Medlock K A, Merrill A H
Department of Biochemistry, Emory University, Atlanta, Georgia 30322.
Biochemistry. 1988 Sep 6;27(18):7079-84. doi: 10.1021/bi00418a061.
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.
在体外使用大鼠肝微粒体以及在体内使用完整的中国仓鼠卵巢（CHO）细胞，研究了β-氯丙氨酸（β-Cl-丙氨酸）对丝氨酸棕榈酰转移酶活性以及鞘氨醇和鞘氨醇从头生物合成的影响。体外抑制作用迅速（5 mM β-Cl-丙氨酸在10分钟内导致完全失活）、不可逆，且具有浓度和时间依赖性，显然涉及活性位点，因为失活仅发生在β-Cl-L-丙氨酸（而非β-Cl-D-丙氨酸）存在时，并被L-丝氨酸阻断。这些是基于机制的（“自杀性”）抑制的特征。当完整的CHO细胞与β-Cl-丙氨酸孵育时，丝氨酸棕榈酰转移酶（SPT）也受到抑制（5 mM时15分钟内发生完全抑制），并且这种处理抑制了完整细胞将[14C]丝氨酸掺入长链碱基。SPT活性丧失和长链碱基合成的浓度依赖性是相同的。β-Cl-L-丙氨酸的作用似乎在对其他细胞功能干扰很小的情况下发生：细胞活力没有损失，[14C]丝氨酸摄取未被阻断，[14C]乙酸的总脂质生物合成没有减少（胆固醇和磷脂酰胆碱中放射性标记的出现也没有减少），并且[3H]胸苷掺入DNA不受影响。基于[3H]亮氨酸的掺入，对蛋白质合成似乎影响很小，其仅减少了14%。尽管已知β-Cl-L-丙氨酸会抑制其他依赖磷酸吡哆醛的酶，但在这些条件下丙氨酸和天冬氨酸转氨酶未受到抑制。这些结果确立了丝氨酸棕榈酰转移酶活性与细胞长链碱基形成速率之间的紧密关联，并表明β-Cl-丙氨酸和其他基于机制的抑制剂可能有助于研究细胞长链碱基合成的变化。
