PROTEIN EXPRESSION AND PURIFICATION 3, 228-235 (19%)

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

U. B. Gebhart, T. L. Maier, S. Stevanovi6, M. G. Bayer, and H. E. A. Schenkl Botanical Institute and Chemical Institute, University of Tiibingen, 7400 Tiibingen, Germany

Received November 1, 1991, and in revised form March 26, 1992

The ferredoxin:NADP+ oxidoreductase of the protist Cyanophoraparadoxa, as a descendant of a former sym- biotic consortium, an important model organism in view of the Endosymbiosis Theory, is the first enzyme purified from a formerly original endocytobiont (cyan- elle) 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 uv- vis spectrum, pH optimum (pH 9), molecular mass of 34 kDa, and an N-terminal amino acid sequence (24 resi- dues). The enzyme shows, as known from other organ- isms, molecular heterogeneity. The N-terminus of a fur- ther ferredoxin:NADP+ oxidoreductase polypeptide represents a shorter sequence missing the first four amino acids of the matUre enzyme. 0 1992 Academic press. h.

Ferredoxin:NADP+ oxidoreductase, an important photosynthetic enzyme, has been independently discov- ered several times, e.g., as “NADPH-dependent diapho- rase” (1) and as “pyridine nucleotide transhydroge- nase” (2). Shin et al. (3) were able to demonstrate that FNR (EC 1.18.1.2) is a flavoprotein with various enzy- matic 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. Ac- cordingly, the enzyme is bound to the outer side of the thylakoid membranes in cyanobacteria (20) and in plas- tids (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)

’ To whom correspondence should be addressed.

228

on its other. The three-dimensional structure of the spinach FNR (carboxyl terminus at residue 314), deter- mined 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 pro- totype 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-en- coded chloroplast proteins in higher plants, synthesized on 80s ribosomes as a preprotein (31,32) and imported post-translationally into chloroplasts. Although the Se- rial Endosymbiosis Theory is now generally accepted, definitive proof is lacking and questions regarding pos- tulated gene transfer and the evolution of the protein- import machinery in organelles remain unanswered.

Cyanophora paradoxa Korsch. (Glaucocystophyceae) (33,34) is a unicellular, biflagellated algal protist with originally symbiotic intracellular cyanobacteria (cya- nelles (35); for details see (36-39)). In general, endocya- nomes (symbiotic consortia of eukaryotes with intracel- lular cyanobacteria) and their descendants, the meta- cyanomes (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 im- ported (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, ferre- doxin:NADP+ oxidoreductase; FPLC, fast protein liquid chromatog- raphy; PAGE, polyacrylamide gel electrophoresis; R,, relative electro- phoretic 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.

and transcriptional and translational inhibition experi- algal cells

ments, we were able to demonstrate that FNR is one of the nucleus-encoded cyanoplast (formerly cyanellar) homogenization

proteins (44). In uitro translation experiments with poly(A)+ RNA corroborated this result (45). The pres- A (crude extract)

ent study describes for the first time the isolation and purification of a protein (FNR) that has suffered a gene ammonium sulfate-precipitation, solubilization of

transfer from a prokaryotic symbiont (cyanobacterium) 1 the pellet and dialysis

to a eukaryotic host nucleus. Furthermore the C. para- B doxa FNR is partially characterized with respect to

(dialysate) I

some molecular properties. anion exchange chromatography (DEAE)

MATERIALS AND METHODS k CI CI cIII (eluates from DEAE column)

Chemicals

I

4 4 dcII dCIII (dialysates of CII and C,,,)

The chemicals used were obtained from different sup- \ pliers, in particular, ammonium peroxosulfate and affinity chromatography (2'5'-ADP-Sepharose 4B) of

TEMED from Bio-Rad (Munich), 2’,5’-ADP-Sepharose CI Or of dCII and dialysis of collected fracti .ons

4B and Mono Q HR 5/5 from Pharmacia (Freiburg), D and DEAE-Cellulose 32 Servacel from Serva (Heidel-

(dialysate D) DII

berg).

Organism, Growth, and Harvest I second anion exchange chromatography (Mono Q)

E (pure FNR)

C. paradoxa B 29.80, Pringsheim strain (Sammlung von Algenkulturen der Universitat Gottingen, Pflan- dialysis

zenphysiologisches Institut, Gottingen, Germany) was continuously grown as previously described (45). The dE (dialysate of E)

relative cell number was controlled by in uiuo vis spec- SCHEMES troscopy using a Beckman Acta V photometer as de- scribed 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

sacculus (47) of the cyanoplasts. Homogenization

cells per milliliter). Cells were harvested by centrifuga- buffer (up to 10 ml/g wet weight) and Triton X-100 (2%,

tion (3 min, 500g) and the pellet was washed five times v/v) were then added and the homogenate was stirred in

by suspension in growth medium and repeated centrifu- the dark for 7 h to solubilize the membrane-bound pro-

gation. After the last centrifugation (3 min, lOOOg), the teins, including FNR (11). Finally, the homogenate was

wet weight was estimated, average values of 0.8 g per centrifuged (20 min, 40,OOOg). The resulting superna-

liter culture were obtained. The harvested cells were tant was designated as crude extract (A) and was used

stored at -25°C. for further purification.

(b) Ammonium sulfate fractionation. Solid ammo-

Purification of FNR nium sulfate was added to A to give 33% saturation. The pH of the solution was kept constant by titration with 1

All preparation steps were performed at 4’C under M Tris. This suspension was incubated for 30 min and green security light or with darkened containers. The then centrifugated (20 min, 40,OOOg). The pellet was dis- procedure is an adaption of different instructions (for carded, and ammonium sulfate was added to the super- other organisms) to the conditions in C. parudoxa sum- natant to give 75% saturation. After 1 h incubation, it marized in Scheme 1. was centrifuged (20 min, 4O,OOOg), and the resulting su-

(a) Crude extract. The frozen cells (10 g wet weight pernatant was discarded. The precipitate was collected, from 12 liter culture) were thawed, suspended in (5 ml/ solubilized in few milliliters of buffer A (50 mM Tris/ g) homogenization buffer (50 mM Tris/HCl, pH 7.5, 5 HCl, pH 7.5,0.1 mM EDTA), dialyzed for approximately mM 2-mercaptoethanol, 0.05 mM phenylmethylsulfonyl 15 h (overnight) against two 5-liter volumes of buffer B fluoride) and equilibrated with dinitrogen (1000 psi) in a (0.1 mM EDTA, 20 mM Tris/HCl, pH 7.5), and desig- Parr bomb for 1 h (4°C). The cells were disrupted by nated dialysate B. forcing them through the outlet valve. The resulting cell (c) Anion-exchange chromatography on DEAE-cellu- homogenate was incubated in the dark with lysozyme lose. Dialysate B (34.5 ml, ca. 300 mg total protein) (3.5 mg/g wet weight) for 30 min to degrade the murein was applied to a DEAE-Cellulose 32 column (2.5 X 18

229 Cyanophora FERREDOXIN-NADP+-OXIDOREDUCTASE

230 GEBHART ET AI,.

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. En- zyme 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 elec- trophoresis and by the specific absorbance spectrum be- tween 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 SDS- PAGE of pure FNR, followed by silver staining, was performed as previously described (44) with the follow- ing 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 mea- suring the spectrum from 250 to 750 nm. The FNR mo-

lar concentration was estimated (9), using the molar ex- tinction 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 of FNR Diaphorase Activity

Two different enzymatic activities were used to local- ize 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-phos- phate (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 con- centrations of the enzyme and is less reproducible than the diaphorase assay. It was therefore used only to con- firm 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 ac- ceptor (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 mix- ture 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 milli- gram of protein.

The pH optimum of diaphorase activity was esti- mated 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 7.0- 9.3), 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 auto- mated Edman degradation in a pulsed-liquid protein se- quencer (Model 477 A) equipped with an on-line phen-

Cyanophora FERREDOXIN-NADP’-OXIDOREDUCTASE 231

ylthiohydantoin amino acid analyzer (Model 120 A, Ap- plied 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 us- ing between 30 and 600 pmol of total protein (FNR pu- rity 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.

RESULTS AND DISCUSSION

Purification

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 puri- fication results of the given methods. Usually we could start with 10 g wet weight of algal cells. This corre- sponds 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 anion- exchange 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 ac- tivity 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 low- ered 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 ne- glected (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% satura- tion) separated most of the pigments, including all chlo- rophyll from the supernatant. With the second precipi- tation (75% saturation) all diaphorase activity was concentrated in the pellet that also contained phycobili- proteins. The phycobiliproteins were completely re- tained 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 non- binding of FNR to the DEAE-cellulose had been previ- ously described only once, in fact for the Nicotiana taba- cum 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 I I 1 1 1

600 - . . . . . . . ..-

.O . ..* . ..-

. ..* . ...**

LOO - I\ . ..- . . . . . ...-*

. l ./

I \ . ..-

..-

. ..*

200 -

. ..* . . . .

/a/..-.“’ \ ..‘.a

**.. . ...-*

*...** i

t 0 - ~.-.-C*--C~ o-o’ “0~>p- CCe-e-*-e-.-m-e-

I I 01 I I 0 5 10 15 20 25

Fraction number ( 2 ml 1

FIG. 1. Affinity chromatography of C. paradoxa ferredoxin:NADP+ oxidoreductase on 2’,5’-ADP-Sepharose 4B: elution profile of diapho- rase 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 af- finity chromatography of C, or of dC,, on 2’,5’-ADP- Sepharose 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 af- finity chromatography was barely detectable. There- fore, these samples were not purified further. Neverthe- less, the affinity chromatography did not bring about satisfactory purification; moreover about f of the en- zyme was lost. So in the final purification step, a second anion-exchange chromatography (Mono Q) of D was es- sential. 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 andpurifi- cation 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 compo- nent (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 AL.

TABLE 1

Purification of Cyanoplast C. paradoxa Ferredoxin:NADP Oxidoreductase: Yield Estimation

Sample Total protein

bg) Total enzyme

activity (U) Specific enzyme activity (U/mg)

Purification factor

Partial Total Total yield (7% )

A 1140 6000 5 1 1 100 B 300 4588 15 2.9 3 77

Cl 64 3866 61 4 12 64 D 5.1 1346 264 4.3 50 23 E 0.9 1158 1331 5.1 253 19.3

Note. For sample designations, refer 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 max- ima 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 dia- lyzed samples were analyzedby native disc electrophore- sis. FNR activity was detected by the diaphorase-tetra- zolium 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 en- zyme activity. The fourth (IV) and uppermost band had the highest enzyme activity. Band III was enriched by the ammonium sulfate precipitation (B). DEAE chroma- tography 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).

3 -

.2 -

.1 -

o-

I I

ow 500 Inml

I I I I I 300 400 500 600 700

Wavelength ( nm I

FIG. 2. Ultraviolet-visual spectrum of C. parudoza ferredox- in:NADP’ oxrdoreductase (I&s, 0.306; E,,,, 0.026; E,,, 0.043; Em, 0.045; the partial spectrum of the Aavin component is shown in the inset).

After the final purification step a portion of sample E was electrophoretically analyzed with regard to the ho- mogeneity of the protein preparation using 1D SDS- PAGE (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 con- sists of various molecular species of FNR with very simi- lar 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 -

FNR-I

a b c d

FIG. 3. C. paradorn Ferredoxin:NADP+ oxidoreductase electropho- retie 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) puri- fied 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 FERREDOXIN-NADP+-OXIDOREDUCTASE 233

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 activ- ity between pH 5.5 and 10.9 is shown. The pH optimum was detected near pH 9.0 with a steep slope on the alka- line side and a flatter slope on the acid side. This obser- vation 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.

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:

I. AVDAK KKGDI PLNLF RPANP YIGK

II. K 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 obser- vation is in agreement with the presence of four to six molecular forms of the enzyme as identified by isoelec- tric focusing (see above). These findings illustrate the

o.4b E z 0.3 c w 0

2 0.2 - d ‘5 E ;1

2 O.l-

2 8

a .HO I Tris / HCI

o- His/KOH

I I I I 6.0 7.0 a.0 io pH Id.0 ll:o

FIG. 4. pH dependence of diaphorase activity of C. paradoxa ferre- doxin:NADP+ oxidoreductase. Buffers are 0.1 M histidine/KOH (pH 5.5-7.5), 0.1 M Tris/HCl (pH 7.0-9.3), 0.1 M glycine/NaOH (pH 8.6- 10.9).

140

120

100

80

60

40

20

0

pmol PTH-Amino Acid

AVDAKKKGDIPLNLFRPANPYIGK

m full-length ~ shortened

FIG. 5. C. paradona ferredoxin:NADP+ oxidoreductase partial se- quence analysis of N-terminal amino acid sequences (from the left; for details see text).

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 en- zyme (32). Admittedly, screening a cDNA bank of C. paradoxa gives no indication of such a gene pair (Jako- witsch et al., in preparation). Alignment and compari- son 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

This work was supported by the Deutsche Forschungsgemeins- chaft (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 (Phar- macia) 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 criti- cal and informative discussions.

REFERENCES

1. Avron, M., and Jagendorf, A. T. (1957) Some further investiga- tions on chloroplast TPNH diaphorase. Arch. Biochn. Biophys. 72,17-24.

234 GEBHART ET AL.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

Keister, D. L., San Pietro, A., and Stolzenbach, F. E. (1960) Pyri- dine nucleotide transhydrogenase from spinach. I. Purification and properties. J. Biol. Chem. 236, 2989-2996.

Shin, M., Tagawa, K., and Arnon, D. L. (1963) Chrystallization of ferredoxin-TPN reductase and its role in the photosynthetic ap- paratus of chloroplasts. Biochem. 2. 338, 84-96.

Bothe, H., and Berzborn, R. J. (1970) Wirkung von Antikorpern gegen die Ferredoxin-NADP-Reduktase aus Spinat auf photo- synthetische Reaktionen in einem zellfreien System aus der Blaualge Anacystis nidulans. Z. Naturforsch. B 25, 529-534.

Masaki, R., Wada, K., and Matsubara, H. (1979) Isolation and characterization of two ferredoxin-NADP+ oxidoreductases from Spirulina platen&. J. B&hem. 86, 951-962.

Masaki, R., Yoshikawa, S., and Matsubara, H. (1982) Steady- state kinetics of reduced ferredoxin with ferredoxin-NADP+ re- ductase Biochim. Biophys. Acta 700, 101-109.

Rowell, P., Diez, J., Apte, S. K., and Stewart, W. D. P. (1981) Molecular heterogeneity of ferredoxin-NADP+ oxidoreductase from the cyanobacterium Anabaena cylindrica. Biochim. Biophys. Acta 657, 507-516.

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

21. Zanetti, G., and Curti, B. (1980) Ferredoxin-NADP+ oxidoreduc- tase, in “Methods in Enzymology” (San Pietro, A., Ed.), Vol. 69, pp. 250-255, Academic Press, San Diego, CA.

22. Carillo, N., and Vallejos, R. H. (1982) Interaction of ferredoxin- NADP oxidoreductase with the thylakoid membrane. Plant Phys- iol. 69, 210-213.

Yao, Y., Tamura, T., Wada, K., Matsubara, H., and Kodo, K. (1984) Spirulina ferredoxin-NADP+ reductase. The complete amino acid sequence. J. Biochem. 95,1513-1516.

Sancho, J., Peleato, M. L., Gomez-Moreno, C., and Edmondson, D. E. (1988) Purification and properties of ferredoxin-NADP+ oxidoreductase from the nitrogen-fixing cyanobacterium Ana- baena variabilis. Arch. Biochem. Biophys. 260, 200-207.

Sancho, J., Medina, M., and Gomez-Moreno, C. (1990) Arginyl groups involved in the binding of Anabaena ferredoxin-NADP+ oxidoreductase to NADP+ and to ferredoxin. Eur. J. Biochem. 187,39-48.

23. Carillo, N., and Vallejos, R. H. (1987) Ferredoxin-NADP+ oxi- doreductase, in “The Light Reactions” (Barber, J., Ed.), pp. 528- 560, Elsevier Science, Amsterdam.

24. Pschorn, R., Riihle, W., and Wild, A. (1988) The influence of the proton gradient on the activation of ferredoxin-NADP+ oxidore- ductase 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 ferredoxin- NADP+ reductase. Plant Cell Physiol. 26, 559-563.

26. Vallejos, R. H., Ceccarelli, E., and Chan, R. (1984) Evidence for the existence of a thylakoid intrinsic protein that binds ferre- doxin-NADP+ oxidoreductase. J. Biol. Chem. 259,8048-8051.

27. Sheriff, S., and Herriott, J. R. (1981) Ferredoxin-NADP+ oxidor- eductase and the location of the NADP binding site. J. Mol. Biol. 145,441-451.

28. Karplus, P. A., Daniels, M. J., and Herriott, J. R. (1991) Atomic structure of ferredoxin-NADP’ reductase: Prototype for a struc- turally novel flavoenzyme family. Science 251, 60-66.

29. Taylor, F. J. R. (1974) Implications and extensions of the serial endosymbiosis theory of the origin of eukaryotes. Tazon 23,229- 258.

Serrano, A., and Rivas, J. (1982) Purification of ferredoxin- NADP’ oxidoreductase from cyanobacteria by affinity chroma- tography on 2’,5’-ADP-Sepharose 4B. Anal. Biochem. 126, 109- 115.

30. Margulis, L. (1981) “Symbiosis in Cell Evolution,” Freeman, San Francisco.

Armstrong, J. J., Surzycki, S. J., Moll, B., and Levine, R. P. (1971) Genetic transcription and translation specifying chloroplast com- ponents in Chlamydomonas reinhardi. Biochemistry 10,692-701. Boger, P. (1971) EinfluI3 von Ferredoxin auf Ferredoxin-NADP- Reduktase. Planta 99, 319-338.

31. Grossmann, A. R., Bartlett, S. G., Schmidt, G. W., Mullet, J. E., and Chua, N.-H. (1982) Optimal conditions for post-translational uptake of proteins by isolated chloroplasts. J. Biol. Chem. 257, 1558-1563.

Smillie, R. M., Graham, D., Dwyer, M. R., Grieve, A., and Tobin, N. F. (1967) Evidence for the synthesis in oiuo of proteins of the Calvin cycle and of the photosynthesis electron-transfer pathway on chloroplast ribosomes. Biochem. Biophys. Res. Commun. 28, 604-610.

32. Jansen, T., Reillnder, H., Steppuhn, J., and Herrmann, R. G. (1988) Analysis of cDNA clones encoding the entire precursor- polypeptide for ferredoxin-NADP+ oxidoreductase from spinach. Curr. Genet. 13,517-522.

33. Kies, L., and Kremer, B. P. (1986) Typification of the Glaucocys- tophyta. Taxon 35, 128-133.

Zanetti, G., Morelli, D., Ronchi, S., Negri, A., Aliverti, A., and Curti, B. (1988) Structural studies on the interaction between ferredoxin and ferredoxin-NADP+ oxidoreductase. Biochemistry 27,3753-3759.

34. Kies, L., and Kremer, B. P. (1990) Phylum Glaucocystophyta, in “Handbook of Protoctists” (Margulis, L., Chapman, D. J., and Corliss, J., Eds.), pp. 152-166, Jones and Bartlett, Boston, MA.

35. Pascher, A. (1929) Studien iiber Symbiosen. I. ijber einige Endo- symbiosen von Blaualgen in Einzellern. Jahrb. Wiss. Bot. 71, 386-462.

Sluiters-Scholten, C. M. T., Moll, W. A. W., and Stegwee, D. (1977) Ferredoxin and ferredoxin-NADP+ oxidoreductase in leaves of Phaseolus vulgaris L. Planta 133, 289-294. Karplus, P. A., Walsh, K. A., and Herriott, J. R. (1984) Amino acid sequence of spinach ferredoxin-NADP+ oxidoreductase. Bio- chemistry 23,6576-6583.

Newman, B. J., and Gray, J. C. (1988) Characterization of a full- length cDNA clone for pea ferredoxin-NADP+ reductase. Plant Mol. Biol. 10, 511-520.

Michalowski, C. B., Schmitt, J. M., and Bohnert, H. J. (1989) Expression during salt stress and nucleotide sequence of cDNA for ferredoxin-NADP+ reductase from Mesembryanthemum crys- tallinum. Plant Physiol. 89, 817-8‘22.

36. Trench, R. K. (1982) Physiology, biochemistry, and ultrastruc- ture of cyanellae, in “Progress in Phycological Research” (Round, E., and Chapman, D. J. Eds.), Vol. I, pp. 257-288, Else- vier Biomedical, Amsterdam/New York.

37. Wasman, C. C., Mffelhardt, W., and Bohnert, H. J. (1987) Cyan- elles: Organization and molecular biology, in “The Cyanobac- teria” (Fay, P., and van Baalen, C., Eds.), Elsevier Science, New York.

38. Schenk, H. E. A. (1990) Cyanophora paradoxa: A short survey, in “Endocytobiology IV, 4th International Colloquium on Endocy- tobiology and Symbiosis” (Nardon, P., Gianinazzi-Pearson, V., Grenier, A. M., Margulis, L., and Smith, D. C., Eds.), pp. 199-209, INRA, Paris.

Serrano, A., Soncini, F. C., and Vallejos, R. H. (1986) Localization 39. Schenk, H. E. A. (1992) Cyanobacterial symbioses, in “The Pro-

Cyanophora FERREDOXIN-NADP+-OXIDOREDUCTASE 235

40.

41.

42.

43.

44.

45.

45.

46.

47.

48.

karyotes” (Ballows et al., Eds.), pp. 3429-3454, Springer-Verlag, niques of Polyacrylamide Gel Electrophoresis,” de Gruyter, Ber- New York. lin/New York.

Schenk, H. E. A., and Hofer, I. (1972) About the light and dark fixation of CO, in the cyanoms Cyanophoraparadona and Glauco- cystis nostochinearum and their endocyanelles, in “Proceedings IInd International Congress on Photosynthesis Research,” (Forti, G., Avron, M., and Melandri, A., Eds.), Vol. III, pp. 2095- 2100, Junk, The Hague.

49. Harris, H., and Hopkins, D. A. (1980) “Handbook of Enzyme Elektrophoresis in Human Genetics,” North-Holland, Amster- dam.

50.

Herdman, M., and Stanier, R. Y. (1977) The cyanelle: Chloro- plast or endosymbiotic prokaryote? FEMS 1, 7-12.

51.

Bayer, M. G., and Schenk, H. E. A. (1986) Biosynthesis of pro- teins in Cyanophoru paradoxa. I. Protein import into the endo- cyanelle analyzed by micro two-dimensional gel electrophoresis. Endocyt. Cell Res. 3, 197-202.

52.

Burnap, R. L., and Trench, R. K. (1989) The biogenesis of the cyanellae of Cyanophora paradoxa. II. Pulse-labelling of cyanellar polypeptides in the presence of transcriptional and translational inhibitors. Proc. R. Sot. London Ser. B 238, 73-87.

53.

54. Bayer, M. G., Maier, T. L., Gebhart, U. B., and Schenk, H. E. A. (1990) Cyanellar ferredoxin-NADP+ oxidoreductase of Cyano- phora paradoxa is encoded by the nuclear genome and synthe- sized on cytoplasmatic 80s ribosomes. Curr. &net. 17,265-267. Jakowitsch, J., Brandtner, M., Loffelhardt, W., Gebhart, U., Bayer, M., Maier, T. L., Schenk, H. E. A., Michalowski, C. and Bohnert, H. J. (1991) Sequence of a precursor polypeptide des- tined to cross and peptidogylcan wall of cyanelles, in “Abstracts of the IIIrd International Congress on Plant Molecular Biology, Tucson, AZ, Oct. 1991.”

55.

56.

Zook, D., and Schenk, H. E. A. (1986) Lipids in Cyanophorapara- dora. III. Lipids in cell compartments. Endocyt. Cell Res. 3,203- 211. Schenk, H. E. A., Hanf, J., and Neu-Miiller, M. (1983) The phy- cobiliproteids in Cyanophoru paradoxa as accessoric pigments and nitrogen storage proteins. 2. Naturforsch. C 34, 972-977.

Schenk, H. E. A. (1970) Nachweis einer lysozymempfindlichen Stutzmembran der Endocyanellen von Cyanophora parudoxa Korsch. 2. Naturforsch. B 25, 640, 656.

Maurer, H. R. (1971) “Disc Electrophoresis and Related Tech-

57.

58.

59.

Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the prin- ciple of protein-dye binding. Anal. Biochem. 72, 248-254. Schmid, G. H., and Radunz, A. (1974) Reactions of a monospe- cific antiserum to ferredoxin-NADP+ reductase with chloroplast preparations. 2. Nuturforsch. C 29, 384-391.

Shin, M., and San Pietro, A. (1968) Complex formation of ferre- doxin-NADP reductase with ferredoxin and with NADP+. Bio- them. Biophys. Res. Commun. 33,38-42.

Spano, A. J., and Schiff, J. A. (1987) Purification, properties and cellular localization of Euglena ferredoxin-NADP+ reductase. Biochim. Biophys. Acta 894,484-498. Bookjans, G., and Boger, P. (1978) Modification of ferredoxin- NADP-reductase from the alga Bumilkriopsis with butanedione and dansyl chloride. Arch. Biochem. Biophys. 190,459-465. Davis, D. J., and San Pietro, A. (1977) Evidence for the role of sulfhydryl groups in a pH-dependent transition of ferredoxin: NADP oxidoreductase. Arch. Biochem. Biophys. 184,572-577. Keirns, J. J., and Wang, J. H. (1972) Studies on nicotineamide adenine dinucleotide phosphate reductase of spinach chloro- plasts. J. Biol. Chem. 247, 7374-7382.

Gozzer, C., Zanetti, G., Galliano, M., Saccbi, G. A., Minchiotti, L., and Curti, B. (1977) Molecular heterogeneity of ferredoxin- NADP+ oxidoreductase from spinach leaves. Biochim. Biophys. Acta 485,278-290.

Ellefson, W. L., and Krogman, D. W. (1979) Studies of the multi- ple forms of ferredoxin-NADP oxidoreductase from spinach. Arch. Biochem. Biophys. 194, 593-599. Schenk, H. E. A., Bayer, M. G., Maier, T. L., Ltittke, A., Gebhart, U. B.. and Stevanovic. S. (1992) Ferredoxin-NADP oxidoreduc- tase of C. paradona nucleus encoded, but cyanobacterial. Gene transfer from symbiont to host, an evolutionary mechanism origi- nating new species. 2. Nuturjorsch. B47, in press.