(Received for publication, October 20, 1994)
From the
9-Demethyl rhodopsin (9dR), an analog of vertebrate rhodopsin,
consists of opsin and a covalently attached chromophore of 11-cis 9-demethylretinal. Electrophysiological evidence that
photoactivated 9dR (9dR*) undergoes abnormally slow deactivation in
salamander rods (Corson, D. W., Cornwall, M. C., and Pepperberg, D.
R.(1994) Visual Neurosci. 11, 91-98) raises the
possibility that opsin phosphorylation, a reaction involved in visual
pigment deactivation, operates abnormally on 9dR*. This possibility was
tested by measuring the light-dependent phosphorylation of 9dR in
preparations obtained from bovine rod outer segments. Outer segment
membranes containing 9dR or regenerated rhodopsin were
flash-illuminated in the presence of
[-
P]ATP and rhodopsin kinase, further
incubated in darkness, and then analyzed for opsin-bound
[
P]P
.
[
P]P
incorporation by 9dR* increased
with both incubation period and bleaching extent but, under all
conditions tested, was less than that measured in rhodopsin controls.
Results obtained with 30-s incubation periods indicated that the
maximal initial rate of incorporation by 9dR* is about 25% of that by
photoactivated rhodopsin. The results imply that the low incorporation
of P
by 9dR* results from a reduced rate of phosphorylation
by rhodopsin kinase and are consistent with the prolonged lifetime of
9dR* determined electrophysiologically.
Photoisomerization of the visual pigment in vertebrate rods
generates metarhodopsin II (R*), ()the bleaching
intermediate that activates transducin and thus induces the
photocurrent response (for review, see (1) ). The deactivation
of metarhodopsin II involves the enzymatic phosphorylation of this
species (2, 3, 4, 5, 6) and the
subsequent non-covalent binding of arrestin to the phosphorylated
pigment(7, 8, 9, 10, 11) .
The 11-cis isomer of 9-demethylretinal, an analog of native retinal chromophore in which a hydrogen atom replaces the methyl group at carbon-9, can combine with opsin to form 9-demethyl rhodopsin (9dR), a light-sensitive pigment(12, 13) . Evidence that light-induced properties of 9dR differ markedly from those of native rhodopsin and porphyropsin (14, 15) emphasizes the importance of retinal's 9-methyl group in visual pigment function and motivates further studies of 9dR. Particular interest in the deactivation of 9dR arises from a recent study of bright flash photocurrent responses in salamander rods (16) . In rods prepared to contain both 9dR and residual porphyropsin, it was shown that responses mediated largely by native pigment are normal, but those mediated by 9dR exhibit sluggish recovery kinetics, i.e. the lifetime of photoactivated 9dR (9dR*) is abnormally long. The present experiments on bovine rod outer segment (ROS) preparations were undertaken to test the possibility that 9dR* functions abnormally in the phosphorylation reaction. Some of our results were reported at the 1993 meetings of the Biophysical Society and the Association for Research in Vision and Ophthalmology(17, 18) .
Some experiments employed rhodopsin
kinase that had been partially purified using procedures similar to
those described by Buczylko et
al.(23) . ROS membranes that had been isolated in the
light were homogenized in 200 ml of buffer (10 mM BTP, 10
mM MgCl, and 0.3 mg/ml each of aprotinin,
benzamidine, and leupeptin, pH 7.5) and incubated for 10 min (4 °C)
in room light. The ROS membranes were pelleted by centrifugation at
46,000
g (10 min, 4 °C). The pellet was mixed with
10 ml of Whatman DE-52 suspended in 10 mM BTP (pH 7.5), and
the mixture was loaded onto a 1.6
10-cm column of Whatman DE-52
that had been pre-equilibrated with 10 mM BTP (pH 7.5). The
column was extensively washed under room light with 10 mM BTP
until the absorbance of the eluent at 280 nm (1-cm path length) was
less than 0.01. The column was then protected from light, and rhodopsin
kinase was eluted with buffer consisting of 10 mM BTP and 110
mM NaCl (pH 7.5). Fractions (1.5 ml) were collected, and
50-µl aliquots were analyzed for activity in light-dependent
rhodopsin phosphorylation(3) . Fractions containing the highest
activity were pooled, and solid adonitol was added to make a 20% (w/v)
solution. The enriched rhodopsin kinase preparation was stored at
-20 °C.
Figure 1:
Bleaching of regenerated rhodopsin (filled circles) and 9dR (open circles). Datapoints and errorbars indicate mean
values ± S.D. (n = 3). Curves fitted to
the data plot text equation(1) , with k = 0.59
(9dR, dashedcurve) or k = 0.53
(rhodopsin, solidcurve). Inset,
hydroxylamine difference spectra for 9dR (opencircles) and rhodopsin (filledcircles). A/
A
,
normalized bleach-induced change in
absorbance.
where r = 2 nmol = the
initial amount of rhodopsin or 9-de-methyl rhodopsin; r* is
the measured amount, in nmol, of bleached pigment (i.e. of
9dR* or R*); and k is a photosensitivity parameter (see, e.g.(25) ). The fitting of to the 9dR
and rhodopsin data yielded, respectively, the dashed (k = 0.59) and solid (k = 0.53) curves. These served as standard curves for determinations of
flash bleaching in the phosphorylation assays.
Figure 2:
Time course of light-induced
[P]P
incorporation by 9dR and
rhodopsin. Following flash illumination, reaction mixtures were
incubated in darkness for the indicated period and then supplemented
with SDS-containing buffer that quenched the phosphorylation reaction. Opensquares, 0.037 nmol of bleached 9dR; filledsquares, 0.033 nmol of bleached rhodopsin; opentriangles, 0.18 nmol of bleached 9dR; filledtriangles, 0.16 nmol of bleached rhodopsin. Datapoints and verticalbars indicate mean
values ± S.D. (n = 3), respectively. Filled and opencircles show data obtained from
unilluminated control samples containing, respectively, rhodopsin and
9dR.
Figure 3:
Initial
rate of [P]P
incorporation
(nmol/liter/min) by illuminated rhodopsin and 9dR. Indicated extents of
flash bleaching are based on use of the curves shown in Fig. 1. Concentrations quoted in the figure and accompanying
text represent molar amounts contained in the 100-µl reaction
mixture. Following illumination, samples were incubated for 30 s and
then analyzed for [
P]P
incorporation. Filledcircles, rhodopsin
samples; opencircles, 9-demethyl rhodopsin samples. Curves are plots of text equation(2) . Inset,
representative autoradiographic data obtained at the higher bleaching
levels. Upperrow (left to right),
rhodopsin (rho) bleaches of 0.7, 1.6, and 3.1 µM; lowerrow (left to right),
9-demethyl rhodopsin (9dR) bleaches of 0.7, 1.8, and 3.4
µM.
where S is the measured concentration of bleached
pigment, v is the measured initial rate of
[P]P
incorporation, v
is the maximal value of v, and K
is a binding constant (curves in Fig. 3). For the rhodopsin data, the fitting of this equation
yielded v
= 60 nmol/liter/min and K
= 0.20 µM; for the 9dR data,
the fit yielded v
= 15 nmol/liter/min and K
= 0.61 µM. The apparent
maximal rate of 9dR* phosphorylation is thus about 25% of that for the
native photoactivated pigment under the present experimental
conditions. The determined values of K
furthermore
indicate that the strength of the interaction between kinase and 9dR*
is considerably weaker than that between kinase and photoactivated
rhodopsin.
Light-dependent phosphorylation of 9dR and rhodopsin was
also examined using a partially purified preparation of rhodopsin
kinase, as described under ``Experimental Procedures.''
Conditions used in these experiments for light/dark incubation of the
final reaction mixtures and procedures for bleaching determinations
differed from those described above (see Fig. 4legend). Shown
in Fig. 4are levels of [P]P
incorporation for rhodopsin and 9dR samples (filled and opencircles, respectively) measured after a 10-min
incubation period that included a bleaching exposure; the abscissa value of each data point indicates the spectrophotometrically
determined extent of bleaching. The general pattern of the Fig. 4data is similar to that seen in the experiment of Fig. 3, which involved flash bleaching and 30-s dark incubation.
That is, in Fig. 4, [
P]P
incorporation by 9dR exhibits a near plateau over the range of
0.17-0.55 nmol of 9dR* (12 samples). Moreover, the average
incorporation represented by these 12 samples, 34.5
10
± 7.1
10
densitometric units (mean
± S.D.), is significantly less than that measured in the
rhodopsin samples over a comparable bleaching range (101.8
10
± 6.4
10
densitometric units
for the 8 samples spanning 0.14-0.60 nmol of R*).
Figure 4:
Bleaching dependence of
[P]P
incorporation in rhodopsin and
9dR samples incubated with partially purified rhodopsin kinase (see
``Experimental Procedures''). Each reaction mixture initially
contained 1 nmol of rhodopsin or 9dR in a total volume of 55 µl.
Each sample received 5 µl of 1 mM [
-
P]ATP; 10 s later, phosphorylation
was initiated by a period of illumination (0-300 s). The light
source was a tungsten-halogen lamp fitted with an infrared cut-off
filter and a broad-band gelatin filter (peak transmittance near 520
nm). Immediately after illumination, an aliquot of the reaction mixture
(30 µl) was removed for spectrophotometric analysis of remaining
bleachable pigment and, thus, of bleaching extent. The remainder of the
sample was incubated in darkness. 10 min after the beginning of
illumination, the phosphorylation reaction was quenched by the addition
of SDS-containing buffer. [
P]P
incorporation was determined by SDS-polyacrylamide gel
electrophoresis and autoradiography and is expressed here in units of
integrated density (D.U.) of the opsin monomer band. Ordinate
values of the data points indicate the mean of results obtained from
duplicate samples. Errorbars represent the range of
the two determinations; this range was in some cases within the
dimension of the data point.
The results show that in bovine ROS preparations, conditions that support the phosphorylation of photoactivated rhodopsin also support the phosphorylation of illuminated 9-demethyl rhodopsin. The level of 9dR* phosphorylation increases with both incubation period (Fig. 2) and bleaching extent (Fig. 3Fig. 4). However, by comparison with phosphorylation levels in the rhodopsin samples, those in the samples of 9dR are lower under all conditions examined.
What property of 9dR underlies its low activity in
light-dependent phosphorylation? The facts that metarhodopsin II is the
principal substrate of rhodopsin kinase and that a metarhodopsin
II-like conformation is not observed spectrally upon illumination of
9dR (14) imply that the low extent of phosphorylation of 9dR*
is linked with the absence of a species that resembles metarhodopsin
II. Precisely how conformational properties of 9dR* affect the course
of the phosphorylation reaction is not resolved by the present study.
The complex formed by 9dR* and rhodopsin kinase may exhibit reduced
phosphorylation rate; alternatively, the 9dR* may exhibit an altered
interaction with the kinase. Some information on this issue comes from
the present Fig. 3data, which indicate a remarkably weak
dependence of [P]P
incorporation
rate on bleaching extent over the range of
0.7-3.0
µM 9dR*. This weak dependence appears not to reflect
either the saturation of kinase activity or the exhaustion of ATP,
based on the high levels of [
P]P
incorporation observed in the rhodopsin controls. The
near-plateau character of the rate data obtained over this range of 9dR
bleaching appears, rather, to reflect a low maximal velocity of
phosphorylation of 9dR*. One possibility consistent with this finding
and with the relatively low phosphorylation of the complex formed
between opsin and all-trans-9-demethylretinal (26) is
that 9dR* forms a non-productive complex with rhodopsin kinase and
thereby reduces maximal kinase activity. Such a possibility is
compatible with the evidence that the binding of kinase by illuminated
rhodopsin does not strictly depend on attainment of the metarhodopsin
II conformation (27, 28, 29) .
Available
information on the properties of 9dR within intact rods comes from
electrophysiological experiments on rods of the
salamander(15, 16) . It was not possible in the
present study to investigate phosphorylation in salamander ROS
preparations. However, both biochemical and electrophysiological
studies suggest a generally similar operation of phototransduction
reactions in amphibian and mammalian rods, including that of R*
phosphorylation(1, 30, 31, 32, 33) .
Furthermore, the deactivation of R*, i.e. the process thought
to be mediated by R* phosphorylation and arrestin binding (2, 3, 4, 5, 6, 7, 8, 9, 10, 11) ,
proceeds with apparently similar kinetics in bovine and salamander rods
at similar temperature. That is, light-scattering data obtained from
intact rods of the bovine retina following flash illumination
(fractional bleaches of <10) imply an
exponential, i.e. first order, decline of R* with a time
constant of 3-5 s(34, 35, 36) ; in
salamander rods, the decline of R* inferred from electrophysiological
data is also exponential, with a time constant of about 2
s(16, 36) . Moreover, the finding that 9dR* in
salamander rods mediates quantal photocurrent responses of low peak
amplitude (15) is compatible with the observation, in bovine
ROS membranes, that 9dR* exhibits a low efficiency of transducin
activation (14) . Based on these considerations, the present
data appear consistent with the notion that the abnormally long 9dR*
lifetime determined electrophysiologically in salamander rods (16) reflects, at least in part, a reduced efficiency of
phosphorylation of the illuminated pigment.