Phenomenon of hot-cold hemolysis: chelator-induced lysis of sphingomyelinase-treated erythrocytes.
Staphylococcus aureus produces a phospholipase C specific for sphingomyelin (beta-hemolysin). Erythrocytes with approximately 50% sphingomyelin in their membranes, e.g., from sheep, have been shown to have up to 60% of this phospholipid hydrolyzed by this enzyme at 37 C in isotonic buffered saline without hemolysis. Cooling of sphingomyelinase C-treated erythrocytes to 4 C causes complete lysis of the cells, a phenomenon known as hot-cold hemolysis. The addition of ethylenediaminetetraacetate (EDTA) to sheep erythrocytes preincubated with sphingomyelinase C was found to induce rapid hemolysis at 37 C. The treated cells became susceptible to chelator-induced hemolysis and to hot-cold hemolysis simultaneously, and the degree of lysis of both mechanisms increased equally with prolonged preincubation with sphingomyelinase C. Erythrocytes of species not readily susceptible to hot-cold hemolysis were equally insusceptible to chelator-induced lysis. Chelators of the EDTA series were the most effective, whereas chelators more specific for Ca2+, Zn2+, Fe2+, Cu2+, and Mg2+ were without effect. The rate of chelator-induced lysis was dependent on the preincubation period with beta-hemolysin and on the concentration of chelator added. The optimal concentration of EDTA was found to equal the amount of exogenously added Mg2+, a cation necessary for sphingomyelinase C activity. Hypotonicity increased the rate of chelator-induced hemolysis, whereas increasing the osmotic pressure to twice isotonic completely inhibited chelator-induced lysis. The data suggest that exogenously added and/or membrane-bound divalent cations are important for the stability of sphingomyelin-depleted membranes. The phenomenon of hot-cold hemolysis may be a consequence of the temperature dependence of divalent ion stabilization.
D015082:2,2'-Dipyridyl / Q000494:pharmacology; D000818:Animals; D002614:Chelating Agents / Q000494:pharmacology*; D004492:Edetic Acid / Q000494:pharmacology; D004912:Erythrocytes / Q000187:drug effects; D006460:Hemolysin Proteins / Q000032:analysis / Q000302:isolation & purification; D006461:Hemolysis / Q000187:drug effects*; D006801:Humans; D009994:Osmolar Concentration; D004369:Pentetic Acid / Q000494:pharmacology; D010618:Phenanthrolines / Q000494:pharmacology; D010727:Phosphoric Diester Hydrolases / Q000494:pharmacology*; D011817:Rabbits; D012756:Sheep; D013053:Spectrophotometry; D013108:Sphingomyelin Phosphodiesterase / Q000494:pharmacology*; D013696:Temperature; D013997:Time Factors
D002614:Chelating Agents; D006460:Hemolysin Proteins; D010618:Phenanthrolines; D015082:2,2'-Dipyridyl; D004369:Pentetic Acid; D004492:Edetic Acid; D010727:Phosphoric Diester Hydrolases; D013108:Sphingomyelin Phosphodiesterase