Kinetics of long-chain (sphingoid) base biosynthesis in intact LM cells: effects of varying the extracellular concentrations of serine and fatty acid precursors of this pathway.
Serine palmitoyltransferase (EC 2.3.1.50) catalyzes the condensation of L-serine and palmitoyl-CoA to yield 3-ketosphinganine in the first unique reaction of long-chain (sphingoid) base biosynthesis. The kinetic effects of changing the extracellular concentrations of the precursors for this pathway were studied with LM cells by following the incorporation of L-[3-14C]serine into the long-chain base (i.e., sphinganine and sphingenine) backbones of complex sphingolipids. [14C]Serine was taken up by the cells and rapidly reached steady-state concentrations similar to those of the medium. From the cellular [14C]serine concentrations and specific activities, the apparent Vmax [14 pmol min-1 (10(6) cells)-1] and Km (0.23 mM) values for long-chain base synthesis were determined and found to be essentially identical with those for serine palmitoyltransferase assayed in vitro [i.e., 13 pmol min-1 (10(6) cells)-1 and 0.27 mM, respectively]. The other precursor, palmitic acid, was also taken up rapidly and increased long-chain base biosynthesis in a concentration-dependent manner. This effect was limited to palmitic acid and matched the known specificity of serine palmitoyltransferase for saturated fatty acyl-CoA's of 16 +/- 1 carbon atoms. These studies delineate the influence of extracellular precursors on the formation of the sphingolipid backbone and suggest that the kinetic properties of serine palmitoyltransferase govern this behavior of long-chain base synthesis in intact cells.
D000217:Acyltransferases / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D002250:Carbon Radioisotopes; D002478:Cells, Cultured; D007700:Kinetics; D051379:Mice; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism*; D010171:Palmitoyl Coenzyme A / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D051102:Serine C-Palmitoyltransferase; D013107:Sphingolipids / Q000096:biosynthesis*
D002250:Carbon Radioisotopes; D010169:Palmitic Acids; D013107:Sphingolipids; D010171:Palmitoyl Coenzyme A; D019308:Palmitic Acid; D012694:Serine; D000217:Acyltransferases; D051102:Serine C-Palmitoyltransferase; C495088:Sptlc1 protein, mouse