The P-700-chlorophyl a-protein complex and two major light-harvesting complexes of Acrocarpia paniculata and other brown seaweeds

Acrocarpia paniculata thylakoids were fragmented with Triton X-100 and the pigment-protein complexes so released were isolated by sucrose density gradient centrifugation. Three main chlorophyll-carotenoid-protein complexes with distinct pigment compositions were isolated.

1. (1) A P-700-chlorophyll a-protein complex, with a ratio of 1 P-700: 38 chlorophyll a: 4 ta-carotene molecules, had similar absorption and fluorescence characteristics to the chlorophyll-protein complex 1 isolated with Triton X-100 from higher plants, green algae and Ecklonia radiata.

2. (2) An orange-brown complex had a chlorophyll a : c₂ : fucoxanthin molar ratio of 2 : 1 : 2. This complex had no chlorophyll c₁ and contained most of the fucoxanthin present in the chloroplasts. This pigment complex is postulated to be the main light-harvesting complex of brown seaweeds.

3. (3) A green complex had a chlorophyll a : c₁ : c₂ : violaxanthin molar ratio of 8 : 1 : 1 : 1. This also is a light-harvesting complex.

The absorption and fluorescence spectral characteristics and other physical properties were consistent with the pigments of these three major complexes being bound to protein. Differential extraction of brown algal thylakoids with Triton X-100 showed that a chlorophyll c₂-fucoxanthin-protein complex was a minor pigment complex of these thylakoids.     

Relative stability of chlorophyll complexes in vivo

The time course of aerobic photobleaching of various chlorophyll-protein complexes in vivo at high light intensities was studied with isolated Aspidistra elatior chloroplasts.

1. 1. C_a680 bleaching starts with the onset of irradiation and, initially, proceeds linearly with time. Washing the chloroplasts causes a nearly constant increase of the bleaching rate throughout the experiment.

2. 2. C_a670 does not appreciably, if at all, bleach initially; subsequently, bleaching proceeds linearly with time and at a slightly higher rate than that for C_a680. Washing makes C_a670 bleach concomitantly with the onset of illumination, and at a nearly constant rate.

3. 3. Bleaching at 665 nm is likely to start only after a relatively long period of illumination. Washing shows no effects during this period. Once bleaching has started, washing causes its rate to increase.

4. 4. No indication of the occurrence of “short-wave” chlorophyll a forms other than C_a670 and C_a665 was obtained.

5. 5. C_b bleaching starts concomitantly with illumination at a low rate. The rate increases more or less exponentially with time. Washing enhances bleaching in two steps.

6. 6. The importance of the results is discussed.

The reduction kinetics of chlorophyll a1 as an indicator for proton uptake between the light reactions in chloroplasts

The flash-induced oxidation kinetics of the primary acceptor of light Reaction II (X-320) and the reduction kinetics of chlorophyll a₁ (P-700) after far-red preilluination have been studied with high time resolution in spinach chloroplasts.

1. 1. The kinetics of chlorophyll a₁ exhibits a pronounced lag phase of 2–3 ms at the onset of reduction as would be expected for the final product of consecutive reactions. Because the oxidation of the plastoquinone pool is the rate-limiting step for the electron transport between the two light reactions, the lag indicates the maximal electron transfer time over all preceding reactions after light Reaction II.

2. 2. The observation that the lag phase decreases with decreasing pH is evidence of an electron transfer step coupled to a proton uptake reaction.

3. 3. Protonation of X-320 after reduction in the flash is excluded because a slight increase of the decay time is found at decreasing pH values.

4. 4. The time course of plastohydroquinone formation is deduced from the first derivative of the reduction kinetics of chlorophyll a₁. This approach covers those plastohydroquinone molecules being available to the electron carriers of System I via the rate-limiting step. Direct measurements of absorbance changes would not allow to discriminate between these and functionally different plastohydroquinone molecules.

5. 5. The derived time course of plastohydroquinone at different pH gives evidence for an additional electron transfer step with a half time of about 1 ms following the proton uptake and preceding the rate-limiting step. It is tentatively attributed to the diffusion of neutral plastohydroquinone across the hydrophobic core of the thylakoid membrane.

6. 6. The lower limit of the rate constant for proton uptake by an electron carrier, consistent with the lag of chlorophyll a₁ reduction, is estimated as > 10¹¹ M⁻¹ · s⁻¹. The value is higher than that of the fastest diffusion controlled protonations of organic molecules in solution.

Possible mechanisms of linear electron transport between light Reaction II and the rate-limiting oxidation of neutral plastohydroquinone are thoroughly discussed.     

Electron transport between plastoquinone and chlorophyll aI in chloroplasts

After preillumination with System I light spinach chloroplasts were excited by one flash or a group of saturating flashes. During the following dark period the time courses of the oxidation of plastohydroquinone and of the simultaneous reduction of oxidized cytochrome f and chlorophyll a_I (P700) have been measured.

1. 1. From a correlation of these kinetics it can be concluded that at least 85% of the electrons from plastohydroquinone are transferred to chlorophyll a_I.

2. 2. After one flash 93% of the oxidized chlorophyll a_I is reduced. This suggests a high equilibrium constant between chlorophyll a_I and its donor as well as an equilibration between different chlorophyll a_I molecules.

3. 3. Cytochrome f is also reduced by plastohydroquinone. A ratio of active cytochrome f to chlorophyll a_I of 0.4:1 is observed. The half-life time of the reduction of cytochrome f is 17 ms. The time course indicates that in the dark cytochrome f does not transfer electrons to chlorophyll a_I and that no more than 15% of the electron transport passes cytochrome f. Therefore cytochrome f should be situated in a side path of the linear electron transport.

4. 4. The electrons which are released from plastohydroquinone and are not accepted by oxidized cytochrome f and chlorophyll a_I have been calculated. From this difference properties of an electron carrier, as yet not identified, between plastoquinone and chlorophyll a_I are predicted.

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.

Inorganic pyrophosphatase and photosynthesis by isolated chloroplasts. II. The controlling influence of orthophosphate

1. 1. It has been proposed that Mg²⁺, inorganic pyrophosphate and a protein fraction which exhibits fructose-1,6-diphosphatase activity may interact to regulate photosynthesis by isolated chloroplasts.

2. 2. Evidence is presented which confirms the interaction and regulation but shows that these effects are indirectly attributable to pyrophosphatase activity rather than fructose-1,6-diphosphatase.

3. 3. When provided with Mg²⁺ and PP_i the pyrophosphatase simply alters the proportions of orthophosphate and PP_i in the reaction mixture. As the P_i concentration is increased, it first stimulates and then inhibits, the degree of inhibition being enhanced by additional Mg²⁺. PP_i ameliorates the inhibition, possibly by chelation of Mg²⁺.

4. 4. It is concluded that the proposed regulation is ultimately governed by the P_i concentration and the known relationship between P_i uptake and triose phosphate export across the chloroplast envelope.

Activation of CO2 fixation in isolated spinach chloroplasts

1. 1. The effect of the Mg²⁺ concentration on the CO₂ fixation activity in situ in isolated and intact spinach chloroplasts upon suspension in hypotonic medium was examined. CO₂ fixation in the dark was activated 25–100 fold by 20 mM Mg²⁺ in the presence of added ATP plus either ribulose 5-phosphate or ribose 5-phosphate. 20 mM Mg²⁺-stimulated fixation only 2–3 fold in the presence of the substrate of fixation, ribulose 1,5-diphosphate. The highest Mg²⁺-stimulated rate of fixation in the dark observed with chloroplasts was 480 μmoles CO₂ fixed per mg chlorophyll per h.

2. 2. The concentration of bicarbonate at half of the maximal velocity (apparent K_m) during the Mg²⁺-stimulated fixation of CO₂ was 0.4 mM in the presence of ATP plus ribose 5-phosphate and 0.6 mM with ribulose 1,5-diphosphate.

3. 3. Dithioerythritol or light enhanced Mg²⁺-stimulated CO₂ fixation 1–3 fold in the presence of ATP plus ribose 5-phosphate but not ribulose 1,5-diphosphate.

4. 4. These results indicate that Mg²⁺ fluxes in the stroma of the chloroplast could control the activity of the phosphoribulokinase with a lesser effect on the ribulosediphosphate carboxylase. An increase in Mg²⁺ of 6–10 mM in the stroma region of the chloroplast would be enough to activate CO₂ fixation during photosynthesis.

Inorganic pyrophosphatase and photosynthesis by isolated chloroplasts. I. Characterisation of chloroplast pyrophosphatase and its relation to the response to exogenous pyrophosphate

1. 1. A soluble, alkaline, Mg²⁺-dependent inorganic pyrophosphatase (EC 3.6.1.1) has been isolated from the stroma of intact spinach and pea chloroplasts and purified some 100-fold. The enzyme has a high specificity for inorganic pyrophosphate and Mg²⁺, and exhibits maximal activity at pH 8.2–8.6. The enzyme shows allosteric characteristics with Mg²⁺ as activator and optimal rates are obtained with a ratio of Mg²⁺ to PP_i of approximately 4 to 1. The enzyme is inhibited by anionic PP_i and by its own reaction product, orthophosphate.

2. 2. If Mg²⁺ is excluded from the medium in which isolated chloroplasts are assayed, active photosynthetic oxygen evolution can still be observed. The addition of P_i, but not PP_i, will then offset a phosphate deficiency. If external Mg²⁺ is present PP_i will also offset a phosphate deficiency and in these circumstances the rapidity and nature of the response is related to the external pyrophosphatase activity.

3. 3. Evidence is presented that the chloroplast envelope is relatively impermeable to PP_i and that the response to added PP_i is due to external hydrolysis followed by entry of P_i to the chloroplast. These results have significance concerning proposed mechanisms for control of photosynthesis.

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.

The site of phosphorylation associated with Photosystem I

1. 1. Small particles prepared from spinach chloroplasts after treatment with digitonin, exhibited Photosystem I reactions, including phosphorylation, at rates as high as those in chloroplasts, whereas electron flow from water to NADP⁺ or ferricyanide through Photosystem II was completely lost. Mediators of cyclic electron flow, such as pyocyanine, or N-methylphenazonium methosulfate in red light, had to be reduced to support photophosphorylation.Diaminodurene at high concentrations catalyzed cyclic phosphorylation under anaerobic conditions without addition of a reductant. In fact, addition of ascorbate gave rise to a marked inhibition which was released by addition of a suitable electron acceptor such as methylviologen.

2. 2. Under aerobic conditions a low O₂ uptake, observed in the presence of diaminodurene, was stimulated several-fold upon addition of methylviologen and was stimulated again several-fold on further addition of ascorbate. The rate of phosphorylation, however, remained the same. The low P/2e ratio obtained under these conditions was not decreased at lower light intensities.

3. 3. These findings suggest a phosphorylation site associated with cyclic electron flow through Photosystem I without participation of the electron carriers of Photosystem II. A non-cyclic electron flow to O₂ can be induced in this system by addition of methylviologen which effectively competes with the electron acceptors of cyclic flow. This non-cyclic electron flow still involves the same phosphorylation site. A scheme for electron transport and for the location of phosphorylation sites in chloroplasts is proposed.

Studies on the mechanism of destabilization of the positive charges trapped in the photosynthetic water-splitting enzyme system Y by a deactivation-accelerating agent

The mechanism of the 2-(3,4,5-trichloro)anilino-3,5 dinitrothiophene (ANT 2S)-induced cyclic electron flow leading to the discharge of the higher-trapped-hole accumulation states S₂ and S₃ in the photosynthetic water-splitting enzyme system Y of chloroplasts has been investigated. It was found:

1. 1. Under normal conditions the ANT 2s-catalyzed cycle includes both light reactions.

2. 2. By selective kinetical inhibition of the electron flow through P700—either by histone treatment or by 2,5-dibromo-3-methyl-6-isopropyl-1,4-benzoquinone blockage—the ANT 2s-induced deactivation of S₂ and S₃ is not significantly changed. Hence, System I activity is not a functional prerequisite for the ANT 2s-catalyzed discharge of S₂ and S₃.

3. 3. The reciprocal half time of the ANT 2s-induced decay of the relative average oxygen yield per flash, as a function of the time t_d between the flashes representing the degree of the Acceleration of the Deactivation Reactions of the water-splitting enzyme system (ADRY) effect, is nearly linearly related to the ANT 2s concentration within the range of 10⁻⁷–10⁻⁶ M.

4. 4. In respect to the mode of action of ANT 2s two different types of mechanism have been discussed: fixed-place mechanism and mobile-catalyst mechanism.

5. 5. Based on the experimental data the conclusion has been drawn that the ADRY agent ANT 2s probably acts as a mobile catalyst.

Properties of protein-chlorophyll complexes from pea (Pisum sativum L.) leaves. The organization of chlorophyll.

Chlorophyll-protein-detergent complexes were prepared from pea chloroplasts by using sodium dodecylbenzenesulphonate and polyacrylamide-gel electrophoresis. Circular-dichroism spectra showed that complex CPI has a dimeric arrangement of chlorophyll a, with additional weaker interactions. Ellipticities were determined for both complexes and for purified chlorophylls in solution, and it is argued that the circular dichroism of complex CPII is derived from chlorophyll-protein interaction rather than from interaction between chlorophylls a and b. The detergent could be removed from the complexes by using urea and gel filtration, leaving the chlorophyll-protein in solution, although in each case a diminished ellipticity indicated some loss of organization. Three-peaked circular-dichroism spectra of chloroplast fragments before and after addition of detergent were compared with a curve obtained by summing graphically the spectra of complexes CPI, CPII and the free-pigment fraction. There was good correspondence at 650 nm, and the longer-wavelength peaks agreed in form and magnitude, but with discrepancies in position. It was concluded that complexes CPI and CPII pre-exist in the original material, but that there is an environmental effect which is destroyed when the complexes are extracted.     

Some effects of thyroidectomy on growth, heat production and the thermoregulatory ability of the immature fowl (Gallus domesticus)

1. 1.|The effect of thyroidectomy at 12 days of age on weight gain, and on heat production and thermoregulatory ability of 4- to 5-week-old chickens at temperatures within and below the thermo-neutral zone was investigated.

2. 2.|Despit the absence of thyroid tissue, as demonstrated with radioiodine, a small amount of thyroxine was found in the plasma of some thyroidectomized (TX) birds.

3. 3.|Thyroidectomy depressed weight gain; pair-fed controls grew significantly faster than TX birds.

4. 4.|Resting heat production of TX birds at thermoneutrality (30°C) was depressed by 18% (P < 0.001) and body temperature by 0.4°C (P < 0.001).

5. 5.|At 12°C heat production of TX birds was similar to that of controls but the body temperature of TX birds was 0.7°C lower (P < 0.001).

6. 6.|Thyroidectomized birds were unable to regulate body temperature at 5°C even if thyroxine was provided on the day before and at the time of cold-exposure. This inability to thermoregulate was probably due to inadequate insulation and poor nutritional status.

Lactoperoxidase-catalyzed iodination of chloroplast membranes. I. Analysis of surface-localized proteins

Lactoperoxidase-catalyzed iodination of chloroplast membranes has been employed to characterize the vectorial distribution of lamellar proteins. The enzymatic reaction is highly specific for only the outermost membrane components (Phillips, D. R. and Morrison, M. (1971) Biochemistry 10, 1766–1771); we have determined the distribution of ¹²⁵I label and changes in photochemical activities after iodination in an effort to identify these components. Three major conclusions are evident:

1. 1. The coupling factor for photophosphorylation is highly exposed and is selectively and rapidly inhibited by the iodination reaction.

2. 2. A loss of Photosystem I activity (NADP reduction) resulted from iodination. Partial reactions indicated the effect was on electron-transport components on the reducing side of Photosystem I. There was also a limited inhibition of methyl viologen reduction.

3. 3. Iodination of intact membranes caused a reduction in rates of Photosystem II-dependent Hill reaction activity. This inhibition could not be explained solely on the basis of iodination effects on electron-transport components involved in the oxidation of water. The implications of these data with respect to previous chloroplast-membrane models are discussed.

The effects of indomethacin on cold-induced vasodilation in the cat

1. 1. Cold-induced vasodilatation (CIVD) was assessed from records of foot-pad temperatures of 6 domestic cats during immersion of both hind feet in a 0°C water bath.

2. 2. All experiments were performed following anaesthesia with sodium pentobarbital.

3. 3. Four days after the control recordings, the animals were given indomethacin, 5 mg/kg intravenously, and the CIVD response was examined once again; a third test of the cold response was performed 4 days after the treatment with indomethacin.

4. 4. Phasic increases in foot-pad temperature (CIVD) during immersion were smaller (P < 0.05) and delayed in onset (P < 0.05) in the animals treated with indomethacin at a dose which inhibits cyclo-oxygenase.

5. 5. It is proposed that CIVD involves a balance between central symphathetic vasoconstrictor tone and periodic prostaglandin-induced vasodilatation.

Comparison studies on the effects of ultraviolet irradiation on photosynthesis

1. 1. A comparison of chloroplasts from which plastoquinone had been extracted with ultraviolet irradiation supports the conclusion that plastoquinone destruction is not the major cause of ultraviolet inhibition of photosynthesis. No photodestruction of chloroplast lipids, carotenoids or soluble proteins by ultraviolet irradiation was detected.

2. 2. Phenazine methosulfate-mediated cyclic photophosphorylation and variable yield fluorescence were inhibited at the same rate as the Hill reaction. Examination of fluorescence emission spectra of chloroplasts and whole algal cells revealed decreases in both the 685-nm and long-wavelength emission peaks.

3. 3. Digestion of chloroplasts with lipase decreased fluorescence in a manner similar to ultraviolet irradiation. Hill reaction activity was also inhibited by lipase digestion.

4. 4. It is concluded that the inhibition of photosynthesis by ultraviolet irradiation is most likely due to a disruption of the structural integrity of the lamellar membranes which results in the loss of System II activity and associated reactions.

Electron paramagnetic resonance saturation studies of P-700 reaction center chlorophyll in plant photosynthesis

Electron paramagnetic resonance (EPR) power saturation and saturation recovery methods have been used to determine the spin lattice, T₁, and spin-spin, T₂, relaxation times of P-700⁺ reaction-center chlorophyll in Photosystem I of plant chloroplasts for 10 K T 100 K. T₁ was 200 μs at 100 K and increased to 900 μs at 10 K. T₂ was 40 ns at 40 K and increased to 100 ns at 10 K. T₁ for 40 K T 100 K is inversely proportional to temperature, which is evidence of a direct-lattice relaxation process. At T = 20 K, T₁ deviates from the 1/T dependence, indicating a cross relaxation process with an unidentified paramagnetic species. The individual effects of ascorbate and ferricyanide on T₁ of P-700⁺ were examined: T₁ of P-700⁺ was not affected by adding 10 mM ascorbate to digitonin-treated chloroplast fragments (D144 fragments). The P-700⁺ relaxation time in broken chloroplasts treated with 10 mM ferricyanide was 4-times shorter than in the untreated control at 40 K. Ferricyanide appears to be relaxing the P-700⁺ indirectly to the lattice by a cross-relaxation process. The possibility of dipolar-spin broadening of P-700⁺ due to either the iron-sulfur center A or plastocyanin was examined by determining the spin-packet linewidth for P-700⁺ when center A and plastocyanin were in either the reduced or oxidized states. Neither reduced center A nor oxidized plastocyanin was capable of broadening the spin-packet linewidth of the P-700⁺ signal. The absence of diplolar broadening indicates that both center A and plastocyanin are located at a distance at least 3.0 nm from the P-700⁺ reaction center chlorophyll. This evidence supports previous hypotheses that the electron donor and acceptor to P-700 are situated on opposite sides of the chloroplast membrane. It is also shown that the ratio of photo-oxidized P-700 to photoreduced centers A and B at low temperature is 2 : 1 if P-700 is monitored at a nonsaturating microwave power.     

Analysis of light-induced proton uptake in isolated chloroplasts

1. 1. A simple kinetic analysis of light-induced proton uptake into chloroplasts is presented. It is derived from a model of the reaction in which the incoming proton is obligatorily bound by an intra-chloroplast component, and allows quantitative analysis of the effect into parameters of light and dark rate constants and the availability of the chloroplast component.

2. 2. The effect of the following agents on the derived parameters has been measured: electron and energy transfer inhibitors, uncouplers, NaCl concentration, light intensity and pH.

3. 3. A maximal ratio of 4 protons taken up per electron transported has been observed, using ferricyanide as an electron acceptor.

4. 4. A stimulation of light-induced proton uptake by phosphate or arsenate, ADP and Mg has been observed. It was not sensitive to concentrations of Dio-9, which eliminated ATP synthesis.

5. 5. The results are seen as inconsistent with the chemiosmotic theory of energy coupling as presently presented. It is suggested that they may be interpreted in terms of a model in which the function of the proton pump is to enable co-transport into the chloroplasts of the negatively charged complex of phosphate, ADP and Mg.

ATPase complex and oxidative phosphorylation in chloramphenicol-induced megamitochondria from mouse liver

1. 1. Functional properties of the ATPase complex are investigated in megamitochondria isolated from livers of weanling mice fed a diet containing 2% chloramphenicol, as an inhibitor of mitochondrial protein synthesis.

2. 2. Whereas the specific activity of ATPase remains unchanged in chloramphenicol-induced megamitochondria, about 40% of the enzyme activity is resistant to inhibition by oligomycin, triethyltin or venturicidin. It is concluded that the ATPase complex lacks one or more components whose synthesis or accumulation is dependent on mitochondrial translation. The inhibitor-resistant ATPase portion appears tightly bound to the mitochondrial membrane.

3. 3. Respiratory chain phosphorylation is tightly coupled in isolated megamitochondria. ATP synthesis and ATP-P_i exchange are diminished by 40%, as compared to control mitochondria, but both processes are sensitive to oligomycin, triethyltin or venturicidin.

4. 4. The decrease in ATP synthesis and ATP-P_i exchange in megamitochondria correlates quite well with the emergence of inhibitor-resistant ATPase.

5. 5. The following electron transport activities in the megamitochondria are reduced: NADH-cytochrome c reductase, by 60%, cytochrome oxidase, by 80%; the amount of antimycin required to gain complete inhibition of the bc₁-segment is diminished by more than 50%. On the other hand succinate dehydrogenase activity is increased by 50%.

6. 6. Chloramphenicol-induced megamitochondria appear to be a useful system for studying the role of mitochondrial translation in the assembly of mammalian mitochondria.

Trypsin inhibition of electron transport

1. 1. A relaxation spectrophotometer was employed to measure the effects of trypsin treatment on electron transport in both cyclic and non-cyclic chloroplast reactions. The parameters measured were electron flow rate through P700 (flux) and the time constant for dark reduction of P700.

2. 2. In the reduction of methyl viologen by the ascorbate-2,6-dichlorophenol-indophenol (DCIP) donor couple, there was no effect of trypsin on P700 flux or on the time constant for dark reduction of P700. In the phenazine methosulfate (PMS) cyclic system, trypsin had either a slightly stimulatory or slightly inhibitory effect on the P700 flux, depending on the presence or absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU): either effect being marginal compared to trypsin effects on Photosystem II.With both ferricyanide and methyl viologen reduction from water, trypsin treament gave a first order decline in P700 flux: which matched the trypsin-induced decline in electron transport with the water to DCIP system, measured by dye reduction. This implies that Photosystem II is inhibited. The inhibition of Photosystem II was up to 90% with a 6–10-min trypsin treatment. This result is consistent with the concept of Photosystem I (P700) being in series with Photosystem II in the electron transfer sequence.

3. 3. Cyclic phosphorylation was severely inhibited (85%) by trypsin treatment which had a somewhat stimulatory effect on P700 flux, indicating uncoupling. Non-cyclic phosphorylation was uncoupled as well as electron flow being inhibited since the P/2e ratio decreased more rapidly as a function of trypsin incubation time than inhibition of electron flow. The two effects, uncoupling and non-cyclic electron flow inhibition, are separate actions of trypsin. It is probably that the uncoupling action of trypsin is due to attack on the coupling factor protein, known to be exposed on the outer surface of thylakoids.

4. 4. Trypsin treatment caused an increase in the rate constant, k_d, for the dark H⁺ efflux, resulting in a decreased steady state level of proton accumulation. The increased proton efflux and the inhibition of phosphorylation are consistent with an uncoupling effect on trypsin.

5. 5. Trypsin treatment did not reduce the manganese content of chloroplasts: as reported by others, Tris washing did remove about 30% of the chloroplast manganese.

6. 6. Electron micrographs of both negatively stained and thin-sectioned preparations showed that, under these conditions, trypsin does not cause a general breakdown of chloroplast lamellae. Inhibition by trypsin must therefore result from attacks on a few specific sites.

7. 7. Both System II inhibition and uncoupling occur rapidly when trypsin treatment is carried out in dilute buffer, a condition which leads to thylakoid unstacking, but both are prevented by the presence of 0.3 M sucrose and 0.1 M KCl, a condition that helps maintain stacked thylakoids. Evidently vulnerability to trypsin requires separation of thylakoids.

8. 8. Since trypsin does not appear to disrupt thylakoids nor prevent their normal aggregation in high sucrose-salt medium and since the trypsin molecule is probably impermeable, it is probable that the site(s) of trypsin attack in System II are exposed on the outer thylakoid surface.