Studies on a mutant form of Escherichia coli citrate synthase desensitised to allosteric effectors.

Naturally occurring citrate synthases fall into distinct molecular and catalytic types. Gram-negative bacteria produce a 'large' enzyme, allosterically inhibited by NADH and, in the facultative anaerobes such as Escherichia coli, also by 2-oxoglutarate. On the other hand, Gram-positive bacteria and all eukaryotes produce a 'small' citrate synthase which is insensitive to these metabolites. As a complement to structure-function studies we have explored the possibility of genetically altering one type of citrate synthase to the other. By mutagenesis and suitable selection we have succeeded in isolating a mutant of E. coli whose citrate synthase is both 'small' and insensitive to NADH and 2-oxoglutarate. Some characteristics of the enzyme are described. Such mutant enzymes offer a novel approach to the study of citrate synthase, its regulation and its natural diversity.     

Spectroscopic properties and chromophore conformations of the photomorphogenic receptor: phytochrome.

Fluorescence lifetimes of 'large (mol. wt. 120,000) and 'small' (mol. wt. 60,000) phytochromes isolated from oat and rye seedlings grown in the dark have been measured at 199 K and 298 K. Phytochrome model compounds have also been studied by phase modulation fluorometrically at 77 K for comparison with lifetime data for phytochrome. It was found that the fluorescence lifetime of 'large' phytochrome was significantly shorter than that of 'small' phytochrome and its chromophore models. The phytochrome chromophore of Pr form has been analyzed by fluorescence polarization, CD, and molecular orbital methods. The fluorescence excitation polarization of 'small' phytochrome and the chromophore model in buffer/glycerol mixture (3 : 1, v/v) at 77 K is very hight (0.4) at the main absorption band and is negative (--0.1) and close to 0 in the near ultraviolet band, respectively. Analyses of the spectroscopic data suggest that the chromophore conformation of Pr and Pfr forms of phytochrome are essentially identical. The induced ellipticity of 'large' rye phytochrome in the blue and near ultraviolet regions was found to be significantly higher than that of 'small' phytochrome, indicating that the binding interaction between the phytochrome chromophore and apoprotein is much tighter in the former than in the latter. In addition, the excitation energy transfer does occur from Trp residue(s) to the chromophore in 'large' phytochrome but not in 'small' Pr. This illustrates one feature of the role played by the large molecular weight apoprotein in the binding site interactions and primary photoprocesses of Pr. Finally, a plausible model for the primary photoprocesses and the mechanism of phytochrome interactions triggered by the Pr leads to Pfr phototransformation have been proposed on the basis of the above results.     

Hydrogenosomal succinate thiokinase in Tritrichomonas foetus and Trichomonas vaginalis

Succinate thiokinase displays a diversity of nucleotide specificity and molecular size throughout Nature. Eukaryotes and Gram-positive bacteria possess distinct 'small' (dimeric) thiokinase enzymes which are specific for adenine (ADP) or guanine (GDP) nucleotides, whereas Gram-negative bacteria contain a single 'large' (tetrameric) enzyme which utilizes both nucleotides. Succinate thiokinase activities, both ADP- and GDP-dependent, were shown to be hydrogenosomal in Tritrichomonas foetus and Trichomonas vaginalis. Surprisingly, the 'small' enzyme was found in T. foetus whereas T. vaginalis contained a 'large' enzyme.     

Heterogeneity of insulin-like growth factor binding proteins between structure and affinity. 2. Forms released by human and rat liver in culture

Since the liver is considered to be the major source both of circulating insulin-like growth factors (IGFs) and of their specific binding proteins (BPs), human and rat liver explants were cultured in serum-free medium with a view to characterizing the binding proteins released into the medium and to comparing them with serum binding proteins. In the culture media, as in the serum, IGFs are associated with their binding proteins in the form of complexes. In gel filtration experiments the liver IGF-BP complexes eluted as a single, homogeneous peak with a relative molecular mass of about 40,000, which is similar to that of the 'small' complex of serum. Their sedimentation coefficient, estimated from sucrose gradient centrifugation, was 2.9 S. Polyacrylamide gel electrophoresis (SDS-PAGE) of human liver culture media, in which the binding proteins were cross-linked to 125I-labelled IGF I revealed molecular heterogeneity. Three specific bands corresponding to Mr 46,000, 40,000 and 37,000 were observed, which resemble those of the serum small complex, but none of the higher-Mr bands seen in serum. SDS-PAGE followed by transfer onto nitrocellulose and incubation with 125I-labelled IGF I (western blot) led to the identification in human liver culture media of five molecular forms of binding protein with Mr of 41,500, 38,500, 34,000, 30,000 and 24,000, identical to those seen in serum. The relative concentrations of the 41,500 and 38,500-Mr forms varied from one medium to another, but the 34,000 and 30,000-Mr forms were regularly more abundant in the liver culture media than in normal serum. The binding proteins produced by the liver therefore represent the native forms in the circulation (although this does not exclude other sources). The absence of high-Mr IGF-BP complexes in the liver culture media, and yet the presence of the 41,500 and 38,500-Mr forms, which are the only binding units of the serum 'large' complex (150,000 Mr), indicates that these two binding proteins are capable of binding IGFs to form 'monomeric' IGF-BP complexes. Western-blot analysis of rat binding proteins revealed a certain analogy with the human proteins, three forms having their Mr between 43,000 and 39,000 and three between 32,000 and 24,000. Liver binding proteins in human adults and foetuses were found to be identical, whereas in the case of serum the 41,500 and 38,500-Mr forms were more abundant in the adult and the 34,000 and 30,000-Mr forms more abundant in the foetus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Laccases produced by lichens of the order Peltigerales

'Large' and 'small' fractions of laccase were found in the thalli of lichens Solorina crocea and Peltigera aphthosa. In both lichens, 'large', possibly dimeric, laccases were determined as 175 and 165 kDa (based on the gel filtration data), and 'small' ones were 76 and 97 kDa (according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis data), respectively. By their substrate specificity, pH optima, and thermostability, they were typical laccases. The fractions of 'small' laccases of 45 kDa from S. crocea and 55 kDa from P. aphthosa consisted of two enzymes.     

Regulatory and molecular properties of citrate synthases from methylotrophs

Abstract Gram-negative methylotrophs contain a high- M _r'large' citrate synthase. Gram-positive methylotrophs, on the other hand, contain a 'small' citrate synthase. These differences in M _r coincided partly with differences in NADH sensitivity. Citrate synthases from obligate Gram-negative and Gram-positive facultative methylotrophs were insensitive to feedback inhibition by NADH; only the enzymes from Gram-negative facultative methylotrophs were inhibited by NADH.     

Nodulating ability of Rhizobium tropici is conditioned by a plasmid-encoded citrate synthase

Rhizobium species elicit the formation of nitrogen-fixing root nodules through a complex interaction between bacteria and plants. Various bacterial genes involved in the nodulation and nitrogen-fixation processes have been described and most have been localized on the symbiotic plasmids (pSym). We have found a gene encoding citrate synthase on the pSym plasmid of Rhizobium tropici, a species that forms nitrogen-fixing nodules on the roots of beans (PhasBoius vuigaris) and trees (Leucaena spp.). Citrate synthase is a key metabolic enzyme that incorporates carbon into the tricarboxylic acid cycle by catalysing the condensation of acetyl-CoA and oxalo-acetic acid to form citrate. R. tropici pcsA (the plasmid citrate synthase gene) is closely related to the corresponding genes of Proteobacteria. pcsA inactivation by a Tn5-mob insertion causes the bacteria to form fewer nodules (30–50% of the original strain) and to have a decreased citrate synthase activity in minimal medium with sucrose. A clone carrying the pcsA gene complemented ail the phenotypic alterations of the pcsA mutant, and conferred Rhizobium iegumino-sarum bv. phaseoli (which naturally lacks a plasmid citrate synthase gene) a higher nodulation and growth capacity in correlation with a higher citrate synthase activity. We have also found that pcsA gene expression is sensitive to iron availability, suggesting a possible role of pcsA in iron uptake.     

Regulation of citrate synthase in facultative methylotrophic bacteria

Commonly the TCA cycle fulfils an anabolic and a catabolic function in case of aerobic chemoorganoheterotrophic nutrition. In methylotrophic growth the TCA cycle is dispensable as a bioenergetic pathway. This is reflected by properties of citrate synthase in facultative methylotrophic bacteria. Two citrate synthases, a "chemoorganoheterotrophic" one, which is inhibited by NADH (or ATP in Acetobacter MB 58), and a "methylotrophic" one, which is not or less affected by energy indicators, were found in Pseudomonas oleovorans, Pseudomonas MS, Pseudomonas MA, and Acetobacter MB 58. The concentration of these citrate synthases depends on the manner of nutrition. Bacteria with ICL-negative-variant of the serine pathway and with ribulosebisphosphate pathway seem to possess only a "chemoorganoheterotrophic" citrate synthase. Possibly the anabolic function of this citrate synthase can be realized by metabolites.     

Cytochemistry of Ca(++)-dependent adenosine triphosphatase (Ca-ATPase) in rat anterior pituitary cells

In the present study, we demonstrate the localization of Ca(++)-ATPase in the anterior pituitary of the male rat. Ca(++)-ATPase was mainly distributed on the membrane system of the granular cells, which included the plasma membrane, the outer mitochondrial membrane, the enveloping membrane of secretory granules, the smooth endoplasmic reticulum and some components of the Golgi complex. No reaction product was detected on the membrane of the rough endoplasmic reticulum or that surrounding the lysosomes. A positive reaction was clearly observed on the membranes surrounding 'large' secretory granules, while that present on the membranes of the 'small' granules was comparatively weak. The cells which contained the 'large' granules were interpreted as growth hormone-secreting cells and those in which the 'small' granules were located as gonadotrophs. There were either no reaction or one that was barely detectable on the plasma membrane of the folliculo-stellate cells. These data along with our previous findings (Soji, 1982, 1984) suggest that the membranous enzymes are not uniformly distributed over all pituitary cells but rather are specific for a given cell population(s).     

Cloning and nucleotide sequence of the gene coding for citrate synthase from a thermotolerant Bacillus sp.

The structural gene coding for citrate synthase from the gram-positive soil isolate Bacillus sp. strain C4 (ATCC 55182) capable of secreting acetic acid at pH 5.0 to 7.0 in the presence of dolime has been cloned from a genomic library by complementation of an Escherichia coli auxotrophic mutant lacking citrate synthase. The nucleotide sequence of the entire 3.1-kb HindIII fragment has been determined, and one major open reading frame was found coding for citrate synthase (ctsA). Citrate synthase from Bacillus sp. strain C4 was found to be a dimer (Mr, 84,500) with a subunit with an Mr of 42,000. The N-terminal sequence was found to be identical with that predicted from the gene sequence. The kinetics were best fit to a bisubstrate enzyme with an ordered mechanism. Bacillus sp. strain C4 citrate synthase was not activated by potassium chloride and was not inhibited by NADH, ATP, ADP, or AMP at levels up to 1 mM. The predicted amino acid sequence was compared with that of the E. coli, Acinetobacter anitratum, Pseudomonas aeruginosa, Rickettsia prowazekii, porcine heart, and Saccharomyces cerevisiae cytoplasmic and mitochondrial enzymes.     

Cloning and nucleotide sequence of the gene coding for citrate synthase from a thermotolerant Bacillus sp.

The structural gene coding for citrate synthase from the gram-positive soil isolate Bacillus sp. strain C4 (ATCC 55182) capable of secreting acetic acid at pH 5.0 to 7.0 in the presence of dolime has been cloned from a genomic library by complementation of an Escherichia coli auxotrophic mutant lacking citrate synthase. The nucleotide sequence of the entire 3.1-kb HindIII fragment has been determined, and one major open reading frame was found coding for citrate synthase (ctsA). Citrate synthase from Bacillus sp. strain C4 was found to be a dimer (Mr, 84,500) with a subunit with an Mr of 42,000. The N-terminal sequence was found to be identical with that predicted from the gene sequence. The kinetics were best fit to a bisubstrate enzyme with an ordered mechanism. Bacillus sp. strain C4 citrate synthase was not activated by potassium chloride and was not inhibited by NADH, ATP, ADP, or AMP at levels up to 1 mM. The predicted amino acid sequence was compared with that of the E. coli, Acinetobacter anitratum, Pseudomonas aeruginosa, Rickettsia prowazekii, porcine heart, and Saccharomyces cerevisiae cytoplasmic and mitochondrial enzymes.     

An investigation of the mechanism of activation of tryptophan by tryptophanyl-tRNA synthetase from beef pancreas

Exposure of dark-grown Euglena to white or red light, but not blue light, produced a twofold increase in the specific activity of citrate synthase. A 400-fold purification of mitochondrial citrate synthase (subunit Mr = 44000) was achieved from cells of Euglena gracilis by affinity chromatography on ATP-activated agarose. Antisera, raised against the homogeneously pure enzyme, were used to demonstrate that the increase in citrate synthase activity on exposure of dark-grown cells to light resulted from an increase in citrate synthase protein. Anti-(citrate synthase) was used to detect precursor citrate synthase resulting from the translation of total polyadenylated RNA from Euglena in a cell-free rabbit reticulocyte lysate system. Citrate synthase mRNA was found to be present in cells at all stages of regreening. However, extraction and translation of polyadenylated RNA from free polysomes isolated from darkgrown and regreening cells demonstrated that appreciable translation of citrate synthase mRNA was only occurring in regreening cells.     

Subunit equilibria of porcine heart citrate synthase. Effects of enzyme concentration, pH, and substrates

Porcine heart citrate synthase, a dimeric protein of Mr = 100,000 composed of two identical subunits, is shown to undergo a monomer-dimer equilibrium. The extent of dimerization is found to be dependent on the concentration of citrate synthase, pH, ionic strength, and the specific buffer system employed. Oxaloacetate and citrate, substrates for the forward and reverse reaction catalyzed by citrate synthase, affect dimerization at concentrations of the protein which exists as monomer in their absence. The dissociation of citrate synthase dimers has been demonstrated utilizing the techniques of gel permeation chromatography, fluorescence polarization, fluorescence energy transfer, and heat denaturation. Earlier studies of citrate synthase quarternary structure found the protein to be nondissociable except under denaturing conditions or extensive modification; however, most former studies were performed at relatively high protein concentration, ionic strength, and pH, conditions which stabilize the dimer. In light of recent evidence derived from x-ray crystallographic studies showing amino acid residues from one subunit contributing to the citrate and CoA binding sites of the other, the dissociation into monomers would be expected to have profound effects on citrate synthase activity and regulation, as well as overall tricarboxylic acid cycle activity.     

Multiple chromatographic forms of ATP citrate lyase from rat liver.

ATP citrate lyase is shown to exist as multiple forms in extracts of rat liver. DEAE-Sephadex ion-exchange chromatography of liver supernatants reveals two peaks of activity. A minor, basic, component, comprising 14% of the recovered activity, is eluted without retention, whereas the major, acidic, form is eluted by a KCl gradient. Gel filtration of similar extracts shows the presence of a high-Mr form of ATP citrate lyase (Mr around 10(7) in addition to the tetrameric enzyme (Mr 4.1 X 10(5). This associated state, which represents 10% of the total activity, is unstable, breaking down to the tetramer, and appears to be disrupted by Mg2+. The basic form changes in the partially purified state to give the acidic form. Most of the high-Mr enzyme is acidic in nature. No evidence could be found for an association of the enzyme with mitochondrial or microsomal membranes. ATP citrate lyase from rat brain also shows two peaks of activity on DEAE-Sephadex ion-exchange chromatography, but the activity is distributed between the peaks in almost equal proportions. However, only the tetrameric enzyme was observed on gel filtration.     

Expression of a Pseudomonas aeruginosa citrate synthase gene in tobacco is not associated with either enhanced citrate accumulation or efflux

Aluminum (Al) toxicity and poor phosphorus (P) availability are factors that limit plant growth on many agricultural soils. Previous work reported that expression of a Pseudomonas aeruginosa citrate synthase gene in tobacco (Nicotiana tabacum; CSb lines) resulted in improved Al tolerance (J.M. de la Fuente, V. Ramírez-Rodríguez, J.L. Cabrera-Ponce, L. Herrera-Estrella [1997] Science 276: 1566-1568) and an enhanced ability to acquire P from alkaline soils (J. López-Bucio, O. Martínez de la Vega, A. Guevara-García, L. Herrera-Estrella [2000] Nat Biotechnol 18: 450-453). These effects were attributed to the P. aeruginosa citrate synthase increasing the biosynthesis and efflux of citrate from roots. To verify these findings we: (a) characterized citrate efflux from roots of wild-type tobacco; (b) generated tobacco lines expressing the citrate synthase gene from P. aeruginosa; and (c) analyzed selected CSb lines described above. Al stimulated citrate efflux from intact roots of wild-type tobacco and root apices were found to be responsible for most of the efflux. Despite generating transgenic tobacco lines that expressed the citrate synthase protein at up to a 100-fold greater level than the previously described CSb lines, these lines did not show increased accumulation of citrate in roots or increased Al-activated efflux of citrate from roots. Selected CSb lines, similarly, failed to show differences compared with controls in either citrate accumulation or efflux. We conclude that expression of the P. aeruginosa citrate synthase gene in plants is unlikely to be a robust and easily reproducible strategy for enhancing the Al tolerance and P-nutrition of crop and pasture species.     

Immunological mapping of fine molecular surface structures of citrate synthase enzymes from different cell types.

Citrate synthase (EC 4.1.3.7), which is present in all living organisms as a key enzyme in aerobic energy metabolism, is one of the most highly phylogenetically conserved enzymes known in terms of its primary and active site structure. However, in terms of other parameters such as in vitro stability, tolerance to changes in pH, degree of self-polymerization, etc., citrate synthases from different sources are markedly different. These divergences can be observed even between isoforms of the enzyme within the same species. Data documenting these diversities suggest that a high degree of difference in tertiary structures may occur. Therefore, the surface profiles of citrate synthase enzymes from yeast, pig, rat, tomato and Escherichia coli were investigated with immunological methods using monoclonal antibody families generated against either pig citrate synthase (alpha-PCS) or yeast citrate synthase-2 (alpha-YCS-2). A high degree of homology of enzyme epitopes was detected on the mitochondrial citrate synthases originating from yeast, tomato, pig and rat cells. Major differences were found between the hexameric citrate synthase originating from E. coli compared with those dimeric forms prepared from eukaryotic cells. Only modest similarities were detected between the highly homologous peroxisomal and mitochondrial yeast citrate synthases. Furthermore, a point mutation of one of the catalytic residues (H274R on recombinant pig and H313R on yeast enzyme) of mitochondrial citrate synthase (CS-1) resulted in a significant increase in immunological similarity with the peroxisomal isoenzyme (CS-2). These findings are discussed in terms of the possible mechanism of evolution of CS-2 in yeast.     

Controlling seed development and seed size in Vicia faba: a role for seed coat-associated invertases and carbohydrate state

We previously provided evidence that seed coat-associated invertase is involved in controlling the carbohydrate state of developing seeds and, by this way, triggering developmental processes (Weber et al . (1995) Plant Cell , 7, 1835–1846). To verify our postulate, we compared seed development of two genotypes of Vicia faba differing in seed weight. The seed coat of the large-seeded genotype formed a higher number of parenchymatous cell layers and matured later. VfCWINV1 encoding a cell wall-bound invertase is expressed in the unloading zone of the seed coat. mRNA levels peaked later in 'large' coats and mRNA was present in more cell layers over a longer time period. Cell wall-bound invertase activity revealed a similar accumulation pattern, obviously generating the high hexose conditions present in the endospermal cavity bathing the premature cotyledons and thus controlling their carbohydrate state. High hexose conditions were correlated with an extended mitotic activity of the 'large' cotyledons. In 'large' and 'small' cotyledons, sucrose levels rose when hexoses decreased apparently terminating cell divisions and initiating differentiation and storage activities. This developmental switch was delayed in 'large' embryos. To prove the outlined relationship, sucrose was added in vitro to mitotically active cotyledons. This treatment favoured nuclear expansion and starch accumulation over cell division. In contrast, a hexose-based medium maintained cell divisions. We conclude that development of the embryo is coordinately regulated with that of the maternal seed coat which controls, by metabolic signals, the phase of cell division of the embryo and consequently also seed size.     

Purification of the mitochondrial citrate transporter in yeast.

We present a method of partial purification of mitochondrial citrate transporter of the yeast Saccharomyces cerevisiae. Based on functional evidence of interaction between citrate transport and citrate synthase, we have used an affinity column containing pig heart citrate synthase (PHCS) for the purification. The purified preparation shows two protein components whose Mr is approximately 50K and 60K. The specific activity of our purest fractions is 2.6 mumoles/min which compares favorably to that of purified beef liver enzyme and purified rat liver enzyme.     

A stable intermediate in the equilibrium unfolding of Escherichia coli citrate synthase.

Urea-induced unfolding of Escherichia coli citrate synthase occurs in two phases, as monitored by circular dichroism at 222 nm (measuring secondary structure) or by tryptophan fluorescence. In this paper we characterize the intermediate state, which retains about 40% of the ellipticity of the native state, and is stable between 2.5 M and 5.5 M urea, approximately. This intermediate binds significant amounts of the probe for hydrophobic surfaces, anilinonaphthalene sulfonate, but forms aggregates at least as high as an octamer, as shown by transverse urea gradient polyacrylamide electrophoresis. Thermal denaturation of E. coli citrate synthase also produces an intermediate at temperatures near 60 degrees C, which also retains about 40% of the native ellipticity and forms aggregates, as measured by electrospray-ionization/time-of-flight mass spectrometry. We have used a collection of "cavity-forming" mutant proteins, in which bulky buried hydrophobic residues are replaced by alanines, to explore the nature of the intermediate state further. A certain amount of these mutant proteins shows a destabilized intermediate, as measured by the urea concentration range in which the intermediate is observed. These mutants are found in parts of the citrate synthase sequence that, in a native state, form helices G, M, N, Q, R, and S. From this and other evidence, it is argued that the intermediate state is an aggregated state in which these six helices, or parts of them, remain folded, and that formation of this intermediate is also likely to be a key step in the folding of E. coli citrate synthase.