Regina Linder

Interaction between sphingomyelin and a cytolysin from the sea anemone Stoichactis helianthus.

The cytolytic toxin from the sea anemone Stoichactis helianthus was inhibited up to 90--95% by suspensions of sphingomyelin but not by phosphatidylcholine or other membrane lipids. When the toxin was incubated with sphingomyelin and the mixture fractionated either by isoelectric focusing or Sephadex gel filtration, the residual hemolytic units migrated together with the lipid and not as free toxin. Incubation with phosphatidylcholine, however, did not shift the toxin peak in either type of column. A toxin-ferritin conjugate retaining hemolytic activity was observed by negative staining to bind to liposomes prepared with sphingomyelin but not with liposomes containing phosphatidylcholine. The results provide evidence that the membrane binding site of the toxin is sphingomyelin.

Enzymatic oxidation of membrane cholesterol in relation to lysis of sheep erythrocytes by corynebacterial enzymes.

Abstract Synergistic hemolysis of sheep erythrocytes brought about by the combined action of Corynebacterium ovis ( C. pseudotuberculosis ) and Corynebacterium equi depends upon the extracellular sphingomyelin-specific phospholipase D of the former species and a partially characterized agent(s) of the latter. Fractionation of the culture supernatant of C. equi revealed a cholesterol oxidase which was purified to near homogeneity by gel filtration and isoelectric focusing. The enzyme was isoelectric at pH 9–10 and had a molecular weight of 61,000. Sheep erythrocytes pretreated with purified sphingomyelinase D of C. ovis were hemolyzed by incubation with C. equi cholesterol oxidase or by the same enzyme from Brevibacterium sp. Lipid analysis revealed complete conversion of membrane cholesterol to cholest-4-en-3-one, the product of cholesterol oxidase action. Cells not pretreated with sphingomyelinase D did not undergo cholesterol oxidation or hemolysis when treated with cholesterol oxidase. Studies with crude culture supernatant of C. equi confirmed the presence of a phospholipase active in hydrolyzing ceramide phosphate generated in the erythrocyte membrane by C. ovis sphingomyelinase. Ceramide thus produced in the membrane is known to make the cells labile to hemolysis. There are, therefore, at least two mechanisms underlying synergistic hemolysis by these coryne-bacteria.

Effect on sphingomyelin-containing liposomes of phospholipase D from Corynebacterium ovis and the cytolysin from Stoichactis helianthus

The toxic, sphingomyelin-specific phospholipase D (phosphatidylcholine phosphatidohydrolase EC 3.1.4.4) from Corynebacterium ovis was purified to near homogeneity. It has a molecular weight of 31 000 and a pI of approx. 9.8. Although not cytolytic itself, it protected red cells from hemolysis by staphylococcal sphingomyelinase (beta-hemolysin) and helianthus toxin. The apparently non-enzymatic cytolysin (helianthus toxin) from the sea anemone Stoichactis helianthus also interacts with membrane sphingomyelin. C. ovis and helianthus toxins were compared with regard to their effects on liposome model membranes, and they were found both to produce changes analogous to those in erythrocytes. Only helianthus toxin caused release of trapped glucose marker, but liposomes could be protected from release by pretreatment with C. ovis toxin. Both toxins demonstrated binding to sphingomyelin-containing liposomes, but only the bacterial sphingomyelinase catalyzed the release of choline from these vesicles.

DISCUSSION PAPER: THE dd‐CARBOXYPEPTIDASE‐TRANSPEPTIDASE SYSTEM IN ESCHERICHIA COLI MUTANT STRAIN 44*

The peptide moiety of the wall peptidoglycan of Escherichia coZi is composed of uncross-linked peptide monomers L-alanyl-y-~glutamyl-(L)-mesodiaminopimelyl-( L) -D-alanine and peptide dimers in which two peptide monomers are linked together through C-terminal D-alanyl( D) -meso-diaminopimelic acid linkages (FIGURE 1). It has been observed previously that cross-linked peptidoglycan that consists of such peptide dimers could be synthesized when the nucleotide precursors UDP-N-acetylglucosamine and UDP-N-acetylmuramyl-L-alanyl-y-D-glutamyl( L) -meso-diaminopimelyl( L) -D-alanyl-D-alanine were combined with Mg*+ ions and a crude disrupted cellular preparation.4-8 These authors also postulated that the soluble enzyme that cleaves the terminal Dalanine residue of UDP-N-acetylmuramyl pentapeptide (see FIGURE 1) in a Dwcarboxypeptidase reaction was distinct from that enzyme in their particulate preparation responsible for transpeptidation. Because of the complexity, however, of such a multienzyme system that did not allow the independent assay of transpeptidase activity, it has not been possible to study the interaction of penicillin with the transpeptidase per se, and thus an understanding at the molecular level of how penicillin kills bacteria has been delayed. Moreover, because two purified single polypeptide chain enzymes isolated from Streptomyces species lo each perform both m-carboxypeptidase and transpeptidase functions, it was decided to reinvestigate such processes in E. coli.

Affinity Chromatography of Succinate Dehydrogenase from the Membranes of Micrococcus Lysodeikticus

Isolated plasma membranes of Micrococcus lysodeikticus were subjected to extraction with n-butanol in a two-phase system. Succinate dehydrogenase obtained in the soluble aqueous phase after high-speed centrifugation was resolved by separation on calcium phosphate gel and affinity chromatography. The affinity ligand used was oxaloacetate and elution from the column was achieved with 0.5 M succinate. In the final product there was an eleven-fold reduction in the 32P-lipid to protein ratio and a fourteen-fold increase in specific activity relative to the high speed supernatant fraction following n-butanol extraction.