Subcellular study of sphingoid base phosphorylation in rat tissues: evidence for multiple sphingosine kinases

The enzymatic phosphorylation of sphingoid bases was analysed in rat tissue s, using D-erythro-[4,5-H-3]sphinganine as substrate. After optimisation of the assay, taking care to block sphingosine-phosphate lyase and sphingosin e phosphatase, highest ATP-dependent kinase activities were present in test is, followed by kidney, and intestinal mucosa. Approximately two thirds of the kidney activity were membrane bound, the remaining being cytosolic. Cla ssical cell fractionation studies of kidney and liver did not allow to iden tify unequivocally the subcellular site of the membrane bound kinase. Separ ation of a particulate fraction from kidney homogenates by Percoll gradient and sucrose density gradient centrifugation revealed that kinase activitie s are associated with vesicles derived from the endoplasmic reticulum and t he plasma membrane. Based on indirect data, such as the effect of detergent s and divalent ions, the cytosolic and both membrane bound activities appea r to reside in different proteins. N,N-Dimethylsphingenine was inhibitory t o all three different kinases, which were mainly active towards the D-eryth ro isomers of sphingenine and sphinganine. (C) 2001 Elsevier Science B.V. A ll rights reserved.