Measurement of the number of ornithine decarboxylase molecules in rat and mouse tissues under various physiological conditions by binding of radiolabelled alpha-difluoromethylornithine.
Measurement of the number of ornithine decarboxylase molecules in rat and mouse tissues under various physiological conditions by binding of radiolabelled alpha-difluoromethylornithine.
复制标题
通过结合放射性标记的α-二氟甲基鸟氨酸，测量各种生理条件下大鼠和小鼠组织中鸟氨酸脱羧酶分子的数量。
DOI：
10.1042/bj2060311
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发表时间：
1982
期刊：
影响因子：
--
通讯作者：
Pegg,AE
中科院分区：
文献类型：
--
作者：
Seely,JE;Pösö,H;Pegg,AE
利用不可逆抑制剂 α-二氟甲基鸟氨酸与鸟氨酸脱羧酶的结合来研究不同条件下大鼠肝脏中以及雄激素处理后小鼠肾脏中存在的酶量。在磷酸吡哆醛存在下，将组织提取物与 3μm-二氟甲基[5-14C]鸟氨酸一起孵育 60 分钟，药物的最大结合发生。在这些条件下，只有一种蛋白质被标记，并且这对应于鸟氨酸脱羧酶，具有约100000和亚基约55000。用硫代乙酰胺或四氯化碳或通过部分肝切除术治疗大鼠产生了鸟氨酸脱羧酶活性的显着增加以及根据二氟甲基[5-14C]鸟氨酸的结合程度确定的酶蛋白量的平行增加。类似地，用放线菌酮或1,3-二氨基丙烷处理大大降低了酶活性和与蛋白质结合的二氟甲基-[5-14C]鸟氨酸的量。在所有情况下，药物与活性的结合比为26fmol/单位，其中1单位对应于30分钟内脱羧的1nmol底物。这些结果表明，即使在大鼠肝脏中最大程度地诱导酶后，每毫克蛋白质中也仅存在约 1 纳克的酶。当用雄激素治疗小鼠时，肾脏鸟氨酸脱羧酶活性显着增加，其幅度取决于菌株。小鼠肾脏提取物中存在的活性量和结合标记的 α-二氟甲基鸟氨酸的能力之间存在极好的对应关系，但在这种情况下，药物与活性的结合比率为 14fmol/单位，表明小鼠酶具有更高的催化中心活性。雄激素诱导后，小鼠肾脏提取物含有约 170 纳克酶/毫克蛋白质。这些结果表明，用 α-二氟甲基鸟氨酸滴定提供了一种有价值的方法来定量哺乳动物组织中存在的活性鸟氨酸脱羧酶的量，并且雄激素处理的小鼠肾脏是比大鼠肝脏更好的酶纯化来源。
The binding of α-difluoromethylornithine, an irreversible inhibitor, to ornithine decarboxylase was used to investigate the amount of enzyme present in rat liver under various conditions and in mouse kidney after treatment with androgens. Maximal binding of the drug occurred on incubation of the tissue extract for 60min with 3μm-difluoromethyl[5-14C]ornithine in the presence of pyridoxal phosphate. Under these conditions, only one protein became labelled, and this corresponded to ornithine decarboxylase, havingMrabout 100000 and subunitMrabout 55000. Treatment of rats with thioacetamide or carbon tetrachloride or by partial hepatectomy produced substantial increases in ornithine decarboxylase activity and parallel increases in the amount of enzyme protein as determined by the extent of binding of difluoromethyl[5-14C]ornithine. Similarly, treatment with cycloheximide or 1,3-diaminopropane greatly decreased both the enzyme activity and the amount of difluoromethyl-[5-14C]ornithine bound to protein. In all cases, the ratio of drug bound to activity was 26fmol/unit, where 1 unit corresponds to 1nmol of substrate decarboxylated in 30min. These results indicate that even after maximal induction of the enzyme in rat liver there is only about 1ng of enzyme present per mg of protein. When mice were treated with androgens there was a substantial increase in renal ornithine decarboxylase activity, the magnitude of which depended on the strain. There was an excellent correspondence between the amount of activity present and the capacity to bind labelled α-difluoromethylornithine in the mouse kidney extracts, but in this case the ratio of drug bound to activity was 14fmol/unit, suggesting that the mouse enzyme has a higher catalytic-centre activity. After androgen induction, the mouse kidney extracts contain about 170ng of enzyme/mg of protein. These results indicate that titration with α-difluoromethylornithine provides a valuable method by which to quantify the amount of active ornithine decarboxylase present in mammalian tissues, and that the androgen-treated mouse kidney is a much better source for purification of the enzyme than is rat liver.