d-Ribulose 1,5-diphosphate carboxylase from the blue-green alga Aphanocapsa 6308

d-Ribulose 1,5-diphosphate carboxylase from extracts of the unicellular blue-green alga Aphanocapsa 6308 has been purified by ammonium sulphate precipitation and linear sucrose density gradient centrifugation. The molecular weight was estimated to be 525 000 and the enzyme consisted of two types of sub-unit of molecular weights 51 000 and 15 000. The small sub-units were not detected after purification involving acid precipitation but were observed if the acid precipitation step was omitted. The Michaelis constants for Mg²⁺ and CO₂, when tested under air, were 0.35 mM and 0.071 mM respectively. Oxygen acted as a competitive inhibitor with respect to CO₂, suggesting that the enzyme also acts as an oxygenase. This was confirmed by measuring ribulose diphosphate-dependent O₂ uptake. A 1:1 stoichiometry between ribulose diphosphate utilization and O₂ consumption was observed. 6-Phosphogluconate inhibited carboxylase activity both at high (20 mM) and low (1 mM) bicarbonate concentrations. The data are compared with the properties of ribulose diphosphate carboxylase from other autotrophic prokaryotes and from chloroplasts.Abbreviations RuDP d-Ribulose 1,5-diphosphate - EDTA ethylene diamine tetraacetic acid - GSH reduced glutathione - SDS sodium dodecyl sulphate - 6PGluc 6-phosphogluconate - STB supplemented Tris buffer     

Bicarbonate stabilization of ribulose 1,5-diphosphate carboxylase.

The carboxylase and oxygenase activities of purified soybean ribulose 1,5-di-P carboxylase (EC4.1.1.39) were unstable when reactions were initiated with enzyme. Time courses of carboxylase and oxygenase activities were curvilinear, approximating hyperbolas. Double reciprocal plots of amount of CO2 incorporated and P-glycolate produced vs. time were constructed to determine a constant representing the half-time of initial enzyme activity, K. K increased with increasing bicarbonate concentration but was independent of O2 tensions between 0.21 and 5 atm. When time courses of carboxylase and oxygenase activities were determined simultaneously, K was identical for both activities. Linear time courses were obtained py preincubation of the enzyme for 10 min in the absence of bicarbonate or by adding 46 mM MgCl2 to the reaction mixture. The observed bicarbonate-dependent decline in ribulose 1,5-di-P carboxylase activity with time is the probable cause for the anomalously high Km(CO2) values previously reported for this enzyme. In the experiments reported here, the apparent Km(CO2) at pH 8.5 increased from 6 muM CO2 at zero time to 78 muM CO2 at 10 min. The corresponding bicarbonate Km values ar 1;3 and 17 mM, respectively, The interaction between bicarbonate and enzyme may be important in the light activation of photosynthetic CO2 fixation in vivo.     

Solubilization of nitrate reductase from the blue-green alga Anabaena cylindrica

Nitrate reductase was solubilized and purified from Anabaenacylindrica by Triton X-100 treatment of particulate preparationsfollowed by adsorption on calcium phosphate gel. Reduced methylviologen, FAD or FMN, but not ferredoxin, served as an effectiveelectron donor for the nitrate reduction by solubilized nitratereductase. ¹This work was supported by a grant (4061) from the Ministryof Education (Received June 25, 1970; )     

Nitrogen fixation and heterocysts in the blue-green alga Anabaena cylindrica

The site of nitrogen fixation in the blue-green alga Anabaenacylindrica Lemra (Fogg strain) was investigated. Less than 4%of the total nitrogen fixed during a relatively short period(5-15 min) was recovered in heterocysts. When estimated on thecellular nitrogen basis, vegetative cells can fix molecularnitrogen at the same rate as do heterocysts. There was no positivecorrelation between nitrogen fixation and heterocyst formation.Results do not support the hypothesis that the heterocyst isthe main site for nitrogen fixation in blue-green algae. ¹ This work was supported by grant (No. 38814) from the Ministryof Education. (Received July 23, 1971; )     

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.     

ATP pools and transients in the blue-green alga,Anabaena cylindrica

Anabaena cylindrica grown in steady state continuous culture has an extractable ATP pool, measured on the basis of the luciferin-luciferase assay of 165±35 nmoles ATP mg chla ^-1. This pool is maintained by a dynamic balance between the rate of ATP synthesis and the rate of ATP utilization. Phosphorylating mechanisms which can maintain the pool in the short term are total photophosphorylation, cyclic photophosphorylation and oxidative phosphorylation. The alga can maintain its ATP pool by switching rapidly from one of these phosphorylating mechanisms to another depending on the environmental conditions. At each switch-over there is a transient drop in the ATP pool for a few seconds. On switching to conditions where only substrate level phosphorylation operates, the ATP pool falls immediately, but takes several hours to recover. The apparent rates of ATP synthesis by total photophosphorylation and by cyclic photophosphorylation are both much higher (210±30 and 250±13 moles ATP mg chla ^-1 h^-1 respectively) than the apparent rate of ATP synthesis by oxidative phosphorylation (22±3 moles ATP mg chla ^-1 h^-1). In long term experiments the ATP pool is maintained when total photophosphorylation is operating. It cannot be maintained in the long term by cyclic photophosphorylation alone in the absence of photosystem II activity or endogenous carbon compounds, or by oxidative phosphorylation in the absence of endogenous carbon compounds. Measurements of ATP, ADP and AMP show that the total pool of adenylates is similar in the light and in the dark in the short term. There is only limited production of ATP under dark anaerobic conditions when glycolysis and substrate phosphorylation can operate which suggests that these processes are of limited significance in providing ATP in Anabaena cylindrica.Abbreviations ADP adenosine 5-diphosphate - AMP adenosine 5-monophosphate - ATP adenosine 5-triphosphate - CCCP carbonyl cyanide m-chlorophenyl hydrazone - DCMU 3-(3,4-dichlorophenyl)1,1-dimethyl urea - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - PEP phosphoenolpyruvate     

Characteristics of the nitrogenase system of the blue-green alga Anabaena cylindrica

Summary The requirements for activity of blue-green algal nitrogenase have been studied. The optimal concentration ranges for ATP and Na₂S₂O₄ are 2-3 mM and 4-10 mM respectively. A magnesium requirement has been confirmed but the enzyme is not specific for Mg²⁺, Co²⁺ and Mn²⁺ will also support activity but Ca²⁺, Cu²⁺ and Zn²⁺ will not. The partially purified enzyme is soluble and specific activities of 50–100 nmoles C₂H₄/mg protein/min have been obtained. The biochemical characteristics of the enzyme, as determined in studies using enzyme inhibitors, are similar to those of bacterial and legume nitrogenases in that the enzyme is a metallo-protein containing iron and reduced thiol groups and the redox capacity of the enzyme involves a possible valency change in the iron. The transfer of electrons from H₂ via a bacterial hydrogenase has been shown to be mediated, at least in part, by ferredoxin. The role of ferredoxin and the interrelationships between photosynthesis, reductant pool and hydrogen metabolism are discussed in the light of recent results obtained by ourselves and other workers.     

Effect of nitrate on nitrogen-fixation by the blue-green alga Anabaena cylindrica

The effect of nitrate on nitrogen-fixation in the blue-greenalga Anabaena cylindrica Lemm (Fogg strain) was investigated.At concentrations up to 2x10^–2 M, nitrate neither inhibitedthe activity of nitrogenase nor repressed its formation. Atthe late logarithmic phase, more than 50% of cell nitrogen wasprovided by nitrogen-fixation when the cells were grown in thepresence of nitrate. Ammonia at a concentration of 1x10^–3M completely repressed the formation of nitrogenase, but hadno effect on its activity. Nitrogen-fixing activity in the algavaried to a considerable extent during growth on N₂ and themaximum activity was attained at the middle logarithmic phase.However, atmospheric nitrogen did not directly affect the inductionof nitrogenase. The formation of nitrogenase in A. cylindricaappears to be controlled by the intracellular level of a certainnitrogenous metabolite. ¹ This work was supported by grant No. 38814 from the Ministryof Education. (Received January 26, 1972; )     

The utilization of molecular hydrogen by the blue-green alga Anabaena cylindrica

The blue-green alga Anabaena cylindrica is found to consume molecular hydrogen in a hydrogenase dependent reaction. This hydrogen uptake proceeds in the dark and is strictly dependent on oxygen, thus representing a Knallgas reactions. Its rate is almost as high as that of the endogenous respiration in Anabaena. Studies with inhibitors reveal that hydrogen is utilized via the complete respiratory chain providing additional energy for the alga. CO plus C₂H₂ completely block the Knallgas reaction which explains the previously reported considerable increase in the total H₂ formation representing the difference between the nitrogenase-dependent H₂-evolution and the reutilization of the gas catalysed by the hydrogenase in intact Anabaena.H₂ is able to support the C₂H₂-reduction in the dark in a reaction again strictly dependent on oxygen. Moreover, H₂ is also consumed in experiments carried out under far red light and in the presence of dichlorophenyl-dimenthyl-urea (DCMU) where the energy for nitrogen fixation is no longer provided by respiration but by cyclic photophosphorylation. Under these conditions, H₂ is found to supply electrons for the formation of C₂H₄ from C₂H₂ in a reaction no longer dependent on the presence of oxygen. Moreover, in these experiments, the presence of H₂ stabilizes the C₂H₂-reduction activity against the deleterious effect of oxygen.Thus, this communication provides evidence for a triplicate function of the H₂-uptake catalysed by hydrogenase in intact Anabaena which is (a) to provide energy by the Knallgas reaction, (b) to supply reducing equivalents for nitrogenase, (c) to protect nitrogenase from damage by oxygen.Abbreviations DCMU N-(3,4-dichlorophenyl)N,N-dimethylurea - DNP 2-4-dinitrophenol - FCCP carbonylcyanid-p-trifluormethoxyphenyl-hydrazone(=p-CF₃-CCP) - Chl chlorophyll     

Activation and inhibition of ribulose 1,5-diphosphate carboxylase by 6-phosphogluconate

Ribulose 1,5-diphosphate carboxylase, when activated by preincubation with 1 mm bicarbonate and 10 mm MgCl₂ in the absence of ribulose 1,5-diphosphate, remains activated for 20 minutes or longer after reaction is initiated by addition of ribulose diphosphate. If as little as 50 μm 6-phosphogluconate is added during this preincubation period, 5 minutes before the start of the reaction, a further 188% activation is observed. However, addition of 6-phosphogluconate at the same time or later than addition of ribulose diphosphate, or at any time with 50 mm bicarbonate, gives inhibition of the enzyme activity. Possible relevance of these effects in vivo regulatory effects is discussed.     

Alanine dehydrogenase of the N2-fixing blue-green alga,Anabaena cylindrica

The l-alanine dehydrogenase (ADH) of Anabaena cylindrica has been purified 700-fold. It has a molecular weight of approximately 270000, has 6 sub-units, each of molecular weight approximately 43000, and shows activity both in the aminating and deaminating directions. The enzyme is NADH/NAD⁺ specific and oxaloacetate can partially substitute for pyruvate. The K _m ^app for NAD⁺ is 14 M and 60 M at low and high NAD⁺ concentrations, respectively. The K _m ^app for l-alanine is 0.4 mM, that for pyruvate is 0.11 mM, and that for oxaloacetate is 3.0 mM. The K _m ^app for NH ₄ ⁺ varies from 8–133 mM depending on the pH, being lowest at high pH levels (pH 8.7 or above). Alanine, serine and glycine inhibit ADH activity in the aminating direction. The enzyme is active both in heterocysts and vegetative cells and activity is higher in nitrogen-starved cultures than in N₂-fixing cultures. The data suggest that although alanine is formed by the aminating activity of ADH, entry of newly fixed ammonia into organic combination does not occur primarily via ADH in N₂-fixing cultures of A. cylindrica. Ammonia assimilation via ADH may be important in cultures with an excess of available nitrogen. The deaminating activity of the enzyme may be important under conditions of nitrogen-deficiency.Abbreviations ADH alanine dehydrogenase - DEAE diethylamino ethyl cellulose - EDTA ethylenediamine tetraacetic acid - GDH glutamic dehydrogenase - GS glutamine synthetase - GOT aspartate-glutamate aminotransferase - NAD⁺ nicotinamide adenine dinucleotide - NADH reduced nicotinamide adenine dinucleotide - NADP⁺ nicotinamide adenine dinucleotide phosphate - NADPH reduced nicotinamide adenine dinucleotide phosphate - SDS sodium dodecyl sulphate - Tris tris(hydroxymethyl) aminomethane     

Effect of ammonia on nitrogen fixation by the blue-green alga Anabaena cylindrica

Ammonia at a concentration of 1 ? 10^–3M completely inhibitednitrogenase activity, as measured by acetylene reduction, inthe blue-green alga Anabaena cylindrica. Free ammonia was undetectablein cells grown either on N₂ or ammonia within the limits ofprecision of the method used. Glutamic acid formed a major aminoacid pool in N₂-grown cells, and basic amino acids, i.e. lysine,histidine and arginine were abundant in ammonia-grown cells.A 10-fold increase in the amounts of labile amino compound(s)was observed when N₂-grown cells were exposed to ammonia. When cells were incubated under anaerobic conditions, the acetylene-reducingactivity increased 2-fold or more; ammonia had no effect. Oxygenwas required for ammonia to inhibit acetylene reduction. Modes of inhibition by ammonia on acetylene reduction were comparedwith those by chloramphenicol, puromycin, cycloheximide, DCMUand CCCP. On the basis of these comparisons we concluded thatammonia not only acts as a suppressor of nitrogenase synthesisbut also inhibits acetylene-reducing activity by lowering thesupply of reductant and/or of energy for the nitrogenase system. ¹This work was supported by grant No. 38814 from the Ministryof Education. (Received July 30, 1973; )     

Pyruvate and nitrogenase activity in cell-free extracts of the blue-green alga Anabaena cylindrica

When extracts of $\text{Anabaena cylindrica}$ are prepared in the absence of dithionite, they catalyze pyruvate-dependent acetylene reduction, a reaction not observable in assays containing dithionite. Ferredoxin and coenzyme-A, but not NADP and ferredoxin-NADP reductase, are required for maximal pyruvate-dependent activity. These acetylene-reducing extracts do not exhibit NADP-pyruvate dehydrogenase activity. However, pyruvate:ferredoxin oxidoreductase is present at levels of activity sufficient to support the $\text{in vitro}$ rate of pyruvate-supported acetylene reduction. These $\text{in vitro}$ data support earlier $\text{in vivo}$ evidence that pyruvate:ferredoxin oxidoreductase transfers electrons from pyruvate to nitrogenase in $\text{A. cylindrica}$.