Quantitation of S-adenosylmethionine decarboxylase protein.
Quantitation of S-adenosylmethionine decarboxylase protein.
复制标题
S-腺苷甲硫氨酸脱羧酶蛋白的定量。
DOI：
10.1021/bi00337a024
复制
发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
Pegg,AE
中科院分区：
文献类型：
--
作者：
Shirahata,A;Christman,KL;Pegg,AE
建立了一种特异性标记S-腺苷甲硫氨酸脱羧酶活性位点的方法。该方法包括在亚精胺合酶抑制剂存在下，将细胞提取物与3H-脱羧S-腺苷甲硫氨酸和氰基硼氢化钠一起孵育。在这些条件下，S-腺苷甲硫氨酸脱羧酶标记特异性和化学计量。该方法用于（a）确定来自大鼠肝、前列腺和腰肌以及来自小鼠SV-3T3细胞的S-腺苷甲硫氨酸脱羧酶的亚单位分子量为32000，（B）滴定各种细胞提取物中S-腺苷甲硫氨酸脱羧酶的活性分子的数量，和（c）提供用于该酶的放射免疫测定的S-腺苷甲硫氨酸脱羧酶的高比活性标记制剂。竞争性放射免疫分析法使用这种标记抗原具有灵敏度，这样，3 fmol（0.1 ng）的酶蛋白可以定量。当SV-3T3细胞暴露于外源性多胺时，S-腺苷甲硫氨酸脱羧酶的迅速丧失被证明是由于活性位点滴定和放射免疫测定的酶蛋白量的迅速下降。哺乳动物细胞中多胺的生物合成和相互转化是高度调节的过程，并且参与该途径的几种酶在广泛的生理条件下表现出显著的活性波动（Janne等人，1978; Pegg & McCann，1982;塔博尔&塔博尔，1984 a）。这些酶[鸟氨酸脱羧酶、亚精胺/精胺N ′-乙酰转移酶和S-腺苷-t]这项工作得到了美国国立卫生研究院研究赠款1P30 CA 18450和CA 18137的部分支持。来信请寄宾夕法尼亚州立大学生理学系。甲硫氨酸脱羧酶（METDC）]即使在最大诱导后也以非常少量存在（Pegg等，1982年）。因此，研究酶活性变化的机制所需的蛋白质量的定量估计方法一直难以开发。最近，通过使用通过与放射性α-（二氟甲基）鸟氨酸（DFMO）反应化学计量地标记酶的技术，在理解鸟氨酸脱羧酶的调节方面取得了进展（Pritchard等人，1981年）。这种标记已被用于滴定细胞提取物中存在的酶分子的数量，以在细胞提取后鉴定蛋白质。
A method for the specific labeling of the active site of S-adenosylmethionine decarboxylase was developed. The method consisted of incubating cell extracts with 3H-decarboxylated S-adenosylmethionine and sodium cyanoborohydride in the presence of a spermidine synthase inhibitor. Under these conditions, S-adenosylmethionine decarboxylase was labeled specifically and stoichiometrically. This procedure was used (a) to establish that the subunit molecular weight of S-adenosylmethionine decarboxylase from rat liver, prostate, and psoas and from mouse SV-3T3 cells was 32000,(b) to titrate the number of active molecules of S-adenosylmethionine decarboxylase in various cell extracts, and (c) to provide a high specific activity labeled preparation of S-adenosylmethionine decarboxylase for use in radioimmunoassay of this enzyme. Competitive radioimmunoassays using this labeled antigenhad a sensitivity such that 3 fmol (0.1 ng) of enzyme protein could be quantitated. The rapid loss of S-adenosylmethionine decarboxylase which occurred when SV-3T3 cells were exposed to exogenous polyamines was shown to be due to a rapid decline in theamount of enzyme protein measured both by titration of the active site and by radioimmunoassay. e biosynthesis and interconversion of polyamines in mammalian cells are highly regulated processes, and several of the enzymes involved in this pathwayexhibit remarkable fluctuations in activity under a wide range of physiological conditions (Janne et al., 1978; Pegg & McCann, 1982; Tabor & Tabor, 1984a). These enzymes [ornithine decarboxylase, spermidine/spermine TV'-acetyltransferase, and S-adenosyl-tThis work was supported in part by Research Grants 1P30 CA 18450 and CA 18137 from the National Institutes of Health.* Correspondence should be addressed to this author at the Depart-ment of Physiology, The Pennsylvania State University. methionine decarboxylase (AdoMetDC)] are present in very small amounts even after maximal induction (Pegg et al., 1982). Therefore, methods for the quantitative estimation of the amount of protein, which are needed to investigate the mechanism by which changes in enzyme activity are brought about, have been difficult to develop. Recently, advances in the understanding of the regulationof ornithine decarboxylase have been made by the use of a technique in which the enzyme is labeled stoichiometrically by reaction with radioactive a-(difluoromethyl) ornithine (DFMO)(Pritchard et al., 1981). Such labeling has been used to titrate the number of enzyme molecules present in cell extracts, to identify the protein after