Laser-flash photolysis of 124 kDa oat phytochrome: Studies concerning the late steps of Pfr formation

Journal of Photochemistry

and Photobiology,

B: Biology, 3 (1989)

209

- 222

209

LASER-FLASH PHOTOLYSIS OF 124 kDa OAT PHYTOCHROME: STUDIES CONCERNING THE LATE STEPS OF Pfr FORMATION PETRA PETER

H. EILFELDa, G. EILFELD’T

JORGEN

VOGELb,

ROBERT

MAURERb

and

aBotanisches Institut der Universitiit Miinchen, Menzingerstr. 67, D-8000 Miinchen 19 (F.R.G.) bZnstitut fiir Physikalische und Theoretische Chemie der Technischen Universitiit Miinchen, Lichtenbergstr. 4, D-8046 Garching (F.R.G.) (Received

March 4, 1988;

accepted

August

Keywords. Phytochrome, oat (Auena tion, reporter group, viscosity.

8, 1988)

satiua L.), kinetics,

chemical

modifica-

Abbreviations ANS FMA HPLC Ibl,

ITOO

h,,

h’,

k,,

k,’

kDa KPB lumi-R meta-Rt,, meta-Rb/Pbi, meta-R,,, meta-Rt,

8-anilino-1-naphthalenesulphonic acid fluorescein mercuric acetate high pressure liquid chromatography intermediates on pathway P, to Pf, rate constants for transformation of intermediates kilodalton potassium phosphate buffer first intermediate on pathway P, to Pf, intermediates on pathway lumi-R to Pf, far-red-absorbing form of phytochrome red-absorbing form of phytochrome

Pfr P*

Summary Phototransformation kinetics of 124 kDa oat phytochrome at 298 K after red (660 nm) laser-flash excitation were recorded at different wavelengths under various conditions. Only the last steps of the phototransformation were investigated. On changing the environment of the apoprotein, especially by increasing the microviscosity, the formation of meta-Rt, and P,, which presumably involve rearrangement of the apoprotein, was retarded. In contrast, the decay of the intermediate meta-R, was not retarded. Using phytochrome specifically labelled by covalent attachment of fluorescein mercuric acetate acting as a reporter group, it was possible to trace changes *Author

to whom

loll-1344/89/$3.50

correspondence

should

be addressed.

0 Elsevier Sequoia/Printed

in The Netherlands

210

in the apoprotein conformation. In this way, it was demonstrated that major changes on exposed sites of the protein take place at the very last step of phototransformation, i.e. during formation of Pi, from the preceding meta-R,. The data are discussed in terms of molecular aspects, such as conformational changes of the protein.

1. Introduction Phytochrome, one of the most interesting and best known photoreceptors in plants, is able to control a variety of important physiological processes [l, 21. Although many of its properties have been investigated in detail, its action at a molecular level is still unclear. Recently, several experiments using monoclonal antibodies [ 3 - 61, circular dichroism in the far-UV spectral region [7], and chemical modification [8,9] point to conformational changes of the protein as a primary signal. However, these conformational changes have not yet been characterized in much detail. In particular, the time scale of their appearance and their correlation with changes of the tetrapyrrolic chromophore have not yet been examined. In the present study, the protein component of 124 kDa phytochrome is investigated during phototransformation by laser-flash excitation and rapid kinetic measurements. This was done by changing the protein environment, as well as by specific labelling of the apoprotein and by monitoring the absorbance changes of a reporter group.

2. Materials and methods 2.1. Phytochrome 124 kDa oat phytochrome was isolated after red irradiation of oat (Auena satiua L., cv. Pirol) tissue according to ref. 8 with omission of the last chromatographic step, i.e. Bio-gel chromatography. By this procedure, phytochrome at concentrations of about 20 PM (based upon monomer molar absorption coefficient of 132 000 1 mol-’ cm-‘, see ref. 10) with a purity index A,,,/A280 up to 0.85 was obtained. The spectral properties of the long-wavelength region were as reported in ref. 11. Additionally, care was taken that for 124 kDa phytochrome the ratio A,s0(Pfr)/A6,s(Pfr) was greater than 1.45. This ratio is a very sensitive test for degradation and denaturation. For 118 kDa phytochrome (obtained by incubation with endogenous proteases according to ref. 12) it drops below 1.27. Phytochrome solutions were prepared in 20 mM KPB, pH 7.8, containing 3% (v/v) glycerol and diluted with the respective agents to the desired concentration (see Table 1). For this, buffer, glycerol (Aldrich) or Ficoll 40 000 (Pharmacia, prepared as 45% (w/v) stock solution in KPB) were used. Glycerol and Ficoll allowed transfer into highly viscous media without

211

denaturation of phytochrome. The relative viscosity (ratio of the viscosity to the viscosity of water [13]) at 293 K of buffer containing 66% (v/v) glycerol was 16 [13], the same as the value found for 33% (w/v) Ficoll 40 000 (P.-S. Song et al., to be published; cf. 8.5 for 10% (w/v) dextran 72 000 U31).

Chemical modification with FMA was carried out as described by Eilfeld et al. [8] by addition of 3.5 equivalents of FMA and preincubation at room temperature for 45 min. By this procedure 2.5 - 3 molecules of FMA were covalently bound to a monomer of phytochrome as determined by gel filtration [8] or native size-exclusion HPLC (Gus et al., to be published). Samples were checked before and after laser flash photolysis by both SDS gel electrophoresis [8] and absorption spectrometry. Neither degradation nor significant spectral changes took place during the measurements. 2.2, Absorption spectra Absorption spectra were recorded on an HP 8451 A Hewlett Packard diode-array photometer with disc storage and processing capability. The shutter time for each spectrum was 0.1 s. If not otherwise noted, spectra were run at 298 K. Cooling of the samples was achieved by a cryostat (Haake, Berlin, F.R.G.). Pulse irradiation of the samples was performed as described in ref. 14. Electronic flash irradiation (duration 10 ms) of P, was done by means of a Metz mecablitz 214 L 28 R flash (Metz, F.R.G.) through a red plastic cut-off filter (50% transmission at 620 nm), giving photoconversion of 15% P, to P, after one flash, as determined from spectra recorded after 5 min. 2.3. Laser flash photolysis

The apparatus for laser flash photolysis has been described in detail in ref. 15. Samples were excited by a 10 ns laser flash at 660 nm. Some additional improvements have been introduced. Monochromatic measuring light was obtained from a halogen lamp through a PMQ II Zeiss monochromator (Zeiss, Oberkochen, F.R.G.). Slit settings were 0.05 - 0.2 mm, giving photon fluxes of about 3 X 1016 quanta m- * s-i. In this way, actinic effects of the measuring light could be completely eliminated for scanning periods up to 20 s. After leaving the sample, the measuring light passed through a second grating monochromator (B&M Spektronik BM 25, 1200 lines mm-‘) with a side-on photomultiplier (Hamamatsu, R 928). This two-monochromator set-up enabled the spectral purity of the measuring light to be significantly increased. The photomultiplier output was digitized and processed according to ref. 16. The sampling times were varied from 2 ps to 10 ms in order to obtain kinetic data from 100 /JSto 10 s. Absorbance changes were averaged over 16 - 32 measurements for wavelengths above 600 nm, and over 128 measurements for wavelengths at around 500 nm. In this way, noise could be reduced to less than 0.0003A (see Fig. 3). The temperature of the cuvette was kept at 297 - 298 K. Further details have been given in ref. 17.

212

2.4. Kinetic analysis The kinetic data were analysed by a multi-exponential curve-fitting procedure [15 - 181 employing a logarithmic time scale (cf. Figs. 1 and 3) with the measuring wavelengths generally set at 667, 695, 715, 730 or 745 nm for monitoring the absorption of the phytochrome chromophore, and at 513 nm for monitoring the reporter group (see Section 3). Fitting of kinetics both for individual wavelengths and for all wavelengths together (“global fit” [16]) gave coincident results. 3. Results 3.1. Phototransformation kinetics of different P, =+Pt, systems The phototransformation rates of laser-flash-irradiated P, were investigated under different environmental conditions. It was ensured that the samples studied showed a P, =+Pf, photoconversion that deviated only slightly from that of 124 kDa oat phytochrome at Amax(Pfr)> 725 nm. The fitting of the experimental curves started at 100 I.CS after irradiation, ensuring that no lumi-R was present, and only meta-R, (calculated from difference spectra; cf. ref. 15). The nomenclature for intermediates in this paper follows that of Kendrick and Spruit [ 19 J, which means that these intermediates represent a generic term for species showing uniform spectral behaviour, i.e. lumi-R represents I,00i, I,$, *a*,meta-R, is I,,il, I,,i2, . . . [20, 211. Fitting was generally carried out up to 5 s, after which no further reaction took place. This time was increased for glycerol samples only (see below). Using these restrictions, only the reactions from meta-Rt, to Pti were analysed. In general, the kinetics were described by the smallest number of exponentials sufficient to fit the experimental traces without showing deviations exceeding the peak-to-peak noise level. With this restriction, it has to be conceded that the given rate constants vary by f 30%, the standard deviation always remaining essentially the same. These intrinsic variations may account for the difference between the rates and those recently reported (see Table 1; ref. 15). Chemical modification of highly reactive cystein groups does not change the kinetics as long as the absorption properties are not altered [8,9] (see Table 1). This holds both for the rates and for the amplitude spectra of the processes with rates around 400 s-l (k,) and around 10 s-i (k,). Especially at 667 nm, no significant change in absorbance takes place for the slow process (k,). This slow process manifests itself essentially as a shift of the farred absorption to longer wavelengths and an increase in absorbance (data not shown; cf. ref. 15). We did not think it worth investigating modified phytochrome showing reduced photoreversibility, as preliminary studies with AN&bleached phytochrome [22] revealed strong deviations from the intermediate pathway of phytochrome, in particular the loss of lumi-R formation [23]. Slight degradation from 124 kDa to 118/124 kDa phytochrome does not alter the kinetics drastically, but significantly lowers the rate of P,

11 (0.01)

- 11(<(0.25) 0.007 )

-

1 667 nm

667 130 513 nm

730nm

meta-Rt,

meta-Re

kz’ kz -

10 (0.02) 10 (0.30) 10 (0.035 )

370 (-0.50) 370 (0.61) 370 (-0.005)

124 kDa FMA d

species kDad*e

< 1 (0.05) - (0)

-

6 (0.02) 6 (0.30)

340 (-0.50) 340 (0.64) -

118/124

-

7 (0.01) 7 (0.35)

-

670 (-0.07) 670 (0.31) -

320 (-0.50) 320 (0.48) -

0.50 (0.09) 0.50 (0.53)

48 (-0.51) 48 (0.17)

124 kDaf 66% (v/v) glycerol

124 kDaf 33% (w/v) FicoJJ

factor@) of different

aRate constants ki and pre-exponential factors (in parentheses) were obtained from global fits. Pre-exponential factors were normalized to final change in absorbance at 667 nm (= -1 .OO). Negative values denote absorbance decreases, and positive values denote absorbance increases. bAe67(Pr) = 1.35 for FMA-modified phytochrome; A,&P,) = 0.50 in all other cases. CCorrelation of a rate constant with transformation of intermediates was achieved by inspection of the difference spectra associated with the process of that rate constant and calculation of pure intermediate spectra. This procedure is described in detail in ref. 15. Thus, processes with rate constants kl (or k,‘) are associated with the decay of meta-Rt,, yielding meta-R,, and those with k2 (or kz’) with formation of Pn. dSamples in KPB, containing 1% (v/v) glycerol. FMA-labelled phytochrome contains 2.5 molecules FMA per monomer of phytochrome. eAccording to SDS gel electrophoresis (not shown), this sample turned out to be a mixture of equal parts of 118 and 124 kDa phytochrome [24]. Nevertheless, its kinetics could be sufficiently described by a simple set of data. fRelative viscosity, 16.

PfI

1

4 kl

I

470 (-0.50) 470 (0.72) - (< 0.007) -

667 nm 730 nm i 513 nm j 667 nm 1 730nm

meta-R, kl

124 kDad

Phytochrome

Wavelength 0 f assay

ReactionC

Kinetic data for reactions involving meta-Rt, decay and Pn appearance (rate constants ki in s-l and pre-exponential oat phytochrome species at 297 K after red laser flash excitation (660 nm, 1 mJ, 10 ns) of the Prb form

TABLE 1

214

formation. This is in agreement with ref. 24, in which the photochemical similarity of 118 and 124 kDa phytochrome is discussed, presumably in contrast to 114 kDa and further degraded phytochrome species. However, the amplitude spectrum for the processes with h, = 300 s-l (ascribed to meta-R, + meta-Rt, for 124 kDa phytochrome) and k, = 6 s-l (ascribed to meta-Rt, + Pr, for 124 kDa phytochrome) are nearly identical in the far-red region in contrast to 124 kDa phytochrome (not shown). Additionally, at 667 nm the absorbance increases 100 ms after the laser flash with a rate of about 1 s-i (&‘). 3.2. Viscosity effects The influence of the viscosity of the external medium on the reaction rates and amplitudes can also be seen in Table 1. The viscosity of the external medium was increased by adding a high molecular weight polydextrane, i.e. Ficoll 40 000 (33% (w/v) final concentration), or a low molecular weight polyhydroxy compound, i.e. glycerol (66% (v/v) final concentration), which exhibit approximately the same macroscopic viscosity. The absorption spectra are generally similar to those in pure KPB, but the Pf, form shows slightly enhanced absorbance in glycerol [ll]. Ficoll only slightly affects rates of phototransformation; the last step is retarded by a factor of 2. Just as with chemically modified phytochrome, the amplitude spectrum remains unchanged (data not shown). However, the situation is different in glycerol (see Fig. 1). In this case, it is no longer possible to describe the kinetics from meta-R, to Pr, by assuming only two intervening processes. This can be seen from the traces at 667 and 715 nm. While the absorbance at 667 nm is nearly constant during 0.2 - 3 ms after the laser flash, the absorbance at 715 nm already increases during this period. Furthermore, at around 50 ms, A,,, is constant but Ab6, continues to decrease. From the difference spectra and the amplitudes of the processes (k, and ki’, see Table l), a species showing the spectral properties of meta-Rt, is formed after about 500 ms. Finally, the process of P, formation (k,) is retarded by a factor of about 25. The overall retardation of the last steps of phototransformation made it possible to run spectra of phytochrome samples at reasonably high temperatures (268 K) after flash excitation using a commercial fast diode-array photometer, which may be of interest for teams with low-cost equipment. The results of such measurements are shown in Fig. 2. The wavelength range of the intermediate spectra could be extended from 250 to 800 nm with 2 nm resolution. The absorption spectra of the respective intermediates in the red spectral region correspond closely to those obtained from laser-flash measurements at room temperature (Fig. 1). In general, the difference spectra (not shown) are similar to those of 60 kDa phytochrome in 0.5 M sucrose at 274 K as reported in ref. 25, especially regarding the evolution of the far-red band. The absorption band in the blue spectral region is monotonically shifted to longer wavelengths on going from P, to Prr, as already observed in cryogenic spectroscopy [ 141. The spectrum of meta-R, is thus

215 AA

1

AA

667 nm

- 0.02

- 0.03

- 0.04 -4

-3

-2

-1

0 log

t Cs3

Fig. 1. Phytochrome phototransformation in highly viscous media. Absorbance changes detected at 715 nm (top) and 667 nm (bottom) after red laser-flash excitation (660 nm) of Pr (A667 = 0.5) at 298 K for 124 kDa oat phytochrome (KPB, diluted with glycerol to a final concentration of 66% (v/v)). Kinetics are shown on a logarithmic time scale (t) from 50 /.& to 5 s. For rate constants and amplitudes, see text and Table 1. For fitting, final absorbance values were read 25 s after the laser flash (&I667 = -0.035, AAvIs = 0.031).

similar to that of the Pt, form of 114/1X3 kDa phytochrome (A,,, = 390 and 722 nm [26]). In the UV spectral region at around 280 nm, in which aromatic amino acids show strong absorption, no measurable absorption changes took place. 3.3. Protein changes as detected by a reporter group In all recent publications, the phototransformation of phytochrome has been followed exclusively by tracing the absorption of the tetrapyrrolic chromophore. Since this chromophore resides in a shielded protein cavern [22], its absorption is influenced by its direct environment, but does not necessarily depend on changes of more remote parts of the protein, like its surface. Kinetic measurements of the protein component by monitoring its

216

300

500

A

CnmI)

000

Fig. 2. Intermediate spectra of P, photoconversion. Time-resolved absorption spectra of 124 kDa phytochrome (KPB, diluted with glycerol to a final concentration of 66% (v/v)) after electronic flash-light excitation of P, at 268 K using a commercial high speed diodearray spectrophotometer (for details see Section 2.2). Absorption spectra of pure intermediates were calculated on the basis of formation of 15% Pe after each flash and no dark reversion of the respective intermediates to P,. . . . . . , P, before flash; - -, formation of meta-Ra, (0.1 s after electronic flash); - - -, formation of meta-R, (1 s); -, formation of Pn (50 s).

UV absorption are extremely difficult owing to overlap with the chromophore absorption and the very small changes in absorbance [27] (cf. also Fig. 2). Recently, the fluorescein derivative FMA has been reported to show different absorption changes (and thus a different environment) when covalently attached to phytochro e in the P, or Pf, form [8]. According to ref. 9, G4.5 cysteins are able to re !rct within seconds with sulfhydryl-modifying chemicals. From this, one can assume that these cysteins reside on the protein surface or within an easily accessible pocket. Additional support for a surface location comes from the observation that reactive cysteins are able to bind covalently to large liposomes (cu. 16 000 kDa) in vitro [28]. Furthermore, the photochemistry of the phytochrome chromophore is not altered upon modification of 2.5 - 3 cysteins [8,9] (Table l), and thus a direct interaction of the (charged) fluorescein chromophore with the tetrapyrrolic chromophore is very unlikely. Besides, this dye has properties that make it a suitable reporter group. It absorbs around 500 nm, where phytochrome shows very low absorption. Even more importantly, the maximum effects of FMA absorption upon phototransformation are observed at 513 nm, which is exactly the isosbestic point for P, + Pf, phototransformation. From Fig. 2, it becomes obvious that this is an isosbestic point for P, + intermediates, too. This had been checked previously by measuring the phototransformation of unlabelled phytochrome at 513 nm, which in fact showed no absorption change from 10 ms to 10 s (data not shown; within experimental limits,

217

peak-to-peak noise +O.O005A).This observation had already been made for degraded phytochrome [ 201. The kinetics of FMA labelled phytochrome are shown in Fig. 3. From the absorbance change at 730 nm (Fig. 3, top), the formation of distinct species, i.e. meta-R,, meta-R, and P,, can be ascribed to definite time intervals. The rate corresponding to the transformation of metaRt, to meta-Rt, (k,) is 370 s-l, and that for meta-Rt, to Pf, (kJ is 12 s-r (obtained from fitting rates for 730 nm only). The similarity with the rates of unmodified phytochrome confirms the idea that the phytochrome phototransformation is not disturbed by labelling. Also shown in Fig. 3 (bottom) are the changes of the FMA chromophore at 513 nm on the same time scale. The absorbance begins to increase at times longer than 20 ms, which only correlates with the transformation meta-R, -+ Pf,. The fitted rate constant at 513 nm is 15 s-l, AA

0.02

AA

I

I

I

I

-3

-2

-1

0 log

t cr J

Fig. 3. Kinetics of fluorescein-labelled phytochrome. Kinetics of phototransformation of 124 kDa oat P, (Ati, = 1.35) lab e11e d with 3.5 equivalents FMA (20 mM KPB, 297 K), monitored at 730 nm (top) and 513 nm (bottom) after red-laser flash excitation (660 nm). Curve fits using a non-linear least-squares-fitting procedure (see Section 2.4) are included in each plot. For rate constants and amplitudes see Table 1.

218

which is in good agreement with the rate at 730 nm and with a global fit (Table 1). The absorption of the FMA chromophore, which is influenced by polarity changes of its (protein) environment [8], changes during P, formation starting from meta-Rt,.

4. Discussion From the results presented, some conclusions can be summarized regarding the mechanism of phytochrome photoconversion. In this work the transformation of meta-Rt, to Pf, was investigated in some more detail. It has shown that the formation of Pf, from meta-R, is associated with at least two different processes, which not only differ with respect to their rate constants (k,, k,) but also regarding their sensitivity upon changes of the protein and its environment. Upon proteolysis, the amplitude spectra of both processes (k,, k,) change, which means that the red shift in going from meta-Rt, to Pf, [15] vanishes. Based on similar findings, parallel pathways in the formation of Pf, have been postulated for degraded phytochrome (for references see ref. 15). In the literature, at 273 - 275 K corresponding values for 12, range from 15 s-l [20] to 50 s-l [25], and for k2 2.8 s-l [20] to 4s’ [25]. Slower processes with k < 1 s-i have been reported too [29]. Evidence for the formation of a bleached intermediate with 118 kDa phytochrome (meta-R,, or P,i) is provided by an additional process with a rate of about 1 s-i (k,‘). The corresponding increase in absorbance at 667 nm might be related to a dark reversion of P,i to P,, which has been observed at 273 - 275 K with half-life times of 300 s and 120 s for phytochrome from pea [20] and oat [30] respectively. More drastic effects can be seen by using glycerol at high concentrations, which is known to increase the microviscosity [31]. The primary decays of meta-Rt, in pure (k, = 470 s-i) and in glycerol containing buffer (k,’ = 670 s-l) (see Table 1) have similar rates. The rate of formation of meta-,R, is changed only in buffer containing 66% (v/v) glycerol, i.e. it is significantly slowed down (kl’ = 48 s-l) under these conditions. Obviously, the process meta-Rt, + meta-R, is split into (at least) two consecutive steps meta-Rt, + meta-Rt,, and meta-Rt,, + meta-Rt,, of which only the latter (k,‘) is strongly retarded in glycerol. Retardation by increased viscosity or degradation of the protein component is most pronounced for the last step, i.e. conversion of meta-Rt, to Pfz,,which is retarded by a factor of about 25 upon increasing the viscosity by a factor of 16. In this respect, the reactions meta-R, to Pf, profoundly differ from those converting P, to meta-Rt,, since the latter reactions have been reported to be quite independent of environmental conditions [ 32 - 341. It should be noted that an increase in macroscopic viscosity, on its own, does not retard significantly the phototransformation, as can be shown by addition of high molecular mass Ficoll 40 000 (40 kDa). This implies that

219

conformational changes of the phytochrome molecule during phototransformation are restricted to sites that are exposed only to a limited degree. As shown in Fig. 3, the conformational changes monitored via FMA take place only during transformation of meta-Rt, to Pt,. This constitutes one more discrimination between the processes meta-Rt, -+ meta-Rt, and metaR, + Pf,. Thus, both processes (k,, k,), which have been reported to describe the evolution of the far-red absorption band of P,, [15], differ clearly in their kinetic behaviour. This indicates a sequential pathway for meta-R, to Pf, [15] rather than parallel pathways [20, 211. Parallel pathways should exhibit uniform behaviour upon environmental changes, as is the case for the decay rates of lumi-R [ 32, 331. At present, it is still speculative that the conformational changes of the protein are restricted to meta-Rt, + Pf, conversion and that this change is responsible for the primary physiological signal of Pi, in Go. But it seems unlikely to be merely coincidental that the label residing on exposed sites does not recognize differences on the protein until after the formation of the active form of the pigment, since it is now quite well accepted that the differences between the protein components initiate the manifold of physiological responses [ 21. All these ideas are summarized in Fig. 4. The scheme supports some concept stated previously for 60 kDa phytochrome [19] and is applicable to the native (124 kDa) pigment too, and includes recently published data [ll, 31, 331. The phototransformation of P, to Pti is separated into three different stages I - III (Fig. 4). Meta-R, is symbolized by a cyclic pool to illustrate its heterogeneity with respect to its formation [20,21,29, 321,

h”

l”mi

Pr c 94 I’ ? ,

_

R

2.5~10'i’,

lumi_R’f5000 1

C______J

I 1

i’3

i) meta-R, met:‘-

470

R,,

s”

-meta-R,a

I

Pf,

II

I

Fig. 4. Model for P, photoconversion. Schematic model of P, phototransformation with proposed molecular actions included (rate constants for dark-relaxation steps at 298 K are given). Step I denotes Z-E isomerization of the tetrapyrrolic chromophore, step II relaxations of the chromophore and its protein cavern, and step III represents conformational changes of the protein as detected by a reporter group attached to exposed sites of the protein. Meta-R, is symbolized by a pool owing to its heterogeneity (see Section 4); meta-R,, has been included in this pool because of its spectral similarity to meta-Rt, (c$ Fig. 2) and as it cannot be resolved kinetically in pure potassium phosphate buffer. +, photochemical process; ---+ and e, dark-relaxation steps, resulting in formation of Pfr;

- - - +, dark reversion

to Pr.

220

photoreactivity [ 351, viscosity dependence (this work), and the possibility that it is a candidate for dark reversion [36]. The viscosity dependence of the rates is most pronounced for step III, which may be associated with the largest conformational changes. This step constitutes the formation of Pfi, the only physiologically active pigment, whose interaction with other cellular components constitutes the beginning of a signal chain. Interaction of phytochrome with both its inhomogeneous and its variable microenvironments in uiuo might result in different rate constants of Pi, appearance in uiuo [37]. Additional environmental factors besides microviscosity must also be considered, since Spruit [ 381 reported formation of Pti to be dependent on the redox state of a plant. Although Pf, formation in pea epicotyl tissue flushed with argon at 294 K took place at 8 s-i (which is close to our values, i.e. for oat in vitro 11 - 20 s-l at 298 K), this rate drops to 3.5 s-l in the presence of oxygen. Such effects, which may be related to yet unknown modifications of the photoreceptor pigment in uiuo, should be topics of further research.

Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (Bonn-Bad Godesberg) and the Fonds der Chemischen Industrie (Frankfurt). We would like to thank Drs. S. E. Braslavsky and P.-S. Song for helpful discussions and Dr. H. Scheer for revising the manuscript. Finally, we are grateful to Mrs. H. Malinowski for carefully preparing the phytochrome samples.

References 1 P. Eilfeld

and W. Haupt, Phytochrome. In M. G. Holmes (ed.), Photoreceptor FuncAcademic Press, London, 1989, in the press. J. C. Lagarias, Progress in the molecular analysis of phytochrome, Photochem. Photobiol., 42 (1985) 811 - 820. M.-M. Cordonnier, H. Greppin and L. H. Pratt, Monoclonal antibodies with differing affinities to the red-absorbing and far-red absorbing forms of phytochrome, Biochemistry, 24 (1985) 3246 - 3253. Y. Shimazaki, M.-M. Cordonnier and L. H. Pratt, Identification with monoclonal antibodies of a second antigenic domain of Aveno phytochrome that changes upon its photoconversion, Plant Physiol., 82 (1986) 109 - 113. B. Thomas, S. E. Penn, G. W. Butcher and G. Galfre, Discrimination between the redand far-red absorbing forms of phytochrome from Arena satiua L. by monoclonal antibodies, Planta, 160 (1984) 382 - 384. B. Thomas and S. E. Penn, Monoclonal antibody ARC MAC 50.1 binds to a site on the phytochrome molecule, which undergoes a photoreversible conformational change, FEBS Lett., 195 (1986) 174 - 178. R. D. Vierstra, P. H. Quail, T.-R. Hahn and P.-S. Song, Comparison of the protein conformations between different forms (P, and Pn) of native (124 kDa) and degraded

tion and Evolution,

2 3

4

5

6

7

221 (118/114 8

9 10

11 12

13

14 15

16

17

18

19 20

21 22 23 24 25 26 27

kDa)

phytochromes

from

Auena

satiua, Photochem.

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