Carbonic anhydrase activities of pea thylakoids as well as thylakoid fragments enriched either in Photosystem 1 (PS1-membranes) or Photosystem 2 (PS2-membranes) were studied. The activity of PS1-membranes if calculated on chlorophyll basis was much higher than the activity of PS2-membranes. Acetazolamide, a non-permeable inhibitor of carbonic anhydrases, increased carbonic anhydrase activity of PS2-membranes at concentrations lower than 10(-6) M and suppressed this activity only at higher concentrations. A lipophilic inhibitor of carbonic anhydrases, ethoxyzolamide, effectively suppressed the carbonic anhydrase activity of PS2-membranes (I50 = 10(-9) M). Carbonic anhydrase activity of PS1-membranes was suppressed alike by both inhibitors (I50 = 10(-6) M). In the course of the electrophoresis of PS2-membranes treated with n-dodecyl-beta-maltoside "high-molecular-mass" carbonic anhydrase activity was revealed in the region corresponding to core-complex of this photosystem. Besides, carbonic anhydrase activity in the region of low-molecular-mass proteins was discovered in the course of such an electrophoresis of both PS2- and PS1-membranes. These low-molecular-mass carbonic anhydrases eluted from corresponding gels differed in sensitivity to specific carbonic anhydrase inhibitors just the same as PS1-membranes versus PS2-membranes. The results are considered as evidence for the presence in the thylakoid membranes of three carriers of carbonic anhydrase activity.

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.

The thylakoid membrane containing photosystem II (PSII membranes) from pea and wheat leaves catalyzed the reaction of CO 2 hydration with low rate, which increased after their incubation either with Triton X-100, up to Triton/chlorophyll ratio 1:1, or 1 M CaCl 2 . The presence of the inhibitor of CAs, p-aminomethylbenzensulfonamide (mafenide), at the start line in the course of electrophoresis of PSII membranes solubilized by n-dodecyl-b-maltoside (DM) decreased the amount of PSII core complex in the gel. The elution of PSII core complex from the column with immobilized mafenide occurred only either by mafenide or another inhibitor of CAs, ethoxyzolamide. The above results led to a conclusion that membrane-bound CA activity associated with PSII is situated in the core complex.

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