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°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. Copyright © 1979, Wiley Blackwell. All rights reserved