Multiple forms of ferredoxin-nicotinamide adenine dinucleotide phosphate reductase from spinach

ARCHIVES

OF

Multiple

BIOCHEMISTRY

AND

BIOPHYSICS

174, 666-674 (1976)

Forms of Ferredoxin-Nicotinamide Adenine Phosphate Reductaae from Spinach’ WALTER Department

W. FREDRICKS

of Biology, Marquette

AND

University,

Received August

Dinucleotide

JUDITH

M. GEHL

Milwaukee,

Wisconsin

53233

25, 1975

Crude extracts of ferredoxin-NADP reductase prepared from spinach by three different methods consistently contained two molecular weight forms of the enzyme: P-l, 117,500, and P-2, 50,000. The lower molecular weight form was purified and shown to consist of two different ionic forms. These three forms of the flavoprotein are immunologically identical. A third molecular weight form of the reductase, excluded by Sephadex G-100, generated P-l and P-2 on rechromatography. Other experiments demonstrated that this enzyme has NADPH-tetrazolium reductase activity and it accounts for essentially all of the tetrazolium reductase activity of isolated chloroplasts.

Ferredoxin-NADP reductase (reduced NADP:ferredoxin oxidoreductase, EC 1.6.99.4) is a membrane-bound component of the photosynthetic electron transport system (2). Berzborn (3) has concluded that the reductase is located in partially protected sites on the thylakoid membrane. Schneeman and Krogmann (4) suggested that the reductase is held to the chloroplast membrane through cationic binding sites. However, neither the precise localization of the reductase on the membrane nor the nature of the forces stabilizing the membrane-reductase association is known. Although it is not known what membrane components are involved in reductase binding in uivo, a number of reductase-protein interactions have been demFerredoxin forms a onstrated in vitro. tight 1:l complex with reductase at low ionic strength (5-7). This complex frequently is cited as the significant catalytic

complex in noncyclic photosynthetic electron transport. Nakamura and Kimura (8), however, have argued that a weakly interacting complex between ferredoxin and reductase is the catalytically effective complex. The NADPH-cytochrome f reductase activity of the flavoprotein has been suggested as potentially important in cyclic electron flow (9). The enzyme also catalyzes the NADPH-dependent reduction of plastocyanin (lo), another electron transport component connecting photosysterns I and II. These catalytic activities indicate that the reductase is capable of forming at least transient complexes with these membrane-bound components. Most of the work characterizing the physical and biochemical properties of the enzyme has been done using a purified preparation with a molecular weight of approximately 50,000. Recently Schneeman and Krogmann (4) have isolated an 85,000-molecular weight form of the enzyme composed of two dissimilar subunits. In this paper we report the existence of several forms of the reductase. A full appreciation of the catalytic roles, structural interactions, and regulatory significance of the reductase warrants consideration of these diverse interactions and multiple forms of the reductase.

’ This investigation was supported in part by Research Grant No. GB-21423 from the National Science Foundation. A preliminary report (1) of this study was presented at the 57th Annual Meeting of the Federation of American Societies for Experimental Biology. L To whom requests for reprints should be addressed. 666 Copyright All rights

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

FERREDOXIN-NADP EXPERIMENTAL

PROCEDURES

Materials. The following reagents were purchased from commercial sources: pig heart isocitric dehydrogenase (L-isocitrate:NADP oxidoreductase (decarboxylating), EC 1.1.1.42), horse heart cytochrome c, the trisodium salt of m-isocitric acid, Tris, crystalline bovine serum albumin, P-mercaptoethanol, Coomassie brilliant blue, INT and MTT” (Sigma); bovine pancreatic trypsin and chymotrypsinogen A (Worthington); human hemoglobin (Mann); NAD and NADP (P-L Biochemicals); and Freund’s complete and incomplete adjuvants (Difco). Other reagents were of the best grade available from commercial sources. Spinach was purchased from a local distributor. Boiled spinach extracts were prepared as previously described (11). Ferredoxin was purified according to the procedure of Tagawa and Arnon (12), and chloroplasts were prepared according to Avron and Jagendorf (2). The chloroplasts were stored at 0°C in the sucrose-phosphate-KC1 isolation medium until assayed. Preparation of ferrodoxin-NADP reductase and specific antiserum. Ferredoxin-NADP reductase was isolated by acetone fractionation (acetone fraction) of crude spinach homogenates according to San Pietro and Lang (13). The acetone fraction was then chromatographed on DEAE-cellulose and fractionated between 40 and 65% saturation with ammonium sulfate (fraction AS-I) after Shin et al. (14). The enzyme was further purified by a sequence of three DEAE-cellulose columns followed by a second ammonium sulfate fractionation (fraction AS-II), according to Shin et al. (14). Preparations of the enzyme at this stage of purification had A436/A27i ratios of from 0.100 to 0.122, were approximately 80 to 90% pure as judged by disc gel electrophoresis, and contained only the low molecular weight (ca. 50,000) form of the enzyme (see below). Specific immune serum was produced in a rabbit by the method of Keister et al. (15) using 0.38 mg of fraction AS-II for the primary injection and 5.54 mg for the secondary injection. Nonimmune serum was collected from this rabbit prior to the primary injection; the immune serum was taken at intervals 1 week after the secondary injection. The sera were not further purified. Crude chloroplast extracts. Three types of spinach extracts were examined for the presence of multiple molecular weight forms of the reductase. The first was the acetone fraction described above. Another was obtained by overnight extraction of isolated chloroplasts with dilute buffer at room temperature after the procedure of Avron and Jagendorf (2). The -___.” Abbreviations used: MTT, 3(4,5dimethylthiamlyl-2).2,5-diphenyl tetrazolium bromide; INT, 2@iodophenyl)-3nitrophenyl&phenyl tetrazolium chloride; DEAE-, diethylaminoethyl.

REDUCTASE

667

third crude extract was obtained by isolating chloroplasts according to the method of Avron et al. (161, but using the sucrose-Tricine-NaCl buffer of McCarty and Racker (17), then precipitating the chloroplasts by adding a suspension of the chloroplasts to 20 volumes of cold acetone after Vambutas and Racker (18). After settling, the supernatant fluid was decanted and discarded and the precipitated chloroplasts allowed to dry on filter paper overnight at 4°C. The dried chloroplast preparation was finally extracted for 15 to 20 min with 50 mM Tris-HCl buffer (pH 8.0) at room temperature. The insoluble sediment was removed by centrifugation for 15 min at 12,700g. Sephadex G-100 column chromatography. Various fractions were analyzed by Sephadex G-100 column chromatography using either 2.5 x 80- or 1.8 x 80-cm columns. The enzyme elution profiles were routinely measured by the ferricyanide diaphorase assay (see below) although on occasion the transhydrogenase assay was employed. Samples of 0.5 or 1 ml were applied to the smaller columns and 2-ml samples to the larger. When chromatographing crude extracts, fraction AS-I or fraction AS-II, the columns were equilibrated and eluted with 50 mM Tris-HCl buffer (pH 8.0). Rechromatography of molecular weight peaks P-V, P-l, or P-2 involved concentrating the pooled fractions 5 or lo-fold using powdered sucrose over a dialysis sac containing the fractions to be concentrated. When chromatographing these pooled, concentrated fractions, the columns were equilibrated and eluted with a buffer composed of Tris-HCl (50 mM, pH 8.0), EDTA (1 mM) and bovine serum albumin (1 mgiml). The columns were calibrated using the following proteins as standards: lactic dehydrogenase (M, 140,000), hemoglobin (M, SS,OOO),chymotrypsinogen A (M, 25,000) and cytochrome c (M, 13,000); dextran blue 2000 was used to determine the void volume. Analytical procedures. The transhydrogenase and NADPH-dependent reductase activities were assayed using the standard reaction system of Keister et al. (19); unless indicated otherwise the pH was 8.6. The electron acceptor for the transhydrogenase assay was NAD (1 pmol), and the reaction was monitored at 340 nm with a Gilford recording spectrophotometer. For reductase activity measurements the acceptors and wavelengths monitored were K,,Fe(CN),, (1.4 /*mol, 420 nm); MTT (0.12 pmol, 560 nm); and INT (0.2 pmol, 500 nm). A unit of enzyme activity is defined as the amount of enzyme that catalyzes the reduction of 1 pmol of K,,Fe(CN),,/mm. The chloroplast-dependent photoreduction of NADP was followed by measuring the absorbance change at 340 nm after a 6-min exposure to white light (1200 fc) filtered through a water heat-shield. The reaction mixture contained in a total volume of

668

FREDRICKS

3 ml: NADP (1 Fmol), MgCl, (10 ymol), NaCl (100 pmol), Tris-HCl buffer, pH 8.0 (150 kmol), purified ferredoxin (3.3 pmol), and chloroplasts as indicated. Ouchterlony double diffusion analyses were done on 3.25 x 4-in. projector slide cover glasses layered with 8 ml of 0.6% ionagar in 50 mM Tris-HCl (pH 7.5), 0.02% sodium azide. Antiserum was added to the center well, and the plates were developed for 1 to 2 days at room temperature, then washed with 50 mM Tris-HCl (pH 7.5), 0.02% sodium azide at 4°C for 2 to 7 days. The washed plates were then stained with the standard reaction mixture with MTT (0.6 pmol) as electron acceptor or with Coomassie brilliant blue. Protein concentrations were estimated by the procedure of Lowry et al. (20) using bovine serum albumin as a standard. Chlorophyll was determined by the procedure of Arnon (21). Polyacrylamide disc gel electrophoresis was performed according to the alternate procedure of Davis (22). RESULTS

The first step in the purification of ferredoxin-NADP reductase is an acetone fractionation of spinach leaf homogenates (see Experimental Procedures). When this crude enzyme preparation was chromatographed on Sephadex G-100, two enzyme peaks were obtained (Fig. 1A). The first peak, P-l, had an apparent molecular weight of 126,000. The second peak, P-2, had an apparent molecular weight of 56,000 which is in the range usually obtained for purified reductase when chromatographed at pH 8.0. After further purification, fraction AS-I still showed both peaks, but the relative amount of P-l had been reduced. The pure enzyme (fraction AS-II) showed only the low molecular weight component (Fig. 1B). Elution patterns similar to those found with the acetone fraction were obtained when aqueous extracts of isolated chloroplasts (see Experimental Procedures) were chromatographed on Sephadex G-100. The relative activities in the peak fractions (i.e., P-I/P-2) in these experiments ranged from 0.36 (seen in Fig. 1A) to 1.12. No major changes in the elution patterns were seen after freezing and thawing the extracts or when the elution buffer was supplemented with 0.1 M NaCl or boiled spinach extract (1 mg/ml), shown previously (11) to contain inhibitors of the transhydrogenase activity of the reductase.

AND

GEHL ’

’

fi

.

A’

FIG. 1. Chromatography of three reductase preparations on Sephadex G-100. Fractions were eluted with 50 mM Tris-HCl (pH 8) and assayed by the standard ferricyanide diaphorase assay. (A), A crude acetone fraction, 24 mg, from spinach was chromatographed on a 2.5 x 80-cm column. (B), The purified enzyme, fraction AS-II, 0.37 mg, was chromatographed on a 1.8 x 77-cm column. (C), Two milliliters of a crude reductase preparation, extracted from acetone-precipitated chloroplasts, was chromatographed on a 2.5 x 83-cm column.

Attempts to rechromatograph P-l, after pooling and concentrating the peak fractions, using the buffer system used for the crude extracts, resulted in loss of enzyme activity. It was found that bovine serum albumin and EDTA stabilized the enzyme. Thus in those experiments where P-l, P-2, and P-V (see below) were rechromatographed, the elution buffer was supplemented with EDTA and bovine serum albumin. When P-2 was rechromatographed on a smaller column, a single enzyme peak was found with essentially the same molecular weight, i.e., 50,000. Rechromatography of P-l showed that 64% of the activity stayed in the high molecular weight form, estimated in this case at 117,500, but a smaller portion appeared in the low molecular weight range, 50,000. Since we pooled those fractions near the center of the peak, it is unlikely that our P-l fraction was contaminated with P-2. It is more likely that P-l broke down to some extent, generating some of the P-2 component. This would indicate that these two enzyme frac-

FERREDOXIN-NADP

REDUCTASE

tions might be structurally related, e.g., P-l a dimer of P-2. Another aliquot of P-l from Fig. 1A was chromatographed after storage for 4 days in a freezer. The elution pattern was unchanged, indicating that no additional breakdown occurred on storage at -17°C. The purified reductase has both diaphorase activity and transhydrogenase activity. To test whether this were also true for both fractions P-l and P-2, crude extracts were again chromatographed on Sephadex G-100, and peak fractions pooled. In this case the diaphorase activities of both peaks were approximately the same per unit volume (Table I). It is further shown that both fractions P-l and P-2 had transhydrogenase activity, and the ratio of activities was about the same. In order to evaluate the structural relationship of fractions P-l and P-2 further, antibody was prepared in a rabbit against purified reductase (fraction AS-II). This enzyme preparation contained only component P-2, the low molecular weight form of the enzyme. It is shown in Fig. 2A that the transhydrogenase and diaphorase activities of both forms of the enzyme were inhibited to the same extent by a given antiserum:enzyme ratio; note that five times as much enzyme was used for the transhydrogenase as for the diaphorase experiments. Nonimmune serum at comparable levels had a slight stimulatory effect on the diaphorase activity and a slight inhibitory effect on the transhydrogenase activity in these experiments, The conclusion drawn from these experiments is that

100

TABLE ENZYME

ACTIVITIES

Fraction

Enzyme activity (+nol reduced h-’ ml -‘i Fe(CN),:’

P-l P-2 P-l/P-2 tio

I

OF FRACTIONS

ra-

20.1 19.4 1.04

NAD

0.275 0.241 1.14

P-l

AND P-2”

Ratio of activities (diaphoraseitranshydrogenase)

73 80 -

” Enzyme activities were measured according to the standard assay procedures using 0.2 ml of fractions P-l or P-2 for determination of diaphorase activity and 2 ml for determination of transhydrogenase activity.

669 r

A

50

c

L

. 0' 0 pl

20 Antlserum/Umt

Enzyme

40 Actwty

FIG. 2. Effect of antiserum on activities of various reductase preparations. The data summarize a number of experiments and have been normalized by expressing the values on the abscissa in units of microliters of antiserum/unit of enzyme activity. The effect of immune serum was corrected for the effect of nonimmune serum. (A), Varying amounts of antiserum were incubated for 5 min with either fraction P-l (open symbols) or P-2 (closed symbols). The mixtures were then assayed for ferricyanide diaphorase (circles) or transhydrogenase activity (sauaresl. (B). Purified reductase was incubated with varying amounts of antiserum as above but supplemented with bovine serum albumin (1 mg/ ml). The NADPH-dependent reductase activities were measured using as acceptors: K,,Fe(CNl, CO), MTT CA), INT (0) and NAD (0).

either P-l and P-2 are immunologically related or undetectable levels of P-l, present in the purified reductase preparation, elicited titers of antibody fortuitously identical to those made against P-2. Table II reports experiments designed to differentiate between these alternatives. Prior incubation of P-2 with antiserum effectively reduced the level of antibody directed against P-l, so the percentage inhibition of a mixture of the two was considerably less than expected if two antibodies were acting independently. These experiments clearly demonstrate that P-l and P2 are immunologically related. A reductase preparation, estimated to be 80 to 90% pure (Fig. 3A), catalyzed the NADPH-dependent reduction of Fe(CN)i’+, NAD, MTT, and INT. The data from several experiments are summarized in Fig. 2B. All four enzyme activities of the puri-

670

FREDRICKS

AND

TABLE ANTIBODY

Antiserum (FlI

GEHL

II

COMPETITION

EXPERIMENTS”

Experiment 1 Diaphorase activity (pmol reduced/h) 0

4.5

Experiment 2 Diaphorase activity (pm01 reduced/h) %I”

0

3.0

%I

4.26 4.26 0.16 96.2 0.56 86.9 3.36 3.36 0.20 94.0 0.06 98.2 7.62 4.42 42.W’ 7.62 1.50 80.3’ .‘I During the first stage of these experiments, 0.2 ml of P-2 or an equal volume of buffer was incubated with and without antiserum for 5 min. During the second stage 0.15 ml of P-l or buffer was added where indicated, incubated for an additional 5 min, and assayed for ferricyanide diaphorase activity. h %I, percentage inhibition of diaphorase activity due to added antiserum. ” ” If P-l and P-2 were inhibited by two separate antibodies in the antiserum preparation, the %I expected would have been: c, 97.1; d, 90.0. Buffer + P-l P-2 + buffer P-2 + P-l

FIG. 3. Analysis of the purified reductase. (A), Disc electrophoresis of purified reductase. The enzyme solution, containing 13.9 pg of protein, was mixed with the marker dye, bromophenol blue, and layered onto the surface of the 2.5% spacer gel (pH 6.7). The 7% separation gel had a pH of 8.9. A current of 2 mA/gel was applied. The gels were stained with a 0.1% solution of Coomassie brilliant blue in methanol:acetic acid:water (1:1:8) and destained with the same solvent without the dye. The gels were analyzed with a Gilford Model 2410s scanner. The marker dye was arbitrarily assigned a relative mobility of 1. (B), Chromatography of purified reductase of DEAE-Sephadex A-50. A column, with 0.055 M Tris2.5 x 80 cm, was equilibrated HCl, pH 7.3. Enzyme equivalent to 122 mg ofprotein was applied. The reductase was eluted with a TrisHCl (pH 7.3) buffer gradient, Three-milliliter fractions were collected. The ferricyanide diaphorase activity of the reductase was assayed by standard procedures.

lied reductase were inhibited by the antiserum, and all four activities showed a similar degree of sensitivity to antiserum.

These data indicate that MTT and INT can serve as electron acceptors in the reductase reaction catalyzed by ferredoxinNADP reductase. Although it had not been previously reported that these tetrazolium salts could serve such a role, it is perhaps not surprising since a large number of acceptors has been demonstrated. The tetrazolium reductase reaction was used to localize the reductase-antibody complex on immunodiffusion analysis of fractions P-l and P-2. A single band was obtained for P-l, for P-2, and for a mixture of P-l and P-2. When P-l and P-2 occupied adjacent wells in the plates, each band fused with the other to give a line of identity, indicating that P-l and P-2 are immunologically identical. We do not know whether P-l was completely dissociated into P-2 during the course of these experiments. In addition to analyzing the acetone fractions of whole leaf homogenates and aqueous extracts of chloroplasts on Sephadex G-100 columns, we did similar experiments using an enzyme extract prepared from isolated chloroplasts which had been precipitated in cold acetone according to the procedure of Vambutas and Racker (18) (see Experimental Procedures). In two experiments we observed an elution pattern similar to that shown in Fig. lA, i.e., two activity peaks. In three experiments a third peak (P-V) was observed which was eluted at the void volume of Sephadex G100 (Fig. 1C). Rechromatography of fraction P-V gave an elution pattern comprised of two peaks corresponding to P-l

FERREDOXIN-NADP

and P-2, suggesting that the larger component dissociated into the smaller components. A cursory survey of enzyme extracts prepared from chloroplasts, as in Fig. 1C but from lettuce, parsley, and tobacco, showed a major peak corresponding to P-2 and very little, if any, P-l or P-V. On the other hand, preliminary experiments with Chlamydomonas reinhardi (strain 137~) indicated the presence of two molecular weight forms, P-V and P-2. Analysis of the purified reductase preparation by acrylamide disc gel electrophoresis showed six protein peaks (Fig. 3A). When a duplicate gel was stained, using the NADPH-tetrazolium reductase reaction to localize the reductase, only peaks 2 and 3 were positive. These results suggested that the reductase exists in two different forms, tentatively designated P2a (peak 2) and P-2b (peak 3). Protein peaks 1, 4, 5, and 6 are unidentified contaminants. Integration of peaks 2 and 3 indicated that the reductase represented approximately 80% of the total protein in this particular preparation. These two forms of the reductase were separated by elution with a shallow buffer gradient from DEAE-Sephadex A-50 (Fig. 3B). In this experiment no enzyme was eluted in the first 400 fractions. Since these two forms of the reductase were not resolved by chromatography on Sephadex G-100 (Fig. 1B) but could be separated by disc electrophoresis or chromatography on DEAE-Sephadex, we suggest that reductase forms P-2a and P-2b have essentially the same molecular weight and are different ionic forms of the flavoprotein. Selected fractions from this chromatographic experiment were further analyzed by disc electrophoresis in acrylamide gels (Fig. 4). The gels were stained for protein components. Fractions 445 and 455 contained a mixture of reductase species P-2a and P-2b as observed in the reductase preparation prior to chromatography on DEAE-Sephadex (Fig. 3A). This is what would be expected since these fractions were taken from the region where there was overlap of the two reductase activity peaks. Since fraction 430 contained only peak 2 and fractions 470, 485, and 500 con-

REDUCTASE

671

tained only peak 3, we concluded that reductase component P-2a was eluted in fractions 415 and 465 and component P-2b was eluted in fractions 440 and 510. The major protein contaminant (peak 4) was completely removed by DEAE-Sephadex chromatography and peaks 1 and 6 were barely detectable. The other contaminant protein (peak 5) was concentrated in fractions eluted just before the reductase components. Duplicates of the gels described in Fig. 4 were stained by the NADPH-tetrazolium reaction to localize reductase activity. Two bands were detected in fractions 445 and 455. A careful analysis of the relative mobilities of these bands clearly demonstrated that the upper band (P-2a) in each case corresponded to protein peak 2 and the lower band (P-2b) to peak 3. Qualitatively, the intensity of staining in these bands correlated with the relative amounts of protein measured in Fig. 4. Fraction 430 gave a single band with the relative mobility of peak 2; the contaminant protein 4 did not catalyze the NADPH-tetrazolium reductase reaction. Likewise, fractions 470, 485, and 500 gave a single band with the relative mobility of peak 3. Ouchterlony double diffusion analysis of the reductase preparation described in Fig. 3A, using antiserum prepared by injection of this enzyme preparation, routinely gave two precipitin lines (data not shown). Only one of these lines, however, had residual NADPH-tetrazolium reductase activity. Using this antiserum preparation, we analyzed the chromatographic fractions for antigen-antibody reactions. Each fraction gave a single precipitin line stainable by the NADPH-tetrazolium reductase reaction. The reductase components of fractions 430 (P-2a) and 500 (P-2b) gave a line of identity. In addition, fractions 430 and 445 have a clearly discernible precipitin line that lacked reductase activity. This would correspond to protein component 5 of Fig. 3A. Since this component in fraction 430 showed a line of nonidentity with the reductase line in fraction 500, we concluded that this component was not a denatured form of the reductase. AS shown in Fig. 5, antiserum prepared

672

FREDRICKS

Relative

Moblllty

AND

GEHL

Relative

Moblllty

FIG. 4. Disc gel electrophoresis of DEAE-Sephadex A-50 fractions. Selected fractions (see arrows in Fig. 3B) were analyzed by disc electrophoresis and stained for protein as in Fig. 3A. The volume of each fraction applied was adjusted to have an amount of enzyme activity equivalent to 13.9 yg of reductase protein. The numbers 1 through 6 on the upper abscissa mark the locations of the six protein peaks seen in Fig. 3A.

against purified reductase inhibited the photoreduction of NADP by chloroplasts in agreement with a previous report by Keister et al. (15); nonimmune serum had a slight simulatory effect. Addition of purified reductase protected the chloroplasts from inactivation by immune serum. These data indicate that the antiserum inhibited the ferredoxin-NADP reductase activity of the chloroplast-bound enzyme.4 Antiserum also inhibited the light-independent reductase activities using ferricyanide or INT as acceptors. Experiments using extracts of whole cells, suggest that there are several enzymes in the cell that can catalyze an NADPH reductase reaction. However, the data in Fig. 5 indicate that at least 90% of the reductase activity of the chloroplast is catalyzed by ferredoxin-NADP reductase. Since the antibody-reductase complex itself can still catalyze INT reduction, one would not expect 100% inhibition of this activity even at high antiserum levels. Thus the reductase probably accounts for more than 90%, and possibly all, of the NADPH-INT reductase activity of the chloroplast. 4 Under the conditions of this experiment, 70 to 60% of the endogenous reductase remained associated with the chloroplasts. The interaction of antibody with chloroplast-bound reductase is also implied in the experiments of Berzborn (31.

DISCUSSION

Three different methods of extracting ferredoxin-NADP reductase from chloroplasts gave extracts containing multiple molecular weight forms of the enzyme. Thus the presence of these various forms of the enzyme does not seem to be simply an artifact of isolation. The 117,500-molecular weight component (P-l) appeared to be more stable than the larger component (PV), but both tended to dissociate to yield the 50,000-molecular weight form (P-2). This observation is consistent with the immunological data which showed that at least components P-l and P-2 were structurally related. Purified preparations of the reductase (P-2) contained two forms of the flavoprotein. These two forms were immunologically indistinguishable and had essentially the same molecular weight. Since they were separable by ion-exchange chromatography and disc electrophoresis, we concluded that they represent two alternate ionic forms. It is not clear what conditions might affect the relative proportions of these components, nor is it clear which component might represent more closely the physiological state of the reductase. It is possible that these various species are a reflection of the two physiological func-

FERREDOXIN-NADP

REDUCTASE

tions proposed for the reductase, i.e., as electron carrier in cyclic and noncyclic photophosphorylation. Avron and Jagenforf (2) showed that the reductase is bound to the chloroplast membrane and is slowly leached from isolated chloroplasts under aqueous conditions. The extraction does not, however, follow simple first-order kinetics (unpublished data), and this observation might be related to the existence of different forms of the reductase, i.e., different molecular associations, within the chloroplast membrane itself. The reductase is known to interact with several membrane components. Its interaction with ferredoxin has received the most attention. The reductase forms a 1:l complex with ferredoxin (6, 7), and this complex is sensitive to ionic strength. It is unlikely that a reductase-ferredoxin complex alone can account for the higher molecular weight forms described in this paper since (a) P-l did not dissociate to a greater extent in the presence of 0.1 M NaCl than in dilute Tris buffer alone, (b) chromatography of P-2 using a buffer supplemented with ferredoxin did not shift the molecular weight to the 117,500 range, and (c) a reductase-ferredoxin complex would be expected to have a molecular weight on the order of only 60,000. Both cytochrome f (23) and plastocyanin (10) can serve as electron acceptors in reactions catalyzed by the reductase and, therefore, can interact with the reductase. Both are also bound to the chloroplast membrane, but there is no evidence of stable complexes between ferredoxin-NADP reductase and these electron carriers. It is conceivable that P-l is a dimer and P-V a higher-order aggregate of P-2, but again there is no evidence to support this contention; and there are no instances in which fraction P-2 or purified reductase has been shown to form higher aggregates, except, of course, on crystallization of the purified enzyme. Recently Schneeman and Krogmann (4) have described the purification of an 85,000-molecular weight form of spinach ferredoxin-NADP reductase, containing 2 equiv of flavine-adenine dinucleotide/mol

100

673 . .

/’

.

. . :I 0) . . 0

02

)J,I Antlserum/pg

04

“I

Chlorophyll

FIG. 5. Effect of antiserum on the photoreduction of NADP by spinach chloroplasts and on light-independent reactions of isolated chloroplasts. This figure summarizes a number of experiments and the data have been normalized by expressing the values on the abscissa in units of microliters of antiserum/ microgram of chlorophyll. Chloroplasts were incubated in the presence (Ml and absence (0) of 280 pg of purified reductase wit,h varying amounts of antiserum for 5 min at room temperature. The photoreduction of NADP was measured at pH 8 as described in Experimental Procedures. Similarly, the effect of antiserum on the NADPH-dependent, light-independent reactions of chloroplasts using K,Fe(CNl,, (0) and INT (01 as acceptors was determined.

of enzyme and composed of nonidentical subunits of 34,000 and 48,000. This form of the enzyme was reported to be a rather stable heterodimer. At this time we are unable to evaluate the relationship between this reductase species and those described in this paper. However, Schneeman and Krogmann did find that their enzyme preparation was inhibited by the same antibody preparation used in these studies. Keirns and Wang (24) reported that reductase, isolated from spinach by a procedure different from that used here, gave one major ionic species and two minor forms on isoelectric focusing. We cannot be sure whether the ionic forms they observed by the exquisitely sensitive isoelectric focusing technique resulted from the same molecular alterations as the ionic forms that we were able to separate by ionexchange chromatography. Keirns and

674

FREDRICKS

Wang implied that the alternate ionic forms they observed have no functional significance. Indeed, we did not find any differences between forms P-2a and P-2b with regard to the types of reactions catalyzed or their interaction with ferredoxin. However, in view of the extensive literature documenting the importance of chemical modification in regulating enzyme activity, we feel it is premature to dismiss as insignificant the manifold forms in which the reductase has been isolated. Finally, we conclude that the NADPHtetrazolium reductase activity observed is a property of ferredoxin-NADP reductase and greater than 90% of the light-independent NADPH-tetrazolium reductase activity of isolated chloroplasts is due to this flavoprotein. ACKNOWLEDGMENTS We would like to thank Dr. J. B. Courtright for his helpful discussions on the immunological experiments and Dr. R. P. Levine of Harvard University for sending us cultures of Chlamydomonas reinhardi. REFERENCES W. W., AND GEHL, J. M. (1973)Fed. Proc. 32, 477. AVRON, M., AND JAGENDORF, A. T. (1956) Arch. Biochem. Biophys. 65, 475-490. BERZBORN, R. J. (1968) in Progress in Photosynthesis Research, Proceedings International Congress on Photosynthesis Research (Metzner, H., ed.), Vol. 1, pp. 106-110, Tubingen. SCHNEEMAN, R., AND KROGMANN, D. W. (1975) J. Biol. Chem. 250, 4965-4971. FOUST, G. P., AND MASSEY, V. (1967) Fed. Proc. 26, 732. SHIN, M., AND SAN PIETRO, A. (1968) Biochem.

1. FREDRICKS, 2.

3.

4. 5.

6.

AND

GEHL

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