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Hydrogen ion changes of rhodopsin I. Proton uptake during the metarhodopsin I478 metarhodopsin II308 reaction
.lRCHIVES
OF
Hydrogen
DIOCHI~;MIS’L’RY
Ion
ANI)
IIIOPHYSICY
Changes
Dcpccrt,mertt
1-i
(1973)
of Rhodopsin
Metarhodopsin .JOSHUA
164,
I. Proton
Id78 Metarhodopsin K. WONG
of Riological
Sciences,
Received
.&XI) SASICORD Purdue
C~niowsitq,
March
Uptake
During
the
IIS,,, Reaction F:. OSTROY I,r~j”u!geite,
IndiarLa
47W7
27, 1072
The hydrogen ion changes resulting frotn the photolysis of the rod visual pigment, rhodopsin, have been investigated. Low temperature was used to isolat,c the mctarhodopsin ITin to 11380 reaction of rhodopsin and indicator dye was used t.o simultattcously measure t,he hydrogen ion changes of the rhodopsitt soluCott. The results indicate that illuminated rhodopsin t,akes up a proton during the tnetarhodopsin I,T8 to II38o reaction and releases protons at later intermediate stages. The results are consistent with data indicating pK changes of rhodopsin as the basis for t,he R, phase of the early receptor potential and hydrogen ion changes of t.he medium or pK changes of rhodopsin as having effects on the late receptor potcttt,ial.
Illumination of t)he visual pigment, rhodopsin, begins a series of spectral changes which are thought to represent distinct conformational states of the lipoprotein. Initiation of the visual process would appear to involve some aspects of these conformational changes and from kinetic evidence indicating rapid decay and major conformational changes it had been suggested that the metarhodopsin 14~~ (Meta 14T8) to metarhodopsin II& (Meta 11380) reaction was the reaction most likely to be the cause of the receptor potentials of the retina (1). Initial direct experiments on the correlation of rhodopsin thermal intermediates and receptor potentials were done by Cone (2), and by Gedney et al. measurements of (3 by simultaneous spectra and electrical signals on excised albino rat and frog retina, respectively. The experiments showed a time course correlation bet\vrcn the Meta IJ7, to IL8,, reaction and the R, phase of the early receptor potential-- the rapid cornea1 negative potent,ial observed on high-intensity illumination of the retina. The basis for the hIet,a 14T8 to Ilag reaction as a c*ausc of the R, phaw of the early receptor potential was suggc>sted by &troy (4) as a Copyright All rights
0 1973 by Academic Press, of reproduction in any form
Ino. reserved
pK change of the rhodopsin. This was based on the pH, salt, and buffer dependence of an early receptor-like potential obtained from rhodopsin during the hleta L8 to in a dialysis II 380 reaction membrane system. There n-as, however, no direct evidence that the pK change of rhodopsin occurred during the Meta IdT8 to IIsso reaction. In a further study of the correlation of the chemical changes of rhodopsin and the retina receptor potentials, Ward and Ostroy (5) investigated the relation between hydrogen ion changes and the la,tc receptor potential-the main potential of the rod cell. By studying the late receptor potential as a function of buffer, D20 and pH, they concluded that the hydrogen ion changes which occur on illumination of the retina may reduce the amplitude of the late receptor potential and could be involved in a process such as adaptation. Again, however, one could not specify the intermediate stages of rhodopsin at’ which t)hc hydrogen ion changes occur. The purpose of this study was to determine directly the rhodopsin intermediate rcnctions nhicah result in hydrogen ion upta1w or rcleaw. That rhotlopsin will
2
WONG
AND
cause hydrogen ion concentration changes upon illumination was first noted by lcukami (6) and Radding and Wald (7) in a study of the pH changes of solutions of cattle rhodopsin. Later Erhardt et al. (S) showed that the hydrogen changes of illuminated rhodopsin did not occur below -29”C, i.e., between the native rhodopsin and the formation of Meta I,,,. Hydrogen ion changes were likewise measured in suspensions of rod outer segments (9-12) and whole retinas (5). In Triton-solubilized (2%) rod outer segment suspensions at pH 5, Rafferty and McConnell (11) suggested that a fraction of the proton upt’ake was associated mth the Meta Id78 decay and in osmotically damaged rod outer segments, Emrich (12) had shown that the rapid hydrogen ion changes had a rate similar to that of the Meta I,,, to II,,, reaction. However, it has not yet been possible to determine unequivocally the relation between the hydrogen ion changes of rhodopsin and the Meta I478 to 113,,, reaction. In previous experiments either the method did not permit isolation of part’icular rhodopsin intermediates, or the sample included disc or plasma membrane components, or both the spectral and pH changes had to be measured or inferred independently. We have overcome these difficulties by: using low t’emperature and low pH to be certain that only the metarhodopsin 14>8 to 11380 reaction would occur; using pure solutions of rhodopsin to be certain that that is the only substance involved; and measuring spectral and pH changes simultaneously. MATERIALS
AND
METHODS
Deionized 27, aqueous digitonin solutions of rhodopsin were prepared from batches of 50 frozen cattle retinas obtained from the Hormel Co., (Austin, MN) using the method of Erhardt et al. (8). In general the rhodopsin solutions had an optical density of 0.8-1.0. The rhodopsin solutions used in t,he low-temperature experiments were further concentrated to an optical density of 1.5-2.5 by placing the rhodopsin solution in dialysis tubing with Sephadex G-200 on the surface. The water-swelled Sephadex grains were removed at 30-min intervals and fresh Sephadex G-200 used until the desired concent,ration was obtained. The following experiments were done to meas-
OSTROY ure the pH changes and photoproducts simultaneously. One milliliter of rhodopsin solution with 150 pM bromocresol green and 5oLj’, glycerol was adjusted to pH 4.50 and placed in a l-cm pathlength jacketed cuvette. The absorbance change of the dye and rhodopsin solution was measured by t,he Cary 14 spectrophotometer. The sample temperature was measured by a thermister placed into the sample (model 42SCYellow Spring Instruments), and regulated by constant-temperature bath (Lauda K2 and KS). The temperature was lowered at 10°C intervals and a spectrum ot the native rhodopsin taken at each interval. When the final temperature of -22°C was reached and spectra taken, the sample was irradiated for 5 min with a fiber optic illuminator (type K150 American Optical Company) containing a CS-3-71 cut off filter (Corning X > 460 nm). After taking a number of spectra to be certain that no changes were occurring, the sample temperature was raised to -15°C and the spectrum followed. Finally, the sample temperature was raised to -5”C, +15”C, and +24”C, respectively with accompanying spectra. To determine the absorbance changes independent of any temperature effects, the difference spectra were taken between the unbleached and bleached sample spectra at the same temperatures. Also, the absorbance due to scattering from condensation was corrected by the following formula:
A scattering at Xz = b4mJ nt;x’ ;yo nm)4> z where it was assumed that any change in absorbance at 300 nm is due to scattering. The bromocresol green was standardized by measuring the 625-nm dye peak in solutions containing rhodopsin (Abs = 0.75) and 150 pM dye. The dye sensitivity is affected by the presence of rhodopsin (pK shift from 4.7 to 5.2) and at pH 4.50 the dye change represents 1.4 pH units/ absorbance unit. At pH G.0 the dye change represented 1.0 pH unit/absorbance unit. Bromothymol blue was standardized by measuring the 620-nm dye peak in solutions containing rhodopsin (Abs = 0.60) and 150 I.~M dye. The pK in the presence of rhodopsin was approximately 7.5 (original pK 7.0). The dye sensitivity was .4 pH units/absorbance unit at pH 6.5-8.0 and 1.4 pH units/absorbance unit at pH 8.5. The pH and absorbance changes were studied at 25°C using the following apparatus and procedure. The rhodopsin solution (1 ml) cont,aining 50~~ bromocresol green was placed in a l-cm pathlength cuvette and irradiated with 50 flashes at 15-set intervals with a stronobar flash (65C Honeywell) containing a 50%nm interference filter (BP
HYDROGEN
ION
CHANGES
Baird-Atomic Inc.). The 380.nm buildup and decay was measured by a Cary 14 spectrophotometer with the phototube protected from flash artifact by a 3800 .i interference filter (Rn/UV type 3 Baird-Atomic: Im,.). 1n a duplicate experiment the dye sbsorbanre change was followed at 6125 :Z using a sllitablc interference filter (B 9 BnildAtomic Inc.). For the charge change study polyacrylamide disc gel electrophoresis (Buchler Tnstrumcnts Inc.) was used. The gels were 3.55; acrylamide with bromophenol blue to mark t,he buffer front and 14; amide black ill 7% acet,ic acid used as the stain. The normal or deionized (8) rhodopsin solutions where adjusted to the desired pH by addition of 0.1 N HCl or SaOH. To provide an intrrnal standard in addition to the dye, a single rhodopsin solution was II& for clach elect rophoresis experimclllt mtd scparatcd into Ilative and illuminatrtl samples. After layering the samples 011 the 12 gels, sis of the samples were kept in the dark while the other six samples were illrlminatctl for 5 mill with a focused 100-W lamp (American Optical) containing a C S 3-71 c,ut off filter (Cornillg X > 460 nm). The electrophoresis was carried out in the dark at 25°C for 25 min at, 6 mA per Illbe for pH 3.5; for 110 min at, 2 mA per tube for pH 6.8; and for 90 min at 2 mA per t,ube for pII 9.3. All gels contained O.OlSti RI acrylamide, 0.00518 11 bisarrylamide, and 0.0278 M TIME11 (?J,N,S,‘N’-t~etramethylethylenediamine). At pH 3.5 and pH 6.8 the gel also contained 0.024 M KOH, 0.0044 M potassium pcrsulfat,e, and O.OOlO(i M riboflavin with the gel adjusted to pH 3.5 or 6.8 with acetic acid. At pH 3.5 and pFI 6.8 the upper buffer contained 0.35 JI beta-alanine and the lower buffer 0.120 M KOH with each adjusted to pH 3.5 or 6.8 with acetic acid. At pII 9.3 the gel also contained 0.149 M Tris (tris(hydroxmethyl)aminomethane), 0.024 M HCL, and 0.0123 M ammonium persulfate. At pH 9.3 the upper buffer consisted of 0.042F ar Tris and 0.0809 JI glycine and the lower buffer 0.119 M Tris and 0.06 JI HCl.
OF
RHODOPSIN
3
I
of the formation of a small amount1 of Rleta II,,,. X1x0 observed is a small dy-absorbancc increase occurring at 630 nm mdicating a slightly higher solution pH (i.e., proton uptnkc by rhodopsin). When t,hc tempcraturo is ruiscd to - 15°C in apc~ctrum 2 the peak at 47s nm decrcascs substantially and thr absorbance of 380 nm increases, indirativr of t#ho Aleta I4T8 to II,,, reaction. Thcl tlyc absorbance at 630 nm also shows :L considerable rise indicating pH incrcnse~ of the solution. With tht* sampIt> kept at - l>“C, the Jlcta I,;, to 11~~ rcaction cwntinucs to procwd (qwetrum :I) and thtl pH continuw to riw. .\s t,hc kmperature is raiwd to -5°C’ (I;&. 2, spectrum 4) and +5”(‘ (spectrum 5) furthw incrcaw are noted at :SSOrun and MO nm indkativc of furthw incrcasw of Jlctn IL,,, and pH changes. I~inall~, as that tcmpcwturn is rakl to + l5Y’ in slwc’trum 6, t,hcl 1lct,:l
RESULTS The spectra preserlted in are the difference between
Figs.
1 and
:!
the unbleachrd and bleached rhodopsin-bromocresol green sample at a series of increasing temperatures. After illumination of the rhodopsin sample at -WC, as presented in Fig. 1 spectrum 1, one notes a substantial dccrease at 520 nm indicative of the rhodopsin bleaching. Also noted is an increase at 478 nm from the formation of Mets IJ7* and a small shoulder around 3S0 nm indicative
I
I
I
I
I
300
400
500
600
700
WAVELENGTH
(nm)
FIG. 1. The blea(,hing of rhodopsin at -22°C to -15°C. pH = 4.50. 150 FM brornocresol green. 50% glycerol. Rhodopsin absorbance (500 nm = 1.25). ljifference spectra between bleached and unbleached sample at the same temperature. (1) -22°C. (2) -15°C. (3) -15°C. (15 min after 2). The absorbance change at 380 nm of .195 and dye change of ,047 (ASH = ,068) at -15°C corresponds to .96 mole II+ per mole of Meta IIs~O (~42,000).
WONG AND
I 300
I 400
/ !300 WAVELENGTH
I 600
I 700 (nm)
FIG. 2. The bleaching of rhodopsin at -22°C to +15”C. Continuation of experiment in Fig. 1. (1) -22°C. (4) -5°C. (5) f5”C. (G) +15oc.
IIs thermally decays as indicated by a decrease in the absorbance at 380 nm and the pH now reverses, becoming more acid, as indicated by a decrease in absorbance at 630 nm. To indicate the results independently of the original rhodopsin bleaching, Fig. 3 presents another experiment similar to the one in Figs. 1 and 2 with the difference spectra now taken between the bleached sample at -22°C and the other temperatures. Spectrum 1 indicates the changes observed in going to -15°C and illustrates the decrease of Meta 1478,the increase of Meta II380 and the dye-absorbance increase at 630 nm. Spectrum 2 indicates the continuing changes at - 15°C illustrating further decreasesof Meta 1478,increases of Meta 11380,and pH increases (630 nm). Further increases at 380 nm and 630 nm are observed at -5°C (spectrum 3) and this time even at +15”C (spectrum 4). Finally, the thermal decay of Meta II380 is observed at +24OC (spectrum 5) accompanied by a decreasein pH. To further correlate the pH changes and
OlciTROY
I 300
400
I
I
I
500
600
700
WAVELENGTH
hm)
FIG. 3. The bleaching of rhodopsin at -22°C to f24”C. pH = 4.50. 150 FM bromocresol green. 50yc glycerol. Rhodopsin absorbance (500 nm = 1.25). Difference spectra between sample bleached at -22°C and the specified temperature. (1) -15°C. (spectra started 24 min after start of -22°C spectra) (2) -15°C (54 min) (3) -5°C (129 min) (4) +15”C (169 min) (5) +24”C (200 min). The total 380-nm absorbance change of 0.172(AA;:?" = .053) and total dye absorbance change at 630 nm of .045 (APH = ,063; AAil:°C = .004) corresponds to 1.04 mole H+ per mole of Meta 1138,,(f42,000).
the Meta IaT8 to Meta II380 reaction a kinetic plot of the hydrogen ion changes and Meta IIsso buildup at -10.5”C is presented in Fig. 4. The rates are comparable with the hydrogen ion changes -40 min) slightly slower than the h2 Meta I&o change (t1,2 -30 min). To provide information on the sequence of pH changes which occur on illumination when the Meta I47B to III,, reaction and later intermediate reactions are permitted, the 380-nm absorbance and dye peak at 612.5 nm were followed at 25°C and pH 5.6. A representative experiment is shown in Fig. 5. A pH increase coinciding with increasing 380 nm absorbance is first observed. Later the pH reverses returning
HYI~ROGI~X /
/WA+ 3636-W / / I I I
ION CHANGES
I
/
OF RHOI)OPSTX
I
.j
/
FIG. 5. pH and 5.60. (A) 380 nm indicates end of sorbance (500 nm)
380.nm changes at ‘&T, pH (0) Bromocresol green. Arrow illumination. Rhodopsin ab= 0.8. 50 PM bromocresol green.
sample but seems to increase t’hc positive charge change observed upon illuminatiotl.
I’rom the expcrimcnt~s in which t.he Met,a I 478to Meta II,*0 reaction is isolated bv uw of low temperature and pH, it is evi60 I20 I80 240 2 dent that a proton uptake process is aeTIME hn) sociated with the Mcta I 178to IIa8, reaction. FIG. -4. Kinetics of Meta 11280and pH changes At -22°C only a small amount of ;\Ieta at -10.5”. pH = 4.5. 150 PM bromocresol green. Il&, is formed and only a small pH change 507; glycerol. Rhodopsin absorbance (500 nm) = is observed (Fig. 1, speckurn 1). When t.hc 0.8. Solution illuminated at -21°C and raised to temperature is raised to - lj°C \\-hich - 10.5’C 17 mill after the end of the 5.min illuniionly permits the Mcta I,,, to IIZ8,, react,ion nntiotl. (as observed in the spectra and kno\vn from previous work (13)), the largest pH almost to thr: initial pH accompanied by change, amounting to approximateI>, one decreasing absorbance at 380 nm. The pH proton per molecule of Meta I1380, occurs decrease was observed at pH 4.5 in 50% (Fig. 1 spectra 2, 3; Fig. 3 spectra 1, 2). The kinetics of the hydrogen ion and glycerol, but not in aqueous solution at that pH. The pH decrease was the dominant Meta IL,, changes arc also comparable process at pH 6.25 in aqueous solution. (Fig. 4) with the slightly slower hydrogen The results of the direct measurement ion changes probably caused by t,he unof the charge change of rhodopsin by disc stirred nature of the solution. That some gel electrophoresis are presented in Table I. nlcta II380 is formed at -??‘C is somewhat All of the results are consistent with the surprising, but both lo\\ pH and glycerol rhodopsin molecule picking up a proton favor Meta IIBsO(13, 14). and becoming more positive (or less negaThe results are N~S(Jconsistent with prctive) upon illumination. At pH 3.5 where vious work done on this problem (7-l”). the rhodopsin molecule is positively charged In those casesa proton uptake process nas the mobility increases upon illumination observed (7-12) and localized to interand at pH 6.8 and 9.3 where the rhodopsin mediates after the formation of Mctu Isis is negatively charged, the mobility of the in rhodopsiu solution (S), and in rod outer illuminntcd sample decreases. The dcioni- segments the proton uptake time course zation of the sample s(‘crns t.o cause litt.le was similar to the Meta IJi8 to IIa,, reaction diff(brcLtlcck in the mobility of the unbk~n~hc~d (12). direct. (YmY’ht~icJIi wa.s cctmplicntc~d, I
L
I
/
I
I
I
1
6
WONG
AND
OSTROY
TABLE THE
Sample
DISC
rhodopsin rhodopsin rhodopsin rhodopsin
(de(de-
I
ELECTROPHOHESIS
~____Moving
Unbleached Bleached Unbleached ionized) Bleached ionized)
GEL
OF RHODOPSIN
-__~ pH 3.5 toward cathode
Mobility
Moving
(R,)
pH 6.8 toward
anode
Moving
pH 9.3 toward
0.058 0.072 0.055
(&0.015) (&0.005) (ZtO.013)
0.99 0.95 0.98
(SO.11) (Z&0.15) (ZtO.07)
0.79 0.75 0.70
(ZtO.03) (rkO.03) (xkO.03)
0.119
(kO.046)
0.98
(f0.19)
0.62
(~tO.06)
however, by observations of proton release above pH 6 after the immediate pH change at 20°C in rhodopsin solution (7), and at 25°C and low temperatures in Tritonsolubilized rod outer segment suspensions (10, 11). We have investigated these observations by studying the hydrogen ion and spectral changes at pH 5.6 and 6.25 at 25°C and at -15°C from pH 4.5-8.5. Because the 25’C experiments may involve intermediates other than Meta ILsO one cannot be certain that that is the only product being observed. From Fig. 5, however, it is clear that at pH 5.6 and 25”C, pH increases are observed consistent with rises in 380-nm absorbance and, at the termination of the illumination, pH decreases occur at the same time as decreases in absorbance at 380 nm. The most reasonable explanation for these results is that the pH increase corresponds to the Meta 14T8 t’o 11380 reaction and that the pH decrease results from later intermediate processes-possibly the decay of Meta II 380. This latter conclusion is reinforced by the lowtemperat’ure experiments which show pH decreases only when the temperature is raised to +24”C (Fig. 2, spectrum 6) or +15”C (Fig. 3, spectrum 5) permitting intermediates after RIcta II,,,. At higher pH values and temperatures, such as pH 6.25 and 25”C, where Meta 11,, would be more favored in the Rleta 14T8 * Meta II,,0 equilibrium, the decay of ;\Ieta 11380 would result in simultaneous Meta I478 to II 380 reaction. Thus, the hydrogen ion concentration changes would be the sum of multiple processes an.d not easily prcdieted. At %“C, pH 6.25, we observed small
anode
decreases in pH during illumination and small increases afterward. Although hydrogen ion release processes occurring after the formation of Meta II,,, seem t’o be responsible for the pH decreases observed on illumination at 20-2.5% (Refs. 7, 10, and Fig. 5), such changes have also been observed at low temperature in Tritonsolubilized rod outer segment suspensions (11) where one expects to prevent the decay of Metma IISgO. We have also observed such pH decreases on illuminat(ion of rhodopsin solutions at pH values from 5.5-8.5 under the same experimental conditions as those of Figs. l-3. Thus, when solutions of rhodopein cont’aining 50% glycerol were illuminated at -15°C pH decreases of the order of -.05 pH units were observed at pH 5.5-6.0; no change or slight increases were observed at pH 6.5-7.5; and decreases of the order of - .09 pH units were observed at pH 8.0-8.5. However, in the thermal decay difference spectra, products in addition to Meta 1478 and Meta 11380 wprc observed. At pH 5.5-7.5 products \\c’re also found in the 440- to 470-nm region (possibly Meta 111~~~ or NROM,) and at pH 8.0-8.5 products were also found in the 360.nm region (possibly ?;R~,G). As noted in Icigs. l-3, this is not thtl case at pH 4.55.0. Thus at, pH values above 5, under the conditions of t,hc>sc experiments, the low temperature is not sufficient to prevent the thermal decay of 1Ieta 11380 and other products will form. The observed pH decrcascs would appear to br a result of these added products. Apparently, thr lower pH of the main experiment not only favors I\Icta TIS8” in the ;\Ieta 1,~~ to 11:~+0
HYI)I:O(;F:N
ION CHANGES
quilibrium, but also stabilizw it, prcwriting further products. The gc4 electrophorc~tic cxperimcwts prescwted in Tablr I ww pcrformtd at ‘25°C and, therefore, involw similar difficulties in not isolating particular intermediates. The results show that in all casw bleached rhodopsin is more positjive than native rhodopsin and indicate an uptake of positivr charge during bleaching. The conclusions of the experiments correlate with certain data 011 the relation between rhodopsin intcrmcdiates and the receptor potentials of the retina. Thus, based 011 a time-course corre4spondenccbetween the R, portion of the early receptor potcntial and the JIeta I478 to IIS80 reaction (2, 3) and the characteristics of a potential obtained from rhodopsin in a dialysis mcm brane system, Ost’rog (4) suggestcld that the early receptor potential is cauwd b\- a pk’ change>of rhodopsin during the AI& I 4id to II,,, reaction. The expwimc~nts presented here do Rho\\. that at low pH a prot,on is taken up by t’he rhodqXiI1 during the ,1Icta Id78 to IIS8,, reaction and thcrcfort indicate that a pK change of rhodopsin dots occur during that reaction. Ward and Ostroy (5) investigated the relation b&\wn hydrogen ion changes and t,hcL late: receptor potential and concluded that the hydrogen ion changes ivhich occur upon illumination oppose the late rcccptor pot’cntial rna,y be involved in a process such as adaptation. The expcrimerits presented here which show proton uptake during the Rlrta 1478to II,,, reaction and then a reversal with proton release during later proccsscs, are the characteristics necessary to implicaatcbhydrogen ion
OF RHOI)OPSIK
changes in both visual excitation.
I
visual adaptation
7
and
This investigntiorl wits supported by Public Health Service Research (irarlt EYOO413 and liesearrh Career 1)evclopment Award (R.lS.0. EY32951) from the Sations Kye Tnstitutc. We thank Clark Grdney and St,cven \:re for theil sssistsnce.
REFERENCES 1. AJJI<.\H.i~iSON, Progr.
E:. W., .\ND OS'TIK)1-, s. 1':. Biophys. Mol. Riol. 17, 179.
(1967)
2. CONE, II. 8. (1969) Proceedings of the Fermi Tnternat,ional School of Physics. Academic Press, New York, p. 187. 3. C;EDN&Y, C., W.~RD, J., AND Osrrwu, S. E. (1971) AlrLer. J. Ph?ysiol. 221, 1754. 4. OSTWOY, S. E. (1971) Pholochem. Photobiol 14, 747. 5. W.utu, J. A., SND OSTROY, S. E:. (1971) Biophys. Sot. Abstr. 11, 46a. 6. Frrriaar, I. (1960) Folk Ophfhalmol. dup. 11, 519. 7. ItaDDING, C. M., AND WaLD, G. (1956) J. Gen.
8. ERH.IRDT,
Physiol.
39, 909.
F., Oslxo~, 8. E., AND AI~I~AH.~~xSON, E. W. (19W) Biochim. BiophFys. Acta 112, 256. 9. FOLK, G., AND Faw, Y. J. (1966) J. Physio/. 183, 211. 10. MCCONNELL, I). C., &FFERTY, C. N., AND DILLEY, R. A. (1968) J. Biol. Chew 243, 5820. 11. R.\FFI~:RTY, C. 11’., .ZND MCCONNELL, I). G. (1969) J. Cell Biol. Abstr. 43, 10Qa. 12. EMRICH, H. M. (1971) 2. Suturforsch. 26, 352. 13. MATTHEWS, R. G., I~UBISUD, R., BROWN, 1’. K., :lND W.4LD, c,. (1963) J. Gen. Physiol. 47, 215. 14. OSTROY, 8. E., ERIURDT, F., ASD AR~:\H~YMSON, E. W. (1966) Biochim. Biophys. Ackr 112, 265.
OF
Hydrogen
DIOCHI~;MIS’L’RY
Ion
ANI)
IIIOPHYSICY
Changes
Dcpccrt,mertt
1-i
(1973)
of Rhodopsin
Metarhodopsin .JOSHUA
164,
I. Proton
Id78 Metarhodopsin K. WONG
of Riological
Sciences,
Received
.&XI) SASICORD Purdue
C~niowsitq,
March
Uptake
During
the
IIS,,, Reaction F:. OSTROY I,r~j”u!geite,
IndiarLa
47W7
27, 1072
The hydrogen ion changes resulting frotn the photolysis of the rod visual pigment, rhodopsin, have been investigated. Low temperature was used to isolat,c the mctarhodopsin ITin to 11380 reaction of rhodopsin and indicator dye was used t.o simultattcously measure t,he hydrogen ion changes of the rhodopsitt soluCott. The results indicate that illuminated rhodopsin t,akes up a proton during the tnetarhodopsin I,T8 to II38o reaction and releases protons at later intermediate stages. The results are consistent with data indicating pK changes of rhodopsin as the basis for t,he R, phase of the early receptor potential and hydrogen ion changes of t.he medium or pK changes of rhodopsin as having effects on the late receptor potcttt,ial.
Illumination of t)he visual pigment, rhodopsin, begins a series of spectral changes which are thought to represent distinct conformational states of the lipoprotein. Initiation of the visual process would appear to involve some aspects of these conformational changes and from kinetic evidence indicating rapid decay and major conformational changes it had been suggested that the metarhodopsin 14~~ (Meta 14T8) to metarhodopsin II& (Meta 11380) reaction was the reaction most likely to be the cause of the receptor potentials of the retina (1). Initial direct experiments on the correlation of rhodopsin thermal intermediates and receptor potentials were done by Cone (2), and by Gedney et al. measurements of (3 by simultaneous spectra and electrical signals on excised albino rat and frog retina, respectively. The experiments showed a time course correlation bet\vrcn the Meta IJ7, to IL8,, reaction and the R, phase of the early receptor potential-- the rapid cornea1 negative potent,ial observed on high-intensity illumination of the retina. The basis for the hIet,a 14T8 to Ilag reaction as a c*ausc of the R, phaw of the early receptor potential was suggc>sted by &troy (4) as a Copyright All rights
0 1973 by Academic Press, of reproduction in any form
Ino. reserved
pK change of the rhodopsin. This was based on the pH, salt, and buffer dependence of an early receptor-like potential obtained from rhodopsin during the hleta L8 to in a dialysis II 380 reaction membrane system. There n-as, however, no direct evidence that the pK change of rhodopsin occurred during the Meta IdT8 to IIsso reaction. In a further study of the correlation of the chemical changes of rhodopsin and the retina receptor potentials, Ward and Ostroy (5) investigated the relation between hydrogen ion changes and the la,tc receptor potential-the main potential of the rod cell. By studying the late receptor potential as a function of buffer, D20 and pH, they concluded that the hydrogen ion changes which occur on illumination of the retina may reduce the amplitude of the late receptor potential and could be involved in a process such as adaptation. Again, however, one could not specify the intermediate stages of rhodopsin at’ which t)hc hydrogen ion changes occur. The purpose of this study was to determine directly the rhodopsin intermediate rcnctions nhicah result in hydrogen ion upta1w or rcleaw. That rhotlopsin will
2
WONG
AND
cause hydrogen ion concentration changes upon illumination was first noted by lcukami (6) and Radding and Wald (7) in a study of the pH changes of solutions of cattle rhodopsin. Later Erhardt et al. (S) showed that the hydrogen changes of illuminated rhodopsin did not occur below -29”C, i.e., between the native rhodopsin and the formation of Meta I,,,. Hydrogen ion changes were likewise measured in suspensions of rod outer segments (9-12) and whole retinas (5). In Triton-solubilized (2%) rod outer segment suspensions at pH 5, Rafferty and McConnell (11) suggested that a fraction of the proton upt’ake was associated mth the Meta Id78 decay and in osmotically damaged rod outer segments, Emrich (12) had shown that the rapid hydrogen ion changes had a rate similar to that of the Meta I,,, to II,,, reaction. However, it has not yet been possible to determine unequivocally the relation between the hydrogen ion changes of rhodopsin and the Meta I478 to 113,,, reaction. In previous experiments either the method did not permit isolation of part’icular rhodopsin intermediates, or the sample included disc or plasma membrane components, or both the spectral and pH changes had to be measured or inferred independently. We have overcome these difficulties by: using low t’emperature and low pH to be certain that only the metarhodopsin 14>8 to 11380 reaction would occur; using pure solutions of rhodopsin to be certain that that is the only substance involved; and measuring spectral and pH changes simultaneously. MATERIALS
AND
METHODS
Deionized 27, aqueous digitonin solutions of rhodopsin were prepared from batches of 50 frozen cattle retinas obtained from the Hormel Co., (Austin, MN) using the method of Erhardt et al. (8). In general the rhodopsin solutions had an optical density of 0.8-1.0. The rhodopsin solutions used in t,he low-temperature experiments were further concentrated to an optical density of 1.5-2.5 by placing the rhodopsin solution in dialysis tubing with Sephadex G-200 on the surface. The water-swelled Sephadex grains were removed at 30-min intervals and fresh Sephadex G-200 used until the desired concent,ration was obtained. The following experiments were done to meas-
OSTROY ure the pH changes and photoproducts simultaneously. One milliliter of rhodopsin solution with 150 pM bromocresol green and 5oLj’, glycerol was adjusted to pH 4.50 and placed in a l-cm pathlength jacketed cuvette. The absorbance change of the dye and rhodopsin solution was measured by t,he Cary 14 spectrophotometer. The sample temperature was measured by a thermister placed into the sample (model 42SCYellow Spring Instruments), and regulated by constant-temperature bath (Lauda K2 and KS). The temperature was lowered at 10°C intervals and a spectrum ot the native rhodopsin taken at each interval. When the final temperature of -22°C was reached and spectra taken, the sample was irradiated for 5 min with a fiber optic illuminator (type K150 American Optical Company) containing a CS-3-71 cut off filter (Corning X > 460 nm). After taking a number of spectra to be certain that no changes were occurring, the sample temperature was raised to -15°C and the spectrum followed. Finally, the sample temperature was raised to -5”C, +15”C, and +24”C, respectively with accompanying spectra. To determine the absorbance changes independent of any temperature effects, the difference spectra were taken between the unbleached and bleached sample spectra at the same temperatures. Also, the absorbance due to scattering from condensation was corrected by the following formula:
A scattering at Xz = b4mJ nt;x’ ;yo nm)4> z where it was assumed that any change in absorbance at 300 nm is due to scattering. The bromocresol green was standardized by measuring the 625-nm dye peak in solutions containing rhodopsin (Abs = 0.75) and 150 pM dye. The dye sensitivity is affected by the presence of rhodopsin (pK shift from 4.7 to 5.2) and at pH 4.50 the dye change represents 1.4 pH units/ absorbance unit. At pH G.0 the dye change represented 1.0 pH unit/absorbance unit. Bromothymol blue was standardized by measuring the 620-nm dye peak in solutions containing rhodopsin (Abs = 0.60) and 150 I.~M dye. The pK in the presence of rhodopsin was approximately 7.5 (original pK 7.0). The dye sensitivity was .4 pH units/absorbance unit at pH 6.5-8.0 and 1.4 pH units/absorbance unit at pH 8.5. The pH and absorbance changes were studied at 25°C using the following apparatus and procedure. The rhodopsin solution (1 ml) cont,aining 50~~ bromocresol green was placed in a l-cm pathlength cuvette and irradiated with 50 flashes at 15-set intervals with a stronobar flash (65C Honeywell) containing a 50%nm interference filter (BP
HYDROGEN
ION
CHANGES
Baird-Atomic Inc.). The 380.nm buildup and decay was measured by a Cary 14 spectrophotometer with the phototube protected from flash artifact by a 3800 .i interference filter (Rn/UV type 3 Baird-Atomic: Im,.). 1n a duplicate experiment the dye sbsorbanre change was followed at 6125 :Z using a sllitablc interference filter (B 9 BnildAtomic Inc.). For the charge change study polyacrylamide disc gel electrophoresis (Buchler Tnstrumcnts Inc.) was used. The gels were 3.55; acrylamide with bromophenol blue to mark t,he buffer front and 14; amide black ill 7% acet,ic acid used as the stain. The normal or deionized (8) rhodopsin solutions where adjusted to the desired pH by addition of 0.1 N HCl or SaOH. To provide an intrrnal standard in addition to the dye, a single rhodopsin solution was II& for clach elect rophoresis experimclllt mtd scparatcd into Ilative and illuminatrtl samples. After layering the samples 011 the 12 gels, sis of the samples were kept in the dark while the other six samples were illrlminatctl for 5 mill with a focused 100-W lamp (American Optical) containing a C S 3-71 c,ut off filter (Cornillg X > 460 nm). The electrophoresis was carried out in the dark at 25°C for 25 min at, 6 mA per Illbe for pH 3.5; for 110 min at, 2 mA per tube for pH 6.8; and for 90 min at 2 mA per t,ube for pII 9.3. All gels contained O.OlSti RI acrylamide, 0.00518 11 bisarrylamide, and 0.0278 M TIME11 (?J,N,S,‘N’-t~etramethylethylenediamine). At pH 3.5 and pH 6.8 the gel also contained 0.024 M KOH, 0.0044 M potassium pcrsulfat,e, and O.OOlO(i M riboflavin with the gel adjusted to pH 3.5 or 6.8 with acetic acid. At pH 3.5 and pFI 6.8 the upper buffer contained 0.35 JI beta-alanine and the lower buffer 0.120 M KOH with each adjusted to pH 3.5 or 6.8 with acetic acid. At pII 9.3 the gel also contained 0.149 M Tris (tris(hydroxmethyl)aminomethane), 0.024 M HCL, and 0.0123 M ammonium persulfate. At pH 9.3 the upper buffer consisted of 0.042F ar Tris and 0.0809 JI glycine and the lower buffer 0.119 M Tris and 0.06 JI HCl.
OF
RHODOPSIN
3
I
of the formation of a small amount1 of Rleta II,,,. X1x0 observed is a small dy-absorbancc increase occurring at 630 nm mdicating a slightly higher solution pH (i.e., proton uptnkc by rhodopsin). When t,hc tempcraturo is ruiscd to - 15°C in apc~ctrum 2 the peak at 47s nm decrcascs substantially and thr absorbance of 380 nm increases, indirativr of t#ho Aleta I4T8 to II,,, reaction. Thcl tlyc absorbance at 630 nm also shows :L considerable rise indicating pH incrcnse~ of the solution. With tht* sampIt> kept at - l>“C, the Jlcta I,;, to 11~~ rcaction cwntinucs to procwd (qwetrum :I) and thtl pH continuw to riw. .\s t,hc kmperature is raiwd to -5°C’ (I;&. 2, spectrum 4) and +5”(‘ (spectrum 5) furthw incrcaw are noted at :SSOrun and MO nm indkativc of furthw incrcasw of Jlctn IL,,, and pH changes. I~inall~, as that tcmpcwturn is rakl to + l5Y’ in slwc’trum 6, t,hcl 1lct,:l
RESULTS The spectra preserlted in are the difference between
Figs.
1 and
:!
the unbleachrd and bleached rhodopsin-bromocresol green sample at a series of increasing temperatures. After illumination of the rhodopsin sample at -WC, as presented in Fig. 1 spectrum 1, one notes a substantial dccrease at 520 nm indicative of the rhodopsin bleaching. Also noted is an increase at 478 nm from the formation of Mets IJ7* and a small shoulder around 3S0 nm indicative
I
I
I
I
I
300
400
500
600
700
WAVELENGTH
(nm)
FIG. 1. The blea(,hing of rhodopsin at -22°C to -15°C. pH = 4.50. 150 FM brornocresol green. 50% glycerol. Rhodopsin absorbance (500 nm = 1.25). ljifference spectra between bleached and unbleached sample at the same temperature. (1) -22°C. (2) -15°C. (3) -15°C. (15 min after 2). The absorbance change at 380 nm of .195 and dye change of ,047 (ASH = ,068) at -15°C corresponds to .96 mole II+ per mole of Meta IIs~O (~42,000).
WONG AND
I 300
I 400
/ !300 WAVELENGTH
I 600
I 700 (nm)
FIG. 2. The bleaching of rhodopsin at -22°C to +15”C. Continuation of experiment in Fig. 1. (1) -22°C. (4) -5°C. (5) f5”C. (G) +15oc.
IIs thermally decays as indicated by a decrease in the absorbance at 380 nm and the pH now reverses, becoming more acid, as indicated by a decrease in absorbance at 630 nm. To indicate the results independently of the original rhodopsin bleaching, Fig. 3 presents another experiment similar to the one in Figs. 1 and 2 with the difference spectra now taken between the bleached sample at -22°C and the other temperatures. Spectrum 1 indicates the changes observed in going to -15°C and illustrates the decrease of Meta 1478,the increase of Meta II380 and the dye-absorbance increase at 630 nm. Spectrum 2 indicates the continuing changes at - 15°C illustrating further decreasesof Meta 1478,increases of Meta 11380,and pH increases (630 nm). Further increases at 380 nm and 630 nm are observed at -5°C (spectrum 3) and this time even at +15”C (spectrum 4). Finally, the thermal decay of Meta II380 is observed at +24OC (spectrum 5) accompanied by a decreasein pH. To further correlate the pH changes and
OlciTROY
I 300
400
I
I
I
500
600
700
WAVELENGTH
hm)
FIG. 3. The bleaching of rhodopsin at -22°C to f24”C. pH = 4.50. 150 FM bromocresol green. 50yc glycerol. Rhodopsin absorbance (500 nm = 1.25). Difference spectra between sample bleached at -22°C and the specified temperature. (1) -15°C. (spectra started 24 min after start of -22°C spectra) (2) -15°C (54 min) (3) -5°C (129 min) (4) +15”C (169 min) (5) +24”C (200 min). The total 380-nm absorbance change of 0.172(AA;:?" = .053) and total dye absorbance change at 630 nm of .045 (APH = ,063; AAil:°C = .004) corresponds to 1.04 mole H+ per mole of Meta 1138,,(f42,000).
the Meta IaT8 to Meta II380 reaction a kinetic plot of the hydrogen ion changes and Meta IIsso buildup at -10.5”C is presented in Fig. 4. The rates are comparable with the hydrogen ion changes -40 min) slightly slower than the h2 Meta I&o change (t1,2 -30 min). To provide information on the sequence of pH changes which occur on illumination when the Meta I47B to III,, reaction and later intermediate reactions are permitted, the 380-nm absorbance and dye peak at 612.5 nm were followed at 25°C and pH 5.6. A representative experiment is shown in Fig. 5. A pH increase coinciding with increasing 380 nm absorbance is first observed. Later the pH reverses returning
HYI~ROGI~X /
/WA+ 3636-W / / I I I
ION CHANGES
I
/
OF RHOI)OPSTX
I
.j
/
FIG. 5. pH and 5.60. (A) 380 nm indicates end of sorbance (500 nm)
380.nm changes at ‘&T, pH (0) Bromocresol green. Arrow illumination. Rhodopsin ab= 0.8. 50 PM bromocresol green.
sample but seems to increase t’hc positive charge change observed upon illuminatiotl.
I’rom the expcrimcnt~s in which t.he Met,a I 478to Meta II,*0 reaction is isolated bv uw of low temperature and pH, it is evi60 I20 I80 240 2 dent that a proton uptake process is aeTIME hn) sociated with the Mcta I 178to IIa8, reaction. FIG. -4. Kinetics of Meta 11280and pH changes At -22°C only a small amount of ;\Ieta at -10.5”. pH = 4.5. 150 PM bromocresol green. Il&, is formed and only a small pH change 507; glycerol. Rhodopsin absorbance (500 nm) = is observed (Fig. 1, speckurn 1). When t.hc 0.8. Solution illuminated at -21°C and raised to temperature is raised to - lj°C \\-hich - 10.5’C 17 mill after the end of the 5.min illuniionly permits the Mcta I,,, to IIZ8,, react,ion nntiotl. (as observed in the spectra and kno\vn from previous work (13)), the largest pH almost to thr: initial pH accompanied by change, amounting to approximateI>, one decreasing absorbance at 380 nm. The pH proton per molecule of Meta I1380, occurs decrease was observed at pH 4.5 in 50% (Fig. 1 spectra 2, 3; Fig. 3 spectra 1, 2). The kinetics of the hydrogen ion and glycerol, but not in aqueous solution at that pH. The pH decrease was the dominant Meta IL,, changes arc also comparable process at pH 6.25 in aqueous solution. (Fig. 4) with the slightly slower hydrogen The results of the direct measurement ion changes probably caused by t,he unof the charge change of rhodopsin by disc stirred nature of the solution. That some gel electrophoresis are presented in Table I. nlcta II380 is formed at -??‘C is somewhat All of the results are consistent with the surprising, but both lo\\ pH and glycerol rhodopsin molecule picking up a proton favor Meta IIBsO(13, 14). and becoming more positive (or less negaThe results are N~S(Jconsistent with prctive) upon illumination. At pH 3.5 where vious work done on this problem (7-l”). the rhodopsin molecule is positively charged In those casesa proton uptake process nas the mobility increases upon illumination observed (7-12) and localized to interand at pH 6.8 and 9.3 where the rhodopsin mediates after the formation of Mctu Isis is negatively charged, the mobility of the in rhodopsiu solution (S), and in rod outer illuminntcd sample decreases. The dcioni- segments the proton uptake time course zation of the sample s(‘crns t.o cause litt.le was similar to the Meta IJi8 to IIa,, reaction diff(brcLtlcck in the mobility of the unbk~n~hc~d (12). direct. (YmY’ht~icJIi wa.s cctmplicntc~d, I
L
I
/
I
I
I
1
6
WONG
AND
OSTROY
TABLE THE
Sample
DISC
rhodopsin rhodopsin rhodopsin rhodopsin
(de(de-
I
ELECTROPHOHESIS
~____Moving
Unbleached Bleached Unbleached ionized) Bleached ionized)
GEL
OF RHODOPSIN
-__~ pH 3.5 toward cathode
Mobility
Moving
(R,)
pH 6.8 toward
anode
Moving
pH 9.3 toward
0.058 0.072 0.055
(&0.015) (&0.005) (ZtO.013)
0.99 0.95 0.98
(SO.11) (Z&0.15) (ZtO.07)
0.79 0.75 0.70
(ZtO.03) (rkO.03) (xkO.03)
0.119
(kO.046)
0.98
(f0.19)
0.62
(~tO.06)
however, by observations of proton release above pH 6 after the immediate pH change at 20°C in rhodopsin solution (7), and at 25°C and low temperatures in Tritonsolubilized rod outer segment suspensions (10, 11). We have investigated these observations by studying the hydrogen ion and spectral changes at pH 5.6 and 6.25 at 25°C and at -15°C from pH 4.5-8.5. Because the 25’C experiments may involve intermediates other than Meta ILsO one cannot be certain that that is the only product being observed. From Fig. 5, however, it is clear that at pH 5.6 and 25”C, pH increases are observed consistent with rises in 380-nm absorbance and, at the termination of the illumination, pH decreases occur at the same time as decreases in absorbance at 380 nm. The most reasonable explanation for these results is that the pH increase corresponds to the Meta 14T8 t’o 11380 reaction and that the pH decrease results from later intermediate processes-possibly the decay of Meta II 380. This latter conclusion is reinforced by the lowtemperat’ure experiments which show pH decreases only when the temperature is raised to +24”C (Fig. 2, spectrum 6) or +15”C (Fig. 3, spectrum 5) permitting intermediates after RIcta II,,,. At higher pH values and temperatures, such as pH 6.25 and 25”C, where Meta 11,, would be more favored in the Rleta 14T8 * Meta II,,0 equilibrium, the decay of ;\Ieta 11380 would result in simultaneous Meta I478 to II 380 reaction. Thus, the hydrogen ion concentration changes would be the sum of multiple processes an.d not easily prcdieted. At %“C, pH 6.25, we observed small
anode
decreases in pH during illumination and small increases afterward. Although hydrogen ion release processes occurring after the formation of Meta II,,, seem t’o be responsible for the pH decreases observed on illumination at 20-2.5% (Refs. 7, 10, and Fig. 5), such changes have also been observed at low temperature in Tritonsolubilized rod outer segment suspensions (11) where one expects to prevent the decay of Metma IISgO. We have also observed such pH decreases on illuminat(ion of rhodopsin solutions at pH values from 5.5-8.5 under the same experimental conditions as those of Figs. l-3. Thus, when solutions of rhodopein cont’aining 50% glycerol were illuminated at -15°C pH decreases of the order of -.05 pH units were observed at pH 5.5-6.0; no change or slight increases were observed at pH 6.5-7.5; and decreases of the order of - .09 pH units were observed at pH 8.0-8.5. However, in the thermal decay difference spectra, products in addition to Meta 1478 and Meta 11380 wprc observed. At pH 5.5-7.5 products \\c’re also found in the 440- to 470-nm region (possibly Meta 111~~~ or NROM,) and at pH 8.0-8.5 products were also found in the 360.nm region (possibly ?;R~,G). As noted in Icigs. l-3, this is not thtl case at pH 4.55.0. Thus at, pH values above 5, under the conditions of t,hc>sc experiments, the low temperature is not sufficient to prevent the thermal decay of 1Ieta 11380 and other products will form. The observed pH decrcascs would appear to br a result of these added products. Apparently, thr lower pH of the main experiment not only favors I\Icta TIS8” in the ;\Ieta 1,~~ to 11:~+0
HYI)I:O(;F:N
ION CHANGES
quilibrium, but also stabilizw it, prcwriting further products. The gc4 electrophorc~tic cxperimcwts prescwted in Tablr I ww pcrformtd at ‘25°C and, therefore, involw similar difficulties in not isolating particular intermediates. The results show that in all casw bleached rhodopsin is more positjive than native rhodopsin and indicate an uptake of positivr charge during bleaching. The conclusions of the experiments correlate with certain data 011 the relation between rhodopsin intcrmcdiates and the receptor potentials of the retina. Thus, based 011 a time-course corre4spondenccbetween the R, portion of the early receptor potcntial and the JIeta I478 to IIS80 reaction (2, 3) and the characteristics of a potential obtained from rhodopsin in a dialysis mcm brane system, Ost’rog (4) suggestcld that the early receptor potential is cauwd b\- a pk’ change>of rhodopsin during the AI& I 4id to II,,, reaction. The expwimc~nts presented here do Rho\\. that at low pH a prot,on is taken up by t’he rhodqXiI1 during the ,1Icta Id78 to IIS8,, reaction and thcrcfort indicate that a pK change of rhodopsin dots occur during that reaction. Ward and Ostroy (5) investigated the relation b&\wn hydrogen ion changes and t,hcL late: receptor potential and concluded that the hydrogen ion changes ivhich occur upon illumination oppose the late rcccptor pot’cntial rna,y be involved in a process such as adaptation. The expcrimerits presented here which show proton uptake during the Rlrta 1478to II,,, reaction and then a reversal with proton release during later proccsscs, are the characteristics necessary to implicaatcbhydrogen ion
OF RHOI)OPSIK
changes in both visual excitation.
I
visual adaptation
7
and
This investigntiorl wits supported by Public Health Service Research (irarlt EYOO413 and liesearrh Career 1)evclopment Award (R.lS.0. EY32951) from the Sations Kye Tnstitutc. We thank Clark Grdney and St,cven \:re for theil sssistsnce.
REFERENCES 1. AJJI<.\H.i~iSON, Progr.
E:. W., .\ND OS'TIK)1-, s. 1':. Biophys. Mol. Riol. 17, 179.
(1967)
2. CONE, II. 8. (1969) Proceedings of the Fermi Tnternat,ional School of Physics. Academic Press, New York, p. 187. 3. C;EDN&Y, C., W.~RD, J., AND Osrrwu, S. E. (1971) AlrLer. J. Ph?ysiol. 221, 1754. 4. OSTWOY, S. E. (1971) Pholochem. Photobiol 14, 747. 5. W.utu, J. A., SND OSTROY, S. E:. (1971) Biophys. Sot. Abstr. 11, 46a. 6. Frrriaar, I. (1960) Folk Ophfhalmol. dup. 11, 519. 7. ItaDDING, C. M., AND WaLD, G. (1956) J. Gen.
8. ERH.IRDT,
Physiol.
39, 909.
F., Oslxo~, 8. E., AND AI~I~AH.~~xSON, E. W. (19W) Biochim. BiophFys. Acta 112, 256. 9. FOLK, G., AND Faw, Y. J. (1966) J. Physio/. 183, 211. 10. MCCONNELL, I). C., &FFERTY, C. N., AND DILLEY, R. A. (1968) J. Biol. Chew 243, 5820. 11. R.\FFI~:RTY, C. 11’., .ZND MCCONNELL, I). G. (1969) J. Cell Biol. Abstr. 43, 10Qa. 12. EMRICH, H. M. (1971) 2. Suturforsch. 26, 352. 13. MATTHEWS, R. G., I~UBISUD, R., BROWN, 1’. K., :lND W.4LD, c,. (1963) J. Gen. Physiol. 47, 215. 14. OSTROY, 8. E., ERIURDT, F., ASD AR~:\H~YMSON, E. W. (1966) Biochim. Biophys. Ackr 112, 265.