THIOREDOXIN SYSTEM 415 and ... - Science Direct

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and there is extensive similarity among the amino acid sequences of cyanobacterial thioredoxin m, other bacterial thioredoxins, and chloroplast thioredoxin m.11,3°,31 Evidence has been obtained for a light-dependent reduction of thioredoxin m in Anabaena cylindrica,32 and the protein is apparently located mainly in the centroplasm of vegetative cells of A. cylindrica and is absent from or present at a reduced level in heterocysts. 29 Several potential functions of cyanobacterial thioredoxin rn have been established, 9-2° although the true physiological function(s) remains to be firmly established. 31 F. K. Gleason, in "Thioredoxin and Glutaredoxin Systems: Structure and Function" (A. Holmgren, C. I. Branden, H. Jornvall, and B.-M. Sjoberg, eds.), p. 21. Raven, New York, 1986. 32 A. J. Darling, P. Rowell, and W. D. P. Stewart, Biochim. Biophys. Acta 850, 116 (1986).

[43] F e r r e d o x i n / T h i o r e d o x i n S y s t e m By NANCY A.

C R A W F O R D , BOIHON C. Y E E , MICHEL DROUX, D O N A L D E . CARLSON, and BoB B. B U C H A N A N

Introduction Light regulates enzymes of oxygenic photosynthesis via several mechanisms.l-4 Important among these is the ferredoxin/thioredoxin system, an enzyme-mediated regulatory mechanism involving ferredoxin, ferredoxin-thioredoxin reductase (FTR), and a thioredoxin. 5 Thioredoxins are proteins, typically of 12,000 molecular weight, that are widely, if not universally, distributed in the animal, plant, and bacterial kingdoms. Thioredoxins undergo reversible reduction and oxidation through changes in thiol groups ( S - - S ~ 2 SH). In the ferredoxin/thioredoxin system, a thioredoxin (Td) is reduced by photoreduced ferredoxin (Fd) via FTR, an iron-sulfur protein 6,7 [Eqs. (1) and (2)]. Thioredoxins can also be chemically reduced in vitro in the dark by the nonphysiological reagent B. B. Buchanan, Annu. Rev. Plant Physiol. 31, 341 (1980). 2 C. Cs6ke and B. B. Buchanan, Biochim. Biophys. Acta 853, 43 (1986). 3 L. E. Anderson, in "Photosynthesis: II. Photosynthetic Carbon Metabolism and Related Processes" (M. Gibbs and E. Latzko, eds.), Vol. 6, p. 271. Springer-Verlag, Berlin, 1979. 4 j. Preiss, Annu. Rev. Plant Physiol. 33, 431 (1982). 5 R. A. Wolosiok and B. B. Buchanan, Nature (London) 266, 565 (1977). 6 M. Droux, J.-P. Jacquot, M. Miginiac-Maslow, P. Gadal, J. C. Huet, N. A. Crawford, B. C. Yee, and B. B. Buchanan, Arch. Biochem. Biophys. 252, 426 (1987). 7 M. Droux, M. Miginiac-Maslow, J.-P. Jacquot, P. Gadal, N. A. Crawford, N. S. Kosower, and B. B. Buchanan, Arch. Biochem. Biophys. 256, 372-380 (1987).

METHODS IN ENZYMOLOGY, VOL. 167

Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

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dithiothreitol (DTT), in the absence of chloroplast membranes, ferredoxin, and FTR [Eq. (3)]. 4 Fdox + 2 H20 Light 4 Fdred + 02 + 4 H + 2 Fdred + Tdox + 2 H + > 2 Fdox + Tdred Tdox + DTTred :' Td~d + DTTox

(1) (2) (3)

Two different thioredoxins, designated thioredoxin f and thioredoxin m, are a part of the ferrodoxin/thioredoxin system which has been found in different types of oxygenic photosynthetic organisms including cyanobacteria, s C3, 9-1~ C4, ~ and crassulacean acid metabolism (CAM) plants. 12,13In the reduced state, the two thioredoxins selectively activate enzymes of carbohydrate biosynthesis, including those of the reductive pentose phosphate cycle [fructose-l,6-bisphosphatase (FBPase), sedoheptulose-l,7-bisphosphatase, phosphoribulokinase, NADP-glyceraldehyde-3-phosphate dehydrogenase] and deactivate glucose-6-phosphate dehydrogenase, L2,14 a key enzyme of the oxidative pentose phosphate cycle, the major pathway for carbohydrate degradation in cyanobacteria. The ferredoxin/thioredoxin system also functions in chloroplasts in regulating other enzymes such as NADP-malate dehydrogenase (NADPMDH) 1,2 and the chloroplast coupling factor (CFrATPase).55 The type of thioredoxin which interacts with each of these enzymes is shown in Table I. In cyanobacteria, the specificity of thioredoxins for some of the target enzymes may not be as rigid as their C3 counterparts. TM Cyanobacteria and certain algae appear to utilize the ferredoxin/thioredoxin system also for regulation of enzymes of sulfur 16 and nitrogen 17 assimilation. This chapter describes procedures for the isolation and assay of thioredoxins m and f a n d of FTR from the cyanobacterium N o s t o c m u s c o r u m .

s B. C. Yee, A. De la Torre, N. A. Crawford, C. Lara, D. E. Carlson, and B. B. Buchanan, Arch Microbiol. 1311, 14 (1981). 9 p. Schiirmann, K. Maeda, and A. Tsugita, Ear. J. Biochem. 116, 37 (1981). 10 R. A. Wolosiuk, N. A. Crawford, B. C. Yee, and B. B. Buchanan, J. Biol. Chem. 254, 1627 (1979). 11 N. A. Crawford, B. C. Yee, S. W. Hutcheson, R. A. Wolosiuk, and B. B. Buchanan, Arch. Biochem. Biophys. 244, 1 (1986). 12 S. W. Hutcheson and B. B. Buchanan, Plant Physiol. 72, 870 (1983). 13 S. W. Hutcheson and B. B. Buchanan, Plant Physiol. 72, 877 (1983). 14 N. A. Crawford, C. W. Sutton, B. C. Yee, T. C. Johnson, D. C. Carlson, and B. B. Buchanan, Arch. Microbiol. 139, 124 (1984). 15 j. D. Mills and P. Mitchell, FEBS Lett. 144, 63 (1982). 16j. D. Schwenn and U. J. Schrieck, FEBS Lett. 170, 76 (1984). 17 R. Tischner and A. Schmidt, Plant Physiol. 70, 113 (1982).

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TABLE I TARGETENZYMESOF THIOREDOXINSfAND ma Thioredoxin type

Target enzymes

Thioredoxin m

NADP-malate dehydrogenase Chloroplast coupling factor (CF~-ATPase) Glucose-6-phosphate dehydrogenaseb

Thioredoxin f

Fructose- 1,6-bisphosphatase Sedoheptulose-1,7-bisphosphatase Phosphoribulokinase NADP-glyceraldehyde-3-phosphatedehydrogenase NADP-malate dehydrogenase Chloroplast coupling factor (CFrATPase)

a Target enzymes of sulfur and nitrogen assimilation are not included.16,t7 b Inhibited by reduced thioredoxin m. Thioredoxin m

Assay Method for Thioredoxin m Principle. Thioredoxin m is assayed by measuring its capacity to promote the reductive activation of N A D P - M D H . H,18 The reducing power needed for activation may be supplied either by photoreduced ferredoxin in the presence o f F T R (see below) or nonphysiologically by DTT. For convenience, we routinely use D T T as the reductant and a N A D P - M D H preparation derived from corn leaves as the target enzyme. The assay is performed in two steps. The first step (activation phase) involves activation o f the N A D P - M D H , and the second step (catalytic or reaction phase) involves spectrophotometrically measuring the resultant activity. Reagents Tris-HC1 buffer (pH 7.9 at 20°), 1 M DTT, 100 m M Corn leaf N A D P - M D H , 0.15 mg/m119 Thioredoxin m fraction N A D P H , 2.5 m M Oxaloacetate, 25 m M Assay Procedure. Both steps of the assay are carried out at room temperature. The activation phase of the assay is carried out in a 5-ml test 18R. A. Wolosiuk, P. Schfirmann, and B. B. Buchanan, this series, Vol. 69, p. 382. 19j._p. Jacquot, B. B. Buchanan, F. Martin, and J. Vidal, Plant Physiol. 68, 300 (1981).

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tube containing an appropriate amount of thioredoxin sample (usually 50 /zl of the column fractions to be analyzed) plus 10/xl each of Tris-HC1 buffer, DTT, and NADP-MDH. The total volume is adjusted to 0.1 ml with water. After a 5-min activation period, a 50-/zl aliquot of the activation mixture is injected into a cuvette with a 1-cm light path containing the following reaction mixture: 100 p.1 each of Tris-HC1 buffer, NADPH, and oxalacetate, plus 650/zl of water. The oxidation of NADPH is followed at 340 nm with a recording spectrophotometer. Purification o f Nostoc Thioredoxin m Chemicals and Materials Nostoc muscorum cells (Anabaena, Sp. 7119), are grown under illumination in liquid culture and N2-CO2 (98 : 2, v/v), as described by Arnon et al.,2° and are harvested 4 days after inoculation. Frozen paste, 250 g, is used below. Tris-HCl buffer (pH 7.9 at 20°), 1 M stock solution; 2 liters 2-Mercaptoethanol, 14 M; 60 ml 2x buffer (I00 m M Tris-HCl, pH 7.9, plus 0.2%, v/v, 2-mercaptoethanol, freshly prepared from stock solutions 1× buffer, half-strength 2x buffer Ammonium sulfate, crystalline, 250 g NaCI, 25 g Sephadex G-100 (Pharmacia Chemical Co., Piscataway, N J), 3500 ml (swollen) DE-52 cellulose, anion exchanger (Whatman Inc., Clifton, N J), 400 ml (wet) Reagent for protein determination, Bradford method (Bio-Rad Laboratories, Richmond, CA) Dialysis tubing, Spectrapor 1, 6,000-8,000 MW cutoff, 40 mm wide (VWR, San Francisco, CA) YM5 ultrafiltration membrane (Amicon Corp., Danforth, MA), 2.5 cm Preparative Procedure for Nostoc Thioredoxin m. All preparative steps are carried out at 4 °. Column fractions are monitored for protein by measuring the absorption at 280 nm. Preparation o f cell-free extract. Cells (250 g) are thawed overnight at 4°, homogenized in 375 ml of 2x buffer, and disrupted in a Ribi cell fractionator under N2 with a breaking pressure of 1150 g/cm 2. Cell debris 20 D. I. Arnon, B. D. McSwain, H. Y. Tsujimoto, and K. Wada, Biochim. Biophys. Acta 357, 231 (1974).

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is removed by centrifugation (40,000 g, 20 min), and the supernatant fraction is used below. Ammonium sulfate fractionation. Solid ammonium sulfate (70.4 g) is added slowly and with stirring to the clarified supernatant fraction (400 ml) to make a 30% saturated solution. Centrifugation of the solution (13,000 g, 15 min) removes the dark green precipitate, which is discarded. The bluish red supernatant fraction (395 ml) is brought to 80% saturation by adding 140.6 g solid ammonium sulfate and is stirred for 30 min. The solution is centrifuged (13,000 g, 20 min), and the supernatant fraction is discarded. The bluish red pellet is resuspended in 100 ml of 2× buffer, added to dialysis tubing, and dialyzed overnight versus 13 liters of l x buffer. In the morning, the sample is clarified by centrifugation (105,000 g, 2 hr). Sephadex G-IO0 chromatography. The clarified bluish red sample is applied to a 5 x 150 cm Sephadex G-100 column previously equilibrated with 1 × buffer. The column is developed with 1 × buffer at a flow rate of 60 ml/hr, and 18-ml fractions are collected with a fraction collector. Fractions are assayed for thioredoxin and FTR (see below). If thioredoxin f activity is also being followed, it is important to include a minus FBPase control to monitor endogenous phosphatases (see below). DE-52 cellulose chromatography. The fractions from the previous step showing thioredoxin activity are pooled and applied to a DE-52 cellulose column (2.2 x 33 cm) previously equilibrated with 1 x buffer. The column is sequentially eluted with 250 ml of buffer, a 500-ml linear gradient of 0-0.2 M NaC1 in buffer, and finally 250 ml of 0.5 M NaC1 in buffer. Fractions (7 ml) are collected and assayed for thioredoxin m activity. Fractions containing thioredoxin m activity are pooled and concentrated by ultrafiltration in an Amicon Diaflo cell fitted with a YM5 membrane, and the protein concentration is determined by the Bradford method using reagent and instructions supplied by Bio-Rad Laboratories. At this point in the procedure, thioredoxin m is not pure but is free of contaminating thioredoxinf, FTR, ferredoxin, and phosphatases. It may be used in studies on the ferredoxin/thioredoxin system of cyanobacteria. Thioredoxin m has been purified to homogeneity in other laboratories. 21,22

Properties of Thioredoxin m The amino acid sequence of Anabaena thioredoxin m reported by Gleason et al. shows 49% similarity with the Escherichia coli thiore21 F. K. Gleason, M. M. Whittaker, A. Holmgren, and H. J6rnvall, J. Biol. Chem. 260, 9567 (1985). 22 S.M" Ip, P. Rowell, A. Aitken, and W. D. P. Stewart, Eur. J. Biochem. 141, 497 (1984).

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doxin 21 and 50% similarity with spinach thioredoxin m (cf. Ref. 23). The active site, Trp-Cys-Gly-Pro-Cys (residues 30-34), is identical to that of all authentic thioredoxins. Thioredoxin m from oxygenic photosynthetic cells differs at residue 29 from E. coli and other aerobic bacteria sequenced to date which have Glu instead of Pro. Although Anabaena or Nostoc thioredoxin rn cross-reacts minimally with anti-E, coli thioredoxin antibodies, 22,24,25 it reacts well with an anti-spinach thioredoxin rn antibody. 24 Thus the thioredoxin m from cyanobacteria shares common characteristics with its bacterial and higher plant counterparts. The thioredoxin m gene from cyanobacteria has been cloned (Ref. 26 and E. Muller, unpublished observations). Thioredoxin f

Assay Method for Thioredoxin f Principle. The principle behind the thioredoxin f a s s a y is the same as in the thioredoxin m assay described. H,~8 Here, any of the known target enzymes of thioredoxinfcould be used to measure its capacity for reductive activation either with DTT or light, thylakoid membranes, and components of the ferredoxin/thioredoxin system. For convenience we routinely use DTT as the reductant and spinach chloroplast fructose-1,6-bisphosphatase (EC 3.1.3.11, FBPase) as the target enzyme. As with thioredoxin m, the assay is performed in two steps involving first activation of FBPase and then measurement of the resulting activity by determining the hydrolysis of fructose 1,6-bisphosphate (FBP) to fructose 6-phosphate and inorganic phosphate. The activity of FBPase can be measured either colorimetrically by analyzing the Pi released or spectrophotometrically by measuring the fructose 6-phosphate formed. Fructose 6-phosphate is determined by following the reduction of NADP in the presence of excess glucose-6-phosphate isomerase and glucose-6-phosphate isomerase and glucose-6-phosphate dehydrogenase. The latter method is useful when the sample contains phosphate buffer. In both cases, assays are carried out in the presence of a limiting concentration of Mg 2+ because higher concentrations (greater than 1-2 mM) partially activate FBPase without thioredoxin. Interfering phosphate and phosphatases in crude extracts make fractionation necessary before a reliable 23 K. Maeda, A. Tsugita, D. Dalzoppo, F. Viibois, and P. Schiirmann, Eur. J. Biochem. 154, 197 (1986). 24 T. C. Johnson, N. A. Crawford, and B. B. Buchanan, J. Bacteriol. 158, 1061 (1984). 25 F. K. Gleason and A. Holmgren, J. Biol. Chem. 256, 8306 (1981). 26 C.-J. Lim, F. K. Gleason, and J. A. Fuchs, J. Bacteriol. 168, 1258 (1986).

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thioredoxin assay can be performed. Even after fractionation, it is important to include a minus FBPase control in order to monitor endogenous phosphatases.

Method I: Colorimetric Assay of Thioredoxin f Reagents Tricine-KOH buffer (pH 7.9 at 20°), 1 M MgSO4, 10 mM DTT, 50 mM Sodium FBP, 60 mM Spinach chloroplast FBPase, 1.6 mg/m127 Thioredoxin f fraction Trichloroacetic acid (TCA), 10% (w/v) Mixture for Pi analysis (see below) Assay Procedure. The reaction is carried out at room temperature in a 10-ml test tube containing 50/xl each of Tricine-KOH buffer, DTT, and MgSO4 ; 10/.d of FBPase; the thioredoxinffraction to be assayed (50/zl in the following purification protocol); and water to bring the volume to 0.45 ml. After a 10-min activation, catalysis of FBPase is initiated by the introduction of 50/~1 of FBP and allowed to continue for 15 min. The reaction is stopped by the addition of 0.5 ml of TCA, and the precipitate is removed by centrifugation. A 0.5-ml aliquot of the supernatant solution is analyzed for Pi by adding 2 ml of the mixture used for Pi analysis. After 10 min, the absorbance at 660 nm is measured. In more highly purified fractions, the TCA precipitation step can be omitted because protein concentrations will be sufficiently low so as not to interfere with P~ measurement.

Reagents for Pi Analysis H2504, 9 N

Ammonium molybdate, 1.65 g in 25 ml hot water FeSO4" 7 H 2 0 , 2.5 g in 25 ml of 9 N H2SO4 Method for Preparing Mixture for Pi Analysis. FeSO4 • 7H20 (2.5 g) is dissolved in 25 ml of 9 N H2504 and added to 150 ml of water. Ammonium molybdate (1.65 g) is dissolved in 25 ml water by gently heating and is slowly added with stirring to the acidic FeSO4 solution. This mixture can be stored for several weeks, but a standard phosphate curve should be included because the mixture changes with time. 27A. N. Nishizawa, B. C. Yee, and B. B. Buchanan, in "Methods in Chloroplast Molecular Biology" (M. Edelman, R. B. Hallick, and N.-H. Chua, eds.), p. 707. Elsevier, New York, 1982.

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Method H: Spectrophotometric Assay of Thioredoxin F Reagents Tris-HCl buffer (pH 7.9 at 20°), 1 M DTT, 100 m M Spinach chloroplast FBPase, 1.6 mg/mF 7 Thioredoxin f fraction MgSO4, 10 mM Glucose-6-phosphate dehydrogenase (Sigma Chemical Co., St. Louis, MO) Glucose-6-phosphate isomerase (Sigma) NADP, 10 m M Sodium FBP, 60 mM Assay Procedure. To a 5-ml test tube are added 10 tzl each of Tris-HCl buffer, DTT, and FBPase; 10-70 tzl of thioredoxin f; and water to bring the volume to 0.1 ml. The mixture is incubated for 5-20 min (depending on thioredoxin f activity) to activate the FBPase. The resulting FBPase activity is measured by injecting a 50-/~1 aliquot of the activation mixture into a cuvette of 1-cm light path, containing the following reaction mixture in a total volume of 0.95 ml: 100 Ixl each of Tris-HC1 buffer, MgSO4, FBP, and NADP plus 0.75 U of glucose-6-phosphate dehydrogenase and 1.8 U of glucose-6-phosphate isomerase. NADP reduction is followed at 340 nm in a recording spectrophotometer. Purification of Nostoc Thioredoxin f Chemicals and Materials. Chemicals and materials needed are identical to those for thioredoxin m purification described above. Preparative Procedure for Nostoc Thioredoxin f. The protocol for purifying Nostoc thioredoxin f is identical to that described for thioredoxin m (see above). The two thioredoxins copurify through the Sephadex G-100 step but are separated on DE-52 cellulose where Nostoc thioredoxin f is eluted at a higher salt concentration than thioredoxin m. A minus FBPase control must be included in the assays at each of the preparative steps to monitor interfering phosphatases. Fractions eluting from the DE-52 cellulose column which contain thioredoxinfactivity are pooled, concentrated, and analyzed for protein concentration as described for thioredoxin m. Thioredoxinfmay be purified to homogeneity using the protocol of Whittaker and Gleason. z8 Properties of Thioredoxin f The thioredoxin f from Anabaena is reported to have a molecular weight of 25,500 z8 when analyzed by sodium dodecyl sulfate-polyacrylM. M. Whittaker and F. K. Gleason, J. Biol. Chem. 259, 14088 (1984).

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amide gel electrophoresis (SDS-PAGE), a value which is more than twice as high as the molecular weight reported for the higher plant equivalent. 9,~1 Furthermore, unlike the case for higher plant thioredoxinf, Whittaker and Gleason 28 report that Anabaena thioredoxin f is a very poor activator of endogenous FBPase and spinach NADP-malate dehydrogenase even at high concentrations (cf. Refs. 9 and 11). Nostoc thioredoxin f is reportedly effective in the activation of endogenous phosphoribulokinase, sedoheptulose-l,7-bisphosphatase, and FBPase. 14 The discrepancy of results in the capability of thioredoxinfto activate FBPase remains to be resolved. Ip et alfl 2 have shown that FBPase from Anabaena cylindrica can be activated by a thioredoxin from the same species and found that the presence of substrate during the activation phase is necessary to see an effect of thioredoxin. 22

Ferredoxin-Thioredoxin Reductase (FTR) Assay Method for FTR Principle. Like thioredoxin, FTR can be assayed by its capacity to activate any of the enzymes targeted by the ferredoxin-thioredoxin system in the presence of ferredoxin, thioredoxin, and illuminated thylakoid membranes. 6,1sAs described for thioredoxin, the FTR assay is carried out in two steps, an activation phase and a catalytic phase. For convenience, we routinely monitor FTR activity using thioredoxin f and FBPase (both from spinach) or thioredoxin m (from spinach) and N A D P - M D H (from corn). Nostoc thioredoxins could be used equally well here. As with the thioredoxinfassay, fractionation of crude extracts prior to assay and the inclusion of a minus FBPase control are important for monitoring FTR in FBPase-linked assays. Method I: Colorimetric FTR Assay (FBPase as Target Enzyme) Reagents Tris-HCl buffer (pH 7.9 at 20°), 1 M M g S O 4 , 10 m M

2,6-Dichlorophenolindophenol (DPIP), 1 mM Sodium ascorbate, 100 mM Spinach chloroplast FBPase, 1.6 mg/m127 Spinach ferredoxin, 2.5 mg/m129 Spinach thioredoxin f, 0.5 mg/mP 1 29 B. B. Buchanan and D. I. Arnon, this series, Vol. 23, p. 413.

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Spinach thylakoid membranes, 1 mg chlorophyll/mP° Sodium FBP salt, 60 mM FTR fractions, 2-10/~g if pure TCA, 10% (w/v) Mixture for Pi analysis (see above) Assay Procedure. The reaction is carried out anaerobically at 20° in Warburg vessels. The main compartment contains 0.1 ml each of TrisHCI buffer, MgSO4, sodium ascorbate, DPIP, and thylakoid membranes; 0.02 ml each of thioredoxin f, ferredoxin, and FBPase; FTR as needed; and water to a total volume of 1.4 ml. The sidearm contains 0. I ml FBP. After equilibration for 5 min with N2, the vessels are incubated for an additional 5 min in 330 ttE/m2/sec light (activation phase). The FBP is added from the sidearm to start the reaction, and the vessels are maintained in the light for the 30-min reaction phase. The reaction is stopped by adding 0.5 ml of 10% TCA after opening the vessels. The precipitate is removed by centrifugation, and 0.5 ml of the supernatant solution is analyzed for Pi a s described in the assay procedure for thioredoxin f. It should be mentioned that, for best results, Tris buffer (Sigma 7-9 grade) should be used in the FTR assay unlike the DTT-linked FBPase assay in which Tricine buffer is preferred. We do not know the reasons for the different buffer sensitivities in the two assays.

Method H: Spectrophotometric FTR Assay (FBPase as Target Enzyme) Reagents Tris-HC1 buffer (pH 7.9 at 20°), 1 M 2,6-Dichlorophenolindophenol (DPIP), 2 mM Sodium ascorbate, 200 mM Spinach ferredoxin, 5 mg/m129 Spinach thioredoxin f , 0.5 mg/ml 1~ Spinach FBPase, 1.6 mg/m127 Spinach thylakoid membranes, 2.4 mg/ml 3° Catalase, 2 mg/ml (Sigma) FTR fraction to be assayed, 2-10/~g if pure MgSO4, 10 m M Sodium FBP, 60 mM Glucose-6-phosphate dehydrogenase (Sigma) Glucose-6-phosphate isomerase (Sigma) NADP, 10 mM 3o j..p. Jacquot, M. Droux, M. Miginiac-Maslow, C. Joly, and P. Gadal, Plant Sci. Lett. 35, 181 (1984).

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Assay Procedure. The reaction is carried out anaerobically at 20° in white 1.5-ml Eppendorf centrifuge tubes fitted with a 11.1-mm-diameter rubber serum stopper pierced with two 20-gauge needles, one of which is connected to a N2 source and the other used as a vent. The tubes contain 10/zl each of Tris-HC1 buffer, thioredoxin f , FBPase, thylakoid membranes, and catalase plus 5 ~1 each of DPIP, sodium ascorbate, and ferredoxin. FTR and water are added as needed to make a final volume of 0.1 ml. The tubes are equilibrated with N2 for 5 min with agitation. The vent needle is removed first and the N2 needle second. The tubes are then illuminated without agitation for 10 min (activation phase). A 20- to 50-/zl aliquot of the activation mixture is removed with a syringe and injected into a cuvette containing 0.95 ml of reaction mixture as described in the spectrophotometric assay (Method II) for thioredoxin f above. Change in absorbance is measured at 340 nm. As with thioredoxin f , this spectrophotometric assay method is useful when samples contain phosphate buffer. The reaction step of the assay is carried out in air.

Method III: Spectrophotometric FTR Assay (NADP-MDH as Target Enzyme) Reagents Tris-HC1 buffer (pH 7.9 at 20°), 1 M DPIP, 2 m M Sodium ascorbate, 200 mM Spinach ferredoxin, 5 mg/m129 Spinach thioredoxin m, 0.5 mg/mP 1 Corn N A D P - M D H , 1.5 mg/m119 Spinach thylakoid membranes, 2.4 mg/ml 3° Catalase, 2 mg/ml (Sigma) FTR fraction to be assayed, 2-10/~g if pure NADPH, 2.5 m M Oxaloacetate (OAA), 25 mM Assay Procedure. The assay is carried out as described for the spectrophotometric FBPase assay except that (1) N A D P - M D P and thioredoxin m are used in the activation mixture in place of FBPase and thioredoxinf, respectively, and (2) the reaction mixture contains 100/xl each of Tris-HCl buffer, NADPH, and OAA plus 650 /zl of water. Here the oxidation of N A D P H is followed by measuring the change in absorbance at 340 nm in a recording spectrophotometer.

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Purification of Nostoc FTR Chemicals and Materials. Chemicals, materials, and Nostoc muscorum cells required are identical to those described for the purification of thioredoxin m (see above) plus the following: Potassium phosphate buffer, pH 7.7, I M stock solution, I00 ml Hydroxylapatite (HTP) (Bio-Rad), 20 g (dry weight) Mono Q, FPLC anion exchanger, and Pharmacia FPLC Unit Reagents for S D S - P A G E 31 Preparative Procedure for Nostoc FTR. The first three steps are identical to the procedure described above (Preparative Procedure for Nostoc Thioredoxin m). If the FBPase-linked FTR assay is used, FTR activity is calculated by subtracting a minus FBPase control from the total activity in the Sephadex G-100 column of the third step. Fractions showing FTR activity are pooled and used in the fourth through sixth steps below. DEAE-cellulose chromatography. The FTR from the Sephadex G-100 column is applied to a 2.2 × 33 cm DE-52 cellulose column previously equilibrated with 1X buffer. After washing the column with 250 ml of buffer, the column is eluted with a l-liter gradient of 0-270 mM NaCI in buffer. Fractions (6 ml) are collected, assayed for FTR, and adjusted for contaminating phosphatase activities as described above. Fractions showing FTR activity are pooled and dialyzed overnight versus 12 liters of buffer. Hydroxylapatite chromatography. The FTR is applied to a 2.2 × 8 cm hydroxyapatite (HTP) column which was previously equilibrated with 2 liters of 1X buffer. The column is eluted sequentially with 150 ml each of 0, 50, 120, and 350 m M potassium phosphate, pH 7.7, in 1X buffer, and 5ml fractions are collected. Absorbance is measured at 280 and 410 nm. The bluish red phycobilin proteins are eluted with 50 mM potassium phosphate and the pale yellow-brown FTR with 350 mM potassium phosphate. FTR may be detected at this stage by measuring absorption at 410 nm and may be assayed using a spectrophotometric assay (Methods II or III). The fractions containing FTR are pooled and concentrated to about 2 ml by ultrafiltration in an Amicon Diaflo cell fitted with a YM5 membrane. The FTR sample is dialyzed overnight versus several liters of 1X buffer. The purity of the FTR may be estimated by measuring its absorption spectrum, i.e., the ratio of its 410 nm and 278 nm absorption peaks (see below). The FTR may be pure at this point or it may require purification by the FPLC Mono Q step described below. With spinach and corn FTR preparations, a ferredoxin-Sepharose 4B chromatography step is per31 U. K. Laemmli, Nature (London) 227, 680 (1970).

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formed at this point. We have little experience with the affinity step in the case of Nostoc FTR, but it would likely be effective here as well. 6 FPLC chromatography, Mono Q. If the FTR from the HTP step is not homogeneous, it may be further purified with a Pharmacia Mono Q (1-ml anion-exchange) column attached to a FPLC apparatus. After a wash with 4 ml of buffer, the column is developed with a 20-ml linear gradient of 80280 mM NaCI in buffer. Fractions (0.5 ml) are collected and absorbance is measured at 410 nm. The FTR elutes at about 195 mM NaC1 and should be pure when analyzed by the SDS-PAGE system of Laemmli. 31

Properties of Nostoc FTR FTR is relatively stable to freezing but should be stored in aliquots to avoid repeated freezing and thawing. The native enzyme has a molecular weight of 28,000, and SDS-PAGE reveals two subunits migrating at 14,000 and 7 , 0 0 0 . 6 FTR is an iron-sulfur protein (4 Fe and S 2- groups/ mol) with absorption peaks at 278 and 410 nm and a n A410/A278 ratio of 0.34-0.40, depending on the integrity of the iron-sulfur cluster. The 14,000-Da subunit (similar subunit) is present in other FTRs examined and cross-reacts with anti-corn FTR polyclonal globulins, whereas the 7,000-Da subunit (variable subunit) is unique to the Nostoc preparation and fails to react with the corn antibody. Likewise, anti-Nostoc FTR polyclonal globulins cross-react only with the similar subunit in FTRs from corn or spinach.

[44] E l e c t r o n P a r a m a g n e t i c R e s o n a n c e C h a r a c t e r i z a t i o n of Iron-Sulfur Proteins

By R. CAMMACK Introduction Iron-sulfur proteins contain clusters of iron and acid-labile sulfide atoms, coordinated to cysteine sulfurs and, occasionally, nitrogen ligands from the protein. In most cases their function is electron transport. In cyanobacteria, as in chloroplasts, the most abundant iron-sulfur clusters are those associated with photosynthetic electron transport. 1,2 They are t M. C. W . E v a n s , in " I r o n - S u l f u r P r o t e i n s " (T. G. Spiro, ed.), p. 249. W i l e y , N e w Y o r k , 1982. 2 R. M a l k i n , Annu. Rev. Plant. Physiol. 33, 455 (1982).

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