Planta

Planta (1988) 176:391-398

9 Springer-Verlag 1988

Polyclonal antibodies raised to phycocyanins contain components specific for the red-absorbing form of phytochrome D.R. Keiller*, G.C. Whitelam**, and H. Smith Department of Botany, University of Leicester, University Road, Leicester LE] 7RH, U K

Abstract. Polyclonal antibodies raised in rabbits to a mixture of sodium-dodecyl-sulphate-denatured C- and allo-phycocyanin, isolated from Anabaena cylindrica, cross-react with 124-kilodalton (kDa) phytochrome from etiolated oats, in enzyme-linked immunosorbent assays and on Western blots. The component(s) of the anti-phycocyanin serum that cross-reacts with phytochrome appears to be specific for the red-absorbing form of phytochrome (Pr). These antibodies can be detached from Pr by irradiation with red light, and thus show photoreversible binding. This property has been used to immunopurify the anti-phytochrome component from the antiserum using red light as the eluting agent. Competition assays and epitope-mapping studies indicate that the anti-phytochrome component may bind to a site located between 6 and 10 kDa from the amino-terminus of etiolated oat phytochrome. Key words: Anabaena (phycocyanin)- Arena (phytochrome) - Phycocyanin - Phytochrome (immunology)

Introduction

The plant photoreceptor, phytochrome, exists in two interconvertible forms, a red-absorbing form, Pr, and a far-red absorbing form, Pfr. Phytochrome mediates many plant developmental responses (see Shropshire and Mohr 1983) and it is generally considered that Pfr is the biologically * Present address: Research Institute for Photosynthesis, University of Sheffield, Sheffield, S10 2TN, U K ** To whom correspondence should be addressed Abbreviations: ELISA=enzyme-linked immunosorbent assay;

kDa = kilodaton; FR = far-red light; Pfr = far-red-light-absorbing form of phytochrome; Pr = red-light-absorbing form ofphytochrome; R = red light; SDS = sodium dodecyl sulphate

active form of the molecule. Despite much investigation the mechanism by which Pfr operates and the molecular differences between Pr and Pfr are obscure. A promising approach to identifying molecular differences between Pr and Pfr is the use of immunological methods. Early attempts used polyclonal sera but after one apparently successful discrimination between red (R)- and far-red (FR)-irradiated phytochrome preparations (Hopkins and Butler 1970), subsequent work failed to confirm the observation (Cundiff and Pratt 1975; Pratt 1973). Work with monoclonal antibodies which recognise single antigenic determinants within the molecule has proved more successful, with numerous reports of monoclonal antibodies that exhibit differential affinities for Pr and Pfr (Thomas et al. 1984; Cordonnier et al. 1985; Shimazaki et al. 1986). However, it is only very recently that an antibody with absolute specificity for one form of phytochrome, Pr, has been reported (Holdsworth and Whitelam 1987). Unlike phytochrome, phycocyanin is a lightharvesting photosynthetic pigment found in bluegreen and red algae (for a review, see Bogorad 1975). Like phytochrome, however, it is a biliprotein having a linear tetrapyrrole prosthetic group. Despite close chemical similarities between the chromophores, and strong similarities in the absorbance spectra (Seigelmann et al. 1966), few studies of immunological similarities between phytochrome and phycocyanin have been made. Berns (1967) reported that polyclonal antibodies to phycocyanins cross-reacted with purified etiolated oat phytochrome in Ouchterlony double-diffusion assays; however, the author suggested that this could have been the consequence of serum non-specificity. Subsequently, Rice and Briggs (1973) were unable to demonstrate cross-reactivity between antisera to phytochrome and phycocyanins.

392

D.R. Keiller et al. : Antibodies to phycocyanin cross-react with phytochrome

There are two possible explanations why antisera raised to phycocyanins do not cross-react with phytochrome and vice-versa. First, they may possess no common antigenic determinants; second, common determinants may exist, but the Ouchterlony diffusion assays used in studies to date are not sensitive enough to detect cross-reacting components. This paper describes the properties of a component(s) of a polyclonal antiserum raised to a mixture of C- and allo-phycocyanins that cross-reacts with 124-kDa etiolated-oat phytochrome and which discriminates between Pr and Pfr. Material and methods Phycocyanin purification. Phycocyanins (C-phycocyanin and allo-phycocyanin) from Anabaena cylindrica were purified by poly(ethyleneimine) precipitation, (NH4)2SO~ precipitation and diethylaminoethyl cellulose ion-exchange chromatography. Pooled fractions containing C- and allo-phycocyanin were mixed and further purified on either 12% sodium dodecyl sulphate (SDS)-polyacrylamide gels or 5% to 10% non-denaturing linear gradient gels to produce pure SDS-denatured and purenative mixtures of C- and allo-phycocyanins. Polyclonal-antibody production. Two male New Zealand white rabbits were injected intramuscularly with either I m g SDSdenatured or I m g native phycocyanins emulsified in Freund's complete adjuvent. This procedure was repeated at three, twoweek intervals after which blood was collected and the immunoglobulin-containing fraction partially purified by repeated precipitation with 40% saturated (NH4)2SO4.

Phytochrome purification. Native phytochrome (124 kDa) was purified from etiolated oat shoots by a modified version of the Vierstra and Quail (1983) procedure (Holdsworth 1987). Except where stated, all phytochrome used had a specific absorbance ratio (A665/A280 nm) of _>0.92.

Protocols for enzyme-linked immunosorbent assay (ELISA). Two bulk-assay procedures were used. In the first, which will be referred to as direct ELISA, flat-bottomed, 96-well vinyl plates (Dynatech, Billingshurst, Sussex, UK) were coated with appropriate amounts of phytochrome in 50 mM bicarbonate buffer, pH 9.2, for 1 h at room temperature. After extensive washing with 1 0 m M Na-phosphate, 0.14 M NaC1, pH 7.4 (PBS), containing 0.05% Tween-20 (Sigma, Poole, Dorset, UK), the remaining protein-binding sites were blocked by filling the wells with PBS-Tween containing 3% (w/v) Marvel skimmed-milk powder (Cadbury, Bournville, UK) and leaving overnight at 4 ~ C. Following a further wash with PBS-Tween, crude anti-phycocyanin immunoglobulin fraction was added at a concentration of 80 ~g.ml-1 in PBS-Tween and incubated for 1 h at room temperature. After extensive washing with PBSTween, anti-rabbit immunoglobulin antibodies, conjugated to peroxidase (Sigma) were added at a dilution of 1 : 500 in PBSTween. Following a 1-h incubation at room temperature the plates were again washed, and bound peroxidase activity determined by adding 1 ~tg. m l - 1 3,3',5,5'-tetramethylbenzidine (Sigma) in 50 mM citrate-acetate buffer, pH 6.0. The second assay procedure used will be referred to as a sandwich ELISA. In this assay, vinyl plates are coated with

8 ~g-ml 1 of LAS 32, a non-discriminating mouse monoclonal antibody raised against undegraded oat (Arena sativa L.) phytochrome (Holdsworth 1987). Washing and blocking was then as described above. All subsequent operations were performed in the dark or under dim green light. Phytochrome was added as Pr and Pfr and the plates incubated for 1 h at room temperature, this was followed by washing, and addition of anti-phycocyanin antibodies, after which the development procedure was identical to that already described. For competition studies the same basic protocol used in the sandwich ELISA was employed with the following modification. An appropriate amount (250ng.wel1-1) of phytochrome was photoconverted to Pr and mixed in darkness with various dilutions of mouse anti-phytochrome monoclonal antibodies. The mixtures were added to ELISA plates which were developed as described above. Two controls were employed, a zero competition control, where phytochrome was mixed with PBS-Tween and a 100% competition control, where PBS-Tween instead of anti-phycocyanin serum was added. All monoclonal antibodies were purified from ascitic fluid by ammonium-sulphate precipitation and cation-exchange chromatography on Zeta Prep 15 SP ion-exchange discs (LKBProdukter AB, Bromma, Sweden) as described by Holdsworth (1987).

Western blotting. For the preparation of crude extracts, 4-d-old, etiolated oat shoots were homogenised in 50 mM 3-(N-morpholino)propanesulphonic acid (Mops), pH 7.8, containing 10 m M ethylenediaminetetraacetic acid (EDTA) and 14 m M fl-mercaptoethanol at a ratio of 1 g of tissue to 1 ml of buffer. Extracts were clarified by centrifugation. For the preparation of phytochrome proteolytic fragments, supernatants of extracts of etiolated oat seedlings were mixed with purified 124-kDa oat phytochrome and the mixtures incubated as described by Jones et al. (1985). Samples were resolved by SDS-polyacrylamide gel electrophoresis on 7% gels and the separated polypeptides electroblotted onto nitrocellulose as detailed by Holdsworth and Whitelain (1987). Immunodevelopment of the blots and visualisation of bands was as described previously (Holdsworth and Whitelam 1987) except that the primary antibodies used were either the crude immunoglobulin G fraction (80 ~g-ml-1) or affinitypurified anti-phycocyanin antibodies (6 ~tg. ml-1). Preparation of immobilised-phytochrome column and immunoaffinity purificationprotocol. LAS32 was coupled to CNBr-activated agarose (Sigma) according to the procedure described by Pratt (1984). After the coupling procedure the column was washed extensively with PBS before adding 124-kDa oat phytochrome in PBS and recirculating it through the column for 2 h at 4 ~ C. After further washing with PBS the column was transferred to darkness and the bound phytochrome converted to either Pr or Pfr. Anti-phycyocanin serum diluted 1 : 10 with PBS was recirculated through the column for 2 h at 4 ~ C. The column was then washed with PBS and the eluent was collected. When the A280 had fallen below 0.01 the column was given a saturating pulse of R and more PBS run through. Once three column volumes had been collected the R-elution procedure was repeated. All fractions collected were screened for antiphycocyanin and anti-phytochrome activity using the direct ELISA system.

Results Direct and sandwich E L I S A . A n t i s e r a SDS-denatured

and

native

raised to phycocyanins were

D.R. Keiller et al, : Antibodies to phycocyanin cross-react with phytochrome

393

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screened by direct ELISA for anti-phytochrome activity. Serum to SDS-denatured phycocyanins cross-reacted strongly with 124-kDa oat phytochrome, whilst that raised to native phycocyanins did not (Fig. J). All subsequent investigations, therefore, were performed using the anti-SDS-denatured phycocyanin serum, henceforth referred to as anti-phycocyanin serum. Using the sandwich ELISA the ability of the anti-phycocyanin serum to discriminate between Pr and Pfr was investigated. Anti-phycocyanin serum discriminated strongly in favour of Pr using this assay (Fig. 2). The signal produced by Pfr is considerably lower than might be expected. Assuming that the mole fraction of Pfr produced by saturating R is 0.874 (Holdsworth and Whitelam 1987), then phytochrome samples irradiated with R should contain 12.6% Pr. To determine whether the low signal produced by Pfr could be the result of a Pfr-specific modification during incubation on the ELISA plate, the following protocol was adopted. Duplicate plates were coated with LAS 32 and blocked as described. Both Pr and Pfr were added to each plate followed by incubation for 60 min in darkness. Anti-phycocyanin antiserum was added and, after a further 45 min, one plate was given saturating R and the other saturating FR. After another 45 rain incubation the plates were washed and developed. Regardless of whether phytochrome was incubated initially as Pr or Pfr the result was identical

I

1~00

Phytochrome tng)

Phytochrome (rig) Fig, 1. Binding of anti-SDS-denatured Anabaena phycocyanin serum ( o - - o ) and anti-native phycocyanin serum ( I - - I ) to 124-kDa etiolated-oat phytochrome as assayed by direct ELISA. Each point is the mean of three replicate assays from a single experiment

.........

Fig. 2. Binding of antibodies to Anabaena phycocyanin to either Pr (o----o) or Pfr ( ~ ) using the sandwich ELISA system. The dotted line is the control which lacked phytochrome. Each point is the mean of four replicates from a single experiment

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Phytochrome (nq) Fig. 3. Photoreversible binding of anti-phycocyanin antibodies to Pr and Pfr in sandwich ELISA. Phytochrome was bound to the ELISA plate via LAS 32 either as Pr (e, o) or Pfr (n, w) and incubated with anti-phycocyanin antibodies before receiving either R (o) FR (I). After a further incubation, binding of anti-phycocyanin antibodies was visualized. Each point is the mean of three replicates

(Fig. 3); Pr was recognised strongly whilst Pfr was not. More surprisingly, binding of the anti-phycocyanin serum was dependent upon the final irradiation. Anti-phycocyanin antibodies bound to Pr became unbound after R irradiation, but were rebound following FR. Thus, binding of anti-phyco-

394

D.R. Keiller et al. : Antibodies to phycocyanin cross-react with phytochrome

0-3

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Phyfochrome (ng) Fig. 4. Binding of anti-phycocyanin antibodies to Pr ( 0 - - o ) and Pfr (n rT) assayed by the direct ELISA. Each point is the mean of four replicates

0-20 Fig. 6. Immunoblots of 124-kDa oat phytochrome stained with either anti-phycocyanin serum (antiphc) or non-immune serum. Within each blot, from left to right, each lane contains 1000, 500 and 250 ng of phytochrome

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Phyfochrome (ng) Fig. 5. Binding of anti-phycocyanin antiserum to partially degraded (118/i 14-kDa) oat phytochrome using the direct ELISA system. Each point is the mean of four replicates

cyanin antibodies to 124-kDa oat phytochrome shows classic R / F R reversibility. When Pr and Pfr were bound to plates using the direct assay system, differential recognition between the two forms of phytochrome was much reduced (Fig. 4). Finally, partially degraded, 118 + 114-kDa oat phytochrome gave no detectable ELISA signal usmg the sandwich system (data not shown), but did, albeit to a lesser degree than 124-kDa phytochrome, in the direct ELISA system (Fig. 5).

Western blotting and epitope location. Anti-phycocyanin antibodies were found to be capable of detecting phytochrome by Western blotting (Fig. 6). The specificity of the interaction was confirmed by the lack of staining observed with a non-immune serum from the same rabbit. Probing blots of phycocyanins with various preparations of antibodies to phytochrome consistently failed to produce detectable staining (data not shown). The ability of the anti-phycocyanin antibodies to immunostain blotted phytochrome raised the possibility that the site(s) on the phytochrome molecule to which the antibodies bind could be determined. Digestions of phytochrome, in the Pr and Pfr forms, by endogenous proteases yield characteristic patterns of proteolytic fragments (Fig. 7). When immunostained with anti-phytochrome antibodies, major proteolytic fragments at 118 and 114 kDa were revealed for digests of Pr, and a fragment at 74-kDa was stained for digests of Pfr. When comparable blots of phytochrome digests were immunodeveloped with anti-phycocyanin an-

D.R. Keiller et al. : Antibodies to phycocyanin cross-react with phytochrome

395

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Fig. 8. Competition ELISA showing the effect of monoclonal antibodies LAS 31 (e e), LAS 33 ( o - - o ) , LAS 35 ( i - - i ) and LAS 41 (rT--D) on binding of anti-phycocyanin antibodies to Pr. Each point is the mean of three replicates Fig. 7. Time course of phytochrome digestion by endogenous proteases, 124-kDa oat phytochrome was added to crude oat extracts and incubated as Pr or Pfr for the times indicated. Each lane contains approx. 2000 ng phytochrome. Blots were probed with either rabbit anti-phytochrome polyclonal antibodies (anti phy), with affinity-purified antibodies to Anabaena phycocyanin (anti phc) or with non-immune serum

1.2

1,000

A E

R

R

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tibodies only the undegraded, 124-kDa species appeared to be stained with no other polypeptides being detectable (Fig. 7).

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Competition ELISA. Possible competitive inhibition of the binding of anti-phycocyanin antibodies to phytochrome by Pr-Pfr-discriminating mouse monoclonal anti-phytochrome antibodies was assayed. The data for four such monoclonal antibodies, designated LAS 31, LAS 33, LAS 35 and LAS 41 are shown in Fig. 8. LAS 31 and LAS 33 bind to epitopes located on the amino-terminal 6kDa domain of the phytochrome molecule, whilst LAS 35 and LAS 41 recognise epitopes located on the adjacent 4-kDa, sub-amino-terminal domain (Holdsworth 1987; Holdsworth and Whitelam 1987). LAS 41 was the only monoclonal antibody to show appreciable inhibition of anti-phycocyanin antibody binding to phytochrome (Fig. 8). This is particularly interesting since LAS 41 shows Prspecificity and photoreversible binding to phytochrome. Despite the observation that LAS 41 competes with the anti-phycocyanin serum, it does not recognise phycocyanins in direct ELISA even when the phycocyanins are added at concentrations of up to 3 ~tg.well- 1 (data not shown). Thus it would

10

20

30 /*0 50 Fraction number

60

Fig. 9. Elution profile of protein ( n - - t z ) , anti-phycocyanin activity (o 9 and anti-phytochrome activity ( o - - o ) from a column containing immobilised 124-kDa phytochrome. Saturating R pulses were given at points designated R

appear that whilst LAS 41 binds closely to the site recognised by a component(s) of the anti-phycocyanin serum is does not bind to the same site.

Affinity purification of anti-phytochrome components. The anti-phytochrome component(s) of the anti-phycocyanin serum bound to immobilised phytochrome in the Pr form, and was released following R irradiation of the column (Fig. 9). The antibodies obtained in this way behaved in an identical manner to the original serum in ELISA and on Western blots, with respect to phytochrome recognition, (data not shown, but see Fig. 9).

396

D.R. Keilleret al. : Antibodiesto phycocyanincross-react with phytochrome

Discussion

This is the first time that unequivocal evidence has been presented for cross-reactivity of anti-phycocyanin serum with phytochrome (Berns 1967; Rice and Briggs 1973) and of a polyclonal serum which is capable of discriminating between Pr and Pfr (Hopkins and Butler 1970). Furthermore, there is evidence to indicate that this discrimination between Pr and Pfr may be absolute. Until recently there were no reports of antibodies, polyclonal or monoclonal, which showed such specificity (Holdsworth and Whitelam 1987). That the anti-phycocyanin antiserum is specific for Pr is based on a number of lines of evidence. Firstly, in sandwich ELISAs the antiserum demonstrates a very large affinity difference in favour of of Pr. On theoretical grounds an antibody showing absolute specificity for Pr would demonstrate a 7.69-fold greater affinity for Pr than Pfr, since Pr always contains approximately 13% Pr even after saturating R (Kelly and Lagarias 1985), as indeed is the case with the Pr-specific antibody, LAS 41, described by Holdsworth and Whitelam (1987). The observation of affinity differences larger than can be expected on theoretical grounds is not unique; both Cordonnier et al. (1985) and Thomas et al. (1984) present data showing larger-than-theoretical maximum differences. Such differences appear to be related to ELISA conformation (Holdsworth and Whitelam 1987); therefore, it is probable that at least some of the observed affinity differences in this study were caused by ELISA conformation. The observation that anti-phycocyanin antibodies show a difference in affinity for Pr and Pfr only in sandwich ELISAs, and not when phytochrome is bound directly to the assay plate, is consistent with previous observations on the differential binding of monoclonal antibodies to Pr and Pfr (Thomas and Penn 1986; Holdsworth 1987). It seems as though the binding of phytochrome directly to the vinyl surface leads to a loss of the conformational differences between Pr and Pfr. Consequently, since a sandwich ELISA has to be employed to maintain the different conformations of Pr and Pfr it is important that the antibody used to bind phytochrome to the assay plate does not compete with the test antibodies for binding to phytochrome and that it does not itself induce changes in the conformations of Pr and Pfr. For these reasons we used monoclonal antibody LAS 32, a non-discriminating antibody that binds to an epitope located in the carboxy-terminal part of the phytochrome molecule (Holdsworth 1987). This antibody does not affect the binding of anti-

phycocyanin antibodies (data not shown) and has no effect on the relative affinities of other monoclonal antibodies that do discriminate between Pr and Pfr (Holdsworth 1987). The possibility that a Pfr-specific modification during incubation caused low Pfr signals can be excluded by the studies demonstrating photoreversibility of binding. This observation coupled with the affinity purification of anti-phytochrome antibodies using R as an eluting agent both indicate strongly that the phytochrome binding components are Pr-specific. Further, indirect evidence indicating Pr-specificity is that the Pr-specific monoclonal antibody, LAS 41 (Holdsworth and Whitelam 1987) competes with the anti-phycocyanin serum for binding to phytochrome. Clearly, the antiphycocyanin binding site(s) is close to an epitope on the phytochrome molecule that is only available in the Pr form. Direct attempts to locate the region(s) of the phytochrome molecule that is being recognised by the anti-phycocanin antibodies involved the use of partial proteolysis and Western blotting. Although the digestion of phytochrome by endogenous proteases yielded a range of polypeptide fragments, the anti-phycocyanin antibodies were apparently only capable of immunostaining the undegraded, 124-kDa polypeptide. At the relatively high loadings of phytochrome that are needed to observe good immunostaining with anti-phycocyanin antibodies it is not possible clearly to resolve the 124-, 118- and i 14-kDa polypeptides. The tentative conclusion that the anti-phycocyanin antibodies immunostain only the 124-kDa species is supported by the selective loss of staining of phytochrome by these antibodies, compared with the anti-phytochrome antibodies, for digests of Pr. As proteolysis proceeds, and the 124-kDa species is cleaved to yield 118-and l l4-kDa polypeptides, staining of phytochrome by anti-phycocyanin antibodies is reduced such that by 2 h immunostaining is barely detectable. For comparable blots developed with anti-phytochrome antibodies there is still strong staining of phytochrome polypeptides at this time, as a result of the appearance of the 118- and 114kDa species (Fig. 7). Assuming that proteolytic clipping of Pr, to produce ll8-kDa and ll4-kDa fragments, occurs exclusively at the amino-terminus of the molecule (Jones et al. 1985), then this observation would seem to indicate that recognition of phytochrome by anti-phycocyanin antibodies is restricted to the amino-terminal 6-kDa domain. This region of the molecule is known to undergo light-induced conformational changes and to possess sites which are preferentially ex-

D.R. Keiller et al. : Antibodies to phycocyanin cross-react with phytochrome

posed on Pr (Jones et al. 1985). However, the 74kDa fragment produced by proteolytic cleavage of Pfr is thought to carry an intact amino-terminus and so it would be anticipated that antibodies to epitopes on the 6-kDa domain should stain this fragment. Since the anti-phycocyanin antibodies do not immunostain the 74-kDa polypeptide, this could mean that either this polypeptide is present at levels below the limits of detection by anti-phycocyanin antibodies or the recognition site(s) is located elsewhere on the molecule. The ability of the anti-phycocyanin antibodies to detect degraded, 118/114-kDa phytochrome by the direct ELISA, though with a much reduced affinity, may tend to support this latter view. However, the possibility that the preparation of degraded phytochrome contained some 124-kDa phytochrome cannot be entirely discounted. Furthermore, anti-phycocyanin antibodies failed to detect degraded phytochrome in the sandwhich ELISA. Grimm et al. (1987) have reported that digestion of phytochrome, in the Pr form, by endogenous proteases can lead to the formation of 118-kDa and 114-kDa peptides that have been clipped at both the aminoand carboxyterminus. If this is the case, then the staining pattern observed for anti-phycocyanin antibodies could be consistent with the recognition of a site(s) at the extreme carboxy-terminus of the phytochrome molecule. Under these circumstances the anti-phycocyanin antibodies would be expected to stain also the 55-kDa peptide, produced by digestion of both Pr and Pfr, since this is thought to represent the complete carboxy-terminal domain (Jones et al. 1985). The failure to detect staining of this fragment as a consequence of its low abundance cannot be ruled out. The observation that the monoclonal antibody LAS 41 can compete, to same extent, with the antiphycocyanin antibodies for binding to phytochrome might tend to indicate that, at least some, binding of anti-phycocyanin antibodies to phytochrome occurs at, or near, the 4-kDa sub-aminoterminal domain. However, competition between LAS 41 and anti-phycocyanin antibodies is only observed in the sandwich ELISA system in which phytochrome will be expected to be in its native state. This raises the possibility that the competition results from the close proximity, in the native, folded state, of two regions of the molecule which are quite separate in terms of the primary structure. Recently, Schneider-Poetsch etal. (1988) have reported that monoclonal antibodies that map to distinct regions of the phytochrome sequence can inhibit each other's binding in ELISA, and conclude that their epitopes are only adjacent

397

in the tertiary structure. Furthermore, these authors observed competition between pairs of monoclonal antibodies in which one antibody recognised an epitope close to the amino-terminus and the other recognised an epitope close to the carboxy-terminus. Although it is not possible to be certain about the region of the phytochrome molecule that is being recognised by the anti-phycocyanin antibodies, it is clear that phytochrome and phycocyanin(s) do share at least one antigenic determinant. However, comparisons of the published amino-acid sequences of phytochrome with those of the subunits of C- and allo-phycoycanins have failed to demonstrate any appreciable regions of homology. This could mean that the shared epitope is not continuous but, rather, is determined to some extent by secondary or higher structure. The findings that the anti-phycocyanin antibodies only recognised native, 124-kDa phytochrome, are Pr-specific and show photoreversible binding raise the possibility of a novel method of phytochrome purification. Previous attempts to immunoaffinity purify phytochrome have produced pure, but spectrally degraded phytochrome as a result of the harsh conditions needed to elute phytochrome in such a system (Pratt 1984). The observation that the anti-phytochrome component(s) could be eluted from immobilised phytochrome by irradiation with R under non-denaturing conditions indicates that that the reverse procedure is feasible. Namely, the R elution of phytochrome from the immobilised antibody. Finally, the finding that phycocyanin and phytochrome share at least one antigenic determinant, which appears to be involved in phytochrome protein-chromophore interactions raises interesting questions concerning the evolution of phytochrome. We are extremely grateful to Dr. Mary Holdsworth for the provision of phytochrome, mouoclonal antibodies and valuable technical advice.

References Berns, D.S. (1967) Immunochemistry of biliproteins. Plant Physiol. 42, i569-1586 Bogorad, L. (1975) Phycobiliproteins and complementary chromatic adaptation. Annu. Rev. Plant Physiol. 26, 369~401 Cordonnier, M-M., Greppin, H., Pratt, L.H. (1985) Monoclonal antibodies with differing affinities to the red-absorbing and far-red absorbing forms of phytochrome. Biochemistry 24, 32463253 Cundiff, S.L., Pratt, L.H. (1975) Phytochrome characterization by rabbit antiserum against high-molecular weight phytochrome. Plant Physiol. 55, 207-211 Grimm, R., Lottspeich, F., Schneider, H.A.W., Rudiger, W.

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D.R. Keiller et al. : Antibodies to phycocyanin cross-react with phytochrome

(1986) Investigations of the peptide chain of 124 kDa phytochrome. Localisation of proteolytic fragments and epitopes for monoclonal antibodies. Z. Naturforsch. 41e, 988 992 Holdsworth, M.L. (1987) Characterization of phytochrome using monoclonal antibodies. Ph.D. Thesis, University of Leicester, UK Holdsworth, M.L., Whitelam, G.C. (1987) A monoclonal antibody specific for the red-absorbing form of phytochrome. Planta 172, 539 547 Hopkins, D.W., Butler, W.L. (1970) Immunochemical and spectroscopic evidence for protein conformational changes in phytochrome transformation. Plant Physiol. 45, 567 570 Jones, AM., Vierstra, R.D., Daniels, S.M., Quail, P.H. (1985) The role of separate molecular domains in the structure of phytochrome from etiolated Arena sativa L. Planta 164, 501 506 Kelly, J.M., Lagarias, J.C. (1985) Photochemistry of 124 kilodalton Arena phytochrome under constant illumination invitro. Biochemistry 24, 6003-6010 Pratt, L.H. (1984) Phytochrome purification In: Techniques in photomorphogenesis, pp. 175-200, Smith, H., Holmes, M.G., eds. Academic Press, London Rice, H.V., Briggs, W.R. (1973) Immunochemistry of phytochrome. Plant Physiol. 51, 939-945 Shimazaki, Y., Cordonnier, MM., Pratt, L.H. (1986) Identification with monoclonal antibodies of a second antigenic do-

main in Arena phytochrome that changes upon its photoconversion. Plant Physiol. 82, 109-113 Shropshire, W., Mohr, H. (1985) (eds.) Encyclopedia of plant physiology, N.S. vol. 16A, B: Photomorphogenesis, Springer, Berlin New York Siegelman, H.W., Turner, B.C., Hendricks, S.B. (1966) The chromophore of phytochrome. Plant Physiol. 41, 1289-1293 Schneider-Poetsch, H.A.W., Schwartz, H., Grimm, R., Rudiger, W. (1988) Cross reactivity of monoclonal antibodies against phytochrome from Zea and Arena. Localisation of epitopes, and an epitope common to monocotyledons, dicotyledons, ferns, mosses and a liverwort. Planta 173, 61-72 Thomas, B., Butcher, G.W., Galfre, G. (1984) Discrimination between the red- and far-red-absorbing forms of phytochrome from Arena sativa L, by monoclonal antibodies. Planta 160, 382-384 Thomas, B., Penn, S.E. (1986) Monoclonal antibody ARC MAC 50.1 binds to a site on the phytochrome molecule which undergoes a photoreversible conformational change. FEBS Lett. 195, 174 178 Vierstra, R.D., Quail, P.H. (1983) Purification and initial characterization of 124 kilodalton phytochrome fi-om Arena. Biochemistry 22, 2498-2505 Received 24 April; accepted 7 July 1988

Polyclonal antibodies raised to phycocyanins contain components specific for the red-absorbing form of phytochrome.

Polyclonal antibodies raised in rabbits to a mixture of sodium-dodecyl-sulphate-denatured C- and allo-phycocyanin, isolated from Anabaena cylindrica, ...
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