Diglyceride kinase from Escherichia coli. Purification in organic solvent and some properties of the enzyme.
The diglyceride kinase activity of membranes from Escherichia coli was extracted into acidic butan-1-ol. The enzyme was purified in organic solvent by precipitation at -20 degrees 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 15400. 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 C55-isoprenoid alcohol, ficaprenol, did not serve as a substrate under the same conditions. Conversely, the butanol-soluble C55-isoprenoid-alcohol kinase from Staphylococcus aureus did not phosphorylate sn-1,2-dipalmitate.
D002462:Cell Membrane / Q000201:enzymology; D004075:Diglycerides; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D010770:Phosphotransferases / Q000302:isolation & purification* / Q000378:metabolism
D004075:Diglycerides; D010770:Phosphotransferases