Abstract Two different glyceraldehyde-3-phosphate (G3P) dehydrogenase (phosphorylating) activities, namely NAD- and NADP-dependent, have been found in ...
Arch Microbiol (1994) 162:14-19
9 Springer-Verlag 1994
Aurelio Serrano 9Wolfgang L6ffelhardt
identification of two different glyceraldehyde-3-phosphate dehydrogenases (phosphorylating) in the photosynthetic protist Cyanophoraparadoxa
Received: 18 November 1993 /Accepted: 11 March 1994 Abstract Two different glyceraldehyde-3-phosphate (G3P) dehydrogenase (phosphorylating) activities, namely NADand NADP-dependent, have been found in cell extracts of the cyanelle-bearing photosynthetic protist Cyanophora paradoxa. Whereas the two G3P dehydrogenase activities were detected with similar specific activity levels (0.1 to 0.2 U/mg of protein) in extracts of the photosynthetic organelles (cyanelles), only the NAD-dependent activity was found in the cytosol. Thus, a differential intracellular localization occurred. The perfect overlapping of the two G3P dehydrogenase activity peaks of the cyanelle in both hydrophobic interaction chromatography and subsequent FPLC (fast protein liquid chromatography) gel filtration indicated that the two activities were due in fact to a single NAD(P)-dependent G3P dehydrogenase (EC 1.2.1.-) with a molecular mass of 148,000. SDS-PAGE of active fractions from FPLC gel filtration showed that the intensity of the major protein band (molecular mass, 38,000) of the enzyme preparation clearly paralleled the activity elution profile, thus suggesting a tetrameric structure for the cyanelle dehydrogenase. On the other hand, FPLC gel filtration analysis of the cytoplasmic fraction revealed a NAD-dependent G3P dehydrogenase with a native molecular mass of 142,000, being equivalent to the classical glycolytic enzyme (EC 1.2.1.12) present in the cytosol of all the organisms so far studied. The significance of these results is discussed taking into account that the cyanobacteria, photosynthetic prokaryotes which share many structural and biochemical features with cyanelles and are considered as their ancestors, have a similar NAD(P)-dependent G3P dehydrogenase.
A. Serrano (N~) Instituto de BioqufmicaVegetal y Fotosfntesis, CSIC y Universidadde Sevilla, Apdo. 1113, E-41080 Sevilla, Spain W. L6ffelhardt Institut f'firBiochemieund Molekulare Zellbiologieder Universit~itWien und Ludwig Boltzmann-Forschungsstellefiir Biochemie, A-1030 Vienna, Austria
Key words Cyanophora paradoxa 9NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase NAD-dependent glyceraldehyde-3-phosphate dehydrogenase 9Cyanelles Abbreviation FPLC Fast protein liquid chromatography
Introduction Cyanophora paradoxa is a photosynthetic biflagellated protist beating photosynthetic organelles termed cyanelles or cyanoplasts (Schenk 1992; Wasmann et al. 1987). Since cyanelles have a fine structure closely resembling that of unicellular cyanobacteria, being even surrounded by a thin peptidoglycan wall layer, they are presumed descendants from endosymbiotic cyanobacteria (Trench 1982). However, cyanelles, which are considered a separate line of plastid evolution, possess only 5-10% of the DNA found in free-living cyanobacteria, their genome being very similar to that of chloroplasts in both size and structure (Wasmann et al. 1987). The glyceraldehyde-3-phosphate (G3P) dehydrogenase (phosphorylating) are outstanding enzymes present in all the organisms so far studied that catalyze key steps in central metabolic pathways like the reductive pentose phosphate cycle and glycolysis (Fotherhill-Gilmore and Michels 1993). The phosphorylating G3P dehydrogenases involved in glycolysis (EC 1.2.1.12) and photosynthetic carbon fixation by eukaryotes (EC 1.2.1.13) exhibit a clear preference for NAD and NADE respectively (Cerff 1982). However, the cyanobacterial G3P dehydrogenase has been reported to be equally active with either coenzyme (EC 1.2.1.-) (Hood and Cart 1967, 1969). The NADP-dependent reverse reaction of the enzyme is thought to be a key step in the reductive pentose phosphate cycle and the NAD-linked forward reaction may function in the two central routes of intermediary carbohydrate metabolism, namely glycolysis and the pentose phosphate pathway (Pelroy et al. 1972; Schrautemeier et al. 1984; McFadden and Shively 1991; Haselkorn and
15 B u i k e m a 1992). However, the suggestion of a single bifunctional cyanobacterial e n z y m e seems to be superseded b y recent findings showing that Anabaena variabilis contains three distinct G 3 P dehydrogenase genes, two of which b e i n g closely related to genes GapC and GapA encoding cytosolic and chloroplast e n z y m e s of higher plants, respectively (Martin et al. 1993). Since it has been recently reported that several G 3 P dehydrogenase activities are present in whole cell extracts of C. paradoxa (Mateos and Serrano 1992), and considering the evident homologies found b e t w e e n cyanelles and cyanobacteria ( W a s m a n n et al. 1987), we focused our work on the identification and intracellular localization of the G3P dehydrogenase (phosphorylating) e n z y m e s of this peculiar photosynthetic eukaryote. Our finding of two dehydrogenases with different c o e n z y m e specificify, one localized in the cyanelles and the other in the cytosol, should be relevant for the clarification of the origin of these plastidlike structures.
Materials and methods Organism and growth The unicellular Glaucocystophycea Cyanophora paradoxa LB555 UTEX was grown photoautotrophically at 25~ under continuous fluorescent white light using a liquid mineral medium continuosly gassed with a mixture of 5% (v/v) CO2 in air as previously described (Mucke et al. 1980). Cell-free extracts Cultures from late exponential phase of C. paradoxa were centrifuged (12,000 rpm) at room temperature using a continuous flow apparatus (Westfalia Separator, Oelde, Germany). The blue-green pellet was then gently resuspended using a Pasteur pipette in 50 mM Tricine-KOH buffer pH 8.5, containing 1 mM EDTA, 2 mM dithiothreitol (DTT), 1 mM phenylmethylsulphonyl fluoride (PMSF) and 10% (v/v) glycerol (rupture buffer), pre-chilled at 4 ~C. Even this gentle treatment selectively disrupted the fragile cell wall without any apparent effect on the cyanelles. Thus, after a subsequent lowspeed centrifugation (2,000 g, 5 min, 4 ~C) a pink-coloured turbid supernatant, containing mostly cytosolic proteins and non-photosynthetic organelles like mitochondria, was separated from the cyanelles, which sedimented as a blue-green pellet. The cytosolic fraction was obtained after high-speed centrifugation (40,000 g, 20 min, 4 ~C) of this turbid supernatant to remove most mitochondrial and other cellular membranes. Cyanelles were twice resuspended and pelleted at low speed as above in order to remove residual cell debris and contaminant cytosolic proteins. Microscopic examination of the final dark blue-green preparation showed that it was formed mainly by apparently intact cyanelles. A yield of about l g (wet weight) of cyanelles per 1 of culture was obtained. The cyanelles were resuspended in rupture buffer at a ratio of 2 ml per g and then disrupted by ultrasonic treatment (30 s per ml, 70W) (Bandelin Electronics, Berlin, Germany) in 30 s pulses at 4 ~C. The dark blue-green soluble cyanellar extract was obtained after centrifugation (40,000 g, 20 rain, 4 ~C) to remove thylakoids and other cyanelle membranes. Enzyme assays and determination of activity G3P dehydrogenase (phosphorylating) assays were performed spectrophotometrically at 25 ~C in the forward direction (oxidation
of G3P) by monitoring NADH, or NADPH, generation at 340 nm in the presence of sodium arsenate. The standard assay medium contained: 50 mM Tricine-KOH buffer pH 8.5, 0.4 mM of NAD(P), 2 units of rabbit muscle aldolase, 1 mM fructose-1,6-bisphosphate, 10 mM sodium arsenate and an adequate quantity of enzyme in a total volume of 1 ml. When both pyridine nucleotide coenzymes were simultaneously present in the assay medium an equimolar amount of each (0.2 mM) was used. Reactions were started by addition of enzyme extracts and the initial rates measured. One unit (U) of enzyme was defined as the amount which catalyzes the formation of 1 gmol of NAD(P)H per min under the conditions used. Protein was determined by the protein-dye binding method (Bradford 1976) using ovalbumin as a standard. Activity levels in cell-free extracts and enzyme preparations were expressed as specific activity (U/mg of protein).
Purification and characterization of G3P dehydrogenases Unless otherwise stated all operations were performed at 0-4 ~C. The cytosolic fraction was subjected to salt fractionation. Solid ammonium sulphate was slowly added with stirring to the extract up to 40% saturation. After 20 min, the protein solution was centrifuged (40,000 g, 20 min), the supernatant was brought to 90% ammonium sulphate saturation, treated as above and centrifuged. The pellet finally obtained, containing virtually all the NAD-dependent G3P dehydrogenase activity, was resuspended in a minimum volume (less than 1 ml) of standard buffer (25 mM Tris-HC1 pH 7.5, 1 mM EDTA, 2 mM DTT, 10% (v/v)glycerol, 1 mM PMSF). Aliquots of 0.5 ml of this preparation were used for analytical fast protein liquid chromatography (FPLC, Pharmacia, Uppsala, Sweden) gel filtration on a Superose 6HR 10/30 column (i cm x 30 cm) previously equilibrated with standard buffer. Isocratic elution was carried out at 25 ~C with equilibrating buffer at a flow rate of 0.5 ml/min by using an automated Pharmacia FPLC system. Absorbance at 280 nm was continuously measured and fractions of 0.5 ml were collected to determine enzyme activity. The fraction of highest activity exhibited a specific activity value of about 7 U/rag of protein. A partial purification of the G3P dehydrogenase of the cyanelle was carried out by using hydrophobic interaction chromatography to remove the huge amount of phycobiliproteins present in crude extracts. Solid ammonium sulphate was added with stirring to the cyanelle extract up to 35% saturation. After 20 min the protein preparation was centrifuged (40,000 g, 20 min) and the supernatant, which contained virtually all the NAD- and NADP-dependent G3P dehydrogenase activities, was applied to a PhenylSepharose CL-4B column (1 cm • 18 cm), previously equilibrated with standard buffer supplemented with 35% ammonium sulphate at a flow rate of 12 ml/h. To remove not adsorbed proteins the column was then washed with 4 bed volumes of the same buffer at a flow rate of 30 ml/h. Under these conditions both G3P dehydrogenase activities remained bound to the column, and were subsequently eluted with a decreasing linear gradient of ammonium sulphate (35-0%; total volume 100 ml) in standard buffer at a flow rate of 6 ml/h. Fractions of 1.9 ml were collected. The active fractions were pooled and concentrated using Amicon YM 30 (Danvers, Mass., USA) membranes. This pink-coloured partially purified preparation, totally devoid of phycobiliproteins, was used for the analytical high performance gel filtration of native proteins on a Superose 6HR column as previously described. The fraction of highest activity exhibited a specific activity value of about 60 U/mg of protein. The samples were either filtered or centrifuged (12,000 g, 5 min) before applying them to the Superose 6HR column, which was previously calibrated with the following 'molecular-mass markers: thyroglobulin, 669,000; ferritin, 440,000; bovine serum albumin (dimer), 134,000; and ribonuclease A, 13,700. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) was carried out in 12% (w/v) acrylamide slab gels (Laemmli 1970). The samples analyzed were the pooled active fractions from hydrophobic chromatography, the cy-
16 tosolic protein fraction precipitated in the 40-90% ammonium sulphate range and, after precipitation with trichloroacetic acid, the fractions collected in the FPLC gel filtrations. The following molecular-mass markers were used: bovine serum albumin, 66,000; ovalbumin, 45,000; pig muscle G3P dehydrogenase, 36,000; bovine carbonic anhydrase, 29,000; bovine trypsinogen, 24,000 and soybean trypsin inhibitor, 20,000. Proteins were stained with 1% (w/v) Coomassie brilliant blue R-250 in acetic acid/methanol/ water (1 : 3 : 6, v/v) for 1 h at room temperature. Column chromatofocusing, used to estimate the isoelectric points (pI) of native G3P dehydrogenases, was carried out in the pH range of 7.5 (or, where indicated, 9.5) to 4.0 with the pH gradient created by washing a Polybuffer Exchanger PBE-94 column (1 c m x 18 cm) at a flow rate of 12 ml/h with 12 bed volumes of eightfold-diluted Polybuffer 74-HC1 (pH 4.0) containing 1 mM EDTA, 2 mM DTT and 10% (v/v) glycerol, according to the instructions of the manufacturer (Chromatofocusing (1980); Pharmacia Fine Chemicals, Uppsala, Sweden). The starting buffer was in all cases 25 mM Tris-HC1 supplemented with 1 mM EDTA, 2 mM DTT and 10% (v/v) glycerol. Fractions of 1.9 ml were collected and checked for G3P dehydrogenase activities, pH and absorbance at 280 nm. ChemicNs NAD, NADP, fructose-l,6-bisphosphate, rabbit muscle aldolase, EDTA, DTT, PMSF, sodium arsenate, Tris, Tricine, and protein standards for SDS-PAGE were purchased from Sigma Chem. Co. (St. Louis, Mo., USA). Phenyl-Sepharose CL-4B, Polybuffer Exchanger PBE-94, Polybuffer 74, and protein standards for gel illtration were from Pharmacia (Uppsala, Sweden). Ingredients for PAGE were from Bio-Rad (Hercules, Calif,, USA). All other chemicals were of analytical grade.
Results and discussion In accordance with a previous report (Mateos and Serrano 1992) the soluble protein fractions of Cyanophora paradoxa cells exhibit the two NAD- and NADP-dependent G3P dehydrogenase (phosphorylating) activities. However, whereas only the NAD-dependent dehydrogenase was found in the cytosol (sp. act,, 0.6-0.8 U/mg of protein) the cyanelle extract was active with either coenzyme, similar specific activity values (0.1-0.2 U/mg of protein) being found in each case. Noteworthy, no significant increase in activity was detected with cyanelle extracts when both coenzymes were simultaneously present during the enzymatic determinations, i.e. the two activities were not additive, strongly suggesting that both should be exerted by the same enzyme. The differential distribution of these enzymatic activities after disruption of C. paradoxa cells and the possible existence of a cyanelle NAD(P)-dependent enzyme similar to that found in cyanobacteria lead us to further investigate the phosphorylating G3P dehydrogenases of this organism. Figure 1A shows that the two G3P dehydrogenase activities of the cyanelles, namely NAD2 and NADP-dependent, were eluted simultaneously from a Phenyl-Sepharose column as a single symmetrical peak by using an inverse linear gradient of ammonium sulphate. The perfect overlapping of these activities indicates that they reside in the same protein. Moreover, this hydrophobic chromatography allows to obtain a complete separation from phyco-
biliproteins, which constitute the bulk of proteins in the cyanelle extract and elute at the end of the gradient. The pooled active peak fractions had a specific activity of about 3 U/mg of protein and in SDS-PAGE exhibited a major protein band of a molecular mass of 38,000. Since all phosphorylating G3P dehydrogenases purified so far are tetrameric enzymes with subunits of molecular masses in the range from 34,000 to 38,000 (Fotherhill-Gilmore and Michels 1993), we tentatively assumed this band to be the subunit of the cyanellar enzyme. To clarify this point we perfol'lned FLPC gel filtration of the active fractions pool from the previous hydropbobic chromatography. The elution profile of FPLC gel filtration (Fig. 1B) clearly shows a perfect superposition of the two activity peaks, which also coincided with the first protein peak eluted from the column. Considering the high resolution separation of proteins achieved with this chromatographic technique, these data suggest that a single enzyme should harbor both dehydrogenase activities. A molecular mass of about 148,000 was estimated for the native cyanelle NAD(P)-dependent dehydrogenase (Fig. 1B), a value that fits well with a tetrameric structure and the assumed subunit size. In accordance with this assumption, SDS-PAGE of fractions from the FPLC gel filtration shows that the intensity of the major protein band of a molecular mass of 38,000 also displays a clear parallelism with the activity peaks (see Fig. 1B). The molecular masses of the native enzyme and the protein subunit are very similar to those estimated for the NAD(P)-dependent G3P dehydrogenase of unicellular cyanobacteria (Udvardy et al. 1982; F. Valverde and A. Serrano, unpublished results). Figure 1C shows the FPLC gel filtration of the host cytoplasmic protein fraction precipitated in the ammonium sulphate saturation range from 40 to 90%. The only symmetrical NAD-dependent G3P dehydrogenase activity peak corresponds to a native enzyme of a molecular mass of about 142,000. This enzyme is therefore very similar to the NAD-dependent G3P dehydrogenase found in the cytoplasm of all organisms which carry out glycolysis (Fotherhill-Gilmore and Michels 1993). SDS-PAGE of the active fractions also showed clearly a protein band of a molecular mass of about 36,000, but in this case the preparation exhibited other major contaminating proteins (not shown). The partially purified cytoplasmic and cyanelle enzyme preparations used for analytical FPLC gel filtration were also subjected to column chromatofocusing to obtain information on the isoelectric points (pI) of the native G3P dehydrogenases of C. paradoxa. This technique, that has been recently employed with success in the separation and characterization of the plastidic and cytoplasmic glutathione reductase isoforms of the unicellular green alga Chlamydomonas reinhardtii (Serrano and Llobell 1993), has been useful only in the case of the cytoplasmic enzyme, which exibits a pI of about 7.5 (Fig. 1D), since the cyanelle G3P dehydrogenase is completely inactivated during this process (data not shown). In this aspect the cyanelle enzyme is very similar to the cyanobacterial NAD(P)-dependent G3P dehydrogenase which, in con-
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