Tag Archives: ATP hydrolysis
Sulfonamides influence on the activity of thylakoid ATPase isolated from spinach chloroplasts
Е. K. Zolotareva, A. P. Khomochkin, O. B. Onoiko
M. G. Kholodhy Institute of Botany, National Academy of Sciences of Ukraine, Kyiv;
e-mail: membrana@ukr.net
Received: 13 September 2019; Accepted: 15 May 2020
The effect of two water-soluble sulfonamides: aromatic 4-aminobenzenesulfonamide (SA) and aliphatic trifluoromethylsulfonamide (TFMSA) on the latent and stimulated ATPase activity of the catalytic part of the thylakoid ATP synthase isolated from spinach (Spinacia oleracea L.) chloroplasts was studied. Three methods of ATPase activity stimulation were applied in the work: brief heating of the enzyme at 62 °С; incubation with 20% methanol; 25 mM sodium sulfite adding to the reaction medium. TFMSA effectively inhibited the ATPase activity of the enzyme at all types of activation. I50 (TFMSA) value for was found to be about 5 μM in the case of enzyme heat treatment and 1 μM in the case of enzyme activation with sulfite or methanol. SA did not affect ATPase activity at stimulation with sulfite and inhibited with I50 about 50 μM at stimulation by heating or methanol. No effect of SA on latent enzyme activity was observed, while TFMSA was shown to activate latent ATPase by about 6 times. The data obtained showed that both sulfonamide compounds, known as carbonic anhydrase inhibitors, are able to modify the activity of chloroplast ATPase.
Influence of sulfite on ATPase activity of coupling factor CF(1) isolated from spinach chloroplasts
O. B. Onoiko, A. P. Khomochkin, O. K. Zolotareva
M. G. Kholodhy Institute of Botany, National Academy of Sciences of Ukraine, Kyiv;
e-mail: membrana@ukr.net
Received: 25 October 2018; Accepted: 13 December 2018
The present study of sulfite participation in the regulation of the chloroplast ATP-synthase complex can help to understand the mechanisms of acid rains toxic effects on the plant photosynthetic apparatus. The aim of the work was to study the effect of sulfite on Ca2+– and Mg2+-dependent ATP hydrolysis catalysed by coupling factor CF1, exposed to preliminary short-term acid treatment. CF1 was isolated from spinach chloroplasts (Spinacia oleracea L.). The latent ATPase activity of CF1 was activated by sodium sulfite addition into incubation media. The rate of ATP hydrolysis was determined by the release of inorganic phosphate. In the presence of 25 mM exogenous sulfite the Mg2+-dependent hydrolytic activity of CF1 was increased 7-fold and Ca2+-dependent activity 3-fold compared to the control. Carbonic anhydrase inhibitors acetazolamide or ethoxyzolamide eliminated sulfite-induced stimulation of ATP hydrolysis. The sulfite stimulating effect on Ca2+– and Mg2+-ATPase activity was increased dramatically after incubation (5 min) of isolated CF1 in a medium with pH 3.5 and its subsequent transition to the alkali medium (pH 8.0). In this case, 1 mM sulfite-induced a 10-fold acceleration of ATP hydrolysis. Carbonic anhydrase specific inhibitors (50 μM) removed the sulfite effect. These data suggest that sulfite was able to replace bicarbonate in the CF1 structure after the release of bound HCO3– during acid treatment.
Reversible pH-dependent activation/inactivation of CF(1)-ATPase of spinach chloroplasts
A. P. Khomochkin, O. B. Onoiko, A. V. Semenikhin, O. K. Zolotareva
M. G. Kholodhy Institute of Botany, National Academy of Sciences of Ukraine, Kyiv;
e-mail: membrana@ukr.net
The aim of the work was to study the reverse pH-dependent regulation of the enzymatic activity of the catalytic part of ATP synthase (EC 3.6.3.14) of chloroplast – coupling factor CF1. It was shown that the short-term incubation of isolated CF1 in the media with pH 4.5 or 3.5 leads to inactivation of Ca2+-ATPase, which is rapidly (t1/2 ~ 1 min) restored in the medium containing 0.5-10 mM bicarbonate at pH 7.8. After acid treatment, the rate of Mg2+-ATPase reaction was also stimulated in the presence of 1 mM bicarbonate (рН 7.8; 37 °С). The increase in Ca2+– and Mg2+-АТР activity of CF1 associated with the addition of NaHCO3 solution was completely eliminated after the introduction of 50 mM acetazolamide – a specific inhibitor of carbonic anhydrase. The obtained results suggest the existence of the bound bicarbonate in the CF1 structure, which apparently participates in proton transfer.