PROTEIN

EXPRESSION

AND

3, 228-235

PURIFICATION

(19%)

Ferredoxin:NADP Oxidoreductase of Cyanophora paradoxa: Purification, Partial Characterization, and N-Terminal Amino Acid Sequence U. B. Gebhart,

T. L. Maier,

Botanical

and Chemical

Received

Institute

November

1, 1991,

and

S. Stevanovi6, Institute,

in revised

form

M. G. Bayer,

University

March

of Tiibingen,

Ferredoxin:NADP+ oxidoreductase, an important photosynthetic enzyme, has been independently discovered several times, e.g., as “NADPH-dependent diaphorase” (1) and as “pyridine nucleotide transhydrogenase” (2). Shin et al. (3) were able to demonstrate that FNR (EC 1.18.1.2) is a flavoprotein with various enzymatic activities. The enzyme has been detected in all investigated oxygen-evolving photoautotrophs such as cyanobacteria (4-ll), algae (12-14), and higher plants (e.g., (15-19)). Its physiological role (in photosystem I) is the reduction of NADP+ with ferredoxin in the final step of the photosynthetic electron transport chain. Accordingly, the enzyme is bound to the outer side of the thylakoid membranes in cyanobacteria (20) and in plastids (21,22), forming a protein complex (23,24) with ferredoxin on its one side and with a lo-kDa connectein (25) and the thylakoid-intrinsic protein (17.5 kDa) (26)

228

correspondence

should

be addressed.

Germany

26, 1992

The ferredoxin:NADP+ oxidoreductase of the protist Cyanophoraparadoxa, as a descendant of a former symbiotic consortium, an important model organism in view of the Endosymbiosis Theory, is the first enzyme purified from a formerly original endocytobiont (cyanelle) that is found to be encoded in the nucleus of the host. This cyanoplast enzyme was isolated by FPLC (19% yield) and characterized with respect to the uvvis spectrum, pH optimum (pH 9), molecular mass of 34 kDa, and an N-terminal amino acid sequence (24 residues). The enzyme shows, as known from other organisms, molecular heterogeneity. The N-terminus of a further ferredoxin:NADP+ oxidoreductase polypeptide represents a shorter sequence missing the first four amino acids of the matUre enzyme. 0 1992 Academic press. h.

’ To whom

and H. E. A. Schenkl 7400 Tiibingen,

on its other. The three-dimensional structure of the spinach FNR (carboxyl terminus at residue 314), determined by X-ray diffraction (27,28), is composed of two domains, the FAD-binding domain (residues 19 to 161) and the NADP-binding domain (residues 162 to 314). The amino acid sequence of these two domains is highly conserved and the atomic structure seems to be the prototype for a structurally novel flavoenzyme family (28). In view of the Serial Endosymbiosis Theory (29,30), it is of interest that this old enzyme is one of the nucleus-encoded chloroplast proteins in higher plants, synthesized on 80s ribosomes as a preprotein (31,32) and imported post-translationally into chloroplasts. Although the Serial Endosymbiosis Theory is now generally accepted, definitive proof is lacking and questions regarding postulated gene transfer and the evolution of the proteinimport machinery in organelles remain unanswered. Cyanophora paradoxa Korsch. (Glaucocystophyceae) (33,34) is a unicellular, biflagellated algal protist with originally symbiotic intracellular cyanobacteria (cyanelles (35); for details see (36-39)). In general, endocyanomes (symbiotic consortia of eukaryotes with intracellular cyanobacteria) and their descendants, the metacyanomes (39), are important model organisms for studying the mechanistic problems of plastid evolution (40). Surprisingly, the genome (127 kb) is reduced to the size of that of the chloroplasts (41) and 80 to 90% of the cyanellar proteins are nucleus-encoded and must be imported (42,43). We wanted therefore to investigate the situation of FNR2 in Cyanophora paradoxa. The purified enzyme has been used as an antigen for preparing a polyclonal monospecific antibody. With this antibody

’ Abbreviations used: DEAE, diethylaminoethyl; FNR, ferredoxin:NADP+ oxidoreductase; FPLC, fast protein liquid chromatography; PAGE, polyacrylamide gel electrophoresis; R,, relative electrophoretic mobility; SDS, sodium dodecyl sulfate. 1046.5928/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

Cyanophora

and transcriptional and translational inhibition experiments, we were able to demonstrate that FNR is one of the nucleus-encoded cyanoplast (formerly cyanellar) proteins (44). In uitro translation experiments with poly(A)+ RNA corroborated this result (45). The present study describes for the first time the isolation and purification of a protein (FNR) that has suffered a gene transfer from a prokaryotic symbiont (cyanobacterium) to a eukaryotic host nucleus. Furthermore the C. paradoxa FNR is partially characterized with respect to some molecular properties. MATERIALS

AND

METHODS

algal

cells homogenization

A

(crude

extract)

1

ammonium the pellet

B

sulfate-precipitation, and dialysis

k

CI

anion

exchange

CI

cIII

chromatography (eluates

4

Organism, Growth, and Harvest C. paradoxa B 29.80, Pringsheim strain (Sammlung von Algenkulturen der Universitat Gottingen, Pflanzenphysiologisches Institut, Gottingen, Germany) was continuously grown as previously described (45). The relative cell number was controlled by in uiuo vis spectroscopy using a Beckman Acta V photometer as described earlier (46). Cultures were diluted or cells were harvested in the late logarithmic growth phase, i.e., on the fourth day of cultivation (approximately 2 to 4 x lo7 cells per milliliter). Cells were harvested by centrifugation (3 min, 500g) and the pellet was washed five times by suspension in growth medium and repeated centrifugation. After the last centrifugation (3 min, lOOOg), the wet weight was estimated, average values of 0.8 g per liter culture were obtained. The harvested cells were stored at -25°C. of FNR

All preparation steps were performed at 4’C under green security light or with darkened containers. The procedure is an adaption of different instructions (for other organisms) to the conditions in C. parudoxa summarized in Scheme 1. (a) Crude extract. The frozen cells (10 g wet weight from 12 liter culture) were thawed, suspended in (5 ml/ g) homogenization buffer (50 mM Tris/HCl, pH 7.5, 5 mM 2-mercaptoethanol, 0.05 mM phenylmethylsulfonyl fluoride) and equilibrated with dinitrogen (1000 psi) in a Parr bomb for 1 h (4°C). The cells were disrupted by forcing them through the outlet valve. The resulting cell homogenate was incubated in the dark with lysozyme (3.5 mg/g wet weight) for 30 min to degrade the murein

\ affinity CI Or of I D

I E

(pure

DEAE

column)

(dialysates

of

chromatography dCII and

dialysis

(2'5'-ADP-Sepharose of collected

(dialysate second

from

(DEAE)

dCIII

dcII

were obtained from different supammonium peroxosulfate and (Munich), 2’,5’-ADP-Sepharose 5/5 from Pharmacia (Freiburg), 32 Servacel from Serva (Heidel-

of

(dialysate)

4

The chemicals used pliers, in particular, TEMED from Bio-Rad 4B and Mono Q HR and DEAE-Cellulose berg).

solubilization

I

Chemicals

Purification

229

FERREDOXIN-NADP+-OXIDOREDUCTASE

D)

anion

CII

and

C,,,) 4B) fracti

of .ons

DII exchange

chromatography

(Mono

Q)

FNR)

dialysis dE

(dialysate

of

E)

SCHEMES

sacculus (47) of the cyanoplasts. Homogenization buffer (up to 10 ml/g wet weight) and Triton X-100 (2%, v/v) were then added and the homogenate was stirred in the dark for 7 h to solubilize the membrane-bound proteins, including FNR (11). Finally, the homogenate was centrifuged (20 min, 40,OOOg). The resulting supernatant was designated as crude extract (A) and was used for further purification. (b) Ammonium sulfate fractionation. Solid ammonium sulfate was added to A to give 33% saturation. The pH of the solution was kept constant by titration with 1 M Tris. This suspension was incubated for 30 min and then centrifugated (20 min, 40,OOOg). The pellet was discarded, and ammonium sulfate was added to the supernatant to give 75% saturation. After 1 h incubation, it was centrifuged (20 min, 4O,OOOg),and the resulting supernatant was discarded. The precipitate was collected, solubilized in few milliliters of buffer A (50 mM Tris/ HCl, pH 7.5,0.1 mM EDTA), dialyzed for approximately 15 h (overnight) against two 5-liter volumes of buffer B (0.1 mM EDTA, 20 mM Tris/HCl, pH 7.5), and designated dialysate B. (c) Anion-exchange chromatography on DEAE-cellulose. Dialysate B (34.5 ml, ca. 300 mg total protein) was applied to a DEAE-Cellulose 32 column (2.5 X 18

230

GEBHART

cm) equilibrated with buffer A. Washing of the column with 300 ml buffer A resulted in elution of most of the enzyme that did not bind to the column, designated eluate Ci. A linear NaCl gradient (0 to 0.5 M NaCl in buffer A; 400 ml), used to ensure that all of the enzyme was eluted, yielded two small peaks of low activity (eluates C,, and C,,,). These two fractions were collected and dialyzed against buffer B, obtaining dialysates dC,, and GII, respectively. (d) Affinity chromatography on 2’,5’-ADP-Sepharose 4B. Eluate Cr (300 ml corresponding to 33 mg protein) was loaded onto an affinity chromatography column (1 X 12.7 cm) equilibrated with buffer C (10 mM Tris/HCl, pH 7.5, and 0.1 mM EDTA). After the column was washed with buffer C, the enzyme was eluted using a NaCl gradient (O-O.6 M NaCl; 50 ml). Fractions 8-14, which contained the most enzyme activity (Fig. l), were combined and dialyzed against buffer D (6 mM Tris/ HCl, pH 7.5, and 1 mM EDTA) (dialysate D). (e) Anion-exchange chromatography on fast protein liquid chromatography (FPLC) Mono Q. Dialysate D was applied to an FPLC Mono Q HR 515 column (0.5 X 5 cm; no more than 3 mg of protein per column run) equilibrated with buffer B and washed with the same buffer. Protein was eluted with a linear gradient of NaCl (O-O.5 M; 10 ml) and l-ml fractions were collected. Enzyme activity was eluted between 80 and 120 mM NaCl. The corresponding fractions (2 X 1 ml) were combined (sample E) and their purity was confirmed by disc electrophoresis and by the specific absorbance spectrum between 250 and 750 nm (see below). The pure enzyme preparation was stored at -25°C. Native Disc Electrophoresis Electrophoretical separation of native protein was performed using flat-bed PAGE as described by Maurer (48). The separation gel (thickness, 2 mm; separation length, ca. 8 cm) contained 10% polyacrylamide and the stacking gel 3% (conditions: 4°C 100 mA, 600 V, 2 h). The native FNR was stained (diaphorase activity) for 30-40 min at room temperature according to the method of Harris and Hopkins (49). SDS-PAGE

and Mole Mass Estimation

One- or two-dimensional ultra-thin-layer SDSPAGE of pure FNR, followed by silver staining, was performed as previously described (44) with the following variations for isoelectric focusing (first dimension): NP-40 was not used, and Servalytes (7%) were added in the ratio (pH 3-lO):(pH 4-6):(pH 5-8) = 1:2:4. Ultraviolet-

Visual Spectrum

The FNR preparation was checked for purity by measuring the spectrum from 250 to 750 nm. The FNR mo-

ET

AI,.

lar concentration was estimated (9), using the molar extinction coefficient of bound flavin and a molecular mass of approximately 34 kDa for the C. paradoxa FNR, obtaining a molar extinction coefficient tdGO= 98OO/(M X cm) for FNR. The purity of FNR preparations can be expressed by a quotient of A,,,IA,,, < 8 (1). Protein

Determination

Protein was determined

by the method of Bradford (50).

Enzyme Assays and the pH Optimum Diaphorase Activity

of FNR

Two different enzymatic activities were used to localize the enzyme in the fractions: transhydrogenase and diaphorase activity. Transhydrogenase activity was measured according to the method of Bijger (13) and modified according to (2). The assay contained 0.1 M Tris/HCl, pH 9 (800 pl), 0.5 mM NADP (20 PI), 50 mM NAD (20 pl), 0.1 M MgCl, (20 pl), 0.25 M glucose-6-phosphate (20 pl), glucose-6-phosphate-dehydrogenase (Boehringer) (20 ~1 = 1.1 U) enzyme sample (50-100 ~1, ca. 5 U), and double-distilled water up to 1 ml. One unit of enzyme activity was defined as E,,, = 0.01/(3 min) (2). The transhydrogenase assay requires higher concentrations of the enzyme and is less reproducible than the diaphorase assay. It was therefore used only to confirm t.he identification of FNR. FNR activity was usually determined by the enzyme’s diaphorase activity, i.e., transfer of electrons from NADPH to dichlorphenol-indophenol, an electron acceptor (5,9). The assay required only small amounts of sample since FNR had a high diaphorase activity and the assay was reproducible. The standard reaction mixture contained 55 mM TrisJHCl, pH 8.5 (900 ccl), 4.25 mM dichlorphenol-indophenol (20 pl), 1.8 mM NADPH (50 pl), enzyme sample (5-30 ~1 = ca. 50 U), and water up to 1 ml. The reaction was initiated by adding enzyme. Reduction of dichlorphenol-indophenol was measured at 600 nm for 60 s. One unit of activity is defined as E,,, = l.O/min (5); specific activity is given in units per milligram of protein. The pH optimum of diaphorase activity was estimated between pH 5.5 and 10.9, using three buffers: 0.1 M histidine/KOH (pH 5.5-7.5), 0.1 M Tris/HCl (pH 7.09.3), and 0.1 M glycine/NaOH (pH 8.6-10.9). Samples of dialysate B were used for the determination. Amino Acid Sequence Analysis For the sequencing procedure, frozen samples of the purified protein (see above) were thawed and desalted against distilled water by using floating membrane filters (Millipore). The analysis was performed by automated Edman degradation in a pulsed-liquid protein sequencer (Model 477 A) equipped with an on-line phen-

Cyanophora

231

FERREDOXIN-NADP’-OXIDOREDUCTASE

ylthiohydantoin amino acid analyzer (Model 120 A, Applied Biosystems). All reagents and solvents were from Applied Biosystems. A trifluoroacetic acid-activated glass fiber filter was coated with 1 mg of BioBrene Plus prior to sequencing. Four analyses were performed using between 30 and 600 pmol of total protein (FNR purity factor, O.D. A,,,lA,,; Pr = 6.8) applied to the filter disk for each run. The standard programs BEGIN-l and NORMAL-l (Applied Biosystems) were used.

I

I

1

1

1

.O LOO -

. I\

...- . ... l

I /a/..-.“’ . .. ...‘.a

200 -

...* *...** . ...-* i 0 - ~.-.-C*--C~ **.. I 0

Purification

. ..*

I 5

....-*

. ..*

....**

. ..*...-

..... .

. ..-

./ . ..-

RESULTS AND DISCUSSION Although some biochemical FNR preparations are described (e.g., (2,3,5,7-9,ll)) no one method achieved a satisfying result when it was used for purification of C. paradoxa FNR. One difficulty lies in the small amount of alga available and the other in the unsatisfactory purification results of the given methods. Usually we could start with 10 g wet weight of algal cells. This corresponds to a maximum of 4.5 mg FNR within the crude extract. For the FNR antibody preparation (44) we needed at least 200 pg purified FNR, so we combined different methods and introduced additionally anionexchange chromatography on a Mono Q column by FPLC. This successful step is now the last in our FNR purification procedure. Estimation of ferredoxin:NADP -oxidoreductase activity in the dark blue-green crude extract was difficult. Transhydrogenase activity was impossible to measure since the optical density at 340 nm of the crude extract was too high. Further dilution of the crude extract lowered the enzyme activity to the point at which it was no longer reproducible. In contrast, the diaphorase assay was reliable and therefore considered the method of choice, although other diaphorase activities also exist in the crude extract (e.g., NADP-dependent diaphorases in the mitochondria). It could be demonstrated that the FNR content in the cytosol is low enough to be neglected (data not shown). The following description of protein purification is summarized in Scheme 1. The results of the FNR purification are as follows: The first ammonium sulfate precipitation (33% saturation) separated most of the pigments, including all chlorophyll from the supernatant. With the second precipitation (75% saturation) all diaphorase activity was concentrated in the pellet that also contained phycobiliproteins. The phycobiliproteins were completely retained on the DEAE column, whereas 84% of the loaded enzyme activity was found in the first eluates of the ion-exchange column (C,). This observation of nonbinding of FNR to the DEAE-cellulose had been previously described only once, in fact for the Nicotiana tabacum FNR (51). Low levels of FNR activity, amounting to approximately 4% of that of Cr, could be eluted from the column by NaCl gradient elution between 75 and

I

600 -

\

..-

\

t o-o’

CCe-e-*-e-.-m-e-

“0~>p-

01 10

15 Fraction

I 20 number

I 25 ( 2 ml 1

FIG. 1. Affinity chromatography of C. paradoxa ferredoxin:NADP+ oxidoreductase on 2’,5’-ADP-Sepharose 4B: elution profile of diaphorase activity during gradient elution of adsorbed samples C, and dCn (see Scheme 1 and text).

125 mM NaCl (Cii; dialyzed dC,,) and between 240 and 290 mM NaCl (Cm; dialyzed d&i). The subsequent affinity chromatography of C, or of dC,, on 2’,5’-ADPSepharose 4B gave the elution profiles as illustrated in Fig. 1. This step reproducibly resulted a great loss of enzyme activity (see Table l), which is in contrast to the results of Serrano and Rivas (ll), who recovered 63% of enzyme activity combined with remarkable purification. Sample dCn, was also similarly chromatographed (data not shown). Enzyme activity of dC,, and dCm after affinity chromatography was barely detectable. Therefore, these samples were not purified further. Nevertheless, the affinity chromatography did not bring about satisfactory purification; moreover about f of the enzyme was lost. So in the final purification step, a second anion-exchange chromatography (Mono Q) of D was essential. To our knowledge Mono Q has not been used in FNR purification yet. This step achieved the highest purification factor of 5.1, resulting in a homogenous sample (purified enzyme E; Fig. 3~). The yield andpurification factor of each FNR purification step are shown in Table 1. The final yield was about 19%. FNR composed nearly 0.4% of all soluble proteins in the crude extract. Partial

Characterization

of FNR

FNR has a typical flavoprotein uv-vis spectrum (Fig. 2). The FNR preparations were checked for purity by the A,,,IA,,, ratio, the ratio of the absorption maxima of the protein component and the enzyme flavin component (1). FNR must have a uv-vis spectrum between 250 and 750 nm with maxima at 280,385, and 458 nm and a marked absorption minimum at 320 nm. Furthermore, the absorption ratio A,,,/A,,, must be equal to or greater than 1. The following A458/A385 values of various FNR preparations were determined from published spectra: 1.03 (l), 1.2 (52), 1.1 (13), 1.07 (5), 1.1 (9). The

232

GEBHART

ET

TABLE Purification

of Cyanoplast

C.

paradoxa

AL.

1

Ferredoxin:NADP

Oxidoreductase:

Yield

Purification Total Sample A B

protein bg)

Total enzyme activity (U)

1140

Cl

D E

Note. For sample

Specific activity

6000

300 64 5.1

4588 3866 1346

0.9

1158

designations,

refer

factor

Partial

Total

1

5 15 61 264

Total 1

2.9 4 4.3

1331

(7% )

100

3 12 50 253

5.1

yield

77 64 23 19.3

to text.

purified C. paradoxa FNR had an A,,,IA,,, value of 1.05. The FNR absorption maxima from various organisms vary (1353). The C. paradoxa FNR has absorption maxima near 276,385, and 460 nm and shoulders near 434 and 494 nm (Fig. 2). As described in the literature, the absorption ratio A,,,/A,, gives values between 7.5 and 9.4: 7.5 (l), 8.0 (5,9), 8.75 (13), 8.9 (53), 9.4 (54). The C. paradoxa FNR has an A,,,IA,, value of 6.8. We assume that this lower relative ratio is an indication of the high purity of our preparation. After the various purification steps, the resulting dialyzed samples were analyzedby native disc electrophoresis. FNR activity was detected by the diaphorase-tetrazolium test. In the crude extract (A), four bands were detected (I to IV with the R, values I, 0.36; II, 0.33; III, 0.3; IV, 0.27), three of which had nearly the same enzyme activity. The fourth (IV) and uppermost band had the highest enzyme activity. Band III was enriched by the ammonium sulfate precipitation (B). DEAE chromatography separates bands III and IV, which eluted in Ci, from bands I and II, which eluted in dC,, (band IV was also enriched in dC,,i).

I

enzyme (U/mg)

Estimation

After the final purification step a portion of sample E was electrophoretically analyzed with regard to the homogeneity of the protein preparation using 1D SDSPAGE (Fig. 3c) and 2D PAGE (Fig. 3d). As detected by silver staining after UTLSDS-PAGE, only one band was visible (Fig. 3~); however, isoelectric focusing (the first dimension of 2D PAGE) allowed the separation of two to six bands in various concentrations as shown in Fig. 3d. The detection depended on the amount of loaded protein and on the sample fraction number. This observation indicates that our FNR preparation consists of various molecular species of FNR with very similar molecular masses and slight differences in charge (see Amino Acid Sequence Analysis). This molecular heterogeneity, earlier described for the enzyme of other species (e.g., (9,58)), could also be demonstrated in the 2D PAGE separation of crude extract (see Figs. la and 2a in (44); for further discussion see below). IEF -

I

FNR-I

3 -

.2 -

ow

.1 -

500 Inml

a o-

FIG. 2. in:NADP’ 0.045; the inset).

I

I

I

300

400

500

Ultraviolet-visual oxrdoreductase partial spectrum

I 600 Wavelength

I 700 ( nm I

spectrum of C. parudoza ferredox(I&s, 0.306; E,,,, 0.026; E,,, 0.043; Em, of the Aavin component is shown in the

b

c

d

FIG. 3. C. paradorn Ferredoxin:NADP+ oxidoreductase electrophoretie separation (1D SDS-PAGE, 2D PAGE). (a) Soluble cyanoplast proteins: 500 ng, SDS-PAGE, silver staining. (b) Soluble cyanoplast proteins: 500 ng, SDS-PAGE, Western blot with anti-FNR (44). (c) Purified FNR: 20 ng sample E, SDS-PAGE, silver staining. (d) purified FNR: 50 ng sample E, 2D PAGE, silver staining. Electrophoretic mole mass estimation: (a) ovalbumin (43 kDa), (b) carboanhydrase (29 kDa), (c) trypsin inhibitor (20.1 kDa), (d) cy-lactalbumin (14.2 kDa), FNR (34 kDa).

Cyanophoro

233

FERREDOXIN-NADP+-OXIDOREDUCTASE

The molecular mass of mature FNR estimated by SDS-PAGE is approximately 34 kDa and lies between the molecular masses of cyanobacterial FNR, 32.6 to 33.3 kDa (7-g), and those of eukaryotes, 34 to 35.7 kDa (l&19,32). In Fig. 4, the pH dependence of FNR diaphorase activity between pH 5.5 and 10.9 is shown. The pH optimum was detected near pH 9.0 with a steep slope on the alkaline side and a flatter slope on the acid side. This observation is in agreement with that for spinach FNR (55). The pH optimum for Spirulina platensis FNR is pH 9.5 (5), but no further information is known.

140

pmol PTH-Amino

Acid

120 100 80

60

40

N-Terminal

Amino Acid Sequence

Partial amino acid sequence analysis of FNR revealed the existence of at least two different N-termini (Fig. 5). In addition to the protein with the N-terminal sequence I, we found a shortened polypeptide chain II that lacked the first four amino acids, thus starting with three lysine residues:

20

0

AVDAKKKGDIPLNLFRPANPYIGK m

I.

AVDAK

II.

K

KKGDI

PLNLF

RPANP

YIGK

KKGDI

PLNLF

RPANP

YIGK.

In two of the sequencing experiments, the shortened polypeptide B was detected in slightly higher amounts than the full-length polypeptide A (Fig. 5); in the other two experiments, the full-length sequence A composed approximately 65% of the total protein. Each pair of experiments was derived from a different isolation batch and also from different neighboring fractions from Mono Q chromatography. This sequencing observation is in agreement with the presence of four to six molecular forms of the enzyme as identified by isoelectric focusing (see above). These findings illustrate the

o.4b E z0.3 c w 0

2

~

FIG. 5.

C. paradona ferredoxin:NADP+ quence analysis of N-terminal amino for details see text).

acid

shortened oxidoreductase sequences

(from

partial the

seleft;

molecular heterogeneity and long-known property of FNR, as previously described ((56-58) and others) for the spinach enzyme and also later for the cyanobacterial enzyme (7,9,20). At present it is not known if our results are indications of protease activity or of the existence of at least two similar genes as found for the spinach enzyme (32). Admittedly, screening a cDNA bank of C. paradoxa gives no indication of such a gene pair (Jakowitsch et al., in preparation). Alignment and comparison of this N-terminus with the corresponding FNR amino acid sequences of cyanobacterial and higher plant’s FNR show a position more similar to that of cyanobacteria than to that of eukaryotes, giving some insight into the evolutionary history of the C. paradoxa FNR (59). ACKNOWLEDGMENTS

d

2‘5 0.2 E ;1 2 8 a

full-length

This work was supported by the Deutsche Forschungsgemeinschaft (SPP Intrazellullre Symbiose, &he 98/10-5/6). We express our thanks to Prof. Dr. H.-A. Bisswanger (Institut ftir Physiologische Chemie, Universitlit Tiibingen) for providing an FPLC system (Pharmacia) in his laboratory, to Prof. Dr. G. Jung (Institut f6r Organische Chemie, Universitlt Tubingen) for direct support of the amino acid sequencing, and also to Fred Kippert (Botanisches Institut) for critical and informative discussions.

O.l.HO o-

I I 6.0

FIG. 4.

Tris / HCI

His/KOH I 7.0

I a.0

pH dependence of diaphorase doxin:NADP+ oxidoreductase. Buffers 5.5-7.5), 0.1 M Tris/HCl (pH 7.0-9.3), 10.9).

I io

pH

Id.0

ll:o

REFERENCES activity of C. paradoxa ferreare 0.1 M histidine/KOH (pH 0.1 M glycine/NaOH (pH 8.6-

1. Avron, M., and Jagendorf, tions on chloroplast TPNH 72,17-24.

A. T. (1957) diaphorase.

Some further Arch. Biochn.

investigaBiophys.

234

GEBHART

ET

2. Keister, D. L., San Pietro, A., and Stolzenbach, F. E. (1960) Pyridine nucleotide transhydrogenase from spinach. I. Purification and properties. J. Biol. Chem. 236, 2989-2996. 3. Shin, M., Tagawa, K., and Arnon, D. L. (1963) Chrystallization ferredoxin-TPN reductase and its role in the photosynthetic paratus of chloroplasts. Biochem. 2. 338, 84-96.

of ap-

4. Bothe, H., and Berzborn, R. J. (1970) Wirkung von Antikorpern gegen die Ferredoxin-NADP-Reduktase aus Spinat auf photosynthetische Reaktionen in einem zellfreien System aus der Blaualge Anacystis nidulans. Z. Naturforsch. B 25, 529-534. 5. Masaki, R., Wada, K., and Matsubara, H. (1979) Isolation characterization of two ferredoxin-NADP+ oxidoreductases Spirulina platen&. J. B&hem. 86, 951-962.

and from

AL. and quantitative determination of ferredoxin-NADP’ ductase, a thylakoid-bound enzyme in the cyanobacterium baena sp. Strain 7119. Plant Physiol. 82, 499-502.

21. Zanetti, G., and Curti, B. (1980) Ferredoxin-NADP+ tase, in “Methods in Enzymology” (San Pietro, pp. 250-255, Academic Press, San Diego, CA.

oxidoreAna-

oxidoreducA., Ed.), Vol. 69,

22. Carillo, N., and Vallejos, R. H. (1982) Interaction of ferredoxinNADP oxidoreductase with the thylakoid membrane. Plant Physiol. 69, 210-213. 23. Carillo, N., doreductase, 560, Elsevier

and Vallejos, R. H. (1987) in “The Light Reactions” Science, Amsterdam.

Ferredoxin-NADP+ (Barber, J., Ed.),

oxipp. 528-

24.

Pschorn, R., Riihle, W., and Wild, A. (1988) The influence of the proton gradient on the activation of ferredoxin-NADP+ oxidoreductase by light. 2. Naturforsch. C 43, 207-212.

25.

Shin, M., Ishida, H., and Nozaki, Y. (1985) A new protein factor, connectein, as a constituent of the large form of the ferredoxinNADP+ reductase. Plant Cell Physiol. 26, 559-563.

26.

Vallejos, R. H., Ceccarelli, the existence of a thylakoid doxin-NADP+ oxidoreductase.

27.

Sheriff, eductase

28.

Karplus, P. A., Daniels, M. J., and Herriott, structure of ferredoxin-NADP’ reductase: turally novel flavoenzyme family. Science

29.

Taylor, F. J. R. (1974) Implications endosymbiosis theory of the origin 258.

30.

Margulis, Francisco.

31.

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