Journal of Weu~ockonrsirp, 1976. Vol. 26, pp 621-623. Pergamon Press. Printed in Great Britain
SHORT COMMUNICATION
Gangliosides in frog retinal rod outer segment membranes (Received 10 June 1975. Accepted 2 September 1975) GANGLIOSIDES were analyzed in preparations of intact retinal rod outer segments of frogs. Such preparations represent as pure a neuronal photoreceptor plasma membrane as can readily be obtained. They are devoid of synapses and have the interesting physiological characteristic of being steadily permeable to sodium ions in the dark; illumination appears to cause the sodium ion channels to close and the membrane to hyperpolarize (BAYLOR& OBRYAN,1971). Calcium ions mimic the effect of light and have been suggested to be the agent involved in clos& YOSHIKAMI, 1974). The ing the sodium channels (HAGINS mixed hydrophilic, lipophilic structure of gangliosides would seem to fit them for a possible role in ion movement through membrane channels. The solubility of gangliosides is influenced by the major physiological cations, the calcium salt being more lipophilic than the salts of sodium, potassium or magnesium, and calcium can effectively compete with the other ions for association with gangliosides 1965). Studies of gangliosides in (QumLes & FOLCH-PI, specialized membranes such as those of the rod outer segment may eventually provide clues to the functions of these glycolipids. MATERIALS AND METHODS
Preparation of intact retinal rod outer segments. Frogs (Rana pipiens) were dark adapted 24 h and the retinas dissected under red light. Rod outer segments were detached by magnetic stirring (LOLLEY& HESS,1969). Ten retinas were placed in SO ml of pH 64 buffer containing 04)059 M-Na,HP04, 0.0108 M-NaH2P04, 0.044 M-NaCl, and 0.2 M-SUCTOSC, in a specially flat-bottomed Bellco trypsinization flask. The retinas floated from one compartment of the flask to another as the fluid was agitated by a magnetic stirring bar and plate. Rod outer segments were thus detached by a gentle procedure that did not disrupt the integrity of the remaining retina. The particular buffer used maintained the retinas intact, the phosphate being the essential ingredient. In 15 min, a yield of 200,000 to 300,000 outer segments per retina was obtained. The retinas and stirring bar were removed by forceps and the outer segment suspension was filtered through 67, 25 and 10 pm nylon mesh, in succession. No neuronal cells or synaptosomes were ever observed in such suspensions by phase contrast or Nomarsky microscopic examination. The outer segments were collected by low speed centrifugation (5 min, 1500 g) and washed twice with sugar-free frog saline solution (70 and 10 ml portions) before the pellet was used for lipid extraction. Supported in part by grants from the National Institutes of Health, NS-07297, NS-06370 and FR05484.
Preparation of ganglioside fraction. The outer segment pellet from 10 retinas was resuspended in 100 pI of frog saline and 2.9 ml of chloroform-methanol (2: 1, v/v) werc added, mixed and allowed to stand at 4°C in the dark for 18 h or longer. Methanol was then added to convert the chloroform-methanol ratio to 1: 1 (v/v); the outer segment residue was readily centrifuged from this lower density solvent, and the supernatant fluid was pipetted off. The residue was extracted for 15 min with chloroforn+ methanol (1:2, v/v) and the first and second extracts were combined. The resulting extract was restored to a volume ratio of 2: 1 chloroform-methanol and partitioned by adding lj5 its volume of water; the upper phase was removed after centrifuging and the lower phase was washed twice with a theoretical upper phase (chloroformmethanol-watcr, 3:48:47, viv) containing a low concentration of KCI (0.015 M), equal in volume to the upper phase removed. The 3 upper phases were combined. A total of 33 pellets from 330 frog retinas were extracted and the upper phases combined for dialysis. The upper phase was concentrated by a rotary evaporator to a volume of 25 ml and dialyzed at 4°C against distilled water, stirred magnetically in a 1 liter glass-stoppered cylinder. The 3 liters of dialysate obtained by 3 days of dialysis were saved and concentrated to check for possible loss of ganglioside from the bag; none was found by thin layer chromatography. The dialysand was concentrated by lyophilization and the gangliosides were dissolved in 2: 1 chloroforn-methanol containing 5% water, for application to thin layer plates. Thin layer chromatography. Plates 3 3/4 inches wide and 20 inches long were coated with Silica Gel G (250 pm thick layer). The tank for development consisted of a 4 liter cylinder, lined with chromatography paper and covered with a single square of heavy duty aluminum foil held snugly with a rubber band. The solvent system was chloroform-methanel-2.5 N-ammonium hydroxide (60:40:9, v/v). The plate was developed once to a height of 11 in (4 h), air dried, and developed again to a height of 18 in (14 h) at a constant temperature of 21°C. Standards consisting of bovine brain gangliosides, Tay Sachs G u l and disialogangliosides (GD,,and ganglioside (GM2), GDlbjfrom Supelco, Inc. (Bellefonte, PA.) were co-chromatoyaphed. Sialic acid-containing spots were detected by resorcinol-HCI spray. A clean cover plate was clamped on the sprayed plate, which was then heated in air in an oven at 110°C until a blue-purple color developed (about 20 minj. Microassay of sialic acid. Sialic acid was determined by & a microversion of the resorcinol method of MIETTINEN TAKKI-MUUKAINEN (1959). Immediately after thin layer chromatography of the outer segment ganglioside fraction,
62 1
622
Short communication
each spot containing sialic acid was scraped off and placed in a 0.5 ml glass-stoppered microtube. Corresponding-sized blank areas of the Silica Gel layer and Calbiochem standards of N-acetylneuraminic acid (the only type found in frog neural tissues) were run for comparison. The samples were reheated with resorcinol reagent as suggested by MACMILLAN & WHERRETT(1969), who found that only 5510% of the sialic acid reacted on the plate, but that reheating gave close to 100% recovery of chromophore; immediate analysis was essential for this good recovery. To the microtubes 100 pI of resorcinol reaction mixture was added and mixed. The tubes were stoppered and placed in a boiling water bath; after 10 min the tubes were removed, remixed, and replaced for 10 min more. The color was extracted with 125 jd butylacetate-butyl alcohol (85: 15, v/v) and read at 580 and 450 nm in a spectrophotometer equipped with pinhole for use of quartz microcuvettes (1.5 x 10 x 25 mm). The same thin layer chromatographic pattern was observed in 2 experiments. RESULTS AND DISCUSSION The dialyzed upper phase solids from 662 million retinal rod outer segments (from 330 frog retinas) were redissolved in 2: 1 chloroform-methanol containing 5% water and run by thin layer chromatography on Silica Gel G. After exposure to Iz vapor, the sample showed 2 small spots migrating parallel to di- and tri-sialogangliosides of bovine brain (GD~,,and GTI). These spots turned blue-purple when sprayed with resorcinol reagent (Fig. l), indicating the presence of sialic acid. The spots were scraped from the plate and sialic acid was determined quantitatively. Sialic acid in the disialoganglioside spot was equivalent to Q46 nmol and that in the trisialoganglioside spot to 0-33 nmol
TABLE1. GANGLIOSIDE SIALIC
ACID I N FROG RETINAL ROD OUTER SEGMENTS
Mode of expression Of
sialic acid ',,
Localization in both outer membrane and disks
Localiaatlon in outer membrane only'
0.00062 0.0062 00124 0.0027 8-6 Y l o - " 1.19 x 10-" 7.17 x 10"
0.ll 11 23
of dry wt'
i w m g dry &ng protein' pgimg wet weight' Mol/Kg wel weight' Molirod outer segment Moleculeshd outer segment
Dry wt or solids are assumed to be 43% of wet wt (or vol) and proteins about 50% of dry wt. Isolated rod outer segments average 594 pg dry wt (LOLLEY& HESS, 1969). The total sample consisted of 66.2 x lo6 rod outer segments (39.3 mg dry wt) and contained 0.79 nmol of ganglioside sialic acid. For determination of the percentage of the total membrane surface that was external membrane, the average rod was assumed to be composed of 1900 disks with a repeat distance of 200 A, a length of 38 pm, a surface area of 150,000 prn' (NILSSON,1965), and a mean dia of 6.5 pm. The external membrane (walls and one end), therefore, had an area of 809 pm2 and represented 054% of the total membrane area.
when calculated for the whole samplc. The original sample (dry wt of 39.3 mg: lower phase lipids, 18.47 mg) thus contained 0.79 nmol of ganglioside sialic acid. The concentration of sialic acid of gangliosides (Table 1) was much lower in retinal rod outer segments than in any other type of nervous tissue in which gangliosides have been reported. The concentration of ganglioside sialic acid was 8.6 pmol/Kg wet wt, or about 11290 as great as the level of rhodopsin (2.5 mmol/L) measured in that organelle
TABLE2. COMPARISON OF GANGLIOSIDE SlALlC ACID CON'CENTRATIONS IN RETINAL ROD OUTER SEGMENTS AND OTHER NEURAL AND MUSCULAR STRUCTURES Siaiic a c d Tissue Rod outer aegrnents Whole brain Rod outer segments Whole retina White matter. corpus callosum Gray matter, cerebral cortex Isolated synaptoromes Synaptic mernbrancs with high AChE activity Muscle Sarcoplastic reticulum Electroplax
FIG.1. Thin layer chromatogram (Silica Gel G )of dialyzed upper phase solids of frog retinal rod outk segments (Lane 4). Ganglioside standards: Lanes (1) G M Z ; (2) G M; ~ (3) GI, and GI,,; and (5) mixed bovine brain gangliosides. The tracing shows sialic acid-containing spots detected by the resorcinol spray. Throughout this paper the ganglioside nomenclature proposed by SWNNERHOLM (1963) has been used.
Reference. species
pg:mp
pghg
protein
wet wt
00062 0.0124
00027
4, frog
090
0083
B. frog C. calf
2 01 057
0.179 0 17
D, human
556
0 57
D, human
pg:mg
dry u't
1.5
C. calf
6-0
E. ox
28.4
E, ox 0-040
0.6I 3
F, rabbit F. rabbit a0148 G. €lei rrophvrus rlr
SHORT COMMUNICATION
Gangliosides in frog retinal rod outer segment membranes (Received 10 June 1975. Accepted 2 September 1975) GANGLIOSIDES were analyzed in preparations of intact retinal rod outer segments of frogs. Such preparations represent as pure a neuronal photoreceptor plasma membrane as can readily be obtained. They are devoid of synapses and have the interesting physiological characteristic of being steadily permeable to sodium ions in the dark; illumination appears to cause the sodium ion channels to close and the membrane to hyperpolarize (BAYLOR& OBRYAN,1971). Calcium ions mimic the effect of light and have been suggested to be the agent involved in clos& YOSHIKAMI, 1974). The ing the sodium channels (HAGINS mixed hydrophilic, lipophilic structure of gangliosides would seem to fit them for a possible role in ion movement through membrane channels. The solubility of gangliosides is influenced by the major physiological cations, the calcium salt being more lipophilic than the salts of sodium, potassium or magnesium, and calcium can effectively compete with the other ions for association with gangliosides 1965). Studies of gangliosides in (QumLes & FOLCH-PI, specialized membranes such as those of the rod outer segment may eventually provide clues to the functions of these glycolipids. MATERIALS AND METHODS
Preparation of intact retinal rod outer segments. Frogs (Rana pipiens) were dark adapted 24 h and the retinas dissected under red light. Rod outer segments were detached by magnetic stirring (LOLLEY& HESS,1969). Ten retinas were placed in SO ml of pH 64 buffer containing 04)059 M-Na,HP04, 0.0108 M-NaH2P04, 0.044 M-NaCl, and 0.2 M-SUCTOSC, in a specially flat-bottomed Bellco trypsinization flask. The retinas floated from one compartment of the flask to another as the fluid was agitated by a magnetic stirring bar and plate. Rod outer segments were thus detached by a gentle procedure that did not disrupt the integrity of the remaining retina. The particular buffer used maintained the retinas intact, the phosphate being the essential ingredient. In 15 min, a yield of 200,000 to 300,000 outer segments per retina was obtained. The retinas and stirring bar were removed by forceps and the outer segment suspension was filtered through 67, 25 and 10 pm nylon mesh, in succession. No neuronal cells or synaptosomes were ever observed in such suspensions by phase contrast or Nomarsky microscopic examination. The outer segments were collected by low speed centrifugation (5 min, 1500 g) and washed twice with sugar-free frog saline solution (70 and 10 ml portions) before the pellet was used for lipid extraction. Supported in part by grants from the National Institutes of Health, NS-07297, NS-06370 and FR05484.
Preparation of ganglioside fraction. The outer segment pellet from 10 retinas was resuspended in 100 pI of frog saline and 2.9 ml of chloroform-methanol (2: 1, v/v) werc added, mixed and allowed to stand at 4°C in the dark for 18 h or longer. Methanol was then added to convert the chloroform-methanol ratio to 1: 1 (v/v); the outer segment residue was readily centrifuged from this lower density solvent, and the supernatant fluid was pipetted off. The residue was extracted for 15 min with chloroforn+ methanol (1:2, v/v) and the first and second extracts were combined. The resulting extract was restored to a volume ratio of 2: 1 chloroform-methanol and partitioned by adding lj5 its volume of water; the upper phase was removed after centrifuging and the lower phase was washed twice with a theoretical upper phase (chloroformmethanol-watcr, 3:48:47, viv) containing a low concentration of KCI (0.015 M), equal in volume to the upper phase removed. The 3 upper phases were combined. A total of 33 pellets from 330 frog retinas were extracted and the upper phases combined for dialysis. The upper phase was concentrated by a rotary evaporator to a volume of 25 ml and dialyzed at 4°C against distilled water, stirred magnetically in a 1 liter glass-stoppered cylinder. The 3 liters of dialysate obtained by 3 days of dialysis were saved and concentrated to check for possible loss of ganglioside from the bag; none was found by thin layer chromatography. The dialysand was concentrated by lyophilization and the gangliosides were dissolved in 2: 1 chloroforn-methanol containing 5% water, for application to thin layer plates. Thin layer chromatography. Plates 3 3/4 inches wide and 20 inches long were coated with Silica Gel G (250 pm thick layer). The tank for development consisted of a 4 liter cylinder, lined with chromatography paper and covered with a single square of heavy duty aluminum foil held snugly with a rubber band. The solvent system was chloroform-methanel-2.5 N-ammonium hydroxide (60:40:9, v/v). The plate was developed once to a height of 11 in (4 h), air dried, and developed again to a height of 18 in (14 h) at a constant temperature of 21°C. Standards consisting of bovine brain gangliosides, Tay Sachs G u l and disialogangliosides (GD,,and ganglioside (GM2), GDlbjfrom Supelco, Inc. (Bellefonte, PA.) were co-chromatoyaphed. Sialic acid-containing spots were detected by resorcinol-HCI spray. A clean cover plate was clamped on the sprayed plate, which was then heated in air in an oven at 110°C until a blue-purple color developed (about 20 minj. Microassay of sialic acid. Sialic acid was determined by & a microversion of the resorcinol method of MIETTINEN TAKKI-MUUKAINEN (1959). Immediately after thin layer chromatography of the outer segment ganglioside fraction,
62 1
622
Short communication
each spot containing sialic acid was scraped off and placed in a 0.5 ml glass-stoppered microtube. Corresponding-sized blank areas of the Silica Gel layer and Calbiochem standards of N-acetylneuraminic acid (the only type found in frog neural tissues) were run for comparison. The samples were reheated with resorcinol reagent as suggested by MACMILLAN & WHERRETT(1969), who found that only 5510% of the sialic acid reacted on the plate, but that reheating gave close to 100% recovery of chromophore; immediate analysis was essential for this good recovery. To the microtubes 100 pI of resorcinol reaction mixture was added and mixed. The tubes were stoppered and placed in a boiling water bath; after 10 min the tubes were removed, remixed, and replaced for 10 min more. The color was extracted with 125 jd butylacetate-butyl alcohol (85: 15, v/v) and read at 580 and 450 nm in a spectrophotometer equipped with pinhole for use of quartz microcuvettes (1.5 x 10 x 25 mm). The same thin layer chromatographic pattern was observed in 2 experiments. RESULTS AND DISCUSSION The dialyzed upper phase solids from 662 million retinal rod outer segments (from 330 frog retinas) were redissolved in 2: 1 chloroform-methanol containing 5% water and run by thin layer chromatography on Silica Gel G. After exposure to Iz vapor, the sample showed 2 small spots migrating parallel to di- and tri-sialogangliosides of bovine brain (GD~,,and GTI). These spots turned blue-purple when sprayed with resorcinol reagent (Fig. l), indicating the presence of sialic acid. The spots were scraped from the plate and sialic acid was determined quantitatively. Sialic acid in the disialoganglioside spot was equivalent to Q46 nmol and that in the trisialoganglioside spot to 0-33 nmol
TABLE1. GANGLIOSIDE SIALIC
ACID I N FROG RETINAL ROD OUTER SEGMENTS
Mode of expression Of
sialic acid ',,
Localization in both outer membrane and disks
Localiaatlon in outer membrane only'
0.00062 0.0062 00124 0.0027 8-6 Y l o - " 1.19 x 10-" 7.17 x 10"
0.ll 11 23
of dry wt'
i w m g dry &ng protein' pgimg wet weight' Mol/Kg wel weight' Molirod outer segment Moleculeshd outer segment
Dry wt or solids are assumed to be 43% of wet wt (or vol) and proteins about 50% of dry wt. Isolated rod outer segments average 594 pg dry wt (LOLLEY& HESS, 1969). The total sample consisted of 66.2 x lo6 rod outer segments (39.3 mg dry wt) and contained 0.79 nmol of ganglioside sialic acid. For determination of the percentage of the total membrane surface that was external membrane, the average rod was assumed to be composed of 1900 disks with a repeat distance of 200 A, a length of 38 pm, a surface area of 150,000 prn' (NILSSON,1965), and a mean dia of 6.5 pm. The external membrane (walls and one end), therefore, had an area of 809 pm2 and represented 054% of the total membrane area.
when calculated for the whole samplc. The original sample (dry wt of 39.3 mg: lower phase lipids, 18.47 mg) thus contained 0.79 nmol of ganglioside sialic acid. The concentration of sialic acid of gangliosides (Table 1) was much lower in retinal rod outer segments than in any other type of nervous tissue in which gangliosides have been reported. The concentration of ganglioside sialic acid was 8.6 pmol/Kg wet wt, or about 11290 as great as the level of rhodopsin (2.5 mmol/L) measured in that organelle
TABLE2. COMPARISON OF GANGLIOSIDE SlALlC ACID CON'CENTRATIONS IN RETINAL ROD OUTER SEGMENTS AND OTHER NEURAL AND MUSCULAR STRUCTURES Siaiic a c d Tissue Rod outer aegrnents Whole brain Rod outer segments Whole retina White matter. corpus callosum Gray matter, cerebral cortex Isolated synaptoromes Synaptic mernbrancs with high AChE activity Muscle Sarcoplastic reticulum Electroplax
FIG.1. Thin layer chromatogram (Silica Gel G )of dialyzed upper phase solids of frog retinal rod outk segments (Lane 4). Ganglioside standards: Lanes (1) G M Z ; (2) G M; ~ (3) GI, and GI,,; and (5) mixed bovine brain gangliosides. The tracing shows sialic acid-containing spots detected by the resorcinol spray. Throughout this paper the ganglioside nomenclature proposed by SWNNERHOLM (1963) has been used.
Reference. species
pg:mp
pghg
protein
wet wt
00062 0.0124
00027
4, frog
090
0083
B. frog C. calf
2 01 057
0.179 0 17
D, human
556
0 57
D, human
pg:mg
dry u't
1.5
C. calf
6-0
E. ox
28.4
E, ox 0-040
0.6I 3
F, rabbit F. rabbit a0148 G. €lei rrophvrus rlr
