Modification of isolated subunit c of the F1Fo-ATPase from Propionigenium modestum by dicyclohexylcarbodiimide.
Subunit c of the F1Fo-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 chloroform/methanol or aqueous buffer containing dodecyl-beta-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.
Kluge
C
C
Mikrobiologisches Institut, Eidgenössische Technische Hochschule, ETH-Zentrum, Zürich, Switzerland.
Dimroth
P
P
D001421:Bacteria, Anaerobic / Q000201:enzymology*; D001665:Binding Sites; D002412:Cations; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid; D006863:Hydrogen-Ion Concentration; D006180:Proton-Translocating ATPases / Q000187:drug effects* / Q000302:isolation & purification / Q000378:metabolism; D012964:Sodium / Q000378:metabolism
D002412:Cations; D005971:Glutamates; D018698:Glutamic Acid; D004024:Dicyclohexylcarbodiimide; D012964:Sodium; D006180:Proton-Translocating ATPases