结肠细胞和红细胞膜中的硫醇甲基转移酶活性。
Thiol methyltransferase activity in colonocytes and erythrocyte membranes.
作者信息
Babidge W J, Millard S H, Roediger W E
机构信息
Department of Surgery, Queen Elizabeth Hospital, Adelaide, Australia.
出版信息
J Clin Pathol. 1995 Jul;48(7):641-4. doi: 10.1136/jcp.48.7.641.
AIMS
To verify the improved thiol methyltransferase (TMT) assay and measure activity in isolated colonocytes and erythrocyte membranes of the same subjects.
METHODS
High performance liquid chromatography with radioactivity detection was used to measure 14C-methylmercaptoethanol formation, the reaction product of cell extracts incubated with mercaptoethanol and 14C-S-adenosylmethionine.
RESULTS
Verification of radiolabelled 14C-methylmercaptoethanol was by exogenous addition of methylmercaptoethanol and simultaneous ultraviolet detection at 214 nm. Using a substrate concentration of 10 mM mercaptoethanol, the Km for S-adenosylmethionine was 25 microM. The sensitivity of the radioactive method was 2 pmol, with coefficients of variation of 7% within assay and 6.4% between assay. TMT activities (mean +/- SE; n = 17) were 471 +/- 64 pmol/hour/mg protein for colonocytes and 73 +/- 7 pmol/hour/mg protein for erythrocyte membranes.
CONCLUSIONS
The direct assay of TMT activity is sensitive, specific and eliminates concern over non-enzymatic methylation of thiol compounds. High activities in colonic epithelial cells deserve evaluation in disease states.
目的
验证改进的硫醇甲基转移酶（TMT）检测方法，并测定同一受试者分离的结肠细胞和红细胞膜中的活性。
方法
采用放射性检测的高效液相色谱法测定14C-甲基巯基乙醇的形成，14C-甲基巯基乙醇是细胞提取物与巯基乙醇和14C-S-腺苷甲硫氨酸孵育后的反应产物。
结果
通过外源添加甲基巯基乙醇并在214nm处同时进行紫外检测来验证放射性标记的14C-甲基巯基乙醇。使用10mM巯基乙醇的底物浓度时，S-腺苷甲硫氨酸的Km为25μM。放射性方法的灵敏度为2pmol，批内变异系数为7%，批间变异系数为6.4%。结肠细胞的TMT活性（平均值±标准误；n = 17）为471±64pmol/小时/毫克蛋白，红细胞膜的TMT活性为73±7pmol/小时/毫克蛋白。
结论
TMT活性的直接检测方法灵敏、特异，消除了对硫醇化合物非酶甲基化的担忧。结肠上皮细胞中的高活性值得在疾病状态下进行评估。
Zotero 插件