Staphylococcal beta-lysin

Low, Duncan K. R (1976) Staphylococcal beta-lysin. PhD thesis, University of Glasgow.

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Abstract

Three strains of S. aureus for the production of beta-lysin (sphingomyelinase C), R-1, G128 and IL7s were examined. Of these, S. aureus G128 was deemed to be the most suitable for further studies. Enzyme production in YDB medium was maximal in the late logarithmic phase of growth, when cell numbers were at a maximum. The cultoore was freed of cells by centrifugation. The supernatant was made 85% with respect to ammtonium sulphate and allowed to stand overnight. The resulting precipitate was fractionated by gel filtration on Biogel P60, ion exchange chromatography on carboxymethyl cellulose and finally electrofocusing in an LKB 110 ml electrofocusing column, pH range 7 - Hj in a sorbitol density gradient. The main peak of g-lysin activity focussed with a pi of 9.3. After removal of ampholines, the product had a specific activity of 62.5 x 106 HU/mg against sheep cells indicating a 38,000 fold purification. The product was free from alpha- and beta-lysin, and protease, nuclease, hyaliironidase, phosphatase, coagulase, lipase and leukocidin activities. It gave a single symmetrical peak in the ultracentrifuge, with an of 3.1, and a single band on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The molecular weight was estimated to be 32,500 by gel chromatography and electrophoresis. The highly purified sphingomyelinase C released water-soluble organic phosphorus from aqueous dispersions of sphingomyelin, and bovine and human erythrocyte ghosts in the presence of Mg2+, in amounts indicating extensive sphingomyelin degradation. Extensive ultrastructural changes were evident in both human and 'bovine freeze- etched ghosts after sphingomyelinase C treatment. Pools of apparently solid particle-free material, possibly the ceramide product of hydrolysis, accumulated in the hydrophobic region of the bilayer. These observations could be explained in terms of a membrane in which sphingomyelin is preferentially located in the outer half of the bilayer. Sphingomyelinase C is an extremely powerful probe of membrane structure. For this purpose it is critical that the degree of freedom from contaminating proteins should be known. The hot-cold lytic activity of sphingomyelinase G was examined using phase-contrast microscopy, and an explanation of this phenomenon in terms of lipid phase transitions was proposed. beta-lysin was found to be non-toxic for mice in doses up to 7.5 mg/kg and was not dermonecrotic. Intravenous injections of Evans blue showed that there was no local increase in vascular permeability to the dye at the site of intracutaneous injection of beta-lysin. The enzyme did not act as a single virulence factor and its role in pathogenicity may well be to increase the sensitivity of normally resistant cells to attack from the other extracellular products of staphylococci.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: John Freer
Keywords: Biochemistry
Date of Award: 1976
Depositing User: Enlighten Team
Unique ID: glathesis:1976-72348
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 24 May 2019 15:12
Last Modified: 24 May 2019 15:12
URI: http://theses.gla.ac.uk/id/eprint/72348

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