Planta (1985)164:501-506
P l a n t a 9 Springer-Verlag 1985
The role of separate molecular domains in the structure of phytochrome from etiolated Avena sativa L. A.M. Jones, R.D. Vierstra*, S.M.Daniels and P. Quail Department of Botany, 139 Birge Hall, University of Wisconsin, Madison, WI 53706, USA
Abstract. The spectral properties of peptides generated from etiolated-Avena, 124-kDa (kilodalton) phytochrome by endogenous protease(s) have been studied to assess the role of the amino-terminal and the carboxyl-terminal domains in maintaining the proper interaction between protein and chromophore. The amino-terminal, 74-kDa chromopeptide, a degradation product of the far-red absorbing form of the pigment (Pfr), is shown to be spectrally similar to the 124-kDa, undegraded molecule. The minimum and maximum of the difference spectrum (Pr-Pfr) are 730 and 665 rim, respectively, and the spectral-change ratio is unity. Also, like undegraded, 124-kDa phytochrome, the 74-kDa peptide exhibits minimal dark reversion. These data indicate that the 55-kDa, carboxyl-terminal half of the polypeptide does not interact with the chromophore and may not have a role in the structural integrity of the amino-terminal domain. The 64-kDa chromopeptide can be generated directly from the 74-kDa species by cleavage of 10 kDa from the amino terminus upon incubation of this species as Pr. Accompanying this conversion are changes in the spectral properties, namely, a shift in the difference spectrum minimum to 722-724 nm and a tenfold increase in the capacity for dark reversion. These data indicate that the 6-10 kDa, amino-terminal segment continues to function in its role of maintaining proper chromophore-protein interactions in the 74-kDa peptide as it does in the undegraded molecule. Conversely, removal of this segment upon proteolysis to the 64-kDa species leads to aberrant spectral proper* Present address: Department of Horticulture, 1575 Linden
Drive, University of Wisconsin, Madison, WI 53706, USA Abbreviations and symbols: Da=dalton; Pfr = far-red-absorb-
ing form of phytochrome; PMSF = phenylmethylsulfonyl fluoride; Pr= red-absorbing form of phytochrome; R = red light; FR = far-red light; AAr/AA~r = spectral change ratio; 2F~ = peak maximum (rim) of Pfr absorbance
ties analogous to those observed when this domain is lost from the full-length, 124-kDa molecule, resulting in the 118/l14-kDa degradation products. The data also show that photoconversion of the 74-kDa chromopeptide from Pfr to Pr exposes proteolytically susceptible sites in the same way as in the 124-kDa molecule. Thus, the separated, 74-kDa amino-terminal domain undergoes a photoinducible conformational change comparable to that in the intact molecule. Key words: Avena (phytochrome) - Immunoblotting - Phytochrome structural domains - Proteolysis.
Introduction The molecular mechanism by which phytochrome elicits photomorphogenic responses is not understood (see Shrophshire and Mohr 1983). One approach toward gaining insight into this mechanism has been to study the structure of the phytochrome molecule (Pratt 1982). Data accumulated using such an approach have permitted a peptide map to be constructed (Daniels and Quail 1984; Vierstra et al. 1984) providing the basis for a further structural analysis which is reported here. Under defined conditions, phytochrome in a crude extract degrades to a group of peptide products (Daniels and Quail 1984; Vierstra et al. 1984). To assess the structural relationship of these peptides, we have followed the changes in absorbance and the capacity for dark reversion that occur as phytochrome degrades. The extent of degradation and the identity of the degradation products were determined using immunoblot analysis with both polyclonal and monoclonal antibodies directed against 124-kDa phytochrome. From a correlation
502
A.M. Jones et al.: Phytochrome structure in etiolated Arena
Fig. 1. Time-course of phytochrome digestion as Pfi- and as Pr. A crude homogenate containing Arena phytochrome was allowed to digest at 20 ~ C for 22 h as either Pfr or Pr. Aliquots were removed at the times indicated, subjected to sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS PAGE, 8% acrylamide) and electrophoretically blotted to nitrocellulose (Western blotting). Blots were probed with polyclonal antibodies prepared in rabbit against 124-kDa, Arena phytochrome and further developed as described in Material and methods
between degradation and the changes in these spectral parameters we have made conclusions about the role of separate domains of the molecule and the protein conformation of the degradation products containing these domains.
Phytochrome preparations and spectral measurements. Undegraded, 124-kDa phytochrome from etiolated Arena was purified as described (Vierstra and Quail 1983b). Partially degraded, 118/114-kDa phytochrome was purified by the method of Hunt and Pratt (1979). Difference spectra were taken by the method described in Vierstra and Quail (1982).
Material and methods
In-vitro digestion. Shoot tissue (50 g) was irradiated with red light (2m,x 666 nm; 5 J m -2 s-1) for 5 rain then homogenized in 37.5 ml of buffer containing 100 mM 3-(N-morpholino)propanesulfonic acid (Mops), pH 7.8, 5 mM tetrasodium ethylenediaminetetraacetic acid (Na 4 EDTA) and 56 mM/~-mercaptoethanol; PMSF was not included. The homogenate was filtered through a nylon cloth, and centrifuged at 48000 g for 10 rain, 2 ~ C. Calcium chloride was added to the supernatant to make a final concentration of 15 m M then the total mixture was stirred on ice for 5 rain and centrifuged at 48 000 g for 20 min, 2 ~ C. The supernatant was divided equally and the aliquots were irradiated for 5 rain with red (R) (16 J m -2 s- 1) or far-red light (FR) (900 J m - 2 s - l ) . U p to this point, special care was taken to keep all buffers ice-cold. After irradiation, the extraction mixture was incubated at 20 ~ C and, at the indicated times, aliquots were taken for sodium dodeeylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and for measurement of the absorbance spectra as previously described (Vierstra and Quail 1982). All manipulations were performed in the dark or under dim green light (Bolton and Quail 1982).
Plant material and chemicals. Oat seeds (Arena sativa L. cv. Garry; Olds Seed Co., Madison, Wis., USA) were sown on wet vermiculite and grown at 28 ~ C in the dark for 4.5 d. Shoots were harvested just below the coleoptilar node and stored up to 7 d in the dark at 4 ~ C. Acrylamide, bisacrylamide, sodium dodecylsulfate (SDS) and agarose were purchased from Biorad Laboratories (Richmond, Cal., USA). Bovine serum albumin (BSA), octyl phenoxy polyethanol (Triton X-100), polyethylene sorbitan monolaurate (Tween 20), phenylmethylsulfonyl fluoride (PMSF), 2-amino-2-(hydroxymethyl)-l,3-propanediol (Tris) and 5-bromo-4-chloro-3-indolyl phosphate were purchased from Sigma Chemical Co. (St. Louis, Mo., USA). Rabbit immunoglobulin (IgG) directed against mouse and goat IgG directed against rabbit IgG and conjugated with alkaline phosphatase were products of Kirkegaard & Perry Laboratories (Gaitherburg, Md., USA). Mouse monoclonal antibodies directed against etiolated, 124-kDa phytochrome were prepared as described in Daniels and Quail (1984).
A.M. Jones et al. : Phytochrome structure in etiolated Arena
503
Sodium dodeeylsulfate-PAGE and immunoblotting were performed with 100-200 ng phytochrome per lane essentially as described previously by Tokuhisa et al. (1985) except for the following modifications. Nonspecific binding on Western blots was saturated with 0.1% BSA in 100 m M Tris (pH 9), 150 m M NaC1 and 0.05% Tween 20 at 2 ~ C overnight. All incubations with antibodies and intermediate washes were with the same buffer except the p H was 7.5. When monoclonal antibodies were used, the primary incubation (monoclonal antibodies) was 3 h, the second incubation with polyclonal, anti-mouse IgG (prepared in rabbit) was 1 h, and the tertiary incubation with polyclonal, anti-rabbit IgG (prepared in goat, conjugated with alkaline phosphatase) was I h. Following each incubation, the blots were rinsed, then washed for 1 h with two changes of buffer. The final wash contained 0.05% SDS.
Results
The different peptide patterns obtained when phytochrome is digested as Pfr and Pr are compared in Fig. 1. As previously shown (Daniels and Quail 1984; Vierstra et al. 1984), the major products of digestion as Pfr are 90-kDa, 74-kDa, and 55-kDa peptides. In addition, 64-kDa and 39-kDa fragments become visible within 2 h of digestion and by 4 h there is little remaining 124-kDa phytochrome. Substantial levels of the Pfr degradation products are visible at time zero (Fig. 1, both panels) illustrating the rapidity of proteolysis when PMSF is excluded from the extraction medium. After 22 h digestion as Pfr the predominant peptide is 74 kDa. Digestion as Pr yields a different pattern. After 4 h of digestion as Pr the predominant peptides are 114 kDa, 64 kDa and 55 kDa. The 114-kDa peptide is more stable than undegraded 124-kDa phytochrome under these conditions but is completely digested by 22 h. The transient appearance of the 55-kDa species indicates that it is rapidly degraded under these conditions. At time zero, 14% of the phytochrome in the sample being digested as Pfr is actually Pr because of incomplete photoconversion (Vierstra and Quail 1983a). This amount of Pr contaminant accounts for the 64-kDa peptide visible throughout Pfr digestion (Fig. 1, left panel). If the sample being digested as Pfr is photoconverted to Pr after 4 h the 74-kDa peptide is lost (Fig. 2). First a 68/64-kDa doublet appears followed by the 64-kDa band which is stable even up to 22 h of digestion. The largest peptide at 4 h from which this 64-kDa peptide could be generated in the amounts shown is the 74-kDa peptide. These data directly confirm, first that the 74-kDa peptide contains the chromophore since susceptibility to proteolysis is induced by FR, and second that the 64-kDa peptide can be derived directly from the 74-kDa peptide by a loss of 10 kDa.
Fig. 2. Time-course of digestion of 74-kDa phytochrome. Phytochrome in a crude extract was allowed to digest as Pfr for 4 h at 20 ~ C to generate a sample enriched in the 74-kDa species and then photoconverted to Pr. The digestion was continued until 22 h. Samples were taken at the times indicated and subjected to SDS-PAGE and Western blot analysis. Blots were probed with polyelonal antibodies prepared in rabbit against 124-kDa, Arena phytochrome and further developed as described in Material and methods
Western blots were probed with monoclonal antibodies to determine from which region of the 74-kDa peptide this 10 kDa was removed. Figure 3 illustrates blots which were probed with a monoclonal antibody specific for an epitope in the first 6 kDa of the amino-terminus (Daniels and Quail 1984; Hershey et al. 1985). This antibody, therefore, does not recognize either the 118-kDa or the 64-kDa peptide generated in Pr digestions. Conversely, both the 124-kDa, undegraded phytochrome and the 74-kDa peptide react positively because they have this amino-terminal epitope (Daniels and Quail 1984). When the sample containing the 74-kDa peptide is photoconverted to Pr after 4 h of Pfr digestion, recognition by the antibody is completely lost within 2 h, indicating that the direct conversion of the 74-kDa fragment to the 64-kDa species (cf Fig. 2) occurs at the amino terminus in the same manner that 124-kDa phytochrome is converted to the l18/114-kDa peptides.
A.M. Jones et al. : Phytochrome structure in etiolated Arena
504
Fig. 3. Immunoblot analysis with Type-1 monoclonal antibodies of peptides generated by proteolytic digestion of Avena phytochrome. A crude extract of phytochrome was digested either as Pfr (left panel) or as Pfr for 4 h then photoconverted to Pr (right panel) as described in Fig. 2. Samples were removed at the times indicated and subjected to SDS-PAGE and Western blot analysis. Blots were probed with a mouse monoclonal antibody that recognizes an epitope located within 6 kDa of the amino-terminus of phytochrome (Daniels and Quail 1984)
732L,,, 750
728
C o n c o m i t a n t w i t h t h e d i g e s t i o n o f the 7 4 - k D a p e p t i d e is a c h a n g e in the p e a k m a x i m u m o f P f r a b s o r b a n c e (2m. Fa x, Fig. 4). V i e r s t r a a n d Q u a i l (1982) p r e v i o u s l y d e m o n s t r a t e d t h a t a 6 - 8 - n m shift in the 2ma FRx is a s s o c i a t e d w i t h the loss o f 10 k D a from 124-kDa phytochrome. I n d i c a t e d here (Fig. 4) is a n identical c h a n g e in the 2ma va x as the t e r m i n a l 10 k D a is lost f r o m 7 4 - k D a p h y t o c h r o m e . S p e c t r a l c h a n g e r a t i o s ( A A r / A A f r ) f o r the 7 4 - k D a a n d 6 4 - k D a p e p t i d e s are 0.98 a n d 1.33, respectively, c o m p a r a b l e to the values f o r 1 2 4 - k D a (1.07) a n d 1 1 8 / 1 1 4 - k D a p h y t o c h r o m e (1.3), respectively (Fig. 5).
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P l a n t a 9 Springer-Verlag 1985
The role of separate molecular domains in the structure of phytochrome from etiolated Avena sativa L. A.M. Jones, R.D. Vierstra*, S.M.Daniels and P. Quail Department of Botany, 139 Birge Hall, University of Wisconsin, Madison, WI 53706, USA
Abstract. The spectral properties of peptides generated from etiolated-Avena, 124-kDa (kilodalton) phytochrome by endogenous protease(s) have been studied to assess the role of the amino-terminal and the carboxyl-terminal domains in maintaining the proper interaction between protein and chromophore. The amino-terminal, 74-kDa chromopeptide, a degradation product of the far-red absorbing form of the pigment (Pfr), is shown to be spectrally similar to the 124-kDa, undegraded molecule. The minimum and maximum of the difference spectrum (Pr-Pfr) are 730 and 665 rim, respectively, and the spectral-change ratio is unity. Also, like undegraded, 124-kDa phytochrome, the 74-kDa peptide exhibits minimal dark reversion. These data indicate that the 55-kDa, carboxyl-terminal half of the polypeptide does not interact with the chromophore and may not have a role in the structural integrity of the amino-terminal domain. The 64-kDa chromopeptide can be generated directly from the 74-kDa species by cleavage of 10 kDa from the amino terminus upon incubation of this species as Pr. Accompanying this conversion are changes in the spectral properties, namely, a shift in the difference spectrum minimum to 722-724 nm and a tenfold increase in the capacity for dark reversion. These data indicate that the 6-10 kDa, amino-terminal segment continues to function in its role of maintaining proper chromophore-protein interactions in the 74-kDa peptide as it does in the undegraded molecule. Conversely, removal of this segment upon proteolysis to the 64-kDa species leads to aberrant spectral proper* Present address: Department of Horticulture, 1575 Linden
Drive, University of Wisconsin, Madison, WI 53706, USA Abbreviations and symbols: Da=dalton; Pfr = far-red-absorb-
ing form of phytochrome; PMSF = phenylmethylsulfonyl fluoride; Pr= red-absorbing form of phytochrome; R = red light; FR = far-red light; AAr/AA~r = spectral change ratio; 2F~ = peak maximum (rim) of Pfr absorbance
ties analogous to those observed when this domain is lost from the full-length, 124-kDa molecule, resulting in the 118/l14-kDa degradation products. The data also show that photoconversion of the 74-kDa chromopeptide from Pfr to Pr exposes proteolytically susceptible sites in the same way as in the 124-kDa molecule. Thus, the separated, 74-kDa amino-terminal domain undergoes a photoinducible conformational change comparable to that in the intact molecule. Key words: Avena (phytochrome) - Immunoblotting - Phytochrome structural domains - Proteolysis.
Introduction The molecular mechanism by which phytochrome elicits photomorphogenic responses is not understood (see Shrophshire and Mohr 1983). One approach toward gaining insight into this mechanism has been to study the structure of the phytochrome molecule (Pratt 1982). Data accumulated using such an approach have permitted a peptide map to be constructed (Daniels and Quail 1984; Vierstra et al. 1984) providing the basis for a further structural analysis which is reported here. Under defined conditions, phytochrome in a crude extract degrades to a group of peptide products (Daniels and Quail 1984; Vierstra et al. 1984). To assess the structural relationship of these peptides, we have followed the changes in absorbance and the capacity for dark reversion that occur as phytochrome degrades. The extent of degradation and the identity of the degradation products were determined using immunoblot analysis with both polyclonal and monoclonal antibodies directed against 124-kDa phytochrome. From a correlation
502
A.M. Jones et al.: Phytochrome structure in etiolated Arena
Fig. 1. Time-course of phytochrome digestion as Pfi- and as Pr. A crude homogenate containing Arena phytochrome was allowed to digest at 20 ~ C for 22 h as either Pfr or Pr. Aliquots were removed at the times indicated, subjected to sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS PAGE, 8% acrylamide) and electrophoretically blotted to nitrocellulose (Western blotting). Blots were probed with polyclonal antibodies prepared in rabbit against 124-kDa, Arena phytochrome and further developed as described in Material and methods
between degradation and the changes in these spectral parameters we have made conclusions about the role of separate domains of the molecule and the protein conformation of the degradation products containing these domains.
Phytochrome preparations and spectral measurements. Undegraded, 124-kDa phytochrome from etiolated Arena was purified as described (Vierstra and Quail 1983b). Partially degraded, 118/114-kDa phytochrome was purified by the method of Hunt and Pratt (1979). Difference spectra were taken by the method described in Vierstra and Quail (1982).
Material and methods
In-vitro digestion. Shoot tissue (50 g) was irradiated with red light (2m,x 666 nm; 5 J m -2 s-1) for 5 rain then homogenized in 37.5 ml of buffer containing 100 mM 3-(N-morpholino)propanesulfonic acid (Mops), pH 7.8, 5 mM tetrasodium ethylenediaminetetraacetic acid (Na 4 EDTA) and 56 mM/~-mercaptoethanol; PMSF was not included. The homogenate was filtered through a nylon cloth, and centrifuged at 48000 g for 10 rain, 2 ~ C. Calcium chloride was added to the supernatant to make a final concentration of 15 m M then the total mixture was stirred on ice for 5 rain and centrifuged at 48 000 g for 20 min, 2 ~ C. The supernatant was divided equally and the aliquots were irradiated for 5 rain with red (R) (16 J m -2 s- 1) or far-red light (FR) (900 J m - 2 s - l ) . U p to this point, special care was taken to keep all buffers ice-cold. After irradiation, the extraction mixture was incubated at 20 ~ C and, at the indicated times, aliquots were taken for sodium dodeeylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and for measurement of the absorbance spectra as previously described (Vierstra and Quail 1982). All manipulations were performed in the dark or under dim green light (Bolton and Quail 1982).
Plant material and chemicals. Oat seeds (Arena sativa L. cv. Garry; Olds Seed Co., Madison, Wis., USA) were sown on wet vermiculite and grown at 28 ~ C in the dark for 4.5 d. Shoots were harvested just below the coleoptilar node and stored up to 7 d in the dark at 4 ~ C. Acrylamide, bisacrylamide, sodium dodecylsulfate (SDS) and agarose were purchased from Biorad Laboratories (Richmond, Cal., USA). Bovine serum albumin (BSA), octyl phenoxy polyethanol (Triton X-100), polyethylene sorbitan monolaurate (Tween 20), phenylmethylsulfonyl fluoride (PMSF), 2-amino-2-(hydroxymethyl)-l,3-propanediol (Tris) and 5-bromo-4-chloro-3-indolyl phosphate were purchased from Sigma Chemical Co. (St. Louis, Mo., USA). Rabbit immunoglobulin (IgG) directed against mouse and goat IgG directed against rabbit IgG and conjugated with alkaline phosphatase were products of Kirkegaard & Perry Laboratories (Gaitherburg, Md., USA). Mouse monoclonal antibodies directed against etiolated, 124-kDa phytochrome were prepared as described in Daniels and Quail (1984).
A.M. Jones et al. : Phytochrome structure in etiolated Arena
503
Sodium dodeeylsulfate-PAGE and immunoblotting were performed with 100-200 ng phytochrome per lane essentially as described previously by Tokuhisa et al. (1985) except for the following modifications. Nonspecific binding on Western blots was saturated with 0.1% BSA in 100 m M Tris (pH 9), 150 m M NaC1 and 0.05% Tween 20 at 2 ~ C overnight. All incubations with antibodies and intermediate washes were with the same buffer except the p H was 7.5. When monoclonal antibodies were used, the primary incubation (monoclonal antibodies) was 3 h, the second incubation with polyclonal, anti-mouse IgG (prepared in rabbit) was 1 h, and the tertiary incubation with polyclonal, anti-rabbit IgG (prepared in goat, conjugated with alkaline phosphatase) was I h. Following each incubation, the blots were rinsed, then washed for 1 h with two changes of buffer. The final wash contained 0.05% SDS.
Results
The different peptide patterns obtained when phytochrome is digested as Pfr and Pr are compared in Fig. 1. As previously shown (Daniels and Quail 1984; Vierstra et al. 1984), the major products of digestion as Pfr are 90-kDa, 74-kDa, and 55-kDa peptides. In addition, 64-kDa and 39-kDa fragments become visible within 2 h of digestion and by 4 h there is little remaining 124-kDa phytochrome. Substantial levels of the Pfr degradation products are visible at time zero (Fig. 1, both panels) illustrating the rapidity of proteolysis when PMSF is excluded from the extraction medium. After 22 h digestion as Pfr the predominant peptide is 74 kDa. Digestion as Pr yields a different pattern. After 4 h of digestion as Pr the predominant peptides are 114 kDa, 64 kDa and 55 kDa. The 114-kDa peptide is more stable than undegraded 124-kDa phytochrome under these conditions but is completely digested by 22 h. The transient appearance of the 55-kDa species indicates that it is rapidly degraded under these conditions. At time zero, 14% of the phytochrome in the sample being digested as Pfr is actually Pr because of incomplete photoconversion (Vierstra and Quail 1983a). This amount of Pr contaminant accounts for the 64-kDa peptide visible throughout Pfr digestion (Fig. 1, left panel). If the sample being digested as Pfr is photoconverted to Pr after 4 h the 74-kDa peptide is lost (Fig. 2). First a 68/64-kDa doublet appears followed by the 64-kDa band which is stable even up to 22 h of digestion. The largest peptide at 4 h from which this 64-kDa peptide could be generated in the amounts shown is the 74-kDa peptide. These data directly confirm, first that the 74-kDa peptide contains the chromophore since susceptibility to proteolysis is induced by FR, and second that the 64-kDa peptide can be derived directly from the 74-kDa peptide by a loss of 10 kDa.
Fig. 2. Time-course of digestion of 74-kDa phytochrome. Phytochrome in a crude extract was allowed to digest as Pfr for 4 h at 20 ~ C to generate a sample enriched in the 74-kDa species and then photoconverted to Pr. The digestion was continued until 22 h. Samples were taken at the times indicated and subjected to SDS-PAGE and Western blot analysis. Blots were probed with polyelonal antibodies prepared in rabbit against 124-kDa, Arena phytochrome and further developed as described in Material and methods
Western blots were probed with monoclonal antibodies to determine from which region of the 74-kDa peptide this 10 kDa was removed. Figure 3 illustrates blots which were probed with a monoclonal antibody specific for an epitope in the first 6 kDa of the amino-terminus (Daniels and Quail 1984; Hershey et al. 1985). This antibody, therefore, does not recognize either the 118-kDa or the 64-kDa peptide generated in Pr digestions. Conversely, both the 124-kDa, undegraded phytochrome and the 74-kDa peptide react positively because they have this amino-terminal epitope (Daniels and Quail 1984). When the sample containing the 74-kDa peptide is photoconverted to Pr after 4 h of Pfr digestion, recognition by the antibody is completely lost within 2 h, indicating that the direct conversion of the 74-kDa fragment to the 64-kDa species (cf Fig. 2) occurs at the amino terminus in the same manner that 124-kDa phytochrome is converted to the l18/114-kDa peptides.
A.M. Jones et al. : Phytochrome structure in etiolated Arena
504
Fig. 3. Immunoblot analysis with Type-1 monoclonal antibodies of peptides generated by proteolytic digestion of Avena phytochrome. A crude extract of phytochrome was digested either as Pfr (left panel) or as Pfr for 4 h then photoconverted to Pr (right panel) as described in Fig. 2. Samples were removed at the times indicated and subjected to SDS-PAGE and Western blot analysis. Blots were probed with a mouse monoclonal antibody that recognizes an epitope located within 6 kDa of the amino-terminus of phytochrome (Daniels and Quail 1984)
732L,,, 750
728
C o n c o m i t a n t w i t h t h e d i g e s t i o n o f the 7 4 - k D a p e p t i d e is a c h a n g e in the p e a k m a x i m u m o f P f r a b s o r b a n c e (2m. Fa x, Fig. 4). V i e r s t r a a n d Q u a i l (1982) p r e v i o u s l y d e m o n s t r a t e d t h a t a 6 - 8 - n m shift in the 2ma FRx is a s s o c i a t e d w i t h the loss o f 10 k D a from 124-kDa phytochrome. I n d i c a t e d here (Fig. 4) is a n identical c h a n g e in the 2ma va x as the t e r m i n a l 10 k D a is lost f r o m 7 4 - k D a p h y t o c h r o m e . S p e c t r a l c h a n g e r a t i o s ( A A r / A A f r ) f o r the 7 4 - k D a a n d 6 4 - k D a p e p t i d e s are 0.98 a n d 1.33, respectively, c o m p a r a b l e to the values f o r 1 2 4 - k D a (1.07) a n d 1 1 8 / 1 1 4 - k D a p h y t o c h r o m e (1.3), respectively (Fig. 5).
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