Abstract
Isolated thylakoid membranes were disrupted by treatment with nonionic detergents digitonin or dodecyl maltoside. Solubilized polypeptide complexes were separated by native gel charge shift electrophoresis. The position of ATP-synthase complex and its isolated catalytic part (CF1) within gel was determined using the color reaction for ATPase activity. Due to the presence of cytochromes, the red band in unstained gels corresponded to the cytochrome b6f complex. Localization of the cytochrome b6f complex, ATP synthase and coupling CF1 in the native gel was confirmed by their subunit composition determined after SDS-electrophoretic analysis. Carbonic anhydrase (CA) activity in polypeptide zones of PS II, cytochrome b6f complex, and ATP-synthase CF1 was identified in native gels using indicator bromothymol blue. CA activity of isolated CF1 in solution was determined by infrared gas analysis as the rate of bicarbonate dehydration. The water-soluble acetazolamide, an inhibitor of CA, unlike lipophilic ethoxyzolamide inhibited CA activity of CF1 Thus, it was shown for the first time that ATP-synthase has a component which is capable of catalyzing the interconversion of forms of carbonic acid associated with proton exchange. The data obtained suggest the presence of multiple forms of carbonic anhydrase in the thylakoid membranes of spinach chloroplasts and confirm their involvement in the proton transfer to the ATP synthase.
Highlights
carbonic anhydrase (CA) activity of isolated CF1 in solution was determined by infrared gas analysis as the rate of bicarbonate dehydration
It was shown for the first time that ATP-synthase has a component which is capable of catalyzing the interconversion of forms of carbonic acid associated with proton exchange
The data obtained suggest the presence of multiple forms of carbonic anhydrase in the thylakoid membranes of spinach chloroplasts and confirm their involvement in the proton transfer to the ATP synthase
Summary
CA activity of isolated CF1 in solution was determined by infrared gas analysis as the rate of bicarbonate dehydration. The water-soluble acetazolamide, an inhibitor of CA, unlike lipophilic ethoxyzolamide inhibited CA activity of CF1 It was shown for the first time that ATP-synthase has a component which is capable of catalyzing the interconversion of forms of carbonic acid associated with proton exchange. The light-dependent reactions of photosynthesis take place on sheets of inner thylakoid membranes of chloroplasts containing 4 functional multiprotein complexes: photosystems I and II, cytochrome b6f, and ATP-synthase These complexes participate in absorption and transformation of light energy to satisfy the metabolic demands of plant cells [1,2,3]. Intracellular proton transport is significantly accelerated through facilitated diffusion with dissolved buffers, like bicarbonate, which is maintained by carbonic anhydrase (CA) This allows assuming the existence of proteins with carbonic anhydrase activity within membrane polypeptide complexes in addition to carbonic anhydrases associated with photosystem II. The detailed study of localization of CA-active proteins has not been conducted yet
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