Effects of inorganic phosphate on the photosynthetic carbon reduction cycle in extracts from the stroma of pea chloroplasts.

1. Turnover of the photosynthetic carbon reduction cycle has been demonstrated in chlorophyll-free reaction mixtures containing chloroplast stromal extract, as evidenced by the fixation of CO2 following addition of small amounts of 3-phosphoglycerate. 2. The activity of the photosynthetic carbon reduction cycle in this system is inhibited by inorganic phosphate (Pi), with activity reduced to 50% by about 6.5 mM Pi. Pi also increased the lag period which elapsed before a steady rate of CO2 fixation was obtained. 3. The effect of Pi on the rate of 3-phosphoglycerate reduction following the addition of substrate amounts of some cycle intermediates was investigated. Substantial inhibition was observed with fructose 1,6-bisphosphate, sedoheptulose 1,7-bisphosphate and erythrose 4-phosphate as substrates. Pi also affected the activity of ribulose-bisphosphate carboxylase, with stimulation at Pi concentrations below 2.5 mM and inhibition at higher concentrations. 4. The results showed that the sedoheptulose bisphosphatase reaction is inhibited more strongly by Pi than the fructose bisphosphatase reaction. 5. It is concluded that the previously established inhibitory effects of Pi on photosynthesis by intact isolated chloroplasts may be partly due to these inhibitory effects of Pi on the reactions of the photosynthetic carbon reduction cycle.

The distribution of metabolites between spinach chloroplasts and medium during photosynthesis in vitro.

1. The formation of metabolites in the stroma compartment of isolated chloroplasts during carbon fixation, and their export to the medium, have been investigated using improved techniques. 2. Rapid separation of photosynthesising chloroplasts from the medium, accompanied by simultaneous quenching of metabolism was achieved by using silicone oil layer filtering centrifugation under illumination. Metabolites were separated by microscale ion-exchange chromatography. Quantitative determination of each metabolite was based on labelling with 32P. 3. It was found that fixed carbon was exported from the chloroplasts only as triose phosphate and phosphoglycerate, and to a minor extent, as pentose monophosphate. The main compounds accumulating in the stroma were hexose and heptose monophosphates and phosphoglycerate. A marked decrease in the concentration of inorganic phosphate in the stroma during the first 5 min of illumination was accompanied by a complementary increase in organic phosphate so that the total amount of phosphate within the chloroplasts remained constant. 4. The concentration difference for phosphoglycerate between the stroma and the medium was much higher than for triose phosphate or inorganic phosphate, although all three compounds are transported across the inner membrane of the chloroplast envelope by the same carrier. It was concluded that the efflux of phosphoglycerate was restricted.

ATP Hydrolysis Activity and Polymerization State of Ribulose-1,5-Bisphosphate Carboxylase Oxygenase Activase (Do the Effects of Mg2+, K+, and Activase Concentrations Indicate a Functional Similarity to Actin?).

The ATPase activity and fluoresence of ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) activase were determined over a range of MgCl2, KCl, and activase concentrations. Both salts promoted ADP release from ATP and intrinsic fluorescence enhancement by adenosine 5[prime]-[[gamma]-thio] triphosphate, but Mg2+ was about 10 times more effective than K+. ATPase and fluorescence enhancement both increased from zero to saturation within the same Mg2+ and K+ concentration ranges. At saturating concentrations (5 mM Mg2+ and 22 mM K+), the specific activity of ATPase (turnover time, about 1 s) and specific intrinsic fluorescence enhancement were maximal and unaffected by activase concentration above 1 [mu]M activase; below 1 [mu]M activase, both decreased sharply. These responses are remarkably similar to the behavior of actin. Intrinsic fluorescence enhancement of Rubisco activase reflects the extent of polymerization, showing that the smaller oligomer or monomer present in low-salt and activase concentrations is inactive in ATP hydrolysis. However, quenching of 1-anilinonapthaline-8-sulfonate fluorescence revealed that ADP and adenosine 5[prime]-[[gamma]-thio] triphosphate bind equally well to activase at low- and high-salt concentrations. This is consistent with an actin-like mechanism requiring a dynamic equilibrium between monomer and oligomers for ATP hydrolysis. The specific activation rate of substrate-bound decarbamylated Rubisco decreased at activase concentrations below 1 [mu]M. This suggests that a large oligomeric form of activase, rather than a monomer, interacts with Rubisco when performing the release of bound ribulose-1,5-bisphosphate from the inactive enzyme.

Subsaturating Ribulose-1,5-Bisphosphate Concentration Promotes Inactivation of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) (Studies Using Continuous Substrate Addition in the Presence and Absence of Rubisco Activase).

We developed a continuous-addition method for maintaining subsaturating concentrations of ribulose-1,5-bisphosphate (RuBP) for several minutes, while simultaneously monitoring its consumption by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). This method enabled us to observe the effects of subsaturating RuBP and CO2 concentrations on the activity of Rubisco during much longer periods than previously studied. At saturating CO2, the activity of the enzyme declined faster when RuBP was maintained at concentrations near its Km value than when RuBP was saturating. At saturating RuBP, activity declined faster at limiting than at saturating CO2, in accordance with previous observations. The most rapid decline in activity occurred when both CO2 and RuBP concentrations were subsaturating. The activity loss was accompanied by decarbamylation of the enzyme, even though the enzyme was maintained at the same CO2 concentration before and after exposure to RuBP. Rubisco activase ameliorated the decline in activity at subsaturating CO2 and RuBP concentrations. The results are consistent with a proposed mechanism for regulating the carbamylation of Rubisco, which postulates that Rubisco activase counteracts Rubisco's unfavorable carbamylation equilibrium in the presence of RuBP by accelerating, in an ATP-dependent manner, the release of RuBP from its complex with uncarbamylated sites.

Isolation of Functionally Intact Rhodoplasts from Griffithsia monilis (Ceramiaceae, Rhodophyta).

A procedure is described for isolating photosynthetically active rhodoplasts ("red algal chloroplasts") from the marine alga Griffithsia monilis. The rhodoplasts exhibited rates of CO(2) fixation and CO(2)-dependent O(2) evolution in the order of 200 micromoles per milligram chlorophyll a per hour when illuminated with red or green light and were approximately 80% intact. The response of the rate of photosynthesis to the inorganic phosphate and pyrophosphate concentrations in the medium was qualitatively similar to that previously reported for spinach chloroplasts. Osmotically shocked rhodoplasts evolved O(2) from ferricyanide in red, but not in green, light and were completely uncoupled. Rhodoplast envelope rupture appeared to be accompanied by phycobilisome loss from the thylakoids.

Reductive pentose phosphate cycle and oxidative carbohydrate metabolic activities in pea chloroplast stroma extracts.

Oxidative and reductive carbohydrate metabolism was studied in reaction mixtures based on chlorophyll-free stromal extracts from chloroplasts of Pisum sativum. A new assay system for the reductive pentose phosphate cycle was characterized.When provided with ATP, an enzymic ATP-regenerating system and reduced pyridine nucleotide, substantial rates of CO(2) fixation and pyridine nucleotide oxidation were observed following the addition of millimolar concentrations of reductive pentose phosphate cycle intermediates. The reduced pyridine nucleotide requirement could be met either by NADPH, or by NADH plus the added enzymes NAD(+)-glyceraldehyde phosphate dehydrogenase and phosphoglycerate kinase. When the assay system was primed with small amounts of reductive pentose phosphate cycle intermediates, lower rates of pyridine nucleotide oxidation were observed, but turnover of the complete cycle was demonstrated. Autocatalytic effects were not evident. The optimum pH and Mg concentrations for cycle turnover were similar to those believed to exist in the stroma of intact chloroplasts in the light.Oxidative carbohydrate metabolism was studied by supplying oxidized pyridine nucleotide and measuring its rate of reduction in the presence of sugar phosphates. Glycolytic activity, estimated as the rate of fructose-6-phosphate entry to the phosphofructokinase reaction was 2.7 micromoles per milligram chlorophyll per hour when fructose-6-phosphate was provided as substrate. Evidence based on glucose-6-phosphate and ribose-5-phosphate-dependent NADP(+) reduction showed that the oxidative pentose phosphate cycle was also active. Apparent oxidative pentose phosphate cycle turnover in the presence of ribose-5-phosphate, estimated as the rate of glucose-6-phosphate entry to the glucose-6-phosphate dehydrogenase reaction, was 1.7 micromoles per milligram chlorophyll per hour.It was concluded that under the defined conditions, reductive pentose phosphate cycle activity could be measured without interference from oxidative carbohydrate metabolism in this experimental system.

Autocatalysis in a reconstituted chloroplast system.

In whole plants and intact chloroplasts, photosynthesis does not reach its full rate immediately upon illumination but only after a lag which is believed to reflect an autocatalytic increase in the concentration of carbon cycle intermediates. Autocatalysis has now been observed in a reconstituted system containing envelope-free chloroplasts augmented with ferredoxin and other stromal proteins but only catalytic amounts of ATP and NADP. With ribose 5-phosphate as substrate, the CO(2) dependent O(2) evolution recorded for such mixtures implies rates of "endogenous" or ferredoxin-dependent photophosphorylation as high as 360 mumoles of orthophosphate esterified mg(-1) chlorophyll hr(-1).

Activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) by rubisco activase : effects of some sugar phosphates.

The activation of purified ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) has been studied in the presence of sugar phosphates, and the effect of rubisco activase on this process determined. During an 11-minute time course at pH 7.7 and 11 micromolar CO(2), the activation of rubisco was strongly inhibited by ribulose-1,5-bisphosphate (4 millimolar), fructose-1,6-bisphosphate (1 millimolar) and ribose 5-phosphate (5 millimolar), but this inhibition was overcome by the addition of rubisco activase and activation then proceeded to a greater extent than spontaneous activation of rubisco. Glycerate 3-phosphate (20 millomolar) slowed the initial rate but not the extent of activation and rubisco activase had no effect on this. The activation of rubisco was shown to be affected by phosphoenolpyruvate (3 millimolar) but not by creatine phosphate (3 millimolar) or ATP (3 millimolar), and the creatine-phosphate/creatine phosphokinase system was used to generate the high ATP/ADP quotients required for rubisco activase to function. ATP was shown to be required for the rubisco activase-dependent rubisco activation in the presence of fructose-1,6-bisphosphate (1 millimolar). It is concluded that rubisco activase has a mixed specificity for some sugar phosphate-bound forms of rubisco, but has low or no activity with others. Some possible bases for these differences among sugar phosphates are discussed but remain to be established.

Carbon dioxide assimilation by leaves, isolated chloroplasts, and ribulose bisphosphate carboxylase from spinach.

The relationship between rate of photosynthesis and CO(2) concentration has been reinvestigated using isolated spinach (Spinacia oleracea) chloroplasts. The apparently low CO(2) concentration required for half-maximal photosynthesis is shown to result partly from a ceiling imposed by electron transport. In double reciprocal plots of rate against CO(2) concentration, this ceiling results in departures from linearity at high CO(2) concentrations. If these rate limitations are disregarded in extrapolation the "true" CO(2) concentration required for half maximal carboxylation by intact chloroplasts is approximately 46 mum (CO(2)).When assayed under comparable conditions, ribulose bisphosphate carboxylase from these chloroplasts also shows an apparent Km (CO(2)) of approximately 46 mum, suggesting that its characteristics are not modified by extraction. An improved assay for ribulose bisphosphate carboxylase yielded rates of carboxylation considerably higher than those previously reported, the highest maximal velocities recorded approaching 1000 mumoles CO(2) fixed mg(-1) chlorophyll hr(-1) at 20 C. With such Km and V(max), values the carboxylase would be able to achieve, at concentrations of CO(2) less than atmospheric, rates of CO(2) fixation equal to those displayed by the parent tissue or by the average plant under favorable conditions in its natural environment.

The simultaneous determination of carbon dioxide release and oxygen uptake in suspensions of plant leaf mitochondria oxidizing glycine.

The construction and operation of a device for continuous measurement of CO(2) release by suspensions of respiring mitochondria is described. A combination of this device with a Clark-type O(2) electrode was used for simultaneous measurement of respiration and of CO(2) release by spinach and pea leaf mitochondria with glycine as substrate. Both mitochondrial preparations showed high rates of respiration and high respiratory control ratios. The addition of oxaloacetate not only inhibited O(2) uptake substantially, but also greatly stimulated glycine oxidation as monitored by CO(2) release. In spinach leaf mitochondria, the maximal rates of glycine oxidation thus obtained, were two times higher than the rate of glycine oxidation required at average rates of photorespiration. It is concluded from these results that under saturating conditions the capacity of glycine oxidation by intact mitochondria exceeds the capacity of glycine-dependent respiration.

The source and characteristics of chemiluminescence associated with the oxygenase reaction catalyzed by Mn(2+)-ribulosebisphosphate carboxylase.

We confirm the observation of Mogel and McFadden (Mogel, S.N., and McFadden, B. A. (1990) Biochemistry 29, 8333-8337) that ribulosebisphosphate carboxylase/oxygenase (rubisco) exhibits chemiluminescence while catalyzing its oxygenase reaction in the presence of Mn2+. However, our results with the spinach and Rhodospirillum rubrum enzymes differ markedly in the following respects. 1) Chemiluminescence intensity was directly proportional to enzyme concentration and behaved as if representing the rate of oxygenase catalysis. 2) The wavelength spectrum peaked at about 770 nm and extended beyond 810 nm. This seems inconsistent with chemiluminescence generated by simultaneous decay of pairs of singlet O2 molecules. It is consistent with manganese(II) luminescence and we discuss its possible sources. The time course of chemiluminescence (resolution, 0.25 s) was distinctively different for spinach and R. rubrum enzymes during the initial 5 s of catalysis, with the bacterial enzyme exhibiting a pronounced initial "burst." Chemiluminescence by the spinach enzyme responded to substrate concentrations in a manner consistent with known oxygenase properties, exhibiting Michaelis-Menten kinetics with ribulose-1,5-bisphosphate (Km 400 nM). Chemiluminescence required carbamylated enzyme with Mn2+ bound at the active site (activation energy, -57.1 KJ.mol-1). As an indicator of oxygenase activity, chemiluminescence represents an improvement over oxygen electrode measurements in response time and sensitivity by factors of at least 100.

Adenine nucleotide levels in the cytosol, chloroplasts, and mitochondria of wheat leaf protoplasts.

Recently, a new method has been described, in which membrane filtration is used to allow the levels of adenine nucleotides in the chloroplast stroma, the cytosol, and the mitochondrial matrix to be measured. This method is now used to investigate the effect of illumination, of respiratory inhibitors, and of uncouplers on the distribution of ATP, ADP, and AMP in wheat (Triticum aestivum var. ;Timmo') leaf protoplasts. (a) The adenine nucleotides are apparently equilibrated by adenylate kinase in the stroma and the cytosol, but not in the mitochondrial matrix. (b) The ATP/ADP quotient in the cytosol is considerably higher than that in the mitochondrial matrix or the chloroplast stroma. (c) A large gradient exists between the ATP/ADP quotients in the cytosol and the mitochondrial matrix in the dark, with a very low ATP/ADP quotient in the mitochondria. This gradient is lowered by uncouplers or respiratory inhibitors showing that, as in animal tissues, it reflects the energization of the mitochondria. (d) In the dark, the stromal ATP/ADP is lower than in the light, and appears to be maintained, at least in part, by import from the cytosol. (e) The cytosolic ATP/ADP, however, actually decreases in the light. This contradicts the widespread assumption, that export of photosynthetically produced ATP from the chloroplast leads to an increase in the cytosolic ATP/ADP, which then inhibits oxidative phosphorylation in the mitochondria. (f) The mitochondrial ATP/ADP increases in the light, and the gradient between the cytosol and mitochondrial matrix falls. This is also difficult to understand in terms of an inhibition of oxidative phosphorylation in the light due to a lack of ADP in the cytosol. (g) The significance of the measured variations in the adenine nucleotide pools are discussed with respect to the diurnal carbohydrate metabolism in a leaf, and to the metabolic function of the chloroplast, the cytosol and the mitochondria.

Inhibition of alcohol dehydrogenase from yeast by pyridine.

1. Inhibition by pyridine of reduction of NAD by ethanol in the presence of yeast alcohol dehydrogenase was studied at 25 degrees in 60mm-glycine buffer (K(+), pH9.3). 2. The apparent Michaelis constant for ethanol increases linearly and that for NAD increases non-linearly with pyridine concentration. 3. Rates, v, observed in the presence of pyridine are lower than the values calculated from the effect of pyridine on the two apparent Michaelis constants and are described by the expression V/v={1+5.8[pyridine]}x{1+0.016(1+124 [pyridine)]/[EtOH]}x{1+0.00019(1+3.3[pyridine]+110 [pyridine](2))/[NAD]}. 4. Mixed inhibitor studies with pyridine and N(1)-methylnicotinamide chloride in 40mm-pyrophosphate buffer (Na(+), pH8.2) indicated little interaction of pyridine with the ;pyridinium site' of the dehydrogenase (interaction constant, alpha, 2.1). 5. The possible competition of ethanol and pyridine for a zinc atom in the active centre of yeast alcohol dehydrogenase is discussed.