ATPase Activity Of Coupling Factor Research Articles

Energy-dependent changes in the conformation of the epsilon subunit of the chloroplast ATP synthase.

Rabbit antiserum raised against the isolated native epsilon subunit of the chloroplast coupling factor 1 activated the ATPase activity of coupling factor 1 in solution by removing the epsilon subunit. Incubation of thylakoid membranes with the antiserum in the dark had no effect on photophosphorylation or on the dithiothreitol-induced Mg2+-ATPase activity. Incubation with the antiserum during illumination, however, strongly inhibited both activities and caused the membranes to become leaky to protons. The results indicate that the formation of a proton gradient across the thylakoid membrane induces a change in conformation of the epsilon subunit of the ATP synthase such that it becomes susceptible to attack and removal by the antibodies. This change may be a part of the mechanism that results in energy-dependent activation of the ATP synthase.

Detergent activation of the ATPase activity of chloroplast coupling factor 1

The activation of the ATPase activity of coupling factor 1 (CF 1) from chloroplasts by several detergents was studied. Further evidence that detergent micelles are important in the activation of Ca 2+-ATPase was obtained. Maximal activation of Ca 2+-ATPase was achieved with short-chain alkyl-β- d-glucopyranoside (alkylglucosides) detergents. Treatment of CF 1 with hexylglucoside or heptylglucoside followed by hydroxylapatite chromatography caused nearly total removal of the ϵ subunit of the enzyme, whereas treatment with decylglucoside caused less ATPase activation and less loss of the ϵ subunit. The ATPase activity of detergent-activated CF 1 was inhibited by purified ϵ subunit. Detergents that form small micelles appear to be most effective in removing the ϵ subunit and in activating the Ca 2+-ATPase of CF 1. When present during assay, the alkylglucosides also induce a Mg 2+-ATPase activity in CF 1. Octyland nonylglucoside are most effective in promoting this reaction. If, however, CF 1 deficient in the ϵ subunit was used, even decylglucoside elicited rapid Mg 2+-ATPase hydrolysis. It is concluded that removal of the ϵ subunit, although necessary for the expression of Mg 2+-ATPase, is not sufficient. The detergents that cause maximal displacement of the ϵ subunit are less effective in inducing Mg 2+-ATPase activity. The selective removal of subunits from CF 1 by specific detergents points to potential problems with the use of these detergents in the solubilization of oligomeric membrane proteins.

Light-induced Mg 2+ ATPase activity of coupling factor in intact chloroplasts

Intense illumination of isolated, intact, spinach chloroplasts triggers the well known proton-pumping Mg 2+ ATPase activity of coupling factor, which can be assayed in subsequently lysed chloroplasts by monitoring ATP-driven quenching of 9-aminoacridine fluorescence. The light-triggered ATPase activity decays slowly in the dark and is inhibited by N, N′-dicyclohexylcarbodiimide. After osmotic lysis and washing of the chloroplasts, preillumination no longer triggers maximal proton-pumping ATPase until methylviologen and dithiothreitol are added to the medium. It is suggested that intact organelles contain soluble or loosely bound cofactors necessary for light-triggering of coupling factor ATPase. On osmotic lysis, these endogenous cofactors are diluted or inactivated and must be replaced by addition of a dithiol reagent and an electron acceptor.

Evidence for a naturally occurring ATPase-inhibitor in Escherichia coli

Stimulation of the Escherichia coli ATPase activity by urea and trypsin shows that there is a latent ATPase activity in particles and in a crude coupling factor of E. coli. Moreover, crude coupling factor, completely dissociated by treatment with 7 M urea, can inhibit the ATPase activity of the crude coupling factor. It is suggested that the latency of the ATPase activity of the coupling factor is due to the presence of a protein, the ATPase-inhibitor.

Partial Resolution of the Enzymes Catalyzing Photophosphorylation: XI. MAGNESIUM-ADENOSINE TRIPHOSPHATASE PROPERTIES OF HEAT-ACTIVATED COUPLING FACTOR I FROM CHLOROPLASTS

Abstract Conditions are reported under which purified coupling factor 1 from spinach chloroplasts, suitably activated, exhibited Mg++-dependent ATPase activity at rates of about 15 µmoles of Pi released per mg of protein per min. The presence of a carboxylic acid and proper MgCl2 concentrations were the main factors determining the rate of hydrolysis. At pH 6, with 8 mm MgCl2, sodium maleate accelerated the Mg++ATPase activity of coupling factor 1 up to 30-fold. At pH 8, 2 mm MgCl2 was optimal and bicarbonate was more effective than maleate as an activator. Higher concentrations of MgCl2 inhibited the reaction. A monospecific antibody against coupling factor 1 inhibited both the Mg++- and the Ca++-dependent ATPase activities. The specificity for triphosphonucleotides was similar for the two reactions. The Km of the Mg++ATPase for ATP was 0.1 mm at pH 6 with 2-(N-morpholino)ethane sulfonic acid buffer and 1.1 mm with maleate buffer. It is concluded that carboxylic acids increase the Vmax by removing inhibitory products of the reaction. From the mode of inhibition by ADP, existence of at least two active sites acting cooperatively, is proposed.

Partial Resolution of the Enzymes Catalyzing Photophosphorylation: VI. Interaction of Coupling Factor 1 from Chloroplasts with a New Coupling Factor for Photophosphorylation

Abstract 1. The ATPase activity of coupling factor 1 from chloroplasts (CF1) after treatment with dithiothreitol was inhibited on addition of CF1-depleted chloroplasts. This inhibition was used as an assay for components of the chloroplast membrane which interact with CF1. 2. The chloroplast membrane contains at least two components that interact with CF1. One component appears to be a lipid which is destroyed by freezing in salt solution. The other component is a protein, which can be extracted from chloroplasts with 0.4 m ammonium hydroxide. 3. The soluble protein component (CF2) inhibits the dithiothreitol-activated ATPase of CF1. CF2 is heat-labile and sensitive to trypsin. CF2 stimulates photophosphorylation in chloroplasts that have been exposed to trypsin.