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

Abstract

The so-called simple proteins isolated from the endotoxin of Serratia marcescens 08 and whole cells Escherichia coli 0 141 :K85(B) by aqueous phenol treatment were characterized by the determination of hydrodynamic properties, electrophoretic behavior, immunochemical specificity and chemical analysis. The chemical composition of both proteins revealed the presence of small amount of lipid A constituents such as glucosamine, phosphorus and fatty acids ; the p-hydroxymyristic acid regarded as the characteristic marker for lipid A was present in all protein preparations. Since the treatment of intact endotoxin or bacterial cells with aqueous phenol results in the formation of two fragments (a lipopolysaccharide and a lipid A-protein), both of which are characterized by the presence of lipid A constituents, this dissociation seems to be caused by cleavage of a phenolsensitive linkage within the molecule of lipid A rather than at its point of attachment to the protein moiety. Thus, the %imple'' protein preparations consist of the protein moiety and a small segment of lipid A. Contrary to previous views, such protein preparations are not simple proteins if this designation implies absence of constituents other than amino acids. However, for a historical reason we suggest that the term simple protein be retained for protein fragments isolated by aqueous phenol treatment of intact endotoxins.The simple protein from Serratia marcescens 0s was treated successively with trypsin and pronase. The resulting trypsin and pronase cores contained increasing amounts of lipid A constituents firmly bound to the residual protein moiety. Since lipid A was separated as an entity from the protein moiety only after acid hydrolysis of the pronase core, it is proposed that in intact endotoxin lipid A is covalently linked to the protein moiety. The absence of any detectable glucosamine and fatty acids in the mixture of peptides and amino acids released by trypsin and pronase, indicated a single point attachment between the lipid A and protein moieties.The protein moieties from Serratia marcemens and Escherichia coli were immunogenic and possessed a common antigenic determinant. Both trypsin and pronase cores retained the immunogenicity and revealed the presence ofa common antigen with the parent simple protein. This second set of antigenic determinant(s) different from the specific 0-antigens seems to be located in the protein moiety close to lipid A.It is generally accepted that the endotoxin or somatic 0-antigen complexes occurring in the cell walls of gram-negative bacteria represent macromolecular entities composed of lipid, polysaccharide and protein moieties [1,2]. Evidence for this structural complexity was presented in early studies by Morgan and Partridge [3-51 who showed that endotoxins isolated from Shigella dysenteria and Salmonella typhosa could be resolved by successive treatments with formamide and 900/, phenol into a

Isolation and Characterization of Lipopolysaccharide Protein from Escherichia coli

“…In order to confirm that the radioactive hexosamine-containing material is covalently bound to protein in the isolated material, the homogeneous preparation was subjected to proteolytic digestion. The homogeneous material and its product of digestion were subjected to electrophoresis on cellulose acetate strips in borate buffer at pH 9 (Fig. 3) and 5,41 of proteolytic digest (600 cpm) were applied to cellulose acetate strips in bands about 0.5 cm in length; three duplicate strips, each 1-inch wide, were prepared in this manner.…”

“…However, in its native state in the outer membrane of the envelope, lipopolysaccharide occurs in close association with protein (6,7). Others (8,9) have reported the occurrence of complexes of lipopolysaccharide components and protein, although these complexes were not thoroughly characterized. In our current studies we considered the possibility that lipopolysaccharide may be covalently attached to a protein of the outer membrane in its native state and that the hot phenol extraction procedure re sults in partial degradation of the native complex macro Glc Purification of Lipopolysaccharide Protein.…”

Degradation of Lipopolysaccharide of Escherichia Coli by a Hot Phenol Extraction

“…It is known that LPS preparations isolated by phenol-water at 5-10° still contain various amounts of protein and those extracted at 65 to 68° are practically free of protein moiety (3, 25). WOBER and ALAUPOVIc isolated the socalled simple protein from the endotoxin of Serratia marcescens 08 and E. coli 0 141: K85 (B) by hot phenol-water treatment (26). It was reported that the native LPS in the outer membrane of the envelope occurred in close association with proteins (27,28).…”

“…Therefore, it may be considered that fatty acids released from LPS by treatment with hot phenol-water are mainly located in 0-acyl-fatty acid in the lipid A portion. WoBER and ALAUPOVIC (26) suggested that the dissociation of endotoxin to LPS and lipid A-protein by hot phenol-water is caused by the cleavage of a phenol-sensitive linkage within the molecule of lipid A rather than at its point of attachment to the protein moiety. The observations by ALAUPOVIC et al (25,26,30) that the hot phenol treatment caused a partial degradation of LPS is consistent with our results described in this paper.…”

Modulation of an Interleukin-12 and Gamma Interferon Synergistic Feedback Regulatory Cycle of T-Cell and Monocyte Cocultures byPorphyromonas gingivalisLipopolysaccharide in the Absence or Presence of Cysteine Proteinases

“…The bacteria at a density of 1.5 g/cm 3 were suspended in saline, stirred for 1 h at 4°C and washed three times with pyrogen-free water, and lyophilized. P. gingivalis LPS was extracted from lyophilized cells by the hot phenol-water method (47,48). Briefly, bacteria suspended in sterile saline were mixed with an equal volume of water-saturated butanol for 15 min at 4°C.…”