Diglyceride Kinase from Escherichia coli

Abstract

The diglyceride kinase activity of membranes from Escherichia coli was extracted into acidic butan-1-01. The enzyme was purified in organic solvent by precipitation at -20 "C, chromatography on DEAE-cellulose and repeated chromatography on Sephadex LH-60. The final 1460-fold purified enzyme preparation gave a single protein band upon isoelectric focusing in the presence of Triton X-100 (PI, 4.0) and upon polyacrylamide-gel electrophoresis in the presence of sodium dodecylsulphate. The latter method as well as gel chromatography on Sephadex LH-60 indicated a molecular weight of about 15 400.The purified enzyme was devoid of lipid, and it required re-addition of lipid for activity. sn-1,2-Dipalmitate and ceramide were phosphorylated, whereas the Css-isoprenoid alcohol, ficaprenol, did not serve as a substrate under the same conditions. Conversely, the butanol-soluble Cssisoprenoid-alcohol kinase from Staphylococcus aureus did not phosphorylate sn-l,2-dipalmitate.

Hexamethylphosphoric triamide as a solubilizing agent

“…A crude 'total' membrane fraction of E. coli K12 and inner membrane vesicles of E. coli ML 308-225 were prepared as in [4] and [8], respectively. Protein was determined in the presence of SDS [3].…”

“…Since E. coli diglyceride kinase is easily purified and reactivated using butanol-1 [4] we have studied the behaviour of this enzyme in HMPT. Conditions allowing solubilization, purification and reactivation of diglyceride kinase in HMPT are reported.…”

“…Due to the high boiling points of >2OO"C of HMPT and MP the previous assay procedure for diglyceride kinase activity [4] could not be used. HMPT-or MP-dissolved enzyme was therefore added directly to the test tube already containing the other required assay components [4] so that a final solvent concentration of 18% (v/v) of HMPT or MP was present.…”

“…HMPT-or MP-dissolved enzyme was therefore added directly to the test tube already containing the other required assay components [4] so that a final solvent concentration of 18% (v/v) of HMPT or MP was present. Addition of cont.…”

“…Two hydrophobic bacterial membrane enzymes, isoprenoid alcohol kinase and diglyceride kinase, have been highly purified and reactivated using butanol-1 [3,4]. The aprotic organic solvents, HMPT and MP, have subsequently been found to be much more effective in solubilizing membrane proteins than chloroform or butanol-1 [5].…”