guanosine-triphosphate and safingol

Clostridium perfringens alpha-toxin induces hemolysis of rabbit erythrocytes through the activation of glycerophospholipid metabolism. Sheep erythrocytes contain large amounts of sphingomyelin (SM) but not phosphatidylcholine. We investigated the relationship between the toxin-induced hemolysis and SM metabolic system in sheep erythrocytes. Alpha-toxin simultaneously induced hemolysis and a reduction in the levels of SM and formation of ceramide and sphingosine 1-phosphate (S1P). N-Oleoylethanolamine, a ceramidase inhibitor, inhibited the toxin-induced hemolysis and caused ceramide to accumulate in the toxin-treated cells. Furthermore, dl-threo-dihydrosphingosine and B-5354c, isolated from a novel marine bacterium, both sphingosine kinase inhibitors, blocked the toxin-induced hemolysis and production of S1P and caused sphingosine to accumulate. These observations suggest that the toxin-induced activation of the SM metabolic system is closely related to hemolysis. S1P potentiated the toxin-induced hemolysis of saponin-permeabilized erythrocytes but had no effect on that of intact cells. Preincubation of lysated sheep erythrocytes with pertussis toxin blocked the alpha-toxin-induced formation of ceramide from SM. In addition, incubation of C. botulinum C3 exoenzyme-treated lysates of sheep erythrocytes with alpha-toxin caused an accumulation of sphingosine and inhibition of the formation of S1P. These observations suggest that the alpha-toxin-induced hemolysis of sheep erythrocytes is dependent on the activation of the SM metabolic system through GTP-binding proteins, especially the formation of S1P.

Topics: 4-Aminobenzoic Acid; ADP Ribose Transferases; Amidohydrolases; Animals; Bacterial Toxins; Botulinum Toxins; Calcium-Binding Proteins; Ceramidases; Chromatography, Thin Layer; Diglycerides; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme Inhibitors; Erythrocytes; Ethanolamines; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Hemolysis; Inositol 1,4,5-Trisphosphate; Lysophospholipids; Oleic Acids; para-Aminobenzoates; Pertussis Toxin; Phosphatidylcholines; Phosphorylcholine; Phosphotransferases (Alcohol Group Acceptor); Rabbits; Sheep; Sphingomyelins; Sphingosine; Time Factors; Toxins, Biological; Type C Phospholipases

The activity of sphinganine kinase, the enzyme catalyzing the first step in the breakdown of the sphingoid long-chain base sphinganine by converting it to sphinganine 1-phosphate, was characterized in microsomes isolated from corn shoots. Activity was assayed by monitoring the conversion of [3H]sphinganine to [3H]sphinganine 1-phosphate, which was recovered in the aqueous phase following lipid extraction. Sphinganine kinase was found to utilize D-erythro-sphinganine and ATP as substrates. Maximum product formation required the presence of Mg2+. The apparent Km for ATP was 0.81 mM. GTP also served as a source of phosphate, whereas CTP and UTP were not effective substrates in this assay. Maximum product formation was observed at sphinganine concentrations of approximately 100 microM. Results of competition experiments suggested that the enzyme could also phosphorylate D-erythro-sphingosine but not DL-threo-sphinganine or D-phytosphingosine. Enzyme activity was greatest in the microsomal fraction obtained by differential centrifugation and was localized to the Golgi and endoplasmic reticulum using marker enzymes. The specific activity of the enzyme under optimal conditions was 1.08 nmol/min x mg protein, a value 25-fold higher than that reported for preparations from brain tissue. Fumonisin, a mycotoxin that disrupts sphingolipid metabolism, did not alter sphinganine kinase activity in vivo or in vitro. The results of this study demonstrate, for the first time, the presence of sphinganine kinase activity in plant tissue and suggest that the properties of the kinase from corn microsomes are distinct from those of the mammalian and protistan enzymes in some respects.

Topics: Adenosine Triphosphate; Cytidine Triphosphate; Golgi Apparatus; Guanosine Triphosphate; Kinetics; Microsomes; Mycotoxins; Phosphotransferases (Alcohol Group Acceptor); Plant Shoots; Sphingosine; Subcellular Fractions; Substrate Specificity; Zea mays