sn-1,2-diacylglycerol kinase of Escherichia coli. Mixed micellar analysis of the phospholipid cofactor requirement and divalent cation dependence

J. P. Walsh, R. M. Bell

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Abstract

Efficient delivery of hydrophobic water-insoluble substrates and cofactors to membrane-bound enzymes is a recurring problem which has impeded kinetic analyses. Kinetic analysis of the Escherichia coli sn-1,2-diacylglycerol kinase, an extremely hydrophobic integral membrane protein of 122 residues, was facilitated by the development of a mixed micellar assay. β-Octyl glucoside micelles quantitatively solubilized diacylglycerol kinase from membranes of strains which overproduced the enzyme up to 250-fold and provided an effective method to disperse and deliver the hydrophobic water-insoluble substrate, sn-1,2-dioleoylglycerol. Diacylglycerol kinase was active in mixed micelles containing octyl glucoside and dioleoylglycerol. Several phospholipids stimulated activity up to 6-fold, suggesting a cofactor function. Activation by phospholipids was not stereospecific and was mimicked partially by fatty acids. Half-maximal activation was observed at 1 mol % cardiolipin, suggesting that a small number of phospholipids are sufficient to activate the enzyme. Activity was dependent on the mole fractions of dioleoylglycerol and phospholipid in the mixed micelles, but independent of micelle number. Several lines of evidence indicated that the transfer of diacylglycerol between micelles was much more rapid than its utilization by the enzyme. Diacylglycerol kinase exhibited Michaelis-Menten kinetics with respect to diacylglycerol and MgATP. A second Mg2+ ion (in addition to MgATP) was required for activity. When Mg2+ was excluded from the assay, Mn2+, Zn2+, Cd2+, and Co2+ supported activity to lesser extents. These data establish a suitable system for in-depth kinetic analysis of the E. coli diacylglycerol kinase and its phospholipid cofactor requirements.

Original languageEnglish (US)
Pages (from-to)6239-6247
Number of pages9
JournalJournal of Biological Chemistry
Volume261
Issue number14
StatePublished - 1986
Externally publishedYes

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Diacylglycerol Kinase
Divalent Cations
Micelles
Escherichia coli
Phospholipids
Kinetics
Diglycerides
Enzymes
Assays
Adenosine Triphosphate
Chemical activation
Membranes
Cardiolipins
Water
Substrates
Membrane Proteins
Fatty Acids
Ions
diolein

ASJC Scopus subject areas

  • Biochemistry

Cite this

sn-1,2-diacylglycerol kinase of Escherichia coli. Mixed micellar analysis of the phospholipid cofactor requirement and divalent cation dependence. / Walsh, J. P.; Bell, R. M.

In: Journal of Biological Chemistry, Vol. 261, No. 14, 1986, p. 6239-6247.

Research output: Contribution to journalArticle

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