Seely J E, Pösö H, Pegg A E
Biochem J. 1982 Aug 15;206(2):311-8. doi: 10.1042/bj2060311.
The binding of alpha-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 3mum-difluoromethyl[5-(14)C]ornithine in the presence of pyridoxal phosphate. Under these conditions, only one protein became labelled, and this corresponded to ornithine decarboxylase, having M(r) about 100000 and subunit M(r) about 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-(14)C]ornithine. Similarly, treatment with cycloheximide or 1,3-diaminopropane greatly decreased both the enzyme activity and the amount of difluoromethyl-[5-(14)C]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 alpha-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 alpha-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.
不可逆抑制剂α-二氟甲基鸟氨酸与鸟氨酸脱羧酶的结合被用于研究在各种条件下大鼠肝脏中以及用雄激素处理后的小鼠肾脏中存在的酶量。将组织提取物与3μmol - 二氟甲基[5-(14)C]鸟氨酸在磷酸吡哆醛存在下孵育60分钟时，药物的结合达到最大值。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，其相对分子质量（M(r)）约为100000，亚基相对分子质量约为55000。用硫代乙酰胺或四氯化碳处理大鼠或进行部分肝切除术后，鸟氨酸脱羧酶活性显著增加，并且通过二氟甲基[5-(14)C]鸟氨酸的结合程度测定的酶蛋白量也相应增加。同样，用环己酰亚胺或1,3 - 二氨基丙烷处理大大降低了酶活性以及与蛋白质结合的二氟甲基[5-(14)C]鸟氨酸的量。在所有情况下，药物结合量与活性的比率为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，即使在大鼠肝脏中酶被最大程度诱导后，每毫克蛋白质中也仅存在约1ng酶。当用雄激素处理小鼠时，肾脏鸟氨酸脱羧酶活性显著增加，其增加幅度取决于品系。在小鼠肾脏提取物中，存在的活性量与结合标记的α-二氟甲基鸟氨酸的能力之间存在良好的对应关系，但在这种情况下，药物结合量与活性的比率为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，小鼠肾脏提取物每毫克蛋白质中含有约170ng酶。这些结果表明，用α-二氟甲基鸟氨酸进行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，并且用雄激素处理的小鼠肾脏是比大鼠肝脏更好的酶纯化来源。
