Inhibition of serine palmitoyltransferase in vitro and long-chain base biosynthesis in intact Chinese hamster ovary cells by beta-chloroalanine.
The effects of beta-chloroalanine (beta-Cl-alanine) on serine palmitoyltransferase activity and the de novo biosynthesis of sphinganine and sphingenine were investigated in vitro with rat liver microsomes and in vivo with intact Chinese hamster ovary (CHO) cells. The inhibition in vitro was rapid (5 mM beta-Cl-alanine caused complete inactivation in 10 min), irreversible, and concentration and time dependent and apparently involved the active site because inactivation only occurred with beta-Cl-L-alanine (not beta-Cl-D-alanine) and was blocked by L-serine. These are characteristics of mechanism-based ("suicide") inhibition. Serine palmitoyltransferase (SPT) was also inhibited when intact CHO cells were incubated with beta-Cl-alanine (complete inhibition occurred in 15 min with 5 mM), and this treatment inhibited [14C]serine incorporation into long-chain bases by intact cells. The concentration dependence of the loss of SPT activity and of long-chain base synthesis was identical. The effects of beta-Cl-L-alanine appeared to occur with little perturbation of other cell functions: the cells exhibited no loss in cell viability, [14C]serine uptake was not blocked, total lipid biosynthesis from [14C]acetic acid was not decreased (nor was the appearance of radiolabel in cholesterol and phosphatidylcholine), and [3H]thymidine incorporation into DNA was not affected. There appeared to be little effect on protein synthesis based on the incorporation of [3H]leucine, which was only decreased by 14%. Although beta-Cl-L-alanine is known to inhibit other pyridoxal 5'-phosphate dependent enzymes, alanine and aspartate transaminases were not inhibited under these conditions. These results establish the close association between the activity of serine palmitoyltransferase and the cellular rate of long-chain base formation and indicate that beta-Cl-alanine and other mechanism-based inhibitors might be useful to study alterations in cellular long-chain base synthesis.
D000217:Acyltransferases / Q000037:antagonists & inhibitors*; D000409:Alanine / Q000031:analogs & derivatives*; D000818:Animals; D001693:Biological Transport, Active; D002478:Cells, Cultured; D006224:Cricetinae; D005260:Female; D066298:In Vitro Techniques; D008055:Lipids / Q000096:biosynthesis; D008862:Microsomes, Liver / Q000378:metabolism; D051381:Rats; D012694:Serine / Q000493:pharmacokinetics; D051102:Serine C-Palmitoyltransferase; D013107:Sphingolipids / Q000096:biosynthesis*; D013110:Sphingosine / Q000031:analogs & derivatives / Q000096:biosynthesis; D015091:beta-Alanine / Q000031:analogs & derivatives* / Q000493:pharmacokinetics / Q000494:pharmacology
D008055:Lipids; D013107:Sphingolipids; D015091:beta-Alanine; C006977:3-chloroalanine; D012694:Serine; D000217:Acyltransferases; D051102:Serine C-Palmitoyltransferase; D013110:Sphingosine; D000409:Alanine; C005682:safingol