The action of sphingomyelinase from Bacillus cereus on ATP-depleted bovine erythrocyte membranes and different lipid composition of liposomes

doi:10.1016/0003-9861(87)90302-X

Archives of Biochemistry and Biophysics

Volume 255, Issue 1, 15 May 1987, Pages 127-135

https://doi.org/10.1016/0003-9861(87)90302-X Get rights and content

Abstract

The presence of cholesterol or phosphatidylethanolamine in sphingomyelin liposomes enhanced 2- to 10-fold the breakdown of sphingomyelin by sphingomyelinase from Bacillus cereus. On the other hand, the presence of phosphatidylcholine was either without effect or slightly stimulative at a higher molar ratio of phosphatidylcholine to sphingomyelin ( $31$ ). In the bovine erythrocytes and their ghosts, the increase by 40–50% or the decrease by 10–23% in membranous cholesterol brought about acceleration or deceleration of enzymatic degradation of sphingomyelin by 50 or 40–50%, respectively. The depletion of ATP (<0.9 mg ATP/100 ml packed erythrocytes) enhanced K⁺ leakage from, and hot hemolysis (lysis without cold shock) of, bovine erythrocytes but decelerated the breakdown of sphingomyelin and hot-cold hemolysis (lysis induced by ice-cold shock to sphingomyelinase-treated erythrocytes), either in the presence of 1 mm MgCl₂ alone or in the presence of 1 mm MgCl₂ and 1 mm CaCl₂. Also, ATP depletion enhanced the adsorption of sphingomyelinase onto bovine erythrocyte membranes in the presence of 1 mm CaCl₂ up to 81% of total activity, without appreciable K⁺ leakage and hot or hot-cold hemolysis. These results suggest that the presence of cholesterol or phosphatidyl-ethanolamine in biomembranes makes the membranes more susceptible to the attack of sphingomyelinase from B. cereus and that the segregation of lipids and proteins in the erythrocyte membranes by ATP depletion causes the deceleration of sphingomyelin hydrolysis despite the enhanced enzyme adsorption onto the erythrocyte membranes.

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When large unilamellar vesicles consisting of sphingomyelin:phosphatidylethanolamine:cholesterol (2:1:1 molar ratio) are treated with sphingomyelinase, production of ceramides in the bilayer is accompanied by leakage of vesicle aqueous contents and by vesicle aggregation in the absence of lipid mixing or vesicle fusion. This is in contrast to the situation of phosphatidylcholine:phosphatidylethanolamine:cholesterol (2:1:1 molar ratio) liposomes when treated with phospholipase C. In that case, in situ generation of diacylglycerol leads to vesicle aggregation followed by vesicle fusion in the absence of leakage (Nieva, J. L., Goñi, F. M., and Alonso, A. (1989) Biochemistry 28, 7364-7367). Moreover, when ceramides (5-10 mol %) are included in the formulation of the phosphatidylcholine-containing vesicles, they reduce the lag time of phospholipase C-induced fusion, although they are less active than diacylglycerols in this respect. ³¹P NMR studies of aqueous lipid dispersions show that diacylglycerols as well as ceramides induce a thermotropic lamellar to non-lamellar phase transition in both phospholipid:cholesterol mixtures under study although sphingomyelin-containing bilayers are more stable than those containing phosphatidylcholine, and ceramide is less active than diacylglycerol in promoting non-lamellar phase formation. These observations are relevant to both the physiological role of ceramides and the current views on the mechanism of membrane fusion.
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1993, Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
By the modification of acidic amino-acid residues with Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3′-sulfonate), the activity of sphingomyelinase of Bacillus cereus was decreased by 80–90%. Also, the reduction of Cys residues in the sphingomyelinase molecule by dithiothreitol cause a drasic decrease in enzymatic activity, whereas the spingomyelinase activity was not affected by treatment with p-chloromercuribenzenesulfonic acid. Actually, no inactivation of sphingomyelinase activity was observed after selective modification of basic amino-acid residues such as Lys, His and Arg, and of the unchanged amino-acid residues Ser and Thr. The treatment of the sphingomyelinase molecule with Woodward's reagent K or dithiothreitol also brought about the inhibition of the specific adsorption of sphingomyelinase toward intact erythrocyte membranes. However, the extent of inhibition in the enzyme adsorption, 20–50%, was less than that observed in the sphingomyelinase activity. These results suggest that acidic amino-acid residues, such as Asp and Glu, in the sphingomyelinase molecule are involved in the catalytic sites and the adsorptive sites. Apparently, the disruption of disulfide linkage in the sphingomyelinase molecule by dithiothreitol destabilized its structure, resulting in a drastic decrease in sphingomyelin-hydrolyzing activity and specific adsorption of sphingomyelinase towards crythrocyte membranes.
Effect of 22R-hydroxycholesterol on the action of sphingomyelinase from Bacillus cereus toward bovine erythrocytes
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The incorporation of 22R-hydroxycholesterol [(22R)-5-cholestene-3β,22-diol] into the bovine erythrocyte membranes remarkably enhanced the degradation of sphingomyelin in erythrocyte membranes by the action of sphingomyelinase from Bacillus cereus, causing much faster hemolysis of erythrocytes. The stimulative effect of 22R-hydroxycholesterol on the breakdown of sphingomyelin was maximal in the presence of Mg²⁺. On the other hand, in spite of the presence of 22R-hydroxycholesterol, the breakdown of sphingomyelin was inhibited by increasing concentrations of Ca²⁺. Also, the incorporation of 22R-hydroxycholesterol into the erythrocyte membranes facilitated the specific adsorption of the enzyme onto the surface of the erythrocyte membranes. The specific adsorption of sphingomyelinase amounted to 20–40% of the total activity in the presence of Mg²⁺ and the absence of divalent metal ions. In the presence of Ca²⁺, the incorporation of 22R-hydroxycholesterol enhanced the enzyme adsorption, exceeding more than 90% of the total activity. Therefore, the incorporation of 22R-hydroxycholesterol into bovine erythrocyte membranes remarkably accelerates the breakdown of sphingomyelin in the presence of Mg²⁺, and the specific adsorption of sphingomyelinase onto erythrocytes in the presence of Ca²⁺.
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^☆: This work was supported in part by a scientific research grant from the Ministry of Education, Science and Culture of Japan.

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The action of sphingomyelinase from Bacillus cereus on ATP-depleted bovine erythrocyte membranes and different lipid composition of liposomes☆

Abstract

Nature (London)

J. Gen. Microbiol

Canad. J. Microbiol

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Arch. Biochem. Biophys

Biochim. Biophys. Acta

Arch. Biochem. Biophys

Biochim. Biophys. Acta

J. Biochem. (Tokyo)

Arch. Biochem. Biophys

J. Biol. Chem

Ann. N.Y. Acad. Sci

Ann. N.Y. Acad. Sci

J. Lab. Clin. Med

Biochem. J

Arch. Biochem. Biophys

J. Biol. Chem