Isolation and purification of dicyclohexylcarbodiimide-reactive proteolipid from Bacillus subtilis membrane.
The membrane-bound ATPase activity of Bacillus subtilis was inhibited by dicyclohexylcarbodiimide (DCCD). The DCCD-reactive proteolipid of B. subtilis was extracted, from labelled or untreated membranes containing F1 or depleted of F1, with neutral or acidic chloroform/methanol. Purification of the [14C]DCCD-binding proteolipid was attempted by column chromatography on methylated Sephadex G-50 and on DEAE-cellulose. The maximal amount of DCCD which could be bound to the purified proteolipid was found to exceed the amount bound by the purified proteolipid extracted from membranes labelled with the lowest [14C]DCCD concentration required for maximal inhibition of the membrane-bound ATPase activity. The radioactive protein peaks eluted by gel filtration and ion-exchange chromatography were analysed by urea-SDS polyacrylamide slab gel electrophoresis and autoradiography. Radioactivity was incorporated into two components of Mr 18 000 and 6000 when proteolipid was purified by methylated Sephadex. The 6000 polypeptide was always present, whatever the extraction and purification procedures. However, the 18 000 polypeptide was present in largest quantity only when proteolipid was extracted from membranes containing F1 and purified by methylated Sephadex. When proteolipid was purified on DEAE-cellulose this [14C]DCCD binding component of Mr 18 000 was absent.
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D001412:Bacillus subtilis / Q000032:analysis*; D002234:Carbodiimides / Q000494:pharmacology*; D002462:Cell Membrane / Q000032:analysis; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D011510:Proteolipids / Q000302:isolation & purification*
D002234:Carbodiimides; D011510:Proteolipids; D004024:Dicyclohexylcarbodiimide; D000251:Adenosine Triphosphatases