- Email: [email protected]
Dynamic processes of visual transduction
Vision Res. Vo!. 24, No. 11, pp. 1445-1454, 1984 Printed in Great Britain. All rights reserved
0042-6989/84 $3.00 + 0.00 Copyright © 1984 Pergamon Press Ltd
DYNAMIC PROCESSES OF VISUAL TRANSDUCTION M. L. APPLEBURY Department of Biological Sciences, Purdue University, W. Lafayette, IN 47907, U.S.A. Abstract--Rhodopsin is one of those rare macromolecules whose inherent chromophore, 11-c/s retinaldehyde, allows one to naturally observe triggered macromolecular changes on the timescale of picoseconds to minutes. Investigations of these molecular processes have been carried out with laser monochromatic light under conditions where the photon flux used for photolysis was carefully measured. The formation of bleaching intermediates has been examined as a function of fluence. Under conditions where the formation of intermediates is unaffected by photon reversal the following observations hold: Upon the absorption of a photon, the initial photochemical event results in production of metastable bathorhodopsin within 6 psec. Artificial rhodopsin regenerated with 9-cis retinal forms a distinct bathorhodopsin which must reflect distortions at the active site differing from those generated with 11-cis retinal. Bathorhodopsin thermally decays through lumirhodopsin and meta I-rhodopsin, to meta II-rhodopsin through a series of coupled equilibria. The final meta I-meta II equilibrium is stable for seconds. The process provides a unique model for utilization of energy to drive (trigger) a biological cascade of events. Rhodopsin
Bathorhodopsin
Lumirhodopsin
Metarhodopsin (I and II)
INTRODUCTION In 1936 and 1938, H. J. A. Dartnall published two papers that laid the foundations for study of the bleaching processes of visual pigments which would govern investigations for the next 50 years (Dartnall et al., 1936; Dartnall et al., 1938). In these works, he discussed three particular principles which were to enable the study of mechanisms of photoreception and transduction by rhodopsin: (I) He pointed out the advantages of use of monochromatic light to photolyze visual pigments. The absolute determination of light intensity is made reasonably feasible enabling one "to apply fundamental photochemical principles to [the process of bleaching]." In this elegant 1936 work, he and his colleagues first determined the photosensitivity, a product of the extinction coefficient and the quantum yield. Had he had a value for the extinction coefficient at that time, the quantum yield would have been determined. In fact, this waited some 33 years (Dartnall, 1968). (2) He carefully considered the levels of intensity (fluence rate) which could be used without leading to artifacts, such as the photoreversal of products of the reaction and pointed out that the apparent bleaching rate (or amplitudes of reaction) should be proportional to the absolute intensity used. And (3) in the 1938 work, he began to examine the effects of temperature upon the reaction.
This presentation was given to celebrate the 70th birthday of Professor H. J. A. Dartnall during the Conference on Visual Pigments held in Bristol, England, 14-16 September 1983.
The studies of temperature dependence of bleaching processes during the 1950's and 1960's led to the identification of the intermediates of bleaching; they continued to govern the nature of investigations in the 1970's and 1980's, both in the study of the primary photochemical processes and in the study of the relaxation of early intermediates. Two examples taken from our own work illustrate the application of these principles and are discussed in the realm of our current understanding of the dynamic processes of bleaching. The first considers the primary photochemical intermediates of bleaching and has implications about the quantum yield of a rhodopsin analogue, 9-cis rhodopsin. The second examines the temperature dependence of the thermally controlled bleaching intermediates, lumirhodopsin (L), meta I-rhodopsin (MI) and meta II-rhodopsin (MII).
METHODS
Picosecond flash photolysis studies The instrumentation for picosecond dynamic studies and the preparation of samples have been fully described in Applebury and Rentzepis (1982) and Spalink et al. (1983). For data shown in this work, 11-cis rhodopsin had a concentration of A (498 nm) = 1.0, 2 m m path and 9-cis rhodopsin A(485nm) = 1.0, 2 mm path.
Microsecond and millisecond flash photolysis studies Flash photolysis was carried out with an apparatus consisting of a tuneable dye laser for initiating 1445
1446
M.L. APPLEBURY
bleaching and a single beam monochromatic light monitoring system coupled to a dual timebase transient recorder (Biomation). The photolysis pulse has a half width of about 200-500 nsec depending on wavelength. Spectral transients with lifetimes of between 10/~sec and minutes can be monitored (Eisenberg-Shinnar et al., 1984). Fresh (or frozen) bovine rod outer segment membranes were prepared in the presence of antioxidants or in deoxygenated solutions. The membranes were washed to deplete them of all peripherally bound proteins in order to study a simplified system in which rhodopsin is the only protein present (minor amounts of an integral membrane protein of --~250 kdaltons are present) and would be unperturbed by interactions with peripheral proteins. The procedures used are fully described in Baehr et al. (1982). The depleted membranes were sonicated and made 100 mM NaC1, 3 mM KC1, 0.5 mM MgC12 and 10 mM HEPES pH 7.
RESULTS AND D I S C U S S I O N
The primary intermediates o f 9-cis rhodopsin
One of the most important studies that examined the mechanisms of phototransduction was the study of rhodopsin in which 11-cis retinal was substituted by 9-cis retinal either by photochemically generating 9-cis in intact rhodopsin or by regenerating opsin with 9-cis retinal. These studies first demonstrated that the product of bleaching of either 11-cis or 9-cis rhodopsin was the all-trans retinal chromophore (Hubbard and Wald, 1952; Hubbard and Kropf, 1958). Later studies demonstrated that a photochemical equilibrium could be established between the 9-cis and 11-cis chromophores in low temperature glasses and indicated that the two chromophores were interconvertible within the environment of the opsin binding site (Yoshizawa and Wald, 1963). Thus, by the early sixties the nature of the primary intermediate in the visual process was described as follows: the absorption of light leads to the rapid isomerization of the 11- or 9-cis chromophore to all-trans retinal leaving the new chromophore in the original l l-cis environment. On a slower thermal timescale, the protein relaxes to the new chromophore conformation as entailed in the following scheme of visual transduction: 11-cis R H O ,
0.67
' all-trans BATHOr----* 9-cis RHO T~ 01 11 all-trans META II RHO
The quantum yields have been determined as 0.67 (Dartnall, 1968) and 0.1-0.3 (Hurley et al., 1977; Hubbard and Kropf, 1958). In studies aimed at establishing the dynamics of bathorhodopsin production, it was observed that the
initial intermediate arose in < 6 psec (the limits of technical resolution) and once established was stable for many nsec (Busch et al., 1972). Studies were carried out with low temperature glasses ( 7 7 4 K) with the hopes of slowing and resolving intermediate steps in the initial production of bathorhodopsin. An initial rate of formation could be observed between 77 and 4 K and it was found to have a marked deuterium isotope effect implicating a role for H + ion translocation in this process (Peters et al., 1977). Combining these results with the elegant studies of the chromophore configuration by resonance Raman spectroscopy (Eyring et al., 1980), the initial photochemical event can currently be described as the photochernically driven isomerization of the chromophore to an extremely distorted transoid structure accompanied by a protein translocation. The proton(s) might be either somewhere in the immediate vicinity of the chromophore (Warshal, 1978; Lewis, 1978) or be particularly involved in Schiff base protonation of the chromophore (Peters et al., 1977). When these studies were extended to the study of 9-cis rhodopsin several observations were inconsistent with the simplistic model given above. As first observed by Green et al. (1977), the rate limiting processes for the formation of bathorhodopsin from 9- or 11-cis rhodopsin are essentially the same, both < 6 psec. These studies were extended to low temperature glasses of 9-cis rhodopsin, under conditions in which a rate limiting step can be resolved (Applebury et al., 1979). The data are illustrated in Fig. 1 and indicate the rates are also similar below 20 K. At temperatures > 20 K, 9-cis appears to have a slower rate, but the error is too large to ascribe significance to this data. As Dartnall (1936) pointed out, in the absence of photolysis of products of bleaching, the production of the initial intermediate should be proportional to the qiiantum yield - d R / d t = [dB/dt] = (oJ,/V
Where R is the molar concentration of rhodopsin undergoing reaction, B is the molar concentration of bathorhodopsin produced, q~ the quantum yield of batho, .It the number (moles) of photons absorbed per unit time t, and V the reaction volume. Clearly, equivalent reaction rates measured for l l-cis and 9-cis rhodopsin are inconsistent with quantum yields of 0.67 and 0.1 respectively, under conditions of equivalent absorption. The above equation holds under conditions in which the excited state R* can be assumed to be a steady state. Under conditions of psec photolysis, the steady state assumption is questionable. The rate of batho production could reflect a more complex mechanism and involve more intermediates, e.g. the rates observed at low temperature studies could reflect some rate limiting relaxation to the ground state bathorhodopsin. Still the amplitude of the reaction should reflect the quantum efficiency
Dynamic processes of visual transduction
26
25
t
•
I
7
24
23
I
50
I
1O0
[
150
I
200
I
250
1 / T x 1000
Fig. 1. Temperature dependence of the rate of formation of 11-cis bathorhodopsin (O) and 9-cis bathorhodopsin (O). Data indicated by the symbol (0) are rate constants for 9-cis bathorhodopsin corrected to that which would be observed for 9-cis rhodopsin with the same initial absorption as the I 1-cis rhodopsin. This is valid only if the rate constant is proportional to the light absorbed. The error in rate constants measured is ~30%.
and the yield of bathorhodopsin from ll-cis and should be ~ 7 times that of 9-cis. In fact, the amplitudes of reaction corrected for absorption are nearly identical (Monger et al., 1979; Applebury et al., 1979). These observations led to an extended study of bleaching with monochromatic light as a function of intensity (fluence rate)--in essence to test the postulates of limiting conditions given by Dartnall et al. (1936). To accomplish the study with reasonable signal to noise, photolysis was carried out with the second harmonic (532 nm) of a N d ( 3 + ) / Y A G laser whose output was a pulse with 25 psec F W H M . The depletion of rhodopsin and production of bathorhodopsin were observed as a difference spectrum recorded at 40 psec, 85 psec or 8 nsec by a double beam spectrophotometer (Spalink et al., 1983). To examine the proportionality of signal amplitude to photons absorbed, where J , = I o [1 -exp(-2.303ebl)] with I 0 as the incident fluence, the molar extinction coefficient, b the molar concentration and l the pathlength, difference spectra data were averaged for low intensities ( < ~ 2 0 #J/mm2), medium fluence (~50/tJ/mm2), and high fluence (>60#J/mm2). Inspection of Fig. 2, indicates that with increasing fluence bathorodopsin formed from 11-cis rhodopsin does not behave as expected. There is no increase in the difference spectra maximum at ~ 5 7 0 n m , which should be proportional to increasing levels of bathorhodopsin produced, although there is an increasing depletion in the appar-
1447
ent levels of rhodopsin bleached. Moreover, for 11-cis rhodopsin, the isosbestic point shifts with increasing fluence indicating more than two species are contributing to the difference spectra. The spectra for 9-cis rhodopsin are more model-like and behave with the appropriate increases in product and decreases in starting material. A more detailed examination of these changes was carried out at the difference spectra wavelength maxima and minima. As shown in Fig. 3, saturation effects occur far below fluence levels which would give total bleach of starting rhodopsin. Under the experimental conditions used, saturation occurs above 10-15~ photolysis. Ostensibly, the products of bleaching are undergoing photoreaction as well. The effect is more pronounced for 11-cis than for 9-cis rhodopsin. For fluence levels over which there is approximate linearity ( < ~ 20 p J/ram 2), the difference spectra can be compared. It is obvious that the ratio of difference spectra maximum to minimum differs for l l-cis and 9-cis. Since 11-cis and 9-cis rhodopsin have nearly the same molar extinction coefficient (~ at 2,,,x differs by a factor of 1.06 and the )'max differ by ~ 13 mm), this ratio should be nearly the same if the products of photolysis were identical. The simplistic deduction is that, in fact, the products are not the same and the product of 9-cis must have a lower extinction coefficient than that of l l-cis rhodopsin. To resolve the difference spectra into component bathorhodopsin produced and rhodopsin bleached, one needs to determine the amount of rhodopsin bleached. In these studies, this was carried out by pooling samples, each of which had been photolyzed with a known low level fluence such that the percent bleach (determined from the residual rhodopsin absorption) could be determined for a given average fluence [Spalink et al. (1983)]. Under conditions of low fluence, the percent bleach of 9-cis or 11-cis rhodopsin is nearly the same when corrected for initial absorption at the 532 nm wavelength of photolysis and indicates that the quantum yields are more nearly equivalent and are unlikely to differ by a factor of 7. Bathorhodopsin spectra resolved under conditions of low fluence (Fig. 2, low) indicate that 9-cis bathorhodopsin has a lower extinction coefficient and that its )'max is red shifted approximately 10nm (Fig. 4) [Spalink et al. (1983)]. Thus, either the 9- and 11-batho products differ in their transoid nature or the protein cannot relax to the same state--or perhaps both play a role at this stage of bleaching. Moreover, spectra compared under the same low fluence photolysis indicate that the bathorhodopsins remain different up to at least 8 nsec (Spalink et al., unpublished results). Thus, either the chromophore or protein (or both) do not relax to a common intermediate over this timescale. Kliger and colleagues (Horwitz et al., 1983) have recently indicated that differences are still apparent on the #sec time-
1448
M . L . APPLEBURY 0.20
.
,
0.15
:.
.
.
.
.
.
.
.
.
.
.
.
i:
:
,
,1 :
..................
i
II
,-;,,~,;;o;;,~
.............................................
, r
,
.~.~'i-.~',
1)~(~;>
,
,
~
. . . .
!
(a)
:
;'~_~17;,I
•
:,
..... i .............................
~.....
0.10
0.05
- .......................... "
- ............................ ~
:
i
~...
i ....................... ~
.~,t-~/,k~)! - ...................
I,
"-.~:x~i~
i! !!
i ......................... ~"i:~
i '
~ ....
,&;,,.~,
ooo li{~,.
o o~ E ii/ -0.10
' "#.-,-:r.a?
: ~
~,. _
"
Low
.,[~tl
~',t
;,' "=
HIGH
•
^,/' ~ mi
-
!
:
::
::
...
........................................................ i:....................................................................................... : LI
-015 (:3 -0.20
I
I
i
I
I
I
I
I
450
400
i
I
I
t
I
500
g
I
I
I
I
550
Wavelength
t
I
I
I
I
600
650
(nm]
(J 0.20
i
i
~
i
I
i
i
i
1
i
t
;
i
i
i
~
i
i
i
i
i
=o g L
9-c/s
o <
Rhodopsin
~4".....
o.,o ........................................................ ............................
"'"',
..... '~'~
-0.o~
.................
-o,o
.......................
-o.~s
LOW
! .......... ;;~.i .......... c! ..................
~
t, ,~;
~':~
:.:',.! ..... !,..%,>/,,'>,.'~,.?.r,..!.~.:::,~. !.:-:.L
...........................
i
i
, ......................... ;., .............. U.>i.l.i.
.............................
i':
"''',,.
~,v.%:..;;:~-
:
......................
............................... .......... :.
:
:
i..........................................................
...........................
............................
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
i...
.
.
.
.
.
.
.
.
.
.
.
.
~" '".',,"w~,~". lG. '
.~
-_:
:......- .....................................................................................................................
: --O,20
•
4o0
1
,l
~
450
-I, I
I
I
I
I
I
I
I
500
I
550
Wavelength
I
I
I
I
I
I
6oo
"3 "I
I
650
(nrn)
Fig. 2. The dependence of difference spectra amplitudes on excitation energy. Samples of A(2,~,x)= 1.0, 2 mm path-length. Excitation was at 532 nrn with fluence ranges low = < 320 #J; mid = 320-480 #J; high = >480 #J. The beam was spread to irradiate an area of ~ 16 mm 2. (a) Bathorhodopsin spectrum minus 11-cis rhodopsin spectrum. (b) Bathorhodopsin spectrum minus 9-cis rhodopsin spectrum. The figure was adapted from Spalink et al., 1983. scale, hence even the l u m i r h o d o p s i n s m a y differ. In s u m m a r y , from studies o f the bleaching process at r o o m temperature, the following scheme seems currently appropriate, where the latter steps are yet to be firmly resolved.
T h e a p p a r e n t difference between these observations m a d e at r o o m t e m p e r a t u r e a n d those originally m a d e with low t e m p e r a t u r e glasses (Yoshizawa a n d Wald, 1963; Yoshizawa, 1972) are n o t obvious. One e x p l a n a t i o n would be t h a t the m o d e s o f protein
11-cis R H O -~ t r a n s o i d B A T H O
LUMIRHO
transoid B A T H O ' ~ 9-cis R H O LUMIRHO'
META I - RHODOPSIN T$
M E T A II -- R H O D O P S I N
T$ RETINAL + OPSIN
Dynamic processes of visual transduction
1449
0.20
0 2 0 -(a)
0.15 --
(b)
i
ii/i~'~
0.15
,/
A/
,/ m 0.10
/
<3
/
0.05
°/° o ~, ?.
(A, 570nm (o) 490nm
/ o.oo
0
<3
o/
/ /o o
/';S °
0.10
/d /o P
i/ i/
Ii/
o / o"" ~ / o
/ o /o 7
(1) 56Ohm (o) 475 nm
lit//
0.05
11-cis Rhodopsin I 1
I 2
I 3
I 4
I 5
Excitation
Rhodopsin
9-c/s
I 6
pulse
o.oo 0
fluence
I 1
I 2
I 3
I 4
I, 6
I 5
(rel.units)
Fig. 3. Bathorhodopsin - rhodopsin difference spectra absorbance changes as a function of excitation fluence. Each point is an average of 4-6 records. One relative unit corresponds to 160-200/xJ for a irradiated area of ~ 16 mm 2. (a) Bathorhodopsin minus 1l-cis rhodopsin spectral amplitudes at 570 nm ( k ) and 490 nm (O). (b) Bathorhodopsin minus 9-eis rhodopsin spectral amplitudes at 560 nm (A) and 475 nm (O). This figure was adapted from Spalink et al., 1983.
relaxation at low temperatures differ from those at room temperature, although it is difficult to intuitively understand why they should be the same for both 11-cis and 9-cis at low temperatures, but differ at more permissible room temperatures. A more recent study of bathorhodopsin production at low temperature under more limiting levels of photolysis does indicate that the products of 11-cis and 9-cis rhodopsin differ, although the extinction coefficient for 9-cis batho was shown to be larger than that of l l - c i s (Mao et al., 1980). No systematic study of bathorhodopsin production in low temperature glasses as a function of fluence has been reported and it is unclear which studies are free of saturation effects and which are not. Low temperature studies are as susceptible to saturation effects as room temperature studies. The ftuence rate experimentally used may differ dramatically, but the total fluence needed is the same for conversion to similar detectable product level. Batho products will build up at the face incident to the photolysis beam and will be distributed in a decreasing exponential manner through the depth of the cell. The actual levels of fluence under which saturation will occur will depend upon the initial absorption as well as the quantum yield of product and cross-sectional absorption of the product, making predictions complex. In approximation, however, for samples with initial absorbances of 0.5-0.8, fluences above 20-30/xJ/mm 2 i.e. 2-3 mJ/cm 2, become suspect. The low extinction values calculated earlier for 11-eis bathorhodopsin at room temperature can be attributed to such saturation effects (Applebury, ]980). The examination of difference spectra for 11-cis rhodopsin questions whether a photoequilibrium between 11-cis, batho and 9-cis is established. Were this
the case, the amplitude of the difference spectra would be expected to maximize at high fluence but the shape of the difference spectra would change relatively little as batho was photoreversed to 11-cis or 9-cis rhodopsin since the absorption of 11-cis and 9-cis differ only modestly. For l l-cis rhodopsin, whatever the nature of the photolysis product contributing to the spectra might be, the change is not appropriate for batho being driven back to the starting 11-cis rhodopsin or to 9-cis rhodopsin. Over the fluence range studied, this problem is not observed for regenerated 9-cis rhodopsin. The differences between the behavior of l l-cis rhodopsin and 9-cis rhodopsin have interesting implications about the way in which the protein controls the chromophore relaxation and the distribution of excess energy. A careful analysis of these two rhodopsins is likely to provide valuable insight about the process of phototransduction.
1.5
Bll R9 Rll / /
o
10
.Q <
29
\
X
l/
\\\
0.5
O.O
I
450
I
500
550
60(3
650
Wavelength ( nm )
Fig. 4. Bathorhodopsin spectra resolved from difference spectra taken at 85 psec, 298 K. R1], parent 11-cisrhodopsin spectrum; Bu, bathorhodopsin of l l-eis rhodopsin; Rg, parent 9-cis spectrum; Bg, bathorhodopsin of 9-cis rhodopsin.
1450
M.L.
APPLEBURY
A study o f the thermal intermediates o f bleaching In 1938 Dartnall's work turned to a study of the effects of temperature, pH and buffer on photosensitivities. He demonstrated and emphasized that there should be little or no temperature effect on the photosensitivity of rhodopsin. As mentioned above and further demonstrated by Peters et al. (1977) this bears out even for picosecond studies of the process. He also predicted the role that temperature might play in controlling the intermediates of bleaching (Dartnall et al., 1938). He mentioned his own observations, based on those of Kuhne, that frozen solutions of visual pigments appear to bleach very slowly. Over the years, of course, the study of the temperature dependencies of the bleaching products enabled investigators to define the series of intermediates that occur in the process of bleaching. The absorption spectra of these intermediates are now well established; the temperatures which permit sequential conversion from one to another are defined; and the kinetics of the MI MII transition have been extensively studied in detergent solubilized solutions. The dynamics of these transitions from batho to meta II are poorly understood, however, particularly for the visual pigment present in its own native environment--the lipid bilayer. In the last few years, we have conducted studies of thermal intermediates of bleaching with rhodopsin in its native lipid environment. The studies were carried out as those discussed above, using monochromatic light of known intensity, defining the photolysis fluence under which artifacts are minimized, and using temperature to selectively study the dynamics of the lumi ~ meta I--. meta II rhodopsin transitions. The following presents our current understanding of these events. The majority of the experiments reported here were carried out with a flash photolysis pulse of 540 nm in order to minimize photoreversals and to avoid, as best possible, pickup of the scattered photolysis light as an artifact in recording the subsequent transition. The use of a dual timebase transient recorder allows transients to be recorded in two modes. (1) The pretrigger mode samples records of the initial rhodopsin absorption and then the transient immediately following the photolysis flash. This is illustrated in Fig. 5(a) where the absorption at 380 nm is recorded prior to the photolysis flash and then during the meta I (~'max 480 rim) to meta II (2~ax 380 rim) transition. The transient change in absorption may be plotted as In (A~ - A , ) vs time to illustrate that the behaviour is that of a first order reaction. It is linear over more than 3 lifetimes as shown in Fig. 5(b), the final state is stable for well over 100 lifetimes ( > 1 sec). On close inspection of early timepoints, however, deviations from linearity can be distinguished which are indicative of an earlier transition of faster rate. This earlier transition may be observed simply by changing the recording timescale or by using the second transient recorder mode. (2) The dual timebase mode records
transients on two time bases, a faster and a slower. As shown in Fig. 5(c), the record tracks the initial fast rise of lumi (2~,x 505 nm) --, meta I (2m~ 480 rim) and then the subsequent meta I--, meta II transition. Thus, the instrumentation enables us to study both transients over a range of temperatures from 0 to 35°C. To avoid artifacts due to sequential photon absorptions, such as photoreversal of intermediates, the amplitudes of formation of meta II signals were measured as a function of fluence of the photolysis pulse. As shown in Fig. 6, the signal amplitude saturates far below the expected maximumly possible amplitude. The figure illustrates the extreme sensitivity of rhodopsin and its intermediates to photoreversal and emphasizes the difficulty of recording true unperturbed transients with good signal to noise. Bleaches of > 15-20~ begin to be subject to photoreversal. This has been demonstrated in more detail by Williams (1975) who used the higher intensities to study the nature of photoreversal. We have limited our studies to transitions carried out at bleaches of 10~o or below. Under these conditions, both the lumi --* meta I and meta I ~ meta II transitions are first order reactions. For the visual pigment transition in digitonin solutions, Matthews et al. (1963) established that the meta I ~-meta II transition is a temperature dependent equilibrium. One of the first questions to ask was whether or not this equilibrium is the same in native membrane bilayers. To resolve this question we attempted to measure the equilibrium constant as a function of temperature in the following way. In the pretrigger mode, the absorbance of the reaction is given by MI e + M I I e + R~h -- Ro = MIe + M i l e - Rb, where absorbances are proportional to intermediates at e, equilibrium concentrations; ub, the unbleached rhodopsin concentrations; o, the initial concentration; and b, the bleached concentration. In the dual timebase mode, the absorbance change (amplitude) of the first to second transition is given by MI e + M I I e -- MI,, where t indicates total meta I. The absorbances recorded are simply the sum of the component concentrations (i) times their known extinction coefficients (ei) at a given wavelength (2) and the appropriate pathlengths (1) Ae(,~ )
= [ m 1 e • ~ml(/~.) -~-
m2 e • e r a 2 ( 2 )
--
rh' e,(2)]l
(pretrigger) Ae(~. )
m2~-era2(2) - m 1," ~,,1(2)]/ (dual timebase)
= [ m 1 e - gml()~) +
since m 1 t = rb and m 1, = m 1 e + m2e, the equations reduce to two equations in two unknowns, m 1e and m2 e. If the values are solved for a series of wavelengths, the concentrations can be reasonably established. Thus, transients were measured as a function of temperature both in pretrigger and dual timebase mode and absorbances were plotted as difference spectra. In practice, the data were fit for a consistent
Dynamic processes of visual transduction
1451
(a)
+ 0.004
, |
0
15
30
45
Time
I
I
60
75
(msec)
(b)
10 T
1801"
I~.LU,I~.LI
.I ]JL. __,iJl L ~ . d ~ 1 = ~ L Jilli, I . L-~d .1 . a t , .~.i J'L~,I
tel 0
_8 E
+ 0.004- V I 2
I-
I
6oo
I
1200
I
;
I
~800
24-0o
3000
Time (msec)
t
I
I
I
1
I
i
,,I
-0.1
-0 . ' I
E c -0.2
I f
-0.3
-0.4
-O.2
-0.3 i I
I I
i
!
t i
-04-
i I
-0.5
-O.5
0 0.5 1.0 10 msec
50
100
0 0.5 1.0 10
50
I00
msec
Fig. 5. Kinetic records showing the meta transition in the bleaching process of rhodopsin in sonicated rod outer segments at 24°C. (a) Data collected by a Biomation transient recorder in the pretrigger mode at 50/1 sec/channel (2048 channels). (b) Data collected in the pretrigger mode at 2 # sec/ch (2048 channels). (c) Data collected in the dual timebase mode, base 1:2 #sec/ch (600 channels), base 2:100/~sec/ch (1448 channels). The left trace was collected without photolysis just prior to the right trace with photolysis. Photolysis wavelength, 500 nm; monitoring absorption 380 nm.
V.R. 24/11--B
1452
M.L. APPLEBURY 0.06
0,05
o//,./ 0.04
<3
0.03
002
i
/////°'g
y
0.01
I 0.2
[ Q4 Relotive
I 0.6
I 08
fluence
Fig. 6. Meta II rhodopsin- rhodopsin difference spectra absorbance changes as a function of excitation fluence at 540 nm. (C)) Amplitudes measured at 500 nm; (0) Amplitudes measured at 380 nm. About 5~o of the incident photolysis beam was deflected and recorded by a photodiode. The relative fluence was measured as the peak amplitude of the pulse traced on an oscilloscope. One relative unit is < 1 mJ per 5 mm diameter irradiated at the sample face. Full bleach would be expected to give an I AA ] about 0.25 for the sample whose absorption was 0.35 A (500 rim).
set of concentration values that satisfied both the pretrigger data and the dual timebase data. The molar extinction coefficients used were taken from spectra where values at 2m,x are rho, 5(498 n m ) = 40,600 M 1cm ~; meta I, e(478 nm) = 44,000 M cm-1; meta II, e ( 3 7 8 n m ) = 3 8 , 0 0 0 M - l c m - ~ ; and when necessary, lumi, e(500 n m ) = 46,700 M -~ cm-L A more detailed account is given in (Eisenberg Shinnar et al., 1984). Figure 7(a) shows the pretrigger data for difference spectra representing concentrations of meta I and meta II at equilibrium minus the concentration of rhodopsin bleached. Comparison of these spectra with model spectra, given in the insert, indicate that they clearly represent at least three species. The isosbestic point is not 0 and can be fairly well represented as the sum of meta I and meta II at equilibrium for any given temperature minus the amount of rhodopsin bleached. At 24°C, the data are adequately fit by 33~o meta I and 67~o meta II. Other values are given in Table 1. Figure 7(b) shows the dual timebase data for difference spectra ostensibly representing the concentration of meta I and meta II at equilibrium minus the maximum meta I concentration. Comparison of the data with model difference curves indicates striking differences. The
hv
< 6psec
- 50nsec
model illustrates a difference curve for two species, meta I and meta II, with a single isosbestic point at 0. Clearly the data have a shifting negative "~maxand no true isosbestic point; they are inconsistent with a simple relaxation of meta I to an equilibrium of meta I and meta II. The spectra can be fit by including a significant amount of lumirhodopsin. This indicates that the first transient observed is that of lumi (or lumi plus batho) to the appropriate equilibrium concentration of lumi + meta I and the second transient is that of lumi + meta I to the equilibrium concentrations of meta I and meta II. At some temperatures these equilibria are fairly well separated; but in general, they must be considered as tightly coupled equilibria. The concentrations of best fit for these intermediates over a series of temperatures are given in Table 1 along with the approximate relaxation times measured. It is important to notice that at lower temperatures the fits indicate that there are significant amounts of lumirhodopsin, which is stable up to msec, and at no temperature does the equilibrium favor one discrete species. Direct comparison of these equilibria states with those of rhodopsin in the intact rod outer segment are of course not possible. Several suggestions have now been made that the interaction of rhodopsin with peripheral membrane proteins may alter the equilibria (Emeis and Hofmann, 1981; Emeis et al., 1982; Bennett, 1982; Parkes et al., 1983). The nature of these equilibria must be reestablished under conditions in which peripheral proteins are present. These results, however, suggest that caution must be taken in making attempts to assign any particular intermediate form to that which is appropriate for interaction with other enzymes, e.g. phosphorylation by opsin kinase or interaction with G-protein. This thermodynamic evaluation of the bleaching process gives some insight into the mechanism of energy redistribution in opsin. Statistically, the coupled equilibria suggest that the molecule fluctuates between a series of states much in the same way as other receptors (e.g. the acetylcholine receptor) or ionic channels (Neher and Stevens, 1977; Heidmann and Changeux, 1978). While the observations in no way imply that rhodopsin is a channel, in analogy to these receptors or channels, it may be suggested that the equilibria may be modified by environmental factors such as phosphorylation, binding of small molecules, or perhaps peripheral proteins. In summary, applying the principles and considerations first set forth by Professor Dartnall, studies of the bleaching of rhodopsin with current state of the art instrumentation lead to a general model which might be summarized as the following. The relaxation times were measured at 24°C; the constants can be extrapolated to be approximately 3-10 times faster at 37°C. ~ 0.3msec
R h o ~ R * - - ~ [ B ] ~- Batho ~ Lumi ~
MetaI
~ ] 0msec
~
MetaII--~
Dynamic
1453
processes of visual transduction
-g0 ~-
~
.~ -~.~
~ ~ -
~N
,/ i
•/,/11
I
Oo
I
J
I
O
~ 0
O 0
I
I
~ 0
o .'~
I
.~
~1
~.~
,
~1 ~ i ~
~.~~+
~ "1"-~ \"
I
I
o
~
0
~ .~ ~ E] ~ , : . ~
I
0
0
I
I
,.~ ~0 ~F7~ ~ o ~ .o ~ ~ . ~+ ~ o ~ . ~ F ~ 7. ~
<1
~o~°~o o o
tO
.~
~
~ . ~
~
.I
.
/
i
0
0 ~
.~ ~ o ~
~
I
-~i
i'
~ o ~~ °o I ~ o + ~='~~ . , o ~ .1:
I ~q o
".~
0..I ~
~
.
~
_
~ : ~
I o o
m o
,
~D 0 0
o
Y
0
I
I
0
o
i
I
• -~ ~1~
<~
~
~
~o
-~N e
1454
M. L. APPLEBURY )d
)-2
Table 1. Transient equilibrium concentrations for sonicated ros pH 7.0 L ~ M1 ~ M2 Temperature K C 278 5 288 15 297 24 (310 37)a aExtrapolated.
2~ 22 Tt M2 M1 L T2 M2 7.2msec -0.475 0.525 500.0msec 0.325 1.8msec 0.05 0.475 0.475 100.0msec 0.50 0.3 msec 0 . 1 0 0.475 0.425 10.0msec 0.675 0.1 --0.7 msec (0.83)
Acknowledgements--The author expresses her appreciation
to J. D. Spalink, B. Weiss, A. Shinnar and K. Savoie-Luisi for their assistance in this work. The project was supported in part by a grant from the National Eye Institute to M.L.A. REFERENCES
Applebury M. L. and Rentzepis P. M. (1982) Picosecond spectroscopy of visual pigments. Meth. Enzymol 81, 354-368. Applebury M. L., Peters K. S. and Rentzepis P. M. (1979) A picosecond kinetic study of isorhodopsin photochemistry. Biophys. J. 25, 316a. Applebury M. L. (1980) The primary processes of vision. A view from the experimental side. Photochem. Photobiol. 32, 425-431. Baehr W., Morita E., Swanson R. and Applebury M. L. (1982) Characterization of bovine rod outer segment G-protein. J. biol. Chem. 257, 6452-6460. Bennett N. (1982) Light-induced interactions between rhodopsin and the GTP-binding protein. Relation with phosphodiesterase activation. Eur. J. Biochem. 123, 133-139. Busch G. E., Applebury M. L., Lamola A. A. and Rentzepis P. M. (1972) The formation and decay of prelumirhodopsin at room temperature. Proc. natn. Acad. Sci. U.S.A. 69, 280~2806. Dartnall H. J. A., Goodeve C. F. and Lythgoe R. J. (1936) The quantitative analysis of the photochemical bleaching of visual purple solutions in monochromatic light. Proc. R. Soc. A, Lond. 156, 158-171. Dartnall H. J. A., Goodeve C. F. and Lythgoe R. J. (1938) The effect of temperature on the photochemical bleaching of visual purple solutions. Proc. R. Soc. A, Lond. 164, 216-230. Dartnall H. J. A. (1968) The photosensitivities of visual pigments in the presence of hydroxylamine. Vision Res. 8, 339-358. Eisenberg Shinnar A., Weiss W. I. and Applebury M. L. (1984) Kinetics of rhodopsin bleaching. 1. The Meta I-II equilibrium in bovine rod outer segment membranes. To be published. Emeis D. and Hofmann K. P. (1981) Shift in the relation between flash-induced metarhodopsin I and metarhodopsin II within the first 10% rhodopsin bleaching in bovine disc membranes. F E B S Lett. 136, 201-207. Emeis D., Kuhn H., Reichert J. and Hofmann K. P. (1982) Complex formation between metarhodopsin II and GTPbinding protein in bovine photoreceptor membranes leads to a shift of the photoproduct equilibrium. F E B S Lett. 143, 29-34. Eyring G., Curry B., Mathies R., Fransen R., Palings I. and Lugtenburg J. (1980) Interpretation of the resonance Raman spectrum of bathorhodopsin based on visual pigment analogues. Biochemistry 19, 2410-2418. Green B. H., Monger T. G., Alfano R. R., Aton B. and
MI 0.625 0.475 0.325 (0.17)
L 0.05 0.025 ---
Callender R. H. (1977) Cis-trans isomerisation in rhodopsin occurs in picoseconds. Nature 269, 179-180. Heidmann T. and Changeux J. P. (1978) Structural and functional properties of the acetylcholine receptor protein in its purified and membrane-bound states. Ann. Rev. Biochem. 47, 317-357. Horwitz J. S., Lewis J. W., Powers M. A. and Kliger D. S. (1983) Nanosecond laser photolysis of rhodopsin and isorhodopsin. Photochem. Photobiol. 37, 181-188. Hubbard R. and Wald G. (1952, 1953) Cis-trans isomer of vitamin A and retinene in the rhodopsin system. J. gen. Physiol. 36, 269-315. Hubbard R. and Kropf A. (1958) The action of light on rhodopsin, proc. natn. Acad. Sci. U.S.A. 44, 130--139. Hurley J. B., Ebrey T. G., Honing B. and Ottolenghi M. (1977) Temperature and wavelength effects on the photochemistry of rhodopsin, isorhodopsin, bacteriorhodopsin and their bathoproducts. Nature 270, 540-543. Lewis A. (1978) The molecular mechanism of excitation in visual transduction and bacteriorhodopsin. Proc. natn. Acad. Sci. U.S.A. 75, 549-553. Mao F,, Ebrey T. G. and Crouch R. (1980) Bathoproducts of rhodopsin, isorhodopsin I and isorhodopsin II. Biophys. J. 29, 247 256. Matthews R. G. Hubbard R., Brown P. K. and Wald G. (1963) Tautomeric forms of metarhodopsin. J. gen. Physiol. 47, 215-240. Monger T. G., Alfano R. R. and Callender R. H. (1979) Photochemistry of rhodopsin and isorhodopsin investigated on a picosecond time scale. Biophys. J. 27, 104-116. Neher E. and Stevens C. F. (1977) Conductance fluctuations and ionic pores in membranes. Ann. Rev. Biophys. Bioengng 6, 345 381. Parkes J. H. and Liebman P. A. (1983) Stabilization of metarhodopsin II by GTP binding protein. Invest. Ophthal. visual Sci., Suppl. 24, 245. Peters K. S., Applebury M. L. and Rentzepis P. M. (1977) Primary photochemical event in vision: Proton translocation. Proc. natn. Acad. Sci. U.S.A. 74, 3119 3123. Spalink J. D., Reynolds A. H., Rentzepis P. M., Sperling W. and Applebury M. L. (1983) Bathorhodopsin intermediates from l l-cis rhodopsin and 9-cis rhodopsin. Proc, natn. Acad. Sci. U.S.A. 80, 1887-1891. Warshal A. (1978) Charge stabilization mechanism in the visual and purple membrane pigments. Proc. natn. Acad. Sci. U.S.A. 75, 2558-2562. Williams T. P. (1975) Dynamics of opsin, a visual protein. Accts. chem. Res. 8, 107-112. Yoshizawa T. and Wald G. (1963) Pre-lumirhodopsin and the bleaching of visual pigments. Nature 197, 1279-1286. Yoshizawa T. (1972) The behavior of visual pigments at low temperatures. In Handbook o f Sensory Physiology (Edited by Dartnall H. J. A.), Vol. 7, Part 1, pp. 146-179. Springer, Berlin.
0042-6989/84 $3.00 + 0.00 Copyright © 1984 Pergamon Press Ltd
DYNAMIC PROCESSES OF VISUAL TRANSDUCTION M. L. APPLEBURY Department of Biological Sciences, Purdue University, W. Lafayette, IN 47907, U.S.A. Abstract--Rhodopsin is one of those rare macromolecules whose inherent chromophore, 11-c/s retinaldehyde, allows one to naturally observe triggered macromolecular changes on the timescale of picoseconds to minutes. Investigations of these molecular processes have been carried out with laser monochromatic light under conditions where the photon flux used for photolysis was carefully measured. The formation of bleaching intermediates has been examined as a function of fluence. Under conditions where the formation of intermediates is unaffected by photon reversal the following observations hold: Upon the absorption of a photon, the initial photochemical event results in production of metastable bathorhodopsin within 6 psec. Artificial rhodopsin regenerated with 9-cis retinal forms a distinct bathorhodopsin which must reflect distortions at the active site differing from those generated with 11-cis retinal. Bathorhodopsin thermally decays through lumirhodopsin and meta I-rhodopsin, to meta II-rhodopsin through a series of coupled equilibria. The final meta I-meta II equilibrium is stable for seconds. The process provides a unique model for utilization of energy to drive (trigger) a biological cascade of events. Rhodopsin
Bathorhodopsin
Lumirhodopsin
Metarhodopsin (I and II)
INTRODUCTION In 1936 and 1938, H. J. A. Dartnall published two papers that laid the foundations for study of the bleaching processes of visual pigments which would govern investigations for the next 50 years (Dartnall et al., 1936; Dartnall et al., 1938). In these works, he discussed three particular principles which were to enable the study of mechanisms of photoreception and transduction by rhodopsin: (I) He pointed out the advantages of use of monochromatic light to photolyze visual pigments. The absolute determination of light intensity is made reasonably feasible enabling one "to apply fundamental photochemical principles to [the process of bleaching]." In this elegant 1936 work, he and his colleagues first determined the photosensitivity, a product of the extinction coefficient and the quantum yield. Had he had a value for the extinction coefficient at that time, the quantum yield would have been determined. In fact, this waited some 33 years (Dartnall, 1968). (2) He carefully considered the levels of intensity (fluence rate) which could be used without leading to artifacts, such as the photoreversal of products of the reaction and pointed out that the apparent bleaching rate (or amplitudes of reaction) should be proportional to the absolute intensity used. And (3) in the 1938 work, he began to examine the effects of temperature upon the reaction.
This presentation was given to celebrate the 70th birthday of Professor H. J. A. Dartnall during the Conference on Visual Pigments held in Bristol, England, 14-16 September 1983.
The studies of temperature dependence of bleaching processes during the 1950's and 1960's led to the identification of the intermediates of bleaching; they continued to govern the nature of investigations in the 1970's and 1980's, both in the study of the primary photochemical processes and in the study of the relaxation of early intermediates. Two examples taken from our own work illustrate the application of these principles and are discussed in the realm of our current understanding of the dynamic processes of bleaching. The first considers the primary photochemical intermediates of bleaching and has implications about the quantum yield of a rhodopsin analogue, 9-cis rhodopsin. The second examines the temperature dependence of the thermally controlled bleaching intermediates, lumirhodopsin (L), meta I-rhodopsin (MI) and meta II-rhodopsin (MII).
METHODS
Picosecond flash photolysis studies The instrumentation for picosecond dynamic studies and the preparation of samples have been fully described in Applebury and Rentzepis (1982) and Spalink et al. (1983). For data shown in this work, 11-cis rhodopsin had a concentration of A (498 nm) = 1.0, 2 m m path and 9-cis rhodopsin A(485nm) = 1.0, 2 mm path.
Microsecond and millisecond flash photolysis studies Flash photolysis was carried out with an apparatus consisting of a tuneable dye laser for initiating 1445
1446
M.L. APPLEBURY
bleaching and a single beam monochromatic light monitoring system coupled to a dual timebase transient recorder (Biomation). The photolysis pulse has a half width of about 200-500 nsec depending on wavelength. Spectral transients with lifetimes of between 10/~sec and minutes can be monitored (Eisenberg-Shinnar et al., 1984). Fresh (or frozen) bovine rod outer segment membranes were prepared in the presence of antioxidants or in deoxygenated solutions. The membranes were washed to deplete them of all peripherally bound proteins in order to study a simplified system in which rhodopsin is the only protein present (minor amounts of an integral membrane protein of --~250 kdaltons are present) and would be unperturbed by interactions with peripheral proteins. The procedures used are fully described in Baehr et al. (1982). The depleted membranes were sonicated and made 100 mM NaC1, 3 mM KC1, 0.5 mM MgC12 and 10 mM HEPES pH 7.
RESULTS AND D I S C U S S I O N
The primary intermediates o f 9-cis rhodopsin
One of the most important studies that examined the mechanisms of phototransduction was the study of rhodopsin in which 11-cis retinal was substituted by 9-cis retinal either by photochemically generating 9-cis in intact rhodopsin or by regenerating opsin with 9-cis retinal. These studies first demonstrated that the product of bleaching of either 11-cis or 9-cis rhodopsin was the all-trans retinal chromophore (Hubbard and Wald, 1952; Hubbard and Kropf, 1958). Later studies demonstrated that a photochemical equilibrium could be established between the 9-cis and 11-cis chromophores in low temperature glasses and indicated that the two chromophores were interconvertible within the environment of the opsin binding site (Yoshizawa and Wald, 1963). Thus, by the early sixties the nature of the primary intermediate in the visual process was described as follows: the absorption of light leads to the rapid isomerization of the 11- or 9-cis chromophore to all-trans retinal leaving the new chromophore in the original l l-cis environment. On a slower thermal timescale, the protein relaxes to the new chromophore conformation as entailed in the following scheme of visual transduction: 11-cis R H O ,
0.67
' all-trans BATHOr----* 9-cis RHO T~ 01 11 all-trans META II RHO
The quantum yields have been determined as 0.67 (Dartnall, 1968) and 0.1-0.3 (Hurley et al., 1977; Hubbard and Kropf, 1958). In studies aimed at establishing the dynamics of bathorhodopsin production, it was observed that the
initial intermediate arose in < 6 psec (the limits of technical resolution) and once established was stable for many nsec (Busch et al., 1972). Studies were carried out with low temperature glasses ( 7 7 4 K) with the hopes of slowing and resolving intermediate steps in the initial production of bathorhodopsin. An initial rate of formation could be observed between 77 and 4 K and it was found to have a marked deuterium isotope effect implicating a role for H + ion translocation in this process (Peters et al., 1977). Combining these results with the elegant studies of the chromophore configuration by resonance Raman spectroscopy (Eyring et al., 1980), the initial photochemical event can currently be described as the photochernically driven isomerization of the chromophore to an extremely distorted transoid structure accompanied by a protein translocation. The proton(s) might be either somewhere in the immediate vicinity of the chromophore (Warshal, 1978; Lewis, 1978) or be particularly involved in Schiff base protonation of the chromophore (Peters et al., 1977). When these studies were extended to the study of 9-cis rhodopsin several observations were inconsistent with the simplistic model given above. As first observed by Green et al. (1977), the rate limiting processes for the formation of bathorhodopsin from 9- or 11-cis rhodopsin are essentially the same, both < 6 psec. These studies were extended to low temperature glasses of 9-cis rhodopsin, under conditions in which a rate limiting step can be resolved (Applebury et al., 1979). The data are illustrated in Fig. 1 and indicate the rates are also similar below 20 K. At temperatures > 20 K, 9-cis appears to have a slower rate, but the error is too large to ascribe significance to this data. As Dartnall (1936) pointed out, in the absence of photolysis of products of bleaching, the production of the initial intermediate should be proportional to the qiiantum yield - d R / d t = [dB/dt] = (oJ,/V
Where R is the molar concentration of rhodopsin undergoing reaction, B is the molar concentration of bathorhodopsin produced, q~ the quantum yield of batho, .It the number (moles) of photons absorbed per unit time t, and V the reaction volume. Clearly, equivalent reaction rates measured for l l-cis and 9-cis rhodopsin are inconsistent with quantum yields of 0.67 and 0.1 respectively, under conditions of equivalent absorption. The above equation holds under conditions in which the excited state R* can be assumed to be a steady state. Under conditions of psec photolysis, the steady state assumption is questionable. The rate of batho production could reflect a more complex mechanism and involve more intermediates, e.g. the rates observed at low temperature studies could reflect some rate limiting relaxation to the ground state bathorhodopsin. Still the amplitude of the reaction should reflect the quantum efficiency
Dynamic processes of visual transduction
26
25
t
•
I
7
24
23
I
50
I
1O0
[
150
I
200
I
250
1 / T x 1000
Fig. 1. Temperature dependence of the rate of formation of 11-cis bathorhodopsin (O) and 9-cis bathorhodopsin (O). Data indicated by the symbol (0) are rate constants for 9-cis bathorhodopsin corrected to that which would be observed for 9-cis rhodopsin with the same initial absorption as the I 1-cis rhodopsin. This is valid only if the rate constant is proportional to the light absorbed. The error in rate constants measured is ~30%.
and the yield of bathorhodopsin from ll-cis and should be ~ 7 times that of 9-cis. In fact, the amplitudes of reaction corrected for absorption are nearly identical (Monger et al., 1979; Applebury et al., 1979). These observations led to an extended study of bleaching with monochromatic light as a function of intensity (fluence rate)--in essence to test the postulates of limiting conditions given by Dartnall et al. (1936). To accomplish the study with reasonable signal to noise, photolysis was carried out with the second harmonic (532 nm) of a N d ( 3 + ) / Y A G laser whose output was a pulse with 25 psec F W H M . The depletion of rhodopsin and production of bathorhodopsin were observed as a difference spectrum recorded at 40 psec, 85 psec or 8 nsec by a double beam spectrophotometer (Spalink et al., 1983). To examine the proportionality of signal amplitude to photons absorbed, where J , = I o [1 -exp(-2.303ebl)] with I 0 as the incident fluence, the molar extinction coefficient, b the molar concentration and l the pathlength, difference spectra data were averaged for low intensities ( < ~ 2 0 #J/mm2), medium fluence (~50/tJ/mm2), and high fluence (>60#J/mm2). Inspection of Fig. 2, indicates that with increasing fluence bathorodopsin formed from 11-cis rhodopsin does not behave as expected. There is no increase in the difference spectra maximum at ~ 5 7 0 n m , which should be proportional to increasing levels of bathorhodopsin produced, although there is an increasing depletion in the appar-
1447
ent levels of rhodopsin bleached. Moreover, for 11-cis rhodopsin, the isosbestic point shifts with increasing fluence indicating more than two species are contributing to the difference spectra. The spectra for 9-cis rhodopsin are more model-like and behave with the appropriate increases in product and decreases in starting material. A more detailed examination of these changes was carried out at the difference spectra wavelength maxima and minima. As shown in Fig. 3, saturation effects occur far below fluence levels which would give total bleach of starting rhodopsin. Under the experimental conditions used, saturation occurs above 10-15~ photolysis. Ostensibly, the products of bleaching are undergoing photoreaction as well. The effect is more pronounced for 11-cis than for 9-cis rhodopsin. For fluence levels over which there is approximate linearity ( < ~ 20 p J/ram 2), the difference spectra can be compared. It is obvious that the ratio of difference spectra maximum to minimum differs for l l-cis and 9-cis. Since 11-cis and 9-cis rhodopsin have nearly the same molar extinction coefficient (~ at 2,,,x differs by a factor of 1.06 and the )'max differ by ~ 13 mm), this ratio should be nearly the same if the products of photolysis were identical. The simplistic deduction is that, in fact, the products are not the same and the product of 9-cis must have a lower extinction coefficient than that of l l-cis rhodopsin. To resolve the difference spectra into component bathorhodopsin produced and rhodopsin bleached, one needs to determine the amount of rhodopsin bleached. In these studies, this was carried out by pooling samples, each of which had been photolyzed with a known low level fluence such that the percent bleach (determined from the residual rhodopsin absorption) could be determined for a given average fluence [Spalink et al. (1983)]. Under conditions of low fluence, the percent bleach of 9-cis or 11-cis rhodopsin is nearly the same when corrected for initial absorption at the 532 nm wavelength of photolysis and indicates that the quantum yields are more nearly equivalent and are unlikely to differ by a factor of 7. Bathorhodopsin spectra resolved under conditions of low fluence (Fig. 2, low) indicate that 9-cis bathorhodopsin has a lower extinction coefficient and that its )'max is red shifted approximately 10nm (Fig. 4) [Spalink et al. (1983)]. Thus, either the 9- and 11-batho products differ in their transoid nature or the protein cannot relax to the same state--or perhaps both play a role at this stage of bleaching. Moreover, spectra compared under the same low fluence photolysis indicate that the bathorhodopsins remain different up to at least 8 nsec (Spalink et al., unpublished results). Thus, either the chromophore or protein (or both) do not relax to a common intermediate over this timescale. Kliger and colleagues (Horwitz et al., 1983) have recently indicated that differences are still apparent on the #sec time-
1448
M . L . APPLEBURY 0.20
.
,
0.15
:.
.
.
.
.
.
.
.
.
.
.
.
i:
:
,
,1 :
..................
i
II
,-;,,~,;;o;;,~
.............................................
, r
,
.~.~'i-.~',
1)~(~;>
,
,
~
. . . .
!
(a)
:
;'~_~17;,I
•
:,
..... i .............................
~.....
0.10
0.05
- .......................... "
- ............................ ~
:
i
~...
i ....................... ~
.~,t-~/,k~)! - ...................
I,
"-.~:x~i~
i! !!
i ......................... ~"i:~
i '
~ ....
,&;,,.~,
ooo li{~,.
o o~ E ii/ -0.10
' "#.-,-:r.a?
: ~
~,. _
"
Low
.,[~tl
~',t
;,' "=
HIGH
•
^,/' ~ mi
-
!
:
::
::
...
........................................................ i:....................................................................................... : LI
-015 (:3 -0.20
I
I
i
I
I
I
I
I
450
400
i
I
I
t
I
500
g
I
I
I
I
550
Wavelength
t
I
I
I
I
600
650
(nm]
(J 0.20
i
i
~
i
I
i
i
i
1
i
t
;
i
i
i
~
i
i
i
i
i
=o g L
9-c/s
o <
Rhodopsin
~4".....
o.,o ........................................................ ............................
"'"',
..... '~'~
-0.o~
.................
-o,o
.......................
-o.~s
LOW
! .......... ;;~.i .......... c! ..................
~
t, ,~;
~':~
:.:',.! ..... !,..%,>/,,'>,.'~,.?.r,..!.~.:::,~. !.:-:.L
...........................
i
i
, ......................... ;., .............. U.>i.l.i.
.............................
i':
"''',,.
~,v.%:..;;:~-
:
......................
............................... .......... :.
:
:
i..........................................................
...........................
............................
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
i...
.
.
.
.
.
.
.
.
.
.
.
.
~" '".',,"w~,~". lG. '
.~
-_:
:......- .....................................................................................................................
: --O,20
•
4o0
1
,l
~
450
-I, I
I
I
I
I
I
I
I
500
I
550
Wavelength
I
I
I
I
I
I
6oo
"3 "I
I
650
(nrn)
Fig. 2. The dependence of difference spectra amplitudes on excitation energy. Samples of A(2,~,x)= 1.0, 2 mm path-length. Excitation was at 532 nrn with fluence ranges low = < 320 #J; mid = 320-480 #J; high = >480 #J. The beam was spread to irradiate an area of ~ 16 mm 2. (a) Bathorhodopsin spectrum minus 11-cis rhodopsin spectrum. (b) Bathorhodopsin spectrum minus 9-cis rhodopsin spectrum. The figure was adapted from Spalink et al., 1983. scale, hence even the l u m i r h o d o p s i n s m a y differ. In s u m m a r y , from studies o f the bleaching process at r o o m temperature, the following scheme seems currently appropriate, where the latter steps are yet to be firmly resolved.
T h e a p p a r e n t difference between these observations m a d e at r o o m t e m p e r a t u r e a n d those originally m a d e with low t e m p e r a t u r e glasses (Yoshizawa a n d Wald, 1963; Yoshizawa, 1972) are n o t obvious. One e x p l a n a t i o n would be t h a t the m o d e s o f protein
11-cis R H O -~ t r a n s o i d B A T H O
LUMIRHO
transoid B A T H O ' ~ 9-cis R H O LUMIRHO'
META I - RHODOPSIN T$
M E T A II -- R H O D O P S I N
T$ RETINAL + OPSIN
Dynamic processes of visual transduction
1449
0.20
0 2 0 -(a)
0.15 --
(b)
i
ii/i~'~
0.15
,/
A/
,/ m 0.10
/
<3
/
0.05
°/° o ~, ?.
(A, 570nm (o) 490nm
/ o.oo
0
<3
o/
/ /o o
/';S °
0.10
/d /o P
i/ i/
Ii/
o / o"" ~ / o
/ o /o 7
(1) 56Ohm (o) 475 nm
lit//
0.05
11-cis Rhodopsin I 1
I 2
I 3
I 4
I 5
Excitation
Rhodopsin
9-c/s
I 6
pulse
o.oo 0
fluence
I 1
I 2
I 3
I 4
I, 6
I 5
(rel.units)
Fig. 3. Bathorhodopsin - rhodopsin difference spectra absorbance changes as a function of excitation fluence. Each point is an average of 4-6 records. One relative unit corresponds to 160-200/xJ for a irradiated area of ~ 16 mm 2. (a) Bathorhodopsin minus 1l-cis rhodopsin spectral amplitudes at 570 nm ( k ) and 490 nm (O). (b) Bathorhodopsin minus 9-eis rhodopsin spectral amplitudes at 560 nm (A) and 475 nm (O). This figure was adapted from Spalink et al., 1983.
relaxation at low temperatures differ from those at room temperature, although it is difficult to intuitively understand why they should be the same for both 11-cis and 9-cis at low temperatures, but differ at more permissible room temperatures. A more recent study of bathorhodopsin production at low temperature under more limiting levels of photolysis does indicate that the products of 11-cis and 9-cis rhodopsin differ, although the extinction coefficient for 9-cis batho was shown to be larger than that of l l - c i s (Mao et al., 1980). No systematic study of bathorhodopsin production in low temperature glasses as a function of fluence has been reported and it is unclear which studies are free of saturation effects and which are not. Low temperature studies are as susceptible to saturation effects as room temperature studies. The ftuence rate experimentally used may differ dramatically, but the total fluence needed is the same for conversion to similar detectable product level. Batho products will build up at the face incident to the photolysis beam and will be distributed in a decreasing exponential manner through the depth of the cell. The actual levels of fluence under which saturation will occur will depend upon the initial absorption as well as the quantum yield of product and cross-sectional absorption of the product, making predictions complex. In approximation, however, for samples with initial absorbances of 0.5-0.8, fluences above 20-30/xJ/mm 2 i.e. 2-3 mJ/cm 2, become suspect. The low extinction values calculated earlier for 11-eis bathorhodopsin at room temperature can be attributed to such saturation effects (Applebury, ]980). The examination of difference spectra for 11-cis rhodopsin questions whether a photoequilibrium between 11-cis, batho and 9-cis is established. Were this
the case, the amplitude of the difference spectra would be expected to maximize at high fluence but the shape of the difference spectra would change relatively little as batho was photoreversed to 11-cis or 9-cis rhodopsin since the absorption of 11-cis and 9-cis differ only modestly. For l l-cis rhodopsin, whatever the nature of the photolysis product contributing to the spectra might be, the change is not appropriate for batho being driven back to the starting 11-cis rhodopsin or to 9-cis rhodopsin. Over the fluence range studied, this problem is not observed for regenerated 9-cis rhodopsin. The differences between the behavior of l l-cis rhodopsin and 9-cis rhodopsin have interesting implications about the way in which the protein controls the chromophore relaxation and the distribution of excess energy. A careful analysis of these two rhodopsins is likely to provide valuable insight about the process of phototransduction.
1.5
Bll R9 Rll / /
o
10
.Q <
29
\
X
l/
\\\
0.5
O.O
I
450
I
500
550
60(3
650
Wavelength ( nm )
Fig. 4. Bathorhodopsin spectra resolved from difference spectra taken at 85 psec, 298 K. R1], parent 11-cisrhodopsin spectrum; Bu, bathorhodopsin of l l-eis rhodopsin; Rg, parent 9-cis spectrum; Bg, bathorhodopsin of 9-cis rhodopsin.
1450
M.L.
APPLEBURY
A study o f the thermal intermediates o f bleaching In 1938 Dartnall's work turned to a study of the effects of temperature, pH and buffer on photosensitivities. He demonstrated and emphasized that there should be little or no temperature effect on the photosensitivity of rhodopsin. As mentioned above and further demonstrated by Peters et al. (1977) this bears out even for picosecond studies of the process. He also predicted the role that temperature might play in controlling the intermediates of bleaching (Dartnall et al., 1938). He mentioned his own observations, based on those of Kuhne, that frozen solutions of visual pigments appear to bleach very slowly. Over the years, of course, the study of the temperature dependencies of the bleaching products enabled investigators to define the series of intermediates that occur in the process of bleaching. The absorption spectra of these intermediates are now well established; the temperatures which permit sequential conversion from one to another are defined; and the kinetics of the MI MII transition have been extensively studied in detergent solubilized solutions. The dynamics of these transitions from batho to meta II are poorly understood, however, particularly for the visual pigment present in its own native environment--the lipid bilayer. In the last few years, we have conducted studies of thermal intermediates of bleaching with rhodopsin in its native lipid environment. The studies were carried out as those discussed above, using monochromatic light of known intensity, defining the photolysis fluence under which artifacts are minimized, and using temperature to selectively study the dynamics of the lumi ~ meta I--. meta II rhodopsin transitions. The following presents our current understanding of these events. The majority of the experiments reported here were carried out with a flash photolysis pulse of 540 nm in order to minimize photoreversals and to avoid, as best possible, pickup of the scattered photolysis light as an artifact in recording the subsequent transition. The use of a dual timebase transient recorder allows transients to be recorded in two modes. (1) The pretrigger mode samples records of the initial rhodopsin absorption and then the transient immediately following the photolysis flash. This is illustrated in Fig. 5(a) where the absorption at 380 nm is recorded prior to the photolysis flash and then during the meta I (~'max 480 rim) to meta II (2~ax 380 rim) transition. The transient change in absorption may be plotted as In (A~ - A , ) vs time to illustrate that the behaviour is that of a first order reaction. It is linear over more than 3 lifetimes as shown in Fig. 5(b), the final state is stable for well over 100 lifetimes ( > 1 sec). On close inspection of early timepoints, however, deviations from linearity can be distinguished which are indicative of an earlier transition of faster rate. This earlier transition may be observed simply by changing the recording timescale or by using the second transient recorder mode. (2) The dual timebase mode records
transients on two time bases, a faster and a slower. As shown in Fig. 5(c), the record tracks the initial fast rise of lumi (2~,x 505 nm) --, meta I (2m~ 480 rim) and then the subsequent meta I--, meta II transition. Thus, the instrumentation enables us to study both transients over a range of temperatures from 0 to 35°C. To avoid artifacts due to sequential photon absorptions, such as photoreversal of intermediates, the amplitudes of formation of meta II signals were measured as a function of fluence of the photolysis pulse. As shown in Fig. 6, the signal amplitude saturates far below the expected maximumly possible amplitude. The figure illustrates the extreme sensitivity of rhodopsin and its intermediates to photoreversal and emphasizes the difficulty of recording true unperturbed transients with good signal to noise. Bleaches of > 15-20~ begin to be subject to photoreversal. This has been demonstrated in more detail by Williams (1975) who used the higher intensities to study the nature of photoreversal. We have limited our studies to transitions carried out at bleaches of 10~o or below. Under these conditions, both the lumi --* meta I and meta I ~ meta II transitions are first order reactions. For the visual pigment transition in digitonin solutions, Matthews et al. (1963) established that the meta I ~-meta II transition is a temperature dependent equilibrium. One of the first questions to ask was whether or not this equilibrium is the same in native membrane bilayers. To resolve this question we attempted to measure the equilibrium constant as a function of temperature in the following way. In the pretrigger mode, the absorbance of the reaction is given by MI e + M I I e + R~h -- Ro = MIe + M i l e - Rb, where absorbances are proportional to intermediates at e, equilibrium concentrations; ub, the unbleached rhodopsin concentrations; o, the initial concentration; and b, the bleached concentration. In the dual timebase mode, the absorbance change (amplitude) of the first to second transition is given by MI e + M I I e -- MI,, where t indicates total meta I. The absorbances recorded are simply the sum of the component concentrations (i) times their known extinction coefficients (ei) at a given wavelength (2) and the appropriate pathlengths (1) Ae(,~ )
= [ m 1 e • ~ml(/~.) -~-
m2 e • e r a 2 ( 2 )
--
rh' e,(2)]l
(pretrigger) Ae(~. )
m2~-era2(2) - m 1," ~,,1(2)]/ (dual timebase)
= [ m 1 e - gml()~) +
since m 1 t = rb and m 1, = m 1 e + m2e, the equations reduce to two equations in two unknowns, m 1e and m2 e. If the values are solved for a series of wavelengths, the concentrations can be reasonably established. Thus, transients were measured as a function of temperature both in pretrigger and dual timebase mode and absorbances were plotted as difference spectra. In practice, the data were fit for a consistent
Dynamic processes of visual transduction
1451
(a)
+ 0.004
, |
0
15
30
45
Time
I
I
60
75
(msec)
(b)
10 T
1801"
I~.LU,I~.LI
.I ]JL. __,iJl L ~ . d ~ 1 = ~ L Jilli, I . L-~d .1 . a t , .~.i J'L~,I
tel 0
_8 E
+ 0.004- V I 2
I-
I
6oo
I
1200
I
;
I
~800
24-0o
3000
Time (msec)
t
I
I
I
1
I
i
,,I
-0.1
-0 . ' I
E c -0.2
I f
-0.3
-0.4
-O.2
-0.3 i I
I I
i
!
t i
-04-
i I
-0.5
-O.5
0 0.5 1.0 10 msec
50
100
0 0.5 1.0 10
50
I00
msec
Fig. 5. Kinetic records showing the meta transition in the bleaching process of rhodopsin in sonicated rod outer segments at 24°C. (a) Data collected by a Biomation transient recorder in the pretrigger mode at 50/1 sec/channel (2048 channels). (b) Data collected in the pretrigger mode at 2 # sec/ch (2048 channels). (c) Data collected in the dual timebase mode, base 1:2 #sec/ch (600 channels), base 2:100/~sec/ch (1448 channels). The left trace was collected without photolysis just prior to the right trace with photolysis. Photolysis wavelength, 500 nm; monitoring absorption 380 nm.
V.R. 24/11--B
1452
M.L. APPLEBURY 0.06
0,05
o//,./ 0.04
<3
0.03
002
i
/////°'g
y
0.01
I 0.2
[ Q4 Relotive
I 0.6
I 08
fluence
Fig. 6. Meta II rhodopsin- rhodopsin difference spectra absorbance changes as a function of excitation fluence at 540 nm. (C)) Amplitudes measured at 500 nm; (0) Amplitudes measured at 380 nm. About 5~o of the incident photolysis beam was deflected and recorded by a photodiode. The relative fluence was measured as the peak amplitude of the pulse traced on an oscilloscope. One relative unit is < 1 mJ per 5 mm diameter irradiated at the sample face. Full bleach would be expected to give an I AA ] about 0.25 for the sample whose absorption was 0.35 A (500 rim).
set of concentration values that satisfied both the pretrigger data and the dual timebase data. The molar extinction coefficients used were taken from spectra where values at 2m,x are rho, 5(498 n m ) = 40,600 M 1cm ~; meta I, e(478 nm) = 44,000 M cm-1; meta II, e ( 3 7 8 n m ) = 3 8 , 0 0 0 M - l c m - ~ ; and when necessary, lumi, e(500 n m ) = 46,700 M -~ cm-L A more detailed account is given in (Eisenberg Shinnar et al., 1984). Figure 7(a) shows the pretrigger data for difference spectra representing concentrations of meta I and meta II at equilibrium minus the concentration of rhodopsin bleached. Comparison of these spectra with model spectra, given in the insert, indicate that they clearly represent at least three species. The isosbestic point is not 0 and can be fairly well represented as the sum of meta I and meta II at equilibrium for any given temperature minus the amount of rhodopsin bleached. At 24°C, the data are adequately fit by 33~o meta I and 67~o meta II. Other values are given in Table 1. Figure 7(b) shows the dual timebase data for difference spectra ostensibly representing the concentration of meta I and meta II at equilibrium minus the maximum meta I concentration. Comparison of the data with model difference curves indicates striking differences. The
hv
< 6psec
- 50nsec
model illustrates a difference curve for two species, meta I and meta II, with a single isosbestic point at 0. Clearly the data have a shifting negative "~maxand no true isosbestic point; they are inconsistent with a simple relaxation of meta I to an equilibrium of meta I and meta II. The spectra can be fit by including a significant amount of lumirhodopsin. This indicates that the first transient observed is that of lumi (or lumi plus batho) to the appropriate equilibrium concentration of lumi + meta I and the second transient is that of lumi + meta I to the equilibrium concentrations of meta I and meta II. At some temperatures these equilibria are fairly well separated; but in general, they must be considered as tightly coupled equilibria. The concentrations of best fit for these intermediates over a series of temperatures are given in Table 1 along with the approximate relaxation times measured. It is important to notice that at lower temperatures the fits indicate that there are significant amounts of lumirhodopsin, which is stable up to msec, and at no temperature does the equilibrium favor one discrete species. Direct comparison of these equilibria states with those of rhodopsin in the intact rod outer segment are of course not possible. Several suggestions have now been made that the interaction of rhodopsin with peripheral membrane proteins may alter the equilibria (Emeis and Hofmann, 1981; Emeis et al., 1982; Bennett, 1982; Parkes et al., 1983). The nature of these equilibria must be reestablished under conditions in which peripheral proteins are present. These results, however, suggest that caution must be taken in making attempts to assign any particular intermediate form to that which is appropriate for interaction with other enzymes, e.g. phosphorylation by opsin kinase or interaction with G-protein. This thermodynamic evaluation of the bleaching process gives some insight into the mechanism of energy redistribution in opsin. Statistically, the coupled equilibria suggest that the molecule fluctuates between a series of states much in the same way as other receptors (e.g. the acetylcholine receptor) or ionic channels (Neher and Stevens, 1977; Heidmann and Changeux, 1978). While the observations in no way imply that rhodopsin is a channel, in analogy to these receptors or channels, it may be suggested that the equilibria may be modified by environmental factors such as phosphorylation, binding of small molecules, or perhaps peripheral proteins. In summary, applying the principles and considerations first set forth by Professor Dartnall, studies of the bleaching of rhodopsin with current state of the art instrumentation lead to a general model which might be summarized as the following. The relaxation times were measured at 24°C; the constants can be extrapolated to be approximately 3-10 times faster at 37°C. ~ 0.3msec
R h o ~ R * - - ~ [ B ] ~- Batho ~ Lumi ~
MetaI
~ ] 0msec
~
MetaII--~
Dynamic
1453
processes of visual transduction
-g0 ~-
~
.~ -~.~
~ ~ -
~N
,/ i
•/,/11
I
Oo
I
J
I
O
~ 0
O 0
I
I
~ 0
o .'~
I
.~
~1
~.~
,
~1 ~ i ~
~.~~+
~ "1"-~ \"
I
I
o
~
0
~ .~ ~ E] ~ , : . ~
I
0
0
I
I
,.~ ~0 ~F7~ ~ o ~ .o ~ ~ . ~+ ~ o ~ . ~ F ~ 7. ~
<1
~o~°~o o o
tO
.~
~
~ . ~
~
.I
.
/
i
0
0 ~
.~ ~ o ~
~
I
-~i
i'
~ o ~~ °o I ~ o + ~='~~ . , o ~ .1:
I ~q o
".~
0..I ~
~
.
~
_
~ : ~
I o o
m o
,
~D 0 0
o
Y
0
I
I
0
o
i
I
• -~ ~1~
<~
~
~
~o
-~N e
1454
M. L. APPLEBURY )d
)-2
Table 1. Transient equilibrium concentrations for sonicated ros pH 7.0 L ~ M1 ~ M2 Temperature K C 278 5 288 15 297 24 (310 37)a aExtrapolated.
2~ 22 Tt M2 M1 L T2 M2 7.2msec -0.475 0.525 500.0msec 0.325 1.8msec 0.05 0.475 0.475 100.0msec 0.50 0.3 msec 0 . 1 0 0.475 0.425 10.0msec 0.675 0.1 --0.7 msec (0.83)
Acknowledgements--The author expresses her appreciation
to J. D. Spalink, B. Weiss, A. Shinnar and K. Savoie-Luisi for their assistance in this work. The project was supported in part by a grant from the National Eye Institute to M.L.A. REFERENCES
Applebury M. L. and Rentzepis P. M. (1982) Picosecond spectroscopy of visual pigments. Meth. Enzymol 81, 354-368. Applebury M. L., Peters K. S. and Rentzepis P. M. (1979) A picosecond kinetic study of isorhodopsin photochemistry. Biophys. J. 25, 316a. Applebury M. L. (1980) The primary processes of vision. A view from the experimental side. Photochem. Photobiol. 32, 425-431. Baehr W., Morita E., Swanson R. and Applebury M. L. (1982) Characterization of bovine rod outer segment G-protein. J. biol. Chem. 257, 6452-6460. Bennett N. (1982) Light-induced interactions between rhodopsin and the GTP-binding protein. Relation with phosphodiesterase activation. Eur. J. Biochem. 123, 133-139. Busch G. E., Applebury M. L., Lamola A. A. and Rentzepis P. M. (1972) The formation and decay of prelumirhodopsin at room temperature. Proc. natn. Acad. Sci. U.S.A. 69, 280~2806. Dartnall H. J. A., Goodeve C. F. and Lythgoe R. J. (1936) The quantitative analysis of the photochemical bleaching of visual purple solutions in monochromatic light. Proc. R. Soc. A, Lond. 156, 158-171. Dartnall H. J. A., Goodeve C. F. and Lythgoe R. J. (1938) The effect of temperature on the photochemical bleaching of visual purple solutions. Proc. R. Soc. A, Lond. 164, 216-230. Dartnall H. J. A. (1968) The photosensitivities of visual pigments in the presence of hydroxylamine. Vision Res. 8, 339-358. Eisenberg Shinnar A., Weiss W. I. and Applebury M. L. (1984) Kinetics of rhodopsin bleaching. 1. The Meta I-II equilibrium in bovine rod outer segment membranes. To be published. Emeis D. and Hofmann K. P. (1981) Shift in the relation between flash-induced metarhodopsin I and metarhodopsin II within the first 10% rhodopsin bleaching in bovine disc membranes. F E B S Lett. 136, 201-207. Emeis D., Kuhn H., Reichert J. and Hofmann K. P. (1982) Complex formation between metarhodopsin II and GTPbinding protein in bovine photoreceptor membranes leads to a shift of the photoproduct equilibrium. F E B S Lett. 143, 29-34. Eyring G., Curry B., Mathies R., Fransen R., Palings I. and Lugtenburg J. (1980) Interpretation of the resonance Raman spectrum of bathorhodopsin based on visual pigment analogues. Biochemistry 19, 2410-2418. Green B. H., Monger T. G., Alfano R. R., Aton B. and
MI 0.625 0.475 0.325 (0.17)
L 0.05 0.025 ---
Callender R. H. (1977) Cis-trans isomerisation in rhodopsin occurs in picoseconds. Nature 269, 179-180. Heidmann T. and Changeux J. P. (1978) Structural and functional properties of the acetylcholine receptor protein in its purified and membrane-bound states. Ann. Rev. Biochem. 47, 317-357. Horwitz J. S., Lewis J. W., Powers M. A. and Kliger D. S. (1983) Nanosecond laser photolysis of rhodopsin and isorhodopsin. Photochem. Photobiol. 37, 181-188. Hubbard R. and Wald G. (1952, 1953) Cis-trans isomer of vitamin A and retinene in the rhodopsin system. J. gen. Physiol. 36, 269-315. Hubbard R. and Kropf A. (1958) The action of light on rhodopsin, proc. natn. Acad. Sci. U.S.A. 44, 130--139. Hurley J. B., Ebrey T. G., Honing B. and Ottolenghi M. (1977) Temperature and wavelength effects on the photochemistry of rhodopsin, isorhodopsin, bacteriorhodopsin and their bathoproducts. Nature 270, 540-543. Lewis A. (1978) The molecular mechanism of excitation in visual transduction and bacteriorhodopsin. Proc. natn. Acad. Sci. U.S.A. 75, 549-553. Mao F,, Ebrey T. G. and Crouch R. (1980) Bathoproducts of rhodopsin, isorhodopsin I and isorhodopsin II. Biophys. J. 29, 247 256. Matthews R. G. Hubbard R., Brown P. K. and Wald G. (1963) Tautomeric forms of metarhodopsin. J. gen. Physiol. 47, 215-240. Monger T. G., Alfano R. R. and Callender R. H. (1979) Photochemistry of rhodopsin and isorhodopsin investigated on a picosecond time scale. Biophys. J. 27, 104-116. Neher E. and Stevens C. F. (1977) Conductance fluctuations and ionic pores in membranes. Ann. Rev. Biophys. Bioengng 6, 345 381. Parkes J. H. and Liebman P. A. (1983) Stabilization of metarhodopsin II by GTP binding protein. Invest. Ophthal. visual Sci., Suppl. 24, 245. Peters K. S., Applebury M. L. and Rentzepis P. M. (1977) Primary photochemical event in vision: Proton translocation. Proc. natn. Acad. Sci. U.S.A. 74, 3119 3123. Spalink J. D., Reynolds A. H., Rentzepis P. M., Sperling W. and Applebury M. L. (1983) Bathorhodopsin intermediates from l l-cis rhodopsin and 9-cis rhodopsin. Proc, natn. Acad. Sci. U.S.A. 80, 1887-1891. Warshal A. (1978) Charge stabilization mechanism in the visual and purple membrane pigments. Proc. natn. Acad. Sci. U.S.A. 75, 2558-2562. Williams T. P. (1975) Dynamics of opsin, a visual protein. Accts. chem. Res. 8, 107-112. Yoshizawa T. and Wald G. (1963) Pre-lumirhodopsin and the bleaching of visual pigments. Nature 197, 1279-1286. Yoshizawa T. (1972) The behavior of visual pigments at low temperatures. In Handbook o f Sensory Physiology (Edited by Dartnall H. J. A.), Vol. 7, Part 1, pp. 146-179. Springer, Berlin.