An improved assay method for the measurement and detection of sphingomyelinase activity.
An improved assay method for the measurement and detection of sphingomyelinase activity.
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用于测量和检测鞘磷脂酶活性的改进测定方法。
DOI：
10.1006/abio.1995.1077
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Chatterjee,S
中科院分区：
文献类型：
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作者：
Taki,T;Chatterjee,S
我们开发了一种改进的测定方法来测量鞘磷脂酶活性并检测在聚丙烯酰胺凝胶上分离的这种酶。鞘磷脂酶活性的测定涉及将[N-甲基-14C]鞘磷脂固定在聚二氟乙烯(PVDF)膜上、与鞘磷脂酶一起孵育以及与[14C]磷酸胆碱相关的放射性测量。酶活性取决于鞘磷脂、酶的浓度、pH 值和温度。三十分钟的孵育时间对于酶活性来说是最佳的。该酶具有双峰 pH 最佳值，最佳酶活性在 pH 5.4 和 7.4 下测量。通过在聚丙烯酰胺凝胶上分离酶并通过暴露于印迹在PVDF膜上的[14C]鞘磷脂来进行酶测定来进行鞘磷脂酶的检测。通过放射自显影观察 PVDF 膜上的酶活性。观察到一条白色条带（表示从 PVDF 中水解去除 [14C] 鞘磷脂。我们通过将鞘磷脂固定在 PVDF 膜上来检测鞘磷脂酶的方法可以作为测定水解产物释放到水相中的各种其他酶的原型。此外，我们在聚丙烯酰胺凝胶上检测鞘磷脂酶的方法可能有助于进一步研究该酶和鞘磷脂的分子生物化学。相关的磷脂酶。
We have developed an improved assay method to measure sphingomyelinase activity and to detect this enzyme separated on polyacrylamide gels. The assay of sphingomyelinase activity involved immobilizing [N-methyl-14C]sphingomyelin on polyvinyldiflouride (PVDF) membrane, incubation with sphingomyelinase, and the measurement of radioactivity associated with [14C]phosphocholine. The enzyme activity was dependent on the concentration of sphingomyelin, enzyme, pH, and temperature. Thirty minutes of incubation time was optimal for enzyme activity. This enzyme had a bimodal pH optima, in that optimum enzyme activity was measured at pH 5.4 and 7.4. The detection of sphingomyelinase was pursued by separating the enzyme on a polyacrylamide gel and carrying out the enzyme assay by exposure to [14C]sphingomyelin blotted on a PVDF membrane. The enzyme activity on the PVDF membrane was visualized by autoradiography. A white band (depicting hydrolytic removal of [14C]sphingomyelin from PVDF was observed. Our method of detecting sphingomyelinase by immobilizing sphingomyelin on PVDF membrane may serve as a prototype for assaying various other enzymes in which the hydrolytic product is released into the aqueous phase. Moreover, our method for detecting sphingomyelinase on polyacrylamide gels may be helpful in further studies on the molecular biochemistry of this and related phospholipases.