ABSTRACT
Pea thylakoids with high carbonic anhydrase (CA) activity (average rates of 5000 micromol H(+) (mg Chl)(-1) h(-1) at pH 7.0) were prepared. Western blot analysis using antibodies raised against the soluble stromal beta-CA from spinach clearly showed that this activity is not a result of contamination of the thylakoids with the stromal CA but is derived from a thylakoid membrane-associated CA. Increase of the CA activity after partial membrane disintegration by detergent treatment, freezing or sonication implies the location of the CA in the thylakoid interior. Salt treatment of thylakoids demonstrated that while one part of the initial enzyme activity is easily soluble, the rest of it appears to be tightly associated with the membrane. CA activity being measured as HCO(3) (-) dehydration (dehydrase activity) in Photosystem II particles (BBY) was variable and usually low. The highest and most reproducible activities (approximately 2000 micromol H(+) (mg Chl)(-1) h(-1)) were observed in the presence of detergents (Triton X-100 or n-octyl-beta-D-glucopyranoside) in low concentrations. The dehydrase CA activity of BBY particles was more sensitive to the lipophilic CA inhibitor, ethoxyzolamide, than to the hydrophilic CA inhibitor, acetazolamide. CA activity was detected in PS II core complexes with average rate of 13,000 micromol H(+) (mg Chl)(-1) h(-1) which was comparable to CA activity in BBY particles normalized on a PS II reaction center basis.
ABSTRACT
Stimulation of the bicarbonate dehydration reaction in thylakoid suspension under conditions of saturating light at pH 7.6-8.0 was discovered. This effect was inhibited by nigericin or the lipophilic carbonic anhydrase (CA) inhibitor ethoxyzolamide (EZ), but not by the hydrophilic CA inhibitor, acetazolamide. It was shown that the action of EZ is not caused by an uncoupling effect. It was concluded that thylakoid CA is the enzyme utilizing the light-generated proton gradient across the thylakoid membrane thus facilitating the production of CO(2) from HCO(3)(-) and that this enzyme is covered from the stroma side of thylakoids by a lipid barrier.
