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Phosphatidylinositol 4-Kinase Associated with Spinach Plasma Membranes. Isolation and Characterization of Two Distinct Forms

Westergren, Tomas LU ; Ekblad, Lars LU ; Jergil, Bengt LU and Sommarin, Marianne LU (1999) In Plant Physiology 121(2). p.507-516
Abstract
Highly purified plasma membranes from spinach (Spinacia oleracea L.) leaves contained phosphatidylinositol (PtdIns) kinase activity that was firmly associated with the membrane. The enzyme was solubilized by detergent treatment (2% [w/v] Triton X-100) and purified by heparin-Sepharose and Q-Sepharose chromatography. Two enzymically active fractions, QI and QII, both exhibiting PtdIns 4-kinase activity, were resolved and purified 100- to 300-fold over the plasma membrane. QI and QII shared similar high apparent Km values for ATP (approximately 0.45 mM) and PtdIns (approximately 0.2 mM) and were insensitive to inhibition by adenosine. While Mg2+ was the preferred divalent cation, Mn2+ could partly substitute in the reaction catalyzed by the... (More)
Highly purified plasma membranes from spinach (Spinacia oleracea L.) leaves contained phosphatidylinositol (PtdIns) kinase activity that was firmly associated with the membrane. The enzyme was solubilized by detergent treatment (2% [w/v] Triton X-100) and purified by heparin-Sepharose and Q-Sepharose chromatography. Two enzymically active fractions, QI and QII, both exhibiting PtdIns 4-kinase activity, were resolved and purified 100- to 300-fold over the plasma membrane. QI and QII shared similar high apparent Km values for ATP (approximately 0.45 mM) and PtdIns (approximately 0.2 mM) and were insensitive to inhibition by adenosine. While Mg2+ was the preferred divalent cation, Mn2+ could partly substitute in the reaction catalyzed by the QII enzyme but not in that catalyzed by QI. Mn2+ acted synergistically with suboptimal Mg2+ concentrations to activate not only the QII enzyme, but also to some extent QI. Both enzymes were inhibited by millimolar concentrations of Ca2+ and micromolar concentrations of wortmannin. The apparent molecular mass for QI was 120 kD, which was determined by SDS-PAGE and western blotting using an antibody against a peptide unique for lipid kinases and the binding of 3H-wortmannin, and for QII 65 kD as determined by immunodetection and renaturation of PtdIns kinase activity in the 65-kD region of polyacrylamide gels. (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Plant Physiology
volume
121
issue
2
pages
507 - 516
publisher
American Society of Plant Biologists
external identifiers
  • pmid:10517842
  • scopus:0032707005
ISSN
1532-2548
language
English
LU publication?
yes
id
71320c29-8a92-472f-8e67-9663d33b81eb (old id 125446)
alternative location
http://www.plantphysiol.org/cgi/content/full/121/2/507
date added to LUP
2007-07-09 10:21:55
date last changed
2017-08-17 14:27:36
@article{71320c29-8a92-472f-8e67-9663d33b81eb,
  abstract     = {Highly purified plasma membranes from spinach (Spinacia oleracea L.) leaves contained phosphatidylinositol (PtdIns) kinase activity that was firmly associated with the membrane. The enzyme was solubilized by detergent treatment (2% [w/v] Triton X-100) and purified by heparin-Sepharose and Q-Sepharose chromatography. Two enzymically active fractions, QI and QII, both exhibiting PtdIns 4-kinase activity, were resolved and purified 100- to 300-fold over the plasma membrane. QI and QII shared similar high apparent Km values for ATP (approximately 0.45 mM) and PtdIns (approximately 0.2 mM) and were insensitive to inhibition by adenosine. While Mg2+ was the preferred divalent cation, Mn2+ could partly substitute in the reaction catalyzed by the QII enzyme but not in that catalyzed by QI. Mn2+ acted synergistically with suboptimal Mg2+ concentrations to activate not only the QII enzyme, but also to some extent QI. Both enzymes were inhibited by millimolar concentrations of Ca2+ and micromolar concentrations of wortmannin. The apparent molecular mass for QI was 120 kD, which was determined by SDS-PAGE and western blotting using an antibody against a peptide unique for lipid kinases and the binding of 3H-wortmannin, and for QII 65 kD as determined by immunodetection and renaturation of PtdIns kinase activity in the 65-kD region of polyacrylamide gels.},
  author       = {Westergren, Tomas and Ekblad, Lars and Jergil, Bengt and Sommarin, Marianne},
  issn         = {1532-2548},
  language     = {eng},
  number       = {2},
  pages        = {507--516},
  publisher    = {American Society of Plant Biologists},
  series       = {Plant Physiology},
  title        = {Phosphatidylinositol 4-Kinase Associated with Spinach Plasma Membranes. Isolation and Characterization of Two Distinct Forms},
  volume       = {121},
  year         = {1999},
}