Thermoluminescence study of charge recombination in Photosystem II at low temperatures. II. Oscillatory properties of the Zv and A thermoluminescence bands in chloroplasts dark-adapted for various time periods

The oscillations of the Z_V and A thermoluminescence bands were investigated in spinach chloroplasts which had been dark-adapted for various time periods and subjected to a series of flashes at +2°C before continuous illumination at various low temperatures. When excited with continuous light below −65°C, the Z_V band exhibited period-4 oscillation, with maxima on preflashes 0, 4 and 8. Above −65°C, the oscillation pattern depended greatly on the dark-adaptation period of the chloroplasts. In preilluminated samples (15 s light followed by 3 min dark), when the Q_B pool is half oxidized, the oscillation of the thermoluminescence intensity measured at −50°C was similar to that observed below −65°C. However, after the thorough dark-adaptation of the chloroplasts (6 h), when the major fraction of the Q_B pool is assumed to be oxidized, a binary oscillation appeared in the oscillation pattern, with maxima at odd flash numbers. Below −65°C, period-2 oscillation of the Z_V band could not be induced by the dark-adaptation of the chloroplasts, suggesting an inhibition of electron exchange between Q_A and Q_B. Upon excitation of the chloroplasts with continuous light at −30°C, the A band oscillated with a periodicity of 4 with maxima at preflash numbers 2 and 6. At pH 7.5, the period-4 oscillation was converted into a period-2 oscillation by thorough dark-adaptation of the chloroplasts (24 h). Model calculations of the oscillatory patterns suggest that the period-4 oscillations of the Z_V and A bands are determined by the concentrations [S₀] + [S₁] and [S₂] + [S₃], respectively, which are present after the preflashes prior to the low-temperature continuous illumination. The period-2 oscillations in the amplitudes of the Z_V and A bands reflect the changes occurring in the redox state of the Q_B pool in a sequence of flashes. The possible relationship between the characteristics of the Z_V and A bands and the temperature-dependence of the S state transitions was investigated. Comparison of the amplitudal changes of the B (S₂Q⁻_B and S₃Q⁻_B recombination) and Q (S₂Q⁻_A recombination) thermoluminescence bands as a function of the excitation temperature suggests that the S₂ → S₃ and S₃ → S₄ transitions are blocked at about −65 and −40°C, respectively. It is also concluded that the thermoluminescence intensity emitted by the reaction center is about twice as high in the S₃ state as in the S₂ state.     

Charge accumulation and recombination in Photosystem II studied by thermoluminescence. I. Participation of the primary acceptor Q and secondary acceptor B in the generation of thermoluminescence of chloroplasts

In the glow curves of chloroplasts excited by a series of flashes at +1°C the intensity of the main thermoluminescence band appearing at +30°C (B band; B, secondary acceptor of Photosystem II) exhibits a period-4 oscillation with maxima on the 2nd and 6th flashes indicating the participation of the S₃ state of the water-splitting system in the radiative charge recombination reaction. After long-term dark adaptation of chloroplasts (6 h), when the major part of the secondary acceptor pool (B pool) is oxidized, a period-2 contribution with maxima occurring at uneven flash numbers appears in the oscillation pattern. The B band can even be excited at ?160°C as well as by a single flash in which case the water-splitting system undergoes only one transition (S₁ → S₂). The experimental observations and computer simulation of the oscillatory patterns suggest that the B band originates from charge recombination of the S₂B^? and S₃B^? redox states. The half-time of charge recombination responsible for the B band is 48 s. When a major part of the plastoquinone pool is reduced due to prolonged excitation of the chloroplasts by continuous light, a second band (Q band; Q, primary acceptor of Photosystem II) appears in the glow curve at +10°C which overlaps with the B band. In chloroplasts excited by flashes prior to DCMU addition only the Q band can be observed showing maxima in the oscillation pattern at flash numbers 2, 6 and 10. The Q band can also be induced by flashes after DCMU addition which allows only one transition of the water-splitting system (S₁ → S₂). In the presence of DCMU, electrons accumulate on the primary acceptor Q, thus the Q band can be ascribed to the charge recombination of either the S₂Q^? or S₃Q^? states depending on whether the water-splitting system is in the S₂ or the S₃ state. The half-time of the back reaction of Q^? with the donor side of PS II (S₂ or S₃ states) is 3 s. It was also observed that in a sequence of flashes the peak positions of the Q and B bands do not depend on the advancement of the water-splitting system from the S₂ state to the S₃ state. This result implies that the midpoint potential of the water-splitting system remains unmodified during the S₂ → S₃ transition.     

Thermoluminescence study of charge recombination in Photosystem II at low temperatures. I. Characterization of the Zv and A thermoluminescence bands

The characteristics of the Z_v and A thermoluminescence bands appearing in the glow curve at about -75 and -30°C, respectively, were investigated in spinach chloroplasts. Inhibitory concentrations of DCMU decreased the amplitude of the Z_v band by half and completely abolished the A band. On the other hand, after two preflashes at +2°C before freezing, the A band could be charged by low-temperature illumination even when the electron transport was interrupted between Q_A and Q_B by DCMU addition after the preflashes. Two-flash preillumination greatly enhanced the amplitude of the A band, but diminished that of the Z_v band. Tris washing and NH₂OH treatment, which inactivated the oxygen-evolving system, almost completely abolished the Z_v band, but did not affect the A band. Severe trypsin treatment, which also impaired the oxygen-evolving system, resulted in a very large intensification of the Z_v band. The half-times of the A and Z_v bands, determined by theoretical analysis of the thermoluminescence data, proved to be about 4 ms and 200–500 μs, respectively. These results, taken together with EPR data from the literature, suggest that the A band arises from charge recombination between a negatively charged acceptor located before the DCMU block (most probably Q⁻_A) and the oxidized donor Z⁺ (which accounts for the EPR Signal II_vf and Signal II_f). The electron carrier responsible for the Z_v band is also a component located prior to the inhibitory site of DCMU (Q⁻_A); its interacting counterpart is an unidentified donor which is involved in charge exchange with the S states.     

Afaltoxin B1 transport in rat blood plasma. Binding to albumin in vivo and in vitro and spectrofluorimetric studies into the nature of the interaction

Binding of [³H]aflatoxin B₁ to rat plasma was investigated in vivo and vn vitro. Column chromatographic and polyacrylamide gel electrophoretic analysis clearly demonstrated that aflatoxin B₁ bound primarily plasma albumin. Very little binding activity was shown by other plasma proteins. Spectrofluorimetric studies were undertaken to gain some insight into the nature of the aflatoxin-albumin interaction. Quenching of the lone tryptophan fluorescence intensity upon aflatoxin binding was due, at least in part, to a ligand-induced conformational change in the albumin molecule. Aflatoxin B₁ binds an apolar site with an association constant of 30 mM⁻¹ at pH 7.4 and 20°C. Neither charcoal treatment of rat albumin nor the presence of 0.15 M NaCl had many significant effect on the interaction. The association constant was pH-dependent, increasing about 1.7-fold as the pH increased from 6.1 to 8.4. This pH dependence is ascribed to a pH-induced conformational change in the albumin molecule. Thermodynamic studies indicated that the aflatoxin-albumin interaction was exothermic (ΔH = −29.3 kJ·mol⁻), with a ΔS value of −13.8 J·mol⁻¹·K⁻¹.     

Thermodynamics of hexokinase-catalyzed reactions.

The enthalpies of the hexokinase-catalyzed phosphorylation or glucose, mannose, and fructose by ATP to the respective hexose 6-phosphates have been measured calorimetrically in TRIS/TRIS HCl buffer at 25.0, 28.5, and 32.0°C. The effects on the measured enthalpy of the glucose/hexokinase reaction due to variation of pH (over the range 6.7 to 9.0) and ionic strength (over the range 0.02 to 0.25) have been examined. Correction for enthalpy of buffer protonation leads to δH^o and δC_p^o values for the processes: eq-D-hexose + ATP⁴⁻ = eq-D-hexose 6-phosphate²⁻ + ADP³⁻+ H⁺. Results are δH^o = −23.8 ± 0.7 kJ · mol⁻¹ and δC_p^o = −156 ± 280 J·mol⁻¹·K⁻¹ for glucose. δH^o = −21.9 ± 0.7 kJ·mol⁻¹ and δC_p^o = 10 ± 140 J·mol⁻¹·K⁻¹ for mannose, and δH^o = −15.0 ± 0.9 kJ·mol⁻¹ and δC_p^o = −41 ± 160 J·mol⁻¹·K⁻¹ for fructose. Combination of these measured enthalpies with Gibbs energy data for hydrolysis of ATP⁴⁻ and that for the hexose 6-phosphates lead to δS^o values for the above hexokinase-catalyzed reactions.     

Kinetics of the formation and hydrolysis reactions of some thiomolybdate(VI) anions in aqueous solution

The kinetics of the formation of the thiomolybdate ions MoOS₃²⁻ and MoS₄²⁻ were determined spectroscopically from the addition of excess sulphide to MoO₂S₂²⁻ in pH buffered media (6–8) at 30 °C. The reverse (hydrolysis) reactions of MoO₂S₂²⁻ and MoOS₃²⁻ were measured under the same conditions. The reaction rates measured are shown below:

Values of the rate-constants (s⁻¹) obtained at pH 7.0 were k₁₀ 2.4 × 10⁻³, k₂₁ 1.5 × 10⁻⁵, k₃₀ 2.1 × 10⁻⁵, k₂₃ 6.0 × 10⁻⁴, and k₃₄ 1.9 × 10⁻⁵; where the results are comparable they are in good agreement with those obtained by earlier workers, although different conditions were used. However, in this work it was found that certain reactions had to be mathematically treated as two consecutively occurring reactions. There is also a difference in interpretation of the mechanism of the hydrolysis reactions of the tri- and tetrathio ions. In general the lability towards further S replacement of O atoms, and the reverse reaction, decreased with increased S substitution. All reaction rates increased with increasing H⁺ ion concentration, mostly this was a linear relationship over the limited pH range examined. The effect of the H⁺ ion is interpreted in terms of protonation of the oxythiomolybdate ions at an O atom leading to increased lability.     

Charge accumulation and recombination in Photosystem II studied by thermoluminescence. II. Oscillation of the C band induced by flash excitation

The characteristics of the thermoluminescence band appearing at +50°C in the glow curve (C band) was investigated in maize chloroplasts. The C band, which had a half-time of 10 min, could be charged in the presence of DCMU, and its amplitude significantly increased if preilluminated chloroplasts were reexcited after DCMU addition. Inactivation of the water-splitting system by hydroxylamine- or Tris-treatment did not abolish the C band. In chloroplasts subjected to various numbers of flashes before DCMU addition, the amplitude of the C band exhibited oscillation patterns which were markedly dependent upon dark adaptation of chloroplasts. Flash excitation of chloroplasts preilluminated by continuous light for 30 s prior to 5 min dark adaptation resulted in a period-4 oscillation with maxima occurring at flash numbers 0, 4, 8, 12. After a 6-h dark-adaptation of chloroplasts the period-4 oscillation was superimposed with a period-2 oscillation. The oscillatory patterns were simulated by model calculations and the possible origin of the C band is discussed.     

Estimation of intracellular pH in muscle of fishes from different thermal environments

A technique based on homogenisation of rapidly frozen tissue was used to investigate the regulation of intracellular pH (pH_i) in freshwater and marine fish from diverse environmental temperatures. The following species were held at ambient temperatures of ca. 1°C (Notothenia coriiceps; Antarctica), 5°C (Pleuronectes platessa, Myoxocephalus scorpius; North Sea), and 26°C (Oreochromis niloticus; African lakes). The effects of seasonal acclimatisation to 4, 11 and 18°C were also examined in rainbow trout in the winter, autumn and summer, respectively. Extracellular (whole blood) pH (pH_e) did not follow the constant relative alkalinity relationship, where pH⁺=pOH⁻ for any particular temperature, over a range of 1–26°C (overall δpH_e/δT=0.009±0.002 U °C⁻¹; P<0.001), apparently being regulated by ionic fluxes and ventilation. Intracellular pH (pH_i) was also regulated independently of pN(=0.5 pK water) in all species of fish examined. The inverse relationship between pH_i and environmental temperature gave an overall δpH_i/δT of −0.010±0.001 U °C⁻¹ (for both white and red muscle) and −0.004±0.003 U °C⁻¹ (cardiac muscle). However, between 1 and 11°C δpH_i/δT was much higher (P<0.001), −0.022±0.003 U °C⁻¹ (white muscle) and −0.022±0.004 U °C⁻¹ (red muscle). The possible adaptive roles for these different acid–base responses to environmental temperature variation among tissues and species, and the potential difficulties of estimating pH_i, are discussed.     

The thermoregulatory limits of an Australian Passerine,the Silvereye (Zosterops lateralis)

(1) The thermal capabilities of Australian silvereyes (Zosterops lateralis, 11 g) were investigated both at low and high ambient temperatures (T_a) during the photophase and scotophase. (2). The peak metabolic rate (PMR) induced by helium–oxygen (79:21 %, He–O₂) exposure during the photophase was 15.64±1.55 mL O₂ g⁻¹ h⁻¹ at an effective lower survival limit T_a (T_pmr) of −39.7±6.1°C. (3). Above the thermoneutral zone (TNZ), metabolic rate, body temperature (T_b), and thermal conductance increased steeply, but they were able to withstand a T_a of 39°C. (4). Our study shows that silvereyes are able to tolerate an impressive range of T_a from about −42°C to at least +39°C and are able to produce enough heat to maintain a thermal difference between T_b and T_a of up to 80°C.     

Studies on the mechanism of ADRY agents (agents accelerating the deactivation reactions of water-splitting enzyme system Y) on thermoluminescence emission

The effect of 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT-2p), known to be the most powerful ADRY agent (Renger, G. (1972) Biochim. Biophys. Acta 256, 428–439), on thermoluminescence has been investigated. Two thermoluminescence bands were analyzed: (a) the emission peaking at about 20–30°C caused by warming up of untreated chloroplasts, illuminated with a single 5 μs flash at room temperature and frozen rapidly to 77 K; and (b) the band emitted in the range of ?10 up 10°C after warming of chloroplast suspensions containing 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) which were illuminated with a single 5 μs flash at ?15°C and frozen rapidly at 77 K. These bands were attributed to the recombination of the B ^?S₂(S₃) and X-320 ^?S₂ states, respectively (Rutherford, A.W., Crofts, A.R. and Inoue, Y. (1982) Biochim. Biophys. Acta 682, 457–465). It was found that: (1) The B ^?S₂(S₃) band is markedly diminished at very low ANT-2p concentrations of less than one molecule per 2000 chlorophylls. (2) The inhibition of the X-320 ^?S₂ band requires significantly higher concentrations of ANT-2p (50% peak reduction at one ANT-2p molecule per 100 chlorophylls). (3) Preflashing at room temperature before cooling to ?15°C diminishes the X-320 ^?S₂ band significantly in the presence of ANT-2p, while almost no effect is observed in its absence. (4) The state X-320 ^?S₂ decays monoexponentially with a half-lifetime of 2 min at ?15°C in the absence of ANT-2p. In the presence of one ANT-2p molecule per 800 chlorophylls the decay becomes biphasic with half-lifetimes of 0.5 and 2 min and an amplitude ratio of 2:3, respectively. The results obtained can be explained consistently by the function of ANT-2p as an ADRY agent acting as a mobile species within the thylakoid membrane at room temperature. At subzero temperatures, a ‘fixed-place’ mechanism appears to be operative. The implications for the ADRY effect and thermoluminescence are discussed.     

Oxidation of (hydroxyethyl)ferrocene by [2-pyridylmethylbis(2-ethylthioethyl)/amine]copper(II)

A kinetic study of the oxidation of (hydroxyethyl)ferrocene (HEF) by [2-pyridylmethylbis(2-ethyl-thioethyl)ainine]copper(II) (Cu(pmas)²⁺) is reported, with the objective of documenting the influence of the two thioether sulfur ligands on the electron transfer rate. Both reactants exhibit a first-order dependence at pH 6, I = 0.1 M(NaNO₃); k(25°C) = 1.3 × 10⁴M⁻¹sec⁻¹, ΔH3 = 10.1 kcal/mole, ΔS3 = −6 eu. The apparent Cu(pmas)^2+/+ self-exchange electron transfer rate constant calculated from this reaction on the basis of relative Marcus theory (4.7 × 10¹M⁻¹ sec⁻¹) agrees well with previous findings on ferrocytochrome c, Fe(CN)₆⁴⁻, and Ru(NH₃)₅py²⁺ oxidations. Spectrophotometric titrations of Cu(pmas)²⁺ and Cu(tmpa)²⁺ (tmpa = tris(2-pyridylmethyl)amine) with azide ion showed that both Cu(pmas)N₃)⁺ (K_f1 = 3.1 × 10³M⁻¹) and Cu(pmas)(N₃)₂ (K_f2 = 3.5 × 10¹M⁻¹) but Cu(tmpa)(N₃)⁺ (K_f = 6.6 × 10²M⁻¹) are formed up to 0.15 M N₃⁻ (25°C, pH 6, I = 0.2 M), suggesting that a thioether sulfur atom is displaced in the uptake of a second N₃⁻ ion by Cu(pmas)(N₃)⁺. The effect of thioether sulfur displacement by azide ion on the HEF-Cu(pmas)²⁺ reaction rate may be understood entirely through the tendency of N₃⁻ to shift the position of the redox equilibrium towards the reactant side, without invoking any special role for the sulfur ligand in promoting electron transfer reactivity.     

Thermodynamics and coordination characteristics of the hydronium-uranium(VI)-dicyclohexano-24-crown-8 extraction complex

The extraction equilibrium of the hydronium-uranium(VI)-dicyclohexano-24-crown-8 complex was carried out in the crown ether1,2-dichloroethaneHCl aqueous solution system at different temperatures. The extraction complex has the overall composition (L)₂·(H₃O⁺·χH₂O)₂·UO₂Cl₄²⁻ (L = dicyclohexano-24-crown-8). The values of the extraction equilibrium constants (K_ex) increase steadily with a decrease in temperature: 13.5 (298 K), 7.96 (301 K), 4.20 (303 K) and 2.07 (305 K). A plot of log K_ex against 1/T shows a straight line. The value of the enthalpy change, ΔH°, was calculated from the slope and equals −212 kJ mol⁻¹. The value of the entropy change, ΔS°, was calculated from ΔH° and K_ex and equals −690 J K⁻¹ mol⁻¹, whereas ΔG° = −6.45 kJ mol⁻¹. Comparing these thermodynamic parameters with those of the dicyclohexano-18-crown-6 isomer A [1] (ΔS° = −314 J K⁻¹ mol⁻¹, ΔH° = −101 kJ mol⁻¹ and ΔG° = −8.37 kJ mol⁻¹), it can be seen that ΔH° and ΔS° are more negative for the former than for the latter, and both are enthalpy-stabilized complexes. The molecular structure of the complex has the feature that there are two H₅O₂⁺ ions in it, in contrast to the H₃O⁺ ions in the dicyclohexano-18-crown-6 isomer A complex [1]. Each of the H₅O₂⁺ ions is held in the crown ether cavity by four hydrogen bonds. The H₅O₂⁺ ion has a central bond. The uranium atom forms UO₂Cl₄²⁻ as a counterion away from the crown ether. The formation of this complex is in good agreement with more negative entropy change and less negative free energy change, as mentioned above.     

Thermoluminescence and fluorescence study of changes in Photosystem II photochemistry in desiccating barley leaves

An effect of desiccation (a decrease of relative water content from 97% to 10% within 35 h) on Photosystem II was studied in barley leaf segments (Hordeum vulgare L. cv. Akcent) using chlorophyll a fluorescence and thermoluminescence (TL). The O-J-I-P fluorescence induction curve revealed a decrease of F_P and a slight shift of the J step to a shorter time with no change in its height. The analysis of the fluorescence decline after a saturating light flash revealed an increased portion of slow exponential components with increasing desiccation. The TL bands obtained after excitation by continuous light were situated at about –27°C (Z_v band – recombination of P680⁺Q_A ⁻), –14 °C (A band – S₃Q_A ⁻), +12 °C (B band – S_2/3Q_B ⁻) and +45 °C (C band – TyrD⁺Q_A ⁻). The bands related to the S-states of oxygen evolving complex (A and B) were reduced by desiccation and shifted to higher and lower temperatures, respectively. In accordance with this, the band observed at about +27 °C (S₂Q_B ⁻) after excitation by 1 flash fired at –10 °C and band at about +20 °C (S_2/3Q_B ⁻) after 2 flashes decreased with increasing water deficit and shifted to lower temperatures. A new band around 5 °C appeared in both regimes of TL excitation for a relative water content of under 42% and was attributed to the Q band (S₂Q_A ⁻). It is suggested that under desiccation, an inhibition of the formation of S₂- and S₃-states in OEC occurred simultaneously with a lowering of electron transport on the acceptor side of PS II. The temperature down-shift of the TL bands obtained after the flash excitation was induced at the initial phases of water stress, indicating a decrease of the activation energy for the S_2/3Q_B ⁻recombination. This revised version was published online in June 2006 with corrections to the Cover Date.     

Morphometric relationship of length–weight and chelae length–width of eastern white river crayfish (Procambarus acutus acutus, Girard, 1852), under culture conditions

Length–weight (TL vs WWT) and chelae length–width (ChL vs ChW) relationships were described for juveniles, males and females, and for form I and form II males of Procambarus acutus acutus. The length–weight relationships for juveniles, form I, form II males, and females could be described as: WWT = 5 × 10⁻³ TL^3.09, WWT = 6 × 10⁻³ TL^3.61, WWT = 6 × 10⁻⁹ TL^3.26, and WWT = 6 × 10⁻⁴ TL^3.5, respectively. In all forms, growth was allometric (P < 0.05). The ancova test indicated that slopes and intercepts of the length–weight regressions were significantly different between sex and sexual stages. The regressions for chelae length–width relationships for form I and form II males, and females were: ChW = −0.81 + 0.27CL, ChW = −0.33 + 0.25CL, and ChW = −0.82 + 0.32CL, respectively. Although the slope and intercepts of regressions for ChL and ChW were similar for those of form I and form II males, the slopes and intercepts of regressions of females were significantly different from form I and form II males. No statistical difference was observed in mean ChL between form II males and females (P > 0.05), but a significant difference was detected in mean ChL between form I and form II males (P < 0.05) and form I and females (P < 0.05). Form I males had longer ChL than form II males and females. The same trend was observed in mean ChW for form I and form II males, but a significant difference was detected between form II males and females (P < 0.05). In addition, results indicated that chelae lengths and widths increased allometrically with total length (TL) for both sex and sexual stages.     

The uncatalysed and the copper(II) promoted hydrolysis of 4-nitrophenyl L-leucinate

The uncatalysed hydrolysis of 4-nitrophenyl L-leucinate has been studied in detail over a range of pH and temperature at I=0.1 M (KNO₃). Base hydrolysis of the ester is strongly promoted by copper(II) ions. Rate constants have been obtained for the following reactions (where EH⁺ is the N- protonated ester and E is the free base form) EH⁺ + OH⁻ → products E + OH⁻ → products E + H₂O → products CuE²⁺ + OH⁻ → products Base hydrolysis of the copper(II) complex CuE²⁺ is 3.8 × 10⁵ times faster than that of E and 75 times faster than that of EH⁺ at 25 °C and I=0.1 M. Activation parameters for these reactions have been determined and possible mechanisms are considered.     

Purification and characterization of polygalacturonase produced by thermophilic Thermoascus aurantiacus CBMAI-756 in submerged fermentation

An extracellular polygalacturonase was isolated from 5-day culture filtrates of Thermoascus aurantiacus CBMAI-756 and purified by gel filtration and ion-exchange chromatography. The enzyme was maximally active at pH 5.5 and 60–65°C. The apparent K _m with citrus pectin was 1.46 mg/ml and the V _max was 2433.3 μmol/min/mg. The apparent molecular weight of the enzyme was 30 kDa. The enzyme was 100% stable at 50°C for 1 h and showed a half-life of 10 min at 60°C. Polygalacturonase was stable at pH 5.0–5.5 and maintained 33% of initial activity at pH 9.0. Metal ions, such as Zn⁺², Mn⁺², and Hg⁺², inhibited 50, 75 and 100% of enzyme activity. The purified polygalacturonase was shown to be an endo/exo-enzyme, releasing mono, di and tri-galacturonic acids within 10 min of hydrolysis.     

Characteristics of Cu deficiency-induced inhibition of photosynthetic electron transport in spinach chloroplasts

In the present work we studied the effect of Cu deficiency on spinach chloroplasts. We found that in spinach the electron transport was inhibited as reported previously for sugar beet (Droppa, M., Terry, N. and Horváth, G. (1984) Proc. Natl. Acad. Sci. USA (1984) 81, 2369–2373). The breakpoint of the Arrhenius plot of the whole electron-transport activity was shifted from +6°C to +12°C in Cu-deficient chloroplasts. A similar effect could be observed with a spin-labelled probe, when the rotational correlation time was plotted vs. the reciprocal temperatures. This indicates that the membrane fluidity might be changed by Cu deficiency. The lipid/protein ratios were similar in both control and deficient chloroplasts. On the other hand, the saturated/unsaturated ratio of phosphatidylcholine (PC), phosphatidylglycerol (PG) and sulpholipids (SL) was increased but that of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) decreased. We conclude that Cu deficiency does not change the entire membrane fluidity but rather the lipid composition of the microenvironment of some electron-transport components. The inhibition of Photosystem II electron transport in Cu-deficient chloroplasts was characterized by thermoluminescence and 2-dimensional gel electrophoresis. It was found that Cu deficiency shifted the main peak of the glow curve from +18°C to +8°C, similar to that of DCMU-poisoned chloroplasts. Two apoproteins of the 29 kDa polypeptide disappeared in Cu-deficient chloroplasts which indicates that this polypeptide has a regulatory role in ensuring the normal electron flow between Q_A and Q_B.     

Turning up the heat on subzero fish: thermal dependence of sustained swimming in an Antarctic notothenioid

We determined the maximum sustained swimming speed (U_crit), and resting and maximum ventilation rates of the Antarctic fish Pagothenia borchgrevinki at five temperatures between −1°C and 8°C. We also determined resting metabolic rate (VO₂) at −1°C, 2°C, and 4°C. U_crit of P. borchgrevinki was highest at −1°C (2.7±0.1 BL s⁻¹) and rapidly decreased with temperature, representing a thermal performance breadth of only 5°C. This narrow thermal performance supports our prediction that specialisation to the subzero Antarctic marine environment is associated with a physiological trade-off in performance at high temperatures. Resting oxygen consumption and ventilation rate increased by more than 200% across the temperature range, which most likely contribute to the decrease in aerobic swimming capabilities at higher temperatures.     

Nicotinamide adenine dinucleotide (NAD+). Formal potential of the NAD+/NAD· couple and NAD· dimerization rate

The rate constant, k_d, for the dimerization of the free radical (NAD^·), produced on the initial one-electron reduction of NAD⁺, was measured by double potential-step chronoamperometry, fast-scan cyclic voltammetry (cathodic-anodic peak current ratio) and slow-scan cyclic voltammetry (peak potential shift) for a medium in which neither NAD⁺ nor its reduction products are adsorbed at the solution/electrode interface. All three methods give concordant values of k_d (approx. 3·10⁷ M^?1·s^?1), which are in reasonable accord with the values determined by pulse radiolysis but are considerably greater than values previously determined electrochemically. For the NAD⁺/NAD^· couple, the heterogeneous rate constant (k_s,h) exceeds 1 cm·s^?1 at 25°C and the formal potential (E⁰_c) vs. sce is ? 1.155 V at 25°C and ? 1.149 V at 1°C at pH 9.1, with an uncertainty of about ±0.005 V.     

The formation constants of dimethylthallium(III)-glutathione complexes in aqueous solution

The complexes formed in the dimethylthallium(III) (Me₂Tl⁺), glutathionate (EGC³⁻) and hydrogen ion system in aqueous solution at 37 °C and I = 150 mmol dm⁻³ (NaCl) have been characterised by means of glass-electrode potentiometry. Glutathione protonation constants were found to be 9.123 ± 0.007, 17.42 ± 0.01, 20.78 ± 0.02, and 22.93 ± 0.02. Formation constants for the complexes [(Me₂Tl)EGCH]⁻ and [(·Me₂Tl) EGC]²⁻ were found to be 11.19 ± 0.03 and 2.39 ± 0.02, respectively. Particular attention has been paid to the evaluation of the effect of possible systematic errors on the constant values determined. Reliable standard deviation estimations have been made by applying a Monte Carlo calculation technique.