaccharide-induced formation of LTC4 in phagocytes.The present paper describes experiments involving lipopolysaccharides from wild-type bacteria (S-form lipopolysaccharides), rough mutants (R-form lipopolysaccharides) and free bacterial, as well as synthetic lipid A (preparation 506). Our results suggest that the physicochemical state of Abbreviations. P,/NaC...
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