Biomedical subjects

A R Portis

Publications and source records attributed to A R Portis.

At least 37 records · Page 2Linked to original sources

PubMed1990

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.

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PubMed1989

Adenosine triphosphate hydrolysis by purified rubisco activase.

Activation of ribulose bisphosphate carboxylase/oxygenase (rubisco) in vivo is mediated by a specific protein, rubisco activase. In vitro, activation of rubisco by rubisco activase is dependent on ATP and is inhibited by ADP. Purified rubisco activase hydrolyzed ATP with a specific activity of 1.5 mumol min-1 mg-1 protein, releasing approximately stoichiometric amounts of ADP and Pi. Hydrolysis was highly specific for ATP-Mg and had a broad pH optimum, with maximum activity at pH 8.0-8.5. ATPase activity was inhibited by ADP but not by molybdate, vanadate, azide, nitrate, or fluoride. Addition of rubisco in either the inactive or activated form had no significant effect on ATPase activity. Incubation of rubisco activase in the absence of ATP resulted in loss of both ATPase and rubisco activation activities. Both activities were also heat labile, with 50% loss in activity after 5 min at 38 degrees C and complete inhibition following treatment at 43 degrees C. Both activities showed a sigmoidal response to ATP concentration, with half-maximal activity at 0.053 mM ATP. Rubisco activation activity was dependent on the concentrations of both ATP and ADP. The results suggest that ATPase activity is an intrinsic property of rubisco activase.

Adenosine Triphosphate↗

PubMed1989

Ribulose-1,5-bisphosphate carboxylase/oxygenase activase protein prevents the in vitro decline in activity of ribulose-1,5-bisphosphate carboxylase/oxygenase.

The rate of CO(2) fixation by ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) following addition of ribulose 1,5-bisphosphate (RuBP) to fully activated enzyme, declined with first-order kinetics, resulting in 50% loss of rubisco activity after 10 to 12 minutes. This in vitro decline in rubisco activity, termed fall-over, was prevented if purified rubisco activase protein and ATP were added, allowing linear rates of CO(2) fixation for up to 20 minutes. Rubisco activase could also stimulate rubisco activity if added after fallover had occurred. Gel filtration of the RuBP-rubisco complex to remove unbound RuBP allowed full activation of the enzyme, but the inhibition of activated rubisco during fallover was only partially reversed by gel filtration. Addition of alkaline phosphatase completely restored rubisco activity following fallover. The results suggest that fallover is not caused by binding of RuBP to decarbamylated enzyme, but results from binding of a phosphorylated inhibitor to the active site of rubisco. The inhibitor may be a contaminant in preparations of RuBP or may be formed on the active site but is apparently removed from the enzyme in the presence of the rubisco activase protein.

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PubMed1988

Involvement of stromal ATP in the light activation of ribulose-1,5-bisphosphate carboxylase/oxygenase in intact isolated chloroplasts.

Light activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) and stromal ATP content were measured in intact isolated spinach chloroplasts. Treatments which decreased stromal ATP, such as incubation with the ATP analog beta,gamma-methylene adenosine triphosphate or with the energy transfer inhibitor phloridzin inhibited the light activation of rubisco. In the absence of added inorganic phosphate (Pi), light activation of rubisco was inhibited, coincident with low stromal ATP. Addition of methyl viologen restored both stromal ATP and rubisco activity to levels observed in the presence of Pi. Activation of rubisco was inhibited in the presence of 2 millimolar dihydroxyacetone phosphate or 3-phosphoglycerate and stromal ATP was also decreased under these conditions. Both were partially restored by increasing the Pi concentration. The strong correlation between activation state of rubisco and stromal ATP concentration in intact chloroplasts under a wide variety of experimental conditions indicates that light activation of rubisco is dependent on ATP and proportional to the ATP concentration. These observations can be explained in terms of the rubisco activase protein, which mediates activation of rubisco at physiological concentrations of CO(2) and ribulose-1,5-bisphosphate and is dependent upon ATP.

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PubMed1988

Protein-bound ribulose bisphosphate correlates with deactivation of ribulose bisphosphate carboxylase in leaves.

Previous reports indicate that ribulose 1,5-bisphosphate (RuBP) binds very tightly to inactive ribulose bisphosphate carboxylase (rubisco) in vitro. Therefore, we decided to investigate whether there was evidence for tight binding of RuBP associated with deactivation of rubisco in vivo. We modified a technique for rapidly separating ;free' metabolites from those bound to high molecular compounds. Arabidopsis thaliana plants were illuminated at various irradiances before freezing the leaves in liquid N(2) and assaying rubisco activity and RuBP. The percentage activation of rubisco varied from 37% at low irradiance (45 micromoles quanta per square meter per second) to 100% at high irradiance (800 micromoles quanta per square meter per second). The total amount of RuBP did not vary much with irradiance, but bound RuBP changed from 36% of the total at low irradiance to none at high irradiance. Bound RuBP was significantly correlated with the estimated number of inactive rubisco sites, with a ratio of about 1:1. After a step increase in irradiance, rubisco activation increased and total RuBP increased transiently, but steady levels of both occurred by 10 minutes. The amount of bound RuBP decreased with a similar time course to the estimated decrease in inactive rubisco sites. After a step decrease in irradiance, rubisco deactivated slowly for at least 25 minutes. Bound RuBP increased gradually but did so more slowly than the estimated increase in inactive rubisco sites.

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PubMed1988

Effects of Irradiance and Methyl Viologen Treatment on ATP, ADP, and Activation of Ribulose Bisphosphate Carboxylase in Spinach Leaves.

Since activation of ribulose bisphosphate carboxylase (rubisco) by rubisco activase is sensitive to ATP and ADP in vitro, we aimed to test the correlation between ATP level and rubisco activation state in intact leaves of Spinacia oleracea L. in response to changes in irradiance and after feeding the electron acceptor methyl viologen. Leaves were exposed to various irradiances for 45 minutes at atmospheric partial pressures of CO(2) and O(2). After measuring the rate of CO(2) assimilation, leaves were freeze-clamped in situ and the punched discs assayed for rubisco activity, and amounts of ribulose bisphosphate (RuBP), ATP, and ADP. The photosynthetic rate and the activation state of rubisco increased with increasing irradiance but the levels of RuBP, ATP, and ADP were not greatly affected. Methyl viologen fed leaves under low irradiance had rubisco activation states of 93% compared to 51% in control leaves. The ATP content of the leaves was also significantly higher and the ratio of ATP to ADP was 4.1 in methyl viologen fed leaves compared to 2.2 in control leaves. From these results and other published results we conclude that a correlation between ATP level and rubisco activation can be observed in intact leaves, but that during changes in irradiance some additional factors are involved in regulating rubisco activation.

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PubMed1988

Purification and assay of rubisco activase from leaves.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) activase protein was purified from spinach leaves by ammonium sulfate precipitation and ion exchange fast protein liquid chromatography. This resulted in 48-fold purification with 70% recovery of activity and yielded up to 18 milligrams of rubisco activase protein from 100 grams of leaves. Based on these figures, the protein comprised approximately 2% by weight of soluble protein in spinach (Spinacia oleracea L.) leaves. The preparations were at least 95% pure and were stable when frozen in liquid nitrogen. Addition of ATP during purification and storage was necessary to maintain activity. Assay of rubisco activase was based on its ability to promote activation of rubisco in the presence of ribulose-1,5-bisphosphate. There was an absolute requirement for ATP which could not be replaced by other nucleoside phosphates. The initial rate of increase of rubisco activity and the final rubisco specific activity achieved were both dependent on the concentration of rubisco activase. The initial rate was directly proportional to the rubisco activase concentration and was used as the basis of activity. The rate of activation of rubisco was also dependent on the rubisco concentration, suggesting that the activation process is a second order reaction dependent on the concentrations of both rubisco and rubisco activase. It is suggested that deactivation of rubisco occurs simultaneously with rubisco activase-mediated activation, and that rubisco activation state represents a dynamic equilibrium between these two processes.

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PubMed1987

Inhibition of the photosynthetic activities of isolated spinach chloroplasts by phosphonate compounds.

The effects of three closely related phosphonate compounds on several photosynthetic activities of isolated chloroplasts were investigated. Phosphonoformic and phosphonopropionic acid were found to inhibit both CO(2) fixation and the reduction of 3-phosphoglyceric acid, with CO(2) fixation being more sensitive. In contrast, phosphonoacetic acid was only slightly inhibitory. The lack of inhibition appeared to be due to its inability to enter the stroma via the phosphate translocator. Measurements of changes in stromal metabolite levels following the inhibition of CO(2) fixation by either phosphonoformic or phosphonopropionic acid indicated that the activity of ribulose bisphosphate carboxylase/oxygenase was reduced. Studies with the isolated enzyme confirmed that both of these compounds were effective competitive inhibitors of the carboxylase activity of the enzyme.

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PubMed1987

Purification and species distribution of rubisco activase.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activase, a soluble chloroplast protein which promotes light-dependent rubisco activation, was partially purified from spinach chloroplasts by ion-exchange and gel-filtration fast protein liquid chromatography. The protein could also be isolated using rate zonal centrifugation in sucrose gradients followed by conventional ion-exchange on DEAE-cellulose. The active enzyme was composed of 44 and 41 kilodalton subunits. Antibodies to the activase polypeptides were produced in tumor-induced mouse ascites fluid and used as probes for activase on immunoblots of soluble proteins from a number of species. One or both of the activase polypeptides were recognized in all higher plant species examined including Arabidopsis thaliana, soybean, kidney bean, pea, tobacco, maize, oat, barley, celery, tomato, pigweed, purslane, dandelion, sorghum, and crabgrass. The polypeptides were not present in a mutant of Arabidopsis which is incapable of activating rubisco in vivo. The activase polypeptides were also detected in cell extracts of the green alga Chlamydomonas reinhardii. Activase activity, which had been demonstrated previously in wild-type Arabidopsis and in spinach, was measured in protoplast extracts of Nicotiana rustica. The results suggest that control of rubisco by activase may be an ubiquitous form of regulation in eucaryotic photosynthetic organisms.

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PubMed1987

Rubisco Activase Mediates ATP-Dependent Activation of Ribulose Bisphosphate Carboxylase.

The activation level of ribulosebisphosphate carboxylase following preincubation with ribulose 1,5-bisphosphate was increased by ATP and ribulosebisphosphate carboxylase activase in the absence of thylakoids or illumination. Maximal activation was obtained with 0.5 millimolar ATP in the presence of an ATP regenerating system (phosphoenolpyruvate and pyruvate kinase). Without the ATP regenerating system, activation was lower, linearly dependent on ATP concentration up to 1.0 millimolar, and was strongly inhibited by ADP.

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PubMed1986

Purification of ribulose-1, 5-bisphosphate carboxylase/oxygenase with high specific activity by fast protein liquid chromatography.

A rapid procedure for the purification of ribulose-1, 5-bisphosphate carboxylase/oxygenase (rubisco) (EC 4.1.1.39) by fast protein liquid chromatography (FPLC) is described. Chloroplasts isolated mechanically from spinach leaves were used as the source of a stromal extract enriched in rubisco. By subsequent fractionation of this extract on ion-exchange FPLC, highly purified rubisco (sp act 2.10-2.76 mumol/mg protein X min) was obtained in less than 30 min. The high specific activity and excellent stability of the final preparation can be attributed to the use of chloroplasts as a starting material and the short time required for the chromatographic separation, both of which minimize proteolytic activity.

Chloroplasts↗

PubMed1986

Light and CO(2) Response of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activation in Arabidopsis Leaves.

The requirements for activation of ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) were investigated in leaves of Arabidopsis wild-type and a mutant incapable of light activating rubisco in vivo. Upon illumination with saturating light intensities, the activation state of rubisco increased 2-fold in the wild-type and decreased in the mutant. Activation of fructose 1,6-bisphosphate phosphatase was unaffected by the mutation. Under low light, rubisco deactivated in both the wild-type and the mutant. Deactivation of rubisco in the mutant under high and low light led to the accumulation of high concentrations of ribulose 1,5-bisphosphate. Inhibiting photosynthesis with methyl viologen prevented ribulose 1,5-bisphosphate accumulation but was ineffective in restoring rubisco activation to the mutant. Net photosynthesis and the rubisco activation level were closely correlated and saturated at a lower light intensity in the mutant than in wild-type. At CO(2) concentrations between 100 and 2000 microliters per liter, the activation state was a function of the CO(2) concentration in the dark but was independent of CO(2) concentration in the light. High CO(2) concentration (1%) suppressed activation in the wild-type and deactivation in the mutant. These results support the concept that rubisco activation in vivo is not a spontaneous process but is catalyzed by a specific protein. The absence of this protein, rubisco activase, is responsible for the altered characteristics of rubisco activation in the mutant.

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PubMed1986

Exchange Properties of the Activator CO(2) of Spinach Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase.

The exchange properties of the activator CO(2) of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase were characterized both in vitro with the purified enzyme, and in situ within isolated chloroplasts. Carboxyarabinitol-1,5-bisphosphate, a proposed reaction intermediate analog for the carboxylase activity of the enzyme, was used to trap the activator CO(2) on the enzyme both in vitro and in situ. Modulation of ribulose-1,5-bisphosphate carboxylase/oxygenase activity in intact chloroplasts during a light/dark cycle was associated with a similar modulation in carboxyarabinitol-1,5-bisphosphate-trapped CO(2). The exchange kinetics of the activator CO(2) were monitored by activation of the enzyme to steady state in the presence of (12)CO(2), followed by addition of (14)CO(2) and determination of the amount of labeled CO(2) trapped on the enzyme by carboxyarabinitol-1,5-bisphosphate. Rate constants (K(obs)) for exchange with both the purified enzyme (0.45 min(-1)) and in illuminated chloroplasts (0.18 min(-1)) were comparable to the observed rate constants for enzyme activation under the two conditions. A similar exchange of the activator CO(2) was not observed in chloroplasts in the dark. Kinetic analysis of the exchange properties of the purified enzyme were consistent with an equilibrium between active and inactive forms of the enzyme during steady state activation.

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PubMed1986

Activation of Ribulosebisphosphate Carboxylase/Oxygenase at Physiological CO(2) and Ribulosebisphosphate Concentrations by Rubisco Activase.

The enzyme-catalyzed activation of ribulosebisphosphate carboxylase/oxygenase (rubisco) was investigated in an illuminated reconstituted system containing thylakoid membranes, rubisco, ribulosebisphosphate (RuBP), MgCl(2), carbonic anhydrase, catalase, the artificial electron acceptor pyocyanine, and partially purified rubisco activase. Optimal conditions for light-induced rubisco activation were found to include 100 micrograms per milliliter rubisco, 300 micrograms per milliliter rubisco activase, 3 millimolar RuBP, and 6 millimolar free Mg(2+) at pH 8.2. The half-time for rubisco activation was 2 minutes, and was 4 minutes for rubisco deactivation. The rate of rubisco deactivation was identical in the presence and absence of activase. The K(act)(CO(2)) of rubisco activation in the reconstituted system was 4 micromolar CO(2), compared to a K(act)(CO(2)) of 25 to 30 micromolar CO(2) for the previously reported spontaneous CO(2)/Mg(2+) activation mechanism. The activation process characterized here explains the high degree of rubisco activation at the physiological concentrations of 10 micromolar CO(2) and 2 to 4 millimolar RuBP found in intact leaves, conditions which lead to almost complete deactivation of rubisco in vitro.

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PubMed1983

Analysis of the role of the phosphate translocator and external metabolites in steady-state chloroplast photosynthesis.

The role of the phosphate translocator and the importance of the extrachloroplastic concentrations of phosphate, 3-phosphoglycerate, and dihydroxyacetone phosphate in steady-state photosynthesis is examined with a kinetic model. The steady-state stromal concentrations of these compounds are calculated as a function of the rate of the various partial reactions of photosynthesis, at various external concentrations which span those likely to occur in vivo. It is shown how the net transport requirements of the various reactions necessitate different adjustments in the stromal concentrations of these compounds, away from the equilibrium values expected in the absence of metabolism. Under most circumstances, the high exchange capacity of the phosphate translocator relative to the transport requirements of CO(2) fixation limits the extent of these displacements, but conditions when the phosphate translocator is limiting photosynthesis are observed and discussed. The model provides a basis for a more quantitative understanding of the role of the phosphate translocator and the external concentrations of phosphate, 3-phosphoglycerate, and dihydroxyacetone phosphate in photosynthesis.

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PubMed1982

A Mutant of Arabidopsis thaliana Which Lacks Activation of RuBP Carboxylase In Vivo.

A mutant of Arabidopsis thaliana has been isolated in which ribulose-1,5-bisphosphate carboxylase is present in a nonactivatable form in vivo. The mutation appears to affect carboxylase activation specifically, and not any other enzyme of the photosynthesis or photorespiratory cycles. The effect of the mutation on carboxylase activation is indirect, inasmuch as the properties of ribulose-1,5-bisphosphate carboxylase purified from the mutant are not distinguishable from those of the wild type enzyme. The mutant requires high levels of atmospheric CO(2) for growth because photosynthesis is severely impaired in atmospheres containing normal levels of CO(2), irrespective of the atmospheric O(2) concentration. In this respect, the mutant is distinguished from previously described high-CO(2) requiring mutants of Arabidopsis which have defects in photorespiratory carbon or nitrogen metabolism.

Journal Article↗

PubMed1982

Effects of the relative extrachloroplastic concentrations of inorganic phosphate, 3-phosphoglycerate, and dihydroxyacetone phosphate on the rate of starch synthesis in isolated spinach chloroplasts.

The effect of external inorganic phosphate (Pi) on starch synthesis in isolated spinach (Spinacia oleracea American Hybrid No. 424) chloroplasts in the presence of millimolar concentrations of 3-phosphoglycerate (PGA) and/or dihydroxyacetone phosphate (DAP) was examined. Whereas CO(2) fixation was relatively constant as the ratio of the external phosphate to the PGA + DAP varied from 1:3 to 3:1, starch synthesis varied from 17% to 2% of the CO(2) fixation rate. With DAP alone, maximal starch synthesis was about 10% of the CO(2) fixation rate. The data demonstrate that the Pi/(PGA + DAP) ratio in the cytoplasm of plant cells could serve to regulate the flow of newly fixed carbon into starch without alterations in the rate of CO(2) fixation.

Journal Article↗

PubMed1981

Evidence of a Low Stromal Mg Concentration in Intact Chloroplasts in the Dark: I. STUDIES WITH THE IONOPHORE A23187.

The loss of Mg(2+) upon the addition of the ionophore A23187 in the dark was prevented by less than 0.1 millimolar MgCl(2) with intact chloroplasts suspended in a sorbitol medium, but required 1 to 3 millimolar MgCl(2) if the chloroplasts were in a K(+) -gluconate medium. Measurements of stromal pH in the dark indicated that, in the K(+) -gluconate medium, the stromal pH is nearly the same as that of the medium, whereas in the sorbitol medium it is much more acidic as reported previously. These observations suggest that the free Mg(2+) concentration in the stroma in the dark is between 1 and 3 millimolar. Other experiments on the inihibition by A23187 of CO(2) fixation in the light and in a system capable of catalyzing CO(2) fixation in the dark, and on the Mg(2+) binding properties of thylakoid membranes, are consistent with this conclusion. The results provide further support for the hypothesis that light-induced Mg(2+) concentration changes occur in the stroma that are important in the light-dark regulation of CO(2) fixation.

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