The dithiothreitol-stimulated dissociation of the chloroplast coupling factor 1 epsilon-subunit is reversible.
The chloroplast coupling factor 1 complex (CF1) contains an epsilon-subunit which inhibits the CF1 ATPase activity. Chloroform treatment of Chlamydomonas reinhardtii thylakoid membranes solubilizes only forms of the enzyme which apparently lack the delta-subunit. Four interrelated observations are described in this paper. (1) The dithiothreitol- (DTT) induced ATPase activation of CF1(-delta) and the DTT-induced formation of a physically resolvable CF1(-delta,epsilon) from the CF1(-delta) precursor are compared. The similar time-courses of these two phenomena suggest that the dissociation of the epsilon-subunit is an obligatory process in the DTT-induced ATPase activation of soluble CF1. (2) The reversible dissociation of the epsilon-subunit of the CF1 is demonstrated by the exchange of subunits between distinguishable oligomers. 35S-labelled chloroplast coupling factor 1 lacking the delta and epsilon subunits [CF1(-delta,epsilon)] was added to a solution of non-radioactive coupling factor 1 lacking only the delta subunit [CF1(-delta)]. After separation of the two enzyme forms, via high resolution anion-exchange chromatography, radioactivity was detected in the chromatographic fractions containing CF1(-delta). (3) epsilon-deficient CF1 can be resolved from DTT pretreated epsilon-containing CF1 for several days after the removal of DTT. On the other hand, brief incubation of the DTT pretreated epsilon-containing CF1 with low concentrations of o-iodosobenzoate results in chromatographs containing only the peak of epsilon-containing CF1. A simple explanation for this phenomenon is that reduction of CF1 with DTT increases the apparent dissociation constant for the epsilon-subunit to an estimated 3.5 x 10(-8) M (+/- 1.0 x 10(-8) M) from a value of less than or equal to 5 x 10(-11) M for the oxidized enzyme. (4) ATPase activity data show that oxidation of the epsilon-deficient enzyme does not completely inhibit its manifest activity, but oxidation of DTT pre-treated CF1 which contains the epsilon-subunit completely inhibits manifest activity. A simple model is proposed for the influence of the oxidation state of the soluble enzyme on the distribution of ATPase-inactive and ATPase-active subunit configurations.
Duhe
R J
RJ
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison 53706.
Selman
B R
BR
D000251:Adenosine Triphosphatases / Q000378:metabolism; D002696:Chlamydomonas / Q000187:drug effects / Q000201:enzymology*; D002845:Chromatography; D004229:Dithiothreitol / Q000494:pharmacology*; D007463:Iodobenzoates / Q000494:pharmacology; D007700:Kinetics; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D012995:Solubility
D007463:Iodobenzoates; C023675:2-iodobenzoic acid; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases; D004229:Dithiothreitol