PERTINENCE OF NUCLEAR-ENVELOPE NUCLEOSIDE TRIPHOSPHATASE-ACTIVITY TO RIBONUCLEIC-ACID TRANSPORT
PERTINENCE OF NUCLEAR-ENVELOPE NUCLEOSIDE TRIPHOSPHATASE-ACTIVITY TO RIBONUCLEIC-ACID TRANSPORT
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DOI：
10.1021/bi00553a033
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发表时间：
1980-01-01
期刊：
影响因子：
2.9
通讯作者：
SMUCKLER, EA
中科院分区：
文献类型：
--
作者：
CLAWSON, GA;JAMES, J;SMUCKLER, EA
用不同方法从纯化的大鼠和小牛肝细胞核中分离核膜。核膜的基本表征表明磷脂分布与微粒体中发现的那些相似，但磷脂酰丝氨酸的酶组成和脂肪酸部分与微粒体中的那些不同。大量的Mg 2+依赖的NTR [核苷三磷酸酶]被发现在核膜从这两个来源。活性与蛋白质浓度呈线性关系，并显示出尖锐的pH依赖性，最大活性接近pH 7.5。在大鼠肝脏制剂中的活性的Arrhenius分析揭示了13.8 kcal/mol的活化能，Lineweaver-Burk图显示Km为1.8 mM ATP。在类似的条件下，小牛肝脏制剂显示出13.3千卡/摩尔的活化能和1.9 mM ATP的Km;与Mg 2+添加5 mM过量（超过核苷酸浓度），他们产生线性Eadie图。两种来源的核膜中的NTR活性显示出广泛的底物特异性，并诱导各种核苷酸添加剂的能荷下降，这些添加剂抑制了RNA转运的体外刺激。两种来源的核膜都能水解二磷酸核苷酸的高能磷酸键。利用ADP的能力并不依赖于偶联氧化磷酸化或电子传递，它显然是通过一种类似肌激酶的活性，使ATP水解。在体外修饰RNA转运的试剂类似地修饰NTR的活性。cAMP增加RNA转运和NTR酶活性，进一步研究表明cAMP使NTR酶活性的Km略有增加，Vmax增加65%。进一步的研究表明，在体内的四氯化碳或硫代乙酰胺治疗大鼠的RNA转运在体内和核膜NTR活性的变化之间的平行性。组织化学研究表明，NTR的活性分布沿着核膜，而不是本地化的核孔在所采用的条件下。硫代乙酰胺诱导的核肿胀产生核膜表面积的变化，这平行增加了由该处理产生的核膜NTR活性。之间的相互作用的NTT活性在核膜和RNA运输方面的底物的行为，激活剂和抑制剂的影响，并在体内治疗引起的扰动，表明这种活动参与RNA运输。
Nuclear envelopes were isolated from purified rat and calf liver nuclei via different methods. Basic characterization of nuclear envelopes demonstrated phospholipid distributions similar to those found in microsomes, but enzymatic compositions and fatty acid moieties of phosphatidylserine differed from those in microsomes. A substantial Mg2+-dependent NTPase [nucleoside triphosphatase] was found in nuclear envelopes from both sources. The activity was linear with protein concentration and showed a sharp pH dependency with maximal activity near pH 7.5. Arrhenius analysis of the activity in rat liver preparations disclosed an activation energy of 13.8 kcal/mol, and Lineweaver-Burk plots showed a Km of 1.8 mM ATP. Under similar conditions, calf liver preparations showed an activation energy of 13.3 kcal/mol and a Km of 1.9 mM ATP; with Mg2+ added in 5 mM excess (over nucleotide concentration) they yielded linear Eadie plots. The NTPase activity in nuclear envelopes from both sources showed a broad substrate specificity, and induced declines in the energy charge of various nucleotide additives that paralleled stimulation of RNA transport in vitro by these additives. Nuclear envelopes from both sources were able to hydrolyze the high-energy phosphate bonds of diphosphate nucleotides. The ability to utilize ADP was not dependent on coupled oxidative phosphorylation or on electron transport; it apparently proceeds via a myokinase-like activity that furnishes ATP. Agents that modify RNA transport in vitro similarly modified the NTPase activity. Cyclic[c]AMP increases RNA transport and the NTPase activity, and further investigation showed that cAMP increased the Km of the NTPase activity slightly and the Vmax by 65%. Further studies in vivo following CCl4 or thioacetamide treatment of rats demonstrated a parallelism between alterations in RNA transport in vivo and nuclear envelope NTPase activity. Histochemical studies demonstrated that the NTPase activity was distributed along the nuclear envelope and was not localized to nuclear pores under the conditions employed. Thioacetamide-induced nuclear swelling produces changes in nuclear envelope surface area, which parallel increases in the nuclear envelope NTPase activity produced by this treatment. The reciprocity between the NTPase activity in nuclear envelopes and RNA transport with regard to substrate behavior, and to effects of activators and inhibitors, and to perturbations induced by in vivo treatments, suggests that this activity participates in RNA transport.