TOPOISOMERASE-I PHOSPHORYLATION INVITRO AND IN RAPIDLY GROWING NOVIKOFF HEPATOMA-CELLS
TOPOISOMERASE-I PHOSPHORYLATION INVITRO AND IN RAPIDLY GROWING NOVIKOFF HEPATOMA-CELLS
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DOI：
10.1002/j.1460-2075.1985.tb04024.x
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发表时间：
1985-01-01
期刊：
影响因子：
11.4
通讯作者：
BUSCH, H
中科院分区：
文献类型：
--
作者：
DURBAN, E;GOODENOUGH, M;BUSCH, H
在体外，磷酸化的变化调节拓扑异构酶I的活性。具体来说，酶活性是通过纯化的蛋白激酶（酪蛋白激酶II型）磷酸化来刺激的。本研究的目的是比较快速生长的诺维科夫肝癌细胞中酪蛋白激酶II型在体外磷酸化的位点与体内磷酸化的位点。体外标记的拓扑异构酶I由三种主要的色氨酸磷酸肽（I- iii）表征。用c18 -反相高效液相色谱柱分离这些肽，分别以17%、22.5%和33%的乙腈在18 (I)、27 （II）和44 （III）馏分洗脱。而在体内标记的拓扑异构体中，hp只鉴定出一个主要的磷酸肽。这个磷酸肽在18段被洗脱，与我在体外标记的磷酸肽的洗脱特性相对应。当在薄层纤维素板上进行高压电泳时，它也与色氨酸磷酸肽I共迁移。初步实验表明，磷酸化发生在距离肽n端6个氨基酸的丝氨酸残基上。这些数据表明，拓扑异构酶I在体内和体外在同一种胰蛋白酶中被磷酸化，并且表明拓扑异构酶I在体内被酪蛋白激酶II磷酸化。
Changes in phosphorylation modulate the activity of topoisomerase I in vitro. Specifically, enzymatic activity is stimulated by phosphorylation with a purified protein kinase (casein kinase type II). The purpose of this study was to compare the sites that are phosphorylated in vitro by casein kinase type II with site(s) phosphorylated in vivo in rapidly growing Novikoff hepatoma cells. Topoisomerase I labeled in vitro was characterized by three major tryptic phosphopeptides (I-III). Separation of these peptides by a C18-reverse phase h.p.l.c. column resulted in their elution at fractions 18 (I), 27 (II) and 44 (III) with 17%, 22.5% and 33% acetonitrile, respectively. In contrast, only one major phosphopeptide was identified by h.p.l.c. in topoisomerae I labeled in vivo. This phosphopeptide eluted at fraction 18 corresponding to the elution properties of phosphopeptide I labeled in vitro. It also co-migrated with tryptic phosphopeptide I when subjected to high-voltage electrophoresis on thin-layer cellulose plates. Preliminary experiments suggest that phosphorylation occurs at a serine residue six amino acids from the N-terminus of the peptide. These data indicate that topoisomerase I is phosphorylated in vivo and in vitro within the same tryptic peptide and suggest that topoisomerase I is phosphorylated in vivo by casein kinase II.