Identification of two cytosolic diacylglycerol kinase isoforms in rat brain, and in NIH-3T3 and ras-transformed fibroblasts.
Identification of two cytosolic diacylglycerol kinase isoforms in rat brain, and in NIH-3T3 and ras-transformed fibroblasts.
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鉴定大鼠脑以及 NIH-3T3 和 ras 转化的成纤维细胞中的两种胞质二酰甘油激酶亚型。
DOI：
10.1042/bj2720569
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发表时间：
1990
期刊：
影响因子：
--
通讯作者：
Macara,IG
中科院分区：
文献类型：
--
作者：
Stathopoulos,VM;Coco-Maroney,A;Wei,CW;Goth,M;Zaricznyj,C;Macara,IG
通过肝素-琼脂糖色谱法从脑胞质溶胶和NIH-3 T3或ras转化的3 T3细胞中分离出两种主要的二酰基甘油激酶（I型和II型）。还通过非变性等电聚焦检测到多种二酰基甘油激酶。两个活性峰大小相似，均在约95 kDa处从Superose f.p.l.c.柱II型酶（PI 8.0）是更活跃的底物时，在脱氧胆酸盐/磷脂酰丝氨酸未定义的环境，而不是辛基葡糖苷/磷脂酰丝氨酸胶束环境。II型活性也增强磷脂酰胆碱作为辅因子的存在。I型酶（PI 4.0）是更活跃的存在下，无论是磷脂酰丝氨酸或磷脂酰肌醇。I型和II型酶具有不同的ATP亲和力。这两种酶都显示出对具有10-12个碳原子的饱和酰基链的二酰基甘油底物的偏好。胞浆酶活性能够结合NIH-3 T3成纤维细胞中富含二酰基甘油的膜，而这种转运在ras转化的3 T3细胞中不受影响。这些结果表明，脑细胞质和NIH-3 T3和ras转化的3 T3细胞中存在多种二酰基甘油激酶。这些酶在辅因子、ATP和底物需求方面不同。这些结果可以解释一些以前的研究之间的矛盾，胞质甘油二酯激酶活性，并建议存在一个家庭的这种激酶的差异调节磷脂辅因子。
Two major species of diacylglycerol kinase (type I and type II) were separated from brain cytosol and from NIH-3T3 or ras-transformed 3T3 cells by heparin-agarose chromatography. Multiple species of diacylglycerol kinase were also detected by non-denaturing isoelectric focusing. The two peaks of activity were of similar size, both co-eluted at approximately 95 kDa from a Superose f.p.l.c. column. Type II enzyme (pI 8.0) was more active when substrate was presented in a deoxycholate/phosphatidylserine undefined environment, as opposed to an octyl glucoside/phosphatidylserine micellar environment. Type II activity was also enhanced by the presence of phosphatidylcholine as cofactor. Type I enzyme (pI 4.0) was more active in the presence of either phosphatidylserine or phosphatidylinositol. Type I and II enzymes had different ATP affinities. Both enzymes showed a preference for diacylglycerol substrates with saturated acyl chains of 10-12 carbon atoms. The cytosolic enzyme activity was able to bind to diacylglycerol-enriched membranes in NIH-3T3 fibroblasts, and this translocation was unaffected in ras-transformed 3T3 cells. These results demonstrate the presence of multiple diacylglycerol kinases in brain cytosol and NIH-3T3 and ras-transformed 3T3 cells. The enzymes differ in cofactor, ATP and substrate requirements. These results can explain some of the contradictions between previous studies of cytosolic diacylglycerol kinase activity, and suggest the presence of a family of such kinases that are differentially regulated by phospholipid cofactors.