The application of purified staphylolytic enzymes to the production of protoplasts and the comparison of protoplast membranes with those of L-forms from the same staphylococci

• Main Author: Ward, John Barrie
• Published: University of Bath 1967
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.767458

1) Two extracellular staphylolytic enzymes have been Isolated and purified from submerged cultures of Streptomyces griseus. 2) The enzymes were electrophoretically homogeneous at several pH values. A single peak was obtained for each protein on ultracentrifugation. Based on gel-filtration molecular weights of 11,800 and 13,000 were determined for enzyme 1 and enzyme 2 respectively. 3) The ionic strength and pH optima for lytic activity of the two enzymes against staphylococcal cells and their isolated cell walls have been detenained. The effects of several cations and certain known enzyme inhibitors have been tested on lytic action of the two enzymes against cell walls. 4) Both enzymes were N-acetylhexosaminidases. Examination of the acid hydrolysis products of fully digested cell walls treated with naBH4 demonstrated that both enzymes were muramidases. 5) Both enzymes caused lysis to varying extents, of the cell walls of a range of gram positive organisms. However, E. coli, the only gram negative organism tested, was only slightly lysed by Enzyme 2; Enzyme 1 had no apparent effect. 6) 'Protoplasts' of Staph. aureus were obtained when cells were treated with Enzyme 2 in a hypertonic sucrose medium. 7) Stable L-form growth has been induced from several strains of Staph. aureus by the action of penicillin. The resultant cultures were free of cell wall material. 8) Membranes obtained from both 'protoplasts' and L-forms were composed predominantly of protein (57-71%) and lipid (22-36%) Small amounts of RNA were also present. On average the lipid content of the L-form membranes was higher than that of the protoplast membranes. 9) Detailed examination of the extracted lipids demonstrated an increased concentration of glycolipid In the L-form membranes. In addition the major phospholipids of the L-form membranes were of the cardiolipin-type rather than the phosphatidylglycerol and lipoamino acids found in the protoplast membranes.