Modification of isolated subunit c of the F 1F 0-ATPase from Propionigenium modestum by dicyclohexylcarbodiimide

Abstract

Subunit c of the F 1F 0-ATPase from Propionigenium modestum was extracted from the particulate cell fraction with chloroform/methanol. The protein was further purified by carboxymethyl cellulose chromatography and anion exchange HPLC in the organic solvent. SDS-PAGE of the purified protein indicated a single stained protein band migrating as expected for the c-subunit. Incubation of isolated subunit c in chlorform/methanol or aqueous buffer containing dodecyl-β- d-maltoside with [ 14C]dicyclohexylcarbodiimide (DCCD) resulted in the incorporation of radioactivity into the protein. The rate of this reaction depended on the external pH; it was significantly faster in the more acidic than in the alkaline pH range. In the presence of Na + subunit c was partially protected from labeling with [ 14C]DCCD at pH 6.1 and at pH 7.5, whereas no protection was evident at pH 5.5. At pH 7.5, the rate of subunit c labeling by [ 14C]DCCD in the presence of 20 mM NaCl was about 50% lower than in the absence of Na + ions. The isolated c-subunit therefore apparently retains in part the Na + binding site which, when occupied, diminishes the reactivity of the protein towards DCCD.