Interaction of sulfite with the noncatalytic and catalytic sites of chloroplast coupling factor cf1.

The interaction between sulfite, an efficient Mg2+-dependent F1-ATPase activator, and chloroplast CF1-ATPase was studied. The sulfite anion was shown to inhibit ADP and ATP binding to the noncatalytic sites of CF1. The stimulating activity of sulfite persists when all noncatalytic sites are nucleotide-occupied. Phosphate, a competing candidate for binding to CF1 catalytic sites, suppresses this activity. These results support the suggestion that the stimulation of Mg2+-dependent ATPase activity of CF1 is caused by sulfite binding to its catalytic sites.

Study of the structure of the thylakoid membrane-bound chloroplast coupling factor CF1.

The structure of thylakoid membrane-bound chloroplast coupling factor CF1 was studied by limited proteolysis followed by sodium dodecylsulfate polyacrylamide gel electrophoresis and N-terminal sequence analysis. The N-terminal fragment of the alpha-subunit was shown to have an exposed area including the peptide bond R21-E22. The cleavage of this peptide bond caused the alphaK24-V25 bond to be exposed to the outside. In the N-terminal fragment of the beta-subunit, the L14-E15 bond was identified and found to be subject to trypsinolysis. Also, the alphaR140-S141, alphaG160-R161, and betaG102-G103 bonds were accessible to the proteolytic attack. In general, the beta-subunit of membrane-bound CF1 is more sensitive to proteolysis than that of solubilized CF1. The products of proteolysis of the alpha-subunit did not contain the polypeptides typical of the reaction of cleavage of the alphaE17-G18 and alphaE22-V23 bonds in isolated CF1. These results suggest a significant structural difference between soluble and membrane-bound CF1. A number of peptide bonds, alphaG160-R161 in particular, were shown to be shielded from proteolytic attack by papain in illuminated thylakoid membranes, probably as a result of membrane energization. In contrast, the light-induced reduction of the gamma-subunit caused an increase in the accessibility of some peptide bonds to this protease, including the alphaG160-R161 bond.

Catalytic properties of ß subunit isolated from chloroplast coupling factor 1.

The chloroplast coupling factor (CF1) was dissociated into subunits by the freezing-thawing procedure in the presence of 0.5 M NaBr and the ß subunit was purified by ion-exchange chromatography on a DEAE-cellulose column. The ß subunit did not catalyze ATP hydrolysis either in the presence or in the absence of reagents known to activate Mg(2+)-dependent ATPase activity of CF1. However, it manifested appreciable adenylate kinase-like and ATP-ADP γ-phosphate exchange activities. The adenylate kinase-like activity only slightly depended on Mg(2+) ions. Ethanol, and especially diadenosine pentaphosphate, inhibited the reaction effectively. In contrast, the ATP-ADP exchange activity was Mg(2+)-dependent. Ethanol and diadenosine pentaphosphate were poor inhibitors. Sulfite, the CF1-ATPase activator, and quercetin, its inhibitor, had a minor effect on catalytic activity of the ß subunit.

pH dependent changes in ADP and ATP affinity for the tight nucleotide-binding site of chloroplast coupling factor 1.

The pH-dependence of ADP and ATP affinity for CF1 tight nucleotide-binding sites was studied under conditions of equilibrium between bound and free labeled nucleotides. With the nucleotide/CF1 ratio>1, the ATP content in tightly bound nucleotides depended only slightly on medium pH. With the nucleotide/CF1 ratio approaching 1, tightly bound ATP content grew rapidly with decreasing pH. Calculations of ADP/ATP ratio in free and tightly bound nucleotides showed that decreasing the pH from 8.0 to 6.0 induced a 150 times greater affinity of the nucleotide-binding site for ATP than for ADP. The data indicates that ATP-ADP equilibrium at the CF1 tight nucleotide-binding site depends on protonation of specific acid-base groups of the enzyme.

[Presteady-state kinetics of ATP hydrolysis by chloroplast CF1-ATPASE]. / Predstatsionarnaia kinetika gidroliza ATP CF1-ATPazoi khloroplastov.

The kinetics of reversible inactivation of chloroplast CF1-ATPase by Mg2+ and ADP was studied. The rate of inactivation obeys the first-order equation and is independent of ADP concentration. An analysis of the dependence of the inactivation rate on Mg2+ concentration demonstrated that the limiting step of inactivation is other than Mg2+ binding, i.e. the subsequent steps which include, in all probability, the conformational changes of the enzyme. The original Mg2+-dependent activity of CF1-ATPase is close to that observed under steady-state conditions in the presence of sulphate and methanol and exceeds the Ca2+-dependent activity approximately 6-fold. Preincubation of CF1-ATPase with Mg2+ results in inhibition of the original activity of the enzyme. This effect is not removed by addition of the ATP-regenerating system (pyruvate kinase + phosphoenol pyruvate) to the preincubation medium but is diminished by sulphite and the non-hydrolyzed analog of ATP--beta, gamma-methyladenosine-5-triphosphate. After addition of AMPPCP to the reaction mixture the initial reaction rate is decreased, while the steady-state rate is increased. It may be concluded that the Mg2+-dependent inactivation of CF1-ATPase is induced by the tightly bound ADP. The latter can be replaced by ATP, which in contrast to ADP does not form an inactive complex with the enzyme. A comparison of experimental results with literature data suggests that the mechanism of "alternating sites" proposed by Boyer et al. for ATP hydrolysis by soluble CF1-ATPase is not realized under the given experimental conditions.