A. Portis - Academia.edu (original) (raw)

Papers by A. Portis

PLANT PHYSIOLOGY, 1983

The role of the phosphate translocator and the importance of the extrachloroplastic concentration... more 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

Plant Physiology, 1987

Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activase, a soluble chloroplast protein... more Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activase, a soluble chloroplast protein which promotes light-dependent rubisco activation, was partialy 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

Plant Physiology, 2001

Anthranilate synthase (AS), the control enzyme of the tryptophan (Trp) biosynthetic pathway, is e... more Anthranilate synthase (AS), the control enzyme of the tryptophan (Trp) biosynthetic pathway, is encoded by nuclear genes, but is transported into the plastids. A tobacco (Nicotiana tabacum) cDNA (ASA2) encoding a feedback-insensitive tobacco AS α-subunit was transformed into two different sites of the tobacco plastid genome through site-specific insertion to obtain transplastomic plants with normal phenotype and fertility. A high and uniform level of ASA2 mRNA was observed in the transplastomic plants but not in the wild type. Although the plants with the transgene insertion atndhF-trnL only expressed one size of theASA2 mRNA, the plants with the transgene incorporated into the region between accD and open reading frame (ORF) 184 exhibited two species of mRNA, apparently due to readthrough. The transplastomic plants exhibited a higher level of AS α-subunit protein and AS enzyme activity that was less sensitive to Trp-feedback inhibition, leading to greatly increased free Trp levels ...

Plant Physiology, 1988

ABSTRACI Ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) activase protein was purified ... more ABSTRACI 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.

The Journal of Membrane Biology, 1981

The aggregation, leakage, and fusion of pure PS (phosphatidylserine) and mixed PS/PC (phosphatidy... more The aggregation, leakage, and fusion of pure PS (phosphatidylserine) and mixed PS/PC (phosphatidylcholine) sonicated vesicles were studied by light scattering, the release of encapsulated carboxyfluorescein, and a new fusion assay which monitors the mixing of the internal compartments of fusing vesicles. On a time scale of 1 min the extent of fusion was considerably greater than leakage. The Ca2+ and Mg2+ concentrations required to induce fusion increased when the PS content of the vesicles was decreased, and/or when the NaCl concentration was increased. Calculations employing a modified Gouy-Chapman equation and experimentally determined intrinsic binding constants of Na+ and Ca2+ to PS were shown to predict correctly the amount of Ca2+ bound in mixed PS/PC vesicles. For vesicles composed of either pure PS or of mixtures with PC in 100 mM NaCl (4:1 and 2:1 PS/PC); the induction of fusion (on a time scale of minutes) occurred when the amount of Ca or Mg bound/PS molecule exceeded 0.35-0.39. The induction of fusion for both pure PS and PS/PC mixed vesicles (with PS exceeding 50%) can be explained by assuming that destabilization of these vesicles requires a critical binding ratio of divalent cations to PS.

Journal of Experimental Botany, 2007

The previous investigations show that the amount and activity of Rubisco appears the major limita... more The previous investigations show that the amount and activity of Rubisco appears the major limitation to effective C 4 photosynthesis at low temperatures. The chilling-tolerant and bioenergy feedstock species Mis-canthus3giganteus (M.3giganteus) is exceptionally productive among C 4 grasses in cold climates. It is able to develop photosynthetically active leaves at temperatures 6°C below the minimum for maize, and achieves a productivity even at 52°N that exceeds that of the most productive C 3 crops at this latitude. This study investigates whether this unusual low temperature tolerance can be attributed to differences in the amount or kinetic properties of Rubisco relative to maize. An efficient protocol was developed to purify large amounts of functional Rubisco from C 4 leaves. The maximum carboxylation activities (V max), activation states, catalytic rates per active site (K cat) and activation energies (E a) of purified Rubisco and Rubisco in crude leaf extracts were determined for M.3giganteus grown at 14°C and 25°C, and maize grown at 25°C. The sequences of M.3giganteus Rubisco small subunit mRNA are highly conserved, and 91% identical to those of maize. Although there were a few differences between the species in the translated protein sequences, there were no significant differences in the catalytic properties (V max , K cat , and E a) for purified Rubisco, nor was there any effect of growth temperature in M.3giganteus on these kinetic properties. Extracted activities were close to the observed rates of CO 2 assimilation by the leaves in vivo. On a leaf area basis the extracted activities and activation state of Rubisco did not differ significantly, either between the two species or between growth temperatures. The activation state of Rubisco in leaf extracts showed no significant difference between warm and cold-grown M.3giganteus. In total, these results suggest that the ability of M.3giganteus to be productive and maintain photosynthetically competent leaves at low temperature does not result from low temperature acclimation or adaptation of the catalytic properties of Rubisco.

Journal of Experimental Botany, 2006

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) activation decreases under moderate hea... more Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) activation decreases under moderate heat stress. This decrease is caused by an impairment of activase function, which is exacerbated by faster rates of Rubisco deactivation at elevated temperatures. To determine if stromal oxidation causes inhibition of activase, transgenic Arabidopsis plants expressing suboptimal amounts of either the redox-regulated 46 kDa a-or non-redox regulated 43 kDa b-isoform of activase were examined. Photosynthesis, as measured by gas exchange and chlorophyll fluorescence, and Rubisco activation were inhibited to a much greater extent by moderately high temperatures in the two transgenic lines expressing suboptimal levels of the individual isoforms of activase compared with wildtype plants or transgenic plants expressing levels of the b-isoform sufficient for wild-type rates of photosynthesis. Net photosynthesis and Rubisco activation in transgenic plants expressing suboptimal amounts of the b-isoform of activase from the Antarctic hairgrass were even more sensitive to inhibition by moderate heat stress than in the transgenic plants containing Arabidopsis activase. The results demonstrate that photosynthesis exhibits a similar sensitivity to inhibition by moderately high temperature in plants expressing either of the two different isoforms of activase. Thus, impairment of activase function under heat stress is not caused by oxidation of the redox-sensitive sulphydryls of the a-isoform of activase. Instead, the results are consistent with thermal denaturation of activase under moderate heat stress, the effects of which on Rubisco activation would be enhanced when activase levels are suboptimal for photosynthesis.

Journal of Biological Chemistry, 2000

In the active form of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39), a c... more In the active form of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39), a carbamate at lysine 201 binds Mg 2؉ , which then interacts with the carboxylation transition state. Rubisco activase facilitates this spontaneous carbamylation/metal-binding process by removing phosphorylated inhibitors from the Rubisco active site. Activase from Solanaceae plants (e.g. tobacco) fails to activate Rubisco from non-Solanaceae plants (e.g. spinach and Chlamydomonas reinhardtii), and non-Solanaceae activase fails to activate Solanaceae Rubisco. Directed mutagenesis and chloroplast transformation previously showed that a proline 89 to arginine substitution on the surface of the large subunit of Chlamydomonas Rubisco switched its specificity from non-Solanaceae to Solanaceae activase activation. To define the size and function of this putative activase binding region, substitutions were created at positions flanking residue 89. As in the past, these substitutions changed the identities of Chlamydomonas residues to those of tobacco. Whereas an aspartate 86 to arginine substitution had little effect, aspartate 94 to lysine Rubisco was only partially activated by spinach activase but now fully activated by tobacco activase. In an attempt to eliminate the activase/Rubisco interaction, proline 89 was changed to alanine, which is not present in either non-Solanaceae or Solanaceae Rubisco. This substitution also caused reversal of activase specificity, indicating that amino acid identity alone does not determine the specificity of the interaction.

Journal of Biological Chemistry, 1997

Annals of the New York Academy of Sciences, 1978

Recent studies with both natural and artificial membrane systems 5-7 have established that lipid ... more Recent studies with both natural and artificial membrane systems 5-7 have established that lipid phase transitions can have a large effect on the activity of various membrane-bound enzymes. It is not clear as yet, however, whether lipid phase transitions play an important role during the normal functioning of membranes in living cells. Since most phospholipids extracted from natural membranes exhibit the solid-to-fluid thermotropic transition at temperatures below the ambient,s-lo it becomes questionable whether such transitions can play a regulatory role in membrane functions during the normal functioning of membranes in living cells. A more plausible case can be made for lipid phase transitions that can be induced isothermally by changes in ionic environment (ionotropic) at ambient temperatures. Such transitions have been described recently with acidic phospholipid membranes following changes in divalent metal concentrations and ~H .~9 1~-~4 Of special interest in this respect is the ability of Ca2+ (but not MgZ+) to induce a complete phase separation from mixed acidic and neutral phospholipids,l2? 15-17 and to produce a drastic shift of the transition point of acidic phospholipids to very high temperatures.l2S 14, l9 Fusion of vesicles composed of acidic phospholipids has also been observed to occur in relation to Ca2+-induced phase changes.15. It is well documented that Ca2+ plays a vital role in natural fusion phenomena,20-22 while Mg2+ is ineffective in most such systems. In view of the same specificity in the induction of phase separations and fusion in pure phospholipid systems,12-15, 23, 24 it is tempting to speculate that Caz+-induced phase changes of acidic phospholipids may be responsible for initiating fusion events in natural membranes.25 In this paper we will examine the available evidence on the interaction of Caz+ and Mg2+ with pure and mixed phospholipid membranes. The interaction will be described in terms of binding, induced phase changes observed by differential scanning calorimetry, structural changes determined by x-ray diffraction, and the release of heat measured in a batch microcalorimeter. The results will be discussed in relation to fusion of the same membranes as observed by various techniques. Finally, the conclusions based on Ca2+-induced fusion of phospho-24

Plant Physiology, 1981

The loss of Mge, upon the addition of the ionophore A23187 in the dark was prevented by less than... more The loss of Mge, upon the addition of the ionophore A23187 in the dark was prevented by less than 0.1 milimolar MgCI2 with intact chloroplasts suspended in a sorbitol medium, but required 1 to 3 miHimolar MgCl2 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+ concentration in the stroma in the dark is between 1 and 3 mllmolar. Other experiments on the inihibition by A23187 of CO2 fixation in the light and in a system capable of catalyzing C02 fixation in the dark, and on the Mge+ binding properties of thylakoid membranes, are consistent with this conclusion. The results provide further support for the hypothesis that light-induced Mge, concentration changes occur in the stroma that are important in the light-dark regulation of CO2 fixation.

Plant Physiology, 1981

The loss of Mge, upon the addition of the ionophore A23187 in the dark was prevented by less than... more The loss of Mge, upon the addition of the ionophore A23187 in the dark was prevented by less than 0.1 milimolar MgCI2 with intact chloroplasts suspended in a sorbitol medium, but required 1 to 3 miHimolar MgCl2 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+ concentration in the stroma in the dark is between 1 and 3 mllmolar. Other experiments on the inihibition by A23187 of CO2 fixation in the light and in a system capable of catalyzing C02 fixation in the dark, and on the Mge+ binding properties of thylakoid membranes, are consistent with this conclusion. The results provide further support for the hypothesis that light-induced Mge, concentration changes occur in the stroma that are important in the light-dark regulation of CO2 fixation.

PLANT PHYSIOLOGY, 1988

Previous reports indicate that ribulose 1,5-bisphosphate (RuBP) binds very tightly to inactive ri... more 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 N2 and assaying rubisco activity and RuBP. The percentage activation of

Archives of Biochemistry and Biophysics, 1978

Planta, 1985

A simple model of photosynthetic CO2 assimilation in Chlamydomonas has been developed in order to... more A simple model of photosynthetic CO2 assimilation in Chlamydomonas has been developed in order to evaluate whether a CO2-concentrating system could explain the photosynthetic characteristics of this alga (high apparent affinity for CO2, low photorespiration, little O2 inhibition of photosynthesis, and low CO2 compensation concentration). Similarly, the model was developed to evaluate whether the proposed defects in the CO2-concentrating system

Plant …, 1992

Purified spinach (Spinacea oleracea L.) and barley (Hordeum vulgare L.) ribulose-1,5-bisphosphate... more Purified spinach (Spinacea oleracea L.) and barley (Hordeum vulgare L.) ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase supported 50 to 100% activation of substrate-bound Rubisco from spinach, barley, wheat (Triticum aestivum L.), soybean (Glycine max L.), pea (Pisum sativum L.), Arabidopsis thaliana, maize (Zea mays L.), and Chiamydomonas reinhardtii but supported only 10 to 35% activation of Rubisco from three Solanaceae species, tobacco (Nicotiana tabacum L.), petunia (Petunia hybrida L.), and tomato (Lycopersicon esculentum L.). Conversely, purified tobacco and petunia Rubisco activase catalyzed 75 to 100% activation of substrate-bound Rubisco from the three Solanaceae species but only 10 to 25% activation of substrate-bound Rubisco from the other species. Thus, the interaction between substrate-bound Rubisco and Rubisco activase is species dependent. The species dependence observed is consistent with phylogenetic relationships previously derived from plant morphological characteristics and from nucleotide and amino acid sequence comparisons of the two Rubisco subunits. Species dependence in the Rubisco-Rubisco activase interaction and the absence of major anomalies in the deduced amino acid sequence of tobacco Rubisco activase compared to sequences in non-Solanaceae species suggest that Rubisco and Rubisco activase may have coevolved such that amino acid

Annual Review of Plant Physiology and Plant Molecular Biology, 1992

The light and CO2 response of (a) photosynthesis, (b) the activation state and total catalytic ef... more The light and CO2 response of (a) photosynthesis, (b) the activation state and total catalytic efficiency (kt,) of ribulose-1,5bisphosphate carboxylase (rubisco), and (c) the pool sizes of ribulose 1,5-bisphosphate, (RuBP), ATP, and ADP were studied in the C3 annuals Chenopodium album and Phaseolus vulgaris at 250C. The initial slope of the photosynthetic CO2 response curve was dependent on light intensity at reduced light levels only (less than 450 micromoles per square meter per second in C. album and below 200 micromoles per square meter per second in P. vulgaris). Modeled simulations indicated that the initial slope of the CO2 response of photosynthesis exhibited light dependency when the rate of RuBP regeneration limited photosynthesis, but not when rubisco capacity limited photosynthesis. Measured observations closely matched modeled simulations. The activation state of rubisco was measured at three light intensities in C. album (1750, 550, and 150 micromoles per square meter per second) and at intercellular CO2 partial pressures (C,) between the CO2 compensation point and 500 microbars. Above a C, of 120 microbars, the activation state of rubisco was light dependent. At light intensities of 550 and 1750 micromoles per square meter per second, it was also dependent on C,, decreasing as the C, was elevated above 120 microbars at 550 micromoles per square meter per second and above 300 microbars at 1750 micromoles per square meter per second. The pool size of RuBP was independent of C, only under conditions when the activation state of rubisco was dependent on C,. Otherwise, RuBP pool sizes increased as C, was reduced. ATP pools in C. album tended to increase as C, was reduced. In P. vulgaris, decreasing C, at a subsaturating light intensity of 190 micromoles per square meter per second increased the activation state of rubisco but had little effect on the k,c. These results support modelled simulations of the rubisco response to light and CO2, where rubisco is assumed to be down-regulated when photosynthesis is limited by the rate of RuBP regeneration.

PLANT PHYSIOLOGY, 1983

The role of the phosphate translocator and the importance of the extrachloroplastic concentration... more 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

Plant Physiology, 1987

Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activase, a soluble chloroplast protein... more Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activase, a soluble chloroplast protein which promotes light-dependent rubisco activation, was partialy 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

Plant Physiology, 2001

Anthranilate synthase (AS), the control enzyme of the tryptophan (Trp) biosynthetic pathway, is e... more Anthranilate synthase (AS), the control enzyme of the tryptophan (Trp) biosynthetic pathway, is encoded by nuclear genes, but is transported into the plastids. A tobacco (Nicotiana tabacum) cDNA (ASA2) encoding a feedback-insensitive tobacco AS α-subunit was transformed into two different sites of the tobacco plastid genome through site-specific insertion to obtain transplastomic plants with normal phenotype and fertility. A high and uniform level of ASA2 mRNA was observed in the transplastomic plants but not in the wild type. Although the plants with the transgene insertion atndhF-trnL only expressed one size of theASA2 mRNA, the plants with the transgene incorporated into the region between accD and open reading frame (ORF) 184 exhibited two species of mRNA, apparently due to readthrough. The transplastomic plants exhibited a higher level of AS α-subunit protein and AS enzyme activity that was less sensitive to Trp-feedback inhibition, leading to greatly increased free Trp levels ...

Plant Physiology, 1988

ABSTRACI Ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) activase protein was purified ... more ABSTRACI 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.

The Journal of Membrane Biology, 1981

The aggregation, leakage, and fusion of pure PS (phosphatidylserine) and mixed PS/PC (phosphatidy... more The aggregation, leakage, and fusion of pure PS (phosphatidylserine) and mixed PS/PC (phosphatidylcholine) sonicated vesicles were studied by light scattering, the release of encapsulated carboxyfluorescein, and a new fusion assay which monitors the mixing of the internal compartments of fusing vesicles. On a time scale of 1 min the extent of fusion was considerably greater than leakage. The Ca2+ and Mg2+ concentrations required to induce fusion increased when the PS content of the vesicles was decreased, and/or when the NaCl concentration was increased. Calculations employing a modified Gouy-Chapman equation and experimentally determined intrinsic binding constants of Na+ and Ca2+ to PS were shown to predict correctly the amount of Ca2+ bound in mixed PS/PC vesicles. For vesicles composed of either pure PS or of mixtures with PC in 100 mM NaCl (4:1 and 2:1 PS/PC); the induction of fusion (on a time scale of minutes) occurred when the amount of Ca or Mg bound/PS molecule exceeded 0.35-0.39. The induction of fusion for both pure PS and PS/PC mixed vesicles (with PS exceeding 50%) can be explained by assuming that destabilization of these vesicles requires a critical binding ratio of divalent cations to PS.

Journal of Experimental Botany, 2007

The previous investigations show that the amount and activity of Rubisco appears the major limita... more The previous investigations show that the amount and activity of Rubisco appears the major limitation to effective C 4 photosynthesis at low temperatures. The chilling-tolerant and bioenergy feedstock species Mis-canthus3giganteus (M.3giganteus) is exceptionally productive among C 4 grasses in cold climates. It is able to develop photosynthetically active leaves at temperatures 6°C below the minimum for maize, and achieves a productivity even at 52°N that exceeds that of the most productive C 3 crops at this latitude. This study investigates whether this unusual low temperature tolerance can be attributed to differences in the amount or kinetic properties of Rubisco relative to maize. An efficient protocol was developed to purify large amounts of functional Rubisco from C 4 leaves. The maximum carboxylation activities (V max), activation states, catalytic rates per active site (K cat) and activation energies (E a) of purified Rubisco and Rubisco in crude leaf extracts were determined for M.3giganteus grown at 14°C and 25°C, and maize grown at 25°C. The sequences of M.3giganteus Rubisco small subunit mRNA are highly conserved, and 91% identical to those of maize. Although there were a few differences between the species in the translated protein sequences, there were no significant differences in the catalytic properties (V max , K cat , and E a) for purified Rubisco, nor was there any effect of growth temperature in M.3giganteus on these kinetic properties. Extracted activities were close to the observed rates of CO 2 assimilation by the leaves in vivo. On a leaf area basis the extracted activities and activation state of Rubisco did not differ significantly, either between the two species or between growth temperatures. The activation state of Rubisco in leaf extracts showed no significant difference between warm and cold-grown M.3giganteus. In total, these results suggest that the ability of M.3giganteus to be productive and maintain photosynthetically competent leaves at low temperature does not result from low temperature acclimation or adaptation of the catalytic properties of Rubisco.

Journal of Experimental Botany, 2006

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) activation decreases under moderate hea... more Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) activation decreases under moderate heat stress. This decrease is caused by an impairment of activase function, which is exacerbated by faster rates of Rubisco deactivation at elevated temperatures. To determine if stromal oxidation causes inhibition of activase, transgenic Arabidopsis plants expressing suboptimal amounts of either the redox-regulated 46 kDa a-or non-redox regulated 43 kDa b-isoform of activase were examined. Photosynthesis, as measured by gas exchange and chlorophyll fluorescence, and Rubisco activation were inhibited to a much greater extent by moderately high temperatures in the two transgenic lines expressing suboptimal levels of the individual isoforms of activase compared with wildtype plants or transgenic plants expressing levels of the b-isoform sufficient for wild-type rates of photosynthesis. Net photosynthesis and Rubisco activation in transgenic plants expressing suboptimal amounts of the b-isoform of activase from the Antarctic hairgrass were even more sensitive to inhibition by moderate heat stress than in the transgenic plants containing Arabidopsis activase. The results demonstrate that photosynthesis exhibits a similar sensitivity to inhibition by moderately high temperature in plants expressing either of the two different isoforms of activase. Thus, impairment of activase function under heat stress is not caused by oxidation of the redox-sensitive sulphydryls of the a-isoform of activase. Instead, the results are consistent with thermal denaturation of activase under moderate heat stress, the effects of which on Rubisco activation would be enhanced when activase levels are suboptimal for photosynthesis.

Journal of Biological Chemistry, 2000

In the active form of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39), a c... more In the active form of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39), a carbamate at lysine 201 binds Mg 2؉ , which then interacts with the carboxylation transition state. Rubisco activase facilitates this spontaneous carbamylation/metal-binding process by removing phosphorylated inhibitors from the Rubisco active site. Activase from Solanaceae plants (e.g. tobacco) fails to activate Rubisco from non-Solanaceae plants (e.g. spinach and Chlamydomonas reinhardtii), and non-Solanaceae activase fails to activate Solanaceae Rubisco. Directed mutagenesis and chloroplast transformation previously showed that a proline 89 to arginine substitution on the surface of the large subunit of Chlamydomonas Rubisco switched its specificity from non-Solanaceae to Solanaceae activase activation. To define the size and function of this putative activase binding region, substitutions were created at positions flanking residue 89. As in the past, these substitutions changed the identities of Chlamydomonas residues to those of tobacco. Whereas an aspartate 86 to arginine substitution had little effect, aspartate 94 to lysine Rubisco was only partially activated by spinach activase but now fully activated by tobacco activase. In an attempt to eliminate the activase/Rubisco interaction, proline 89 was changed to alanine, which is not present in either non-Solanaceae or Solanaceae Rubisco. This substitution also caused reversal of activase specificity, indicating that amino acid identity alone does not determine the specificity of the interaction.

Journal of Biological Chemistry, 1997

Annals of the New York Academy of Sciences, 1978

Recent studies with both natural and artificial membrane systems 5-7 have established that lipid ... more Recent studies with both natural and artificial membrane systems 5-7 have established that lipid phase transitions can have a large effect on the activity of various membrane-bound enzymes. It is not clear as yet, however, whether lipid phase transitions play an important role during the normal functioning of membranes in living cells. Since most phospholipids extracted from natural membranes exhibit the solid-to-fluid thermotropic transition at temperatures below the ambient,s-lo it becomes questionable whether such transitions can play a regulatory role in membrane functions during the normal functioning of membranes in living cells. A more plausible case can be made for lipid phase transitions that can be induced isothermally by changes in ionic environment (ionotropic) at ambient temperatures. Such transitions have been described recently with acidic phospholipid membranes following changes in divalent metal concentrations and ~H .~9 1~-~4 Of special interest in this respect is the ability of Ca2+ (but not MgZ+) to induce a complete phase separation from mixed acidic and neutral phospholipids,l2? 15-17 and to produce a drastic shift of the transition point of acidic phospholipids to very high temperatures.l2S 14, l9 Fusion of vesicles composed of acidic phospholipids has also been observed to occur in relation to Ca2+-induced phase changes.15. It is well documented that Ca2+ plays a vital role in natural fusion phenomena,20-22 while Mg2+ is ineffective in most such systems. In view of the same specificity in the induction of phase separations and fusion in pure phospholipid systems,12-15, 23, 24 it is tempting to speculate that Caz+-induced phase changes of acidic phospholipids may be responsible for initiating fusion events in natural membranes.25 In this paper we will examine the available evidence on the interaction of Caz+ and Mg2+ with pure and mixed phospholipid membranes. The interaction will be described in terms of binding, induced phase changes observed by differential scanning calorimetry, structural changes determined by x-ray diffraction, and the release of heat measured in a batch microcalorimeter. The results will be discussed in relation to fusion of the same membranes as observed by various techniques. Finally, the conclusions based on Ca2+-induced fusion of phospho-24

Plant Physiology, 1981

The loss of Mge, upon the addition of the ionophore A23187 in the dark was prevented by less than... more The loss of Mge, upon the addition of the ionophore A23187 in the dark was prevented by less than 0.1 milimolar MgCI2 with intact chloroplasts suspended in a sorbitol medium, but required 1 to 3 miHimolar MgCl2 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+ concentration in the stroma in the dark is between 1 and 3 mllmolar. Other experiments on the inihibition by A23187 of CO2 fixation in the light and in a system capable of catalyzing C02 fixation in the dark, and on the Mge+ binding properties of thylakoid membranes, are consistent with this conclusion. The results provide further support for the hypothesis that light-induced Mge, concentration changes occur in the stroma that are important in the light-dark regulation of CO2 fixation.

Plant Physiology, 1981

The loss of Mge, upon the addition of the ionophore A23187 in the dark was prevented by less than... more The loss of Mge, upon the addition of the ionophore A23187 in the dark was prevented by less than 0.1 milimolar MgCI2 with intact chloroplasts suspended in a sorbitol medium, but required 1 to 3 miHimolar MgCl2 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+ concentration in the stroma in the dark is between 1 and 3 mllmolar. Other experiments on the inihibition by A23187 of CO2 fixation in the light and in a system capable of catalyzing C02 fixation in the dark, and on the Mge+ binding properties of thylakoid membranes, are consistent with this conclusion. The results provide further support for the hypothesis that light-induced Mge, concentration changes occur in the stroma that are important in the light-dark regulation of CO2 fixation.

PLANT PHYSIOLOGY, 1988

Previous reports indicate that ribulose 1,5-bisphosphate (RuBP) binds very tightly to inactive ri... more 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 N2 and assaying rubisco activity and RuBP. The percentage activation of

Archives of Biochemistry and Biophysics, 1978

Planta, 1985

A simple model of photosynthetic CO2 assimilation in Chlamydomonas has been developed in order to... more A simple model of photosynthetic CO2 assimilation in Chlamydomonas has been developed in order to evaluate whether a CO2-concentrating system could explain the photosynthetic characteristics of this alga (high apparent affinity for CO2, low photorespiration, little O2 inhibition of photosynthesis, and low CO2 compensation concentration). Similarly, the model was developed to evaluate whether the proposed defects in the CO2-concentrating system

Plant …, 1992

Purified spinach (Spinacea oleracea L.) and barley (Hordeum vulgare L.) ribulose-1,5-bisphosphate... more Purified spinach (Spinacea oleracea L.) and barley (Hordeum vulgare L.) ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase supported 50 to 100% activation of substrate-bound Rubisco from spinach, barley, wheat (Triticum aestivum L.), soybean (Glycine max L.), pea (Pisum sativum L.), Arabidopsis thaliana, maize (Zea mays L.), and Chiamydomonas reinhardtii but supported only 10 to 35% activation of Rubisco from three Solanaceae species, tobacco (Nicotiana tabacum L.), petunia (Petunia hybrida L.), and tomato (Lycopersicon esculentum L.). Conversely, purified tobacco and petunia Rubisco activase catalyzed 75 to 100% activation of substrate-bound Rubisco from the three Solanaceae species but only 10 to 25% activation of substrate-bound Rubisco from the other species. Thus, the interaction between substrate-bound Rubisco and Rubisco activase is species dependent. The species dependence observed is consistent with phylogenetic relationships previously derived from plant morphological characteristics and from nucleotide and amino acid sequence comparisons of the two Rubisco subunits. Species dependence in the Rubisco-Rubisco activase interaction and the absence of major anomalies in the deduced amino acid sequence of tobacco Rubisco activase compared to sequences in non-Solanaceae species suggest that Rubisco and Rubisco activase may have coevolved such that amino acid

Annual Review of Plant Physiology and Plant Molecular Biology, 1992

The light and CO2 response of (a) photosynthesis, (b) the activation state and total catalytic ef... more The light and CO2 response of (a) photosynthesis, (b) the activation state and total catalytic efficiency (kt,) of ribulose-1,5bisphosphate carboxylase (rubisco), and (c) the pool sizes of ribulose 1,5-bisphosphate, (RuBP), ATP, and ADP were studied in the C3 annuals Chenopodium album and Phaseolus vulgaris at 250C. The initial slope of the photosynthetic CO2 response curve was dependent on light intensity at reduced light levels only (less than 450 micromoles per square meter per second in C. album and below 200 micromoles per square meter per second in P. vulgaris). Modeled simulations indicated that the initial slope of the CO2 response of photosynthesis exhibited light dependency when the rate of RuBP regeneration limited photosynthesis, but not when rubisco capacity limited photosynthesis. Measured observations closely matched modeled simulations. The activation state of rubisco was measured at three light intensities in C. album (1750, 550, and 150 micromoles per square meter per second) and at intercellular CO2 partial pressures (C,) between the CO2 compensation point and 500 microbars. Above a C, of 120 microbars, the activation state of rubisco was light dependent. At light intensities of 550 and 1750 micromoles per square meter per second, it was also dependent on C,, decreasing as the C, was elevated above 120 microbars at 550 micromoles per square meter per second and above 300 microbars at 1750 micromoles per square meter per second. The pool size of RuBP was independent of C, only under conditions when the activation state of rubisco was dependent on C,. Otherwise, RuBP pool sizes increased as C, was reduced. ATP pools in C. album tended to increase as C, was reduced. In P. vulgaris, decreasing C, at a subsaturating light intensity of 190 micromoles per square meter per second increased the activation state of rubisco but had little effect on the k,c. These results support modelled simulations of the rubisco response to light and CO2, where rubisco is assumed to be down-regulated when photosynthesis is limited by the rate of RuBP regeneration.