Characterization of serine palmitoyltransferase activity in Chinese hamster ovary cells.
Serine palmitoyltransferase (palmitoyl-CoA: L-serine C-palmitoyltransferase (decarboxylating) EC 2.3.1.50) catalyzes the first unique and regulatory reaction of sphingolipid biosynthesis. Its activity was demonstrated in Chinese hamster ovary cells (CHO-K1) by measuring the incorporation of radiolabel from L-[3H]serine into 3-ketosphinganine, which was found to be the predominant chloroform-soluble product under optimal assay conditions. Most of the total activity (14.8 +/- 4.2 pmol/min per 10(6) cells, measured with sonicated cells) was recovered in particulate fractions, with the highest percentage (54%) and specific activity (102 pmol/min per mg) in the high-speed (airfuge) pellet. The greatest activity was obtained with palmitoyl-CoA; however, other fatty acyl-CoA thioesters were also utilized. Serine palmitoyltransferase required pyridoxal 5'-phosphate for activity, but was apparently fully saturated with this coenzyme when assayed with sonicated cells. Regardless of whether the CHO cells were grown in culture medium containing whole serum with or without sphinganine addition, lipoprotein-depleted serum, lipid-extracted serum, low-density lipoproteins, or no serum, the activities of this enzyme were identical. This finding was confirmed using human fibroblasts. Hence, these results establish that CHO cells, and probably others, are engaged in long-chain base synthesis de novo and that serine palmitoyltransferase activity is not regulated by the availability of such compounds in the culture media.
D000217:Acyltransferases / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010053:Ovary; D051102:Serine C-Palmitoyltransferase; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity
D000217:Acyltransferases; C495089:SPTLC2 protein, human; D051102:Serine C-Palmitoyltransferase