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Two light-harvesting proteins associated with photosystem II of higher plants, namely the major antenna complex LHCIIb and the minor Lhcb4 protein (CP29), have been investigated by resonance Raman spectroscopy. One of the two chlorophylls b and up to five of the six chlorophylls a present in Lhcb4 are shown to adopt similar binding conformations to the (presumably) corresponding molecules in LHCIIb, whereas at least two chlorophylls in the former protein assume unique conformations relative to the bulk complex. The overall conformation of bound xanthophyll molecules is identical in the two antenna proteins, although some small differences are apparent. The pigment binding properties of these two LHCs are discussed, with particular reference to possible structural motifs within this extended family of proteins.

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A spectroscopic characterization is presented of the minor photosystem II chlorophyll a/b-binding protein CP29 (or the Lhcb4 protein) from spinach, prepared by a modified form of a published protocol [Henrysson, T., Schroder, W. P., Spangfort, M. & Akerlund, H.-E. (1989) Biochim. Biophys. Acta 977, 301-308]. The isolation procedure represents a quicker, cheaper means of isolating this minor antenna protein to an equally high level of purity to that published previously. The pigment-binding protein shows similarities to other related light-harvesting complexes (LHCs), including the bulk complex LHCIIb but more particularly another minor antenna protein CP26 (Lhcb5). It is also, in the main, similar to other preparations of CP29, although some significant differences are discussed. In common with CP26, the protein binds about six chlorophyll a and two chlorophyll b molecules. Two chlorophyll b absorption bands are present at 638 and 650 nm and they are somewhat more pronounced than in a recent report [Giuffra, E., Zucchelli, G., Sandonà, D., Croce, R., Cugini, D., Garlaschi, F.M., Bassi, R. & Jennings, R.C. (1997) Biochem. 36, 12984-12993]. The bands give rise to positive and negative linear dichroism, respectively; both show negative CD bands (cf. bands with similar properties at 637 and 650 nm in CP26). Chlorophyll a absorption is dominated by a large contribution at 674 nm which also shows similarities to the major band in LHCIIb and CP26, while (as for CP26) a reduction in absorption around 670 nm is observed relative to the bulk complex. Principal differences from LHCIIb and CP26, and from other CP29 preparations, occur in the carotenoid region.

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The relationship between the binding of dicyclohexylcarbodiimide (DCCD) to isolated light-harvesting proteins of photosystem II and the inhibition of chlorophyll fluorescence quenching by DCCD have been investigated. For a range of different complexes an approximately linear relationship was obtained between the efficiency of DCCD binding and the DCCD-dependent reversal of fluorescence quenching. The most efficient labeling was found for the minor light-harvesting complexes, CP29 and CP26. In the case of the former, five different preparations were compared including two reconstituted complexes in which a putative DCCD-binding site had been mutagenized. Again, an approximately linear relationship between DCCD binding and the extent of reversal of fluorescence quenching was found. However, the binding of DCCD was found to occur at least an order of magnitude faster than the change in fluorescence. The results are discussed in terms of the multiplicity of DCCD-binding sites and the influence of protein structure on both the binding of DCCD and the fluorescence quenching mechanism.

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CLIPs (cytoplasmic linker proteins) are a class of proteins believed to mediate the initial, static interaction of organelles with microtubules. CLIP-170, the CLIP best characterized to date, is required for in vitro binding of endocytic transport vesicles to microtubules. We report here that CLIP-170 transiently associates with prometaphase chromosome kinetochores and codistributes with dynein and dynactin at kinetochores, but not polar regions, during mitosis. Like dynein and dynactin, a fraction of the total CLIP-170 pool can be detected on kinetochores of unattached chromosomes but not on those that have become aligned at the metaphase plate. The COOH-terminal domain of CLIP-170, when transiently overexpressed, localizes to kinetochores and causes endogenous full-length CLIP-170 to be lost from the kinetochores, resulting in a delay in prometaphase. Overexpression of the dynactin subunit, dynamitin, strongly reduces the amount of CLIP-170 at kinetochores suggesting that CLIP-170 targeting may involve the dynein/dynactin complex. Thus, CLIP-170 may be a linker for cargo in mitosis as well as interphase. However, dynein and dynactin staining at kinetochores are unaffected by this treatment and further overexpression studies indicate that neither CLIP-170 nor dynein and dynactin are required for the formation of kinetochore fibers. Nevertheless, these results strongly suggest that CLIP-170 contributes in some way to kinetochore function in vivo.

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The effect of the reductant hydrazine on the flash-induced oxygen oscillation patterns of spinach thylakoids was used to characterize a new super-reduced redox state of the water oxidase in photosystem II. The formation of a discrete S(-3) state is evident from the shift of the first maximum of oxygen evolution from the 3rd flash through the 5th flash to the 7th flash during a 90 min incubation of dark-adapted thylakoids with 10 mM hydrazine sulfate at pH 6.8 on ice. A distinct period four oscillation with further maxima on the 11th and 15th flashes is still observed at this stage of the incubation. The data analysis within the framework of an extended Kok model reveals that a S(-3) state population of almost 50% can be achieved by this treatment. A prolonged incubation of the S(-3) sample with 10 mM hydrazine (and even 100 mM) does not lead to a further shift of the first maximum toward the 9th flash that could reflect the formation of the S(-5) state. Instead, a slow oxidation of S(-3) to S(-2) takes place by an as yet unidentified electron acceptor. A consistent simulation of all the measured oxygen oscillation patterns of this study could, however, only be achieved by including the formal redox states S(-4) and S(-5) in the fits (S(-4) + S(-5) up to 35%). The implications of these findings for the oxidation states of the manganese in the tetranuclear cluster of the water oxidase are discussed.

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In the present study the light induced formation of superoxide and intrinsic superoxide dismutase (SOD) activity in PS II membrane fragments and D1/D2/Cytb559-complexes from spinach have been analyzed by the use of ferricytochrome c (cyt c(III)) reduction and xanthine/xanthine oxidase as assay systems. The following results were obtained: 1.) Photoreduction of Cyt c (III) by PS II membrane fragments is induced by addition of sodium azide, tetracyane ethylene (TCNE) or carbonylcyanide-p-trifluoromethoxy-phenylhydrazone (FCCP) and after removal of the extrinsic polypeptides by a 1M CaCl2-treatment. This activity which is absent in control samples becomes completely inhibited by the addition of exogenous SOD. 2.) The TCNE induced cyt c(III) photoreduction by PS II membrane fragments was found to be characterized by a half maximal concentration of c1/2=10 µM TCNE. Simultaneously, TCNE inhibits the oxygen evolution rate of PS II membrane fragments with c1/2≈ 3 µM. 3.) The photoproduction of O2 (-) is coupled with H(+)-uptake. This effect is diminished by the addition of the O2 (-)-trap cyt c(III). 4.) D1/D2/Cytb559-complexes and PS II membrane fragments deprived of the extrinsic proteins and manganese exhibit no SOD-activity but are capable of producing O2 (-) in the light if a PS II electron donor is added.Based on these results the site(s) of light induced superoxide formation in PS II is (are) inferred to be located at the acceptor side. A part of the PS II donor side and Cyt b559 in its HP-form are proposed to provide an intrinsic superoxide dismutase (SOD) activity.

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The effect of redox-active amines NH2R (R = OH or NH2) on the period-four oscillation pattern of oxygen evolution has been analyzed in isolated spinach thylakoids as a function of the redox state Si (i = 0, ..., 3) of the water oxidase. The following results were obtained: (a) In dark-adapted samples with a highly populated S1 state, NH2R leads via a dark reaction sequence to the formal redox state "S-1"; (b) the reaction mechanism is different between the NH2R species; NH2OH acts as a one-electron donor, whereas NH2NH2 mainly functions as a two-electron donor, regardless of the interacting redox state Si (i = 0, ..., 3). For NH2NH2, the modified oxygen oscillation patterns strictly depend upon the initial ratio [S0(0)]/[S1(0)] before the addition of the reductant; while due to kinetic reasons, for NH2OH this dependence largely disappears after a short transient period. (c) The existence of the recently postulated formal redox state "S-2" is confirmed not only in the presence of NH2NH2 [Renger, G., Messinger, J., & Hanssum, B. (1990) in Current Research in Photosynthesis (Baltscheffsky, M., Ed.) Vol. 1, pp 845-848, Kluwer, Dordrecht] but also in the presence of NH2OH. (d) Activation energies, EA, of 50 kJ/mol were determined for the NH2R-induced reduction processes that alter the oxygen oscillation pattern from dark-adapted thylakoids. (e) Although marked differences exist between NH2OH and NH2NH2 in terms of the reduction mechanism and efficiency (which is about 20-fold in favor of NH2OH), both NH2R species exhibit the same order of rate constants as a function of the redox state Si in the nonperturbed water oxidase: kNH2R(S0) greater than kNH2R(S1) much less than kNH2R(S2) much greater than kNH2R(S3) The large difference between S2 and S3 in their reactivity toward NH2R is interpreted to indicate that a significant change in the electronic configuration and nuclear geometry occurs during the S2----S3 transition that makes the S3 state much less susceptible to NH2R. The implications of these findings are discussed with special emphasis on the possibility of complexed peroxide formation in redox state S3 postulated previously on the basis of theoretical considerations [Renger, G. (1978) in Photosynthetic Water Oxidation (Metzner, H., Ed.) pp 229-248, Academic Press, London].

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The protolytic reactions of PSII membrane fragments were analyzed by measurements of absorption changes of the water soluble indicator dye bromocresol purple induced by a train of 10 µs flashes in dark-adapted samples. It was found that: a) in the first flash a rapid H(+)-release takes place followed by a slower H(+)-uptake. The deprotonation is insensitive to DCMU but is completely eliminated by linolenic acid treatment of the samples; b) the extent of the H(+)-uptake in the first flash depends on the redox potential of the suspension. In this time domain no H(+)-uptake is observed in the subsequent flashes; c) the extent of the H(+)-release as a function of the flash number in the sequence exhibits a characteristic oscillation pattern. Multiphasic release kinetics are observed. The oscillation pattern can be satisfactorily described by a 1, 0, 1, 2 stoichiometry for the redox transitions Si → Si+1 (i=0, 1, 2, 3) in the water oxidizing enzyme system Y. The H(+)-uptake after the first flash is assumed to be a consequence of the very fast reduction of oxidized Q400(Fe(3+)) formed due to dark incubation with K3[Fe(CN)6]. The possible participation of component Z in the deprotonation reactions at the PSII donor side is discussed.

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In isolated, blood perfused, supramaximally stimulated, isotonically working gastrocnemii of dogs lactic acid (LA) output and O2-consumption (V O2) were measured according to the Fick principle. Simultaneously concentration of muscle tissue was determined at rest and at different times during exercise. In one series of experiments metabolic alkalosis was induced by infusions of THAM of Na bicarbonate. As a result arterial pH increased to about 7.5 and standard [HCO3-1] to 31-35 mmol per 1. In another group of experiments metabolic acidosis was induced by HCl infusions. In these experiments pH decreased to 7.0-7.1 and standard [HO301] to 8-11 mmol per 1. During the first 3-4 min after the onset of exercise LA concentration of muscle tissue rose to 18-19 mumol per g wet weight in both series of experiments. During acidosis the highest average values for LA release from the muscle were about 1.1 mumoles per g per minute. During alkalosis LA permeation rate was nearly three times as high. As a consequence of increased rate of permeation, LA concentration of muscle tissue decreased more rapidly in alkalosis than in acidosis. In both series of experiments work per time and VO2 were practically equal during the first 5-6 min of exercise. Thereafter work per time and VO2 decreased more rapidly in acidosis than in alkalosis, a result which probably is due to higher LA concentration in muscle at this time in acidosis. It is concluded that LA permeation rate across muscle cell membrane is increased by high extracellular HCO3- concentration in combination with low H+ activity and vice versa.

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