Phosphorylation of rat liver nuclear envelopes. II. Characterization of in vitro lipid phosphorylation.

Charles Smith, W. W. Wells

Research output: Contribution to journalArticle

153 Citations (Scopus)

Abstract

Incubation of nuclear envelopes isolated from normal rat liver with [gamma-32P]ATP resulted in the rapid labeling of chloroform-soluble products. These products were identified as phosphatidic acid (PA), phosphatidylinositol 4-phosphate (DPI), and phosphatidylinositol 4,5-bisphosphate (TPI) based on their chromatographic mobilities on Silica Gel H and cellulose thin layer plates, and by analysis of their deacylation products by high pressure liquid chromatography. The extent of phosphorylation of these products was dependent on the method used to isolate the nuclear envelopes. Relatively gentle isolation methods, such as lysis of purified nuclei with heparin or digestion with deoxyribonuclease I, produce nuclear envelopes which possess significantly higher lipid kinase activity than do envelopes isolated by sonication. Incorporation of 32P into PA, DPI, and TPI in 2 min under standard assay conditions was 13, 152, and 22 pmol/mg of membrane protein, respectively. Degradation of labeled DPI and TPI was evident after 2-5 min of incubation. Nuclear envelope-associated diacylglycerol kinase, phosphatidylinositol kinase, and DPI kinase are characterized with regard to their pH and Mg2+ requirements. The effects of metals, phospholipids, and sulfhydryl reagents on these kinase activities are also described.

Original languageEnglish (US)
Pages (from-to)9368-9373
Number of pages6
JournalJournal of Biological Chemistry
Volume258
Issue number15
StatePublished - Aug 10 1983

Fingerprint

Phosphorylation
Nuclear Envelope
Liver
Rats
Phosphotransferases
Lipids
Phosphatidic Acids
Phosphatidylinositols
High pressure liquid chromatography
Diacylglycerol Kinase
Sulfhydryl Reagents
Sonication
Deoxyribonuclease I
Silica Gel
Chloroform
Cellulose
Labeling
Heparin
Digestion
Assays

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

@article{65a13227dbd74122be35f447663cb93f,
title = "Phosphorylation of rat liver nuclear envelopes. II. Characterization of in vitro lipid phosphorylation.",
abstract = "Incubation of nuclear envelopes isolated from normal rat liver with [gamma-32P]ATP resulted in the rapid labeling of chloroform-soluble products. These products were identified as phosphatidic acid (PA), phosphatidylinositol 4-phosphate (DPI), and phosphatidylinositol 4,5-bisphosphate (TPI) based on their chromatographic mobilities on Silica Gel H and cellulose thin layer plates, and by analysis of their deacylation products by high pressure liquid chromatography. The extent of phosphorylation of these products was dependent on the method used to isolate the nuclear envelopes. Relatively gentle isolation methods, such as lysis of purified nuclei with heparin or digestion with deoxyribonuclease I, produce nuclear envelopes which possess significantly higher lipid kinase activity than do envelopes isolated by sonication. Incorporation of 32P into PA, DPI, and TPI in 2 min under standard assay conditions was 13, 152, and 22 pmol/mg of membrane protein, respectively. Degradation of labeled DPI and TPI was evident after 2-5 min of incubation. Nuclear envelope-associated diacylglycerol kinase, phosphatidylinositol kinase, and DPI kinase are characterized with regard to their pH and Mg2+ requirements. The effects of metals, phospholipids, and sulfhydryl reagents on these kinase activities are also described.",
author = "Charles Smith and Wells, {W. W.}",
year = "1983",
month = "8",
day = "10",
language = "English (US)",
volume = "258",
pages = "9368--9373",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "15",

}

Phosphorylation of rat liver nuclear envelopes. II. Characterization of in vitro lipid phosphorylation. / Smith, Charles; Wells, W. W.

In: Journal of Biological Chemistry, Vol. 258, No. 15, 10.08.1983, p. 9368-9373.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Phosphorylation of rat liver nuclear envelopes. II. Characterization of in vitro lipid phosphorylation.

AU - Smith, Charles

AU - Wells, W. W.

PY - 1983/8/10

Y1 - 1983/8/10

N2 - Incubation of nuclear envelopes isolated from normal rat liver with [gamma-32P]ATP resulted in the rapid labeling of chloroform-soluble products. These products were identified as phosphatidic acid (PA), phosphatidylinositol 4-phosphate (DPI), and phosphatidylinositol 4,5-bisphosphate (TPI) based on their chromatographic mobilities on Silica Gel H and cellulose thin layer plates, and by analysis of their deacylation products by high pressure liquid chromatography. The extent of phosphorylation of these products was dependent on the method used to isolate the nuclear envelopes. Relatively gentle isolation methods, such as lysis of purified nuclei with heparin or digestion with deoxyribonuclease I, produce nuclear envelopes which possess significantly higher lipid kinase activity than do envelopes isolated by sonication. Incorporation of 32P into PA, DPI, and TPI in 2 min under standard assay conditions was 13, 152, and 22 pmol/mg of membrane protein, respectively. Degradation of labeled DPI and TPI was evident after 2-5 min of incubation. Nuclear envelope-associated diacylglycerol kinase, phosphatidylinositol kinase, and DPI kinase are characterized with regard to their pH and Mg2+ requirements. The effects of metals, phospholipids, and sulfhydryl reagents on these kinase activities are also described.

AB - Incubation of nuclear envelopes isolated from normal rat liver with [gamma-32P]ATP resulted in the rapid labeling of chloroform-soluble products. These products were identified as phosphatidic acid (PA), phosphatidylinositol 4-phosphate (DPI), and phosphatidylinositol 4,5-bisphosphate (TPI) based on their chromatographic mobilities on Silica Gel H and cellulose thin layer plates, and by analysis of their deacylation products by high pressure liquid chromatography. The extent of phosphorylation of these products was dependent on the method used to isolate the nuclear envelopes. Relatively gentle isolation methods, such as lysis of purified nuclei with heparin or digestion with deoxyribonuclease I, produce nuclear envelopes which possess significantly higher lipid kinase activity than do envelopes isolated by sonication. Incorporation of 32P into PA, DPI, and TPI in 2 min under standard assay conditions was 13, 152, and 22 pmol/mg of membrane protein, respectively. Degradation of labeled DPI and TPI was evident after 2-5 min of incubation. Nuclear envelope-associated diacylglycerol kinase, phosphatidylinositol kinase, and DPI kinase are characterized with regard to their pH and Mg2+ requirements. The effects of metals, phospholipids, and sulfhydryl reagents on these kinase activities are also described.

UR - http://www.scopus.com/inward/record.url?scp=0021099767&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0021099767&partnerID=8YFLogxK

M3 - Article

C2 - 6308005

AN - SCOPUS:0021099767

VL - 258

SP - 9368

EP - 9373

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 15

ER -