Pyrenoid protein from the brown alga Pilayella littoralis

Characterization by peptide mapping and amino acid analysis of the two major pyrenoid polypeptides from the brown alga Pilayella littoralis shows that they are very similar to the subunits of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) from this alga. The observed similarities are discussed in relation to previous pyrenoid protein characterization from members of the Chlorophyceae.Abbreviations DTT dithiothreitol - EDTA Na₂ ethylenediamine tetraacetic acid (disodium salt) - PMFS phenylmethylsul-phonylfluoride - PVPP polyvinylpyrrolidone - RuBP ribulose-1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate carboxylase - SDS sodium dodecyl sulphate - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - TRIS 2-amino-2-(hydroxymethyl) propane-1,3-diol - TPCK L-1-tosylamido-2-phenylethylchoromethyl ketone     

Characterization of the pyrenoid isolated from unicellular green algaChlamydomonas reinhardtii: Particulate form of RuBisCO protein

Summary The pyrenoid is a protein complex in the chloroplast stroma of eukaryotic algae. After the treatment with mercury chloride, pyrenoids were isolated by sucrose density gradient centrifugation from cell-wall less mutant cells, CW-15, as well as wild type cells, C-9, of unicellular green algaChlamydomonas reinhardtii. Pyrenoids were characterized as a fraction whose protein/chlorophyll ratio was very high, and also examined by Nomarski differential interference microscopy. Most of the components consisted of 55 kDa and 16 kDa polypeptides (11) which were immunologically identified as the large and small subunit of RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) protein, respectively. Some minor polypeptides were also detected. Substantial amount of RuBisCO protein is present as a particulate form in the pyrenoid in addition to the soluble form in algal chloroplast stroma.Abbreviations BPB bromophenol blue - DAB 3,3-diaminobenzidine - DTT dithiothreitol - ELISA enzyme-linked immunosorbent assay - High-CO₂ cells cells grown under air enriched with 4% CO₂ - Low-CO₂ cells cells grown under ordinary air (containing 0.04% CO₂) - NP-40 nonionic detergent (Nonidet) P-40 - PAGE polyacrylamide gel electrophoresis - PAP peroxidase-antiperoxidase conjugate - RuBisCO ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - SDS sodium dodecylsulfate     

Differences in amino acid sequence of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from two species of Dasycladaceae

In contrast to other plants the plastid genome of Acetabularia is larger in size and shows a high degree of variability. This study on the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase demonstrates that strongly conserved areas also exist in the plastid genome of the Dasycladaceae. Searching for differences in the amino acid sequence of the large subunit from Acetabularia mediterranea and Acicularia schenckii, proteolytic peptides which differ in their elution behaviour in reverse-phase high-performance liquid chromatography were sequenced. Only six amino acids were found to be exchanged in the large subunit from these two species. Since these two species diverged approx. 150 million years ago, these results imply that 0.84 amino-acid exchanges per 100 amino acids have occurred in 10⁸ years, underlining the strong conservatism of the large subunit.Abbreviations A Acetabularia mediterranea - Ac. Acicularia schenckii - HPLC high-performance liquid chromatography - LSU large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase - PAGE polyacrylamide gel electrophoresis - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase - SDS sodium dodecyl sulfate     

Comparison of Properties of the Proteolytic Degradation of Unassembled Nuclear-encoded Subunits of Ribulose-1,5-bisphosphate Carboxylase and of the Coupling Factor of Photophosphorylation CF1*

The proteolytic degradation of unassembled small subunit polypeptides of ribulose-1,5-bisphosphate carboxylase and of the δ-subunit of the coupling factor of photophosphorylation CF₁ were analyzed and compared in vitro in the presence of stroma or membrane preparations from ribosome-deficient plastids isolated from 32°C-grown rye leaves (Secale cereale L.). Extracts obtained from 70S ribosome-deficient rye leaves after radioactive labeling were used as substrate source for the unassembled polypeptides. Soluble stroma as well as membrane preparations from isolated plastids contained proteolytic activities catalyzing the degradation of both the small subunits of ribulose-1,5-bisphosphate carboxylase and CF₁-δ in vitro. Maximal in vitro degradation was observed at pH 2–3 for the unassembled small subunits, but at pH 6–7 for the purified holoprotein of ribulose-1,5-bisphosphate carboxylase, and at pH 6.0 for unassembled CF₁-δ. Degradation of unassembled small subunits of ribulose-1,5-bisphosphate carboxylase at pH 3.0 was stimulated by Cu²⁺ but not by Ca²⁺, Mg²⁺ or ATP. At pH 3.0 the degradation of unassembled small subunits of ribulose-1,5-bisphosphate carboxylase was not inhibited by various protease inhibitors but was even stimulated. At pH 7.0 its degradation was inhibited by HgCl₂ and diazoacetyl nor-leucine methyl ester + Cu-acetate. The degradation of CF₁-δ was markedly inhibited by phenylmethylsulphonyl fluoride (PMSF) and to a lesser extent by 1,10-phenanthroline. According to present results different proteolytic systems appear to be involved in the degradation of unassembled small subunits of ribulose-1,5-bisphosphate carboxylase and of unassembled CF₁-δ.     

Phycoerythrin is absent from the pyrenoid of Porphyridium cruentum: photosynthetic implications

The thylakoid lamellae which traverse the pyrenoid of the unicellular red alga Porphyridium cruentum (Agardh) Nägeli appear to lack phycobilisomes. We have confirmed by immuno-electron microscopy that phycoerythrin (PE), an important structural component of the phycobilisomes of red algae, is absent from the pyrenoid. To characterize pyrenoid thylakoids further, electron-microscopic cytochemical methods were employed to detect photosystem activity. Photosystem (PS) I activity was demonstrated in both stromal and pyrenoid thylakoids by the photooxidation of 3,3-diaminobenzidine. In contrast, the localization of photoreduced distyryl nitroblue tetrazolium demonstrated that PSII activity was restricted to stromal thylakoids. The observed partitioning of PE and PSII activity within the plastid may be related to another observation, that being the localization of nearly all ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) within the pyrenoid of this alga. It is possible that the pyrenoid of P. cruentum functions as a specific metabolic compartment where CO₂ fixation is enhanced by the absence of photosynthetic O₂ evolution.Abbreviations DAB 3,3-diaminobenzidine-4HCl - DS-NBT distyryl nitroblue tetrazolium chloride - EF exoplasmic face - LSU large subunit of RuBisCO - PE phycoerythrin - PS photosystem - RuBisCO ribulose-1,5-bisphosphate carboxylase/oxygenase We thank Drs. Jacqueline Fleck (CNRS, Strasbourg) and Robert MacColl (New York State Department of Health, Albany) for providing us with the antibodies used in this study. We also thank Dr. C.E. Smith for use of the Zeiss MOP-3 digital analyser and Dr. Geneviève Bricheux for kindly providing Lowicryl-embedded samples of P. cruentum. Aatrex^® was kindly donated by Ciba-Geigy. This research was supported by the Natural Sciences and Engineering Research Council of Canada (grant No. A-2921).     

How various factors influence the CO2/O2 specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase

Temperature, activating metal ions, and amino-acid substitutions are known to influence the CO₂/O₂ specificity of the chloroplast enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase. However, an understanding of the physical basis for enzyme specificity has been elusive. We have shown that the temperature dependence of CO₂/O₂ specificity can be attributed to a difference between the free energies of activation for the carboxylation and oxygenation partial reactions. The reaction between the 2,3-enediolate of ribulose 1,5-bisphosphate and O₂ has a higher free energy of activation than the corresponding reaction of this substrate with CO₂. Thus, oxygenation is more responsive to temperature than carboxylation. We have proposed possible transition-state structures for the carboxylation and oxygenation partial reactions based upon the chemical natures of these two reactions within the active site. Electrostatic forces that stabilize the transition state of the carboxylation reaction will also inevitably stabilize the transition state of the oxygenation reaction, indicating that oxygenase activity may be unavoidable. Furthermore, the reduction in CO₂/O₂ specificity that is observed when activator Mg²⁺ is replaced by Mn²⁺ may be due to Mg²⁺ being more effective in neutralizing the negative charge of the carboxylation transition state, whereas Mn²⁺ is a transition-metal ion that can overcome the triplet character of O₂ to promote the oxygenation reaction.Abbreviations CABP 2-carboxyarabinitol 1,5-bisphosphate - enol-RuBP 2,3-enediolate of ribulose 1,5-bisphosphate - K_c K_mfor CO₂ - K_o K_mfor O₂ - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate - V_c V _max for carboxylation - V_o V _max for oxygenation     

Carbon assimilation by different developmental stages of Laminaria saccharina

Various stages of the life cycle of the marine brown alga Laminaria saccharina (L.) Lamour. (Laminariales, Phaeophyta) including male and female gametophytes, female gametes, zygotes and young sporophytes of different age were investigated for their potentials of carbon dioxide (¹⁴CO₂) fixation. Rates of photosynthesis attain the same order of magnitude in all stages. Photosynthetic ¹⁴CO₂-fixation is accompanied by a substantial light independent carbon assimilation. This is confirmed by rate determinations of the equivalent carboxylating enzymes present in the plants, ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) and phosphoenolpyruvate carboxokinase (EC 4.1.1.32) as well as by chromatographic analyses of the appropriate [¹⁴C]-assimilate patterns.Abbreviations RuBP-C ribulose-1,5-bisphosphate carboxylase - PEP-CK phosphoenolpyruvate carboxykinase - PEP phosphoenolpyruvate - PS photosynthesis - DF dark fixation     

Active-site histidines in recombinant cyanobacterial ribulose-1,5-bisphosphate carboxylase/oxygenase examined by site-directed mutagenesis

The functions of His²⁹¹, His²⁹⁵ and His³²⁴ at the active-site of recombinant A. nidulans ribulose-1,5-bisphosphate carboxylase/ oxygenase have been explored by site-directed mutagenesis. Replacement of His²⁹¹ by K or R resulted in unassembled proteins, while its replacement by E, Q or N resulted in assembled but inactive proteins. These results are in accord with a metal ion-binding role of this residue in the activated ternary complex by analogy to x-ray crystallographic analyses of tobacco and spinach enzymes.His³²⁴ (H327 in spinach), which is located within bonding distance of the 5-phosphate of bound bi-substrate analog 2-carboxyarabinitol 1,5-bisphosphate in the crystal structures, has been substituted by A, K, R, Q and N. Again with the exception of the H324K and R variants, these changes resulted in detectable assembled protein. The mutant H324A protein exhibited no detectable carboxylase activity, whereas the H324Q and H324N changes resulted in purifiable holoenzyme with 2.0 and 0.1% of the recombinant wild-type specific carboxylase activity, respectively. These results are consistent with a phosphate binding role for this residue.The replacement of His²⁹⁵, which has been suggested to aid in phosphate binding, with Ala in the A. nidulans enzyme leads to a mutant with 5.8% of the recombinant wild-type carboxylase activity. All other mutations at this position resulted in unassembled proteins. Purified H295A and H324Q enzymes had elevated K_m(RuBP) values and unchanged CO₂/O₂ specificity factors compared to recombinant wild-type.Abbreviations CABP D-2-carboxyarabinitol 1,5 bisphosphate - IPTG isopropyl-b-d-thiogalactopyranoside - L large subunit of rubisco - PAGE polyacrylamide gel electrophoresis - rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-P₂, ribulose 1,5 bisphosphate - S small subunit of rubisco - SDS sodium dodecyl sulfate - X-gal 5-bromo-4-chloro-3-indolyl-b-d-galactoside     

Suitable conditions for characterization,identification, and isolation of the mRNA of the small subunit of ribulose-1,5-bisphosphate carboxylase from Nicotiana sylvestris

The products synthesized in vitro by messenger RNA (mRNA) extracted from Nicotiana sylvestris were analyzed by electrophoresis on polyacrylamide slab gels. Only three of the major polypeptides synthesized are considered here: P₅₅, P₃₂, and P₂₀. P₅₅ and P₃₂ were translated from chloroplast mRNA. P₅₅ corresponds to the large subunit of ribulose-1,5-bisphosphate (RuP₂) carboxylase; P₃₂ is probably a chloroplast membrane protein. P₂₀, the polypeptide synthesized from cytoplasmic poly(A)⁺ RNA, is the precursor of the small subunit of RuP₂ carboxylase. The balance between P₂₀ and P₃₂, in which their relative proportions varied inversely, was regulated by the age of the leaves and the time of illumination; we took advantage of this phenomenon to isolate the mRNA from the small subunit in relatively large amounts. This mRNA has a molecular weight of 350,000.Abbreviations RuP₂ ribulose-1,5-bisphosphate - mRNA messenger RNA - SDS sodium dodecyl sulfate     

Amino acid sequence of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit from Euglena

Summary The amino acid sequence of the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) small subunit (SSU) from Euglena has been established by alignment of the sequence of peptides obtained by cleavage with chymotrypsin, trypsin, Staphylococcus aureus protease or formic acid. The Euglena SSU has 138 amino acids and thus represents longest SSU sequence described so far. Homology is only 41% with cyanobacteria SSU and about 51% with higher plant SSU, whereas it is around 75% between higher plants. The largest homologous portion between all the known SSU sequences is localized in the second half and covers about 20 amino acids. The phylogenetic tree based on known SSU sequences has been established and the rate of amino acid substitution for SSU is estimated to be about 1.35×10^-9 per year and per site. Despite heterogeneity in amino acid sequence, we found that the overall secondary structure is fairly well conserved.Abbreviations DABITC Dimethyl amino azobenzene isothiocyanate - HPLC high pressure liquid chromatography - Kd Kilo daltons - LSU large subunit - PITC phenyl isothiocyanate - RuBisCO ribulose-1,5-bisphosphate carboxylase/oxygenase - SDS sodium dodecyl sulfate - SSU small subunit - TFA trifluoric acetic acid     

Protein composition of the carboxysomes of Thiobacillus neapolitanus

For purifying carboxysomes of Thiobacillus neapolitanus an isolation procedure was developed which resulted in carboxysomes free from whole cells, protoplasts and cell fragments. These purified carboxysomes are composed of 8 proteins and at the most of 13 polypeptides. The two most abundant proteins which make up more than 60% of the carboxysomes, are ribulose-1,5-bisphosphate carboxylase and a glycoprotein with a molecular weight of 54,000. The shell of the carboxysomes consists of four glycoproteins, one also with a molecular weight of 54,000. The other proteins are present in minor quantities. Ribulose-1,5-bisphosphate carboxylase is the only enzyme which could be detected in the carboxysomes and 3-phosphoglycerate was the only product formed during incubation with ribulose-1,5-diphosphate and bicarbonate. The supernatant of a broken and centrifuged carboxysome suspension contained the large subunit of ribulose-1,5-bisphosphate carboxylase. The small subunit of ribulose-1,5-bisphosphate carboxylase was found in the pellet together with the shell proteins which indicates that the small subunit of ribulose-1,5-bisphosphate carboxylase is connected to the shell.Abbreviations RuBisCO ribulose-1,5-bisphosphate carboxylase - PMSF phenylmethylsulfonyl fluoride - PAA gelectrophoresis, polyacrylamide gelelectrophoresis - SDS sodium dodecyl sulphate - CIE crossed immunoelectrophoresis - IEF isoelectric focusing     

Reversible heat-inactivation of the calvin cycle: A possible mechanism of the temperature regulation of photosynthesis

Photosynthetic CO₂ fixation rates in leaves and intact chloroplasts of spinach measured at 18°–20° C are substantially decreased by pretreatment at temperatures exceeding 20° C. Mild heating which causes 80% inhibition of CO₂ fixation does not affect phosphoglyceroacid reduction and causes increases in the ATP/ADP ratio and the light-induced transthylakoid proton gradient. The inactivation of the CO₂ fixation is completely reversible with half-times of recovery in the order of 15–20 min. Comparison of steady-state patterns of ¹⁴C labeled Calvin cycle intermediates of heat-treated and control samples reveals a large increase in the ribulose-1,5-bisphosphate/phosphoglyceroacid ratio and a large decrease in the phosphoglyceroacid/triosephosphate ratio. It is concluded that inactivation of CO₂ fixation occurring at elevated temperatures is caused by inhibition of the ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39). Measurements of light-induced light scattering changes of thylakoids and of the light-induced electrochromic absorption shift show that these signals are affected by mild heating in a way which is strictly correlated with the inactivation of the CO₂ fixation. It is proposed that the function of the ribulose-1,5-bisphosphate carboxylase in vivo requires a form of activation that involves properties of the thylakoid membrane which are affected by the heat treatment. The fact that these changes in thylakoid membrane properties and of ribulose-1,5-bisphosphate carboxylase activity are already affected at elevated temperatures which can still be considered physiological, and the reversible nature of these changes, suggest that they may play a role in temperature regulation of the overall photosynthetic process.Abbreviations 9-AA 9-aminoacridine - DMO 5,5-dimethyloxazolidine-2,4-dione - FBP fructose-1,6-bisphosphate - HEPES N-2-hydroxyethylpiperazine N-2-ethane sulfonic acid - HMP hexose monophosphates - PGA 3-phosphoglycerate - PMP pentose monophosphates - RuBP ribulose-1,5-bisphosphate - SBP seduheptulose-1,7-bisphosphate - TP triose monophosphates     

Involvement of abscisic acid in photosynthetic process in Hordeum vulgare L. during salinity stress

In Hordeum vulgare L. plants, NaCl stress imposed through the root medium for a period of 8 days decreased the rate of CO₂ assimilation, the chlorophyll and protein leaf content, and the activity of ribulose-1,5-bisphosphate carboxylase. The activity of phosphoenolpyruvate carboxylase was twofold over the control. Pretreatment with abscisic acid (ABA) for 3 days before salinization diminished the inhibitory effect of NaCl on the rate of CO₂ fixation. The leaf Na⁺ and Cl^– content decreased in ABA-pretreated plants. Both ABA and NaCl treatments led to an increase in the endogenous level of ABA in the plant leaves. Patterns of total proteins extracted from the leaves of control or ABA- and salt-treated plants were compared. Both ABA and NaCl induced marked quantitative and qualitative changes in the polypeptide profiles concerning mainly the proteins with approximately equal mobility. The results are discussed in terms of a possible role of ABA in increasing the salt tolerance when ABA is applied to the plants for a short period before exposure to salinity stress, thus improving the invulnerability to unfavorable conditions.Abbreviations RuBPC ribulose-1,5-bisphosphate carboxylase - PSII photosystem II - ABA abscisic acid - PEPC phosphoenolpyruvate carboxylase - DTTr dithiothreitol - BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay - SDS sodium dodecyl sulfate - PAGEr polyacrylamide gel electrophoresis     

Inactivation of the photosynthetic carbon reduction cycle in isolated mesophyll protoplasts subjected to freezing stress

Isolated mesophyll protoplasts from Valerianella locusta L. were subjected to freeze-thaw cycles. Subsequently, steady-state pool sizes of ¹⁴C-labeled intermediates of the photosynthetic carbon reduction cycle were determined by high performance liquid chromatography. Protoplasts in which CO₂ fixation was inhibited by preceding freezing stress, showed a strong increase in the proportion of fructose-1,6-bisphosphate, sedoheptulose-1,7-bisphosphate and triose phosphates. These results indicate an inhibition of the activities of stromal fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase. Furthermore, freezing stress caused a slight increase in the proportion of labeled ribulose-1,5-bisphosphate, which may be based on an inhibition or ribulose bisphosphate carboxylase activity. It was shown earlier (Rumich-Bayer and Krause 1986) that freezing-thawing readily affects photosynthetic CO₂ assimilation independently of thylakoid inactivation. The present results are interpreted in terms of an inhibition of the light-activation system of the photosynthetic carbon reduction cycle, caused by freezing stress.Abbreviations FBP Fructose-1,6-bisphosphate - HMP Hexose Monophosphates - PGA 3-phosphoglycerate - PMP Pentose Monophosphates - RBP Ribulose-1,5-bisphosphate - SBP Sedoheptulose-1,7-bisphosphate - TP Triose Phosphates     

Mass-spectrometric evidence for the double-carboxylation pathway of malate synthesis by Crassulacean acid metabolism plants in light

Phyllodia of the Crassulacean acid metabolism (CAM) plant Kalanchoë tubiflora were allowed to fix ¹³CO₂ in light and darkness during phase IV of the diurnal CAM cycle, and during prolongation of the regular light period. After ¹³CO₂ fixation in darkness, only singly labelled [¹³C]malate molecules were found. Fixation of ¹³CO₂ under illumination, however, produced singly labelled malate as well as malate molecules which carried label in two, three or four carbon atoms. When the irradiance during ¹³CO₂ fixation was increased, the proportion of singly labelled malate decreased in favour of plurally labelled malate. The irradiance, however, did not change either the ratio of labelled to unlabelled malate molecules found in the tissue after the ¹³CO₂ application, or the magnitude of malate accumulation during the treatment with label. The ability of the tissue to store malate and the labelling pattern changed throughout the duration of the prolonged light period. The results indicate that malate synthesis by CAM plants in light can proceed via a pathway containing two carboxylation steps, namely ribulose-1,5-bisphosphate-carboxylase/oxygenase (EC 4.1.1.39) and phosphoenolpyruvate carboxylase (EC 4.1.1.31) which operate in series and share common intermediates. It can be concluded that, in light, phosphoenolpyruvate carboxylase can also synthesize malate independently of the proceeding carboxylation step by ribulose-1,5-bisphosphate carboxylase/oxygenase.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase (EC 4.1.1.31) - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) - TMS trimethylsilyl     

Water stress effects on photosynthesis in different mulberry cultivars

The effect of water stress on photosynthesis was determined in five mulberry cultivars (Morus alba L. cv. K-2, MR-2, BC2-59, S-13 and TR-10). Drought was imposed by withholding water and the plants were maintained at different water potentials ranging from 0.5 -MPa to 2.0 -MPa. Photosynthetic rates, activities of ribulose-1,5-bisphosphate carboxylase and sucrose phosphate synthase, photosystem II activity and chlorophyll content were used as key parameters to assess photosynthetic performance. There was a marked variation in the photosynthetic rates and ribulose-1,5-bisphosphate carboxylase activity among the five mulberry cultivars subjected to water stress. Photosystem II (PSII) and sucrose phosphate synthase activities were also severely reduced as measured by drought conditions. Of the five mulberry cultivars, S-13 and BC2-59 showed higher photosynthetic rates, ribulose-1,5-bisphosphate carboxylase activity, high sucrose phosphate synthase activity and photochemical efficiency of PSII compared to the other varieties.     

Proteolysis and transition-state-analogue binding of mutant forms of ribulose-1,5-bisphosphate carboxylase/oxygenase from Chlamydomonas reinhardtii

Trypsin digestion reduces the sizes of both the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) from the green alga Chlamydomonas reinhardtii. Incubation of either CO₂/Mg²⁺ -activated or nonactivated enzyme with the transition-state analogue carboxyarabinitol bisphosphate protects a trypsin-sensitive site of the large subunit, but not of the small subunit. Incubation of the nonactivated enzyme with ribulosebisphosphate (RuBP) provided the same degree of protection. Thus, the very tight binding that is a characteristic of the transitionstate analogue is apparently not required for the protection of the trypsin-sensitive site of the large subunit. Mutant enzymes that have reduced CO₂/O₂ specificities failed to bind carboxyarabinitol bisphosphate tightly. However, their large-subunit trypsin-sensitive sites could still be protected. The K _m values for RuBP were not significantly changed for the mutant enzymes, but the V _max values for carboxylation were reduced substantially. These results indicate that the failure of the mutant enzymes to bind the transition-state analogue tightly is primarily the consequence of an impairment in the second irreversible binding step. Thus, in all of the mutant enzymes, defects appear to exist in stabilizing the transition state of the carboxylation step, which is precisely the step proposed to influence the CO₂/O₂ specificity of Rubisco.Abbreviations and Symbols CABP 2-carboxyarabinitol 1,5-bisphosphate - enol-RuBP 2,3-enediolate of ribulose 1,5-bisphosphate - K _c K _m for CO₂ - K _o K _m for O₂ - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate - V _c V _max for carboxylation - V _o V _max for oxygenation Paper No. 9313, Journal Series, Nebraska Agricultural Research DivisionThis work was supported by National Science Foundation grant DMB-8703820. We thank Drs. Archie Portis and Raymond Chollet for their helpful comments, and also thank Dr. Chollet for graciously providing CABP and [¹⁴C]CABP.     

The drelationship between CO2-assimilation rate,Rubisco carbamylation and Rubisco activase content in activase-deficient transgenic tobacco suggests a simple model of activase action

Transgenic tobacco (Nicotiana tabacum L. cv. W38) plants with an antisense gene directed against the mRNA of ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco) activase were used to examine the relationship between CO₂-assimilation rate, Rubisco carbamylation and activase content. Plants used were those members of the r₁ progeny of a primary transformant with two independent T-DNA inserts that could be grown without CO₂ supplementation. These plants had from < 1% to 20% of the activase content of control plants. Severe suppression of activase to amounts below 5% of those present in the controls was required before reductions in CO₂-assimilation rate and Rubisco carbamylation were observed, indicating that one activase tetramer is able to service as many as 200 Rubisco hexadecamers and maintain wild-type carbamylation levels in vivo. The reduction in CO₂-assimilation rate was correlated with the reduction in Rubisco carbamylation. The anti-activase plants had similar ribulose-1,5-bisphosphate pool sizes but reduced 3-phosphoglycerate pool sizes compared to those of control plants. Stomatal conductance was not affected by reduced activase content or CO₂-assimilation rate. A mathematical model of activase action is used to explain the observed hyperbolic dependence of Rubisco carbamylation on activase content.Abbreviations CA1P 2-carboxyarabinitol-1-phosphate - P_ip_a intercellular, ambient partial pressure of CO₂ - PGA 3-phospho-glycerate - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - SSU small subunit of Rubisco     

Photosynthetic characteristics of coccoid marine cyanobacteria

Two strains of marine Synechococcus possessed a much greater potential for photorespiration than other marine algae we have studied. This conclusion was based on the following physiological and biochemical characteristics: a) a light-dependent O₂ inhibition of photosynthetic CO₂ assimilation at atmospheric O₂ concentrations. The degree of inhibition was dependent on the relative concentrations of dissolved O₂ and CO₂, being greatest at 100% O₂ with no extra bicarbonate added to the medium; b) actively photosynthesizing cells had high levels of ribulose-1,5-bisphosphate carboxylase compared with phosphoenolpyruvate carboxylase; ribulose-1,5-bisphosphate oxygenase activities were three times greater than ribulose-1,5-bisphosphate carboxylase activities; c) cells photosynthesizing in 21% O₂, showed significant ¹⁴C-labelling of phosphoglycolate and glycolate and the percentage of total carbon-14 incorporated into these two compounds increased when the O₂ concentration was 100%; d) at 100% O₂, there was a post-illumination enhanced rate of O₂ consumption, which was three times greater than dark respiration, and the rate declined with increasing bicarbonate concentrations. The inhibitory effect of O₂ on photosynthesis did not appear to be solely due to photorespiration, since O₂ inhibition of photosynthetic O₂ evolution was much greater than that of photosynthetic CO₂ assimilation. Also, O₂ inhibition of photosynthetic O₂ evolution declined only slightly with decreasing light intensities, while the inhibition of CO₂ assimilation declined rapidly with decreasing light intensity.     

Purification and properties of ribulose 1,5-bisphosphate carboxylase from sunflower leaves

Extracts from sunflower leaves possess a high ribulose-1,5-bisphosphate (RuBP) carboxylase capacity but this enzyme activity is not stable. A purification procedure, developed with preservation of carboxylase activity by MgSO₄, yielded purified RuBP carboxylase with high specific activity (40 nkat mg^-1 protein). Measurement of kinetic parameters showed high Km values (RuBP, HCO ₃ ^- ) and high Vmax of the reaction catalyzed by this sunflower enzyme; the results are compared with those obtained for soybean carboxylase. Enzyme characteristics are discussed in relation to stabilization and activation procedures and to the high photosynthesis rates of this C₃ species.