Abstract
Several enzymes involved in the phosphoinositide metabolism have been shown to be present in nuclei of rat liver and Friend cells. In this paper we demonstrate that nuclear matrices of mouse NIH 3T3-fibroblasts and rat liver cells, isolated by nuclease treatment and high salt extraction, contain phosphatidylinositol 4-kinase (PdtIns 4-kinase), phosphatidylinositol 4-phosphate 5-kinase (PtdIns(4)P 5-kinase), diacylglycerol kinase, and phospholipase C. By a selective extraction the nucleus can be dissected in the peripheral matrix (lamina-pore complex) and the internal matrix as shown by using marker antibodies. Surprisingly, PtdIns 4-kinase was found exclusively in the peripheral nuclear matrix, whereas PtdIns(4)P 5-kinase was found to be associated to internal matrix structures. Diacylglycerol kinase and phospholipase C activities were also preferentially detected in the internal matrix. These data demonstrate a differential localization of the phosphoinositide kinases in the nucleus and suggest that the phosphoinositide metabolism may play a specific role in the nucleus.
Highlights
Several enzymes involved in the phosphoinositide have been demonstrated to activate in vitro the low specific metabolism have been shown to be present in nuclei of activity form of DNA polymerase a [5], while an inositol rat liverand Friend cells
In this study we have investigated in more detail the association of enzymes involved in the phosphoinositide metabolism to the nuclear matrix
We demonstrate in this paper that the nuclear matrix of mammalian cells contains phosphoinositide kinase, DAG kinase,and PLC activities
Summary
Zsolation of Nuclei-Membrane depleted nuclei from mouse NIH 3T3-fibroblasts were isolated as described under "Experimental Procedures." Purity of these nuclei was analyzed using different enzyme markers as LDH activity as cytosolic marker, 5"nucleotidase as plasma membrane marker, and Phosphoinositide Metabolism and Nuclear Matrices antimycine A-insensitive NADH-cytochrome c reductase as nuclei isolation.For this purposwee chose rat liver cells which marker for the endoplasmic reticulum. Incubationof lipid memthan 95% of cellular proteinsand more than 98% of the brane-free nucleiisolatedfrom NIH3T3fibroblasts with nucleic acids are removed [42] The nuclear matrix was still able to phosnuclear proteins laminA and C and p160 are concentratedby phorylate theexogenous lipids (Fig. 2, lane M ) .Similar results this extraction. By isolating nuclei in the Kinase activities in nuclei isolated from NIH 3T3 cells was absence of Triton X-100 we could check the effect of this 3.1 f0.8% for the PtdIns kinase1, 3.7 f2.1% for the PtdInsP detergenton a possible reallocation of thekinasesduring kinase, and 9.6 f 2.2% for the DAG kinase. Obtained in thisway were 1.9 +: 0.2% and 2.5 f 0.1% of total cellular protein for the peripheral and internnaulclear matrix,
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