SUMMARYPurified gamma haemolysin of Staphylococcus a u r a was characterized in relation to the alpha, beta and delta haemolysins. The sedimentation coefficient of the gamma lysin was 2.65, somewhat higher than the s , , values of 1-4 for freshly purified alpha lysin and 1.8 for the beta lysin. The molecular weight of gamma lysin determined by gel filtration was 45000 daltons. The PI of gamma lysin was 6-0, while that of the alpha, beta and delta lysins ranged from 8-5 to 9-6.The amino acid analysis of gamma lysin was characterized by low levels of methionine and histidine. Methionhe was, however, the N-terminus, which suggested that all of the amino acid might be involved in the N-terminal group.The gamma lysin was immunologically distinct from the alpha, beta and delta lysins by quantitative precipitin tests; in Ouchterlony agar gel diffusion tests, single lines of precipitation were observed which showed no evidence of cross-reactions amongst the four haemolysins.Gamma, beta and delta lysins had no effect in mice when injected at increasing doses ranging from o to 100 pg. The alpha lysin killed mice, the LD, dose being 0.68 & 0.12 pg, or 27 to 34 pg/kg mouse tissue. Gamma lysin was, however, lethal for guinea pigs when 5opg quantities were injected intracardially. Gamma lysin also lysed human leucocytes and destroyed C-6 (human lymphoblast) cells. Some nitrogen and phosphorus was released from human erythrocyte membranes treated with gamma lysin, when compared to untreated cells, and the rate of this release was linear over a 3 h period. Gamma lysin had no detectable effect on phospholipids extracted from erythrocytes or on lipid-free membrane protein. However, the haemolytic reaction was inhibited by erythrocyte membranes when these were added to lysin-red cell suspensions. Furthermore, human redcell phospholipids competitively inhibited haemolysis. Haemolysis was also inhibited by EDTA and activity could be restored by dialysis. The haemolytic reaction required sodium ions.
PLATE XIXAT T E M P,T s by several investigators have failed to demonstrate the immunogenicity of the 6-haemolysin of StaphyZococcus aureus. Gladstone and Yoshida (1967) did not convincingly demonstrate that 8-lysin was immunogenic and they showed that inhibitors of its haemolytic activity were present in serum. Recent evidence suggests that these serum inhibitors are lipoproteins (Donahue, 1969;Kantor, Temples and Shaw, 1972).In this study, the immunogenicity of 8-lysin has been demonstrated by the use of purified antibody preparations. MATERIALS AND METHODSPreparation of 6-lysin The strain employed, methods of production and purification of 6-lysin were those of Caird and Wiseman (1970). Purified S-lysin was also kindly sent to us by Dr Arnold Kreger. Haemolysin titrations were performed as described by Wiseman and Caird (1972). Buffers were prepared according to Gomori (1955) and, where required, 0 . 0 1~ phosphate buffer at pH 7 was supplemented with 0.85% NaCl. Nitrogen content was assayed by the microKjeldahl technique as described by Markham (1942) and an estimate of protein content was derived from the assay value multiplied by 6.25.Preparation of u-, ,8-and 7-lysins u-and y-Haemolysins were prepared and purified as described elsewhere (Fackrell and Wiseman, 1974; Wiseman, Caird and Fackrell, 1975). Purified ,!I-lysin was used in one immunodiffusion study and was prepared by a modification of the method of Wiseman and Caird (1967) in which the haemolysin was passed through Sephadex G-75 equilibrated with Hallander's (1963) buffer. The active lysin was pooled when removed from the column, dialysed against distilled water for 48 hours, centrifuged and the supernatant fluid lyophilised.Serological procedures Immunodi'usion, immunoelectrophoresis and quantitative precipitin methods were those of Campbell et al. (1970). The blood agarose required in some immunodiffusion tests was prepared by the addition of 1.0 ml of packed, washed human group-0 RBC to 100 ml of a 1 % (w/v) solution of Difco agarose in phosphate-buffered saline held at 50°C.Production of antisera. Antibodies to the haemolysins were prepared in 2-month-old New Zealand white rabbits purchased from the Canadian Research Animal Farm, Bradford,
Enzymes known to specifically cleave the band 3 component of the rabbit erythrocyte membrane were found to reduce both the hemolytic sensitivity to and the binding of the alpha toxin of Staphylococcus aureus. Lectins which bind to band 3 also inhibited the toxin. Lectins which do not bind to band 3 have no effect. Purified band 3, isolated by affinity chromatography on a concanavalin A column, was homogeneous by polyacrylamide gel electrophoresis, had a molecular weight of 100 000, and inhibited the hemolytic activity of alpha toxin. Antibodies to the toxin-toxoid receptor were serologically indistinguishable from antiband 3.
In murine muscular dystrophy, hindlimb muscle contains a functionally defective thiol protease inhibitor (TPI) which has been implicated in the onset and progression of the disease in mice. More recently, this protease inhibitor has been identified as parvalbumin, a calcium binding protein. In this study, a polyclonal antibody against mouse muscle parvalbumin was used to study the concentration and distribution of this protein in normal and dystrophic male mice at various ages. Immunodetection assays were used to screen extracts of hindlimb, forelimb, brain, heart, lung, liver, and kidney in 60-day-old normal and dystrophic male mice for parvalbumin content. Parvalbumin was detected in relatively high amounts in both hindlimb and forelimb muscle extracts, while much lower concentrations were detected in brains of normal and dystrophic animals. No parvalbumin was detected in the lung, liver, heart, or kidney extracts using the immunoassay. With aging, the parvalbumin concentration in hindlimb muscle of normal mice remained fairly constant for 90 days, whereupon the level increased at 120 days. In contrast, the parvalbumin concentration in hindlimb muscle of dystrophic mice decreased steadily with age to about 22%% of normal animals at 120 days. The parvalbumin content was also reduced in dystrophic brain.
Bdellovibrio bacteriovorus 6-5-S releases at least two enzymes into the culture fluid when grown on autoclaved cells of Spirillum serpens VHL. One of the enzymes is bacteriolytic and the other is proteolytic. The lytic enzyme was purified by a factor of 3000 using ammonium sulfate fractionation, Sephadex G-100 gel filtration, and the anion exchanger DEAE-Sephadex. The lytic enzyme degrades peptidoglycan of S. serpens by hydrolyzing the diaminopimelic acid – alanine bond in the tetrapeptide chain. Ca2+ or Mg2+ exerted little or no effect on the activity of the lytic enzyme. The enzyme had a molecular weight of 40 000 as indicated by gel filtration. Crude preparations were unstable at 4C. The second enzyme, a protease that digested Azocoll, was purified by a factor of 7 by ammonium sulfate fractionation followed by gel filtration with Sephadex G-100. The protease was eluted in the void volume from a Sephadex G-100 column and therefore may have a molecular weight of at least 100 000. Its activity was enhanced by additions of Ca2+ and Mg2+. The enzyme was stable at 4C.
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