Rabbit Erythrocytes by Staphylococcal Alpha-Toxin

Abstract

In our previous papers (6, 7), alpha-toxin from the Wood-46 strain of Staphylococcus aureus was isolated in a crystalline form, and the physicochemical properties and biological activities were described. Among the many biological activities, alpha-toxin has a lytic effect on rabbit erythrocytes and artificial membranes (1, 2). Although the specific mechanism of the action of alpha-toxin is still unknown, Cassidy et al. (4), using [1251]alpha-toxin and liposomes, have recently suggested that specific receptor substances for alpha-toxin may exist in rabbit erythrocyte membranes. In this study we present data indicating that the inhibition of alphatoxin hemolysis by flavin mononucleotide (FMN) is due to competition with staphylococcal alpha-toxin for a site on the rabbit erythrocyte membranes. The crystalline staphylococcal alpha-toxin was obtained from the procedures of the previous paper (6, 7). ['251]alpha-toxin was prepared by the method of Kato and Watanuki (5). Iodinated toxin retained 82% of its original hemolytic activity for rabbit erythrocytes as assayed by the method of Bernheimer and Schwartz (3). Table 1 shows the relative sensitivity of enzyme-treated and normal erythrocytes to hemolysis by alpha-toxin. Pronase-treated erythrocytes (15% protein, 10% sugar components released) were more resistant to the hemolytic activity than those treated with amino sugarsplitting enzymes. The binding of [125I]toxin to the Pronase-treated cells was greatly decreased to less than 3% of that of nontreated, normal cells. Neuramidase treatment (10% sialic acid released) caused no alteration in the sensitivity to hemolysis by the alpha-toxin or the extent of binding of [125IJtoxin (Table 1). A comparison of the concentrations needed for 50% hemolysis inhibition shows that FMN is an order of magnitude more active than riboflavin or flavin adenine dinucleotide. Riboflavin is at least an order of magnitude more active than the phospholipids lecithin, phosphorylcholine, and phosphatidyl ethanolamine (Table 2). The effect of FMN upon alpha-toxin hemolysis was studied further to ascertain the nature of the inhibition. Reciprocal plots of hemolysis against alpha-toxin concentrations (Fig. 1) suggested that the inhibition of the initial rate of

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Cite this paper

@inproceedings{KatoRabbitEB, title={Rabbit Erythrocytes by Staphylococcal Alpha-Toxin}, author={Iwao Kato and K Sakoda and Masaru Saito and Yoshie Suzuki} }