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Studies on the Isoforms of Isocitrate Dehydrogenase from Chlamydomonas reinhardtii
r. Plant Physiol. VoL 143. pp. 129-134 {1994}
Studies on the lsoforms of lsocitrate Dehydrogenase from Chlamydomonas reinhardtii JosE. M. MARTINEZ-RivAs1 and JosE. M. VEGA Departamento de Bioqu1mica Vegetal y Biologfa Molecular, Facultad de Qu1mica, Universidad de Sevilla, Apartado 553, 41080-Sevilla, Spain 1 To whom correspondence should be addressed Received August 8, 1993 · Accepted September 15, 1993
Summary
Two isocitrate dehydrogenase enzymes (IDH) specific for NAD+ or NADP+ can be separated from
Chlamydomonas reinhardtii crude extract by hydrophobic chromatography on phenyl-sepharose, with
NADP-IDH activity 5-fold higher than NAD-IDH. The NAD-IDH (EC 1.1.1.41) showed sigmoidal kinetic for D,L-isocitrate (So.s = 370 llM) and normal Michaelis-Menten kinetic for NAD+ (Km = 150 IJ.M) and Mn2+ (Km = 30 IJ.M). It was inhibited by 2-oxoglutarate, citrate, glyoxylate or adenine nucleotides, and also by sulphydryl, amino or carboxyl group reagents. The NADP-IDH (EC 1.1.1.42) activity fraction contained two isoenzymes, with an IDH/IDH 2 ratio of approximately 3.0. These isoenzymes have been separated and purified and their physicochemical and kinetic parameters were very similar. The main difference between IDHt and IDH2 was the in vitro activation of IDH 2 by thiols (1 mM), which may reach 2.5-fold. IDHt activity remains unaffected by thiols at this concentration.
Key words: Chlamydomonas reinhardtii, isocitrate dehydrogenase, isocitrate metabolism. Abbreviations: EDC .. 1-Ethyl-3-(3-dimethyl aminopropyl)-carbodiimide; GEE = Glycine ethyl ester; GOGAT =Glutamate synthase; GS = Glutamine synthetase; IAA = Iodoacetamide; IDH = Isocitrate dehydrogenase; MMTS = Methyl methane thiosulfonate; TNBS = 2,4,6-Trinitrobenzene sulphonic acid.
Introduction
Two enzymes with IDH activity have been described in photosynthetic organisms, NAD-IDH (EC 1.1.1.41) and NADP-IDH (EC 1.1.1.42), which are highly specific for their corresponding electron acceptor. The NAD-IDH activity has been located inside the mitochondria of higher plants and has a catabolic role in the Krebs cycle (Chen and Gadal, 1990). However its presence in microalgae has not been yet reported. On the other hand, the NADP-IDH activity has been studied in cyanobacteria (Muro-Pastor and Florencio, 1992}, eukaryotic algae (Foo and Badour, 1977; Ramaley and © 1994 by Gustav Fischer Verlag, Stuttgart
Hudock, 1973), and higher plants (Chen and Gadal, 1990; Ni et al., 1987}. Two isoenzymes with NADP-IDH activity have been purified and characterized from higher plants; the IDH 1 was located in the cytosol, while the IDH2 appears to be in the chloroplast stroma. Both isoenzymes seem to be involved in the 2-oxoglutarate supply required for ammonium assimilation inside the chloroplast (Chen et al., 1989, 1990}. No information is available concerning the possible presence of NADP-IDH isoenzymes in microalgae. This paper reports the separation of one NAD-IDH enzyme and two NADP-IDH isoenzymes from C. reinhardtii
130
Josf M. M.u:Iim:z-RivAS and Josf M. VEGA and then were broken by freezing in liquid nitrogen for 1 min and thawing in SOmM K-phosphate buffer (pH 7.5) containing 14mM 2-mercaptoethanol (standard buffer). The resulting homogenate was centrifuged at 16,000 x g for 30 min, and the supernatant was used as crude extract. All the subsequent operations were performed at 0-4 °C. A solution of 2% (w/v) protamine sulfate (pH 7.5) was slowly added to the crude extract up to a final concentration of 0.16% (w/v). After 10min incubation with gentle stirring, the suspension was centrifuged at 27,000 x g for 5 min and the pellet discarded. 2. Phenyl sepharose chromatography. The resulting supernatant was supplemented with ammonium sulfate up to 15% saturation before it was applied to a column packed with phenyl-sepharose and equilibrated with the standard buffer, containing similar ionic strength. Then the column bed was washed with 300 mL of the same buffer, and the NADP-IDH activity was eluted from the column with 300 mL buffer containing ammonium sulfate to a saturation of 5% (Fig. 1). Fractions with high activity were pooled, concentrated to a volume of 10 mL and dialyzed overnight against 1,000 volumes of 10mM K-phosphate buffer (pH 7.5) supplemented with 14mM 2-mercaptoethanol. The NAD-IDH activity was eluted with 300mL 50% (v/v) ethyleneglycol in standard buffer (see also Fig. 1) and fractions containing high activity were pooled, dialyzed overnight against 1,000 volumes of buffer and concentrated by ultrafiltration. This enzymatic preparation was used for further studies. 3. Blue-sepharose chromatography. The resulting NADP-IDH enzyme preparation was then passed through a blue-sepharose column equilibrated with 10 mM K-phosphate buffer (pH 7.5) containing 14mM 2-mercaptoethanol. Figure 2 shows that IDH 1 activity was eluted with 50 mM phosphate, while the IDH 2 elution required a final phosphate concentration of 100mM in the buffer. The two peaks of activity were pooled separately, concentrated by ultrafiltration and dialyzed overnight against standard buffer. 4. Sephadex G-150 chromatography. The obtained IDH 1 and IDH 2 preparations (2 mL each) were separately applied to a Sephadex G-150 column equilibrated with standard buffer and eluted with the same buffer in fractions of 1.5 mL. Two separate pools were obtained, concentrated and kept at 4 °C. Before use, the corresponding enzyme preparation was dialyzed overnight against 1,000 volumes of 50mM K-phosphate buffer (pH 7.5) to remove 2-mercaptoethanol.
crude extract. They have been purified and physicochemically characterized. Materials and Methods
Chemicals Amino acids, nucleotides, substrates, metabolites and specific reagents for functional groups of proteins were purchased from Sigma (St. Louis, U.S.A.). Standard proteins, phenyl sepharose, blue sepharose and Sephadex G-150 were from Pharmacia (Uppsala, Sweden). All other chemicals were reagents of analytical grade and used as supplied by Merck (Darmstadt, Germany).
Organism and culture conditions The unicellular green alga Chlamydomonas reinhardtii, wild type, strain 21 gr, was grown at 25 °C in liquid medium (Mardnez-Rivas et al., 1991) with 10mM NH4Cl as nitrogen source. The cultures were bubbled with air supplemented with 5% (v/v) C02 and continuously illuminated with white light from fluorescent lamps
(5owm-2).
Enzyme assay and protein determination NAD-IDH activity was determined at 30 °C in 1 mL reaction mixture containing: 50 Jlmol K-phosphate buffer (pH 7.5), 0.5 Jlmol MnCb, 1.5 Jlmol NAD+, 4 Jlmol D,L-isocitrate and the appropriate amount of enzyme. The mixture to measure NADP-IDH activity was similar, but containing 0.15Jlmol ofNADP+ instead ofNAD•, and only 2 Jlmol of D,L-isocitrate. In both cases the reaction was started by addition of isocitrate and it was followed spectrophotometrically at 340 nm. One unit of activity was the amount of enzyme that catalyzed the production of 1 Jlmol NAD(P)H min - 1• Protein was determined by the Bradford (1976) method with bovine serum albumin as standard.
Purification ofNAD· and NADP-isocitrate dehydrogenase enzymes 1. Extraction and protamine sulfate treatment. Cells were harvested during the logarithmic phase (A 660 = 1.5-2.0) of growth,
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ous
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FRACTION
NUMBER
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Fig. 1: Phenyl-sepharose hydrophobic chromatography of IDH activity from C reinhardtii. Arrows indicate the addition of ammonium sulfate (AS) or ethyleneglycol (EG) to the standard buffer. Fractions (3 mL) were collected at a flow rate of 30 mL h - 1 and A280 {.&---- -.&), NAD-IDH (e-e) and NADP-IDH (0-0) activities were determined.
Isocitrate dehydrogenase from Chlamydomonas IDmlll K·P1
~
..
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IDDml K·P1
5Dml K·P1
~
. Fig. 2: Blue-Sepharose chromatography of NADP-isocitrate dehydrogenase activity from C. reinhardtii. The enzymatic preparation was applied to a column (1.6x20cm) packed with blue-sepharose and previously washed at a flow rate of 15 mL h- \ with 10mM K-phosphate (pH 7.5) containing 14mM 2-mercaptoethanol. Arrows indicate the points of which the washing buffer was changed (K-Pi • K-phosphate). Fractions of 1.5 mL were collected and IDH activity and A2so were determined.
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Electrophoretic analysis Analytical separations were performed in 10% acrylamide with the discontinuous gel system of Jovin et al. (1964). Proteins were located by staining with 1 % Coomassie brilliant blue in 7% acetic acid for 30 min. NADP-IDH was located in the electrophoresis gds by submerging them in a reaction mixture containing per mL: 100 14mol Tris-HCl buffer (pH 8.0), 10 14mol MnCh, 2 14mol NADP+, 10 14mol D,L-isocitrate, 0.05% (w/v) nitroblue tetrazolium and 0.005% (v/v) phenazine methosulfate. After 30min at room temperature in the dark with gentle stirring, the activity was shown by a blue hand. The gels were then washed with water and stored in 7% acetic acid.
Determination ofstokes radius The stokes radius was determined as described by Siegel and Monty (1966) using a Sephadex G-150 column (1.6x100cm) equilibrated with 50 mM K-phosphate buffer (pH 7.5).
FRACTIDI
lUMBER
Table 1: Purification of the NADP-isocitrate dehydrogenase isoenzymes from C. reinhardtii. The NADP-isocitrate dehydrogenase activity was purified from the crude extract obtained from 42 g of cells grown under standard conditions with ammonium as nitrogen source. Step and fraction
Volume
(mL)
(U)
Activity
Protein (mg)
Specif. Act. (U mg- 1)
Crude extract
182
29
522
0.05
Protamine sulfate supernatant
192
29
407
0.07
Phenyl-sepharose eluate
75
18
64
0.28
Blue-sepharose eluate IDHt IDH2
75 23
5.5 3.5
3.9 2.6
1.41 1.35
Sephadex G-150 eluate IDH 1 21 18 IDH2
4.7 1.4
1.1 0.5
4.27 2.80
Results
Separation and Purification oflsocitrate dehydrogenases from C reinhardtii The partially purified NAD-IDH preparation had a specific activity of 100 mU mg- 1 protein, a purification factor of about 6-fold and a yield of 94 %. The purification procedure for NADP-IDH isoenzymes, summarized in Table 1, yielded IDHt and IDH2 preparations with specific activities of 4.3 and 2.8 U mg- 1 protein, respectively. The purity of these preparations was tested by polyacrylamide gel electrophoresis. The main protein band (about 80% total protein) was due to IDH 1• Two minor protein bands lacked IDH activity. On the other hand, the IDH2 preparation showed a protein band (about 50% of total protein) containing the activity with minor bands representing impurities. Both IDH isoenzymes showed similar electrophoretic mobility, with Rf = 0.46 (data not shown).
At 4 °C the NAn-dependent activity showed a half-life of 60h as compared with 96h shown by NADP-IDH; moreover, 2-mercaptoethanol resulted in efficient protection of the activity during this manipulation, indicating that sulphydryl groups are essential for activity (data not shown). Spectrophotometric studies of purified IDH isoenzymes did not reveal the presence of any prosthetic group with light absorption in the visible region.
Kinetic properties of NAD-IDH from C. reinhardtii The purified NAD-IDH preparation showed a high specificity for NAD+ and D,L-isocitrate as substrates, and required a divalent cation for maximum activity, as in other organisms (Chen and Gadal, 1990). The enzyme showed partial activity with Co2+, Cd2 +, Mgl+ and Zn2+; however, Mn2+ was the best cofactor. The NAD-IDH enzyme shows
132
Josf M. MAR'liNEz-RivAS andJosf M. VEGA
Table 2: Effect of metabolites and chemical reagents on the isocitrate dehydrogenase activity from C. reinhardtii. Assays for rn·H activity were carried out using purified preparations of each enzyme and including in the reaction mixture the corresponding chemicals as indicated; 100% of activity for NAD-IDH, NADP-IDH1 and NADP-IDH2was 140, 52 and 57 mU mL -I, respectively. Metabolite None 2-0xogluarate NAD• EDTA ATP ADP
AMP
Citrate Succinate Fumarate Glycolate Glyoxylate pHMB 1NBS EDC +GEE
Activity {%)
Concentration
(mM) 10 10 1 5 5 5 10 10 10 10 10 0.01 1 1; 500
NAD-IDH
NADP-IDH 1
NADP-IDH2
100 67 95 11 49 62 67 62
100 76 46 0
100 73
98 2 55 52 52
22
55 90 60 68 63 55 40 10 10 80
70
0 45 68 80 63 88 93 83 45 5 5 65
pH and temperature optima of 8.0 and 40 °C, with an activation energy of 78.1 KJ mol- 1• Particularly interesting is the sigmoidal kinetic observed with respect to D,L-isocitrate, showing a So.s of 370 JJ.M, as deduced from Hill plots. The Km for NAD+ was 150 JJ.M and that for Mn2+ 30 JJ.M. NAD-IDH activity was not inhibited by an excess of substrates, or NADP+. However, adenine nucleotides, particularly ATP, 2-oxoglutarate, the product of the reaction, and citrate, a substrate analoge, inhibit it slightly. Glyoxylate, the product of isocitrate lyase {EC 4.1.3.1) activity, is an effective inhibitor of the NAD-IDH (Table 2), which is consistent with the antagonistic functionality between the Krebs and glyoxylate cycles. The inhibition by pHMB, a sulphydryl reagent, or by TNBS, an antagonist of amino groups, or EDC in the presence of an excess of GEE, which blocks carboxyl groups, indicate the essential nature of these functional groups for the enzyme catalytic cycle.
Physico-chemical and kinetic properties ofthe IDH1 and IDH2 isoenzymes from C. reinhardtii The NADP-IDH activity was inhibited by EDTA, an ion chelating agent, 2-oxoglutarate, the product of the reaction, and NAD+ {see Table 2). No significant effect was observed with any of the proteinogenic amino acids tested, at a final concentration of 10 mM, but adenine nucleotides, particularly ATP, may inhibit both isoenzymes. Significant inhibition of IDH 1 and IDH 2 by glyoxylate and citrate was observed, while succinate, fumarate or glycolate inhibit only IDH 1• Treatment with pHMB or TNBS causes a complete inactivation of IDH 1 and IDH2 isoenzymes after 2 h. On the other hand, if we add EDC to the purified isoenzymes in the presence of an excess of GEE, only 25 % inactivation was observed with each isoenzyme {see Table2). Table 3 shows that IDH 1 and IDHz are very similar in their molecular parameters, optimal temperature, activation energy and apparent Km values for isocitrate, NADP+ and Mn2+. IDH 1 and IDH 2 were very specific for D,L-isocitrate
and NADP+ as substrates, showing no activity with NAD+. To display their activity both isoenzymes require a divalent cation, Mn2+ being the most effective one. Mgl+, when used as the cation, allowed only 60% of the maximum activity.
Requirement ofsulpbydryl and amino groups for the catalysis ofiDH1 and IDHJ/rom C. reinhardtii Incubation of IDH isoenzymes with 1 mM MMTS or 1 mM IAA produces about 50 and 90% inhibition of the activity, respectively. However, this inactivation was effectively protected by the presence of substrates, especially isocitrate (data not shown), which suggests the involvement of sulphydryl groups in the substrate active sites of both isoenzymes. The requirement of sulphydryl groups for the catalytic activity of NADP-IDH isoenzymes from C. reinhardtii has also been shown by their activation with thiol compounds.
Table 3: Physicochemical characterization of the NADP-isocitrate dehydrogenase isoenzymes from C. reinhardtii. Molecular weight was determined by gel filtration. The corresponding Km values were determined using saturating concentration of all substrates, except for the concentration of interest, which was varied. Parameter Stokes radius (nm) Molecular weight (kDa) Optimal pH Optimal temperature (0 C) Activation energy (KJ mol- 1) Km D, L-isocitrate (J.LM) Km NADP• (J.LM) KmMn2 + (J.LM)
....... ..,.... ......
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3.6 70 7.5 55 28.5 12 15 12
100 0
3.6 70 8.0 55 24.0 8 20 18
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15
0
200
400
2·1ERCAPTDETHAIDL (ml)
Fig. 3: Activation by thiols on the activity of IDH, and IDH2 isoenzymes from C. reinhardtii. Aliquots of 1 mL of purified IDH, and IDH2 were incubated at 30 °C in 50 mM K-phosphate buffer (pH 7.5), containing 2-mercaptoethanol at the indicated concentrations. After 20 min, the NADP-IDH activity was determined by adding 0.5 mL of the corresponding preincubation mixture to the reagents of the standard assay; 100% of activity was 8 mU mL -I in each case.
Isocitrate dehydrogenase from Chlamydomonas Incubation of IDH 1 or IDH 2 with increasing concentrations of 2-mercaptoethanol produces a parallel stimulation of their activity, reaching 1.5 and 2.5-fold of the original value, respectively, at 10 mM of the thiol. Particularly interesting is the stimulation effect observed with 1 mM thiol, which is maximum for IDH 2 and non-perceptible for IDH1 (Fig. 3). Similar results were obtained when the isoenzymes were incubated with a dithiol, like dithioerythritol (data not shown). The activation kinetics of IDH 2 with 10 mM thiol increase the enzyme activity by 100% after 5 min.
Discussion In this paper we show the separation of NAD-IDH and NADP-IDH activities from Chlamydomonas reinhardtii crude extract using phenyl-sepharose chromatography. Similar IDH activity separation was obtained from pea using ion exchange chromatography on DEAE cellulose (Cox and Davies, 1967), and from the yeast Candida tropicalis using differential fractionation with ammonium sulfate (N abeshima et al., 1977). In C. reinhardtii the NADP-IDH activity level was 5-fold higher than NAD-dependent activity, according to data reported in higher plants (Chen and Gadal, 1990). To date, only the NADP-IDH activity from C. segnis (Foo and Badour, 1977) and C. reinhardtii, strain Y-2 (Ramaley and Hudock, 1973), have been described but no isoenzyme separation has been reported in eukaryotic algae. The IDH/ IDH 2 activity ratio found in C. reinhardtii is about 3.0, which is in line with that published for higher plants (Chen et al., 1989). According to the purification method reported here for the NADP-IDH isoenzymes, isolation of intact chloroplasts was not required to purify the IDH 2 , as previously established for pea leaves (Chen et al., 1989). The kinetic parameters of C. reinhardtii NAD-IDH are very similar to those for the enzyme from higher plants (Coultate and Dennis, 1969; Tezuka and Laties, 1983). The physicochemical and kinetic properties of the IDH 1 and IDH 2 from C. reinhardtii have been studied with the idea to establish differences between the isoenzymes. The data reported in this paper led us to suggest for these isoenzymes the following: a) They are slightly smaller (Mr = 70 kDa) than IDH from other eukaryotic algae with 95 kDa protein (Ramaley and Hudock, 1973) or Synechocystis sp. PCC 6803 with 108 kDa protein (Muro-Pastor and Florencio, 1992), and very much smaller than IDH2 from pea leaves with 152kDa protein (Chen et al., 1990); b) The kinetic properties, including affinity for substrates, requirement of Mn2+ for maximum activity, inhibition by ATP, or Krebs cycle or photorespiratory intermediates, indicate that both NADP-IDH isoenzymes are similar to other NADPIDH from cyanobacteria (Muro-Pastor and Florencio, 1992) and higher plants (Henson et al., 1986); and c). The essential nature of sulphydryl groups for NADP-IDH activity shown in this paper and its relation with substrate active sites are in good agreement with previous reports in other photosynthetic organisms (Gupta and Singh, 1988). The requirement of free amino groups for the activity of IDH 1 and IDH 2 is also shown. No similar information has been reported for any NADP-IDH from photosynthetic organisms.
133
The in vitro stimulation of IDH2 activity by thiols parallel the data reported for GS (Florencio and Vega, 1983) and GOGAT (Gotor et al., 1990) from C. reinhardti~ and suggest the possibility of common regulatory mechanisms. Consistent with this idea, it is interesting to mention that in Chlo· retia fusca the GS-GOGAT pathway seems to be light-dependent controlled throughout the thioredoxin-thioredoxin reductase system (Tischner and Schmidt, 1982). Acknowledgements
The authors wish to thank for financial support given by Direcci6n General de lnvestigaci6n Ciendfica y Tecnica (DGICYT, Research Grant No. PB90-0880) and Plant Andaluz de Investigaci6n (PAl, Research Group No. 3213). One of us Q.M.M-R) also thanks Junta de Andaluda (Spain) for a fellowship.
References BRADFORD, M. M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248-254 (1976). CHEN, R. D. and P. GADAL: Structure, functions and regulation of NAD and NADP-dependent isocitrate dehydrogenase in higher plants and in other organisms. Plant Physiol. Biochem., 28, 411-427 (1990). CHEN, R. D., E. BISMUTH, M. L. CHAMPIGNY, and P. GADAL: Chromatographic and immunological evidence that chloroplastic and cytosolic pea (Pisum sativum L.) NADP-isocitrate dehydrogenases are distinct isoenzymes. Planta, 178, 157-163 (1989). CHEN, R. D., P. GADAL, E. BISMUTH, and M. L. CHAMPIGNY: NADPisocitrate dehydrogenases in higher plants. In: ULLRICH, W. R., C. RIGANO, A. FuGGI, and P. J. APARICIO (eds.): Inorganic Nitrogen in Plants and Microorganisms, pp. 229-232. Springer-Verlag, Berlin. ISBN 3-540-52773-7 (1990). CouLTATE, T. P. and D. T. DENNIS: Regulatory properties of a plant NAD: isocitrate dehydrogenase. The effect of inorganic ions. Eur. J. Biochem., 7, 153-158 (1969). Cox, G. F. and D. D. DAVIES: Nicotinamide-adenine dinucleotidespecific isocitrate dehydrogenase from pea mitochondria. Purification and properties. Biochem. J., 105, 729-734 (1967). FLORENCIO, F. J. and J. M. VEGA: Separation, purification and characterization of two isoforms of glutamine synthetase from Chla· mydomonas reinhardtii. Z. Naturforsch., 38 c, 531-538 (1983). Foo, S. S. K. and S. S. BADouR: Regulation of isocitrate metabolism in Chlamydomonas segnis. Can. J. Bot., 55, 2178-2185 (1977). GoToR, C., J. M. MARTINEz-RivAs, A. J. MARQUEZ, and J. M. VEGA: Functional properties of purified ferredoxin-glutamate synthase from Chlamydomonas reinhardtii. Phytochemistry, 29, 711-717 (1990). GuPTA, V. K. and R. SINGH: Partial purification and characterization of NADP+-isocitrate dehydrogenase from immature pod walls of chickpea (Cicer arietinum L.). Plant Physiol., 87, 741744 (1988). HENSON, C. A., S. H. DuKE, and M. CoLLINs: Characterization of NADP+-isocitrate dehydrogenase from host plant cytosol of lucerne (Medicago sativa) root nodules. Physiol. Plant., 67, 538544 (1986). JoviN, T., A. CHARAMBACK, and M. A. NAuGHTON: Apparatus for preparative temperature regulated polyacrylamide gel electrophoresis. Anal. Biochem., 9, 351-369 (1964).
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Jos£M.Mu:IiNEz-RrvAsandJos£M. VEGA
MARTINEz-RrvAs, J. M., J. M. VEGA, and A. J. MARQUEZ: Differential regulation of the nitrate-reducing and ammonium-assimilatory systems in synchronous cultures of Chlamydomonas reinhardtii. FEMS Microbial. Lett., 78, 85-88 (1991). Mmw-PASTOR, M. I. and F. J. FLORENCIO: Purification and properties of NADP-isocitrate dehydrogenase from the unicellular cyanobacterium Synechocystis sp. PCC 6803. Eur. J. Biochem., 203, 99-105 (1992). NABESHIMA, S., S. lsHIYAMA, A. TANAKA, and S. FUKUI: Partial purification and some kinetic properties of NAD-linked and NADPlinked isocitrate dehydrogenase from Candid:z tropicalis. Agric. Bioi. Chern., 41, 509-516 (1977). NI, W., E. F. RoBERTSON, and H. C. REEvES: Purification and characterization of cytosolic NADP specific isocitrate dehydrogenase from Pisum sativum. Plant Physiol., 83, 785-788 (1987).
RAMALEY, R. F. and M. 0. HunocK: Purification and properties of isocitrate dehydrogenase (NADP) from Thermus aquaticus YT-1, Bacillus subtilis-168 and Chlamydomonas reinhardtii-Y-2. Biochim. Biophys. Acta., 315, 22-36 (1973). SIEGEL, L. M. and K. J. MoNTY: Determination of molecular weight and frictional ratios of proteins in impure systems by use of filtration and density gradient centrifugation. Application to crude preparation of sulfite and hydroxylamine reductants. Biochim. Biophys. Acta, 112, 346-362 (1966). TEZUKA, T. and G. G. LATIES: Isolation and characterization of inner membrane-associated and matrix NAD-specific isocitrate dehydrogenase in potato mitochondria. Plant Physiol., 72, 959963 (1983). . TISCHNER, R. and A. ScHMIDT: A thioredoxin-mediated activation of glutamine synthetase and glutamate synthase in synchronous Chlorella sorokiniana. Plant Physiol., 70, 113-116 (1982).
Studies on the lsoforms of lsocitrate Dehydrogenase from Chlamydomonas reinhardtii JosE. M. MARTINEZ-RivAs1 and JosE. M. VEGA Departamento de Bioqu1mica Vegetal y Biologfa Molecular, Facultad de Qu1mica, Universidad de Sevilla, Apartado 553, 41080-Sevilla, Spain 1 To whom correspondence should be addressed Received August 8, 1993 · Accepted September 15, 1993
Summary
Two isocitrate dehydrogenase enzymes (IDH) specific for NAD+ or NADP+ can be separated from
Chlamydomonas reinhardtii crude extract by hydrophobic chromatography on phenyl-sepharose, with
NADP-IDH activity 5-fold higher than NAD-IDH. The NAD-IDH (EC 1.1.1.41) showed sigmoidal kinetic for D,L-isocitrate (So.s = 370 llM) and normal Michaelis-Menten kinetic for NAD+ (Km = 150 IJ.M) and Mn2+ (Km = 30 IJ.M). It was inhibited by 2-oxoglutarate, citrate, glyoxylate or adenine nucleotides, and also by sulphydryl, amino or carboxyl group reagents. The NADP-IDH (EC 1.1.1.42) activity fraction contained two isoenzymes, with an IDH/IDH 2 ratio of approximately 3.0. These isoenzymes have been separated and purified and their physicochemical and kinetic parameters were very similar. The main difference between IDHt and IDH2 was the in vitro activation of IDH 2 by thiols (1 mM), which may reach 2.5-fold. IDHt activity remains unaffected by thiols at this concentration.
Key words: Chlamydomonas reinhardtii, isocitrate dehydrogenase, isocitrate metabolism. Abbreviations: EDC .. 1-Ethyl-3-(3-dimethyl aminopropyl)-carbodiimide; GEE = Glycine ethyl ester; GOGAT =Glutamate synthase; GS = Glutamine synthetase; IAA = Iodoacetamide; IDH = Isocitrate dehydrogenase; MMTS = Methyl methane thiosulfonate; TNBS = 2,4,6-Trinitrobenzene sulphonic acid.
Introduction
Two enzymes with IDH activity have been described in photosynthetic organisms, NAD-IDH (EC 1.1.1.41) and NADP-IDH (EC 1.1.1.42), which are highly specific for their corresponding electron acceptor. The NAD-IDH activity has been located inside the mitochondria of higher plants and has a catabolic role in the Krebs cycle (Chen and Gadal, 1990). However its presence in microalgae has not been yet reported. On the other hand, the NADP-IDH activity has been studied in cyanobacteria (Muro-Pastor and Florencio, 1992}, eukaryotic algae (Foo and Badour, 1977; Ramaley and © 1994 by Gustav Fischer Verlag, Stuttgart
Hudock, 1973), and higher plants (Chen and Gadal, 1990; Ni et al., 1987}. Two isoenzymes with NADP-IDH activity have been purified and characterized from higher plants; the IDH 1 was located in the cytosol, while the IDH2 appears to be in the chloroplast stroma. Both isoenzymes seem to be involved in the 2-oxoglutarate supply required for ammonium assimilation inside the chloroplast (Chen et al., 1989, 1990}. No information is available concerning the possible presence of NADP-IDH isoenzymes in microalgae. This paper reports the separation of one NAD-IDH enzyme and two NADP-IDH isoenzymes from C. reinhardtii
130
Josf M. M.u:Iim:z-RivAS and Josf M. VEGA and then were broken by freezing in liquid nitrogen for 1 min and thawing in SOmM K-phosphate buffer (pH 7.5) containing 14mM 2-mercaptoethanol (standard buffer). The resulting homogenate was centrifuged at 16,000 x g for 30 min, and the supernatant was used as crude extract. All the subsequent operations were performed at 0-4 °C. A solution of 2% (w/v) protamine sulfate (pH 7.5) was slowly added to the crude extract up to a final concentration of 0.16% (w/v). After 10min incubation with gentle stirring, the suspension was centrifuged at 27,000 x g for 5 min and the pellet discarded. 2. Phenyl sepharose chromatography. The resulting supernatant was supplemented with ammonium sulfate up to 15% saturation before it was applied to a column packed with phenyl-sepharose and equilibrated with the standard buffer, containing similar ionic strength. Then the column bed was washed with 300 mL of the same buffer, and the NADP-IDH activity was eluted from the column with 300 mL buffer containing ammonium sulfate to a saturation of 5% (Fig. 1). Fractions with high activity were pooled, concentrated to a volume of 10 mL and dialyzed overnight against 1,000 volumes of 10mM K-phosphate buffer (pH 7.5) supplemented with 14mM 2-mercaptoethanol. The NAD-IDH activity was eluted with 300mL 50% (v/v) ethyleneglycol in standard buffer (see also Fig. 1) and fractions containing high activity were pooled, dialyzed overnight against 1,000 volumes of buffer and concentrated by ultrafiltration. This enzymatic preparation was used for further studies. 3. Blue-sepharose chromatography. The resulting NADP-IDH enzyme preparation was then passed through a blue-sepharose column equilibrated with 10 mM K-phosphate buffer (pH 7.5) containing 14mM 2-mercaptoethanol. Figure 2 shows that IDH 1 activity was eluted with 50 mM phosphate, while the IDH 2 elution required a final phosphate concentration of 100mM in the buffer. The two peaks of activity were pooled separately, concentrated by ultrafiltration and dialyzed overnight against standard buffer. 4. Sephadex G-150 chromatography. The obtained IDH 1 and IDH 2 preparations (2 mL each) were separately applied to a Sephadex G-150 column equilibrated with standard buffer and eluted with the same buffer in fractions of 1.5 mL. Two separate pools were obtained, concentrated and kept at 4 °C. Before use, the corresponding enzyme preparation was dialyzed overnight against 1,000 volumes of 50mM K-phosphate buffer (pH 7.5) to remove 2-mercaptoethanol.
crude extract. They have been purified and physicochemically characterized. Materials and Methods
Chemicals Amino acids, nucleotides, substrates, metabolites and specific reagents for functional groups of proteins were purchased from Sigma (St. Louis, U.S.A.). Standard proteins, phenyl sepharose, blue sepharose and Sephadex G-150 were from Pharmacia (Uppsala, Sweden). All other chemicals were reagents of analytical grade and used as supplied by Merck (Darmstadt, Germany).
Organism and culture conditions The unicellular green alga Chlamydomonas reinhardtii, wild type, strain 21 gr, was grown at 25 °C in liquid medium (Mardnez-Rivas et al., 1991) with 10mM NH4Cl as nitrogen source. The cultures were bubbled with air supplemented with 5% (v/v) C02 and continuously illuminated with white light from fluorescent lamps
(5owm-2).
Enzyme assay and protein determination NAD-IDH activity was determined at 30 °C in 1 mL reaction mixture containing: 50 Jlmol K-phosphate buffer (pH 7.5), 0.5 Jlmol MnCb, 1.5 Jlmol NAD+, 4 Jlmol D,L-isocitrate and the appropriate amount of enzyme. The mixture to measure NADP-IDH activity was similar, but containing 0.15Jlmol ofNADP+ instead ofNAD•, and only 2 Jlmol of D,L-isocitrate. In both cases the reaction was started by addition of isocitrate and it was followed spectrophotometrically at 340 nm. One unit of activity was the amount of enzyme that catalyzed the production of 1 Jlmol NAD(P)H min - 1• Protein was determined by the Bradford (1976) method with bovine serum albumin as standard.
Purification ofNAD· and NADP-isocitrate dehydrogenase enzymes 1. Extraction and protamine sulfate treatment. Cells were harvested during the logarithmic phase (A 660 = 1.5-2.0) of growth,
'
'
5US
15'1i AS
1.0
~·-
i
A•
,._......
ous
'
'
t.
o.&
I I
I
I
\
I I
•
0.4
I
I
I
I
I
I I
0.2
'
...' I I
I
FRACTION
NUMBER
.'E
.... ......'"' .c..
I
I
~
0.!
50UB
a:
Fig. 1: Phenyl-sepharose hydrophobic chromatography of IDH activity from C reinhardtii. Arrows indicate the addition of ammonium sulfate (AS) or ethyleneglycol (EG) to the standard buffer. Fractions (3 mL) were collected at a flow rate of 30 mL h - 1 and A280 {.&---- -.&), NAD-IDH (e-e) and NADP-IDH (0-0) activities were determined.
Isocitrate dehydrogenase from Chlamydomonas IDmlll K·P1
~
..
'
IDDml K·P1
5Dml K·P1
~
. Fig. 2: Blue-Sepharose chromatography of NADP-isocitrate dehydrogenase activity from C. reinhardtii. The enzymatic preparation was applied to a column (1.6x20cm) packed with blue-sepharose and previously washed at a flow rate of 15 mL h- \ with 10mM K-phosphate (pH 7.5) containing 14mM 2-mercaptoethanol. Arrows indicate the points of which the washing buffer was changed (K-Pi • K-phosphate). Fractions of 1.5 mL were collected and IDH activity and A2so were determined.
J
131
.
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·~
e
.....•... ............... ... ... ....= ...... .• e
0
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,_
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Electrophoretic analysis Analytical separations were performed in 10% acrylamide with the discontinuous gel system of Jovin et al. (1964). Proteins were located by staining with 1 % Coomassie brilliant blue in 7% acetic acid for 30 min. NADP-IDH was located in the electrophoresis gds by submerging them in a reaction mixture containing per mL: 100 14mol Tris-HCl buffer (pH 8.0), 10 14mol MnCh, 2 14mol NADP+, 10 14mol D,L-isocitrate, 0.05% (w/v) nitroblue tetrazolium and 0.005% (v/v) phenazine methosulfate. After 30min at room temperature in the dark with gentle stirring, the activity was shown by a blue hand. The gels were then washed with water and stored in 7% acetic acid.
Determination ofstokes radius The stokes radius was determined as described by Siegel and Monty (1966) using a Sephadex G-150 column (1.6x100cm) equilibrated with 50 mM K-phosphate buffer (pH 7.5).
FRACTIDI
lUMBER
Table 1: Purification of the NADP-isocitrate dehydrogenase isoenzymes from C. reinhardtii. The NADP-isocitrate dehydrogenase activity was purified from the crude extract obtained from 42 g of cells grown under standard conditions with ammonium as nitrogen source. Step and fraction
Volume
(mL)
(U)
Activity
Protein (mg)
Specif. Act. (U mg- 1)
Crude extract
182
29
522
0.05
Protamine sulfate supernatant
192
29
407
0.07
Phenyl-sepharose eluate
75
18
64
0.28
Blue-sepharose eluate IDHt IDH2
75 23
5.5 3.5
3.9 2.6
1.41 1.35
Sephadex G-150 eluate IDH 1 21 18 IDH2
4.7 1.4
1.1 0.5
4.27 2.80
Results
Separation and Purification oflsocitrate dehydrogenases from C reinhardtii The partially purified NAD-IDH preparation had a specific activity of 100 mU mg- 1 protein, a purification factor of about 6-fold and a yield of 94 %. The purification procedure for NADP-IDH isoenzymes, summarized in Table 1, yielded IDHt and IDH2 preparations with specific activities of 4.3 and 2.8 U mg- 1 protein, respectively. The purity of these preparations was tested by polyacrylamide gel electrophoresis. The main protein band (about 80% total protein) was due to IDH 1• Two minor protein bands lacked IDH activity. On the other hand, the IDH2 preparation showed a protein band (about 50% of total protein) containing the activity with minor bands representing impurities. Both IDH isoenzymes showed similar electrophoretic mobility, with Rf = 0.46 (data not shown).
At 4 °C the NAn-dependent activity showed a half-life of 60h as compared with 96h shown by NADP-IDH; moreover, 2-mercaptoethanol resulted in efficient protection of the activity during this manipulation, indicating that sulphydryl groups are essential for activity (data not shown). Spectrophotometric studies of purified IDH isoenzymes did not reveal the presence of any prosthetic group with light absorption in the visible region.
Kinetic properties of NAD-IDH from C. reinhardtii The purified NAD-IDH preparation showed a high specificity for NAD+ and D,L-isocitrate as substrates, and required a divalent cation for maximum activity, as in other organisms (Chen and Gadal, 1990). The enzyme showed partial activity with Co2+, Cd2 +, Mgl+ and Zn2+; however, Mn2+ was the best cofactor. The NAD-IDH enzyme shows
132
Josf M. MAR'liNEz-RivAS andJosf M. VEGA
Table 2: Effect of metabolites and chemical reagents on the isocitrate dehydrogenase activity from C. reinhardtii. Assays for rn·H activity were carried out using purified preparations of each enzyme and including in the reaction mixture the corresponding chemicals as indicated; 100% of activity for NAD-IDH, NADP-IDH1 and NADP-IDH2was 140, 52 and 57 mU mL -I, respectively. Metabolite None 2-0xogluarate NAD• EDTA ATP ADP
AMP
Citrate Succinate Fumarate Glycolate Glyoxylate pHMB 1NBS EDC +GEE
Activity {%)
Concentration
(mM) 10 10 1 5 5 5 10 10 10 10 10 0.01 1 1; 500
NAD-IDH
NADP-IDH 1
NADP-IDH2
100 67 95 11 49 62 67 62
100 76 46 0
100 73
98 2 55 52 52
22
55 90 60 68 63 55 40 10 10 80
70
0 45 68 80 63 88 93 83 45 5 5 65
pH and temperature optima of 8.0 and 40 °C, with an activation energy of 78.1 KJ mol- 1• Particularly interesting is the sigmoidal kinetic observed with respect to D,L-isocitrate, showing a So.s of 370 JJ.M, as deduced from Hill plots. The Km for NAD+ was 150 JJ.M and that for Mn2+ 30 JJ.M. NAD-IDH activity was not inhibited by an excess of substrates, or NADP+. However, adenine nucleotides, particularly ATP, 2-oxoglutarate, the product of the reaction, and citrate, a substrate analoge, inhibit it slightly. Glyoxylate, the product of isocitrate lyase {EC 4.1.3.1) activity, is an effective inhibitor of the NAD-IDH (Table 2), which is consistent with the antagonistic functionality between the Krebs and glyoxylate cycles. The inhibition by pHMB, a sulphydryl reagent, or by TNBS, an antagonist of amino groups, or EDC in the presence of an excess of GEE, which blocks carboxyl groups, indicate the essential nature of these functional groups for the enzyme catalytic cycle.
Physico-chemical and kinetic properties ofthe IDH1 and IDH2 isoenzymes from C. reinhardtii The NADP-IDH activity was inhibited by EDTA, an ion chelating agent, 2-oxoglutarate, the product of the reaction, and NAD+ {see Table 2). No significant effect was observed with any of the proteinogenic amino acids tested, at a final concentration of 10 mM, but adenine nucleotides, particularly ATP, may inhibit both isoenzymes. Significant inhibition of IDH 1 and IDH 2 by glyoxylate and citrate was observed, while succinate, fumarate or glycolate inhibit only IDH 1• Treatment with pHMB or TNBS causes a complete inactivation of IDH 1 and IDH2 isoenzymes after 2 h. On the other hand, if we add EDC to the purified isoenzymes in the presence of an excess of GEE, only 25 % inactivation was observed with each isoenzyme {see Table2). Table 3 shows that IDH 1 and IDHz are very similar in their molecular parameters, optimal temperature, activation energy and apparent Km values for isocitrate, NADP+ and Mn2+. IDH 1 and IDH 2 were very specific for D,L-isocitrate
and NADP+ as substrates, showing no activity with NAD+. To display their activity both isoenzymes require a divalent cation, Mn2+ being the most effective one. Mgl+, when used as the cation, allowed only 60% of the maximum activity.
Requirement ofsulpbydryl and amino groups for the catalysis ofiDH1 and IDHJ/rom C. reinhardtii Incubation of IDH isoenzymes with 1 mM MMTS or 1 mM IAA produces about 50 and 90% inhibition of the activity, respectively. However, this inactivation was effectively protected by the presence of substrates, especially isocitrate (data not shown), which suggests the involvement of sulphydryl groups in the substrate active sites of both isoenzymes. The requirement of sulphydryl groups for the catalytic activity of NADP-IDH isoenzymes from C. reinhardtii has also been shown by their activation with thiol compounds.
Table 3: Physicochemical characterization of the NADP-isocitrate dehydrogenase isoenzymes from C. reinhardtii. Molecular weight was determined by gel filtration. The corresponding Km values were determined using saturating concentration of all substrates, except for the concentration of interest, which was varied. Parameter Stokes radius (nm) Molecular weight (kDa) Optimal pH Optimal temperature (0 C) Activation energy (KJ mol- 1) Km D, L-isocitrate (J.LM) Km NADP• (J.LM) KmMn2 + (J.LM)
....... ..,.... ......
...a: ......'"' ...
200
___...--c--o
/0.
a:
...
c:;
.•... ~
a..
-------·--. ·---._
•I
= ,_ I
3.6 70 7.5 55 28.5 12 15 12
100 0
3.6 70 8.0 55 24.0 8 20 18
o-o IDH, •-• IDH 2
......
~--0
lp' 5
15
0
200
400
2·1ERCAPTDETHAIDL (ml)
Fig. 3: Activation by thiols on the activity of IDH, and IDH2 isoenzymes from C. reinhardtii. Aliquots of 1 mL of purified IDH, and IDH2 were incubated at 30 °C in 50 mM K-phosphate buffer (pH 7.5), containing 2-mercaptoethanol at the indicated concentrations. After 20 min, the NADP-IDH activity was determined by adding 0.5 mL of the corresponding preincubation mixture to the reagents of the standard assay; 100% of activity was 8 mU mL -I in each case.
Isocitrate dehydrogenase from Chlamydomonas Incubation of IDH 1 or IDH 2 with increasing concentrations of 2-mercaptoethanol produces a parallel stimulation of their activity, reaching 1.5 and 2.5-fold of the original value, respectively, at 10 mM of the thiol. Particularly interesting is the stimulation effect observed with 1 mM thiol, which is maximum for IDH 2 and non-perceptible for IDH1 (Fig. 3). Similar results were obtained when the isoenzymes were incubated with a dithiol, like dithioerythritol (data not shown). The activation kinetics of IDH 2 with 10 mM thiol increase the enzyme activity by 100% after 5 min.
Discussion In this paper we show the separation of NAD-IDH and NADP-IDH activities from Chlamydomonas reinhardtii crude extract using phenyl-sepharose chromatography. Similar IDH activity separation was obtained from pea using ion exchange chromatography on DEAE cellulose (Cox and Davies, 1967), and from the yeast Candida tropicalis using differential fractionation with ammonium sulfate (N abeshima et al., 1977). In C. reinhardtii the NADP-IDH activity level was 5-fold higher than NAD-dependent activity, according to data reported in higher plants (Chen and Gadal, 1990). To date, only the NADP-IDH activity from C. segnis (Foo and Badour, 1977) and C. reinhardtii, strain Y-2 (Ramaley and Hudock, 1973), have been described but no isoenzyme separation has been reported in eukaryotic algae. The IDH/ IDH 2 activity ratio found in C. reinhardtii is about 3.0, which is in line with that published for higher plants (Chen et al., 1989). According to the purification method reported here for the NADP-IDH isoenzymes, isolation of intact chloroplasts was not required to purify the IDH 2 , as previously established for pea leaves (Chen et al., 1989). The kinetic parameters of C. reinhardtii NAD-IDH are very similar to those for the enzyme from higher plants (Coultate and Dennis, 1969; Tezuka and Laties, 1983). The physicochemical and kinetic properties of the IDH 1 and IDH 2 from C. reinhardtii have been studied with the idea to establish differences between the isoenzymes. The data reported in this paper led us to suggest for these isoenzymes the following: a) They are slightly smaller (Mr = 70 kDa) than IDH from other eukaryotic algae with 95 kDa protein (Ramaley and Hudock, 1973) or Synechocystis sp. PCC 6803 with 108 kDa protein (Muro-Pastor and Florencio, 1992), and very much smaller than IDH2 from pea leaves with 152kDa protein (Chen et al., 1990); b) The kinetic properties, including affinity for substrates, requirement of Mn2+ for maximum activity, inhibition by ATP, or Krebs cycle or photorespiratory intermediates, indicate that both NADP-IDH isoenzymes are similar to other NADPIDH from cyanobacteria (Muro-Pastor and Florencio, 1992) and higher plants (Henson et al., 1986); and c). The essential nature of sulphydryl groups for NADP-IDH activity shown in this paper and its relation with substrate active sites are in good agreement with previous reports in other photosynthetic organisms (Gupta and Singh, 1988). The requirement of free amino groups for the activity of IDH 1 and IDH 2 is also shown. No similar information has been reported for any NADP-IDH from photosynthetic organisms.
133
The in vitro stimulation of IDH2 activity by thiols parallel the data reported for GS (Florencio and Vega, 1983) and GOGAT (Gotor et al., 1990) from C. reinhardti~ and suggest the possibility of common regulatory mechanisms. Consistent with this idea, it is interesting to mention that in Chlo· retia fusca the GS-GOGAT pathway seems to be light-dependent controlled throughout the thioredoxin-thioredoxin reductase system (Tischner and Schmidt, 1982). Acknowledgements
The authors wish to thank for financial support given by Direcci6n General de lnvestigaci6n Ciendfica y Tecnica (DGICYT, Research Grant No. PB90-0880) and Plant Andaluz de Investigaci6n (PAl, Research Group No. 3213). One of us Q.M.M-R) also thanks Junta de Andaluda (Spain) for a fellowship.
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Jos£M.Mu:IiNEz-RrvAsandJos£M. VEGA
MARTINEz-RrvAs, J. M., J. M. VEGA, and A. J. MARQUEZ: Differential regulation of the nitrate-reducing and ammonium-assimilatory systems in synchronous cultures of Chlamydomonas reinhardtii. FEMS Microbial. Lett., 78, 85-88 (1991). Mmw-PASTOR, M. I. and F. J. FLORENCIO: Purification and properties of NADP-isocitrate dehydrogenase from the unicellular cyanobacterium Synechocystis sp. PCC 6803. Eur. J. Biochem., 203, 99-105 (1992). NABESHIMA, S., S. lsHIYAMA, A. TANAKA, and S. FUKUI: Partial purification and some kinetic properties of NAD-linked and NADPlinked isocitrate dehydrogenase from Candid:z tropicalis. Agric. Bioi. Chern., 41, 509-516 (1977). NI, W., E. F. RoBERTSON, and H. C. REEvES: Purification and characterization of cytosolic NADP specific isocitrate dehydrogenase from Pisum sativum. Plant Physiol., 83, 785-788 (1987).
RAMALEY, R. F. and M. 0. HunocK: Purification and properties of isocitrate dehydrogenase (NADP) from Thermus aquaticus YT-1, Bacillus subtilis-168 and Chlamydomonas reinhardtii-Y-2. Biochim. Biophys. Acta., 315, 22-36 (1973). SIEGEL, L. M. and K. J. MoNTY: Determination of molecular weight and frictional ratios of proteins in impure systems by use of filtration and density gradient centrifugation. Application to crude preparation of sulfite and hydroxylamine reductants. Biochim. Biophys. Acta, 112, 346-362 (1966). TEZUKA, T. and G. G. LATIES: Isolation and characterization of inner membrane-associated and matrix NAD-specific isocitrate dehydrogenase in potato mitochondria. Plant Physiol., 72, 959963 (1983). . TISCHNER, R. and A. ScHMIDT: A thioredoxin-mediated activation of glutamine synthetase and glutamate synthase in synchronous Chlorella sorokiniana. Plant Physiol., 70, 113-116 (1982).