(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 [γ-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 [γ-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 [γ-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 [γ-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 4ʹ-phosphate group of PtdIns(4)P and the individual 4ʹ- and 5ʹ-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.
Skip Nav Destination
Article navigation
October 1997
-
Cover Image
Cover Image
- PDF Icon PDF LinkFront Matter
- PDF Icon PDF LinkTable of Contents
Research Article|
October 15 1997
Metabolism and possible compartmentalization of inositol lipids in isolated rat-liver nuclei
R. Lewis VANN;
R. Lewis VANN
1
*Department of Signalling, Babraham Institute, Babraham, Cambridge CB2 4AT, U.K.
1To whom correspondence should be addressed, at the present address: Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, U.S.A.
Search for other works by this author on:
Peter F. B. WOODING;
Peter F. B. WOODING
*Department of Signalling, Babraham Institute, Babraham, Cambridge CB2 4AT, U.K.
Search for other works by this author on:
F. Robin IRVINE;
F. Robin IRVINE
†Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QJ, U.K.
Search for other works by this author on:
Nullin DIVECHA
Nullin DIVECHA
*Department of Signalling, Babraham Institute, Babraham, Cambridge CB2 4AT, U.K.
Search for other works by this author on:
Publisher: Portland Press Ltd
Received:
March 27 1997
Revision Received:
June 06 1997
Accepted:
June 19 1997
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1997
1997
Biochem J (1997) 327 (2): 569–576.
Article history
Received:
March 27 1997
Revision Received:
June 06 1997
Accepted:
June 19 1997
Citation
R. Lewis VANN, Peter F. B. WOODING, F. Robin IRVINE, Nullin DIVECHA; Metabolism and possible compartmentalization of inositol lipids in isolated rat-liver nuclei. Biochem J 15 October 1997; 327 (2): 569–576. doi: https://doi.org/10.1042/bj3270569
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Captcha Validation Error. Please try again.