- Email: [email protected]
Characterization of the light-induced increase in the Michaelis constant of the cGMP phosphodiesterase in frog rod outer segments
Biochimica et Biophysica Acta 870 (1986) 256-266
256
Elsevier BBA 32479
C h a r a c t e r i z a t i o n o f t h e light-induced i n c r e a s e in t h e M i c h a e l i s c o n s t a n t o f the c G M P p h o s p h o d i e s t e r a s e in f r o g r o d o u t e r s e g m e n t s Satoru Kawamura * and Motohiko Murakami Department of Physiology, Keio University School of Medicine, Shinjuku, Tokyo 160 (Japan) (Received October 23rd, 1985)
Key words: Phosphodiesterase; Michaelis constant; Light-induced K m increase; Km-increase factor; Phototransduction; (Frog photoreceptor disk membrane)
Activation of cGMP phosphodiesterase in rod disk membrane in the light is thought to be an intermediary process of phototransduction. In various preparations of frog rod outer segments, the Michaelis constant (K~,) of the phosphodiesterase was measured with pH assay method. On illumination, the K m increased from the value of the dark (130 pM) by about 8-fold (1 mM) in crude preparations, but did not change appreciably in purified disk membranes, confirming the previous finding by Robinson et al. (Robinson, P.R., Kawamura, S., Abramson, B. and Bownds, M.D. (1980) J. Gen. Physiol. 76, 631-645). The present work further showed that the light-induced K a increase is labile to various experimental manipulations such as sonication, freeze-thawing, etc. However, the K m in the light was relatively high in a crude disk membrane preparation and in a lyzed preparation. In addition, reconstitution experiments revealed that the K a increase does not require soluble components. Both proteolytic digestion and phospholipase treatment reduced the light K a of the phosphodiesterase in crude disk membranes. The above results suggest that there is a labile factor(s) responsible for the light-induced K a increase and that the factor is a membrane-bound protein and requires structural integrity of the disk membrane to exert its function. The latency of the K a increase after light stimulation was less than 2 s.
Introduction
is about 100 #M [1-3]. However, the values of the in the light were dependent on the type of the preparation: in purified disk membranes, the K m was little influenced by illumination [2,3], while it increased about 10-fold (1 mM) in crude rod outer segments [1]. Since the phosphodiesterase activity in vivo should be determined by both the maximal enzyme activity (Vma~) and the Kin, the light-induced K m increase must have an important physiological role in in vivo cGMP metabolism. The purpose of the present experiment has been to contribute to the study of phototransduction a n d / o r adaptation mechanisms of the rod photoreceptors by providing some basic information about the characteristics of the light-induced K m increase. The phosphodiesterase activity was deKm
On exposure to light, cGMP phosphodiesterase in the disk membrane of rod outer segment is activated [1-4] through several steps of reactions (see Refs. 5 and 6 for review). This activation possibly leads to the reduction of sodium conductance of the plasma membrane by reducing internal cGMP concentration [7-10]. The Michaelis constant of the phosphodiesterase has been measured by many investigators and it is generally agreed that the K m in the dark * To whom correspondence should be addressed. Abbreviation: Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.
0167-4838/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
257
termined from pH decrease caused by hydrolysis of cGMP (pH assay method) [3,4,11]. Because the light-induced K m increase has been demonstrated only in one report [1], we first tried to confirm it in crude preparation of frog rod outer segments. It was then suggested that the light-induced K m increase could be explained by assuming the presence of a protein factor(s) which is labile to various manipulations usually used in the biochemical experiment of the phosphodiesterase. The loss of the activity of the factor during the purification process seemed to be caused by denaturation of the factor a n d / o r changes in structural organization of the disk membranes. Materials and Methods
Retinas were obtained from dark-adapted bullfrogs (Rana catesbeiana). In the present study, after isolation from retina, rod outer segments were used without further purification, and in addition to this, they were made permeable or reduced into disk membranes so that cGMP in the external solution could have access to the phosphodiesterase residing in the disk membrane. Therefore, a retina was immersed in 2 ml of a low-sodium, low-calcium solution (110 mM KC1/ 10 mM NaC1/2 mM MgC12/0.1 mM CAC12/2.78 mM EGTA/10 mM Hepes/1 mM dithiothreitol, (pH 7.8)) which simulated the intracellular medium. Then, in order to detach the rod outer segments, the retina was gently sucked into, and ejected from a 1-ml pipet tip, the opening of which had been cut by a razor blade and made 3-4 mm in diameter. This manipulation was repeated about 50 times. After a brief centrifugation (5 × g, 5 s), the supernatant containing rod outer segments was isolated. Similarly prepared outer segments were all shown to be permeable, since they were stained by a fluorescent probe [1]. Hereafter, this permeabilized preparation is given the term 'crude rod outer segments'. It has been reported that Vm~x of the phosphodiesterase in the crude outer segments gradually increases in the dark for 2 h after their isolation until it reaches a steady value [4]. For quantitative measurement of the phosphodiesterase activity, Vm~ had to be the same during the experiment. Therefore, the crude rod outer segments
were kept at room temperature at least for 3 h or stored overnight at 4°C before use. Light microscopic observation revealed that some of the rod outer segments retained their intact structure, but most of the rod outer segments were reduced into disk membranes in these preparations. Therefore, these preparations are hereafter called crude disk membranes. Though the light-induced K m increase was originally observed in crude rod outer segments [1], we realized during the course of the experiment that it occurs also in crude disk membranes. Since this preparation had an advantage over crude rod outer segments in the constancy of Vm~x,we used mainly crude disk membranes in the present study, unless otherwise stated. In some experiments, however, crude disk membranes were purified with the sucrose floatation method [3] using 38% (w/w) sucrose (purified disk membranes). For each measurement of the phosphodiesterase activity, a 200-#1 portion of a suspension of crude disk membranes was made 0.5 mM in GTP and 0.5 mM in ATP by adding 5 #1 of a mixture of 20 mM GTP and 20 mM ATP. The suspension was continuously stirred and was supplemented with cGMP [4]. All the above manipulations were carfled out in the dark with the aid of an infrared image converter (NVR 2015, NEC, Tokyo). When continuous light stimulation was given, the pH of the solution decreased, because hydrolysis of cGMP accompanied proton liberation [3]. The pH decrease was monitored with a pH electrode (MI410, Microelectrodes Inc., Londonderry, NH) and displayed on a pen chart recorder. Time resolution of the measurement was about 2 s. The amount of liberated protons was calibrated by back-titrating the suspension with known amounts of NaOH. The phosphodiesterase activity was calculated from a tangent of the pH trace and expressed as mol of cGMP hydrolyzed per mol of rhodopsin present per min (cGMP/rh per rain). In the working pH range of the present study (pH 7.4-7.8), the pH change was almost proportional to the concentration change of cGMP (for details, see Fig. 1 in Ref. 4). Rhodopsin concentration was 10-20 /~M. Light source was a 200 W tungsten lamp equipped with a cut-off filter passing light longer than 460 nm. The light bleached 3.7.107 rhodopsin molecules/outer segment per s.
258 c G M P , cAMP, ATP, GTP, polyphosphoinositides, phospholipases, trypsin, chymotrypsin and trypsin inhibitor were supplied by Sigma (St. Louis, MO); leupeptin was from Peptide Institute, Inc. (Osaka, Japan) and the other chemicals from Nakarai (Kyoto, Japan). Sucrose laurylmonoester was a generous gift from Dr. N. Kawase at R y o t o Co. (Tokyo).
Results
Measurement of kinetic parameters in the light with the initial velocity method Since the light-induced K m increase has been reported only in one report [1], first of all, we measured the K m of the phosphodiesterase in the light in crude disk membranes. Conventionally, the kinetic parameters are determined from the measurement of initial velocities of product formation at different substrate concentrations [1-3]. Therefore, we added c G M P in the dark to five portions of crude disk m e m b r a n e suspensions to yield five different concentrations of c G M P . In each portion, phosphodiesterase activation was initiated by light stimulation, as indicated by a d o w n w a r d arrow (Fig. la) and the p H decrease was monitored. In Fig. la, five recordings are superimposed. F r o m these recordings, the initial velocities of the phosphodiesterase activity in the light were determined about 2 s after the onset of the light stimulation at five substrate concentrations. The Lineweaver-Burk plot of the phosphodiesterase light activity gave Vmax = 830 c G M P / r h per min and g m = 710 # M (initial velocity method, closed circles and dotted line in Fig. lc). The dark K m w a s also determined (data not shown) and was 130 ~tM, in agreement with reported values [1-3]. Therefore, the above experiments confirmed the previous finding that there is a light-induced K m increase in a crude preparation [11. Phosphodiesterase light activities are usually calculated by subtracting dark activities [1-4]. However, this subtraction is reasonable only when the dark activity derives from an enzyme different from that activated in the light. Instead, we assumed that, in our preparation, there was only one kind of phosphodiesterase of which Vmax and K m were increased in the light. In this case, the dark
activity cannot be separated from the light activity. This assumption seemed to be reasonable, because the rate of c G M P hydrolysis in the dark was low (for example, see the p H recordings in Fig. la). The light-induced K m increase might take place on the phosphodiesterase which is activated in the dark with some treatment, for example, by proteinases (Fig. 6). In this case also, the dark activity cannot be separated from the light activity. For these reasons, the dark activity
• b•y
lmM cGMP
, 30sec
]
--
O
-.~
0 tl.
"
,/
_~~.r
"'"
"00 2 .
i
-2
Z
~"
-1
0
I/cGMP
i
I
|
1
2
3
(mM} -1
Fig. 1. Measurement of the K m of the phosphodiesterase with the pH assay method, (a) Initial velocity method. The pH decrease (upward shift) caused by cGMP hydrolysis was recorded in a suspension of crude disk membranes in the presence of 0.493, 0.833, 1.38, 1.82 and 2.67 mM cGMP (from bottom to top) by initiating the reaction with continuous illumination (downward arrow). Five recordings are superimposed. (b) Continuous recording method. The pH decrease was continuosuly measured until the pH change ceased and all the added cGMP (2.67 mM) was hydrolyzed. (c) Lineweaver-Burk plots of the reactions shown in (a) and (b). The initial phosphodiesterase activity was calculated at each cGMP concentration from the trace in (a) (O) or from the data obtained at 15 s (©), 35 s (A) and 65 s (D) after the onset of light illumination (see text). The phosphodiesterase activities in (b) (×) were calculated from tangents of the pH trace at the points indicated by upward arrows (remaining cGMP concentrations: 1.05, 0.700 and 0.351 mM from bottom to top). Assuming linear relation, best-fit lines were determined. Calculated kinetic parameters are given in the text.
259 was not subtracted from the light activity in the present work. The light-induced K m increase might be timedependent. To test this possibility, known amounts of cGMP were added to five portions of crude disk membranes in a similar way as in Fig. la, but at a fixed illumination period (original recordings not shown). The activities measured at 15 s (open circles in Fig. lc), 35 s (triangles) and 65 s (squares) after the stimulation seemed to be indistinguishable. When those data were combined for the determination of the kinetic parameters, the Lineweaver-Burk plot gave Vmax = 710 c G M P / r h per rain and K m = 740 #M (Fig. lc, straight line). The above results indicated that the Vmax in the light was 830 c G M P / r h per rain immediately after the light stimulation (dotted line), but in the light, it decreased to 710 c G M P / r h per rain (straight line), that is, about 86% of the maximum value. Similar decrease in Vm~x was observed in four separate experiments. In contrast to the decrease in Vm~x, the g m w a s constantly high during illumination. Measurement of kinetic parameters in the light with a continuous p H recording Since the pH assay method provides a real time recording of the phosphodiesterase activity [3,4], a single continuous recording of the p H could be used for determination of the kinetic parameters. In Fig. lb, 15/~1 of 40 mM c G M P was added to a portion of crude disk membrane suspension which was taken from the same source used in the experiment in Fig. la. The reaction was initiated by a continuous illumination (Fig. lb, downward arrow). The pH was allowed to decrease from pH 7.8 to 7.45 until the pH decrease ceased and all the added c G M P was hydrolyzed. At time longer than 15 s after the onset of light stimulation (upward arrows), the phosphodiesterase activities were determined from tangents of the pH recording. Since the pH change was almost proportional to the cGMP concentration change in the present study (see Materials and Methods), we calculated the remaining c G M P concentration from the pH difference between the steady final level and the point where the activity was measured. The Lineweaver-Burk plot (crosses and broken line in Fig. lc) gave Vm~x = 710 c G M P / r h per rain and g m = 790 #M. This type of measurement (continuous
recording method) also showed a high light Km, and the kinetic parameters obtained by this method were similar to those determined by the initial velocity method at 15-65 s after the light stimulation. The continuous pH recording method was more readily achieved than the initial velocity method. Furthermore, it was completed within several minutes at most. This rapidity made the kinetic measurement possible even in crude rod outer segments in which steady increase in Vm~x progressed for 2 h in the dark (see Materials and Methods). For these reasons, the continuous recording method was employed in the following study. However, it must be mentioned that we had to choose data points recorded at least 15 s after a light stimulation, since the Vmax changes at the beginning of illumination but stays at a constant level after 15 s (Fig. l a and c). The light K m value was similar in each experiment when the disk membranes were taken from the same source. However, when the source was different, it varied, probably because of the difference in experimental manipulation during preparation (see below). Light-induced K m increase is confined in disk membranes Since the light-induced K m increase was observed in a crude preparation but not in a purified preparation, one of the possible mechanisms of the light-induced K m increase is that a soluble factor(s) modifies the K m of the phosphodiesterase. Previous work [1] showed that the light-induced K m increase was observed not only in crude rod outer segments but also in a preparation washed by a modified Ringer solution. However, in that work, washing of the crude rod outer segments was brief (1200 × g, 1 rain) and the soluble fraction might not be eliminated completely. Therefore, we systematically re-examined whether a factor(s) in the soluble fraction was involved in the light-induced K m increase. As shown in Fig. 1, even in a suspension of crude disk membranes, the light K m of the phosphodiesterase was high. Therefore, if the factor responsible for the light-induced K m increase is soluble, it should be contained in the soluble fraction of the crude disk membrane suspension. To
260
test this possibility, a reconstitution experiment was performed. After addition of cGMP, the pH decrease after light stimulation was measured in a suspension of crude disk membranes (trace 1 in Fig. 2) as well as in a purified disk membrane suspension (trace 2). The Lineweaver-Burk plot showed a high light K m (1.16 raM) in crude disk membranes (open circles in Fig. 2b) and a low light K m (190 #M) in purified disk membranes (closed circles). Though the light K m of the purified disk membranes was slightly higher than the reported value (about 100 #M; Refs. 1-3), the Km value in purified disk membranes differed in different preparations. This is probably because the experimental manipulation was slightly different in each preparation (see below). The soluble fraction of crude disk membranes was prepared by centrifuging (20000 × g, 15 min) a portion of the suspension of crude disk membranes which was used in the measurement in trace 1. Then, the soluble fraction was combined with pelletted purified disk membranes so that the rhodopsin concentration in this preparation was similar to that used in the experiment of trace 2. After addition of cGMP, the pH decrease was recorded (trace 3 in Fig. 2a) and the light Km was
a
~ ~ I
mu I
1
r 6 "2
"E >--.4-
ltxj
°°TI : -
I
1 rain
j
O~
O o
2
-4
-2
12,
I
I
2
I
1/cGMP CraM)-1
Fig. 2. Indication that light-induced K m increase does not require soluble components. (a) The p H decreases after light stimulation (arrows) were continuously monitored in suspensions of crude disk membranes (trace 1), purified disk membranes (trace 2) and purified disk membranes supplemented with the soluble fraction of the crude disk membranes (trace 3). (b) Lineweaver-Burk plots of the reactions shown in (a). The phosphodiesterase activity was calculated in the same way as shown in Fig .lb and c. Open circles, closed circles and crosses were obtained from traces 1, 2 and 3, respectively. Assuming linear relation, best-fit lines were determined.
determined. The Lineweaver-Burk plot (crosses in Fig, 2b) showed that the light g m w a s low (230 /~M) in the combined preparation, indicating that the light-induced K m increase does not require soluble fractions. Then, the purified disk membranes were mixed with various supernatant or precipitate fractions which were obtained at each step of the purification process. The light K m was determined in the same way as in Fig. 2. The results showed that the value in the mixture was always low (data not shown). In the experiment in Fig. 2, the phosphodiesterase dark activity in trace 2 was higher than that in trace 1. This observation was consistent with the finding by Yee and Liebman [3] that the phosphodiesterase dark activity increases by floatation and washes. The dark activity in trace 2 was suppressed with the addition of the soluble fraction of the crude disk membranes (trace 3), suggesting the presence of the inhibitor of the phosphodiesterase [12] in the soluble fraction. It was then possible that the increase in the dark activity was caused by dilution of the inhibitor during the preparation of the purified disk membranes. Though the result in Fig. 2 left the possibility that the soluble factor was easily denatured, t h e experiment in Fig. 3 unequivocally showed that soluble factors are not involved in the light-induced K m increase. Crude disk membranes which preserved the, activity of the light-induced Km increase were combined with purified disk membranes (Fig. 3). The mixing ratio of the two types of preparations were varied, and in each mixture the kinetic parameters were determined with the continuous pH recording method. Though the preparation was a mixture of phosphodiesterases of low and high light K~, the Lineweaver-Burk plot gave a fairly straight line, and we could determine the Vm~x and apparent light K m in each mixture. In Fig. 3, the obtained light K m is plotted as a function of the content of the high light K m phosphodiesterase which was determined from the percentage of the crude disk membranes in the mixture. It can be seen from the figure that the apparent light g m in a mixture increased as the content of the high light g m phosphodiesterase increased. Since it was not known whether or not
261 this increase in the apparent light K m was due to restoration of the activity of the light-induced K m increase in the purified disk membranes, the result was theoretically analyzed. In the following analysis, the expected K m value in a mixture was calculated under the assumption that the lightinduced K m increase took place only in crude disk membranes. Then, we compared the calculated K m value with the experimental result in Fig. 3 to find out whether the assumption was reasonable or not. If low- and high-K m phosphodiesterases in a mixture contribute to the observed phosphodiesterase activity in proportion to their contents, the phosphodiesterase activity (o) in the light in the mixture should be expressed as follows: v = pV~a ~
S - -S S + K ~ + ( i - p)V'~x S + K~
(1)
where S is the concentration of cGMP, superscripts H and L designate high- and low-K m phosphodiesterases, respectively, and p is the content of the h i g h - K m phosphodiesterase. We calculated VHx and K H from the experiment done in crude disk membranes (corresponding to the data at 100% high-K m phosphodiesterase in Fig. 3), and V~Lax and K L from
/,
1.0
/
E /
i
t'
p
E
.¢-
/tt
D., ,~
0 -I
0
I 50
I
100
High K m PDE content (*/,)
Fig. 3. Kin-increasefactor is confined in the disk membrane. Purified disk membranes (containing low-Km phosphodiesterase) were mixed with crude disk membranes (cont a i n i n g h i g h - K m phosphodiesterase)with various ratios. In the same way as in Fig. lb and c, light Km was determinedin each mixture and plotted against the content of high-Kin phosphodiesterase. The broken fine shows the theoretically expected Km values which were obtained under the assumption that the light-induced K m increase took place only in crude disk membranes (see text). PDE, phosphodiesterase.
purified membranes (corresponding to the data at 0%). The obtained values were: VmHax= 1080 c G M P / r h per min, KmH = 1.08 mM, V ~ = 690 c G M P / r h per min and K L = 0 . 1 9 0 mM as an average of two determinations. Taking three cGMP concentrations (three levels of S) which were actually chosen to determine the g m values in the experiment in Fig. 3, corresponding values of v were calculated from Eqn. 1 at a fixed value of p. From the relation of v and S at the above three different substrate concentrations, K m was determined from the Lineweaver-Burk plot. The value of p was changed from zero to 1, and at each value of p, the above calculation was repeated to obtain a theoretical curve (dashed line in Fig. 3). The consistency between the experiment and the theory in Fig. 3 indicated that the high light K m phosphodiesterase and t h e low light K m phosphodiesterase were activated independently. This result showed that the light-induced K m increase is confined to the disk membrane. L i g h t - i n d u c e d K m i n c r e a s e is labile
The reduction of the light K m during purification of disk membranes suggested that the light-induced K m increase is labile. This was further demonstrated in Fig. 4, where the light K m w a s measured in various types of preparations which were usually used in biochemical experiments of rod outer segments. Crude rod outer segments were freshly prepared ('crude ros' preparation). Some of them were pelletted by centrifugation (20000 × g, 15 min). The pellet was resuspended in the low-sodium, lowcalcium solution ('washed'). Some of the washed preparation were partially purified with sucrose floatation ('purified'). Crude rod outer segments were fragmented with vigorous pipeting with a syringe (' fragmented') or sonicated for 5 s (' sonic.') or frozen overnight and then thawed (' f. thawed'). In all these preparations, the light K m values were determined and all the above manipulations were found to reduce the light K m of the phosphodiesterase (Fig. 4), though the extent of the reduction depended on the type of the manipulation. However, in crude disk membranes ('crude disk') which were obtained by storing 'crude ros' preparation overnight at 4°C, the light K m value was close to that of crude rod outer segments. The
262
1.0
T
E E v
0.5
o
l
~
~'-~
Fig. 4. Light-induced K , - i n c r e a s e is labile. Crude rod outer segments (crude ros) were washed by centrifugation (washed), partially purified with sucrose floatation (purified), fragmented by pipeting (fragmented), sonicated briefly (sonic.) freezethawed (f. thawed), kept at 4°C overnight (crude disk) or sedimented and resuspended in a hypotonic buffer (lyzed). Light K m values were determined as in Fig. l b and c from 10-20 determinations in each type of preparation. Vertical bars show standard deviations.
above result indicated that the level of the light K m of the phosphodiesterase depends on how the phosphodiesterase or the disk membrane is manipulated• The result in Fig. 4 suggested that, as degeneration of the structure of rod outer segment proceeded the light K m value decreased. In fact, under a light microscope, we observed rod structure and stacks of disks in the suspension ot~ crude rod outer segments (top panel, Fig. 5) but not in the freeze-thawed preparation (middle panel, Fig. 5). However, though the light K m of the crude disk membrane was high (Fig. 4), the main constituent of this preparation was disk membranes (see Materials and Methods). Therefore, there seemed to be no simple correlation between rod structure and the activity of the light-induced g m increase. This point was further examined in a 'lyzed' preparation which was obtained after centrifugation (500 × g, 2 min) of crude rod outer segments by resuspending the precipitate in a hypotonic buffer (10 mM H e p e s / 2 mM MgC12/0.1 mM CAC12/2.78 mM EGTA (pH 7.8)). In the 'lyzed' preparation, the rod structure and stacks of
disks both disappeared completely (bottom panel, Fig. 5), while the light K m was relatively high ('lyzed' in Fig. 4), indicating that the rod structure is not necessary to induce the light-induced K m increase. When a 'lyzed' preparation was further sedimented (500 x g, 2 rain) and resuspended gently in the hypotonic buffer either in the light or in the dark, the light K m did not decrease appreciably (data not shown). This observation strengthened the conclusion obtained in Figs. 2 and 3 that the light-induced K m increase does not require soluble fractions, because most of the peripheral proteins in the frog disk membrane are soluble and should be eliminated by washing under the condition [13]. Since a mixture of the phosphodiesterases of high K m and low K m gave an intermediate K m value (Fig. 3), the intermediate K m value in the
,t'x.-.,_,e~ all" " - , ~ ~T..-~.j.-.-~,O:
•
.',
).1~.~ •
..~',~ ~ - ~ ."..--i ~'~
~'~ ~ .• d ~.". ~ . - : a ~ l,~
•
•
":: •
'~-,
.. ~ , . ~ .,..~,,,
-r~ ~-.
,.o,.... "..
"-" :s,~.'~',,, .~',~_ ~. "~.~ ~ , I ~'"
" ...'1"
',
~.. -~,
,~
~'~ 4:'-"~ ~" ~ . : -)',~. ,' -~~ " -
,--~',v'gllz~
...,. • x~,:,-....~,~
.
.~
f ,~. .,r ~' ~ " -
..
. |,
"
•
•
: ~1~ .'~' .
(
~.~. ~,, • ~
.
",r/~'.~ .~,
"~r.~B,';. ""~-~ ,-, ~.,~....'~...¢z~ff" * ;.:..-,.,~.,~,,t,z; ,~.,~.~.
¢ g, @
Fig. 5. Morphological appearance of some of the preparations. U n d e r a light microscope, morphological appearance was observed in crude rod outer segments (top), frceze-thawed preparation (middle) and lyzed preparation (bottom). The measured K m values in the preparations shown in this figure were 1.2 mM, 250 /tM and 940 # M , from top to bottom, respectively. Magnifications are the same from top to bottom, and a calibration bar in the top panel indicates 100 #m.
263
various types of the preparation in Fig. 4 could be explained by the notion that the preparation was a mixture of high- and low-K m phosphodiesterases. By adding various chemicals to sonicated preparations, we tried to find out the condition which could restore the activity of the light-induced K m increase. However, cAMP, cGMP, polyphosphoinositides, isobuthylmethylxantine, ATP, GTP, S-adenosylmethionine, S-adenosylhomocysteine and dithiothreitol were all ineffective when these chemicals were incubated with sonicated preparations. Calcium ions rather enhanced the reduction of the light Km.
Light-induced K m increase is affected by proteinases and phospholipases To characterize the light-induced K m increase, the effects of proteinases and phospholipases were investigated (Fig. 6 and Table I). In Fig. 6a, tryp-
0.8
1000
EO.4
sin was added to a suspension of crude disk membranes to give a final concentration of 1 0 / ~ g / m l . Then, single portions were withdrawn at various time intervals and were mixed with soybean trypsin inhibitor (final concentration 0.5 m g / m l ) . In each portion, the light K m and the phosphodiesterase activity were determined and the time-courses of the changes in kinetic parameters were measured. In agreement with previous reports [12,14], trypsin digestion increased both phosphodiesterase dark activity (open triangles in Fig. 6a) and l/m,X (data not shown). In addition, trypsin reduced the light K m of the phosphodiesterase (open circles in Fig. 6a) in the crude disk membranes, suggesting that the light-induced K m increase is associated with a protein(s). Though trypsin affected both the phosphodiesterase activity and the light Km, the timecourse of reduction of the light K m was slightly faster than that of the phosphodiesterase dark activity increase. This raised the possibility that the process responsible for the reduction of the light K m is different from that for the phosphodiesterase dark activity increase. Since trypsin
500
v
TABLE I
0 -, 0
~
0.8-' b
i i
1'0 i
; i
2;0
; '/
"
'_
I 0 60 ^ L
o '-" .--
000 .~
E
** l.H
E 0.4
i00
0-,
,
0
,
2LO
~
410
T i m e of i n c u b a t i o n
a rl
0
610 (min)
Fig. 6. Proteolytic digestion reduces light Km. (a) Crude disk membranes were incubated with trypsin (final concentration; 10 /~g/ml) at 30°C for various periods and the reaction was terminated with addition of trypsin inhibitor (0.5 mg/ml). Light K m (O) and phosphodiesterase dark activity (A) were determined, and they were plotted against incubation time. Filled circle and filled triangle show light K m and phosphodiesterase dark activity, respectively, in the presence of soybean trypsin inhibitor throughout the incubation period. (b) Same as in (a), but the crude disk membranes were digested with chymotrypsin (30 /~g/ml). The reaction was terminated with the addition of soybean trypsin inhibitor (0.5 mg/ml). PDE, phosphodiesterase.
EFFECT OF PHOSPHOLIPASES ON L I G H T K m A N D PHOSPHODIESTERASE ACTIVITIES IN C R U D E DISK MEMBRANES Crude disk membranes were incubated at 30°C for 30-60 min with phospholipases A 2 (final concentration: 1.5 mg/ml), C (0.6 m g / m l ) or D (1.5 mg/ml). As a control, the outer segments were incubated at 30°C for 60 rain without enzymes. Each value shown is a mean of duplicate experiments_+ the range of the values about the mean. The experiments were performed in the presence of 1 mM calcium (see text). For comparison, the effect of trypsin is listed. PDE, phosphodiesterase. Addition
K m (/tM)
PDE activity ( c G M P / r h per min) dark
None Phospholipase A 2 Phospholipase C Phospholipase D Trypsin a
light
530 + 10 100 _+10 220 + 30 200 + 10
190_+ 20 70 + 10 130 -]- 10 100 ± 10
730 + 20 500 _+10 390 + 50 410 + 20
60 4- 2
800 4- 30
1060 + 70
a The measurement was performed in the low-sodium, lowcalcium solution.
264
cleaves the peptide bond at the carboxyl terminal of lysine or arginine, it was expected that other proteinases showed different selectivity between the reduction of the light K m and the increase in the phospliodiesterase dark activity. Chymotrypsin is known to act on the carboxyl terminal of aromatic amino acid residues. This enzyme was added to a suspension of crude disk membranes (final concentration: 30 # g / m l ) in the presence of leupeptin (10 # g / m l ) which selectively inhibits the activity of trypsin, a contaminant in the commercially available chymotrypsin (see Sigma catalog, 1985). Single portions were withdrawn at various incubation periods and mixed with soybean trypsin inhibitor (0.5 m g / m l ) which also inhibits chymotrypsin activity. The enzyme reduced the light g m of the phosphodiesterase (open circles in Fig. 6b) and increased the phosphodiesterase dark activity (open triangles in Fig. 6b). Contrary to the effect of trypsin (Fig. 6a), the time-course of the reduction of the light g m by chymotrypsin was slower than that of the dark activity increase (open triangles in Fig. 6b). Because trypsin and chymotrypsin showed different selectivity between the reduction of the light K m and the dark activity increase, it was concluded that the site of the phosphodiesterase molecule responsible for the light-induced K m increase is different from that for the activation. Filled symbols in Fig. 6a and b sh.ow the controls in which proteinase activity was inhibited throughout the incubation period. In Table I, the effects of phospholipases A 2 (from bee venom), C (from Clostridium perfringens) and D (from peanut) are summarized. Since these enzymes are active only in the presence of calcium [15,16], crude disk membranes were pelletted by centrifugation and resuspended in a solution of which calcium concentration was made 1 mM by replacing 0.1 mM CaC12 plus 2.78 mM EGTA with 1 mM CaC12. During the course of the preparation, the light K m decreased from 750 to 530 #M (control) probably because of repetitive pipeting. As shown in Table I, all the phospholipases examined in the present study reduced the light K m of the phosphodiesterase. However, in contrast to proteinases used in Fig. 6, the phospholipases reduced the light K m a s well as the dark and light activities of the phosphodiesterase
!
[J
!
!
!
,
,
I
|
,
,
i
0.8 ~
0.6
~0.4 0.2 0
. . ,
0
0.01
0.1
SLME eoncentration
1 (%}
Fig. 7. Effect of sucrose laurylmonoesteron light K m of the phosphodiesterase.The light Km in crude disk membraneswas measured in the presence of various concentrationsof sucrose laurylmonoester (SLME). Each closed circle shows a single determination. (Table I). In control experiments, the effects of these phospholipases were measured at low calcium concentration where the phospholipase activities were inhibited [15,16]. In the low-sodium, lowcalcium solution, the phospholipases affected neither the light g m nor the Irmax. For comparison, the effect of trypsin was also listed in Table I, though the trypsin digestion was carried out in the low-sodium, low-calcium solution.
Effect of detergent on light-induced K,, increase The reduction of the light K m by phospholipases (Table I) suggested the necessity of some structural organization of membrane lipid for the light-induced K m increase. To test this possibility, we examined the effect of detergent which was expected to modify the molecular environment of the phosphodiesterase and its associated molecules. Though many detergents inhibit the activation of the phosphodiesterase, sucrose laurylmonoester is known to solubilize the phosphodiesterase without affecting the phosphodiesterase activity appreciably [17]. In Fig. 7, the light K m was measured at various concentrations of sucrose laurylmonoester. The detergent reduced the light g m , and half-maximal effect was observed at about 0.03% (w/w) of sucrose laurylmonoester. Though the incubation period of the disk membranes with sucrose laurylmonoester was varied (5-30 rain), no significant effect was observed.
265 Discussion
Possible mechanisms which underlie the light-induced K m increase In the present experiment, the light-induced increase in K m was characterized. There would be at least two possible mechanisms which account for the light-induced K m increase. One mechanism assumes the presence of a factor (Km-increase factor) which increases the K m of the activated phosphodiesterase. The other assumes the contribution of the rod structure: saturation of the phosphodiesterase requires a higher concentration of cGMP in the space between stacks of disks, because a local depletion of cGMP in the space occurs more rapidly than new cGMP can diffuse in from the medium. However, the latter possibility could be excluded from the experiment showing that the light K~ still retained a relatively high livel in the 'lyzed' preparation in which the rod structure and stacks of disks both disappeared (Figs. 4 and 5). Therefore, it would be most reasonable to assume the presence of the Kin-increase factor. According to the Michaelis-Menten equation, the K m is expressed by the equation: K~ = (k_ 1 + k+2)/k+l, where k÷a, k_ 1 and k+2 are the rate constants in the reaction scheme shown in the following: k+l
k+2
E + S ~ ES ~ E+P k_j
where E is the phosphodiesterase, S is cGMP and P is 5'-GMP and H +. Therefore, the Km-increase factor increases the K m value in the light by acting on any of the rate constants in the above scheme. Further kinetic analysis is necessary to elucidate the action mechanism of the Km-increase factor.
Characteristics of the Kin-increase factor The present experiment showed that the Kin-increase factor easily loses its activity with various experimental manipulations (Fig. 4) or addition of a detergent (Fig. 7). The activity of the factor is not found in the soluble fraction (Figs. 2 and 3), but is retained under hypotonic shock (Fig. 4). Proteinases and phospholipases reduce the light g m (Fig. 6 and Table I).
A simple picture deduced from the above experiments is as follows. The Kin-increase factor is a protein (Fig. 6). The factor is membrane-bound and exerts its effect without participation of soluble components (Figs. 2 and 3). It is labile and requires structural integrity of the disk membrane to cause the light-induced K m increase. Therefore, the factor loses its activity by mechanical treatment (Fig. 4), detergent (Fig. 7) and enzymatical degradation of membrane phospholipid (Table I). Since the Kin-increase factor has not been isolated, it is not known whether or not the factor is a protein different from the phosphodiesterase molecule itself. However, the factor is not associated with the phosphodiesterase inhibitor for the following reason. One suggestion has been that the increase in phosphodiesterase dark activity caused by proteolytic digestion is due to the degradation of the phosphodiesterase inhibitor [12,18]. If the factor is asosciated with the inhibitor, the increase in the phosphodiesterase dark activity caused by proteinases should proceed in parallel with the reduction of the light Km. Apparently, this was not the case (Fig. 6). One of the possible sources of the Kin-increase factor was a contaminant from the retinal neurons other than photoreceptors. However, this possibility was excluded: if this was the case, the mixing experiment in Fig. 3 should have always yielded the high light K~, since all the factors necessary for the light-induced K m increase was in the fraction of crude disk membranes.
Physiological function of the light-induced K m increase In vivo phosphodiesterase activity (v) would be determined by both the maximal velocity, Vmax, and the K m in the Miachaelis-Menten equation: s O=Vm~,S+Km
(2)
where S is the cytoplasmic concentration of cGMP. The effect of the light-induced K m increase should be reflected in the second term of Eqn. 2, that is, S / ( S + Kin). Though the cytoplasmic cGMP concentration in intact frog rod outer segments has not been directly measured, it is estimated to be 60-80 #M [19,20] or possibly about 6 gM [10]. Assuming these values and taking 100/~M and 1
266 m M for the K m in the dark and light, respectively, the effect of light-induced K m increase was calculated. The result showed that the second term decreases to 11-17% of the value in the dark. Therefore, the light-induced K m increase modulates the in vivo phosphodiesterase activity by reducing the effect of the increase in Vmax which is caused by light illumination. One of the possible ways by which the Km-increase factor exerts its physiological effect is to increase the K m in a time-dependent manner. If the K m increase takes place gradually after light stimulation, it means that the phosphodiesterase activity in vivo once becomes high on exposure to light and then gradually decreases during light stimulation. This decrease could be one of the mechanisms of the light adaptation of the rod photoreceptors. However, as shown in Fig. 1, the light K m appears to be constant from the beginning of the activation at least within the time resolution of the present p H assay method (about 2 s). Obviously, detailed experiment with faster time resolution is necessary. Besides the light-induced K m increase, it is noteworthy that V~oaxof the phosphodiesterase decreases when crude disk membranes were continuously exposed to light (Fig. 1). T h o u g h the details are not k n o w n yet, the result suggests the presence of some inhibitory mechanism of the phosphodiesterase activation after light exposure. It is possible that this time-dependent decrease in Vmax has some role in the desensitization of the photoreceptor during light adaptation.
Acknowledgements This work was supported by a Grants-in-Aid from the Ministry of Education, Science and Culture of Japan (No. 59770103) to S.K. The authors
thank Professor Y. Tsukada and Dr. S. Kohsaka for use of laboratory equipment and for helpful discussion.
References 1 Robinson, P.R., Kawamura, S., Abramson, B. and Bownds, M.D. (1980) J. Gen. Physiol. 76, 631-645 2 Miki, N., Keirns, J.J., Marks, F.R., Freeman, J, and Bitensky, M.W. (1973) Proc. Natl. Acad. Sci. USA 70, 3820-3824 3 Yee, R. and Liebman, P.A. (1978) J. Biol. Chem. 253, 8902-8909 4 Kawamura, S. and Bownds, M.D. (1981) J. Gen. Physiol. 77, 571-591 5 Stryer, L. (1983) Cold Spring Harbor Symla. Quant. Biol. Vol. 48, 841-852 6 Yamazaki, A., Uchida, S., Stein, P.J., Wheeler, G.L. and Bitensky, M.W. (1984) Adv. Cyclic Nucleotide Prot. Phospho. Res. 16, 381-392 7 Miller, W.H. and Nicol, G.D. (1979) Nature (Lond.) 280, 64-66 8 Kawamura, S. and Murakami, M. (1983) Jpn. J. Physiol. 33, 789-800 9 Fesenko, E.E., Kolensnikov, S.S. and Lyubarsky, A.L. (1985) Nature (Lond.) 313, 310-313 10 Yau, K.-W. and Nakatani, K. (1985) Nature (Lond.) 317, 252-255 11 Kawamura, S. (1983) Biochim. Biophys. Acta 732, 276-281 12 Hurley, J.B. and Stryer, L. (1982) J. Biol. Chem. 257, 11094-11099 13 Kuhn, H. (1980) Nature (Lond.) 283, 587-589 14 Miki, N., Baraban, J.M., Keirns, J.J., Boyce, J.J. and Bitensky, M.W. (1975) Biochemistry 250, 6320-6327 15 Munjal, D. and Elliot, W.B. (1971) Toxicon 9, 403-409 16 Yamakawa, Y. and Ohsaka, A. (1977) J. Biochem. 81, 115-126 17 Fukada, Y,, Kawamura, S., Yoshizawa, T. and Miki, N. (1981) Biochim. Biophys. Acta 675, 188-194 18 Yamazaki, A., Stein, P., Chernoff, N. and Bitensky, M.W. (1983) J. Biol. Chem. 258, 8188-8194 19 Woodruff, M.L. and Fain, G. (1982) J. Gen. Physiol. 80, 533-555 20 Cote, R.H., Biernbaum, M.S., Nicol, G.D. and Bownds, M.D. (1984) J. Biol. Chem. 259, 9635-9641
256
Elsevier BBA 32479
C h a r a c t e r i z a t i o n o f t h e light-induced i n c r e a s e in t h e M i c h a e l i s c o n s t a n t o f the c G M P p h o s p h o d i e s t e r a s e in f r o g r o d o u t e r s e g m e n t s Satoru Kawamura * and Motohiko Murakami Department of Physiology, Keio University School of Medicine, Shinjuku, Tokyo 160 (Japan) (Received October 23rd, 1985)
Key words: Phosphodiesterase; Michaelis constant; Light-induced K m increase; Km-increase factor; Phototransduction; (Frog photoreceptor disk membrane)
Activation of cGMP phosphodiesterase in rod disk membrane in the light is thought to be an intermediary process of phototransduction. In various preparations of frog rod outer segments, the Michaelis constant (K~,) of the phosphodiesterase was measured with pH assay method. On illumination, the K m increased from the value of the dark (130 pM) by about 8-fold (1 mM) in crude preparations, but did not change appreciably in purified disk membranes, confirming the previous finding by Robinson et al. (Robinson, P.R., Kawamura, S., Abramson, B. and Bownds, M.D. (1980) J. Gen. Physiol. 76, 631-645). The present work further showed that the light-induced K a increase is labile to various experimental manipulations such as sonication, freeze-thawing, etc. However, the K m in the light was relatively high in a crude disk membrane preparation and in a lyzed preparation. In addition, reconstitution experiments revealed that the K a increase does not require soluble components. Both proteolytic digestion and phospholipase treatment reduced the light K a of the phosphodiesterase in crude disk membranes. The above results suggest that there is a labile factor(s) responsible for the light-induced K a increase and that the factor is a membrane-bound protein and requires structural integrity of the disk membrane to exert its function. The latency of the K a increase after light stimulation was less than 2 s.
Introduction
is about 100 #M [1-3]. However, the values of the in the light were dependent on the type of the preparation: in purified disk membranes, the K m was little influenced by illumination [2,3], while it increased about 10-fold (1 mM) in crude rod outer segments [1]. Since the phosphodiesterase activity in vivo should be determined by both the maximal enzyme activity (Vma~) and the Kin, the light-induced K m increase must have an important physiological role in in vivo cGMP metabolism. The purpose of the present experiment has been to contribute to the study of phototransduction a n d / o r adaptation mechanisms of the rod photoreceptors by providing some basic information about the characteristics of the light-induced K m increase. The phosphodiesterase activity was deKm
On exposure to light, cGMP phosphodiesterase in the disk membrane of rod outer segment is activated [1-4] through several steps of reactions (see Refs. 5 and 6 for review). This activation possibly leads to the reduction of sodium conductance of the plasma membrane by reducing internal cGMP concentration [7-10]. The Michaelis constant of the phosphodiesterase has been measured by many investigators and it is generally agreed that the K m in the dark * To whom correspondence should be addressed. Abbreviation: Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.
0167-4838/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
257
termined from pH decrease caused by hydrolysis of cGMP (pH assay method) [3,4,11]. Because the light-induced K m increase has been demonstrated only in one report [1], we first tried to confirm it in crude preparation of frog rod outer segments. It was then suggested that the light-induced K m increase could be explained by assuming the presence of a protein factor(s) which is labile to various manipulations usually used in the biochemical experiment of the phosphodiesterase. The loss of the activity of the factor during the purification process seemed to be caused by denaturation of the factor a n d / o r changes in structural organization of the disk membranes. Materials and Methods
Retinas were obtained from dark-adapted bullfrogs (Rana catesbeiana). In the present study, after isolation from retina, rod outer segments were used without further purification, and in addition to this, they were made permeable or reduced into disk membranes so that cGMP in the external solution could have access to the phosphodiesterase residing in the disk membrane. Therefore, a retina was immersed in 2 ml of a low-sodium, low-calcium solution (110 mM KC1/ 10 mM NaC1/2 mM MgC12/0.1 mM CAC12/2.78 mM EGTA/10 mM Hepes/1 mM dithiothreitol, (pH 7.8)) which simulated the intracellular medium. Then, in order to detach the rod outer segments, the retina was gently sucked into, and ejected from a 1-ml pipet tip, the opening of which had been cut by a razor blade and made 3-4 mm in diameter. This manipulation was repeated about 50 times. After a brief centrifugation (5 × g, 5 s), the supernatant containing rod outer segments was isolated. Similarly prepared outer segments were all shown to be permeable, since they were stained by a fluorescent probe [1]. Hereafter, this permeabilized preparation is given the term 'crude rod outer segments'. It has been reported that Vm~x of the phosphodiesterase in the crude outer segments gradually increases in the dark for 2 h after their isolation until it reaches a steady value [4]. For quantitative measurement of the phosphodiesterase activity, Vm~ had to be the same during the experiment. Therefore, the crude rod outer segments
were kept at room temperature at least for 3 h or stored overnight at 4°C before use. Light microscopic observation revealed that some of the rod outer segments retained their intact structure, but most of the rod outer segments were reduced into disk membranes in these preparations. Therefore, these preparations are hereafter called crude disk membranes. Though the light-induced K m increase was originally observed in crude rod outer segments [1], we realized during the course of the experiment that it occurs also in crude disk membranes. Since this preparation had an advantage over crude rod outer segments in the constancy of Vm~x,we used mainly crude disk membranes in the present study, unless otherwise stated. In some experiments, however, crude disk membranes were purified with the sucrose floatation method [3] using 38% (w/w) sucrose (purified disk membranes). For each measurement of the phosphodiesterase activity, a 200-#1 portion of a suspension of crude disk membranes was made 0.5 mM in GTP and 0.5 mM in ATP by adding 5 #1 of a mixture of 20 mM GTP and 20 mM ATP. The suspension was continuously stirred and was supplemented with cGMP [4]. All the above manipulations were carfled out in the dark with the aid of an infrared image converter (NVR 2015, NEC, Tokyo). When continuous light stimulation was given, the pH of the solution decreased, because hydrolysis of cGMP accompanied proton liberation [3]. The pH decrease was monitored with a pH electrode (MI410, Microelectrodes Inc., Londonderry, NH) and displayed on a pen chart recorder. Time resolution of the measurement was about 2 s. The amount of liberated protons was calibrated by back-titrating the suspension with known amounts of NaOH. The phosphodiesterase activity was calculated from a tangent of the pH trace and expressed as mol of cGMP hydrolyzed per mol of rhodopsin present per min (cGMP/rh per rain). In the working pH range of the present study (pH 7.4-7.8), the pH change was almost proportional to the concentration change of cGMP (for details, see Fig. 1 in Ref. 4). Rhodopsin concentration was 10-20 /~M. Light source was a 200 W tungsten lamp equipped with a cut-off filter passing light longer than 460 nm. The light bleached 3.7.107 rhodopsin molecules/outer segment per s.
258 c G M P , cAMP, ATP, GTP, polyphosphoinositides, phospholipases, trypsin, chymotrypsin and trypsin inhibitor were supplied by Sigma (St. Louis, MO); leupeptin was from Peptide Institute, Inc. (Osaka, Japan) and the other chemicals from Nakarai (Kyoto, Japan). Sucrose laurylmonoester was a generous gift from Dr. N. Kawase at R y o t o Co. (Tokyo).
Results
Measurement of kinetic parameters in the light with the initial velocity method Since the light-induced K m increase has been reported only in one report [1], first of all, we measured the K m of the phosphodiesterase in the light in crude disk membranes. Conventionally, the kinetic parameters are determined from the measurement of initial velocities of product formation at different substrate concentrations [1-3]. Therefore, we added c G M P in the dark to five portions of crude disk m e m b r a n e suspensions to yield five different concentrations of c G M P . In each portion, phosphodiesterase activation was initiated by light stimulation, as indicated by a d o w n w a r d arrow (Fig. la) and the p H decrease was monitored. In Fig. la, five recordings are superimposed. F r o m these recordings, the initial velocities of the phosphodiesterase activity in the light were determined about 2 s after the onset of the light stimulation at five substrate concentrations. The Lineweaver-Burk plot of the phosphodiesterase light activity gave Vmax = 830 c G M P / r h per min and g m = 710 # M (initial velocity method, closed circles and dotted line in Fig. lc). The dark K m w a s also determined (data not shown) and was 130 ~tM, in agreement with reported values [1-3]. Therefore, the above experiments confirmed the previous finding that there is a light-induced K m increase in a crude preparation [11. Phosphodiesterase light activities are usually calculated by subtracting dark activities [1-4]. However, this subtraction is reasonable only when the dark activity derives from an enzyme different from that activated in the light. Instead, we assumed that, in our preparation, there was only one kind of phosphodiesterase of which Vmax and K m were increased in the light. In this case, the dark
activity cannot be separated from the light activity. This assumption seemed to be reasonable, because the rate of c G M P hydrolysis in the dark was low (for example, see the p H recordings in Fig. la). The light-induced K m increase might take place on the phosphodiesterase which is activated in the dark with some treatment, for example, by proteinases (Fig. 6). In this case also, the dark activity cannot be separated from the light activity. For these reasons, the dark activity
• b•y
lmM cGMP
, 30sec
]
--
O
-.~
0 tl.
"
,/
_~~.r
"'"
"00 2 .
i
-2
Z
~"
-1
0
I/cGMP
i
I
|
1
2
3
(mM} -1
Fig. 1. Measurement of the K m of the phosphodiesterase with the pH assay method, (a) Initial velocity method. The pH decrease (upward shift) caused by cGMP hydrolysis was recorded in a suspension of crude disk membranes in the presence of 0.493, 0.833, 1.38, 1.82 and 2.67 mM cGMP (from bottom to top) by initiating the reaction with continuous illumination (downward arrow). Five recordings are superimposed. (b) Continuous recording method. The pH decrease was continuosuly measured until the pH change ceased and all the added cGMP (2.67 mM) was hydrolyzed. (c) Lineweaver-Burk plots of the reactions shown in (a) and (b). The initial phosphodiesterase activity was calculated at each cGMP concentration from the trace in (a) (O) or from the data obtained at 15 s (©), 35 s (A) and 65 s (D) after the onset of light illumination (see text). The phosphodiesterase activities in (b) (×) were calculated from tangents of the pH trace at the points indicated by upward arrows (remaining cGMP concentrations: 1.05, 0.700 and 0.351 mM from bottom to top). Assuming linear relation, best-fit lines were determined. Calculated kinetic parameters are given in the text.
259 was not subtracted from the light activity in the present work. The light-induced K m increase might be timedependent. To test this possibility, known amounts of cGMP were added to five portions of crude disk membranes in a similar way as in Fig. la, but at a fixed illumination period (original recordings not shown). The activities measured at 15 s (open circles in Fig. lc), 35 s (triangles) and 65 s (squares) after the stimulation seemed to be indistinguishable. When those data were combined for the determination of the kinetic parameters, the Lineweaver-Burk plot gave Vmax = 710 c G M P / r h per rain and K m = 740 #M (Fig. lc, straight line). The above results indicated that the Vmax in the light was 830 c G M P / r h per rain immediately after the light stimulation (dotted line), but in the light, it decreased to 710 c G M P / r h per rain (straight line), that is, about 86% of the maximum value. Similar decrease in Vm~x was observed in four separate experiments. In contrast to the decrease in Vm~x, the g m w a s constantly high during illumination. Measurement of kinetic parameters in the light with a continuous p H recording Since the pH assay method provides a real time recording of the phosphodiesterase activity [3,4], a single continuous recording of the p H could be used for determination of the kinetic parameters. In Fig. lb, 15/~1 of 40 mM c G M P was added to a portion of crude disk membrane suspension which was taken from the same source used in the experiment in Fig. la. The reaction was initiated by a continuous illumination (Fig. lb, downward arrow). The pH was allowed to decrease from pH 7.8 to 7.45 until the pH decrease ceased and all the added c G M P was hydrolyzed. At time longer than 15 s after the onset of light stimulation (upward arrows), the phosphodiesterase activities were determined from tangents of the pH recording. Since the pH change was almost proportional to the cGMP concentration change in the present study (see Materials and Methods), we calculated the remaining c G M P concentration from the pH difference between the steady final level and the point where the activity was measured. The Lineweaver-Burk plot (crosses and broken line in Fig. lc) gave Vm~x = 710 c G M P / r h per rain and g m = 790 #M. This type of measurement (continuous
recording method) also showed a high light Km, and the kinetic parameters obtained by this method were similar to those determined by the initial velocity method at 15-65 s after the light stimulation. The continuous pH recording method was more readily achieved than the initial velocity method. Furthermore, it was completed within several minutes at most. This rapidity made the kinetic measurement possible even in crude rod outer segments in which steady increase in Vm~x progressed for 2 h in the dark (see Materials and Methods). For these reasons, the continuous recording method was employed in the following study. However, it must be mentioned that we had to choose data points recorded at least 15 s after a light stimulation, since the Vmax changes at the beginning of illumination but stays at a constant level after 15 s (Fig. l a and c). The light K m value was similar in each experiment when the disk membranes were taken from the same source. However, when the source was different, it varied, probably because of the difference in experimental manipulation during preparation (see below). Light-induced K m increase is confined in disk membranes Since the light-induced K m increase was observed in a crude preparation but not in a purified preparation, one of the possible mechanisms of the light-induced K m increase is that a soluble factor(s) modifies the K m of the phosphodiesterase. Previous work [1] showed that the light-induced K m increase was observed not only in crude rod outer segments but also in a preparation washed by a modified Ringer solution. However, in that work, washing of the crude rod outer segments was brief (1200 × g, 1 rain) and the soluble fraction might not be eliminated completely. Therefore, we systematically re-examined whether a factor(s) in the soluble fraction was involved in the light-induced K m increase. As shown in Fig. 1, even in a suspension of crude disk membranes, the light K m of the phosphodiesterase was high. Therefore, if the factor responsible for the light-induced K m increase is soluble, it should be contained in the soluble fraction of the crude disk membrane suspension. To
260
test this possibility, a reconstitution experiment was performed. After addition of cGMP, the pH decrease after light stimulation was measured in a suspension of crude disk membranes (trace 1 in Fig. 2) as well as in a purified disk membrane suspension (trace 2). The Lineweaver-Burk plot showed a high light K m (1.16 raM) in crude disk membranes (open circles in Fig. 2b) and a low light K m (190 #M) in purified disk membranes (closed circles). Though the light K m of the purified disk membranes was slightly higher than the reported value (about 100 #M; Refs. 1-3), the Km value in purified disk membranes differed in different preparations. This is probably because the experimental manipulation was slightly different in each preparation (see below). The soluble fraction of crude disk membranes was prepared by centrifuging (20000 × g, 15 min) a portion of the suspension of crude disk membranes which was used in the measurement in trace 1. Then, the soluble fraction was combined with pelletted purified disk membranes so that the rhodopsin concentration in this preparation was similar to that used in the experiment of trace 2. After addition of cGMP, the pH decrease was recorded (trace 3 in Fig. 2a) and the light Km was
a
~ ~ I
mu I
1
r 6 "2
"E >--.4-
ltxj
°°TI : -
I
1 rain
j
O~
O o
2
-4
-2
12,
I
I
2
I
1/cGMP CraM)-1
Fig. 2. Indication that light-induced K m increase does not require soluble components. (a) The p H decreases after light stimulation (arrows) were continuously monitored in suspensions of crude disk membranes (trace 1), purified disk membranes (trace 2) and purified disk membranes supplemented with the soluble fraction of the crude disk membranes (trace 3). (b) Lineweaver-Burk plots of the reactions shown in (a). The phosphodiesterase activity was calculated in the same way as shown in Fig .lb and c. Open circles, closed circles and crosses were obtained from traces 1, 2 and 3, respectively. Assuming linear relation, best-fit lines were determined.
determined. The Lineweaver-Burk plot (crosses in Fig, 2b) showed that the light g m w a s low (230 /~M) in the combined preparation, indicating that the light-induced K m increase does not require soluble fractions. Then, the purified disk membranes were mixed with various supernatant or precipitate fractions which were obtained at each step of the purification process. The light K m was determined in the same way as in Fig. 2. The results showed that the value in the mixture was always low (data not shown). In the experiment in Fig. 2, the phosphodiesterase dark activity in trace 2 was higher than that in trace 1. This observation was consistent with the finding by Yee and Liebman [3] that the phosphodiesterase dark activity increases by floatation and washes. The dark activity in trace 2 was suppressed with the addition of the soluble fraction of the crude disk membranes (trace 3), suggesting the presence of the inhibitor of the phosphodiesterase [12] in the soluble fraction. It was then possible that the increase in the dark activity was caused by dilution of the inhibitor during the preparation of the purified disk membranes. Though the result in Fig. 2 left the possibility that the soluble factor was easily denatured, t h e experiment in Fig. 3 unequivocally showed that soluble factors are not involved in the light-induced K m increase. Crude disk membranes which preserved the, activity of the light-induced Km increase were combined with purified disk membranes (Fig. 3). The mixing ratio of the two types of preparations were varied, and in each mixture the kinetic parameters were determined with the continuous pH recording method. Though the preparation was a mixture of phosphodiesterases of low and high light K~, the Lineweaver-Burk plot gave a fairly straight line, and we could determine the Vm~x and apparent light K m in each mixture. In Fig. 3, the obtained light K m is plotted as a function of the content of the high light K m phosphodiesterase which was determined from the percentage of the crude disk membranes in the mixture. It can be seen from the figure that the apparent light g m in a mixture increased as the content of the high light g m phosphodiesterase increased. Since it was not known whether or not
261 this increase in the apparent light K m was due to restoration of the activity of the light-induced K m increase in the purified disk membranes, the result was theoretically analyzed. In the following analysis, the expected K m value in a mixture was calculated under the assumption that the lightinduced K m increase took place only in crude disk membranes. Then, we compared the calculated K m value with the experimental result in Fig. 3 to find out whether the assumption was reasonable or not. If low- and high-K m phosphodiesterases in a mixture contribute to the observed phosphodiesterase activity in proportion to their contents, the phosphodiesterase activity (o) in the light in the mixture should be expressed as follows: v = pV~a ~
S - -S S + K ~ + ( i - p)V'~x S + K~
(1)
where S is the concentration of cGMP, superscripts H and L designate high- and low-K m phosphodiesterases, respectively, and p is the content of the h i g h - K m phosphodiesterase. We calculated VHx and K H from the experiment done in crude disk membranes (corresponding to the data at 100% high-K m phosphodiesterase in Fig. 3), and V~Lax and K L from
/,
1.0
/
E /
i
t'
p
E
.¢-
/tt
D., ,~
0 -I
0
I 50
I
100
High K m PDE content (*/,)
Fig. 3. Kin-increasefactor is confined in the disk membrane. Purified disk membranes (containing low-Km phosphodiesterase) were mixed with crude disk membranes (cont a i n i n g h i g h - K m phosphodiesterase)with various ratios. In the same way as in Fig. lb and c, light Km was determinedin each mixture and plotted against the content of high-Kin phosphodiesterase. The broken fine shows the theoretically expected Km values which were obtained under the assumption that the light-induced K m increase took place only in crude disk membranes (see text). PDE, phosphodiesterase.
purified membranes (corresponding to the data at 0%). The obtained values were: VmHax= 1080 c G M P / r h per min, KmH = 1.08 mM, V ~ = 690 c G M P / r h per min and K L = 0 . 1 9 0 mM as an average of two determinations. Taking three cGMP concentrations (three levels of S) which were actually chosen to determine the g m values in the experiment in Fig. 3, corresponding values of v were calculated from Eqn. 1 at a fixed value of p. From the relation of v and S at the above three different substrate concentrations, K m was determined from the Lineweaver-Burk plot. The value of p was changed from zero to 1, and at each value of p, the above calculation was repeated to obtain a theoretical curve (dashed line in Fig. 3). The consistency between the experiment and the theory in Fig. 3 indicated that the high light K m phosphodiesterase and t h e low light K m phosphodiesterase were activated independently. This result showed that the light-induced K m increase is confined to the disk membrane. L i g h t - i n d u c e d K m i n c r e a s e is labile
The reduction of the light K m during purification of disk membranes suggested that the light-induced K m increase is labile. This was further demonstrated in Fig. 4, where the light K m w a s measured in various types of preparations which were usually used in biochemical experiments of rod outer segments. Crude rod outer segments were freshly prepared ('crude ros' preparation). Some of them were pelletted by centrifugation (20000 × g, 15 min). The pellet was resuspended in the low-sodium, lowcalcium solution ('washed'). Some of the washed preparation were partially purified with sucrose floatation ('purified'). Crude rod outer segments were fragmented with vigorous pipeting with a syringe (' fragmented') or sonicated for 5 s (' sonic.') or frozen overnight and then thawed (' f. thawed'). In all these preparations, the light K m values were determined and all the above manipulations were found to reduce the light K m of the phosphodiesterase (Fig. 4), though the extent of the reduction depended on the type of the manipulation. However, in crude disk membranes ('crude disk') which were obtained by storing 'crude ros' preparation overnight at 4°C, the light K m value was close to that of crude rod outer segments. The
262
1.0
T
E E v
0.5
o
l
~
~'-~
Fig. 4. Light-induced K , - i n c r e a s e is labile. Crude rod outer segments (crude ros) were washed by centrifugation (washed), partially purified with sucrose floatation (purified), fragmented by pipeting (fragmented), sonicated briefly (sonic.) freezethawed (f. thawed), kept at 4°C overnight (crude disk) or sedimented and resuspended in a hypotonic buffer (lyzed). Light K m values were determined as in Fig. l b and c from 10-20 determinations in each type of preparation. Vertical bars show standard deviations.
above result indicated that the level of the light K m of the phosphodiesterase depends on how the phosphodiesterase or the disk membrane is manipulated• The result in Fig. 4 suggested that, as degeneration of the structure of rod outer segment proceeded the light K m value decreased. In fact, under a light microscope, we observed rod structure and stacks of disks in the suspension ot~ crude rod outer segments (top panel, Fig. 5) but not in the freeze-thawed preparation (middle panel, Fig. 5). However, though the light K m of the crude disk membrane was high (Fig. 4), the main constituent of this preparation was disk membranes (see Materials and Methods). Therefore, there seemed to be no simple correlation between rod structure and the activity of the light-induced g m increase. This point was further examined in a 'lyzed' preparation which was obtained after centrifugation (500 × g, 2 min) of crude rod outer segments by resuspending the precipitate in a hypotonic buffer (10 mM H e p e s / 2 mM MgC12/0.1 mM CAC12/2.78 mM EGTA (pH 7.8)). In the 'lyzed' preparation, the rod structure and stacks of
disks both disappeared completely (bottom panel, Fig. 5), while the light K m was relatively high ('lyzed' in Fig. 4), indicating that the rod structure is not necessary to induce the light-induced K m increase. When a 'lyzed' preparation was further sedimented (500 x g, 2 rain) and resuspended gently in the hypotonic buffer either in the light or in the dark, the light K m did not decrease appreciably (data not shown). This observation strengthened the conclusion obtained in Figs. 2 and 3 that the light-induced K m increase does not require soluble fractions, because most of the peripheral proteins in the frog disk membrane are soluble and should be eliminated by washing under the condition [13]. Since a mixture of the phosphodiesterases of high K m and low K m gave an intermediate K m value (Fig. 3), the intermediate K m value in the
,t'x.-.,_,e~ all" " - , ~ ~T..-~.j.-.-~,O:
•
.',
).1~.~ •
..~',~ ~ - ~ ."..--i ~'~
~'~ ~ .• d ~.". ~ . - : a ~ l,~
•
•
":: •
'~-,
.. ~ , . ~ .,..~,,,
-r~ ~-.
,.o,.... "..
"-" :s,~.'~',,, .~',~_ ~. "~.~ ~ , I ~'"
" ...'1"
',
~.. -~,
,~
~'~ 4:'-"~ ~" ~ . : -)',~. ,' -~~ " -
,--~',v'gllz~
...,. • x~,:,-....~,~
.
.~
f ,~. .,r ~' ~ " -
..
. |,
"
•
•
: ~1~ .'~' .
(
~.~. ~,, • ~
.
",r/~'.~ .~,
"~r.~B,';. ""~-~ ,-, ~.,~....'~...¢z~ff" * ;.:..-,.,~.,~,,t,z; ,~.,~.~.
¢ g, @
Fig. 5. Morphological appearance of some of the preparations. U n d e r a light microscope, morphological appearance was observed in crude rod outer segments (top), frceze-thawed preparation (middle) and lyzed preparation (bottom). The measured K m values in the preparations shown in this figure were 1.2 mM, 250 /tM and 940 # M , from top to bottom, respectively. Magnifications are the same from top to bottom, and a calibration bar in the top panel indicates 100 #m.
263
various types of the preparation in Fig. 4 could be explained by the notion that the preparation was a mixture of high- and low-K m phosphodiesterases. By adding various chemicals to sonicated preparations, we tried to find out the condition which could restore the activity of the light-induced K m increase. However, cAMP, cGMP, polyphosphoinositides, isobuthylmethylxantine, ATP, GTP, S-adenosylmethionine, S-adenosylhomocysteine and dithiothreitol were all ineffective when these chemicals were incubated with sonicated preparations. Calcium ions rather enhanced the reduction of the light Km.
Light-induced K m increase is affected by proteinases and phospholipases To characterize the light-induced K m increase, the effects of proteinases and phospholipases were investigated (Fig. 6 and Table I). In Fig. 6a, tryp-
0.8
1000
EO.4
sin was added to a suspension of crude disk membranes to give a final concentration of 1 0 / ~ g / m l . Then, single portions were withdrawn at various time intervals and were mixed with soybean trypsin inhibitor (final concentration 0.5 m g / m l ) . In each portion, the light K m and the phosphodiesterase activity were determined and the time-courses of the changes in kinetic parameters were measured. In agreement with previous reports [12,14], trypsin digestion increased both phosphodiesterase dark activity (open triangles in Fig. 6a) and l/m,X (data not shown). In addition, trypsin reduced the light K m of the phosphodiesterase (open circles in Fig. 6a) in the crude disk membranes, suggesting that the light-induced K m increase is associated with a protein(s). Though trypsin affected both the phosphodiesterase activity and the light Km, the timecourse of reduction of the light K m was slightly faster than that of the phosphodiesterase dark activity increase. This raised the possibility that the process responsible for the reduction of the light K m is different from that for the phosphodiesterase dark activity increase. Since trypsin
500
v
TABLE I
0 -, 0
~
0.8-' b
i i
1'0 i
; i
2;0
; '/
"
'_
I 0 60 ^ L
o '-" .--
000 .~
E
** l.H
E 0.4
i00
0-,
,
0
,
2LO
~
410
T i m e of i n c u b a t i o n
a rl
0
610 (min)
Fig. 6. Proteolytic digestion reduces light Km. (a) Crude disk membranes were incubated with trypsin (final concentration; 10 /~g/ml) at 30°C for various periods and the reaction was terminated with addition of trypsin inhibitor (0.5 mg/ml). Light K m (O) and phosphodiesterase dark activity (A) were determined, and they were plotted against incubation time. Filled circle and filled triangle show light K m and phosphodiesterase dark activity, respectively, in the presence of soybean trypsin inhibitor throughout the incubation period. (b) Same as in (a), but the crude disk membranes were digested with chymotrypsin (30 /~g/ml). The reaction was terminated with the addition of soybean trypsin inhibitor (0.5 mg/ml). PDE, phosphodiesterase.
EFFECT OF PHOSPHOLIPASES ON L I G H T K m A N D PHOSPHODIESTERASE ACTIVITIES IN C R U D E DISK MEMBRANES Crude disk membranes were incubated at 30°C for 30-60 min with phospholipases A 2 (final concentration: 1.5 mg/ml), C (0.6 m g / m l ) or D (1.5 mg/ml). As a control, the outer segments were incubated at 30°C for 60 rain without enzymes. Each value shown is a mean of duplicate experiments_+ the range of the values about the mean. The experiments were performed in the presence of 1 mM calcium (see text). For comparison, the effect of trypsin is listed. PDE, phosphodiesterase. Addition
K m (/tM)
PDE activity ( c G M P / r h per min) dark
None Phospholipase A 2 Phospholipase C Phospholipase D Trypsin a
light
530 + 10 100 _+10 220 + 30 200 + 10
190_+ 20 70 + 10 130 -]- 10 100 ± 10
730 + 20 500 _+10 390 + 50 410 + 20
60 4- 2
800 4- 30
1060 + 70
a The measurement was performed in the low-sodium, lowcalcium solution.
264
cleaves the peptide bond at the carboxyl terminal of lysine or arginine, it was expected that other proteinases showed different selectivity between the reduction of the light K m and the increase in the phospliodiesterase dark activity. Chymotrypsin is known to act on the carboxyl terminal of aromatic amino acid residues. This enzyme was added to a suspension of crude disk membranes (final concentration: 30 # g / m l ) in the presence of leupeptin (10 # g / m l ) which selectively inhibits the activity of trypsin, a contaminant in the commercially available chymotrypsin (see Sigma catalog, 1985). Single portions were withdrawn at various incubation periods and mixed with soybean trypsin inhibitor (0.5 m g / m l ) which also inhibits chymotrypsin activity. The enzyme reduced the light g m of the phosphodiesterase (open circles in Fig. 6b) and increased the phosphodiesterase dark activity (open triangles in Fig. 6b). Contrary to the effect of trypsin (Fig. 6a), the time-course of the reduction of the light g m by chymotrypsin was slower than that of the dark activity increase (open triangles in Fig. 6b). Because trypsin and chymotrypsin showed different selectivity between the reduction of the light K m and the dark activity increase, it was concluded that the site of the phosphodiesterase molecule responsible for the light-induced K m increase is different from that for the activation. Filled symbols in Fig. 6a and b sh.ow the controls in which proteinase activity was inhibited throughout the incubation period. In Table I, the effects of phospholipases A 2 (from bee venom), C (from Clostridium perfringens) and D (from peanut) are summarized. Since these enzymes are active only in the presence of calcium [15,16], crude disk membranes were pelletted by centrifugation and resuspended in a solution of which calcium concentration was made 1 mM by replacing 0.1 mM CaC12 plus 2.78 mM EGTA with 1 mM CaC12. During the course of the preparation, the light K m decreased from 750 to 530 #M (control) probably because of repetitive pipeting. As shown in Table I, all the phospholipases examined in the present study reduced the light K m of the phosphodiesterase. However, in contrast to proteinases used in Fig. 6, the phospholipases reduced the light K m a s well as the dark and light activities of the phosphodiesterase
!
[J
!
!
!
,
,
I
|
,
,
i
0.8 ~
0.6
~0.4 0.2 0
. . ,
0
0.01
0.1
SLME eoncentration
1 (%}
Fig. 7. Effect of sucrose laurylmonoesteron light K m of the phosphodiesterase.The light Km in crude disk membraneswas measured in the presence of various concentrationsof sucrose laurylmonoester (SLME). Each closed circle shows a single determination. (Table I). In control experiments, the effects of these phospholipases were measured at low calcium concentration where the phospholipase activities were inhibited [15,16]. In the low-sodium, lowcalcium solution, the phospholipases affected neither the light g m nor the Irmax. For comparison, the effect of trypsin was also listed in Table I, though the trypsin digestion was carried out in the low-sodium, low-calcium solution.
Effect of detergent on light-induced K,, increase The reduction of the light K m by phospholipases (Table I) suggested the necessity of some structural organization of membrane lipid for the light-induced K m increase. To test this possibility, we examined the effect of detergent which was expected to modify the molecular environment of the phosphodiesterase and its associated molecules. Though many detergents inhibit the activation of the phosphodiesterase, sucrose laurylmonoester is known to solubilize the phosphodiesterase without affecting the phosphodiesterase activity appreciably [17]. In Fig. 7, the light K m was measured at various concentrations of sucrose laurylmonoester. The detergent reduced the light g m , and half-maximal effect was observed at about 0.03% (w/w) of sucrose laurylmonoester. Though the incubation period of the disk membranes with sucrose laurylmonoester was varied (5-30 rain), no significant effect was observed.
265 Discussion
Possible mechanisms which underlie the light-induced K m increase In the present experiment, the light-induced increase in K m was characterized. There would be at least two possible mechanisms which account for the light-induced K m increase. One mechanism assumes the presence of a factor (Km-increase factor) which increases the K m of the activated phosphodiesterase. The other assumes the contribution of the rod structure: saturation of the phosphodiesterase requires a higher concentration of cGMP in the space between stacks of disks, because a local depletion of cGMP in the space occurs more rapidly than new cGMP can diffuse in from the medium. However, the latter possibility could be excluded from the experiment showing that the light K~ still retained a relatively high livel in the 'lyzed' preparation in which the rod structure and stacks of disks both disappeared (Figs. 4 and 5). Therefore, it would be most reasonable to assume the presence of the Kin-increase factor. According to the Michaelis-Menten equation, the K m is expressed by the equation: K~ = (k_ 1 + k+2)/k+l, where k÷a, k_ 1 and k+2 are the rate constants in the reaction scheme shown in the following: k+l
k+2
E + S ~ ES ~ E+P k_j
where E is the phosphodiesterase, S is cGMP and P is 5'-GMP and H +. Therefore, the Km-increase factor increases the K m value in the light by acting on any of the rate constants in the above scheme. Further kinetic analysis is necessary to elucidate the action mechanism of the Km-increase factor.
Characteristics of the Kin-increase factor The present experiment showed that the Kin-increase factor easily loses its activity with various experimental manipulations (Fig. 4) or addition of a detergent (Fig. 7). The activity of the factor is not found in the soluble fraction (Figs. 2 and 3), but is retained under hypotonic shock (Fig. 4). Proteinases and phospholipases reduce the light g m (Fig. 6 and Table I).
A simple picture deduced from the above experiments is as follows. The Kin-increase factor is a protein (Fig. 6). The factor is membrane-bound and exerts its effect without participation of soluble components (Figs. 2 and 3). It is labile and requires structural integrity of the disk membrane to cause the light-induced K m increase. Therefore, the factor loses its activity by mechanical treatment (Fig. 4), detergent (Fig. 7) and enzymatical degradation of membrane phospholipid (Table I). Since the Kin-increase factor has not been isolated, it is not known whether or not the factor is a protein different from the phosphodiesterase molecule itself. However, the factor is not associated with the phosphodiesterase inhibitor for the following reason. One suggestion has been that the increase in phosphodiesterase dark activity caused by proteolytic digestion is due to the degradation of the phosphodiesterase inhibitor [12,18]. If the factor is asosciated with the inhibitor, the increase in the phosphodiesterase dark activity caused by proteinases should proceed in parallel with the reduction of the light Km. Apparently, this was not the case (Fig. 6). One of the possible sources of the Kin-increase factor was a contaminant from the retinal neurons other than photoreceptors. However, this possibility was excluded: if this was the case, the mixing experiment in Fig. 3 should have always yielded the high light K~, since all the factors necessary for the light-induced K m increase was in the fraction of crude disk membranes.
Physiological function of the light-induced K m increase In vivo phosphodiesterase activity (v) would be determined by both the maximal velocity, Vmax, and the K m in the Miachaelis-Menten equation: s O=Vm~,S+Km
(2)
where S is the cytoplasmic concentration of cGMP. The effect of the light-induced K m increase should be reflected in the second term of Eqn. 2, that is, S / ( S + Kin). Though the cytoplasmic cGMP concentration in intact frog rod outer segments has not been directly measured, it is estimated to be 60-80 #M [19,20] or possibly about 6 gM [10]. Assuming these values and taking 100/~M and 1
266 m M for the K m in the dark and light, respectively, the effect of light-induced K m increase was calculated. The result showed that the second term decreases to 11-17% of the value in the dark. Therefore, the light-induced K m increase modulates the in vivo phosphodiesterase activity by reducing the effect of the increase in Vmax which is caused by light illumination. One of the possible ways by which the Km-increase factor exerts its physiological effect is to increase the K m in a time-dependent manner. If the K m increase takes place gradually after light stimulation, it means that the phosphodiesterase activity in vivo once becomes high on exposure to light and then gradually decreases during light stimulation. This decrease could be one of the mechanisms of the light adaptation of the rod photoreceptors. However, as shown in Fig. 1, the light K m appears to be constant from the beginning of the activation at least within the time resolution of the present p H assay method (about 2 s). Obviously, detailed experiment with faster time resolution is necessary. Besides the light-induced K m increase, it is noteworthy that V~oaxof the phosphodiesterase decreases when crude disk membranes were continuously exposed to light (Fig. 1). T h o u g h the details are not k n o w n yet, the result suggests the presence of some inhibitory mechanism of the phosphodiesterase activation after light exposure. It is possible that this time-dependent decrease in Vmax has some role in the desensitization of the photoreceptor during light adaptation.
Acknowledgements This work was supported by a Grants-in-Aid from the Ministry of Education, Science and Culture of Japan (No. 59770103) to S.K. The authors
thank Professor Y. Tsukada and Dr. S. Kohsaka for use of laboratory equipment and for helpful discussion.
References 1 Robinson, P.R., Kawamura, S., Abramson, B. and Bownds, M.D. (1980) J. Gen. Physiol. 76, 631-645 2 Miki, N., Keirns, J.J., Marks, F.R., Freeman, J, and Bitensky, M.W. (1973) Proc. Natl. Acad. Sci. USA 70, 3820-3824 3 Yee, R. and Liebman, P.A. (1978) J. Biol. Chem. 253, 8902-8909 4 Kawamura, S. and Bownds, M.D. (1981) J. Gen. Physiol. 77, 571-591 5 Stryer, L. (1983) Cold Spring Harbor Symla. Quant. Biol. Vol. 48, 841-852 6 Yamazaki, A., Uchida, S., Stein, P.J., Wheeler, G.L. and Bitensky, M.W. (1984) Adv. Cyclic Nucleotide Prot. Phospho. Res. 16, 381-392 7 Miller, W.H. and Nicol, G.D. (1979) Nature (Lond.) 280, 64-66 8 Kawamura, S. and Murakami, M. (1983) Jpn. J. Physiol. 33, 789-800 9 Fesenko, E.E., Kolensnikov, S.S. and Lyubarsky, A.L. (1985) Nature (Lond.) 313, 310-313 10 Yau, K.-W. and Nakatani, K. (1985) Nature (Lond.) 317, 252-255 11 Kawamura, S. (1983) Biochim. Biophys. Acta 732, 276-281 12 Hurley, J.B. and Stryer, L. (1982) J. Biol. Chem. 257, 11094-11099 13 Kuhn, H. (1980) Nature (Lond.) 283, 587-589 14 Miki, N., Baraban, J.M., Keirns, J.J., Boyce, J.J. and Bitensky, M.W. (1975) Biochemistry 250, 6320-6327 15 Munjal, D. and Elliot, W.B. (1971) Toxicon 9, 403-409 16 Yamakawa, Y. and Ohsaka, A. (1977) J. Biochem. 81, 115-126 17 Fukada, Y,, Kawamura, S., Yoshizawa, T. and Miki, N. (1981) Biochim. Biophys. Acta 675, 188-194 18 Yamazaki, A., Stein, P., Chernoff, N. and Bitensky, M.W. (1983) J. Biol. Chem. 258, 8188-8194 19 Woodruff, M.L. and Fain, G. (1982) J. Gen. Physiol. 80, 533-555 20 Cote, R.H., Biernbaum, M.S., Nicol, G.D. and Bownds, M.D. (1984) J. Biol. Chem. 259, 9635-9641