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1.
The binding of endogenous manganese (Mn) to proteins released from spinach grana-thylakoid membranes by 2% cholate detergent or by osmotic shock is investigated. A mixture of 15–20 proteins is released by cholate and has been separated by isoelectric focusing in a sucrose gradient or by chromatofocusing. Mn coelutes with several proteins, but is lost upon dialysis. A dramatic redistribution of this Mn occurs in proteins released by osmotic shock in the presence of hydrophobic and hydrophilic oxidants. Maintaining an oxidizing solution potential during extraction apparently precludes reduction of the higher oxidation states of Mn to the labile Mn(II) state by reducing agents released from the membranes during lysing. This allows proteins to be separated which bind non-labile Mn ions. Under these extraction conditions, a protein is isolated which has an apparent molecular weight ( Mr) of 65 000 or 56 000 on SDS-polyacrylamide gel electrophoresis depending on the sample buffer system used. The nondissociated protein occurs as a monomer of 58 kDa (90%) and an apparent dimer of 112 kDa (10%) by gel filtration. This protein binds little Mn if extracted by cholate and separated by isoelectric focusing. However, extraction by osmotic shock in the presence of oxidants and separation by chromatofocusing results in the retention of 1.9 ± 0.3 Mn ions per monomer. This protein is identical to that reported by Spector and Winget (Spector, M., and Winget, G.D. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 957–959). Contrary to their result, this protein does not reconstitute O 2 evolution when added to depleted membranes. Rabbit antibody to this purified protein inhibits O 2 evolution by 20% when incubated with intact grana-thylakoid membranes or 10–20% with partially inverted, French-pressed thylakoids. This inhibition is completely removed by 10 ?3 M NH 3Cl as an uncoupler of photophosphorylation. These results support a role in Phosphorylation and a location on the outer surface of the thylakoids. This antibody also selectively binds purified coupling factor, CF 1, the multisubunit phosphorylation enzyme which is located on the outer thylakoid surface and which is known to bind two Mn ions tightly (Hochman, Y. and Carmeli, C. (1981) Biochemistry 20, 6293–6297). Thus the β-subunit of CF 1, which has a molecular weight of 56 kDa, can be identified as the locus of Mn binding in CF 1 and as the Mn protein isolated by Spector and Winget. This protein plays no role on O 2 evolution. 相似文献
2.
When spinach chloroplast membranes were exposed to osmotic stress in vitro, by incubation in 1.0 M sorbitol + 10 mM MgCl 2 their oxygen evolving system was suppressed. The possible reasons for such inactivation of PS II mediated oxygen evolution were examined. There were conformational changes in the chloroplast membranes, as indicated by their absorption spectra. The pattern of sensitivity to DCMU was not altered. The sensitivity of PS II to water stress remained, even after a pre-wash treatment with NaCI (which removed 18 and 24 kD proteins) but not when the thylakoids were pretreated with NH 20H or CaCl 2 (removed manganese and 33 kD). The manganese content of thylakoid membranes was markedly reduced under osmotic stress in presence of magnesium. We suggest that exposure of chloroplasts to 1.0 M sorbitol in presence of Mg 2+ released manganese from thylakoid membranes, thereby leading to a suppression in oxygen evolution. 相似文献
3.
In weak yet optimal light intensity, complete photoactivation of the water-oxidizing enzyme in NH 2OH-extracted wheat ( Triticum aestivum, var Oasis) leaf segments could be obtained only after long dark preincubation. Photoactivation was not affected by ethylenediaminetetraacetate or inhibitors of photophosphorylation and protein synthesis, but was partially inhibited by a divalent cation ionophore. Complete photoactivation required ligation of ~4 Mn by the water oxidizing enzyme. Without dark preincubation, photosystem II (PSII) was susceptible to weak light photoinhibition resulting in: (a) 50% maximum decrease in photooxidation of artificial electron donors by PSII: (b) increased times for the variable fluorescence rise (with 3-(3,4-dichlorophenyl)-1,1-dimethyl urea): (c) abolishment of photoactivation: and (d) the imposition of sensitivity to inhibitors of photophosphorylation and 70S but not 80S protein synthesis on subsequent light-dependent recovery from photoinhibition and recovery of O2 evolution. Decrease in susceptibility to photoinhibition and increase in rates of photoactivation resulting from dark preincubations proved closely correlated. Neither protein synthesis nor increases in abundances of thylakoid Mn2+ and Ca2+ were required for escape from photoinhibition. However, photoactivation of the wateroxidizing enzyme in NH2OH-extracted Chlamydomonas occurred in absence of dark preincubation and protein synthesis. Results are discussed in the context of disassembly/reassembly/resynthesis of specific PSII polypeptides. 相似文献
4.
Cyclic and square wave voltammetry (−1500 to 1500 mV) of {Mn[μ-(C 6H 5) 2NC 4O 3] 2[H 2O] 4} n [manganese(II) diphenylaminosquarate] ( 1) and [Mn(μ-C 6H 5C 4O 3)(C 6H 5C 4O 3)(H 2O) 3] n [manganese(II) phenylsquarate] ( 2) at a gold disk electrode in dimethylsulfoxide (DMSO) and dimethylformamide (DMF), reveal several couples attributable to both ligand and metal-based redox processes. For the manganese(II) phenylsquarate in DMF, the metal-based peaks are more numerous and readily discernible than in DMSO. In either of the solvents, the ligand-based peaks always occur at more positive or more negative potentials than the metal-based ones. In 1 and 2, Mn(II)/Mn(0), Mn(III)/Mn(II), Mn(IV)/Mn(III) and Mn(V)/Mn(IV) couples are observed. However, the manganese redox peaks appear at more negative potentials in 1. 相似文献
5.
Proton spin-lattice relaxation rates ( R1) have been measured in a variety of dark-adapted chloroplast suspensions over a range of field strengths between 1 and 15 kG (4–5 MHz). When the effects of EDTA or Tris washing on chloroplast relaxivities are compared, the pool of Mn associated with oxygen evolution is seen not to contribute significantly to relaxivity. Instead, nearly all of the observed relaxivity, which is characterized by a paramagnetic maximum near 20.7 MHz in the field dispersion profile of R1, appears to arise from contaminating non-functional Mn(II) that can be removed by EDTA during the isolation procedure. These observations, which contradict previous reports ascribing chloroplast relaxivity to the water-oxidizing system, require a reevaluation of proposed models, derived from NMR studies, of the state of Mn in the water-splitting reaction.Chloroplasts from which loosely bound non-functional Mn has been removed by EDTA washing do show an enhancement of relaxivity when exposed to NH 2OH at concentrations known to inactivate water oxidation. This NH 2OH-induced relaxivity is comprised of Mn(II) in two distinct paramagnetic sites. One site is chelatable by EDTA, whereas the other site is not. This finding suggests that some Mn(II) tightly bound to thylakoid membranes can contribute to relaxivity after inactivation of the oxygen-evolving reaction. 相似文献
6.
The effect of high salt concentration on photosystem II (PS II) electron transport rates and chlorophyll a fluorescence induction kinetics was investigated in coupled and uncoupled spinach thylakoid membranes. With increase in salt
concentration, the rates of electron transport mediated by PS II and the F
v/ F
m ratio were affected more in uncoupled thylakoids as compared to coupled thylakoid membranes. The uncoupled thylakoid membranes
seemed to behave like coupled thylakoid membranes at high NaCl concentration (∼1 M). On increasing the salt concentration,
the uncoupler was found to be less effective and Na + probably worked as a coupling enhancer or uncoupling suppressor. We suggest that positive charge of Na + mimics the function of positive charge of H + in the thylakoid lumen in causing coupled state. The function of NaCl (monovalent cation) could be carried out by even lower
concentration of Ca 2+ (divalent cation) or Al 3+ (trivalent cation). We conclude that this function of NaCl as coupling enhancer is not specific, and in general a positive
charge is required for causing coupling in uncoupled thylakoid membranes.
Published in Russian in Biokhimiya, 2009, Vol. 74, No. 6, pp. 761–767. 相似文献
7.
Extraction conditions have been found which result in the retention of managanese to the 33–34 kDa protein, first isolated as an apoprotein by Kuwabara and Murata (Kuwabara, T. and Murata, N. (1979) Biochim. Biophys Acta 581, 228–236). By maintaining an oxidizing-solution potential, with hydrophilic and lipophilic redox buffers during protein extraction of spinach grana-thylakoid membranes, the 33–34 kDa protein is observed to bind a maximum of 2 Mn/protein which are not released by extended dialysis versus buffer. This manganese is a part of the pool of 4 Mn/Photosystem II normally associated with the oxygen-evolving complex. The mechanism for retention of Mn to the protein during isolation appears to be by suppression of chemical reduction of natively bound, high-valent Mn to the labile Mn(II) oxidation state. This protein is also present in stoichiometric levels in highly active, O 2-evolving, detergent-extracted PS-II particles which contain 4–5 Mn/PS II. Conditions which result in the loss of Mn and O 2 evolution activity from functional membranes, such as incubation in 1.5 mM NH 2OH or in ascorbate plus dithionite, also release Mn from the protein. The protein exists as a monomer of 33 kDa by gel filtration and 34 kDa by gel electrophoresis, with an isoelectric point of 5.1 ± 0.1. The protein exhibits an EPR spectrum only below 12 K which extends over at least 2000 G centered at g = 2 consisting of non-uniformly separated hyperfine transitions with average splitting of 45–55 G. The magnitude of this splitting is nominally one-half the splitting observed in monomeric manganese complexes having O or N donor ligands. This is apparently due to electronic coupling of the two 55Mn nuclei in a presumed binuclear site. Either a ferromagnetically coupled binuclear Mn 2(III,III) site or an antiferromagnetically coupled mixed-valence Mn 2(II,III) site are considered as possible oxidation states to account for the EPR spectrum. Qualitatively similar hyperfine structure splittings are observed in ferromagnetically coupled binuclear Mn complexes having even-spin ground states. The extreme temperature dependence suggests the population of low-lying excited spin states such as are present in weakly coupled dimers and higher clusters of Mn ions, or, possibly, from efficient spin relaxation such as occurs in the Mn(III) oxidation state. Either 1.5 mM NH 2OH or incubation with reducing agents abolishes the low temperature EPR signal and releases two Mn(II) ions to solution. This is consistent with the presence of Mn(III) in the isolated protein. The intrinsically unstable Mn 2(II,III) oxidation state observed in model compounds favors the assignment of the stable protein oxidation state to the Mn 2(III,III) formulation. This protein exhibits characteristics consistent with an identification with the long-sought Mn site for photosynthetic O 2 evolution. An EPR spectrum having qualitatively similar features is observable in dark-adapted intact, photosynthetic membranes (Dismukes, G.C., Abramowicz, D.A., Ferris, F.K., Mathur, P., Upadrashta, B. and Watnick, P. (1983) in The Oxygen-Evolving System of Plant Photosynthesis (Inoue, Y., ed.), pp. 145–158, Academic Press, Tokyo) and in detergent-extracted, O 2-evolving Photosystem-II particles (Abramowicz, D.A., Raab, T.K. and Dismukes, G.C. (1984) Proceedings of the Sixth International Congress on Photosynthesis (Sybesma, C., ed.), Vol. I, pp. 349–354, Martinus Nijhoff/Dr. W. Junk Publishers, The Hague, The Netherlands), thus establishing a direct link with the O 2 evolving complex. 相似文献
8.
The role of Cl ? in the electron transfer reactions of the oxidizing side of Photosystem II (PS II) has been studied by measuring the fluorescence yield changes corresponding to the reduction of P +-680, the PS II reaction center chlorophyll, by the secondary PS II donor, Z. In Cl ?-depleted chloroplasts, a rapid rise in fluorescence yield was observed following the first and second flashes, but not during the third or subsequent flashes. These results indicate that there exists an additional endogenous electron donor beyond P-680 and Z in Cl ?-depleted systems. In contrast, the terminal endogenous donor on the oxidizing side of PS II in Tris-washed preparations has previously been shown to be Z, the component giving rise to EPR signals II f and II vf. The rate of reduction of P +-680 in the Cl ?-depleted chloroplasts was as rapid as that measured in uninhibited systems, within the time resolution of our instrument. Again, this is in contrast to Tris-washed preparations in which a dramatic decrease in the rate if this reaction has been previously reported. We have also carried out a preliminary study on the rate of rereduction of Z + in the Cl ?-depleted system. Under steady-state conditions, the reduction half-time of Z + in uninhibited systems was about 450 μs, while in the Cl ?-depleted chloroplasts, the reduction of Z + was biphasic, one phase with a half-time of about 120 ms, and a slower phase with a half-time of several seconds. The appearance of the quenching state due to P +-680 observed following the third flash on excitation of Cl ?-depleted chloroplasts was delayed by two flashed when low concentrations of NH 2OH (20–50 μM) were included in the medium. Hydrazine at somewhat higher concentrations showed the same effect. This is taken to indicate that the reactions leading to PS II oxidation of NH 2OH or NH 2NH 2 are uninhibited by Cl ? depletion. Addition of NH 2OH at low concentrations to Tris-washed chloroplasts did not alter the pattern of the fluorescence yield, indicating that the reactions leading to the NH 2OH oxidation present in Cl ?-depleted systems are absent following Tris inhibition. The results are discussed in terms of an inhibition by Cl ? depletion of the reactions of the oxygen-evolving complex. It is suggested that no intermediary redox couple exists between the oxygen-evolving complex and Z, and that Z + is reduced directly by Mn of the complex. In terms of the S-state model, Cl ? depletion appears to inhibit the advancement of the mechanism beyond S 2, but not to inhibit the transitions from S 0 to S 1, or from S 1 to S 2. 相似文献
9.
Divalent salt-washing of O 2-evolving PS II particles caused total liberation of 33-, 24- and 16-kDa proteins, but the resulting PS II particles retained almost all amounts of Mn present in initial particles. The retained Mn was EPR-silent when the particles were kept in high concentrations of divalent salt. By divalent salt-washing, the activity of diphenylcarbazide (DPC) photooxidation was not affected at all, neither suppressed nor enhanced, while O 2 evolution was totally inactivated. These results indicate that Mn can be kept associated with PS II particles even after liberation of the 33-kDa protein, and suggest that the 33-kDa protein is probably not responsible for binding Mn onto membranes, but is possibly responsible for maintaining the function of Mn atoms in the O 2-evolving center. 相似文献
10.
The mechanism of the fact that manganese deprivation and cerium addition affect the photochemical efficiency of plants is
unclear. In this study, we investigated the improvement by cerium of the damage of the photochemical function of maize chloroplasts
under manganese-deprived stress. Chlorophyll fluorescence induction measurements showed that the ratio of variable to maximum
fluorescence (Fv/Fm) underwent great decreases under manganese deficiency, which was attributed to the reduction of intrinsic
quantum efficiency of the photosystem II units. The electron flow between the two photosystems, activities of Mg 2+–ATPase and Ca 2+–ATPase, and rate of photophosphorylation on the thylakoid membrane of maize chloroplasts were reduced significantly by exposure
to manganese deprivation. Furthermore, the inhibition of cyclic photophosphorylation was more severe than non-cyclic photophosphorylation
under manganese deficiency. However, added cerium could relieve the inhibition of the photochemical reaction caused by manganese
deprivation in maize chloroplasts. It implied that manganese deprivation could disturb photochemical reaction of chloroplasts
strongly, which could be improved by cerium addition. 相似文献
11.
Mesophyll protoplasts were isolated from unhardened and cold-acclimated leaves of Valerianella locusta L. and subjected to freeze-thaw treatment. To evaluate the extent and course of freezing injury, photosynthetic reactions of whole protoplasts and of free thylakoid membranes, liberated from protoplasts by osmotic lysis, were measured. In addition, the integrity of the protoplasts was determined by microscopy. The results reveal an increased frost tolerance of protoplasts isolated from acclimated leaves with respect to all parameters measured. CO 2-dependent O 2 evolution (representing net photosynthetic CO 2 fixation of protoplasts) was the most freezing-sensitive reaction; its inhibition due to freeze-thaw treatment of protoplasts was neither correlated with disintegration of the plasma membrane, nor was it initiated by inactivation of the thylakoid membranes. The frost-induced decline of protoplast integrity was not closely correlated to thylakoid damage either. Freezing injury of the thylakoid membranes was manifested by inhibition of photosynthetic electron transport and photophosphorylation. Both photosystems were affected by freezing and thawing with strongest inhibition occurring in the water-oxidation system or at the oxidizing site of photosystem II. Photophosphorylation responded more sensitively to freezing stress than electron transport, although uncoupling (increased permeability of the thylakoid membranes to protons) was not a conspicuous effect. The data are discussed in relation to freezing injury in leaves and seem to indicate that frost damage in vivo is initiated at multiple sites.Abbreviations Chl
chlorphyll
- DCMU
3-(3,4-dichlorophenyl)-1,1-dimethylurea
- DCIP
2,6-dichlorophenolindophenol
- DPC
1,5-diphenylcarbazide
- Hepes
2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid
- MES
2-(N-morpholino)-ethanesulfonic acid
- PS I
photosystem I
- PS II
photosystem II 相似文献
12.
Treatment of intact thylakoid membranes with Triton X-100 at pH 6 produces a preparation of the PS II complex capable of high rates of O 2 evolution. The preparation contains four managanese, one cytochrome b-559, one Signal II f and one Signal II s per 250 chlorophylls. By selective manipulation of the preparation polypeptides of approximate molecular weights of 33, 23 and 17 kDa can be removed from the complex. Release of 23 and 17 kDa polypeptides does not release functional manganese. Under these conditions Z + is not readily and directly accessible to an added donor (benzidine) and it appears as if at least some of the S-state transitions occur. Evidence is presented which indicates that benzidine does have increased access to the oxygen-evolving complex in these polypeptide depleted preparations. Conditions which release the 33 kDa species along with Mn and the 23 and 17 kDa polypeptides generate an alteration in the structure of the oxidizing side of PS II, which becomes freely accessible to benzidine. These findings are examined in relationship to alterations of normal S-state behavior (induced by polypeptide release) and a model is proposed for the organization of functional manganese and polypeptides involved in the oxygen-evolving reaction. 相似文献
13.
Thylakoid membranes were treated with either pancreatic or snake venom phospholipase A 2, and the residual phospholipid content of these membranes was determined and compared to the rates of Photosystem II and/or Photosystem I electron transports. The hydrolysis curves of both phosphatidylglycerol and phosphatidylcholine displayed a first, rapid phase which was almost temperature-insensitive, followed by a second, slower phase which depended strongly on the temperature. When pancreatic phospholipase A 2 had access either to the outer face or to both faces of the thylakoid membrane, either only part of or all the phospholipids, respectively, could be hydrolysed. These results were interpreted as indicating an asymmetric distribution of phospholipids across the thylakoid membrane, phosphatidylglycerol and phosphatidylcholine being preferentially located in the outer and the inner layer, respectively. When acting on uncoupled thylakoid membranes, phospholipase A 2 exerted an inhibitory effect on Photosystem II activity and a stimulatory effect on Photosystem I activity. The involvement of phosphatidylcholine and of phosphatidylglycerol in electron transport activities of Photosystem II and of Photosystem I are discussed with special reference to the role of the external and internal pools of these phospholipids. 相似文献
14.
Removal of coupling factor protein (CF 1) from spinach thylakoid membranes results in an enhancement of proton permeability but has no effect on chloride or potassium permeability. Anion permeability was measured by the rate of thylakoid packed volume changes. Potassium permeability was monitored by turbidity changes, packed thylakoid volume changes and ion flux studies using 86Rb + as a tracer. 45Ca 2+ was used to measure divalent cation fluxes. CF 1-depleted chloroplasts had an unaltered rate of Ca 2+ uptake, but the rate of Ca 2+ efflux appeared to be increased. Calcium efflux rates could also be increased by the addition of a proton specific uncoupler, FCCP. 相似文献
15.
The effects of nano-TiO 2 (rutile) on the photochemical reaction of chloroplasts of spinach were studied. The results showed that when spinach was
treated with 0.25% nano-TiO 2, the Hill reaction, such as the reduction rate of FeCy, and the rate of evolution oxygen of chloroplasts was accelerated
and noncyclic photophosphorylation (nc-PSP) activity of chloroplasts was higher than cyclic photophosphorylation (c-PSP) activity,
the chloroplast coupling was improved and activities of Mg 2+-ATPase and chloroplast coupling factor I (CF 1)-ATPase on the thylakoid membranes were obviously activated. It suggested that photosynthesis promoted by nano-TiO 2 might be related to activation of photochemical reaction of chloroplasts of spinach. 相似文献
16.
The role of carboxylic residues at the high-affinity, Mn-binding site in the ligation of iron cations blocking the site [Biochemistry 41 (2000) 5854] was studied, using a method developed to extract the iron cations blocking the site. We found that specifically bound Fe(III) cations can be extracted with citrate buffer at pH 3.0. Furthermore, citrate can also prevent the photooxidation of Fe(II) cations by Y Z. Participation of a COOH group(s) in the ligation of Fe(III) at the high-affinity site was investigated using 1-ethyl-3-[(3-dimethylamino)propyl] carbodiimide (EDC), a chemical modifier of carboxylic amino acid residues. Modification of the COOH groups inhibits the light-induced oxidation of exogenous Mn(II) cations by Mn-depleted photosystem II (PSII[−Mn]) membranes. The rate of Mn(II) oxidation saturates at ≥10 μM in PSII(−Mn) membranes and ≥500 μM in EDC-treated PSII (−Mn) samples. Intact PSII(−Mn) membranes have only one site for Mn(II) oxidation via Y Z (dissociation constant, Kd = 0.64 μM), while EDC-treated PSII(−Mn) samples have two sites ( Kd = 1.52 and 22 μM; the latter is the low-affinity site). When PSII(−Mn) membranes were incubated with Fe(II) before modifier treatment (to block the high-affinity site) and the blocking iron cations were extracted with citrate (pH 3.0) after modification, the membranes contained only one site ( Kd = 2.3 μM) for exogenous Mn(II) oxidation by Y Z radical. In this case, the rate of electron donation via Y Z saturated at a Mn(II) concentration ≥15 μM. These results indicate that the carboxylic residue participating in Mn(II) coordination and the binding of oxidized manganese cations at the HA Z site is protected from the action of the modifier by the iron cations blocking the HA Z site. We concluded that the carboxylic residue (D1 Asp-170) participating in the coordination of the manganese cation at the HA Z site (Mn4 in the tetranuclear manganese cluster [Science 303 (2004) 1831]) is also involved in the ligation of the Fe cation(s) blocking the high-affinity Mn-binding site. 相似文献
17.
The kinetics of Mn release during NH 2OH inactivation of the water oxidizing reaction is largely insensitive to the S-state present during addition of NH 2OH. This appears to reflect reduction by NH 2OH of higher S-states to a common more reduced state (S 0 or S ?1) which alone is susceptible to NH 2OH inactivation. Sequences of saturating flashes with dark intervals in the range 0.2–5 s ?1 effectively prevent NH 2OH inactivation and the associated liberation of manganese. This light-induced protection disappears rapidly when the dark interval is longer than about 5 s. Under continuous illumination, protection against NH 2OH inactivation is maximally effective at intensities in the range 10 3–10 4 erg · cm ?2 · s ?1. This behavior differs from that of NH 2OH-induced Mn release, which is strongly inhibited at all intensities greater than 10 3 erg · cm ?2 · s ?1. This indicates that two distinct processes are responsible for inactivation of water oxidation at high and low intensities. Higher S-states appear to be immune to the reaction by which NH 2OH liberates manganese, although the overall process of water oxidation is inactivated by NH 2OH in the presence of intense light. The light-induced protection phenomenon is abolished by 50 μM DCMU, but not by high concentrations of carbonyl cyanide m-chlorophenylhydrazone, which accelerates inactivation reactions of the water-splitting enzyme, Y (an ADRY reagent). The latter compound accelerates both inactivation of water oxidation and manganese extraction in the dark. 相似文献
19.
We used Triton-prepared PS II membranes in studies of the inactivation of O 2 evolution and solubilization of Mn and specific PS II polypeptides by NH 2OH, N- and O-substituted NH 2OH derivatives, NH 2NH 2 and NH 4Cl. The inactivation of O 2-evolution, solubilization of Mn and the solubilization of the extrinsic PS II polypeptides (17, 23 and 33 kDa) proved closely correlated, half-maximal effects occurring with only 100 μM NH 2OH. NH 2OH (2 mM) and NaCl (1 M) extractions solubilized about one-half the amount of protein solubilized by 0.8 M Tris-HCl (pH 8.0). The inactivation of the Mn-S-state complex proceeded by apparent first-order kinetics, the rate constant dependent on NH 2OH (CH 3NHON) concentration and pH. In the range of micromolar concentrations of NH 2OH, this inactivation did not occur via a cooperative type mechanism. Depletion of the 17 and 23 kDa proteins modified the pH dependency of inactivation (from pH 7.8 to 6.5) and also resulted in an approx. 2-fold maximum increase in the inactivation rate constant. Significantly, reconstitution of such NaCl-TMF-2 membranes with the 17 and 23 kDa proteins reverted both the pH dependency and the inactivation rate constant to that of TMF-2. A hierarchy of effectivity for solubilization of Mn and protein, which was highly correlated with inactivation of the Mn-S-state enzyme, was observed among NH 2OH and its derivatives. This same hierarchy was observed irrespective of prior depletion of the 17 and 23 or the 17, 23 and 33 kDa proteins from TMF-2. The hierarchy of effectivity among derivatives was: NH 2OH > CH 3NHOH > NH 2NH 2, NH 2OSO 3 > NH 2OCH 3 ⪢ CH 3NHOCH 3, NH 4Cl. The function(s) of the extrinsic PS II proteins as determinants of the reactivity of the Mn-S-state complex with polar amine vs other type compounds is discussed. 相似文献
20.
(1) In isolated chloroplasts (class B) electron flow is controlled mainly by the intrathylakoid pH (pH in). A decrease in pH in due to the light-driven injection of protons inside the thylakoid leads to the retardation of electron flow between two photosystems. This effect can be abolished by uncouplers or under photophosphorylation conditions (addition of Mg 2+-ADP with P i); Mg 2+-ATP does not influence the steady-state rate of electron flow, (2) The steady-state pH difference, ΔpH, across the thylakoid membrane was estimated from quantitative analysis of the rate of P-700 + reduction. In chloroplasts, without adding Mg 2+-ADP, ΔpH increases from 1.6 to 3.2 as the external pH rises from 6 to 9.5. Under the photophosphorylation conditions, ΔpH decreases showing a minimum at the external pH 7.5 ( ). (3) The value of photosynthetic control, K, measured as the ratio of the steady-state rates of P-700 + reduction in the presence of Mg 2+-ADP (with P i) and without adding Mg 2+-ADP is dependent on external pH variations, showing a maximum value of at pH out 7.5. This pH dependence coincides with that of the ADP-stimulated ΔpH decrease. (4) Experiments with spin labels provide evidence that the light-induced changes in the thylakoid membrane are sensitive to the addition of uncouplers and are affected only slightly by the addition of Mg 2+-ADP and P i. 相似文献
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