Studies on the Protein Moiety of Endotoxin from Gram‐Negative Bacteria

Abstract

Conjugated protein isolated from the endotoxin complex of Xerratia marcescens 08 by lo/, acetic acid hydrolysis was characterized by analytical ultracentrifugation, electrophoretic and immunological properties and chemical analysis. The chemical composition of conjugated protein and other degraded fragments of endotoxin showed that all characteristic constituents of lipid A were present exclusively in conjugated protein. Successive treatment of conjugated protein with trypsin and pronase resulted in the isolation of corresponding tryptic and pronase cores which were characterized by an increased content of lipid A constituents. Lipid A was separated as an entity from the conjugated protein only after hydrolysis with 0.1 N hydrochloric acid. This result confirms our previous proposal that lipid A is covalently linked to the protein moiety in whole endotoxin of S. marcescens 08. "Conjugated" protein isolated by acetic acid hydrolysis and "simple" protein prepared by hot aqueous phenol treatment of whole endotoxin differ from one another not by the presence or absence of lipid A but rather by the presence of the entire lipid A moiety in the conjugated protein and only a fragment of lipid A in the simple protein.Conjugated protein and its tryptic and pronase cores were immunogenic and possessed a common antigenic determinant with simple protein and its corresponding cores. Toxicity studies revealed that both conjugated and simple proteins retained to different degrees the toxic properties of whole endotoxin. Sodium dodecyl sulfate had an enhancing rather than diminishing effect on the toxicity of endotoxin and its various fragments. The importance of lipid A as the toxic factor of endotoxins was established not only indirectly by determining the toxicity of each fragment containing lipid A, but also directly by demonstrating that lipid A dispersed in a Tris-dodecyl sulfate buffer displayed toxic properties similar to those of the lipopolysaccharide preparation.Morgan and Partridge [l --31 demonstrated that hydrolysis of whole endotoxin of Shigella dysenteriae by lo/, acetic acid resulted in the isolation of the so-called conjugated protein which may be converted into simple protein by treatment with either goo/,, phenol or 0.1 N sodium hydroxide. Because conjugated protein from Shigella pradysenteriae retained the typical toxic properties, Goebel and his coworkers [4,5] have referred to protein preparations isolated by lo/, acetic acid hydrolysis as toxic proteins. Since tryptic digestion of the intact endotoxin and of toxic protein did not destroy the toxic component, authors concluded that the toxin is either not a protein or that it represents a portion of the protein resistant to the action of proteolytic enzymes. The suggested existence of a toxic component other A preliminary report of this study was presented at the 158th National Meeting of the American Chemical Society, New York, September, 1969. than protein and polysaccharide marked the beginning not only of an extensive search for but also of a lastin...

Characteristics of lipid A-protein complex from endotoxin of Shigella dysenteriae type 1 (S and R strains)

“…Thus, taken together, the results obtained indicate that the protein present in LPSP and, in particular, associated with the lipid A-protein complex is a constituent part thereof. This is fully confirmed by some earlier studies (16,26,29,36) concerned with the significance of protein in preparations of endotoxin, LPSP, or lipid A of some other bacteria (34,35). This protein moiety, which seems to be covalently bound to lipid A (26), may be released from the complex in a number of ways.…”

“…The principal portion of lipid A, 70 to 80%, is fatty acids, and the remainder is the sugar moiety (amino sugar) and phosphorus. However, a different situation arises if, instead of LPS, the starting material has been LPSP complex (which is the case herein with S. dysenteriae type 1 since phenol-water extraction was not used in order to avoid possible degradation of some endotoxin component [29,35]). It is then not surprising that treatment with a weak acid yields a lipid A-protein complex with a relatively high proportion of protein (31).…”

Biological Activities of Lipid A Complexed with Bovine‐Serum Albumin

“…Moreover, physicochemical factors such as the solubility and state of aggregation of the molecules may play a role in endotoxicity [33]. I n free lipid A, for example, active structures may be masked by a high degree of aggregation [35]. The exposure of a "toxic conformation" may be induced by the polar polysaccharide moiety (as in lipopolysaccharides) [34] or by a protein carrier such as bovine serum albumin.…”

Isolation and Characterization of 2-Keto-3-Deoxyoctonate-Lipid A from a Heptose-Deficient Mutant of Escherichia coli

“…It is possible that part of the glycolipid is bound to some cell wall component other than polysaccharide. Wober and Alaupovit (42,43) presented data suggesting that lipid A in E. coli and Serratia marcescens is bound not only to polysaccharide but also to protein. However, their results do not indicate how much of the total lipid A is bound to protein.…”