Chlorophyll a fluorescence and photochemical activities of chloroplast fragments

1. Comparative studies were made on the fluorescence characteristics of chlorophyll a at 20° and −193°, and quantum efficiencies for P 700 oxidation and NADP⁺ reduction were measured in chloroplasts and chloroplast fragments obtained after incubation with 0.5% digitonin.

2. Differences in the flurescence yield of chlorophyll a in flowing and stationary suspensions of untreated chloroplasts and of the large fragments are indicative of light-induced photoreduction of the quencher Q of chlorophyll a, associated with pigment System 2 (chlorophyll a₂). The relatively low constant fluorescence yield of chlorophyll a in the small fragments indicates the absence of fluorescent chlorophyll a₂ from these fragments and suggests that the low fluorescence is due to chlorophyll a, associated with pigmen System 1 (chlorophyll a₁). The ratio of the fluorescence yields of chlorophyll a₁ and chlorophyll a₂ is 0.45:1. In the large particles the concentration ratio of pigment System 1 and System 2 is 1:3.

3. The efficiencies of quanta absorbed at 673, 683 and 705 nm for NADP⁺ reduction and P 700 oxidation in untreated chloroplasts and chloroplast fragments indicate that digitonin treatment results in a separation of System 2 from System 1 in the small fragments. Sonication does not cause such a separation. Under the conditions used P 700 oxidation and NADP⁺ reduction in the small fragments separated after digitonin treatment, occurred with maximal efficiency of 0.7 to 1.0 and 0.7, respectively.

4. The constancy of the fluorescence yield of chlorophyll a₁ in the small fragments, under conditions at which P 700 is oxidized and NADP⁺ is reduced, is interpreted as evidence either for the hypothesis that the fluorescence of chlorophyll a₁ is controlled by the redox state of the primary photoreductant XH, or alternatively for the hypothesis that energy transfer from fluorescent chlorophyll a₁ to P 700 goes via an intrinsically weak fluorescent, still unknown, chlorophyll-like pigment.

5. The low-temperature emission band around 730 nm is argued not to be due to excitation by System 1 only; the relatively large half width of the band, as compared to the emission bands at 683 and 696 nm, suggests that it is possibly due to overlapping emission bands of different pigments.     

Light-dependent changes of the Mg concentration in the stroma in relation to the Mg dependency of CO2 fixation in intact chloroplasts

(1) Light-dependent changes of the Mg²⁺ content of thylakoid membranes were measured at pH 8.0 and compared with earlier measurements at pH 6.6. In a NaCl and KCl medium, the light-dependent decrease in the Mg²⁺ content of the thylakoid membranes at pH 8.0 is found to be 23 nmol Mg²⁺ per mg chlorophyll, whereas in a sorbitol medium it is 83 nmol Mg₂₊ per mg chlorophyll.

(2) A light dependent increase in the Mg²⁺ content of the stroma was detected when chloroplasts were subjected to osmotic shock, amounting to 26 nmol/mg chlorophyll. Furthermore, a rapid and reversible light-dependent efflux of Mg²⁺ has been observed in intact chloroplasts when the divalent cation ionophore A 23 187 was added, indicating a light-dependent transfer of about 60 nmol of Mg²⁺ per mg chlorophyll from the thylakoid membranes to the stroma.

(3) CO₂ fixation, but not phosphoglycerate reduction, could be completely inhibited when A 23 187 was added to intact chloroplasts in the absence of external Mg²⁺. If Mg²⁺ was then added to the medium, CO₂ fixation was restored. Half of the maximal restoration was achieved with about 0.2 mM Mg²⁺, which is calculated to reflect a Mg²⁺ concentration in the stroma of 1.2 mM. The further addition of Ca²⁺ strongly inhibits CO₂ fixation.

(4) The results suggest that illumination of intact chloroplasts causes an increase in the Mg²⁺ concentration of 1–3 mM in the stroma. Compared to the total Mg²⁺ content of chloroplasts, this increase is very low, but it appears to be high enough to have a possible function in the light regulation of CO₂ fixation.     

Biochemical and biophysical studies on cytochrome c oxidase XIV. The reaction with cytochrome c as studied by pulse radiolysis

1. The reduction of cytochrome c oxidase by hydrated electrons was studied in the absence and presence of cytochrome c.

2. Hydrated electrons do not readily reduce the heme of cytochrome c oxidase. This observation supports our previous conclusion that heme a is not directly exposed to the solvent.

3. In a mixture of cytochrome c and cytochrome c oxidase, cytochrome c is first reduced by hydrated electrons (k = 4 · 10¹⁰ M⁻¹ · s⁻¹ at 22 °C and pH 7.2) after which it transfers electrons to cytochrome c oxidase with a rate constant of 6 · 10⁷ M⁻¹ · s⁻¹ at 22 °C and pH 7.2.

4. It was found that two equivalents of cytochrome c are oxidized initially per equivalent of heme a reduced, showing that one electron is accepted by a second electron acceptor, probably one of the copper atoms of cytochrome c oxidase.

5. After the initial reduction, redistribution of electrons takes place until an equilibrium is reached similar to that found in redox experiments of Tiesjema, R. H., Muijsers, A. O. and Van Gelder, B. F. (1973) Biochim. Biophys. Acta 305, 19–28.     

Net movements of monovalent and bivalent cations, and their relation to energy metabolism, in slices of hepatoma 3924A and of a mammary tumour

1. During incubation at 1° in saline medium buffered either with phosphate or bicarbonate, slices of Morris hepatoma 3924A, and of a chemically induced tumour of rat mammary gland, lost K⁺ and gained Na⁺, Ca²⁺ and water.

2. Upon subsequent incubation at 38° in oxygenated medium, these changes were partially reversed. In the hepatoma, the reaccumulation of K⁺ was equally efficient in phosphate or bicarbonate medium, and in the presence and absence of glucose. Ca²⁺ was extruded in bicarbonate, but not in phosphate medium, and its extrusion was reduced in the presence of glucose.

3. When respiration was inhibited in the presence of glucose, K⁺ transport by the hepatoma continued to an extent which varied with the glycolytic activity of the slices, suggesting that the rate of ATP synthesis was a limiting factor under these conditions.

4. In the absence of glucose, the transport of Na⁺ and K⁺ was completely stopped by respiratory inhibition. However, more than 50% of the O₂ uptake had to be inhibited before any effect on transport was observed, suggesting that the rate of synthesis of ATP from endogenous respiration is in excess of that required to maintain transport.

5. Inhibition of transport by ouabain was accompanied by a 30% fall in the rate of endogenous respiration, and by a fall of 33% in the rate of glycolysis in the presence of cyanide plus glucose.

6. Comparison of the minimum rates of respiration and of glycolysis (in the presence of glucose plus cyanide) required to maintain the maximal extent of K⁺ transport in the hepatoma slices, suggests that ATP derived from oxidative phosphorylation or from anaerobic glycolysis is equally efficient as a source of energy for ion transport.     

Studies on the exchange of G-actin-bound calcium with bivalent cations

1. The possibility of the replacement of G-actin-bound calcium by various bivalent cations has been investigated. After the reaction with all cations studied, with the exception of Cu²⁺, action remains active, i.e., contains bound ATP and polymerizes in 0.1 M KCl.

2. The amount of G-actin-bound calcium, as well as the sum of bivalent cation after replacement, not removable by short-time Dowex-50 treatment, accounts to about 1 mole per 50000 g of G-actin.

3. The rate of exchange is of the same order for bivalent cations studied, including calcium.

4. G-actin-bound Ca²⁺ is fully replaced, besides free Ca²⁺, by free Mn²⁺ and Cd²⁺. The replacement with Mg²⁺, Co²⁺, Ni²⁺ and Zn²⁺ is not complete, and there is practically no reaction with Ba²⁺ and Sr²⁺.

5. Assuming the affinity constant of Ca²⁺ as 1, the following affinity constants for other bivalent cations were obtained: Mn²⁺, 0.90; Cd²⁺, 1.07; Mg²⁺, 0.27; Zn²⁺, 0.22; Co²⁺, 0.18; Ni²⁺, 0.08.

6. The results obtained show that there exists a close correlation between the ionic radius of a particular bivalent cation, and its ability to replace bound Ca²⁺.     

A convenient electrochemical preparation of reduced methyl viologen and a kinetic study of the reaction with oxygen using an anaerobic stopped-flow apparatus

1. Single reduced methyl viologen (MV^.+) acts as an electron donor in a number of enzyme systems. The large changes in extinction coefficient upon oxidation (λ_max 600 nm; MV^.+, = 1.3 · 10⁴ M⁻¹ · cm⁻¹; oxidised form of methyl viologen (MV²⁺), = 0.0) make it ideally suited to kinetic studies of electron transfer reactions using stopped-flow and standard spectrophotometric techniques.

2. A convenient electrochemical preparation of large amounts of MV^.+ has been developed.

3. A commercial stopped-flow apparatus was modified in order to obtain a high degree of anaerobicity.

4. The reaction of MV^.+ with O₂ produced H₂O₂ (k > 5 · 10⁶ M⁻¹ · s⁻¹, pH 7.5, 25 °C). H₂O₂ subsequently reacted with excess MV^.+ (k = 2.3 · 10³ M⁻¹ · s⁻¹, pH 7.5, 25 °C) to produce water. The kinetics of this reaction were complex and have only been interpreted over a limited range of concentrations.

5. The results support the theory that the herbicidal action of methyl viologen (Paraquat, Gramoxone) is due to H₂O₂ (or radicals derived from H₂O₂) induced damage of plant cell membrane.     

Actomyosin-like protein from brain separation and characterization of the actin-like component

1. Actin-like protein (neurin) has been separated from actomyosin-like protein (neurostenin) isolated from bovine brain. This was accomplished by gel filtration chromatography (Sephadex G-200) and by ultracentrifugation in a continuous sucrose gradient containing 0.6 M KI.

2. The actin-like protein stimulated the Mg²⁺-ATPase activity of muscle myosin.

3. It contained bound nucleotide which exchanged with free [¹⁴C]ATP.

4. It polymerized in the presence of 0.1 M KCl and 0.1 mM Mg²⁺ with release of P_i; increase in viscosity occurred upon dilution of the 0.6 M KI to 0.1 M.

5. The neurin reacted immunologically to form a single band with antiserum to neurostenin.

6. The neurin, similar to muscle actin, contained 3-methylhistidine.

7. The sedimentation constant of the protein was 2.8 S.     

Studies on the energy coupling sites of photophosphorylation IV. The relation of proton fluxes to the electron transport and ATP formation associated with Photosystem II

1. By using dibromothymoquinone as the electron acceptor, it is possible to isolate functionally that segment of the chloroplast electron transport chain which includes only Photosystem II and only one of the two energy conservation sites coupled to the complete chain (Coupling Site II, observed P/e₂ = 0.3–0.4). A light-dependent, reversible proton translocation reaction is associated with the electron transport pathway: H₂O → Photosystem II → dibromothymoquinone. We have studied the characteristics of this proton uptake reaction and its relationship to the electron transport and ATP formation associated with Coupling Site II.

2. The initial phase of H⁺ uptake, analyzed by a flash-yield technique, exhibits linear kinetics (0–3 s) with no sign of transient phenomena such as the very rapid initial uptake (“pH gush”) encountered in the overall Hill reaction with methylviologen. Thus the initial rate of H⁺ uptake obtained by the flash-yield method is in good agreement with the initial rate estimated from a pH change tracing obtained under continuous illumination.

3. Dibromothymoquinone reduction, observed as O₂ evolution by a similar flash-yield technique, is also linear for at least the first 5 s, the rate of O₂ evolution agreeing well with the steady-state rate observed under continuous illumination.

4. Such measurements of the initial rates of O₂ evolution and H⁺ uptake yield an H⁺/e⁻ ratio close to 0.5 for the Photosystem II partial reaction regardless of pH from 6 to 8. (Parallel experiments for the methylviologen Hill reaction yield an H⁺/e⁻ ratio of 1.7 at pH 7.6.)

5. When dibromothymoquinone is being reduced, concurrent phosphorylation (or arsenylation) markedly lowers the extent of H⁺ uptake (by 40–60%). These data, unlike earlier data obtained using the overall Hill reaction, lend themselves to an unequivocal interpretation since phosphorylation does not alter the rate of electron transport in the Photosystem II partial reaction. ADP, P_i and hexokinase, when added individually, have no effect on proton uptake in this system.

6. The involvement of a proton uptake reaction with an H⁺/e⁻ ratio of 0.5 in the Photosystem II partial reaction H₂O → Photosystem II → dibromothymoquinone strongly suggests that at least 50% of the protons produced by the oxidation of water are released to the inside of the thylakoid, thereby leading to an internal acidification. It is pointed out that the observed efficiencies for ATP formation (P/e₂) and proton uptake (H⁺/e⁻) associated with Coupling Site II can be most easily explained by the chemiosmotic hypothesis of energy coupling.     

The activation by Ca of the ATPase of extracted muscle fibrils with variation of ionic strength, pH and concentration of MgATP

1. The alteration of the Ca²⁺ requirements of the ATPase activity of fibrils from rabbits and crabs at varying ionic strength, pH and concentration of MgATP (i.e. MgATP²⁻ + MgHATP⁻) was investigated.

2. Under physiological conditions, it was found that the ATPase activity of rabbit and crab fibrils after an initial increase decreased steeply when the Ca²⁺ concentration is raised above 1×10⁻⁴ M. This is a primary effect of the over-optimal Ca²⁺ concentration and not a secondary one caused by the influence of accompanying ions.

3. The Ca²⁺ requirements for ATP splitting by rabbit fibrils remain constant at an ionic strength from 0.1 to 0.2 and for a MgATP concentration in the range from 0.5 to 10 mM. At I = 0.05 it is about 5 times smaller than at 0.1. When the pH is decreased from 8 to 7, the Ca²⁺ requirements are increased some 10 times but only 3 times when the pH is varied between 7 and 6.

4. In crab fibrils, there is no alteration of the Ca²⁺ requirements when the ionic strength is varied between 0.05 and 0.2, but a reduction of the pH from 8.0 to 6.0 raises the Ca²⁺ requirements for half activation and for threshold by a factor of 10. Changing the MgATP concentration increases the Ca²⁺ requirements only in the range from 1 to 5 mM, while the concentration required in 0.5 mM is identical with that at 1 mM, and 10 mM corresponds to 5 mM.

5. It can be deduced from the experimental results that at a pH above 6.0 maximal activation is always obtained if the Ca²⁺ concentration is 5×10⁻⁵ M. By contrast, relaxation is only achieved when the Ca²⁺ concentration is below 1×10⁻⁷ M for pH 7.0 and I > 0.1 or below 1×10⁻⁸ for pH > 7.0 or I < 0.1.

6. To achieve complete relaxation, an ethyleneglycoldiaminotetraacetate (EGTA) concentration of 1 mM is sufficient, even when there is a large degree of contamination by Ca²⁺ as long as the pH stays above 6.5.     

Control of excitation transfer in photosynthesis V. Correlation of membrane structure to regulation of excitation transfer between two pigment systems in isolated spinach chloroplasts

1. Changes in fluorescence yield of chlorophyll a in isolated chloroplasts have been interpreted by means of regulation of excitation transfer between two pigment systems of photosynthesis^5–7. In order to investigate the relationship between the membrane structure of chloroplasts and the regulation of excitation transfer, changes of light scattering and chlorophyll a fluorescence of isolated spinach chloroplasts were measured upon addition of cations, Mg²⁺ and Na⁺. The cations increased the intensities of both light scattering and fluorescence yield. The changes showed similar time courses and concentration dependences. These facts suggest that modification of membrane structure produced by the cations suppresses the excitation transfer between the two pigment systems.

2. In another case of structural change which is induced by light in the presence of N-methylphenazonium methosulfate, there was little correlation between light-scattering and fluorescence changes.

3. Changes in fluorescence yield induced by the addition of Mg²⁺ were measured in disintegrated chloroplasts and fractionated particles. The effects of Mg²⁺ on fluorescence were observed only in preparations of grana stacks, but not in preparations of stroma lamellae. These findings suggest that the excitation transfer is regulated between the two pigment systems located in the grana thylacoid membranes.     

Dialysis Culture for the Production of Extracellular Protein A from Staphylococcus aureus A676

Different nutrient media have been tested for the production of extracellular protein A from Staphylococcus aureus A676. High yields were obtained in trypticase soy broth with glucose as the main energy source. In aerated fermenter cultures the amount of protein A obtained was 0·2–0·3 gl⁻¹, as compared to reported yields of 0·03 gl⁻¹. Yields were further increased in dialysis cultures, where protein A concentrations of 2 gl⁻¹. were achieved.     

Chlorophyll a fluorescence as a monitor of nanosecond reduction of the photooxidized primary donor P-680 of Photosystem II

1. Changes in the fluorescence yield of aerobic Chlorella vulgaris have been measured in laser flashes of 15 ns, 30 ns and 350 ns half time. The kinetics after the first flash given after a 3 min dark period could be simulated on a computer using the hypothesis that the oxidized acceptor Q and primary donor P⁺ are fluorescence quenchers, and Q⁻ is a weak quencher, and that the reduction time for P⁺ is 20–35 ns.

2. The P⁺ reduction time for at least an appreciable part of the reaction centers was found to be longer after the second and subsequent flashes. In the first 5 flashes an oscillation was observed. Under steady state conditions, with a pulse separation of 3 s, a reduction time for P⁺ of about 400 ns for all reaction centers gave the best correspondence between computed and experimental fluorescence kinetics.     

Studies on the mechanism of inhibition of the mitochondrial electron transport by antimycin. III. Binding of antimycin to sub-mitochondrial particles and to complex III

1. Extraction by ether removes only about one-half of antimycin added to sub-mitochondrial particles, independently of the amount of antimycin added up to that necessary for 100% inhibition.

2. The amount of antimycin extractable with ether remains the same even when the antimycin is redistributed between an antimycin-inhibited preparation and an untreated. The antimycin remaining after ether extraction is redistributed between ether-accessible and ether-inaccessible sites when the preparation is incubated on its own or with an untreated preparation.

3. Low concentrations of cholate increase the extractability of the antimycin by ether.

4. Complex III binds antimycin more firmly than sub-mitochondrial particles. However, antimycin is readily extracted by ether, leading to restoration of enzymic activity and cleavage of the complex by bile salts.

5. The results are consistent with an explanation of the sigmoidal inhibition curve with antimycin and preparations of the intact respiratory chain in terms of an allosteric model.     

Association and dissociation of the thick and thin filaments within myofibrils in conditions of “contraction” and “relaxation”

1. The current assumption that the low ATPase activity of relaxed myofibrils is represented by the ATPase activity of myosin which has been set free during the dissociation of actomyosin was investigated. For this purpose, the ATPase activity of relaxed skeletal myofibrils of the rabbit and of the crab Maia squinado has been compared with the activity of contracted fibrils and of purified rabbit myosin in conditions of varying ionic strength, pH and concentrations of MgATP (i.e. MgATP²⁻ + MgHATP⁻) and Mg²⁺.

2. Contraction and relaxation of the fibrils was induced by changing the concentration of Ca²⁺ from about 5×10⁻⁵ to below 1×10⁻⁸ M.

3. In all conditions studied, the ATPase activity of relaxed fibrils was about 6–8 times less than that of the contracted fibrils, but it remained a typical actomyosin ATPase.

4. Quantitatively and qualitatively, this ATPase differs from the ATPase of myosin. For instance, its dependence on pH is the reverse of that of the myosin ATPase.

5. Calculation showed that the fibrils are dissociated by 90% in conditions of relaxation. Since the ATPase activity of myosin was merely some 2% of the actomyosin activity, the major part of the ATPase of fibrils, even at a dissociation of 90%, is bound to show the properties of the ATPase of actomyosin.

6. However, a dissociation of 90% cannot be distinguished from a dissociation of 100% by means of physical methods (viscosity, superprecipitation, resistance to stretch, etc.). This explains why physical methods indicate a “full” dissociation of actomyosin although, enzymatically, the ATPase is still of the actomyosin type.

7. The possible reasons are discussed for the discrepancy between the 100-fold increase in the ATP turnover and the 1000-fold increase in energy turnover of the living muscle during the transition from relaxed to active state. The most probable explanation seems to be an ATPase activity of myosin which is too high by a factor of ten as compared to the energy turnover of living muscle at the resting state. This high activity cannot be caused by a contamination of the myosin by Ca²⁺-insensitive actomyosin.     

Avian embryonic thermoregulation: role of Q10 in interpretation of endothermic reactions

1. Incubated chicken eggs (D14 to D21) were placed separately in transparent acrylic glass chambers which were immersed in a 37.5°C water bath for 3 h. The chambers were then moved for 3 h to another water bath controlled at 34.5°C. Oxygen consumption and temperature of the allantoic fluid (T_af) were measured at 5-min intervals for the whole experiment using an oxygen analyzer and CrNi–Ni-thermocouples, respectively. Heat production (HP) was calculated, using an assumed RQ of 0.72.

2. At 37.5°C the relationship between HP and embryonic age follows a sigmoid curve. Between D18 (HP 7.25 J g⁻¹ h; 2.01 W kg⁻¹) and D19 (HP 7.21 J g⁻¹ h⁻¹; 2.00 W kg⁻¹) this function had a plateau phase with a duration of about 24 h.

3. During the cooling process, HP decreased continuously and the relationship between T_a and HP could be described by an exponential function. From the results, it was possible to calculate the relationships between T_af, as a measure of body core temperature, and Q₁₀; the lower the T_af the higher the Q₁₀.

4. Because the actual measured HP is the result of the negative effect of Q₁₀ on HP and the stimulating influence of the CNS-generated HP, a Q₁₀ of more than 2.0 demonstrates the absence of endothermy. Chicken embryos aged between 14 and 21 d have a Q₁₀ of less than 2.0 at body temperatures (T_af) between 34 and 30°C. It is postulated that in chicken embryos of this age endothermic reactions may occur.

5. The Q₁₀-method is suitable for investigating the prenatal development of endothermic reactions in avian embryos.     

The respiratory chain of Azotobacter vinelandii II. The effect of cyanide on cytochrome d

1. Cyanide causes a slow disappearance of the oxidized band (648 nm) of cytochrome d in particles of Azotobacter vinelandii and inhibits the appearance of the reduced band (631 nm). No effect of cyanide is found on the reduced band of cytochrome d.

2. The kinetics of the disappearance of the 648-nm band of cytochrome d with excess cyanide deviates from first-order kinetics at lower temperatures (22 °C) indicating that at least two conformations of the enzyme are involved. At higher temperatures (32 °C) the observed kinetics of the cyanide reaction are first order with a k_on = 0.7 M⁻¹·s⁻¹ and with an estimated k_off of approximately 5·10⁻⁵ s⁻¹.

3. The value of the k_off (7·10⁻⁴−14·10⁻⁴ s⁻¹ at 32 °C) determined from the rate of reduction of cyanocytochrome d by Na₂S₂O₄ or NADH is one order of magnitude larger than the k_off value found when the enzyme is in its oxidized state.

4. No effect of cyanide is found on the spectrum of cytochrome a₁.     

Relative tolerance of cirripede larval stages to acute thermal shock: A laboratory study

(1) Meroplankters drawn into once-through cooling circuits of coastal power plants are subjected to transient thermal stress. The effect of such acute thermal shock on the development of barnacle larvae was studied in the laboratory.

(2) The response of the barnacle larvae (naupliar and cyprid stages) to elevated temperature was dependent on exposure time and their stage of development.

(3) Among the stages tested, N-6 larvae showed maximum tolerance. Exposure to 37°C did not affect larval survival, but delayed development of N-2 larva to cypris by one day.

(4) Exposure at 40°C delayed, hastened or did not affect the development time of N-2 and N-4 larvae through cypris, depending on exposure time.

(5) Ten mins exposure at 43°C proved lethal to all larval stages with mortality ranging from 20 to 86%.

(6) Development success of the surviving larvae, measured in terms of cypris yield, showed no significant difference from controls, at temperatures below 40°C.

(7) Settlement activity was significantly affected in only those cyprid larvae which were exposed to 43°C for 10 min.

(8) Results of the present study indicate that thermal stress experienced in the once-through cooling system does not have significant impact on survival and development of the barnacle larvae at temperatures of 37–40°C.     

Transfer and trapping of excitation energy in Photosystem II as studied by chlorophyll a2 fluorescence quenching by dinitrobenzene and carotenoid triplet. The matrix model

1. The curves representing the reciprocal fluorescence yield of chlorophyll a of Photosystem II (PS II) in Chlorella vulgaris as a function of the concentration of m-dinitrobenzene in the states P Q and P Q^-, are found to be straight parallel lines; P is the primary donor and Q the primary acceptor of PS II. In the weakly trapping state P Q^- the half-quenching of dinitrobenzene is about 0.2 mM, in vitro it is of the order of 10 mM. The fluorescence yield as a function of the concentration of a quencher is described for three models for the structure of pigment systems: the model of separate units, the model of limited energy transfer between the units, and the matrix model. If it is assumed that the rate constant of quenching by dinitrobenzene is high and thus the number of dinitrobenzene molecules per reaction center low, it can be concluded that the pigment system of PS II in C. vulgaris is a matrix of chlorophyll molecules in which the reaction centers are embedded. Theoretical and experimental evidence is consistent with such an assumption.

For Cyanidium caldarium the zero fluorescence yield Ф₀ and its quenching by dinitrobenzene were found to be much smaller than the corresponding quantities for C. vulgaris. Nevertheless, our measurements on C. caldarium could be interpreted by the assumption that the essential properties (rate constants, dinitrobenzene quenching) of PS II are the same for these two species belonging to such widely different groups.

2. The measured dinitrobenzene concentrations required for half-quenching in vivo and other observations are explained by (non-rate-limiting) energy transfer between the chlorophyll a molecules of PS II and by the assumptions that dinitrobenzene is approximately distributed at random in the membrane and does not diffuse during excitation.

3. The fluorescence kinetics of C. vulgaris during a 350 ns laser flash of variable intensity could be simulated on a computer using the matrix model. From the observed fluorescence quenching by the carotenoid triplet (C^T) and the measurement of the number of C^T per reaction center via difference absorption spectroscopy, the rate constant for quenching of C^T is calculated to be k_T = 3.3 · 10¹¹ s⁻¹ which is almost equal to the rate constant of trapping by an open reaction center (Duysens, L.N.M. (1979) CIBA Foundation Symposium 61 (New Series), pp. 323–340).

4. The fluorescence quenching by C^T in non-treated spinach chloroplasts after a 500 ns laser flash (Breton, J., Geacintov, N.E. and Swenberg, C.E. (1979) Biochim. Biophys. Acta 548, 616–635) could be explained within the framework of the matrix model when the value for k_T is used as given in point 3.

5. The observations mentioned under point 1 indicate that the fluorescence yield Ф₀ for centers in trapping state P Q is probably for a fraction exceeding 0.8 emitted by PS II.     

Differential ozone sensitivity of rice cultivars as indicated by visible injury and grain yield

Surface ozone pollution may cause reductions in rice ( Oryza sativa L.) yield. Ozone sensitivity in rice cultivars is often evaluated based on visible leaf injury at an early growth stage. However, it is not clear whether reduction in grain yield is related to visible injury. Therefore, visible damage and grain yield reduction were examined in Japanese and Asian rice cultivars exposed to ozone. In experiment 1, 3-week-old rice seedlings were exposed to ozone (min.: 20 nl·l⁻¹, max.:120 nl·l⁻¹) for 12 h in open-top chambers (OTCs). Visible leaf injury was quantified according to a leaf bronzing score. In experiment 2, rice plants were exposed to ozone in OTCs throughout the cropping season until grain harvest. Daily mean ozone concentrations were maintained at 2, 23, 28, 42, and 57 nl·l⁻¹ with a regular diurnal pattern of exposure. After harvest, grain yield was determined. Based on visible injury to the uppermost fully expanded leaf, the indica cultivar 'Kasalath' was most tolerant, and the japonica cultivar 'Kirara 397' was most sensitive to ozone. However, grain yields for both 'Kasalath' and 'Kirara 397' were significantly decreased after ozone exposure. The indica cultivar 'Jothi' suffered severe injury after ozone exposure but had no reduction in grain yield. Therefore, ozone sensitivity of rice cultivars evaluated by visible injury did not coincide with that evaluated by the reductions in grain yield. These results suggest that mechanisms that induce acute leaf injury do not relate to chronic ozone toxicity that reduces yield.     

Studies on the activation of purified mitochondrial ATPase by phospholipids

1. An ATPase which is activated by phospholipids and inhibited by oligomycin, has been purified from beef heart submitochondrial particles using affinity chromatography. Phospholipid and detergent are removed by washing the enzyme with a solution of serum albumin while it is attached to the biospecific adsorbent.

2. The ATPase is activated up to 18-fold by lysolecithin and to a smaller extent by cardiolipin, phosphatidylinositol and phosphatidylethanolamine. The amount required of each of these phospholipids to give half-maximal activation is apparently inversely related to the number of fatty acid chains in the lipid. Lecithin, which is a poor activator of the ATPase, competitively inhibits the activation by cardiolipin.

3. The activation of the ATPase consists of an increase in both the maximal velocity of the reaction and the affinity for substrate ATP. The pH optimum of the reaction is not influenced by the charge of the lipid.

4. Arrhenius plots of ATPase activated with lysolecithin show a transition to a higher activation energy at temperatures below 19 °C. The sensitivity of the lysolecithin-activated enzyme to oligomycin is markedly reduced below the same temperature. With cardiolipin the transition is observed at 13 °C.

5. ADP, Mg²⁺ and to a smaller extent ATP, Mg²⁺ enhance the activation of ATPase by suboptimal amounts of phospholipid.