Characterization of the A Component of Streptococcus zymogenes Lysin

Paul A. Granato, Robert W. Jackson
1971 Journal of Bacteriology  
By utilizing conventional techniques of pressure ultrafiltration, gel filtration chromatography, diethylaminoethyl cellulose chromatography, and preparative polyacrylamide electrophoresis, the A component of the group D lysin produced by Streptococcus zymogenes has been purified to a state of apparent homogeneity when determined by the techniques of anionic and cationic disc gel electrophoresis. The A component was found to be a protein possessing a molecular weight of 27,000, a sedimentation
more » ... , a sedimentation coefficient approximating 3.2S, and a net negative charge at physiological pH. Brock and Davie (4) showed that the lysin excreted by a group D streptococcus, Streptococcus zymogenes X-14, during logarithmic growth was both bacteriolytic and hemolytic. In addition, they presented evidence that the lysin probably achieved bacteriolysis of certain grampositive organisms by attacking the cell membrane (5). The lytic effect was demonstrated on spheroplasts after removal of membrane-associated teichoic acids. The latter were demonstrated to be inhibitors of the lysin (6). This laboratory has since isolated mutants of S. zymogenes X-14 which demonstrated that the lysin was composed of at least two different macromolecular components (7). The L component appeared to possess the catalytic activity, whereas the A component behaved in the manner of a typical enzyme activator. Hemolytic activity was linearly dependent on the amount of L substance present, whereas activity increased hyperbolically with increasing A substance until a saturation plateau was reached. When the A/L ratio became sufficiently large, activity was inhibited. We speculated that lysin may serve some in vivo function for the organism in regard to membrane biogenesis, maintenance, or transport. Since the in vitro activity of the lysin could apparently be regulated by varying concentrations of the A component and is subject to control by teichoic acids, it seemed possible that in vivo activity may be responsive to similar regulatory mechanisms. With this in mind, the physical and chemical characterization of these components I Predoctoral trainee of National Science Foundation (grant GZ-914). became of great interest to this laboratory. This report is concerned with the purification and the physical and chemical characterization of the A component. MATERIALS AND METHODS Media. Brain Heart Infusion (BHI) broth (Difco) was used as the growth medium for all strains. These were routinely carried on BHI agar plates containing 5% sheep erythrocytes. Buffers. Phosphate-buffered saline (PBS-A) was used in a concentration of 0.01 M phosphate containing 0.145 M NaCl (pH 6.8) for all hemolytic assays. Phosphate buffer (PBS-B) at the same phosphate concentration and pH but with 0.125 M NaCI was used as the Bio-Gel P-30 elution buffer. PBS-B containing 0 to 0.3 M NaCl was used for the diethylaminoethyl (DEAE) cellulose elution. Preparation of rabbit erythrocytes. Rabbit erythrocytes were obtained by cardiac puncture and suspended in an equal volume of Alsever's solution. Before use, the cells were washed four times with PBS-A and suspended at 2% by volume in the same buffer. Preparation of A and L supernatant fluids. The A supernatant fluid was prepared by adding a 10% inoculum of an overnight culture of an A-producing mutant (A18) into sterile BHI medium and incubating at 37 C with slow shaking. When the culture turbidity reached 150 Klett units (no. 54 filter), the cells were removed by centrifugation at 4 C for 30 min at 20,000 x g. (The yield was approximately 0.2 mg, dry weight, of cells/ml.) The L supernatant fluid was prepared in a similar manner by using an L-producing mutant (L16), except that culture was grown to about 190 Klett units (corresponding to approximately 0.5 mg, dry weight, of cells/ml). The supernatant fluids from each mutant were then refrigerated at 4 C until required for use. Chromatography materials. All filtration gels were purchased from Bio-Rad Laboratories. DEAE cellulose with a capacity of 0.90 meq/g was obtained from 551 on May 9, 2020 by guest
doi:10.1128/jb.107.2.551-556.1971 fatcat:4nyri5rcargmvnd4u66otbq75m