CarboniC anhydrase aCtivity of integral-funCtional Complexes of thylakoid membranes of spinaCh Chloroplasts

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.


CarboniC anhydrase aCtivity of integral-funCtional Complexes of thylakoid membranes of spinaCh Chloroplasts
A. V. SemeNIhIN, O. K. ZOlOtAreVA m.G. Kholodny Institute of Botany, National Academy of Sciences of Ukraine, Kyiv; e-mail: membrana@ukr.netIsolated 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 (CF 1 ) 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 CF 1 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 CF 1 was identified in native gels using indicator bromothymol blue.CA activity of isolated CF 1 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 CF 1 .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.K e y w o r d s: thylakoid membranes, ATP-synthase, coupling factor СF 1 , carbonic anhydrase, proton transport.
T he 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].These complexes, together with other components of thylakoid membranes, carry out photosynthetic electron transfer and proton transfer coupled with it.This process creates transmembrane proton gradient that is the driving force of ATP synthesis.There is a couple of ways by which the protons may enter into thylakoid lumen.The first happens in plastoquinone protonation-deprotonation cycle, which acquires protons from the outer side of the membrane in Q B center of photosystem II and releases them after oxidation reaction in cytochrome b 6 center.The second occurs when water is decomposed in photosystem II complex.Protons leave thylakoids via proton transport of ATP-synthase coupled with synthesis of ATP [4].Thus, at least three out of four main multiprotein complexes of photosynthetic membranes are involved in light-dependent proton exchange.According to analysis [5] the required rate of proton transport from sites where they are generated on thylakoid membrane to ATP-synthase cannot be maintained by free diffusion.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.
CA (carbonate hydrolyase, EC 4.2.1.1)is a zinccontaining metalloenzyme that catalyzes interconversion of CO 2 and HCO 3 -.CA accelerates the rate of the equilibrium in this reaction by tens of thousand times.Without CA in solution, the equilibrium is attained very slowly.There are both soluble and membrane-bound forms of this enzyme in cells of higher plants [6].It has been demonstrated that CA molecules in thylakoid membranes of chloroplasts are associated with at least three different components of electron-transport chain.Two proteins with carbonic anhydrase activity have been identified in monomeric and dimeric forms of photosystem II (PS II) [7].It has been also demonstrated that PS I-enriched membrane samples contain CA [8].There is an evidence that PS II-associated CA generates bicarbonate ions at a rate sufficient to bound protons released in the inner space of thylakoid in the process of photosynthetic oxidation of water [9].Hence, CA prevents acidic denaturation of manganese cluster of PS II, as critical increase in local H + concentration may damage the active centers of the complex and result in their destruction with manganese release in liquid environment [10,11].
The existence of multiple forms of CA in spinach chloroplasts has been confirmed [12].Nevertheless, the detailed study of localization of CA-active proteins has not been conducted yet.
The aim of the present work is to modify native gel charge shift electrophoresis and two-dimensional electrophoresis methods for separation and identification of native pigment-protein and protein complexes of spinach and study of their carbonic anhydrase activity.
To this end, we developed a method for solubilization of functional multiprotein complexes of thylakoid membranes with nonionic detergent digitonin.The complexes were identified through 2-D electrophoresis (by their molecular mass and peptide composition), and their carbonic anhydrase and ATPase activity was assayed by enzyme-specific reactions.

materials and methods
The thylakoids were extracted from fresh spinach leaves homogenized on ice in buffer (40 mM tris-HCl (pH 7.8), 400 mM sorbitol, 5 mM EDTA, 1 mM MgCl 2 , 10 mM NaCl, 5 mM sodium ascorbate, 0.05% BSA).The suspension was filtered through double layer of tissue and centrifuged at 2500 g at 4 ºC for 4 min.The sediment was resuspended in hypotonic buffer (20 mM tris-HCl (pH 7.5), 5 mM sorbitol and 5 mM MgCl 2 ), followed by centrifugation at 2500 g at 4 ºC for 4 min.The sediment of isolated thylakoids was washed with storage buffer (200 mM sorbitol, 10 mM NaCl, 10 mM kCl, 2.5 mM MgCl 2 , 10 mM tris-HCl, pH 7.8).The procedure was repeated twice, then the sediment was resuspended in small volume of storage buffer to chlorophyll concentration of 4 mg/ml.Chlorophyll concentration in thylakoid membrane samples was assayed after Arnon [13].
In order to solubilize protein complexes the thylakoid membranes were incubated for 30 min at 4 ºC in 10% solution of digitonin (at detergent:protein ratio of 1:1; 2:1; 4:1; 8:1) in the presence of protease inhibitors 0.75 M aminocaproic acid and 1 mM phenylmethylsulfonyl fluoride (PMSF).The soluble proteins of thylakoid membranes were then separated by centrifugation at 140,000 g at 4 ºC for 45 min.Protein content in supernatant was assayed after Lowry et al. [14].
Native gel charge shift electrophoresis of membrane protein complexes was done after Andersson et al. [15] with modifications, Kolesnichenko et al. [16] in PAA gel slabs (70×80×1.5mm) in acrylamide gradient (4-11%) in 0.38 M tris-HCl buffer (pH 8.8).Stacking gel contained 3.75% of acrylamide in 0.06 M tris-HCl buffer (pH6.8).Cathode and anode buffers were glycine and tris solutions (25 mM tris-HCl, 192 mM glycine, pH 8.3).To create charge shift, SDS was added to cathode buffer to 0.005% concentration.The electrophoresis was conducted at 4 ºC in order to maintain the native conditions of multiprotein functional complex of thylakoids.The protein bands were visualized with the help of coomassie G-250 dye.
The gel strips containing protein bands were excised and used for SDS denaturating electrophoresis in second direction in modified Laemmli system [17] in PAA gel slabs (70×80×1.5mm) in order to analyze the peptide composition of complexes.The electrophoretic separation of proteins was conducted in acrylamide concentration gradient from 10% to 20% in 0.38 M tris-HCl buffer (pH 8.8), 0.1% SDS.
ATPase activity was assayed after Allen and Hyncik [18] and Gomori [19].The gels after native electrophoresis were incubated for 10-12 hours in solution containing 10 mM ATP, 10 mM CaCl 2 in 100 mM tris-HCl (pH 9.5).The gels were then washed with water and incubated for 20 min in 3 mM solution of Pb(NO 3 ) 2 in 80 mM malate buffer (pH 7.0).After washing the gels were placed in 0.2% solution of Na 2 S. ATP synthase localization was visua lized as dark red bands of insoluble PbS.
CA activity was visualized according to Edwards and Patton [20].The gels were incubated for 30 min in 0.2% of bromothymol blue in 50 mM veronal buffer (pH 8.1).The gel was then placed in water saturated with CO 2 at 0 ºC.CA spots were visualized where indicator color changed from blue to yellow.
CF 1 coupling factor was extracted from cationfree thylakoids with 1 mM EDTA solution and purified after [21] with some modifications.ATPase activity of isolated CF 1 was assayed by phosphate generation measured after Lowry and Lopes [22].Rate of CA reaction catalyzed by isolated CF 1 was assayed by CO 2 generation from dehydration of bicarbonate.CO 2 content was determined with infrared gas analyzer (S151, Qubit Systems Inc., Canada).

results and discussion
The thylakoid membranes are distinct in their heterogeneous localization and their association with numerous and diverse functional protein complexes.In accordance with their protein composition, there exist terminal membranes, lateral (marginal) membranes of stromal (lamellar) thylakoids and intragranal (stacked) thylakoids, which are in tight junctions with each other.PS II is localized mostly within grana in the regions of tight contacts of thylakoids.PS I is generally found in stromal thylakoids and in marginal and terminal regions of grana.This system is absent in stacked regions of grana.On the other hand, cytochrome complexes are distributed more or less evenly throughout the thylakoid membranes, with somewhat higher density within regions of tight thylakoid contacts in grana.ATP synthase complex is predominantly localized of stromal thylakoids, and in marginal and terminal regions of grana [3,4].
Thylakoid membrane disintegration and native functional complexes separation are achieved with application of neutral (nonionic) detergents.The most efficient detergent or a mix of detergents and protein/detergent ratio in incubation medium has to be selected experimentally for solubilization of complexes from each particular membrane type.The native pigment-protein complexes of thylakoids may be extracted with dodecylmaltoside (DM), tridecylmaltoside (TM), digitonin or triton X-100 [23], which disrupt bonds between polypeptide complexes and lipid matrix of membranes and preserve the native composition and functional activity of photosynthetic complexes.The full extraction of all the multiprotein complexes from various thylakoid parts under mild solubilization is unlikely to be achievable due to heterogeneous nature of protein and lipid thylakoid components and due to irregularities in localization of membrane complexes.So, the solubilization of particular pigment-protein complex is dependent on the nature of detergent used as well as on the ratio of detergent to protein in incubation medium.According to studies [23], native protein complexes of grana are extracted with DM or TM, and polypeptide complexes of thylakoid stroma may be extracted successfully by digitonin application to target membranes.
The number of protein bands in an electrophoretic line is dependent on detergent:protein ratio, and the complexes with high molecular mass appear at higher concentrations of detergent.It is apparent that lower concentrations of detergent cannot provide for separation of these complexes from lipid matrix.As can be seen on Fig. 1, the most effective solubilization of native thylakoid protein complexes is achieved at digitonin:protein ratio 8:1.This solubilization procedure allows for detection of protein structures with molecular mass over 900 kDa.According to published data, the mass of the main functional protein complexes of photosynthetic membranes are approx.400 kDa (cytochrome b6f), 600 kDa (ATP synthase), and 700 kDa (PS I) [23].These complexes are organized as dimers within membranes (cytochrome b6f complex, PS II) [1,4] or trimers (light-harvesting complex, LHC).The identification of high molecular mass components (over 900 kDa) on electrophoretic plates affirms that this method is suitable for solubilization of oligomeric membrane components with digitonin.
Therefore, the chosen concentrations of digitonin allowed for efficient separation of primary polypeptide complexes of spinach thylakoids.Previously, dodecylmaltoside application allowed for a fairly clear resolution of thylakoid membrane complexes from Arabidopsis [23] and rice [24].In con-
On unstained electrophoretic plates of digitonin-solubilized thylakoid complexes there is a clear red band within the region corresponding to the marker of 440 kDa molecular mass.This band is conceivably related to cytochrome b6f complex, which has a natural red color due to the presence of cytochromes.
We analyzed the specific enzymatic activity in PAA gels in order to identify protein bands with ATPase activity that may be part of native ATP-synthase complex.As can be seen on Fig. 2, the ATPase activity in gels incubated in buffer system with ATP and Pb(NO 3 ) 2 is detected within a zone that corresponds to 700 kDa of molecular mass, which is consensually viewed as the full mass of the ATPsynthase complex.Moreover, the ATPase activity was detected in zones corresponding to molecular masses of over 900 kDa, which probably signifies oligomeric enzyme forms, and 400 kDa, which is approximately the molecular mass of coupling factor CF 1 .The latter is the catalytic hydrophilic part of the ATP-synthase complex containing no hydrophobic subunits responsible for anchoring the complex in membrane.CF 1 retains the ability to catalyze ATP hydrolysis, which permits its easy detection after electrophoresis.The second dimension electrophoresis in denaturing conditions in the presence of SDS (Laemmli system) may also be used to identify protein zones associated with ATP synthase.
The two-dimensional protein electrophoresis in PAA gel, which combines the potentials of native gel and denaturing gel electrophoresis (in the presence of SDS), is widely used for analysis of protein and peptide composition of biological objects [23].As the native proteins and protein complexes have been separated (the first dimension), the gel strip is cut out, incubated in the presence of SDS and mercaptoethanol and then separated in denaturing SDS system (Laemmli system) in the second dimension.This approach makes possible the analysis of peptide composition of protein complexes that exhibit certain specific enzymatic activity.
As the number, stoichiometry, and molecular mass of all the ATP-synthase subunits are known, it is possible to identify certain protein zones of ATPase activity with full ATP-synthase complex or with its catalytic part CF 1 , in accordance with their polypeptide composition.The electrophoretic plate of polypeptide zones with ATPase enzymatic activi ty (Fig. 3) demonstrates that protein content associated with the polypeptide zone of molecular mass approximating 700 kDa corresponds the polypeptide composition of the full ATP-synthase complex -CF 0 CF 1 (of nine polypeptide component : CF 0 subunits I (19 kDa), II (16.5 kDa), III (8 kDa), IV (25 kDa); and CF 1 subunits α (60 kDa), β (56 kDa), γ (39 kDa), δ (20.5 kDa), ε (14.7 kDa)) [25].Thus, the zone of native gel that corresponds to molecular експериментальні роботи

Fig. 2. electrophoretic analysis of solubilized native protein complexes of spinach thylakoid membranes in PAA gel: A -the gel was stained with coomassie G-250 (1 -marker proteins); B -the gel was incubated in
AtPase activity detection medium mass of 700 kDa may be identified (according to its protein composition) as the full ATP-synthase complex, and the zone corresponding to molecular mass of 400 kDa (Fig. 2 and 3) as the coupling factor CF 1 .

electrophoretic analysis of peptide composition of protein complexes from spinach thylakoid membranes (second dimension): 1 -peptide composition of AtP-synthase complex; 2 -peptide composition of functional complexes; 3 -marker proteins; 4 -PAA gel strip after native electrophoresis
The most pronounced CA activity out of those detected in native gel polypeptides was associated with PS ii protein complex (Fig. 4).As mentioned above, there are two proteins with CA activity in PS  The protein zones of native gel identified as ATP synthase complex and coupling factor CF 1 as per their ATPase activity and subunit composition were catalytically active in CA reaction under our experimental conditions (Fig. 4).

Fig. 4. electrophoretic analysis of solubilized native protein complexes of spinach thylakoid membranes in PAA gel: A -the gel was stained with coomassie G-250 (1 -marker proteins); B -the gel was incubated in carbonic anhydrase activity detection medium
We extracted and purified coupling factor CF 1 from thylakoid membrane components in order to define the specific localization of CA activity.The results of electrophoretic analysis of isolated CF 1 demonstrate its peptide composition (Fig. 5).Apparently, under native conditions the sample contains single polypeptide with approximate molecular mass of 400 kDa, which corresponds to molecular mass of CF 1 as described elsewhere.The purified CF 1 polypeptide is degraded under the effect of SDS and mercaptoethanol during second dimension electrophoresis into five protein zones with molecular masses close to those of coupling factor's subunits [25].The sample was catalytically active both in ATPase reaction and in CA reaction (Fig. 5 and 6; Table ).
As can be seen, the CA activity of isolated CF 1 is inhibited by water-soluble acetazolamide (AzA), and is not significantly inhibited by lipophilic ethoxyzolamide (ezA).This is in contrast to the results of the studies of effects by these inhibitors on CA activity of PS ii, where ezA had been more efficient in inhibition than AzA [7,8].

A
II complex[7,8], which may explain the more intensive coloration of PS II protein zone.

Fig. 5 .CF1
Fig. 5. Electrophoretic analysis of purified native coupling factor CF 1 in PAA gel: A -the gel was stained with coomassie G-250 (1 -marker proteins, 2 -native protein complexes of thylakoid membranes); B -the gel was incubated in AtPase activity detection medium; C -the gel was incubated in carbonic anhydrase activity detection medium