Enzyme levels in relation to obligate phototrophy in chlamydobotrys

During the transition from photoheterotrophic growth on acetate to phototrophic growth on carbon dioxide, there is a decrease in isocitrate lyase and increase in ribulose-1,5-diphosphate carboxylase activity in Chlamydobotrys stellata cultures. The increase in ribulose-1,5-diphosphate carboxylase activity is the result of protein synthesis, there being a close correlation between increase in enzyme activity and protein precipitated by antibody to ribulose-1,5-diphosphate carboxylase. The purified ribulose-1,5-diphosphate carboxylase was similar to the constitutive enzyme from other green algae having a molecular weight of 530,000 and composed of two types of subunit of molecular weight 53,000 and 14,000.     

Specific inhibition of oxygenase activity of ribulose-1,5-diphosphate carboxylase by hydroxylamine

Ribulose-1,5-diphosphate oxygenase activity of ribulose-1,5-diphosphate carboxylase was completely inhibited by preincubation of the enzyme with 5mM hydroxylamine in presence of the substrate ribulose-1,5-diphosphate. Inhibition by hydroxylamine was uncompetitive with respect to ribulose-1,5-diphosphate and noncompetitive with respect to magnesium. Carboxylase activity was not affected by hydroxylamine. These results suggest that the two activities of the enzyme can be regulated differentially and that inhibiting the oxygenase activity does not stimulate the carboxylase activity of the enzyme. The data further suggest that the inhibition by hydroxylamine may be through its interaction with carbonyl groups of the enzyme exposed on the binding of ribulose-1,5-diphosphate to the protein.     

Salt responses of carboxylation enzymes from species differing in salt tolerance

This paper reports effects of salts on in vitro activity of phosphoenolpyruvate carboxylase and ribulose-1,5-diphosphate carboxylase, isolated from species differing in salt tolerance.     

Early effects of illumination on the activity of some photosynthetic enzymes

Leaves of dark-grown corn (Zea mays) were illuminated for periods ranging from 3 minutes to 12 hours. The changes in the activities of ribose-5-phosphate isomerase, ribulose-5-phosphate kinase, and ribulose-1,5-diphosphate carboxylase were followed.

The activity of ribose-5-phosphate isomerase did not change significantly until between 12 and 24 hours of illumination. An increase in ribulose-5-phosphate kinase activity occurred after a lag of about 6 hours. The increase in carboxylase activity began after 3 minutes of illumination and increased until after 3 to 6 hours in the light, after which it began to decline. The increases in these enzymes appear to be the result of protein synthesis.

     

Control of Ribulose-1,5-diphosphate Carboxylase Activity During Expansion of Leaves of Capsicum frutescens L.

The activity of ribulose-1,5-diphosphate carboxylase per unitlaminar area increases rapidly during early stages of leaf expansionin Capsicum frutescens L. cv. California Wonder. This is followedby a decrease to a level that is constant until expansion stops. A previous suggestion that the expansion of younger leaves inthe same phyllotactic sector controlled the decrease in enzymeactivity in older expanding leaves has not been verified byexperiments involving the selective excision of leaves and cotyledons.Decrease in enzyme activity was accompanied by a fall in FractionI protein content but another chloroplastic enzyme, -aminolevulinicacid dehydrase, did not exhibit a decrease in activity. Intra-chloroplasticmechanisms, rather than the influence of other plant organs,are suggested as controlling ribulose-1,5-diphosphate carboxylaseactivity during later stages of leaf expansion.     

CHLOROPLAST STRUCTURE AND FUNCTION IN ac-20, A MUTANT STRAIN OF CHLAMYDOMONAS REINHARDI : I. CO2 Fixation and Ribulose-1,5-Diphosphate Carboxylase Synthesis

A mutant strain of the green alga Chlamydomonas reinhardi, ac-20, is described in which both the rate of CO₂ fixation by whole cells and the rate of carboxylation of ribulose-1,5-diphosphate in cell-free extracts are reduced, particularly when sodium acetate is present in the growth medium. Of the enzymes of the reductive pentose phosphate cycle tested, only ribulose-1,5-diphosphate carboxylase activity is reduced in the mutant strain, and it appears that the low carboxylase activity limits the strain's rate of photosynthetic carbon metabolism. Evidence is presented to show that the fluctuation in the level of the enzyme activity in the presence or absence of acetate results from the fluctuation in the level of some factor(s) limiting the rate of synthesis of the protein.     

Chlorophyll Accumulation, CO2 Fixation Enzymes, and Hill Activity, in Greening Roots of Lens culinaris

The carboxylation of ribulose-1,5-diphosphate was demonstrated in vitro with extracts of ctiolated seedling roots. The presence of ribulose-1,5-diphosphate carboxylase was characterized in the subcellular fraction enriched in amyloplasts. Synthesis of chlorophyll, development of CO² fixation capacities and of Hill activity upon illumination have been studied with roots of Lens culinaris seedlings. The marked increases in CO² fixation with ribulose-1,5-diphosphate as the substrate and in Hill activity that occur after a lag phase seem to be related to cytological changes during the greening of roots.     

Further proof for the catalytic role of the larger subunit in the spinach leaf ribulose-1,5-diphosphate carboxylase

The catalytically active oligomeric form of the larger subunit, A_m, obtained from spinach leaf ribulose-1,5-diphosphate carboxylase by pretreatment with p-mercuribenzoate at pH 7.5 followed by incubation at pH 9.0, was free of the smaller subunit based on C-terminal amino acid analyses. Valine was the predominant C-terminus of the A_m preparations, the release of tyrosine being negligibly small [cf. Sugiyama and Akazawa, $\text{Biochemistry}$ 9 (1970) 4499]. The pH optimum of the ribulose-1,5-diphosphate carboxylase reaction by A_m was about 8.5, in comparison to the native enzyme which showed an alkaline pH optimum only in the absence of Mg²⁺. The substrate saturation curve of the catalytic subunit with respect to bicarbonate followed the Michaelis-Menten equation, as contrasted to the anomalous reaction kinetics of the native ribulose-1,5-diphosphate carboxylase molecule reported previously. These overall results indicate that the allosteric properties of spinach ribulose-1,5-diphosphate carboxylase are possibly conveyed by a unique structural conformation that requires the presence of the smaller subunit in association with the larger catalytic subunit component of the enzyme molecule.     

Evidence for in vivo Light-induced Synthesis of Ribulose-1,5-diP Carboxylase and Phosphoribulokinase in Greening Barley Leaves

When actinomycin D, puromycin, streptomycin, chloramphenicol, and cycloheximide, known inhibitors of protein synthesis, were applied to leaves of intact seedlings or detached leaves of barley prior to their greening, the same general response resulted: the light-induced increase in activity of ribulose 1,5-diphosphate carboxylase was prevented while that of phosphoribulokinase was only partially suppressed; synthesis of chlorophyll was arrested. This is taken as preliminary evidence that de novo synthesis of protein may be responsible for the observed increase in ribulose-1,5-diphosphate carboxylase activity during greening. However, other factors may be involved with the light-induced stimulation of phosphoribulokinase.

Carbohydrate metabolites and substrates of the enzymes failed to induce the formation of ribulose-1,5-diphosphate carboxylase and phosphoribulokinase in the dark. No evidence was found for the presence of inhibitors in etiolated seedlings or activators in illuminated leaves of barley. Carboxylase activity almost equal to that of the illuminated water control was stimulated by MgCl₂ in the dark; MgCl₂ had no effect on the activity of the kinase.

     

The presence of ribulose 1,5-diphosphate carboxylase in the nonphotosynthetic endosperm of germinating castor beans

Ribulose 1,5-diphosphate carboxylase was detected in extracts of germinating castor bean (Ricinus communis var. Hale) endosperms. This is the first report of this enzyme in a nonphotosynthetic (no chlorophyll) plant tissue. Radioactive 3-phosphoglyceric acid has been identified as the principle product resulting from the enzymatic condensation of ¹⁴C-bicarbonate and ribulose-1,5-diP in endosperm extracts. The Km values of bicarbonate and ribulose-1,5-diP for the endosperm carboxylase are 1.14 × 10⁻²m and 7.5 × 10⁻⁵m, respectively. The carboxylase activity peaks at 4 days in endosperms of castor beans germinated in the dark. The specific activity of the carboxylase at this stage of germination is 4.3 μmoles of 3-phosphoglycerate formed/mg protein·hr. The presence of ribulose-1,5-diP carboxylase and other enzymes of the reductive pentose phosphate pathway show the potential of this pathway in castor bean endosperms.     

Loss of Ribulose 1,5-Diphosphate Carboxylase and Increase in Proteolytic Activity during Senescence of Detached Primary Barley Leaves

Symptoms typical of senescence occurred in green detached primary barley (Hordeum vulgare L.) leaves placed in darkness and in light. Chlorophyll, total soluble protein, ribulose 1,5-diphosphate carboxylase protein and activity each progressively decreased in darkness and to a lesser extent in light. In all treatments most of the total soluble protein lost was accounted for by a decrease in ribulose 1,5-diphosphate carboxylase protein, suggesting that the chloroplast was a major site of degradation early in senescence.     

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     

The Association of d-Ribulose- 1,5-Bisphosphate Carboxylase/Oxygenase with Phosphoribulokinase

When Ribulose- 1,5-bisphosphate carboxylase/oxygenase was purified from spinach leaves (Spinacia oleracea) using precipitation with polyethylene glycol and MgCl₂ followed by DEAE cellulose chromatography, 75% of phosphoribulokinase and 7% of phosphoriboisomerase activities copurified with ribulose- 1,5-bisphosphate carboxylase/oxygenase. This enzyme preparation showed ribose-5-phosphate and ribulose-5-phosphate dependent carboxylase and oxygenase activities which were nearly equivalent to its corresponding ribulose- 1,5-bisphosphate dependent activity. The ribose-5-phosphate and ribulose-5-phosphate dependent reaction rates were stable and linear for much longer time periods than the ribulose- 1,5-bisphosphate dependent rates. When sucrose gradients were used to purify ribulose- 1,5-bisphosphate carboxylase/oxygenase from crude stromal extracts, phosphoribulokinase was found to cosediment with ribulose- 1,5-bisphosphate carboxylase. Under these conditions most of the phosphoriboisomerase activity remained with the slower sedimenting proteins. Ammonium sulfate precipitation resulted in separation of the ribulose- 1,5-bisphosphate carboxylase peak from phosphoribulokinase peak. Crude extracts of peas Pisum sativum and spinach contained 0.725 to 0.730 milligram of phosphoribulokinase per milligram of chlorophyll, respectively, based on an enzyme-linked immunosorbent assay.     

Ribulose-1,5-diphosphate carboxylase and oxygenase from green plants are two different enzymes.

Ribulose-1,5-diphosphate oxygenase is shown to be an enzyme different from ribulose-1,5-diphosphate carboxylase. The enzymes can be separated from each other by a preparation method employing gel filtration at a pH of 8.3 which is higher than that previously used. The oxygenase activity is correlated with a blue color and a characteristic Cu EPR signal. This indicates that the oxygenase is a copper-containing enzyme.     

Inhibition of ribulose 1,5-diphosphate carboxylase by 6-phosphogluconate

6-Phosphogluconate is a much more effective inhibitor of the photosynthetic carboxylation enzyme, ribulose-1, 5-diphosphate carboxylase, than other sugar phosphates and sugar acids of the reductive and oxidative pentose phosphate cycles. The inhibition appears to be noncompetitive with ribulose 1,5-diphosphate. Since 6-phosphogluconate is unique to the oxidative cycle and inhibits at concentrations comparable to those found in vivo, it is proposed that its inhibition of the carboxylase may be a regulatory factor. If so, it would operate during darkness as a different control factor from those factors postulated to activate the carboxylase during photosynthesis.     

Protein synthesis in plant leaf tissue. The sites of synthesis of the major proteins.

Protein synthesis in the leaves of green pea seedlings (Pisum sativum) is examined by short term labeling with [35S]methionine and autoradiography of the labeled proteins after fractionation by sodium dodecyl sulfate-acrylamide gel electrophoresis. The two subunits of ribulose-1,5-diphosphate carboxylase and the chloroplast lamellar proteins are identified as the major proteins being synthesized. Three protein chlorophyll complexes are characterized by sodium dodecyl sulfate-acrylamide gel electrophoresis; all three complexes are disrupted by heating to 100 degrees in sodium dodecyl sulfate solution. Studies with inhibitors of protein synthesis indicate that the large subunit of ribulos-1,5-diphosphate carboxylase is synthesized in the chloroplast, in contrast to the majority of the soluble proteins, including the small subunit of ribulose-1,5-diphosphate carboxylase, which is synthesized in the cytoplasm. PII protein, the major lamellar protein associated with photosystem II, is also synthesized on cytoplasmic ribosomes. However, many of the lamellar proteins are synthesized within the chloroplast. Integration into the lamellar system of at least one of the chloroplast-synthesized proteins is shown to be dependent on cytoplasmic protein synthesis.     

Properties of ribulose diphosphate carboxylase immobilized on porous glass

Ribulose-1,5-diphosphate carboxylase from spinach has been bound to arylamine porous glass with a diazo linkage and to alklamine porous glass with glutaraldehyde. Stability at elevated temperatures and responses to changes of pH and ribulose-1,5-diphosphate, Mg²⁺, and dithiothreitol concentrations were not significantly different from the soluble enzyme, though stability at 4°C was somewhat improved.     

Interactions between ribulose-1,5-bisphosphate carboxylase and stromal metabolites. II. Corroboration of the role of this enzyme as a metabolite buffer

The capacity of ribulose-1,5-bisphosphate carboxylase to bind reversibly chloroplast metabolites which are the substrates for both thylakoid and stromal enzymes was assessed using spinach chloroplasts and chloroplast extracts and with pure wheat ribulose-1,5-bisphosphate carboxylase. Measurements of the rate of coupled electron flow to methyl viologen in ‘leaky’ chloroplasts (which retained the chloroplast envelope and stromal enzymes but which were permeable to metabolites) and also with broken chloroplasts and washed thylakoids were used to study the effects of binding ADP and inorganic phopshate to ribulose-1,5-bisphosphate carboxylase. The presence of ribulose-1,5-bisphosphate carboxylase significantly altered the values obtained for apparent K_m for inorganic phosphate and ADP of coupled electron transport. The K_m (P_i) in washed thylakoids was 60–80 μM, in ‘leaky’ chloroplasts it was increased to 180–200 μM, while in ‘leaky’ chloroplasts preincubated with KCN and ribulose 1,5-bisphosphate the value was decreased to 40–50 μM. Similarly, the K_m (ADP) of coupled electron transport in washed thylakoids was 60–70 μM, in ‘leaky’ chloroplasts it was 130–150 μM and with ‘leaky’ chloroplasts incubated in the presence of KCN and ribulose 1,5-bisphosphate a value of 45–50 μM was obtained. The ability of ribulose 1,5-bisphosphate carboxylase to reduce the levels of free glycerate 3-phosphate in the absence of ribulose 1,5-bisphosphate was examined using a chloroplast extract system by varying the concentrations of stromal protein or purified ribulose 1,5-bisphosphate carboxylase. The effect of binding glycerate 3-phosphate to ribulose-1,5-bisphosphate carboxylase on glycerate 3-phosphate reduction was to reduce both the rate an the amount of NADPH oxidation for a given amount of glycerate 3-phosphate added. The addition of ribulose 1,5-bisphosphate reinitiated NADPH oxidation but ATP or NADPH did not. Incubation of purified ribulose-1,5-bisphosphate carboxylase with carboxyarabinitolbisphosphate completely inhibited the catalytic activity of the enzyme and decreased inhibition of glycerate-3-phosphate reduction. Two binding sites with different affinities for glycerate 3-phosphate were observed with pure ribulose-1,5-bisphosphate carboxylase.     

CHLOROPLAST STRUCTURE AND FUNCTION IN ac-20, A MUTANT STRAIN OF CHLAMYDOMONAS REINHARDI : II. Photosynthetic Electron Transport

Photosynthetic electron transport is markedly affected in mixotrophic cells of ac-20 because they lack the capacity to form the wild-type level of cytochrome 559, as well as Q, the quencher of fluorescence of photochemical system II. The other components of the electron-transport chain, as well as reactions dependent upon photochemical system I, are unaffected in the mutant strain. These observations are discussed in terms of the previously reported effects of the ac-20 mutation on CO₂ fixation and ribulose-1,5-diphosphate carboxylase activity.     

Photosynthesis and photorespiration in presenescent, senescent, and rejuvenated soybean cotyledons

Various growth and physiological parameters were measured in germinating, presenescent, and senescing soybean (Glycine max [L.] Merr.) cotyledons and in cotyledons rejuvenated by epicotyl removal 18 days after planting. The maximal measured carbon dioxide exchange rates (CER) in the cotyledons were in the range of those reported for field-grown soybean leaves. Rejuvenated cotyledons accumulated total chlorophyll in excess of the maximum observed in presenescent cotyledons. When photosynthetic rates were expressed per cotyledon, the CER in rejuvenated tissue recovered to the maximal rates observed in presenescent cotyledons. Ribulose-1,5-bisphosphate carboxylase/oxygenase in rejuvenated cotyledons also recovered to the maximal amount seen in presenescent cotyledons so that CER appeared to be a function of ribulose-1,5-bisphosphate carboxylase/oxygenase content during most of the period studied. Observations of the postillumination outburst of CO₂ and ¹⁴C label in glycine indicated that photorespiration was occurring in the cotyledons and that photorespiration relative to photosynthesis was different in rejuvenated compared with presenescent cotyledons.