Metabolism and possible compartmentalization of inositol lipids in isolated rat-liver nuclei.
Metabolism and possible compartmentalization of inositol lipids in isolated rat-liver nuclei.
复制标题
分离的大鼠肝核中肌醇脂质的代谢和可能的区室化。
作者：
R. Vann;P. Wooding;F. Irvine;N. Divecha
(1)用Triton X-100（TX-100）清洗离体大鼠肝细胞核，通过电子显微镜观察核膜的去除情况。0.04%（w/v）TX-100可去除所有包膜。(2)在这种去除之后，尽管几乎完全不存在可检测的（通过质量测定）DAG和PtdIn，但是仍然发生来自[γ-32 P]ATP的肌醇脂质和二酰基甘油（DAG）的磷酸化。这表明核中这两种脂质的大部分存在于核膜中，但提取后剩余的少量（定义为核内）可用于脂质激酶的磷酸化（DAG为36%，PtdIns为24%，以完整核的掺入百分比表示）。(3)PtdIns（4，5）P2不遵循与PtdIns和DAG相同的模式;在去除核膜后，40%的这种脂质质量留在细胞核中。此外，类似量的PtdIns（4，5）P2也抵抗用甚至更高浓度的去污剂提取，表明PtdIns（4，5）P2具有离散的核内位置，可能与核蛋白结合。(4)外源性底物PtdIns、PtdIns（4）P和DAG添加到膜耗尽的细胞核中导致[γ-32 P]ATP的大部分脂质磷酸化重建（分别为完整细胞核的70%、90%和94%），表明相应激酶主要位于核内。(5)细胞核还显示磷酸单酯酶和磷脂酸水解酶活性;当[γ-32 P]ATP被去除时，观察到放射性标记前的PtdIns（4）P、PtdIns（4，5）P2和磷脂酸的去磷酸化。然而，一些放射性明显对这些酶具有抗性，表明存在多个这些脂质池。(6)将过量的非放射性标记的ATP添加到用[γ-32 P]ATP预标记的细胞核中，导致PtdIns（4，5）P2标记的初始增加，这意味着PtdIns（4）P和PtdIns（4，5）P2的放射性标记池之间存在标记物-产物关系。这通过分析32 P掺入PtdIns（4）P的4 '-磷酸基团和PtdIns（4，5）P2的单个4'-和5 '-磷酸基团得到证实。来自这些实验的数据还表明，PtdIns（4，5）P2可以从预先存在的PtdIns（4）P库产生，以及从PtdIns从头产生。(7)综上所述，我们的数据表明，离体大鼠肝细胞核有一个核内肌醇脂质代谢机制，利用酶和底物中发现的细胞质和质膜，可能有一些，但不完全，区室化的核肌醇循环的组成部分。
(1) The removal of the nuclear envelope from isolated rat-liver nuclei by washing with Triton X-100 (TX-100) was assessed by electron microscopy. All the envelope was removed by 0.04% (w/v) TX-100. (2) After this removal, phosphorylation of inositol lipids and diacylglycerol (DAG) from [gamma-32P]ATP still occurs, despite the near complete absence of detectable (by mass assay) DAG and PtdIns. This suggests that the majority of these two lipids in nuclei are present in the nuclear membrane, but the small amounts remaining after extraction, defined as intranuclear, are available for phosphorylation by lipid kinases (36% for DAG and 24% for PtdIns respectively, when expressed as a percentage of incorporation of intact nuclei). (3) PtdIns(4,5)P2 did not follow the same pattern as PtdIns and DAG; after removal of the nuclear membrane, 40% of the mass of this lipid was left in the nucleus. Moreover, a similar amount of PtdIns(4,5)P2 was also resistant to extraction with even higher concentrations of detergent, suggesting that PtdIns(4,5)P2 has a discrete intranuclear location, probably bound to nuclear proteins. (4) Addition of exogenous substrates, PtdIns, PtdIns(4)P and DAG, to membrane-depleted nuclei resulted in reconstitution of the majority of lipid phosphorylations from [gamma-32P]ATP (70%, 90% and 94% of intact nuclei respectively), suggesting a predominantly intranuclear location for the respective kinases. (5) Nuclei also showed phosphomonoesterase and phosphatidic acid hydrolase activity; dephosphorylation of pre-radiolabelled PtdIns(4)P, PtdIns(4,5)P2 and phosphatidic acid was observed when [gamma-32P]ATP was removed. However, some of the radioactivity was apparently resistant to these enzymes, suggesting the existence of multiple pools of these lipids. (6) Addition of excess non-radiolabelled ATP to nuclei pre-labelled with [gamma-32P]ATP resulted in an initial increase in the label in PtdIns(4,5)P2, implying a precursor-product relationship between the radiolabelled pools of PtdIns(4)P and PtdIns(4,5)P2. This was confirmed by analysis of the incorporation of 32P into the 4'-phosphate group of PtdIns(4)P and the individual 4'- and 5'-phosphate groups of PtdIns(4,5)P2. The data from these experiments also indicated that PtdIns(4,5)P2 can be produced from a pre-existing pool of PtdIns(4)P, as well as de novo from PtdIns. (7) Taken together our data suggest that isolated rat-liver nuclei have an intranuclear inositol lipid metabolism mechanism utilizing enzymes and substrates equivalent to those found in cytosol and plasma membrane, and that there may be some, but not complete, compartmentalization of the components of the nuclear inositol cycle.
人红细胞含有两种形式的 4-磷酸磷脂酰肌醇 5-激酶，它们对细胞膜的活性不同。
DOI：
--
发表时间：
1990
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Bazenet,CE;Ruano,AR;Brockman,JL;Anderson,RA
通讯作者：
Anderson,RA
α-凝血酶诱导的核 sn-1,2-二酰甘油源自培养的成纤维细胞中的磷脂酰胆碱水解。
影响因子：
2.9
作者：
Jarpe,MB;Leach,KL;Raben,DM
通讯作者：
Raben,DM