Extrinsic Polypeptides Research Articles

Acid rain: Acidic mist-induced response in growth and photosynthetic activities on crop plants

Experiments were conducted in the field to investigate the influence of acidic mists treatment on height, biomass, leaf chlorophyll, net photosynthesis, and photosystem activities in Vigna sinensis L. and Phaseolus mungo L. Plants exposed to different acidic mists (pH 5, 4, 2) for 5 days showed significant reduction in height and biomass accumulation. The level of chlorophyll on a unit fresh weight basis showed progressive reduction upon increasing acidity of mists treatment; the reduction was due to the loss of Chl a and Chl b. Leaf-soluble protein was not significantly reduced. The increased stomatal diffusive resistance and reduced photosynthetic pigments lowered the net photosynthetic rate. However, when various photosynthetic activities were followed in isolated chloroplast, a decrease in the rates was obtained in the seedlings exposed to pH 4 and 2. No significant change in photosystem I activity was observed at various acidic mists treatments. Analysis of chloroplast polypeptides indicates specific loss of 33, 23, and 17 kDa extrinsic polypeptides.

The origin of 40–50°C thermoluminescence bands in Photosystem II

We have used thermoluminescence (TL) and EPR measurements of Photosystem II (PS II) from spinach in order to identify charge pairs responsible for TL bands in the region of 40–50°C including the ‘C-band’ (peak V) and the TL band from PS II depleted of calcium. In intact PS II membrane preparations, in the presence of DCMU, a TL band at 50°C is induced following illumination at 77 K. This band decays, at 30°C, with a half-time of 10 min. This decay corresponds to the disappearance of the EPR signal arising from Q A − and an accelerated decay of the organic free radical Tyr D +. It is concluded that recombination of this charge pair is probably responsible for the thermoluminescence emission. In PS II preparations that have been depleted of calcium using a salt/EGTA wash followed by rebinding of the extrinsic polypeptides, a TL band is produced at around 45–50°C following 198 K illumination. In such samples a stable S 2 state of the water-splitting complex is present, giving rise to a modified form of the EPR multiline signal. During incubation at 30°C in the dark this signal decays with a half-time around 20–25 min. This decay is not accelerated by the presence of Q A − induced by low-temperature illumination of the sample. In contrast, low-temperature illumination does result in an acceleration in the decay of Tyr D +, indicating that Tyr D +/Q − A recombination is again the dominant origin of thermoluminescence. In PS II depleted of calcium by incubation at pH 4.0, the possibility that TL emission temperature is determined by a change in the mid-point redox potential of Q A (Krieger, A. and Weis, E. (1992) Photosynthetica 27, 89–98) was investigated by comparing TL from equivalent samples of control and Ca 2+-depleted PS II. It was shown that the emission temperature of the high temperature TL band induced by illumination at 77 K did not differ significantly between control and treated samples, suggesting that, under the conditions used, the potential of Q A does not change significantly.

The distribution of Photosystem I and Photosystem II polypeptides between the cytoplasmic and thylakoid membranes of cyanobacteria

In a previous study we found that the 33 kDa extrinsic polypeptide of Photosystem II is present in both the cytoplasmic and thylakoid membranes of cyanobacteria, but forms part of a functional complex only in the latter [Smith et al. (1987) Mol. Microbiol. 6, 1821–1827]. In order to determine if this phenomenon is restricted to the 33 kDa polypeptide we have extended this study in Anacystis nidulans to include a number of other polypeptides of Photosystem I and Photosystem II. We have found that D1 and possibly PsaC are present in both membranes, CP43 and CP47 are confined to the thylakoid membranes, and the distribution of PsaD and PsaE is dependent upon the growth stage of the cyanobacteria.

The distribution of Photosystem I and Photosystem II polypeptides between the cytoplasmic and thylakoid membranes of cyanobacteria

In a previous study we found that the 33 kDa extrinsic polypeptide of Photosystem II is present in both the cytoplasmic and thylakoid membranes of cyanobacteria, but forms part of a functional complex only in the latter [Smith et al. (1987) Mol. Microbiol. 6, 1821–1827]. In order to determine if this phenomenon is restricted to the 33 kDa polypeptide we have extended this study in Anacystis nidulans to include a number of other polypeptides of Photosystem I and Photosystem II. We have found that D1 and possibly PsaC are present in both membranes, CP43 and CP47 are confined to the thylakoid membranes, and the distribution of PsaD and PsaE is dependent upon the growth stage of the cyanobacteria.

Topological study of PSI-A and PSI-B, the large subunits of the photosystem-I reaction center.

The core of the photosystem-I reaction center is formed by polypeptides PSI-A and PSI-B, the products of the homologous psaA and psaB genes. Based on hydropathy analyses, models have been proposed for the folding of these polypeptide chains in the membrane [Fish, L. E., Kück, U. & Bogorad, L. (1985), in Molecular biology of the photosynthetic apparatus, pp. 111-120, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY]. To test these models, we have tried to identify regions of PSI-A that are exposed to the surrounding medium, on the stromal or lumenal surface of the membrane. Immunogold labeling of thylakoid vesicles, with antibodies to synthetic peptides, shows that residues 413-421 of PSI-A are exposed on the stromal surface of the membrane, and that the accessibility of this region is enhanced by NaSCN treatment, which removes extrinsic polypeptides. This treatment also enhances a trypsin-cleavage site which may lie just after residues 413-421. Immunogold labeling also indicates that residues 371-379 and 497-505 are exposed on the lumenal surface. These results establish the conformation of the central portion of the polypeptide. Assuming that the transmembrane regions are correctly predicted by the 11-helix model, the N-terminal domain, as well as the conserved region proposed to bind the iron-sulfur center FX, would be expected to be on the stromal surface.

The manganese stabilising protein (MSP) of Prochlorothrix hollandica is a hydrophobic membrane-bound protein

The oxygen-evolving Photosystem II complex of the chlorophyll-a b- containing prokaryote Prochlorothrix hollandica is similar to that of cyanobacteria in terms of the absence of the 23 kDa extrinsic polypeptide. In contrast to cyanobacteria and green chloroplasts, the manganese stabilizing protein (MSP) of Prochlorothrix is a hydrophobic protein as detected by Triton X-114 phase partitioning and has an apparent molecular mass of 37 kDa. The catalytic site of water oxidation in Prochlorothrix thylakoids is significantly more resistant to inhibition by hydroxylamine as compared with thylakoids of Chlamydomonas or Synechocystis 6803. These results indicate that the properties of the donor side of Photosystem II complex of Prochlorothrix differ from that of the related cyanobacteria.

Modification of the thermoluminescence properties of Ca2+ depleted Photosystem II membranes by the 23 kDa extrinsic polypeptide and by oligocarboxylic acids

Photosystem II membranes were isolated from chloroplasts of pokeweed (Phytolacca americana) and rendered deficient in Ca(2+), an inorganic cofactor of photosynthetic water oxidation. The thermoluminescence properties of such membranes were found to depend on the Ca(2+)-depleting method used. This feature was analyzed with respect to the thermoluminescence emission that accompanied the recombination reaction between the reduced acceptor QA (-) and the oxidant of the S2 state. It was determined that the differences observed among various preparations of Ca(2+)-depleted membranes were attributable to the presence or absence of the extrinsic 23 kDa polypeptide on the membranes. The binding of this polypeptide to Ca(2+)-depleted membranes devoid of the 17 and 23 kDa extrinsic polypeptides caused the thermoluminescence to be emitted at a higher temperature due to a further stabilization of an already abnormally stable S2 state. Addition of the chelators EDTA or EGTA and of citrate brought about a similar response. The conditions required for the upshift of the emission temperature of thermoluminescence strongly resembled those identified by Boussac et al. (FEBS Lett. 277 (1990) 69-74) as responsible for modifying the EPR multiline signal from the S2 state of Ca(2+)-depleted PS II membranes. Consistent with the authors' interpretation of the reason for this modification, we conclude that the elevated emission temperature of the thermoluminescence emission reflects an abnormal ligand environment of the Mn-center in PS II that may be created by a direct ligation of the added agents to Mn. Evidence is also presented that the return to a normal S2 after an addition of Ca(2+) occurs via yet another condition of S2 which, in terms of its thermoluminescence properties, resembles that of Ca(2+)-depleted membranes before addition of modifying agents, but is not identical to it.

Thermoluminescence properties of the S2-state in chloride-depleted water oxidizing complexes after reconstituting treatments with various monovalent anions

Under conditions that assured rebinding of the extrinsic 17 and 23 kDa polypeptides, Cl(-)-depleted Photosystem II membranes isolated from spinach chloroplasts were subjected to reconstituting treatments in media containing NaF, NaCl, NaBr, NaI or NaNO3, or they were kept in a medium without any added salt other than the buffer. After removing most of the unbound reconstituting anions by washing, the O2-evolution activities and thermoluminescence properties of the membranes were compared. While the temperature of maximal thermoluminescence emission was lowest for membranes treated with Cl(-), no uniform correlation was evident between the temperature profile of the thermoluminescence emission and the apparent activating effectiveness of the anions in the membranes' water oxidizing machinery. However, the differences between the thermoluminescence features did conform to a trend according to which the emission temperatures were upshifted as the size of the activating anion increased, and its hydration energy decreased, i.e. Cl(-)<Br(-)<NO3 (-)<I(-). The inactive F(-) anions were not well retained by the membranes. To explain the experimental data it is suggested that the structural environment of the charge accumulating Mn-center is influenced by the ionic conditions encountered by the Photosystem II membranes after Cl(-) removal, further enforced by the binding of compatible anions, and then stabilized by the 17 and 23 kDa extrinsic polypeptides. If, as some concepts imply, the anion binding sites are located at or near the functional Mn, only very exceptional characteristics of the water-oxidizing mechanism may account for the observation that the potentially electron-donating I(-) anion can serve as activator and that it stabilizes rather than destabilizes the S2-state.

NMR paramagnetic relaxation enhancements due to manganese in the S0 and S2 states of Photosystem II-enriched membrane fragments and in the detergent-solubilized Photosystem II complex

The NMR paramagnetic relaxation enhancement (NMR-PRE) produced in the solvent proton resonance by manganese in the S0 and S2 states of the oxygen evolving center (OEC) has been recorded for three Photosystem II (PS II)-enriched preparations: (1) PS II-enriched thylakoid membrane fragments (TMF-2 particles); (2) salt-washed (2M NaCl) TMF-2 particles; and (3) the octylglucopyranoside (OGP)-solubilized PS II complex. The second and third preparations, but not the first, are depleted of the peripheral 17 and 23 kD polypeptides associated with the OEC. It has been proposed that depletion of these polypeptides increases the exposure of OEC manganese to the aqueous phase. The NMR-PRE response measures the quantity (T1m+τm)(-1), where T1m is the spin relaxation time and τm is the mean residence time with respect to chemical exchange reactions of solvent protons in the manganese coordination sphere, and, thus, the NMR-PRE provides a direct measure of the solvent proton chemical exchange rate constant τm (-1). This study tested whether the 17 and 23 kD polypeptides shield the OEC from the solvent phase and whether their depletion enhances the S2 and S0 NMR-PRE signals by removing a kinetic barrier to the solvent proton chemical exchange reaction. The amplitude of the S2 NMR-PRE signal, measured in its chemical exchange-limited regime (τm>T1m), is slightly decreased, rather than increased, in preparations (2) and (3) relative to (1), indicating that removal of the 17 and 23 kD polypeptides slightly slows, rather than accelerates, the rate-limiting steps of the solvent proton chemical exchange reactions. In addition, the lifetime of the S2 state was shortened several-fold in the solubilized PS II complex and in salt-washed TMF-2 membranes relative to untreated TMF-2 control samples. The S0 NMR-PRE signal, which is present in TMF-2 suspensions, was not detected in suspensions of the solubilized PS II complex, even though these samples contained high concentrations of active manganese centers (approximately double those of the TMF-2 control) and exhibited an S2 NMR-PRE signal of comparable amplitude to that of the TMF-2 preparation. These results suggest that the 17 and 23 kD extrinsic polypeptides do not shield the NMR-visible water binding site in the OEC from the aqueous phase, although their removal substantially alters the proton relaxation efficiency by shortening T1m.

Studies of the slowly exchanging chloride in Photosystem II of higher plants

(36)Cl(-) was used to study the slow exchange of chloride at a binding site associated with Photosystem II (PS II). When PS II membranes were labeled with different concentrations of (36)Cl(-), saturation of binding at about I chloride/PS II was observed. The rate of binding showed a clear dependence on the concentration of chloride approaching a limiting value of about 3·10(-4) s(-1) at high concentrations, similar to the rate of release of chloride from labeled membranes. These rates were close to that found earlier for the release of chloride from PS II membranes isolated from spinach grown on (36)Cl(-), which suggests that we are observing the same site for chloride binding. The similarity between the limiting rate of binding and the rate of release of chloride suggests that the exchange of chloride with the surrounding medium is controlled by an intramolecular process. The binding of chloride showed a pH-dependence with an apparent pKa of 7.5 and was very sensitive to the presence of the extrinsic polypeptides at the PS II donor side. The binding of chloride was competitively inhibited by a few other anions, notably Br(-) and NO3 (-). The slowly exchanging Cl(-) did not show any significant correlation with oxygen evolution rate or yield of EPR signals from the S2 state. Our studies indicate that removal of the slowly exchanging chloride lowers the stability of PS II as indicated by the loss of oxygen evolution activity and S2 state EPR signals.

Photocontrol of the Accumulation of Plastid Polypeptides during Greening of Tomato Cotyledons

A pulse of red light acting through phytochrome accelerates the formation of chlorophyll upon subsequent transfer of dark-grown seedlings to continuous white light. Specific antibodies were used to follow the accumulation of representative subunits of the major photosynthetic complexes during greening of seedlings of tomato (Lycopersicon esculentum). The time course for accumulation of the various subunits was compared in seedlings that received a red light pulse 4 h prior to transfer to continuous white light and parallel controls that did not receive a red light pulse. The light-harvesting chlorophyll-binding proteins of photosystem II (LHC II), the 33-kD extrinsic polypeptide of the oxygen-evolving complex (OEC1), and subunit II of photosystem I (psaD gene product) all increased in the light, and did so much faster in seedlings that received the inductive red light pulse. The red light pulse had no significant effect on the abundance of the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), nor on several plastid-encoded polypeptides: the large subunit of Rubisco, the beta subunit of the CF(1) complex of plastid ATPase, and the 43- and 47-kD subunits of photosystem II (CP43, CP47). Subunits I (cytochrome b(6)f) and III (Rieske Fe-S protein) of the cytochrome b(6)f complex showed a small or no increase as a result of the red pulse. The potentiation of greening by a pulse of red light, therefore, is not expressed uniformly in the abundance of all the photosynthetic complexes and their subunits.

Specific release of a 9-kDa extrinsic polypeptide of photosystem I from spinach chloroplasts by salt washing

The newly reported 9-kDa polypeptide in photosystem I [(1991) FEBS Lett. 280, 332–334) is an extrinsic component located on the lumenal side of the thylakoid membrane. This subunit can be solubilized with high salt buffer and does not bind any cofactors. The photosystem I electron transfer chain remains intact and functional in the absence of this component as characterized by the photoreduction of NADP*.

Stabilization of the water oxidizing polypeptide assembly on Photosystem II membranes by osmolytes and other solutes

The integrity of Photosystem II membranes isolated from chloroplast thylakoids is profoundly affected by the solute environment. Examples are given for stabilizing effects various solutes have on the binding of the 17 and 23 kDa extrinsic polypeptides under conditions conductive to their dissociation. It is concluded that these and many other solute effects on Photosystem II membranes can be accommodated readily in a concept developed by Timasheff and his coworkers according to which the responses of proteins to their solute environment are consequences of interaction preferences among the constituents of the solvent-protein-solute systems.

Requirement of manganese for the photooxidation of hydroxylamine by photosystem II

The relation of hydroxylamine photooxidation by photosystem II (PSII) and the presence of manganese was studied in PSII membranes either native or depleted in extrinsic polypeptides (17, 23 and 33 kDa) or depleted in both polypeptides and manganese. It was observed that the PSII catalyzed oxidation of hydroxylamine was inhibited if manganese was absent. We showed with various types of PSII preparations that the presence of manganese, endogenous or added, was essential for the photooxidation of hydroxylamine by photosystem II.

Interaction of Zn2+ with the donor side of Photosystem II

The inhibitory effect of Zn(2+) on photosynthetic electron transport was investigated in native and CaCl2-treated (depleted in extrinsic polypeptides) Photosystem II (PS II) submembrane preparations. Inhibition of 2,6-dichlorophenolindophenol photoreduction by Zn(2+) was much stronger in protein-depleted preparations in comparison to the native form. It was found that Ca(2+) significantly reduced the inhibition in the native PS II preparations, as did Mn(2+) in a combination with H2O2 in the protein-depleted counterparts. No other tested monovalent or divalent cations could replace Ca(2+) or Mn(2+) in the respective experiments. Diphenylcarbazide could partially relieve (40-45%) the inhibition in both types of preparations. The above indicates the presence of an active Zn(2+) inhibitory site on the donor side of PS II. However, neither Ca(2+) nor Mn(2+) could completely prevent inhibition by high concentrations of Zn(2+) (>1 mM). We propose that elevated levels of Zn(2+) strongly perturb the conformation of the PS II core complex and might also affect the acceptor side of the photosystem.

Electric parameters of photosystem II particles — electric light scattering study

Electric light scattering and microelectrophoresis were applied to investigate the electric moments (permanent dipole moment and electric polarizability and electrophoretic mobility of envelope-free chloroplasts and photosystem II (PS II particles. The effect of the removal of the extrinsic polypeptides (18, 24 and 33 kDa) on the electric moments was also studied. A significant difference was observed between the orientation behaviour of chloroplasts and PS II preparations. The data indicate that the permanent and induced dipole moments contribute to the orientation of the PS II particles, whereas chloroplasts possess induced dipole moment only. NaCl and Tris treatments of PS II preparations influence both the transverse permanent dipole moment and the electric polarizability of PS II particles. The increase in the electrophoretic mobility of PS II particles on removal of the extrinsic proteins corresponds to an increase in the electric polarizability value, demonstrating its interfacial nature.

Calcium retards hydroxylamine inhibition of oxygen evolution activity by stabilization of manganese(2+) binding to photosystem II

Calcium is required for oxidation of water to molecular oxygen by photosystem II; the Ca2+ demand of the reaction increases upon removal of 23- and 17-kDa extrinsic polypeptides from detergent-derived preparations of the photosystem. Employing the manganese reductant NH2OH as a probe to examine the function of Ca2+ in photosystem II reveals that (1) Ca2+ slows the rate of NH2OH inhibition of O2 evolution activity, but only in photosystem II membranes depleted of extrinsic proteins, (2) other divalent cations (Sr2+, Cd2+) that compete for the Ca2+ site also slow NH2OH inhibition, (3) Ca2+ is noncompetitive with respect to NH2OH, (4) in order to slow inhibition, Ca2+ must be present prior to the initiation of NH2OH reduction of manganese, and (5) Ca2+ appears not to interfere with NH2OH reduction of manganese. We conclude that the ability of Ca2+ to slow the rate of NH2OH inhibition arises from the site in photosystem II where Ca2+ normally stimulates O2 evolution and that the mechanism of this phenomenon arises from the ability of Ca2+ or certain surrogate metals to stabilize the ligation environment of the manganese complex.

Pancreatic Polypeptide and Peptide YY Inhibit the Denervated Canine Pancreas

Pancreatic polypeptide and peptide YY are inhibitors of pancreatic exocrine secretion in vivo but not in vitro, which suggests secondarily mediated mechanism(s) of action. To determine the role of extrinsic neural and intrinsic cholinergic elements on this inhibitory effect, a total of nine dogs underwent two-stage extrapancreatic denervation and creation of a chronic pancreatic fistula. After recovery, pancreatic polypeptide or peptide YY (400 pmol/kg/h) was administered during the intermediate hour of a 3-hour secretion (125 ng/kg/h)/cholecystokinin (50 ng/kg/h) infusion. Exocrine secretion pancreatic polypeptide or peptide YY hours was compared with that of the first and third hours. The studies were then repeated during infusion of atropine (10 micrograms/kg/h). Despite extrinsic denervation, pancreatic polypeptide and peptide YY significantly inhibited secretin/cholecystokinin-induced pancreatic output. Although less profound, significant inhibition persisted in the presence of an atropine background. Pancreatic polypeptide or peptide YY infusion also decreased the exocrine response to meal stimulation. We conclude that the inhibitory effects of pancreatic polypeptide and peptide YY are not mediated by extrapancreatic, and possibly not by intrapancreatic cholinergic, neural pathways.

Effect of linolenic acid on release of the 43 kDa polypeptide and manganese from photosystem II membranes depleted of the 33 kDa extrinsic polypeptide

The effect of linolenic acid (18:3) on release of the 43 kDa polypeptide and manganese from photosystem II (PS II) membranes depleted of extrinsic polypeptides was studied. In both control and NaCl‐washed particles which were depleted of the extrinsic 23 and 16 kDa polypeptides, the 18:3 treatment caused a 20% release of the 33 and 43 kDa polypeptides. In CaCl2, (or urea + NaCl)‐washed particles, which were depleted of the 33 kDa polypeptide in addition to the 23 and 16 kDa polypeptides, the release of the 43 kDa polypeptide increased to 70%, whereas only 25% of the 47 kDa polypeptide was removed. These findings suggest (i) that the 33 and the 43 kDa polypeptides are neighbows in the photosynthetic membrane and (ii) that the 33 kDa polypeptide shields the 43 kDa polypeptide against the action of 18:3. Incubation of CaCl2, or (urea + NaCI)‐treated PSII particles in the presence or absence of 18:3 resulted in the loss of only 2 of the 4 Mn atoms present per reaction center. this indicates that the 2 Mn atoms more firmly associated with PSII are not affected by the removal of the extrinsic 16, 23 and 33 kDa polypeptides, and the intrinsic 43 kDa polypeptide. nor by the treatment with linolenic acid.

Effect of linolenic acid on release of the 43 kDa polypeptide and manganese from photosystem II membranes depleted of the 33 kDa extrinsic polypeptide

Effect of linolenic acid on release of the 43 kDa polypeptide and manganese from photosystem II membranes depleted of the 33 kDa extrinsic polypeptide