Purification and properties of L-aspartate aminotransferase of Chlamydomonas reinhardtii

An enzyme which catalyzes the transamination of L-aspartate with 2-oxoglutarate has been purified 400-fold to electrophoretic homogeneity from the unicellular green alga Chlamydomonas reinhardtii 6145c. An apparent relative molecular mass of 138,000 was estimated by gel filtration. The enzyme is a dimer consisting of two identical subunits of Mr 65,000 each as deduced from PAGE/SDS studies. A stoichiometry of two molecules pyridoxal 5-phosphate/enzyme molecule was calculated. The enzyme has an isoelectric point of 8.48 and its absorption spectrum exhibits a maximum at 412 nm which is shifted to 330 nm upon addition of L-aspartate. L-Aspartate or pyridoxal 5-phosphate, but not 2-oxoglutarate, protected the enzyme from heat inactivation. The purified enzyme was able to transaminate, although to a low extent, L-phenylalanine and L-tyrosine with 2-oxoglutarate, and L-serine, L-alanine and L-glutamine with oxaloacetate. L-Aspartate aminotransferase exhibited hyperbolic kinetics for 2-oxoglutarate and oxaloacetate, and nonhyperbolic behaviour for L-aspartate and L-glutamate. Apparent Km values were 0.55 mM for 2-oxoglutarate, 0.044 mM for oxaloacetate, 2.53 mM for L-aspartate and 3.88 mM for L-glutamate. Transamination of L-aspartate in C. reinhardtii is a bisubstrate reaction with a bi-bi ping-pong mechanism, and is not inhibited by substrates.     

Purification and functional characterization of glutamate-1-semialdehyde aminotransferase from Chlamydomonas reinhardtii

The formation of delta-aminolevulinic acid, the first committed precursor of chlorophyll biosynthesis, occurs in the chloroplast of plants and algae by the C5-pathway, a three-step, tRNA-dependent transformation of glutamate. Previously, we reported the purification and characterization of the first two enzymes of this pathway, glutamyl-tRNA synthetase and Glu-tRNA reductase from the green alga Chlamydomonas reinhardtii (Chen, M.-W., Jahn, D., Sch?n, A., O'Neill, G. P., and S?ll, D. (1990) J. Biol. Chem. 265, 4054-4057 and Chen, M.-W., Jahn, D., O'Neill, G. P., and S?ll, D. (1990) J. Biol. Chem. 265, 4058-4063). Here we present the purification of the third enzyme of the pathway, the glutamate-1-semialdehyde aminotransferase from C. reinhardtii. The enzyme was purified from the membrane fraction of a whole cell extract employing four different chromatographic separations. The apparent molecular mass of the protein was approximately 43,000 Da as analyzed by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, by nondenaturing rate zonal sedimentation on glycerol gradients, and by gel filtration. By these criteria, the enzyme in its active form is a monomer of 43,000 Da. In the presence of pyridoxal 5'-phosphate, purified glutamate-1-semialdehyde aminotransferase converts synthetic glutamate 1-semialdehyde to delta-aminolevulinic acid. The enzyme is inhibited by gabaculine and aminooxyacetate, both typical inhibitors of aminotransferases. The purified glutamate-1-semialdehyde aminotransferase successfully reconstitutes the whole C5-pathway in vitro from glutamate in the presence of purified glutamyl-tRNA synthetase, glutamyl-tRNA reductase, Mg2+, ATP, NADPH, tRNA, and pyridoxal 5'-phosphate.     

Purification and properties of L-alanine:4,5-dioxovalerate aminotransferase from Chlorella regularis

The enzyme L-alanine:4,5-dioxovalerate aminotransferase (EC 2.6.1.43), which catalyzes the synthesis of 5-aminolevulinic acid, was purified 161-fold from Chlorella regularis. The enzyme also showed L-alanine:glyoxylate aminotransferase activity (EC 2.6.1.44). The activity of glyoxylate aminotransferase was 56-fold greater than that of 4,5-dioxovalerate aminotransferase. The ratio of the two activities remained nearly constant during purification, and when the enzyme was subjected to a variety of treatments. 4,5-Dioxovalerate aminotransferase activity was competitively inhibited by glyoxylate, with a Ki value of 0.5 mM. Double-reciprocal plots of velocity versus 4,5-dioxovalerate with varying L-alanine concentrations indicate a ping-pong reaction mechanism. The apparent Km values for 4,5-dioxovalerate and L-alanine were 0.12 and 3.5 mM, respectively. The enzyme is an acidic protein having an isoelectric point of 4.8. The molecular weight of the enzyme was estimated to be 126,000, with two identical subunits. These results suggest that, in Chlorella, as in bovine liver mitochondria and Euglena, both 4,5-dioxovalerate and glyoxylate aminotransferase activities are associated with the same protein. From the activity ratio of transamination and catalytic properties, it is concluded that this enzyme does not function primarily as a part of the 5-carbon pathway to 5-aminolevulinic acid synthesis.     

Purification of Hydrogenase from Chlamydomonas reinhardtii

A method is described which results in a 2750-fold purification of hydrogenase from Chlamydomonas reinhardtii, yielding a preparation which is approximately 40% pure. With a saturating amount of ferredoxin as the electron mediator, the specific activity of pure enzyme was calculated to be 1800 micromoles H₂ produced per milligram protein per minute. The molecular weight was determined to be 4.5 × 10⁴ by gel filtration and 4.75 × 10⁴ by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has an abundance of acidic side groups, contains iron, and has an activation energy of 55.1 kilojoules per mole for H₂ production; these properties are similar to those of bacterial hydrogenases. The enzyme is less thermally stable than most bacterial hydrogenases, however, losing 50% of its activity in 1 hour at 55°C. The K_m of purified hydrogenase for ferredoxin is 10 micromolar, and the binding of these proteins to each other is enhanced under slightly acidic conditions. Purified hydrogenase also accepts electrons from a variety of artificial electron mediators, including sodium metatungstate, sodium silicotungstate, and several viologen dyes. A lag period is frequently observed before maximal activity is expressed with these artificial electron mediators, although the addition of sodium thiosulfate at least partially overcomes this lag.     

Purification and substrate inactivation of xanthine dehydrogenase from Chlamydomonas reinhardtii.

Xanthine dehydrogenase (XDH) from the unicellular green alga Chlamydomonas reinhardtii has been purified to electrophoretic homogeneity by a procedure which includes several conventional steps (gel filtration, anion exchange chromatography and preparative gel electrophoresis). The purified protein exhibited a specific activity of 5.7 units/mg protein (turnover number = 1.9 .10(3) min-1) and a remarkable instability at room temperature. Spectral properties were identical to those reported for other xanthine-oxidizing enzymes with absorption maxima in the 420-450 nm region and a shoulder at 556 nm characteristic of molybdoflavoproteins containing iron-sulfur centers. Chlamydomonas XDH was irreversibly inactivated upon incubation of enzyme with its physiological electron donors xanthine and hypoxanthine, in the absence of NAD+, its physiological electron acceptor. As deduced from spectral changes in the 400-500 nm region, xanthine addition provoked enzyme reduction which was followed by inactivation. This irreversible inactivation also took place either under anaerobic conditions or whenever oxygen or any of its derivatives were excluded. Adenine, 8-azaxanthine and acetaldehyde which could act as reducing substrates of XDH were also able to inactivate it upon incubation. The same inactivating effect was observed with NADH and NADPH, electron donors for the diaphorase activity associated with xanthine dehydrogenase. In addition, partial activities of XDH were differently affected by xanthine incubation. We conclude that xanthine dehydrogenase inactivation by substrate is due to an irreversible process affecting mainly molybdenum center and that sequential and uninterrupted electron flow from xanthine to NAD+ is essential to maintain the enzyme in its active form.     

Partial Purification of Intact Chloroplasts from Chlamydomonas reinhardtii

Partially purified intact chloroplasts were prepared from batch cultures of both wild type (Wt) and a mutant strain of Chlamydomonas reinhardtii. Protoplasts were generated from log phase cultures of Wt (137c) and the phosphoribulokinase-deficient mutant F60 by incubation of the cells in autolysine. These protoplasts were suspended in an osmoticum, cooled, and then subjected to a 40 pounds per square inch pressure shock using a Yeda pressure bomb. The resulting preparation was fractionated on a Percoll step gradient which separated the intact chloroplasts from both broken chloroplasts and protoplasts.

The chloroplast preparation was not significantly contaminated with the cytoplasmic enzyme activity phosphoenolpyruvate carboxylase (>5%), and contained (100%) stromal enzyme activity ribulose-1,5-bisphosphate carboxylase. The chloroplast preparation is significantly contaminated by mitochondria, as determined by succinate dehydrogenase activity. Chloroplasts prepared from Wt cells retained CO₂-dependent O₂ photoevolution at rates in excess of 60 micromoles per milligram chlorophyll per hour, an activity which is severely inhibited by the addition of 10 millimolar KH₂PO₄. The chloroplasts are osmotically sensitive as determined by ferricyanide-dependent O₂ photoevolution.

     

Purification and Characterization of UDP-Arabinopyranose Mutase from Chlamydomonas reinhardtii

Chlamydomonas reinhardtii cells are surrounded by a mixture of hydroxyprolin-rich glycoproteins consisting of L-arabinose, D-galactose, D-glucose, and D-mannose residues. The L-arabinose residue is thought to be attached by a transfer of UDP-L-arabinofuranose (UDP-Araf), which is produced from UDP-L-arabinopyranose (UDP-Arap) by UDP-arabinopyranose mutase (UAM). UAM was purified from the cytosol to determine the involvement of C. reinhardtii UAM (CrUAM) in glycoprotein synthesis. CrUAM was purified 94-fold to electrophoretic homogeneity by hydrophobic and size-exclusion chromatography. CrUAM catalyzed the reversible conversion between UDP-Arap and UDP-Araf and exhibited autoglycosylation activity when UDP-D-[¹⁴C]glucose was added as substrate. Compared to the properties of native and recombinant CrUAM overexpressed in Escherichia coli, native CrUAM showed a higher affinity for UDP-Arap than recombinant CrUAM did. This increased affinity for UDP-Arap might have been caused by post-translational modifications that occur in eukaryotes but not in prokaryotes.     

Purification and characterization of chloroplastic NADP-isocitrate dehydrogenase from Chlamydomonas reinhardtii

Chloroplastic NADP-isocitrate dehydrogenase isoenzyme (NADP-IDH₂; EC 1.1.1.42) from the eukaryotic microalga Chlamydomonas reinhardtii was purified to electrophoretic homogeneity by a procedure which included affinity chromatography on Red-Sepharose as the key step. The 70-kDa isoenzyme was found to be a dimer formed by 40-kDa subunits. Antibodies raised against a recombinant tobacco cytosolic NADP-IDH cross-reacted strongly with the cytosolic NADP-IDH₁ and weakly with the NADP-IDH₂ isoenzyme from this alga. NADPH and GTP were found to inhibit both isoenzymes, whereas intermediates of the tricarboxylic acid cycle, glycolysis or reductive pentose phosphate cycle had no significant effect. The simultaneous presence of isocitrate and Mn²⁺ protected NADP-IDH₂ against thermal inactivation or inhibition by reagents specific for arginine or lysine.     

Purification and Characterization of a Membrane-Bound Protease from Chlamydomonas reinhardtii

In Chlamydomonas reinhardtii y-1, newly synthesized chlorophyll a/b-binding apoproteins are degraded when chlorophylls are not present for assembly of stable light-harvesting complexes. A protease was purified from the membrane fraction of degreened y-1 cells, which digested chlorophyll a/b-binding proteins in membranes from C. reinhardtii pg-113, a protease-deficient strain. This protease was active with p-nitroanilides of nonpolar amino acids (Leu and Phe), but not of basic amino acids (Lys and Arg). The apparent molecular weight of the enzyme is 38,000 ± 2,000 as determined by electrophoresis in the presence of sodium dodecyl sulfate. Typical inhibitors of the major classes of proteases were ineffective with this enzyme. Protease activity was constant from pH 7.5 to 9; a plot of log V versus pH suggested that deprotonation of an ionizable group with a pK value of 6.0 to 6.5 is required for activity. The protease was inactivated by diethylpyrocarbonate and by photooxidation sensitized by rose bengal. These results suggested that a histidyl residue is required for catalysis. Although very sensitive to photodynamic conditions in vitro, the enzyme was not inactivated in vivo when cells were exposed to light.     

Characterization of an l-aspartate aminotransferase activity in Chlamydomonas reinhardtii

The isolation and characterization of an l -aspartate aminotransferase (AAT) activity (EC 2.6.1.1) in the unicellular green alga Chlamydomonas reinhardtii 6145c are reported for the first time. The enzyme transaminates aspartate with the 2-oxoglutarate-glutamate system, and exhibits maximum aminotransferase activity at pH 7.8 and 37°C. It has an Mr of 138 kDa, contains pyridoxal 5'-phosphate, and has a K_m apparent for oxalacetate of 0.55 m M and exhibits positive co-operativity with l -aspartate with an S_0.5 of 2.53 m M and a Hill coefficient of 1.57. In vivo, activity levels were affected by the carbon and nitrogen sources and by the change in the dark-light conditions. All these responses are interpreted in terms of a possible physiological regulation of AAT activity to keep the intracellular pools of glutamate and aspartate within margins compatible with environmental fluctuations.     

Purification of His6-tagged Photosystem I from Chlamydomonas reinhardtii

We have developed a rapid method for isolation of the Photosystem I (PS1) complex from Chlamydomonas reinhardtii using epitope tagging. Six histidine residues were genetically added to the N-terminus of the PsaA core subunit of PS1. The His₆-tagged PS1 could be purified with a yield of 80–90% from detergent-solubilized thylakoid membranes within 3 h in a single step using a Ni-nitrilotriacetic acid (Ni-NTA) column. Immunoblots and low-temperature fluorescence analysis indicated that the His₆-tagged PS1 preparation was highly pure and extremely low in uncoupled pigments. Moreover, the introduced tag appeared to have no adverse effect upon PS1 structure/function, as judged by photochemical assays and EPR spectroscopy of isolated particles, as well as photosynthetic growth tests of the tagged strain. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Purification and characterization of a gamma-like DNA polymerase from Chlamydomonas reinhardtii

A crude in vitro system which initiates chloroplast DNA synthesis near the D-loop site mapped by electron microscopy [Wu, M., Lou, J. K., Chang, D. Y., Chang, C. H., & Nie, Z. Q. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6761-6765] consists of soluble proteins and proteins extracted from purified thylakoid membrane. In this paper, a DNA polymerase activity was purified to near homogeneity from the soluble protein fraction of this in vitro system by sequential chromatographic separations on heparin-agarose, DEAE-cellulose, and single-stranded DNA-agarose columns and sedimentation in a glycerol gradient. In the glycerol gradient, the enzyme activity sedimented at a position corresponding to a 110-kDa protein. Electrophoretic analysis of the highly purified fraction on SDS-polyacrylamide gel revealed a major polypeptide band with an apparent molecular mass of approximately 116 kDa. In situ DNA polymerase activity assay shows that the DNA polymerization function is associated with the 116-kDa band and an 80-kDa band which could be a subunit of the enzyme. Polymerization activity is inhibited by N-ethylmaleimide, ethidium bromide, and dideoxycytosine triphosphate and is relatively resistant to aphidicolin. Poly(dA).(dT)10 and gapped double-stranded DNA are preferred templates. The purified enzyme contains no exonuclease activity and can initiate DNA replication in a supercoiled plasmid DNA template containing the chloroplast DNA replication origin.     

Isolation,Purification, and Characterization of Mitochondria from Chlamydomonas reinhardtii

Mitochondria were isolated from autotrophically grown Chlamydomonas reinhardtii cell-wall-less mutant CW 92. The cells were broken by vortexing with glass beads, and the mitochondria were collected by differential centrifugation and purified on a Percoll gradient. The isolated mitochondria oxidized malate, pyruvate, succinate, NADH, and [alpha]-ketoglutarate. Respiratory control was obtained with malate (2.0) and pyruvate (2.2) but not with the other substrates. From experiments with KCN and salicylhydroxamic acid, it was estimated that the capacity of the cytochrome pathway was at least 100 nmol O2 mg-1 protein min-1 and the capacity of the alternative oxidase was at least 50 nmol O2 mg-1 protein min-1. A low sensitivity to oligomycin indicates some difference in the properties of the mitochondrial ATPase from Chlamydomonas as compared to higher plants.     

Purification and Characterization of NAD-Isocitrate Dehydrogenase from Chlamydomonas reinhardtii

NAD-isocitrate dehydrogenase (NAD-IDH) from the eukaryotic microalga Chlamydomonas reinhardtii was purified to electrophoretic homogeneity by successive chromatography steps on Phenyl-Sepharose, Blue-Sepharose, diethylaminoethyl-Sephacel, and Sephacryl S-300 (all Pharmacia Biotech). The 320-kD enzyme was found to be an octamer composed of 45-kD subunits. The presence of isocitrate plus Mn²⁺ protected the enzyme against thermal inactivation or inhibition by specific reagents for arginine or lysine. NADH was a competitive inhibitor (K_i, 0.14 mm) and NADPH was a noncompetitive inhibitor (K_i, 0.42 mm) with respect to NAD⁺. Citrate and adenine nucleotides at concentrations less than 1 mm had no effect on the activity, but 10 mm citrate, ATP, or ADP had an inhibitory effect. In addition, NAD-IDH was inhibited by inorganic monovalent anions, but l-amino acids and intermediates of glycolysis and the tricarboxylic acid cycle had no significant effect. These data support the idea that NAD-IDH from photosynthetic organisms may be a key regulatory enzyme within the tricarboxylic acid cycle.IDH catalyzes the oxidative decarboxylation of isocitrate to produce 2-oxoglutarate. According to the specificity for the electron acceptor, two enzymes with IDH activity are known, NAD-IDH (EC 1.1.1.41) and NADP-IDH (EC 1.1.1.42) (Chen and Gadal, 1990a).In photosynthetic organisms NADP-IDH has been detected in the cytosol, chloroplasts, mitochondria, and peroxisomes. Cytosolic NADP-IDH has been purified from higher plants (Chen et al., 1988) and eukaryotic algae (Martínez-Rivas et al., 1996), and its cDNA has been cloned from alfalfa (Shorrosh and Dixon, 1992), soybean (Udvardi et al., 1993), potato (Fieuw et al., 1995), and tobacco (Gálvez et al., 1996). This 80-kD isoenzyme is a dimer, and it is likely to be involved in the synthesis of NADPH for biosynthetic purposes in the cytosol (Chen et al., 1988), in the synthesis of 2-oxoglutarate for ammonium assimilation (Chen and Gadal, 1990b), and in the cycling, redistribution, and export of amino acids (Fieuw et al., 1995). Chloroplastic NADP-IDH has been studied in higher plants (Gálvez et al., 1994) and eukaryotic algae (Martínez-Rivas and Vega, 1994). It is a 154-kD dimer that has been proposed to be involved in the supply of NADPH for biosynthetic reactions in the chloroplast when photosynthetic NADPH production is low (Gálvez et al., 1994). The mitochondrial NADP-IDH of higher plants may have a physiological role in the production of NADPH, which can be converted to NADH by a transhydrogenase or used to reduce glutathione in the mitochondrial matrix (Rasmusson and Møller, 1990). NADP-IDH activity has also been detected in peroxisomes from spinach leaves (Yamazaki and Tolbert, 1970).NAD-IDH is localized exclusively in the mitochondria in association with the TCA cycle. This enzyme has been purified from several nonphotosynthetic eukaryotes such as fungi (Keys and McAlister-Henn, 1990; Alvarez-Villafañe et al., 1996) and animals (Giorgio et al., 1970), in which it appears to be a 300-kD octamer. Its key regulatory role in the TCA cycle is well documented. The NAD-IDH from yeast is activated by AMP and citrate (Hathaway and Atkinson, 1963), whereas the animal enzyme is activated by ADP and citrate (Cohen and Colman, 1972). In addition, the NAD-IDH cDNAs have been cloned from yeast (Cupp and McAlister-Henn, 1991, 1992) and animals (Nichols et al., 1995; Zeng et al., 1995). In these organisms, the enzyme is composed of two (yeast) or more (animals) different subunits encoded by different genes.To our knowledge, no NAD-IDH from photosynthetic organisms has yet been purified to homogeneity, mainly because of the low stability of the enzyme (Oliver and McIntosh, 1995). However, partial purifications have been reported from pea (Cox and Davies, 1967; Cox, 1969; McIntosh and Oliver, 1992), potato (Laties, 1983), spruce (Cornu et al., 1996), and the eukaryotic microalga Chlamydomonas reinhardtii (Martínez-Rivas and Vega, 1994). Matrix and membrane forms of the enzyme have been detected in potato (Tezuka and Laties, 1983) and pea (McIntosh, 1997). Although it is an allosteric enzyme that exhibits sigmoidal kinetics with respect to isocitrate (Cox and Davies, 1967; McIntosh and Oliver, 1992) and is activated in vitro by ABA (Tezuka et al., 1990), the regulatory importance of NAD-IDH in photosynthetic organisms is still under debate.To elucidate the regulatory significance of NAD-IDH in photosynthetic organisms and its apparent contribution to the 2-oxoglutarate supply for ammonium assimilation, we have purified and characterized the NAD-IDH from C. reinhardtii.     

Purification of highly active oxygen-evolving photosystem II from Chlamydomonas reinhardtii

A method is described for the isolation and purification of active oxygen-evolving photosystem II (PS II) membranes from the green alga Chlamydomonas reinhardtii. The isolation procedure is a modification of methods evolved for spinach (Berthold et al. 1981). The purity and integrity of the PS II preparations have been assesssed on the bases of the polypeptide pattern in SDS-PAGE, the rate of oxygen evolution, the EPR multiline signal of the S₂ state, the room temperature chlorophyll a fluorescence yield, the 77 K emission spectra, and the P700 EPR signal at 300 K. These data show that the PS II characteristics are increased by a factor of two in PS II preparations as compared to thylakoid samples, and the PS I concentration is reduced by approximately a factor ten compared to that in thylakoids.Abbreviations BSA bovine serum albumin - Chl chlorophyll - DCBQ 2,6-dichloro-p-benzoquinone - DCMU (diuron) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMQ 2,5-dimethyl-p-benzoquinone - EDTA ethylenediamine tetraacetic acid - EPR electron paramagnetic resonance - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MES 2-[N-Morpholino]ethanesulfonic acid - OEE oxygen evolving enhancer - PS II photosystem II - SDS-PAGE sodium dedocyl sulfate polyacrylamide gel electrophoresis