Department of Physiology, College of Medicine, University of Kentucky, Lexington, KY, USA.
Methods Mol Biol. 2021;2187:113-129. doi: 10.1007/978-1-0716-0814-2_7.
This chapter will discuss methods for analyses of the rates of sphingomyelin synthesis and turnover associated with lipid rafts or plasma membrane. These methods involve the use of fluorescently (NBD-C6-ceramide or NBD-C6-Sphingomyelin)) or radioactively labeled substrates ([H-methyl]-phosphatidylcholine, [H-acyl]-ceramide, [C-methyl]-sphingomyelin) to quantify in vitro the activity of the sphingomyelin synthase (SMS) (also known as phosphatidylcholine:ceramide phosphocholine transferase), acid sphingomyelinase (the endosomal/lysosomal (L-SMase) and the secretory (S-SMase) forms) and neutral sphingomyelinase-2 (nSMase-2). These methods allow to quantify changes in the activity of enzymes that affect the SM-to-ceramide ratio on the plasma membrane, and consequently, the lipid rafts biophysical properties, dynamics, and raft-associated receptor clustering and signaling events. Specific attention is paid to challenges caused by the fact that SMS and nSMase-2 are integral/membrane bound proteins and how to avoid the use of detergent that suppress their specific activities.
本章将讨论分析与脂筏或质膜相关的神经鞘磷脂合成和周转率的方法。这些方法涉及使用荧光标记（NBD-C6-神经鞘氨醇或 NBD-C6-神经鞘磷脂）或放射性标记的底物（[H-甲基]-磷酸胆碱、[H-酰基]-神经酰胺、[C-甲基]-神经鞘磷脂），体外定量测定神经鞘磷脂合成酶（SMS）（也称为磷酸胆碱：神经酰胺磷酸胆碱转移酶）、酸性神经鞘磷脂酶（内体/溶酶体（L-SMase）和分泌（S-SMase）形式）和中性神经鞘磷脂酶-2（nSMase-2）的活性。这些方法可定量测定影响质膜上神经鞘氨醇与神经酰胺比率的酶活性的变化，从而影响脂筏的物理特性、动态变化以及与脂筏相关的受体聚集和信号事件。特别关注 SMS 和 nSMase-2 是完整/膜结合蛋白这一事实所带来的挑战，以及如何避免使用抑制其特定活性的去污剂。
