POLYAMINE-MEDIATED TURNOVER OF ORNITHINE DECARBOXYLASE IN CHINESE-HAMSTER OVARY CELLS
POLYAMINE-MEDIATED TURNOVER OF ORNITHINE DECARBOXYLASE IN CHINESE-HAMSTER OVARY CELLS
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DOI：
10.1042/bj2360351
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发表时间：
1986-06-01
影响因子：
4.1
通讯作者：
GERNER, EW
中科院分区：
文献类型：
--
作者：
GLASS, JR;GERNER, EW
本文用化学成分确定的培养基培养的中国仓鼠卵巢（CHO）细胞，研究了多胺对鸟氨酸脱羧酶（ODC）的调节作用。维持在限定培养基中的细胞没有可检测的腐胺，并且约为100%。1-3单位的ODC活性/106个细胞，其中1个单位对应于1纳摩尔的底物脱羧在30分钟。确定的培养基是鸟氨酸缺乏，从而限制了ODC的外源性底物，并随后减少细胞内多胺的积累。通过加入外源性鸟氨酸或腐胺，细胞内腐胺的恢复和亚精胺形成的增加导致ODC活性的显著降低，这被α-氨基葡萄糖的降低所抵消。DL-二氟甲基[3，4 - 3 H]鸟氨酸（DFMO）结合蛋白的Mr约为53，000，其可用抗ODC抗体沉淀。每单位活性的DFMO结合的计算显示酶的比活性没有变化。我们确定了[35 S]蛋氨酸标记的肽对应于ODC的放射性标记的全细胞蛋白的免疫沉淀。只有一种蛋白质被沉淀，大约先生。53，000，其与DFMO结合蛋白共迁移。免疫沉淀的放射性标记的蛋白质从细胞中孵育的存在下，外源性鸟氨酸表明，所观察到的活性降低是不是由于抑制ODC蛋白质的合成。对来自用[35 S]甲硫氨酸脉冲标记，然后用100 μ M-鸟氨酸、-Purtescine或-亚精胺处理5小时的细胞的免疫可沉淀ODC蛋白的分析显示，在细胞内多胺库恢复后，标记的ODC蛋白明显消失。在没有外源性多胺处理的情况下，没有观察到ODC的可检测的营业额。这些数据支持的假设，ODC蛋白，和随后的活动，是由细胞内多胺含量通过影响营业额的酶的机制进行调节。
We have used Chinsee-hamster ovary (CHO) cells maintained in a chemically defined mediumn to study the regulation of ornithine decarboxylase (ODC) by polyamines. Cells maintained in the defined medium had no detectable putrescine, and approx. 1-3 units of ODC activity/106 cells, where 1 unit corresponds to 1 nmol of substrate decarboxylated in 30 min. The defined medium is ornithine-deficient, thus limiting the exogenous substrate for ODC, and subsequently decreasing intracellular polyamine accumulation. Restoration of intracellular putrescine and increased formation of spermidine by addition of exogenous ornithine or putescine led to a marked decrease in ODC activity, which was paralleled by a decrease in a .alpha.-DL-difluoromethyl[3,4-3H]ornithine (DFMO)-binding protein of Mr approx. 53,000, which is precipitable with anti-ODC antibody. Calculation of DFMO binding per unit of activity showed no change in the specific activity of the enzyme. We identified [35S]methionine-labelled peptides corresponding to ODC by immunoprecipitation of radiolabeled whole cell proteins. Only one protein was precipitated, of Mr approx. 53,000, which co-migrated with the DFMO-binding protein. Immunoprecipitation of radiolabelled proteins from cells incubated in the presence of exogenous ornithine indicted that the observed activity decrease was not due to an inhibition of ODC protein synthesis. Analysis of immunoprecipitable ODC protein from cells that had been pulse-labelled with [35S]methionine, and then treated for 5 h with 100 .mu.M-ornithine, -purtescine or -spermidine, revealed a distinct disappearance of labelled ODC protein after restoration of intracellular polyamine pools. No detectable turnover of ODC was observed in the absence of exogenous polyamine treatment. These data support the hypothesis that ODC protein, and subsequent activity, is regulated by intracellular polyamine content through mechanisms that influence turnover of the enzyme.