Alfalfa Root Nodule Carbon Dioxide Fixation : III. Immunological Studies of Nodule Phosphoenolpyruvate Carboxylase

Antiserum was prepared in rabbits against purified alfalfa (Medicago sativa L.) nodule phosphoenolpyruvate carboxylase (PEPC). Immunotitration assays revealed that the antiserum recognized the enzyme from alfalfa nodules, uninoculated alfalfa roots, and from soybean nodules. Tandem-crossed immunoelectrophoresis showed that the PEPC protein from alfalfa roots and nodules was immunologically indistinguishable. The 101 kilodalton polypeptide subunit of alfalfa nodule PEPC was identified on Western blots. The PEPC polypeptide was detected in low quantities in young alfalfa roots and nodules but was present at increased levels in mature nodules. Senescent nodules appeared to contain a reduced amount of the PEPC polypeptide. PEPC was also detected by western blot in some plant- and bacterially-conditioned ineffective alfalfa nodules but was not detected in bacteroids isolated from effective nodules. Alfalfa nodule PEPC is constitutively expressed in low levels in roots. In nodules, expression of PEPC polypeptide increases several-fold, resulting in increased PEPC activity. Antiserum prepared against the C₄ PEPC from maize leaves recognized the PEPC enzyme in all legume nodules and roots tested, while the antiserum prepared against alfalfa nodule PEPC also recognized the leaf PEPC of several C₄ plant species. Neither antiserum reacted strongly with any C₃ leaf proteins. The molecular weight of the PEPC polypeptide from C₄ leaves and legume nodules appears to be similar.     

Alfalfa Root Nodule Carbon Dioxide Fixation : II. Partial Purification and Characterization of Root Nodule Phosphoenolpyruvate Carboxylase

A nonphotosynthetic phosphoenolpyruvate carboxylase (EC 4.1.1.31) was partially purified from the cytosol of root nodules of alfalfa. The enzyme was purified 86-fold by ammonium sulfate fractionation, DEAE-cellulose, hydroxylapatite chromatography, and reactive agarose with a final yield of 32%. The enzyme exhibited a pH optimum of 7.5 with apparent K_m values for phosphoenolpyruvate and magnesium of 210 and 100 micromolar, respectively. Two isozymes were resolved by nondenaturing polyacrylamide disc gel electrophoresis. Subsequent electrophoresis of these isozymes in a second dimension by sodium dodecyl sulfate slab gel electrophoresis yielded identical protein patterns for the isozymes with one major protein band at molecular weight 97,000. Malate and AMP were slightly inhibitory (about 20%) to the partially purified enzyme. Phosphoenolpyruvate carboxylase comprised approximately 1 to 2% of the total soluble protein in actively N₂-fixing alfalfa nodules.     

Carbon Dioxide Fixation by Barley Roots

The non-volatile, 80 per cent.ethanol-soluble products of fixationhave been investigated in excised roots, using C^14O₂ and radiochromatography. The main radioactive compounds separated were malic, citric(or iso-citric), aspartic, and glutamic acids, asparagine andglutamine. Less activity was present in serine, tyrosine, -ketoglutaricacid, and alanine, and in a number of unidentified compounds. The uptake of C^14O₂ was inhibited by virtually anaerobic conditions. From the above observations it is considered likely that C¹⁴is transformed through the reactions of the tricarboxylic acidcycle. C¹⁴ in the soluble fraction was markedly increased by maintainingthe root material in water rather than in a nutrient solutionprior to exposure to C^14O₂ This increase was chiefly in malicacid.     

Nitrogen Fixation and Carbon Dioxide Assimilation in Rhizobium japonicum

In free-living Rhizobium japonicum cultures, the stimulatory effect of CO₂ on nitrogenase (acetylene reduction) activity was mediated through ribulose bisphosphate carboxylase activity. Two mutant strains (CJ5 and CJ6) of R. japonicum defective in CO₂ fixation were isolated by mitomycin C treatment. No ribulose bisphosphate carboxylase activity could be detected in strain CJ6, but a low level of enzyme activity was present in strain CJ5. Mutant strain CJ5 also exhibited pleiotropic effects on carbon metabolism. The mutant strains possessed reduced levels of hydrogen uptake, formate dehydrogenase, and phosphoribulokinase activities, which indicated a regulatory relationship between these enzymes. The CO₂-dependent stimulation of nitrogenase activity was not observed in the mutant strains. Both mutant strains nodulated soybean plants and fixed nitrogen at rates comparable to that of the wild-type strain.     

Carbon Dioxide Fixation by Lupin Root Nodules: I. Characterization, Association with Phosphoenolpyruvate Carboxylase, and Correlation with Nitrogen Fixation during Nodule Development

In vivo CO₂ fixation and in vitro phosphoenolpyruvate (PEP) carboxylase levels have been measured in lupin (Lupinus angustifolius L.) root nodules of various ages. Both activities were greater in nodule tissue than in either primary or secondary root tissue, and increased about 3-fold with the onset of N₂ fixation. PEP carboxylase activity was predominantly located in the bacteroid-containing zone of mature nodules, but purified bacteroids contained no activity. Partially purified PEP carboxylases from nodules, roots, and leaves were identical in a number of kinetic parameters. Both in vivo CO₂ fixation activity and in vitro PEP carboxylase activity were significantly correlated with nodule acetylene reduction activity during nodule development. The maximum rate of in vivo CO₂ fixation in mature nodules was 7.9 nmol hour⁻¹ mg fresh weight⁻¹, similar to rates of N₂ fixation and reported values for amino acid translocation.     

Carbon Dioxide Fixation in Neuronal and Astroglial Cells in Culture

The concentration of potassium in the extracellular fluid has been found to stimulate the rate of CO2 fixation by astroglial cells grown in primary culture. Raising the concentration of extracellular potassium increased both the initial rate of formation of the 14C-labeled products of 14CO2 fixation and the final steady-state level of these products within the cells. In contrast, neither veratridine nor L-glutamate affected the rate of CO2 fixation in astroglial cells. The very low rate of CO2 fixation found in primarily neuronal cultures was unaffected by increased extracellular potassium as was CO2 fixation in fibroblasts. When cultured alone, astroglial cells release a large fraction of the 14C-labeled products of CO2 fixation into the surrounding medium. Mixed cultures of astroglia and neurons also fix CO2 but, in contrast to astroglia cultured alone, release only a small fraction of the 14C-labeled products into the culture medium.     

Carbon Dioxide Fixation and Ribulose-1, 5-bisphosphate Carboxylase Activity in Intact Leaves of Sugar Beet Plants Exposed to Salinity and Water Stress

The influence of salinity in the growing media on ribulose-1,5-bisphosphate (RuBP) carboxylase and on CO₂ fixation by intactsugar beet (Beta vulgaris) leaves was investigated. RuBP carboxylase activity was mostly stimulated in young leavesafter exposure of plants for 1 week to 180 mM NaCl in the nutrientsolution. This stimulation was more effective at the higherNaHCO₂ concentrations in the reaction medium. Salinity also enhanced CO₂ fixation in intact leaves mostlyat rate-limiting light intensities. A 60 per cent stimulationin CO₂ fixation rate was obtained by salinity under 450 µEm^–2 s^–1. At quantum flux densities of 150 µEm^–2 s^–1 (400–700 nm) this stimulation was280 per cent. Under high light intensities no stimulation bysalinity was found. In contrast, water stress achieved by directleaf desiccation or by polyethylene glycol inhibited enzymeactivity up to fourfold at –1.2 MPa. Beta vulgaris, sugar beet, ribulose-1, 5-bisphosphate carboxylase, salt stress, water stress, carbon dixoide fixation, salinity     

Regulation of Autotrophic and Heterotrophic Carbon Dioxide Fixation in Hydrogenomonas facilis

After growth on various carbon sources, sonic extracts of Hydrogenomonas facilis contained ribulosediphosphate (RuDP) carboxylase and phosphoribulokinase (Ru5-P kinase). After very short sonic treatment, a reductive adenosine triphosphate (ATP)-dependent incorporation of (14)CO(2) was also detectable. Reduced nicotinamide adenine dinucleotide (NADH(2)) served as reductant 30-fold more effectively than reduced nicotinamide adenine dinucleotide phosphate (NADPH(2)). Adenosine 5'-phosphate (AMP) and adenosine 5'-pyrophosphate (ADP) inhibited Ru5-P kinase and NADH(2)-, ATP-dependent CO(2) fixation. The levels and duration of CO(2) fixation suggested that it is a cyclic process. The requirement of reduced pyridine nucleotide and ATP and the sensitivity of fixation to AMP and ADP support the conjecture that it occurs via the Calvin cycle. After thorough study of variables affecting catalysis, specific activities (millimicromoles of substrate disappearing per milligram of protein) at 30 C were determined for RuDP carboxylase (C), Ru5-P kinase (K) and ATP-, NADH(2)- dependent CO(2) fixation (CO(2) F) after growth autotrophically on fructose, glucose, ribose, glutamate, lactate, succinate, and acetate. Values for these growth modes were, respectively-for C: 67.3, 51.1, 51.4, 24.6, 2.05, 10.2, 2.25, 1.4; for K: 24.7, 24.0, 23.2, 14.2, 12.8, 12.9, 13.4, 2.8; and for CO(2) F: 4.54, 4.83, 3.10, 2.87, 0.85, 1.51, 0.24, 0.41. The qualitative parallel between values for RuDP carboxylase and CO(2) fixation suggests that one major control point in fixation is the step catalyzed by RuDP carboxylase.     

Enzymic Fractionation of the Stable Carbon Isotopes of Carbon Dioxide by Ribulose-1,5-bisphosphate Carboxylase

The enzymic fractionation of the stable carbon isotopes of CO₂ (Δco₂) was determined using a purified preparation of ribulose-1,5-bisphosphate (RuBP) carboxylase isolated from cotton (a C₃ plant) leaves. The bicarbonate concentration in the reaction mixture saturated the enzyme and furnished an infinite pool of ¹²CO₂ and ¹³CO₂ for enzyme fractionation. The RuBP was 96 to 98% pure. The phosphoglycerate synthesized in the reaction mixtures was purified free of RuBP, phosphoglycolate, and other phosphate esters by column chromatography on Dowex 1-Cl⁻ resin. The average Δco₂ value of −27.1% was determined from five separate experiments. A discussion of the isotope fractionation associated with photosynthetic CO₂ fixation in plants shows that the enzymic fractionation of stable carbon isotopes of CO₂ by RuBP carboxylase is of major importance in determining the δ¹³C values of C₃ plants.     

Photosynthesis in Rhodospirillum rubrum. II. Photoheterotrophic Carbon Dioxide Fixation

The contribution of the reductive pentose phosphate cycle to the photometabolism of carbon dioxide and to carbon metabolism in Rhodospirillum rubrum grown photoheterotrophically with l-malate as the carbon source is nil, unlike autotrophically grown R. rubrum. Glycolic acid appears to be the first stable product of CO(2) fixation in R. rubrum cultured photoheterotrophically on l-malate. The results obtained in (14)CO(2) fixation experiments suggest that the photometabolism of CO(2) through glycolate into malate is a major pathway of CO(2) fixation in such cells. However, l-malate was a much more efficient precursor of phosphate esters, and of glutamic acid, than was carbon dioxide; l-malate is therefore, in this case, a far more important source of cell carbon than is carbon dioxide.The products of the light-dependent incorporation of CO(2) and of acetate were investigated in R. rubrum grown photoheterotrophically on acetate. Carboxylation reactions and the reductive pentose phosphate cycle are apparently of greater significance in the photometabolism of acetate heterotrophs than in malate heterotrophs; the photometabolism of the acetate photoheterotrophs seems to be intermediate between the photoheterotrophy of malate heterotrophs and strict autotrophy.     

面包酵母细胞出芽率与产品质量关系研究

速用压榨酵母(Compressed yeast,CY)和活性干酵母(Active dry yeast,ADY)是用于面点制作的主要两类酵母产品。发酵力则是其共同质量指标。目前,国产压榨酵母的发酵力波动很大,活性干酵母不仅发酵力低,而且保质期短。面包酵母的发酵力定义为由一定数量的酵母样品,面粉和水制成的面团在恒温下产生CO_2的能力。本文采用Burrows法测量面包酵母发酵力,面团由0.15克干酵母,20克面粉和15mol脱离子水混合而成,并以该面团在30℃、180min内产生的CO₂总量作为发酵力的表征,文献^[2]表明,面包酵母的发酵力与终细胞群体中的带芽细胞分率(Fraction of budding cells,FBC)有密切的关系,传统的概念是,为提高酵母产品质量,FBC越低愈好。经研究发现,压榨酵母的耐贮存力及活性干酵发酵力与FBC成反比,但对速用压榨酵母而言,其发酵力FBC的正相关关系甚为明显,这与传统有着根本的差异。     

Photosynthesis in Rhodospirillum rubrum. I. Autotrophic Carbon Dioxide Fixation

The incorporation and distribution of activity from ¹⁴CO₂ was investigated under autotrophic conditions in the facultative photoautotroph, Rhodospirillum rubrum, with cells cultured on hydrogen, carbon dioxide, and ammonium sulfate. In 1 second ¹⁴CO₂ fixation experiments essentially all of the activity was found in 3-phosphoglyceric acid: plotted against time percent incorporation into phosphate esters has a strikingly negative slope. These results suggest that under autotrophic conditions the reductive pentose phosphate cycle or the key reactions of the cycle play a major role in carbon metabolism in this photosynthetic bacterium. Incorporation into amino acids and into intermediates of the tricarboxylic acid cycle was quite low.