Sites of function of manganese within photosystem II. Roles in O2 evolution and system II

The Mn content of spinach chloroplasts has been decreased by growth deficiency, extraction and by ageing at 35°. We studied the effect of subnormal Mn content upon the chloroplasts capacity to evolve O₂ and to photooxidize electron donors other than water via Photosystem II. We observed the following:

1. 1. In fresh chloroplasts ascorbate and other reducing agents, if present in sufficient concentration, fully replace water as the System II oxidant and can sustain maximum rates of 1000–1200 equiv/chlorophyll per h.

2. 2. None of the studied donors proved entirely specific for System II; to a variable extent all could react with the oxidant of System I. We therefore considered only the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-(DCMU)-sensitive fraction of the observed rates as pertinent.

3. 3. Normal fresh chloroplasts contained 3 Mn/200 chlorophylls_II and showed flash yields of approx. 1 O₂/1600 chlorophylls. This indicates that each System II trapping and O₂-evolving center contains three Mn atoms.

4. 4. O₂ evolution capacity is abolished when about 2/3 of the total Mn pool is removed by way of Tris or hydroxylamine extraction, i.e. upon removal of two of the three Mn atoms normally present per reaction center. Between the limits of 1 Mn per trap and 3 Mn per trap O₂ evolution capacity is linear with Mn content.

5. 5. Mn removal affects the rates of O₂ evolution in strong light and in weak light (quantum yield) in the same fashion. This indicates that complete O₂ reaction centers are inactivated.

6. 6. With Mn removal the capacity for donor (ascorbate or p-phenylenediamine) photooxidation in strong light declines in a very similar fashion as the O₂ evolving capacity. However, after removal of 2/3 of the Mn pool (by Tris or hydroxylamine extraction) 15–20% of the maximum rate remains (100–250 equiv/chlorophyll per h) as previously noticed by other workers. Secondly, the rate in weak light (quantum yield) of these photooxidations remains unaffected by Mn removal. This shows that for donor photooxidation the larger of the two Mn pools is not essential.

7. 7. Complete removal of Mn (< 1 Mn/4000 chlorophylls) led to 90–95% loss of donor photooxidation in strong light.

8. 8. Removal of 2/3 of the Mn left a low fluorescence yield (variable fraction = 0) which could be fully restored by adding DCMU. After complete removal of Mn (< 1 Mn/4000 chlorophylls) DCMU enhanced the yield of the variable fluorescence to only 1/2 the maximum level but the full maximum could be restored by chemical reduction. This indicates that fluorescence quencher of System II, Q, is not affected by Mn removal.

9. 9. Of the three Mn associated with each trapping center, one is linked more closely to the center than the other two. While all three are essential for O₂ evolution, artificial donors can enter with various rate constants at several loci on the oxidant side of System II.

Rheological studies of synergistic kappa carrageenan-carob galactomannan gels

The mechanical properties, Young's modulus and yield stress of different carob galactomannan-kappa carrageenan blends have been examined using an Instron testing machine as a function of the following parameters:

1. (1) Constant total polysaccharide concentration.

2. (2) Galactomannan concentration for a given carrageenan concentration.

3. (3) Carrageenan concentration for a given galactomannan concentration.

4. (4) Galactomannan molecular weight.

5. (5) Carrageenan molecular weight.

The properties of the binary gels were compared to the kappa carrageenan gels. From the results, two types of gel structure could occur: a coupled network defined by specific junction zones (and involving unsubstituted mannose residues of the galactomannan chains) and also a gel structure with both polysaccharides associating independently.     

The high-energy state of the thylakoid system as indicated by chlorophyll fluorescence and chloroplast shrinkage

Certain long-term fluorescence phenomena observed in intact leaves of higher plants and in isolated chloroplasts show a reverse relationship to light-induced absorbance changes at 535 nm (“chloroplast shrinkage”).

1. 1. In isolated chloroplasts with intact envelopes strong fluorescence quenching upon prolonged illumination with red light is accompanied by an absorbance increase. Both effects are reversed by uncoupling with cyclohexylammonium chloride.

2. 2. The fluorescence quenching is reversed in the dark with kinetics very similar to those of the dark decay of chloroplast shrinkage.

3. 3. In intact leaves under strong illumination with red light in CO₂-free air a low level of variable fluorescence and a strong shrinkage response are observed. Carbon dioxide was found to increase fluorescence and to inhibit shrinkage.

4. 4. Under nitrogen, CO₂ caused fluorescence quenching and shrinkage increase at low concentrations. At higher CO₂ levels fluorescence was increased and shrinkage decreased.

5. 5. In the presence of CO₂, the steady-state yield of fluorescence was lower under nitrogen than under air, whereas chloroplast shrinkage was stimulated in nitrogen and suppressed in air.

6. 6. These results demonstrate that the fluorescence yield does not only depend on the redox state of the quencher Q, but to a large degree also on the high-energy state of the thylakoid system associated with photophosphorylation.

Mechanisms of heat protection by cycloheximide or puromycin: Involvement of polyamines?

1. 1.|When Chinese hamster ovary cells are treated with cycloheximide (10 μg/ml) or puromycin (100 μg/ml) for 2 h before and during heating at 43°C for 3 h, there is protection from hyperthermic killing; i.e. the plating efficiency increases 2000-fold from 3.7 × 10⁻⁵ to (6–9) × 10⁻².

2. 2.|The total intracellular levels of spermidine and spermine are not altered by the hyperthermic or drug treatments.

3. 3.|The small 30% decrease in intracellular putrescine observed after heating is not altered by drug treatment.

4. 4.|Heat protection by treatment with cycloheximide or puromycin cannot be attributed to changes in levels of total intracellular polyamines.

Electrical skin resistance and rectal temperature changes in resting human subjects exposed to heat

1. 1.|Fourteen male volunteers were examined under passive heating.

2. 2.|Electrical skin resistance (ESR) and rectal temperature (T_re) were measured during the whole period of exposure.

3. 3.|It was found that:

• —|ESR decreases rapidly with increasing air temperature. Assuming an exponential curve yields a mean time constant of 14 min.

• —|There is a correlation between the individual ESR time constants and T_re increases (r = 0.695, P < 0.005).

• —|Additional changes of ESR were noted in 8 subjects at a constant air temperature of 42°C.

4. 4.|It is concluded that ESR may be a useful indicator of the sweating response of the human thermoregulatory system during exogenous heat load.

Hatching of freshwater sponge gemmules after low temperature exposure: Ephydatia mülleri (Porifera: Spongillidae)

1. 1.|Gemmules of Ephydatia mülleri can withstand exposure to temperatures down to −80°C for 63 days without loss of hatchability.

2. 2.|Hatching is slowed following exposure to temperatures below −27°C.

3. 3.|There is a slight but significant relationship between gemmule size and the time to hatch.

4. 4.|This species can withstand long-term exposure to winter air temperatures occurring within its known geographic range.

Effect of low temperature (−30 to −60°C) on the reoxidation of the Photosystem II primary electron acceptor in the presence and absence of 3(3,4-dichlorophenyl)-1,1-dimethylurea

The fluorescence yield has been measured on spinach chloroplasts at low temperature (−30 to −60°C) for various dark times following a short saturating flash. A decrease in the fluorescence yield linked to the reoxidation of the Photosystem II electron acceptor Q is still observed at −60°C. Two reactions participate in this reoxidation: a back reaction or charge recombination and the transfer of an electron from Q⁻ to Pool A. The relative competition between these two reactions at low temperature depends upon the oxidation state of the donor side of the Photosystem II center:

1. (1) In dark-adapted chloroplasts (i.e. in States S₀+S₁ according to Kok, B., Forbush, B. and McGloin, M. (1970) Photochem. Photobiol. 11, 457–475), Q, reduced by a flash at low temperature, is reoxidized by a secondary acceptor and the positive charge is stabilized on the Photosystem II donor Z. Although this reaction is strongly temperature dependent, it still occurs very slowly at −60°C.

2. (2) When chloroplasts are placed in the S₂+S₃ states by a two-flash preillumination at room temperature, the reoxidation of Q⁻ after a flash at low temperature is mainly due to a temperature-independent back reaction which occurs with non-exponential kinetics.

3. (3) Long continuous illumination of a frozen sample at −30°C causes 6–7 reducing equivalents to be transferred to the pool. Thus, a sufficient number of oxidizing equivalents should have been generated to produce at least one O₂ molecule.

4. (4) A study of the back reaction in the presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) shows the superposition of two distinct non-exponential reactions one temperature dependent, the other temperature independent.

Comparative aspects of the thermal biology of the short-tailed gerbil, Desmodillus auricularis, and the bushveld gerbil, Tatera leucogaster

1. 1. The response of oxygen consumption (VO₂), thermal conductance (C_d and C_min, body temperature (T_b), and evaporative water loss (EWL) of Tatera leucogaster and Desmodillus auricularis were measured over the range of ambient temperatures (T_a) from 5–35°C.

2. 2. Basal metabolic rate (BMR) of T. leucogaster was 0.841 ± 0.049 ml O₂ g⁻¹ h⁻¹ and lower than predicted, while that of D. auricularis was similar to the expected value (1.220 ± 0.058 ml O₂ g⁻¹ h⁻¹). D. auricularis had a high, narrow thermoneutral zone (TNZ) typical of nocturnal, xerophilic, burrowing rodents.

3. 3. D. auricularis and T. leucogaster regulated T_b over the range T_a = 5–35°C and kept EWL and dry thermal conductance at a minimum below the TNZ. However, the EWL of T. leucogaster increased rapidly above T_a = 30°C.

4. 4. After comparison with data from other species, it was concluded that there is an optimum size for xeric, nocturnal, burrowing rodents.

The ability of isolated myocardial cells of the rat to contract at temperatures near freezing point

1. 1.|Single spontaneously-beating myocardial cells were prepared by enzymatic isolation from rat heart ventricles. Using a TV camera and videotape recorder, beating parameters were characterized with respect to temperature.

2. 2.|The cells obtained showed two types of contractions: (a) action-potential-coupled, fast contractions, which could be triggered electrically but sometimes also occurred spontaneously; and (b) a phasic type of contraction which occurs spontaneously as a slowly (25–200 μm s⁻¹) proceedings wave through the cell and is not coupled to the action potential.

3. 3.|Both the spontaneous phasic and electrically-triggered fast contractions continued down to about 0°C, where the cells went into an irreversible contracture, but recovered if the temperature was raised before the contracture occurred.

4. 4.|In physically-contracting cells, the beat rate and the velocity of the contraction wave were temperature dependent showing a linear relationship on an Arrhenius plot; apparent activation energies were 31 and 54 kJ mol⁻¹, respectively.

5. 5.|When fast contractions were triggered by electric field stimulation, the threshold of cells was reduced as temperature rose. The rheobase values showed that cell excitability decreased more steeply when the temperature fell below 20°C. This change could not be seen in the values of chronaxie, which showed a linear relationship on the Arrhenius plot within the whole temperature range used (6–35°C).

6. 6.|It is concluded that the lower temperature limit is not the same for all structural levels of the rat heart. Myocardial cells are able to contract still at the freezing point while mechanisms initiating the heart beat require higher temperatures to be functional.

Interaction of oxidized and reduced N-methylphenazonium methosulfate (PMS) with Photosystem II

In 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) poisoned chloroplasts, the restoration of the fluorescence induction is presumed to be due to a back reaction of the reduced primary acceptor (Q⁻) and the oxidized primary donor (Z⁺) of Photosystem II. Carbonylcyanide m-chlorophenylhydrazone (CCCP) is known to inhibit this back reaction. The influence of reduced N-methylphenazonium methosulfate (PMS) in the absence of CCCP and of oxidized PMS in the presence of CCCP on the back reaction was investigated and the following results were obtained:

1. (1) Reduced PMS at the concentration of 1 μM inhibits the back reaction as effectively as hydroxylamine, suggesting an electron donating function of reduced PMS for System II.

2. (2) The inhibition of the back reaction by CCCP is regenerated to a high degree by oxidized PMS which led to assume a cyclic System II electron flow catalysed by PMS.

3. (3) At concentrations of reduced PMS higher than 1 μM it is shown that both the fast initial emission and more significantly the variable emission are quenched.

Accumulation of bicarbonate in intact chloroplasts following a pH gradient

With silicone layer filtering centrifugation the uptake of radioactively labelled bicarbonate into isolated spinach chloroplasts was followed. This uptake was shown to have the following properties:

1. (a) It is so rapid that the kinetics of uptake usually cannot be resolved.

2. (b) Bicarbonate is accumulated in the stroma. The factor between the internal and external concentrations increases greatly when the pH of the medium is lowered from pH 8.5 to pH 7.0.

3. (c) The accumulation factor is independent of the concentration in the medium for a long concentration range.

4. (d) The accumulation of bicarbonate is increased when the chloroplasts are illuminated. This increase is abolished by the addition of uncoupler.

5. (e) Diamox, an inhibitor of carbonic anhydrase, inhibits the rate of bicarbonate uptake.

The activity of carbonic anhydrase was assayed in isolated chloroplasts and in leaf homogenates. In agreement with earlier reports the main activity was found to be located in the chloroplasts. This activity is latent; it can be only assayed if the chloroplasts are osmotically shocked.

From these results the following conclusions have been drawn:

1. (a) The inner membrane is impermeable to protons. Light-driven proton transport into the thylakoid space causes an alkalisation of the stroma.

2. (b) The uptake of bicarbonate proceeds via diffusion of CO₂ across the inner membrane. There are no indications for a specific transport of bicarbonate.

3. (c) The CO₂ concentration in the chloroplasts may be equal to the CO₂ concentration in the external space. The distribution of bicarbonate between the two compartments is inversely proportional to the distribution of protons.

A possible involvement of carbonic anhydrase and the bicarbonate pool in the stroma in increasing the CO₂ affinity of CO₂ fixation is discussed.     

Light-induced changes of fluorescence and absorbance in spinach chloroplasts at −40 °C

1. 1. Spinach chloroplasts were stored in the dark for at least 1 h, rapidly cooled to −40 °C, and illuminated with continuous light or short saturating flashes. In agreement with the measurements of Joliot and Joliot, chloroplasts that had been preilluminated with one or two flashes just before cooling showed a less efficient increase in the yield of chlorophyll a fluorescence upon illumination at −40 °C than dark-adapted chloroplasts. The effect disappeared below −150 °C, but reappeared again upon warming to −40 °C. Little effect was seen at room temperature in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), added after the preillumination.

2. 2. Light-induced absorbance difference spectra at −40 °C in the region 500–560 nm indicated the participation of two components, the socalled 518-nm change (P518) and C-550. After preillumination with two flashes the absorbance change at 518 nm was smaller, and almost no C-550 was observed. After four flashes, the bands of C-550 were clearly visible again.

3. 3. The fluorescence increase and the absorbance change at 518 nm showed the same type of flash pattern with a minimum after the second and a maximum at the fourth flash. In the presence of 100 μM hydroxylamine, the fluorescence response was low after the fourth and high again after the sixth flash, which confirmed the hypothesis that the flash effect was related to the so-called S-state of the electron transport pathway from water to Photosystem 2.

4. 4. The kinetics of the light-induced absorbance changes were the same at each wavelength, and, apart from the size of the deflection, they were independent of preillumination. Flash experiments indicated that the absorbance changes were a one-quantum reaction. This was also true for the fluorescence increase in dark-adapted chloroplasts, but with preilluminated chloroplasts several flashes were needed to approximately saturate the fluorescence yield.

5. 5. The results are discussed in terms of a mechanism involving two electron donors and two electron acceptors for System 2 of photosynthesis.

Thermal responses of the fresh water turtle Mauremys caspica to step-function changes in the ambient temperature

1. 1.|The turtle Mauremys caspica cools significantly faster than it heats in air. The heating/cooling ratio is 0.49.

2. 2.|The variation of body temperature in relation to time-course in response to a step-function change of environmental temperature, fitted to a second-order system improves that of a first-order system.

3. 3.|The gradient between ambient temperature (T_a) and equilibrium body temperature (T_b) increases significantly and progressively when ambient temperature rises over 25°C.

4. 4.|At 40°C thermoregulatory hyperventilation was detected, implying an increase in air convection requirement (ventilation relative to O₂ consumption, ).

Binding of radioactively labeled carboxyatractyloside, atractyloside and bongkrekic acid to the ADP translocator of potato mitochondria

1. 1. The inhibition of the ADP-stimulated respiration of potato mitochondria by carboxyatractyloside is relieved by high concentration of ADP or by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Atractyloside is a much less potent inhibitor than carboxyatractyloside. The inhibition of the ADP-stimulated respiration required about 60-times more atractyloside than carboxyatractyloside.

2. 2. [³⁵S]carboxyatractyloside and [³H]bongkrekic acid bind to potato mitochondria with high affinity (K_d = 10 to 20 nM, n = 0.6–0.7 nmol per mg protein). Added ADP competes with carboxyatractyloside for binding; on the contrary ADP increases the amount of bound bongkrekic acid. [³H]atractyloside binds to potato mitochondria with a much lower affinity (K_d = 0.45 μM) than carboxyatractyloside or bongkrekic acid.

3. 3. Bound [³H]atractyloside is displaced by ADP, carboxyatractyloside and bongkrekic acid. The displacement of bound [³⁵S]carboxyatractyloside by bongkrekic acid and of bound [³H]bongkrekic acid by carboxyatractyloside is markedly increased by ADP.

4. 4. Bongkrekic acid competes with [³⁵S]carboxyatractyloside for binding. Addition of a small concentration of ADP considerably enhances the inhibitory effect of bongkrekic acid on [³⁵S]carboxyactratyloside binding.

5. 5. The adenine nucleotide content of potato mitochondria is of the order of 1 nmol per mg protein. ADP transport in potato mitochondria is inhibited by atractyloside 30- to 40-times less efficiently than by carboxyatractyloside.

The mechanism of the oxidation of ascorbate and Mn by chloroplasts: The role of the radical superoxide

1. 1. The mechanism of the photooxidation of ascorbate and of Mn²⁺ by isolated chloroplasts was reinvestigated.

2. 2. Our results suggest that ascorbate or Mn²⁺ oxidation is the result of the Photosystem I-mediated production of the radical superoxide, and that neither ascorbate nor Mn²⁺ compete with water as electron donors to Photosystem II nor affect the rate of electron transport through the two photosystems: The radical superoxide is formed as a result of the autooxidation of the reduced forms of low potential electron acceptors, such as methylviologen, diquat, napthaquinone, or ferredoxin.

3. 3. In the absence of ascorbate or Mn²⁺ the superoxide formed dismutases either spontaneously or enzymatically producing O₂ and H₂O₂. In the presence of ascorbate or Mn²⁺, however, the superoxide is reduced to H₂O₂ with no formation of O₂. Consequently, in the absence of reducing compounds, in the reaction H₂O to low potential acceptor one O₂ (net) is taken up per four electrons transported where as in the presence of ascorbate, Mn²⁺ or other suitable reductants up to three molecules O₂ can be taken up per four electrons transported.

4. 4. This interpretation is supported by the following observations: (a) in a chloroplast-free model system containing NADPH and ferredoxin-NADP reductase, methylviologen can be reduced to a free radical which is autooxidizable in the presence of O₂; the addition of ascorbate or Mn²⁺ to this system results in a two fold stimulation of O₂ uptake, with no stimulation of NADPH oxidation. The stimulation of O₂ uptake is inhibited by the enzyme superoxide dismutase; (b) the stimulation of light-dependent O₂ uptake in the system H₂O → methylviologen in chloroplasts is likewise inhibited by the enzyme superoxide dismutase.

5. 5. In Class II chloroplasts in the system H₂O → NADP upon the addition of ascorbate or Mn²⁺ an apparent inhibition of O₂ evolution is observed. This is explained by the interaction of these reductants with the superoxide formed by the autooxidation of ferredoxin, a reaction which proceeds simultaneously with the photoreduction of NADP. Such an effect usually does not occur in Class I chloroplasts in which the enzyme superoxide dismutase is presumably more active than in Class II chloroplasts.

6. 6. It is proposed that since in the Photosystem I-mediated reaction from reduced 2,4-dichlorophenolindophenol to such low potential electron acceptor as methylviologen, superoxide is formed and results in the oxidation of the ascorbate present in the system, the ratio ATP/2e in this system (when the rate of electron flow is based on the rate of O₂ uptake) should be revised in the upward direction.

Effect of temperature on growth and bioenergetics of a tropical moth

1. 1.|Larval development and metamorphosis of Achaea junta were prolonged at 22°C, compared to 27, 32 and 35°C.

2. 2.|Overall rates of consumption, assimilation, production and metabolism of the larvae increased with temperature.

3. 3.|Efficiencies of assimilation and conversion of the digested food were significantly altered by life stage and temperature.

4. 4.|About 60% of the pupal energy was transferred to the imago at the tested temperatures.

Effect of hyperthermia on blood constituents in the domestic rabbit (Lepus cuniculus)

1. 1. Effects of exposing rabbits to temperatures 37–50°C on body-core temperature and some blood constituents were investigated.

2. 2. Heat stroke death occurred at or above a critical core-temperature of 43.0°C.

3. 3. Plasma osmolality and levels of glucose, urea and lactate were significantly elevated in hyperthermia.

4. 4. Widespread tissue damage was indicated by increases in plasma activities of glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT) and creatinine phosphokinase (CPK).

5. 5. The most sensitive indicators of impending heat stroke in heat stressed rabbits were plasma levels or urea, lactate and CPK.

Work at cold storage

1. 1. Work activities in cold storage rooms were assessed by a mailed questionnaire survey of cold storage facilities in Japan.

2. 2. There are nearly 4000 cold storage facilities and about 80% are being kept at temperatures below −20°C.

3. 3. The chief items of stock in storage were marine products, livestock products, frozen food and agricultural products.

4. 4. Methods used for loading and unloading in cold storage rooms are forklift, manual handling, and automatic machines.

5. 5. Use of forklifts appeared to be widespread.

6. 6. Working time differed according to the ambient temperature of the cold storage rooms.

7. 7. Common ailments of cold storage workers are lumbago, bronchitis, neuralgia etc.

Affinities of ATP for the dinitrophenol-induced ATPase

1. 1. The effect of Mg²⁺ on ATP-dependent processes catalysed by intact rat-liver mitochondria can be explained quantitatively by the formation of Mg-ATP complexes that cannot act as a substrate for the adenine nucleotide translocator.

2. 2. The dinitrophenol-induced ATPase is characterized by two affinities of ATP: K_m(1) = 6.7 μM and K_m(2) = 63 μM, which contribute to the extent of 70% and 30%, respectively, to the total ATPase activity under the standard conditions employed.

3. 3. K_m(1) of ATP is competitively increased by atractyloside, and is insensitive to changes in cation concentration or to oligomycin or aurovertin.

4. 4. K_m(2) is as sensitive to atractyloside as the K_m(1) and is also insensitive to oligomycin. However, it is increased by decreasing the cation concentration, and disappears in the presence of aurovertin.

5. 5. It is proposed that two conformations of the adenine nucleotide translocator exist, characterized by their different affinities for ATP. The distribution of the enzyme over these two conformations appears to be a function of the energy state of the mitochondria (coupled or uncoupled).

A relationship between heat load and plasma enzyme concentration

1. 1. The sensitivity of serum enzyme levels as indicators of tissue damage is less well established in the prodromal period of heatstroke, especially for sub-lethal stress conditions.

2. 2. Anaesthetized rats were exposed to two different sets of thermal conditions.

3. 3. Plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK) and lactate dehydrogenase (LD) were assayed in each group upon termination of stress, 6 h post-stress and 24 h post-stress.

4. 4. The tissue “damage” sustained was mild to moderate and completely reversible.

5. 5. The rate of rise in body temperature may constitute an important factor in the ultimate pathology.

6. 6. CK proved to be the most sensitive parameter of tissue “damage”.