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Influence of temperature changes on the ability of gangliosides to complex with Ca2+
J. therm. BioL Vol. 5. pp. 243 to 247 Pergamon Press Ltd 1980. Printed in Great Britain
INFLUENCE OF TEMPERATURE CHANGES ON THE ABILITY OF GANGLIOSIDES TO COMPLEX WITH Ca 2+ W. PROBS~rand H. R,~aCMA~.'~ Zoological Institute, University of Stuttgart-Hohenheim, 7000 Stuttgart 70 (Hohenheim), Federal Republic of Germany (Received 22 March 1980; accepted in revised form 31 May 1980)
Al~tract--l. The influence of temperature changes on Ca2+-binding to brain ganglioside mixtures of different polarity, to single gangliosides (Gas, G,,sa, GT,s) and to their deceramide was investigated potentiometrically by means of ion-selective electrodes. 2. Following cooling (3.5°C/min.)from 37 to 13°C the CaZ+-binding to gangliosides, except GM,, was increased (7-30%). 3. Subsequent rewarming from 13 to 37°C resulted in up to 100% release of previously-bound Ca'+. 4. When comparing the maximal absolute binding difference of Ca' + to gangliosides during temperature changes a decrease of these differences could be stated which corresponds to an increase in the polarity of the gangliosides. 5. From these experiments it is concluded that a higher polarity of neuronal gangliosides is responsible for a lower thermal sensitivity of Ca2+-binding to these compounds. This may be involved in the process of thermal adaptation of ¢ctothermic vertebrates.
INTRODUCTION
taken for experiments. The absence of metallic cations (concentration below 10- ~ M) in these substances had been proved by atomic absorption spectrometry (Perkin-Elmer, type 703).
GANGLIOSIDES,glycosphingolipids containing different amounts of sialic = neuraminic acid (NeuAc), are highly enriched in neuronal elements of the CNS, parExtraction and purification of oangliosides ticularly in its synaptic membranes (Eichbcrg et al., In order to obtain ganglioside mixtures of different com1964; Wiegandt, 1976; Lapetina et al., 1968; Whittaker & Greengard, 1971 ; Morgan et ai., 1973, 1976). position and polarity, brain gangliosides were extracted from I day and 3 week old chicken brain (Gallus doraesThese gangliosides have been shown previously to ticus). According to Rgsner et al. (1979) the portion of possess a pronounced ability to bind calcium (Behr & polar gangliosides decreases concomittantly with an inLehn, 1973; Probst et al., 1979) making these glyco- crease of less polar fractions during chicken development. lipids candidates for a possible specific participation Brain gangliosides from rainbow trout (5almo gairdneri) in Ca2+-dependcnt events of synaptic transmission captured at summer and winter, respectively, were also and thermal adaptative processes (Rahmann et al., analyzed since these also show large differences in their 1975; 1976; Rahmann, 1978, 1979). Brain gangliosides pattern configurations (Hilbig et al., 1979). For extraction, participate in the acclimatization of poikilothermic 30-50 animals were decapitated, the brains being quickly vertebrates to lowered environmental temperatures by removed, deep frozen and stored at -20°C. Gangliosides were extracted according to the method of Tettamanti et means of the formation of more polar, polysialilated al. (1973). After dialysing (3 days against 4°C water) purififractions, which probably enables a more effective cation of gangliosides was carried out by means of silica transmission process to occur under low temperature gel columns according to the method of Holm (1968). The conditions (Hilbig et al., 1979; Hilbig & Rahmann, purified ganglioside mixtures, as well as single gangliosides, 1979; RiSsner et al., 1979). Nevertheless the molecular their sialo-oligu-saccaddes and free NeuAc, were finally basis of these adaptative mechanisms, up to now, is chromatographed down ion-exchange columns (Dowex 50W x 8.5-100 mesh, protonized; Serva, Heidelberg) and still unknown. In order to elucidate these phenomena in the present paper the influence of temperature lyophilized. The concentration of ganglioside-NeuAc was measured changes on the Ca'+-binding abilities of differently according to the method of Jourdian et al. (1971) and the composed brain gangliosides, single ganglioside composition of gangliosid© mixtures determined by TLCspecies and their deceramide derivatives were investi- analysis as described by Hilbig et al. (1979; compare Fig. I gated in vitro by means of potentiometrical pro- and Table 1). cedures. The arrangement of the four ganglioside mixtures according to an increase of their polarity yields the following sequence: brain gangliosides from: 3 week old chicken MATERIALS AND METHODS < summer trout < i day old chicken < winter trout. Substances Different ganglioside mixtures (see below), single ganglioside fractions (Gul, Gal=, GTlb; Sup61co Inc., Bellefonte), deceramide derivatives of Gut and Gas, (DcC-Gu~, DeC-Gas.; g/fts from Professor Dr "H. Wiegandt, University of Marburg) and free NeuAc (Merck, Darmstadt) were
243
Potentiometry Ganglioside-Ca'÷ "cocktails" (0.1 mM gangliosideNeuAc; 0.1 mM CaCI~ in 5raM triethanolamine-HCl buffer, pH 7.3) were taken for experiments. The ganglioside concentration used here is greater than the critical micellar concentration (Formisano et aL, 1979). In the above experi-
W. PROBSTand H. RAHMA~N
244
Table 1, Relative composition of the ganglioside mixtures from adult and I day old chicken brain (Gallus domesticus). Single ganglioside fractions are listed as percentage of total ganglioside-NeuAc
Ganglioside fraction
GMa Gas GMI Go3
Gvl, GD2 GDIb GTlb GQt~, Oe Gx
Chicken 1 day 3 weeks (%) (%) 3.6 -15.8
1.7 8.0 16.4
25.8 5.8 7.1 23.5 13.4
25.4 5.9 7.6 20.8 11.5 2.7
4.3 0.7
merits this was established using miceliar induced toluidin blue-metachromas~¢ (unpublished results). The concentration of free Ca z* was measured potcntiometricaUy by means of a Ca 2*-se|ective fluid membrane electrode (Orion type 93-20) connected to a highly sensitive mV-meter (Orion model 901). A silver-silver chloride electrode (Orion 90-01), containing 1 M triethanolamine-hydrochloride saturated with Ag* as bridge, was used for reference. The mV-meter was connected to a muitichannel recorder (Kontron W + W Recorder 316) by means of a thermogouple (0.5 mm ¢, Thermocoax, Philips). 5 ml of the gangliosidc-Ca z" "cocktaiis" described above were measured, at 37 + 0.2°C, into a double-sided tempered polypropylen¢ beaker mounted on a magnetic stirrer * IUPAC-IUB nomenclature of lipids (1976). Biochem. J. (1978) 171, 21-35.
with constant stirring rate. The whole measuring equipment was electrically grounded and screened by a Faraday cage. For each measurement a calibration curve was carried out by adding known concentrations of Ca 2 ÷ to buffer without Ca 2+-chelator. A programmable calculator (Hewlett Packard 9825 A) with peripheral apparatus (printer, plotter, floppy disk) was connected to the measuring equipment so that data could be handled "on line".
Temperature influence The ganglioside-Ca 2+ "cocktails" were cooled by a cryothermostat with a rate of 3.5°C/min from 37 to 13°C and rewarmed again to 37°C at the same rate. Ca~+-conccntration expressed in mV, was registered every 3°C, and converted into tool/! (M) after subtracting the temperature effect by means of a calibration curve over the full temperature range as seen above.
Nomenclature of gangliosides The fractions were named according to Svennerholm (1963). The corresponding nomenclature according to IUPAC-IUB* is given below: GMI = II~NeuAc-GgOse4-Cer Gnl= = IV3NeuAc, lI3NeuAc--GgOs¢4-Cer Gr Ib = IVaNeuAc, II3(NeuAc)z-GgOse4-Cer D¢C--G~I = [13NeuAc-g3gOse,L DeC-Gal, = IV3NeuAc, II3NeuAc-GgOse4 NeuAc = N-acetyl neuraminic acid
RESULTS In a first set of experiments the binding abilities of the four different ganglioside mixtures, single ganglioside fractions, their deeeramide-dcdvatives and free N e u A c to Ca 2+ were tested for a Ca :+- and ganglioside-NeuAc concentration bf 0.1 m M at 37:1: 0.2°C. As can be seen from Table 2 the ganglioside mixture 10
3
5
/
summer trout ,,
summer trout
.
winter trout
Fig. 1. Thin.layer chromatogram and densitogram from brain ganglioside mixtures of summer and winter trout (Salmo gairdneri). Ganglioside bands with identical migration rates to standards were additionally named according to Svennerholm (1963).
Temperature changes with
Amount of Ca "+ bound to ganglioside (x 10 -~ M)
Ganglioside mixtures of" 1 day old chicken 3 week old chicken Winter trout Summer trout
7.4 6.6 5.4 2.6
Single compounds: NeuAc DeC-GM~ DeC-Go~.
6.4 0.8 6.9
Gu~ Go~.
4.5
TTZ b
l.l
245
gangliosides
tures of 3 week old chicken, winter and summer trout. Among single gangliosides Got, is found to bind most Ca 2*. The large differences concerning the binding abilities of the various compounds to Ca' + demonstrate the effect of different molecular configuration, since the Ca 2 +- and NeuAc-concentrations of all substances investigated was the same. The results confirm very well previous data of Probst et al. (1979). In a further set of experiments the influence of changing temperatures on the Ca2*-binding ability of the various substances was tested. As can be seen from Fig. 2 the CaZ+-binding abilities of the single ganglioside fractions, their deceramide-derivatives and also of free NeuAc showed only slight effects (Fig. 2a). Nevertheless cooling caused an increase of Ca2+-binding to DeC-Go~, and also to GT=b with an amount of 7 and 50~/~ respectively, and a decrease of about 50 and 100% in the case of Gu, and DeC--Gut, respectively. The binding properties of Go~, did not change at all. By subsequent warming Ca 2* was released from all gangliosides and their derivatives except DeC-Gu=
Table 2. Amount of Ca ~+ bound to gangliosides at 37°C (NeuAc-Ca ~÷ isomolar at 0.1 mM) Compounds
Ca 2 ÷
1.0
of I day old chicken brain showed the greatest Ca 2+binding ability with a value of 7.4 x 10 - s M of bound Ca 2+ followed by the brain ganglioside mix-
8
DeCTGDI a .+.......+..._+.......+.._.+_.....÷.~...e..~,.g...~.. ........ ÷ . . , , . . ÷ , . . . . . + . . . . . . + .....
6
I
NeuAc
o c.) "--. . . . . . . . . . . . . . . . .
-... GDIo
'
GM1
2
'
,~--~,P~*"'~ !\m...GTIb DeC-GMI o/
37
. . . . 31
~ 25
19
,
+ -~,'--~,-'~.~"~,: 13 19 25 31
+-
37
time [mini
,0J bc h i c k e n ( 3 w e e k s o l d ) , i "1
~
.f
.~ . chicken (l day old)
.j'%uA
:
\'"
} -"-÷-- -,. ~ -÷ ---.÷-.,..-'÷.... .+ .............. -t,I .............~ ............+ .............. +
X
I rainbow trout(winter} I
zl
roinbow trout (summe~
0l
. . . . . . .
",7
31
2s
! 19
13
%-
,
. 19
, t.empem.turet'C! 2s
3t
37
time [mini Fig. 2. Influence of temperature changes on Ca 2 *-binding abilities of gangliosides (0. l mM gangliosideNeuAc, 0.1 mM Ca z÷ m triethanolamine-HCI buffer, pH 7.3; cooling from 37 to 13°C and subsequent rewarming with a rate of 3.5°C/min). (a) Single ganglioside species and deceramide derivates (b) Ganglioside mixtures. Mean values of 2-3 measurements each. T.B. 514--E
W. PROBSTand H. RAHMANN
246
with amounts of about 10% in the case of DeC-Got,, 20% in that of Gn~,, up to 70% for GMI and 90% for Grlb whereas the binding to free NeuAc did not change at all. Comparing the maximal absolute binding differences of Ca 2+ to the single gangliosides (except to the Dec-derivatives and to free NeuAc), following temperature changes, a decrease of binding could be seen corresponding to an increase in the polarity of the gangliosides. In this connection it was of special interest to investigate the influence of changing temperatures on the Ca2+-binding to natural brain ganglioside mixtures with different polarity (Fig. 2b). Generally their Ca 2 +binding abilities showed the same tendency as seen above for single gangliosides, giving evidence for an increase of Ca2÷-binding by cooling and a Ca 2+release by subsequent warming. The relatively nonpolar ganglioside mixture from the brain of 3 week old chicken, however, was influenced most markedly by temperature changes: in this case up to 30% of additional Ca 2 + was bound to this mixture after cooling and up to 65% was released following subsequent warming. The values for the other ganglioside mixtures were lower according to their increased polarity. With regard to the thermal sensitivity the registration of the maximal percental and absolute variations of Ca2+-binding to the four different mixtures reveals the sequence: brain ganglioside mixture of: 3 week old chicken > summer trout > 1 day old chicken > winter trout. Thus it was demonstrated that higher polarity of gangliosides is responsible for lower thermal sensitivity of Ca2+-binding to these neuronal compounds. DISCUSSION Summarizing the results of this in vitro-study for both ganglioside mixtures and single ganglioside species it can be said that the mieellar configuration of the gangliosides, which is related to the composition, molecular constitution and polarity (Formisann et al. 1977), is assumed to be responsible for the effects of temperature on Ca z +-binding. This assumption is supported by the fact that, the Ca 2+-binding to free NeuAc and the deceramide-derivatives of gangliosides (with exception to DeC-Gul) which are soluble and therefore do not form micelles, was modified only slightly. Taking into consideration that the degree of association can be changed by altered molecular mobility following temperature changes these last slight effects are quite understandable (De Fontaine et al., 1977). By cooling micelles their molecular packing may become more solid and also they may fuse to larger aggregates (De Fontaine et al., 1977). Thus more Ca 2+ may be bound or associated to gangliosid¢ micelles. By rewarming the cooling effects are reversed, the molecular mobility rises again and weakly bound Ca 2+ is released partially above the original level. For more polar gangliosides these effects are not so pronounced, because the higher negative changes and their molecular configuration may prevent greater variations of molecular packing following temperature changes. In vivo gangliosides are probably incorporated in synaptic membranes in the form of clusters (Sharom
et al., 1977, 1978), which may have similar configurations and therefore react in a similar way to micelles thought to be formed in the present experiments. The data demonstrate, that an increase in the polarity of gangliosides (more NeuAc per ganglioside molecule or in the case of ganglioside mixtures a higher portion of more polar fractions) leads to a decrease in the thermal sensitivity of Ca 2 Z-binding to these molecules. Accordingly the previously shown polysialilation of neuronal membranes of ectothermic vertebrates in adaptation to lowered ambient temperature (Hilbig et al., 1979), may be considered to be part of a molecular adaptation mechanism (Rahmann, 1978, 1979). This may not be particularly important in regulating CaZ+-related processes at synapses as, for instance, the process of neuronal transmission (Reckhaus et al., 1979). Acknowledgement--Supported by Deutsche Forschungsgemeinschaft.
a
grant of the
REFERENCES BeHR J.-P. & LEtlN J.-M. (1973) The binding of divalent cations by purified ganglios~des. FEBS Lett. 31,297-300. DE FONTAtNED. L., RO~ D. K. & TeltN/d B. (1977) Two improved methods for the determination of association constants and thermo dynamic parameters. The inter. actions of adenosine 5-monophosphate and tryptophan. Physiol. Chem. gl, 792-793. EICHBERGJ., WHITTAKERV. P. & DAW.~g3NR. M. C. (1964) The distribution of lipide in subcellular particles of guinea-pig brain. Biochem. 2. 92, 91-100. FORMlSANOS., JOHNSONM. L., LEEG., ALOJS. M. & EDELHOCH H. (1979) Critical micelle concentrations of gangliosides. Biochemistry !$, I 119-1124. HILmG R. & R . ~ N N H. (1979) Changes in brain ganglioside composition of normothermic and hibernating golden hamster (Mesocricetus auratus). Comp. Biochem. Physiol. 61B, 527-531. HILmG R., RAH~NN H. & R6SNER H. (1979) Brain gangliosides and temperature adaptation in eury- and stenothermic teleost fish (carp and rainbow trout). J. therm. Biol. 4, 29-34. JOURDEAN G., D ~ L. & ROSF.S~N S. (1971) The sialic acids: XL A perjodate-resorcinol method for the quantitative estimation of free sialic acids and their glycosides. 2. biol. Chem. 246, 430-435. LAP~"nNAE. G., SOTOE. F. & DE ROB~TlS E. (1968) Lipids and proteolipids in isolated subcellular membranes of rat brain cortex. J. Neurochem. 15, 437-445. MORGANI. G., ZANETTAJ. P., BRECKENRIDGEW. C., VINC~qDON G. & GOMZ~SG. (1973) The chemical structure of synaptic membranes. Brain Res. 62, 405-41 I. MORGAN I. G., TETTX~N~ G. & GOMaOSG. (1976) Bioch,anical evidence on the role of gangliosides in nerve ending. Adv. exp. Med. Biol. 71, 137-150. PROI~T W., R~NER H., WIEOANDT H. & R ~ N N H. (1979) The complexing ability of gangliosides for Ca 2+, I. Influence of mono- and divalent cations and of acetylcholine. Hoppe-Seyler's Z. physiol. Chem. 360, 979-986. R~a~tAm~ H., R ~ E a H. & BtumR H. (1976) A functional model of sialo-glycomacromol¢cules in synaptic transmission and memory formation. J. theor. 8ioL 57, 231-23"/. RAHU~t,m H. (1976) Possible functional role of gangliosides. Adv. exp. Med. Biol. 71, 151-161. RAHUXNN H. (1978) Gangliosides and thermal adaptation in vertebrates. ,lap. J. exp. Med. 48, 85-96.
Temperature changes with Ca' + gangliosides
247
RAHMANNH. (1979) Gangliosides and thermal adaptation. SVENNERHOLM L. (1963) Chromatographic separation of human brain gangliosides. J. Neurochem. 10, 613-623. In Structure and Function of Gangliosides (Edited by DREYFUS H., MANDEL P., SVENNERHOLMF. • URBAN TE'I'I'AMANTIG., BONALLI F., MARCHE$1NIS. £" ZAMBOTTI V. (1973) A new procedure for the extraction, purificaP. F.) Plenum, New York. in press. tion and fractionation of brain gangliosides. Biochim. REC~AUS W., R6~.R H. & RAH~t~NN H. (1979) Effect of biophys. Acta 296, 160-170. cooling on photic evoked responses in the optic tectum WIEGANDTH. (1976) The subcellular localization of gangof teleosts. J. therm. Biol. 4, 209-212. liosides in the brain. J. Neurocher~ 14, 671-674. R6SNER H., SrGLER K. & RAHMANNH. (1979) Changes of brain gangliosides in chicken and mice during hetero- WmTTAKERV. P. & GREENGARDP. (1971) The isolation of synaptosomes from the brain of a teleost fish. J. Neurothermic development. J. therm. Biol. 4, 121-124. chem. 18, 173-176. SHAROMF. J. & Gt)o~NTC. W. M. (1979) A ganglioside spin label: ganglioside headgroup interactions. Biochem. biophys. Res. Commun. 74, 1039-1045. Key Word lndex--Ganglioside-Ca 2 + complex; temperaSH^ROM F. J. & GRANT C. W. M. (1978) A model for ganglioside behaviour in cell membranes. Biochim. bio- ture; potentiometry; Gallus domesticus; Salmo gairdneri; brain. phys. Acta 507, 280-293.
INFLUENCE OF TEMPERATURE CHANGES ON THE ABILITY OF GANGLIOSIDES TO COMPLEX WITH Ca 2+ W. PROBS~rand H. R,~aCMA~.'~ Zoological Institute, University of Stuttgart-Hohenheim, 7000 Stuttgart 70 (Hohenheim), Federal Republic of Germany (Received 22 March 1980; accepted in revised form 31 May 1980)
Al~tract--l. The influence of temperature changes on Ca2+-binding to brain ganglioside mixtures of different polarity, to single gangliosides (Gas, G,,sa, GT,s) and to their deceramide was investigated potentiometrically by means of ion-selective electrodes. 2. Following cooling (3.5°C/min.)from 37 to 13°C the CaZ+-binding to gangliosides, except GM,, was increased (7-30%). 3. Subsequent rewarming from 13 to 37°C resulted in up to 100% release of previously-bound Ca'+. 4. When comparing the maximal absolute binding difference of Ca' + to gangliosides during temperature changes a decrease of these differences could be stated which corresponds to an increase in the polarity of the gangliosides. 5. From these experiments it is concluded that a higher polarity of neuronal gangliosides is responsible for a lower thermal sensitivity of Ca2+-binding to these compounds. This may be involved in the process of thermal adaptation of ¢ctothermic vertebrates.
INTRODUCTION
taken for experiments. The absence of metallic cations (concentration below 10- ~ M) in these substances had been proved by atomic absorption spectrometry (Perkin-Elmer, type 703).
GANGLIOSIDES,glycosphingolipids containing different amounts of sialic = neuraminic acid (NeuAc), are highly enriched in neuronal elements of the CNS, parExtraction and purification of oangliosides ticularly in its synaptic membranes (Eichbcrg et al., In order to obtain ganglioside mixtures of different com1964; Wiegandt, 1976; Lapetina et al., 1968; Whittaker & Greengard, 1971 ; Morgan et ai., 1973, 1976). position and polarity, brain gangliosides were extracted from I day and 3 week old chicken brain (Gallus doraesThese gangliosides have been shown previously to ticus). According to Rgsner et al. (1979) the portion of possess a pronounced ability to bind calcium (Behr & polar gangliosides decreases concomittantly with an inLehn, 1973; Probst et al., 1979) making these glyco- crease of less polar fractions during chicken development. lipids candidates for a possible specific participation Brain gangliosides from rainbow trout (5almo gairdneri) in Ca2+-dependcnt events of synaptic transmission captured at summer and winter, respectively, were also and thermal adaptative processes (Rahmann et al., analyzed since these also show large differences in their 1975; 1976; Rahmann, 1978, 1979). Brain gangliosides pattern configurations (Hilbig et al., 1979). For extraction, participate in the acclimatization of poikilothermic 30-50 animals were decapitated, the brains being quickly vertebrates to lowered environmental temperatures by removed, deep frozen and stored at -20°C. Gangliosides were extracted according to the method of Tettamanti et means of the formation of more polar, polysialilated al. (1973). After dialysing (3 days against 4°C water) purififractions, which probably enables a more effective cation of gangliosides was carried out by means of silica transmission process to occur under low temperature gel columns according to the method of Holm (1968). The conditions (Hilbig et al., 1979; Hilbig & Rahmann, purified ganglioside mixtures, as well as single gangliosides, 1979; RiSsner et al., 1979). Nevertheless the molecular their sialo-oligu-saccaddes and free NeuAc, were finally basis of these adaptative mechanisms, up to now, is chromatographed down ion-exchange columns (Dowex 50W x 8.5-100 mesh, protonized; Serva, Heidelberg) and still unknown. In order to elucidate these phenomena in the present paper the influence of temperature lyophilized. The concentration of ganglioside-NeuAc was measured changes on the Ca'+-binding abilities of differently according to the method of Jourdian et al. (1971) and the composed brain gangliosides, single ganglioside composition of gangliosid© mixtures determined by TLCspecies and their deceramide derivatives were investi- analysis as described by Hilbig et al. (1979; compare Fig. I gated in vitro by means of potentiometrical pro- and Table 1). cedures. The arrangement of the four ganglioside mixtures according to an increase of their polarity yields the following sequence: brain gangliosides from: 3 week old chicken MATERIALS AND METHODS < summer trout < i day old chicken < winter trout. Substances Different ganglioside mixtures (see below), single ganglioside fractions (Gul, Gal=, GTlb; Sup61co Inc., Bellefonte), deceramide derivatives of Gut and Gas, (DcC-Gu~, DeC-Gas.; g/fts from Professor Dr "H. Wiegandt, University of Marburg) and free NeuAc (Merck, Darmstadt) were
243
Potentiometry Ganglioside-Ca'÷ "cocktails" (0.1 mM gangliosideNeuAc; 0.1 mM CaCI~ in 5raM triethanolamine-HCl buffer, pH 7.3) were taken for experiments. The ganglioside concentration used here is greater than the critical micellar concentration (Formisano et aL, 1979). In the above experi-
W. PROBSTand H. RAHMA~N
244
Table 1, Relative composition of the ganglioside mixtures from adult and I day old chicken brain (Gallus domesticus). Single ganglioside fractions are listed as percentage of total ganglioside-NeuAc
Ganglioside fraction
GMa Gas GMI Go3
Gvl, GD2 GDIb GTlb GQt~, Oe Gx
Chicken 1 day 3 weeks (%) (%) 3.6 -15.8
1.7 8.0 16.4
25.8 5.8 7.1 23.5 13.4
25.4 5.9 7.6 20.8 11.5 2.7
4.3 0.7
merits this was established using miceliar induced toluidin blue-metachromas~¢ (unpublished results). The concentration of free Ca z* was measured potcntiometricaUy by means of a Ca 2*-se|ective fluid membrane electrode (Orion type 93-20) connected to a highly sensitive mV-meter (Orion model 901). A silver-silver chloride electrode (Orion 90-01), containing 1 M triethanolamine-hydrochloride saturated with Ag* as bridge, was used for reference. The mV-meter was connected to a muitichannel recorder (Kontron W + W Recorder 316) by means of a thermogouple (0.5 mm ¢, Thermocoax, Philips). 5 ml of the gangliosidc-Ca z" "cocktaiis" described above were measured, at 37 + 0.2°C, into a double-sided tempered polypropylen¢ beaker mounted on a magnetic stirrer * IUPAC-IUB nomenclature of lipids (1976). Biochem. J. (1978) 171, 21-35.
with constant stirring rate. The whole measuring equipment was electrically grounded and screened by a Faraday cage. For each measurement a calibration curve was carried out by adding known concentrations of Ca 2 ÷ to buffer without Ca 2+-chelator. A programmable calculator (Hewlett Packard 9825 A) with peripheral apparatus (printer, plotter, floppy disk) was connected to the measuring equipment so that data could be handled "on line".
Temperature influence The ganglioside-Ca 2+ "cocktails" were cooled by a cryothermostat with a rate of 3.5°C/min from 37 to 13°C and rewarmed again to 37°C at the same rate. Ca~+-conccntration expressed in mV, was registered every 3°C, and converted into tool/! (M) after subtracting the temperature effect by means of a calibration curve over the full temperature range as seen above.
Nomenclature of gangliosides The fractions were named according to Svennerholm (1963). The corresponding nomenclature according to IUPAC-IUB* is given below: GMI = II~NeuAc-GgOse4-Cer Gnl= = IV3NeuAc, lI3NeuAc--GgOs¢4-Cer Gr Ib = IVaNeuAc, II3(NeuAc)z-GgOse4-Cer D¢C--G~I = [13NeuAc-g3gOse,L DeC-Gal, = IV3NeuAc, II3NeuAc-GgOse4 NeuAc = N-acetyl neuraminic acid
RESULTS In a first set of experiments the binding abilities of the four different ganglioside mixtures, single ganglioside fractions, their deeeramide-dcdvatives and free N e u A c to Ca 2+ were tested for a Ca :+- and ganglioside-NeuAc concentration bf 0.1 m M at 37:1: 0.2°C. As can be seen from Table 2 the ganglioside mixture 10
3
5
/
summer trout ,,
summer trout
.
winter trout
Fig. 1. Thin.layer chromatogram and densitogram from brain ganglioside mixtures of summer and winter trout (Salmo gairdneri). Ganglioside bands with identical migration rates to standards were additionally named according to Svennerholm (1963).
Temperature changes with
Amount of Ca "+ bound to ganglioside (x 10 -~ M)
Ganglioside mixtures of" 1 day old chicken 3 week old chicken Winter trout Summer trout
7.4 6.6 5.4 2.6
Single compounds: NeuAc DeC-GM~ DeC-Go~.
6.4 0.8 6.9
Gu~ Go~.
4.5
TTZ b
l.l
245
gangliosides
tures of 3 week old chicken, winter and summer trout. Among single gangliosides Got, is found to bind most Ca 2*. The large differences concerning the binding abilities of the various compounds to Ca' + demonstrate the effect of different molecular configuration, since the Ca 2 +- and NeuAc-concentrations of all substances investigated was the same. The results confirm very well previous data of Probst et al. (1979). In a further set of experiments the influence of changing temperatures on the Ca2*-binding ability of the various substances was tested. As can be seen from Fig. 2 the CaZ+-binding abilities of the single ganglioside fractions, their deceramide-derivatives and also of free NeuAc showed only slight effects (Fig. 2a). Nevertheless cooling caused an increase of Ca2+-binding to DeC-Go~, and also to GT=b with an amount of 7 and 50~/~ respectively, and a decrease of about 50 and 100% in the case of Gu, and DeC--Gut, respectively. The binding properties of Go~, did not change at all. By subsequent warming Ca 2* was released from all gangliosides and their derivatives except DeC-Gu=
Table 2. Amount of Ca ~+ bound to gangliosides at 37°C (NeuAc-Ca ~÷ isomolar at 0.1 mM) Compounds
Ca 2 ÷
1.0
of I day old chicken brain showed the greatest Ca 2+binding ability with a value of 7.4 x 10 - s M of bound Ca 2+ followed by the brain ganglioside mix-
8
DeCTGDI a .+.......+..._+.......+.._.+_.....÷.~...e..~,.g...~.. ........ ÷ . . , , . . ÷ , . . . . . + . . . . . . + .....
6
I
NeuAc
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time [mini Fig. 2. Influence of temperature changes on Ca 2 *-binding abilities of gangliosides (0. l mM gangliosideNeuAc, 0.1 mM Ca z÷ m triethanolamine-HCI buffer, pH 7.3; cooling from 37 to 13°C and subsequent rewarming with a rate of 3.5°C/min). (a) Single ganglioside species and deceramide derivates (b) Ganglioside mixtures. Mean values of 2-3 measurements each. T.B. 514--E
W. PROBSTand H. RAHMANN
246
with amounts of about 10% in the case of DeC-Got,, 20% in that of Gn~,, up to 70% for GMI and 90% for Grlb whereas the binding to free NeuAc did not change at all. Comparing the maximal absolute binding differences of Ca 2+ to the single gangliosides (except to the Dec-derivatives and to free NeuAc), following temperature changes, a decrease of binding could be seen corresponding to an increase in the polarity of the gangliosides. In this connection it was of special interest to investigate the influence of changing temperatures on the Ca2+-binding to natural brain ganglioside mixtures with different polarity (Fig. 2b). Generally their Ca 2 +binding abilities showed the same tendency as seen above for single gangliosides, giving evidence for an increase of Ca2÷-binding by cooling and a Ca 2+release by subsequent warming. The relatively nonpolar ganglioside mixture from the brain of 3 week old chicken, however, was influenced most markedly by temperature changes: in this case up to 30% of additional Ca 2 + was bound to this mixture after cooling and up to 65% was released following subsequent warming. The values for the other ganglioside mixtures were lower according to their increased polarity. With regard to the thermal sensitivity the registration of the maximal percental and absolute variations of Ca2+-binding to the four different mixtures reveals the sequence: brain ganglioside mixture of: 3 week old chicken > summer trout > 1 day old chicken > winter trout. Thus it was demonstrated that higher polarity of gangliosides is responsible for lower thermal sensitivity of Ca2+-binding to these neuronal compounds. DISCUSSION Summarizing the results of this in vitro-study for both ganglioside mixtures and single ganglioside species it can be said that the mieellar configuration of the gangliosides, which is related to the composition, molecular constitution and polarity (Formisann et al. 1977), is assumed to be responsible for the effects of temperature on Ca z +-binding. This assumption is supported by the fact that, the Ca 2+-binding to free NeuAc and the deceramide-derivatives of gangliosides (with exception to DeC-Gul) which are soluble and therefore do not form micelles, was modified only slightly. Taking into consideration that the degree of association can be changed by altered molecular mobility following temperature changes these last slight effects are quite understandable (De Fontaine et al., 1977). By cooling micelles their molecular packing may become more solid and also they may fuse to larger aggregates (De Fontaine et al., 1977). Thus more Ca 2+ may be bound or associated to gangliosid¢ micelles. By rewarming the cooling effects are reversed, the molecular mobility rises again and weakly bound Ca 2+ is released partially above the original level. For more polar gangliosides these effects are not so pronounced, because the higher negative changes and their molecular configuration may prevent greater variations of molecular packing following temperature changes. In vivo gangliosides are probably incorporated in synaptic membranes in the form of clusters (Sharom
et al., 1977, 1978), which may have similar configurations and therefore react in a similar way to micelles thought to be formed in the present experiments. The data demonstrate, that an increase in the polarity of gangliosides (more NeuAc per ganglioside molecule or in the case of ganglioside mixtures a higher portion of more polar fractions) leads to a decrease in the thermal sensitivity of Ca 2 Z-binding to these molecules. Accordingly the previously shown polysialilation of neuronal membranes of ectothermic vertebrates in adaptation to lowered ambient temperature (Hilbig et al., 1979), may be considered to be part of a molecular adaptation mechanism (Rahmann, 1978, 1979). This may not be particularly important in regulating CaZ+-related processes at synapses as, for instance, the process of neuronal transmission (Reckhaus et al., 1979). Acknowledgement--Supported by Deutsche Forschungsgemeinschaft.
a
grant of the
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RAHMANNH. (1979) Gangliosides and thermal adaptation. SVENNERHOLM L. (1963) Chromatographic separation of human brain gangliosides. J. Neurochem. 10, 613-623. In Structure and Function of Gangliosides (Edited by DREYFUS H., MANDEL P., SVENNERHOLMF. • URBAN TE'I'I'AMANTIG., BONALLI F., MARCHE$1NIS. £" ZAMBOTTI V. (1973) A new procedure for the extraction, purificaP. F.) Plenum, New York. in press. tion and fractionation of brain gangliosides. Biochim. REC~AUS W., R6~.R H. & RAH~t~NN H. (1979) Effect of biophys. Acta 296, 160-170. cooling on photic evoked responses in the optic tectum WIEGANDTH. (1976) The subcellular localization of gangof teleosts. J. therm. Biol. 4, 209-212. liosides in the brain. J. Neurocher~ 14, 671-674. R6SNER H., SrGLER K. & RAHMANNH. (1979) Changes of brain gangliosides in chicken and mice during hetero- WmTTAKERV. P. & GREENGARDP. (1971) The isolation of synaptosomes from the brain of a teleost fish. J. Neurothermic development. J. therm. Biol. 4, 121-124. chem. 18, 173-176. SHAROMF. J. & Gt)o~NTC. W. M. (1979) A ganglioside spin label: ganglioside headgroup interactions. Biochem. biophys. Res. Commun. 74, 1039-1045. Key Word lndex--Ganglioside-Ca 2 + complex; temperaSH^ROM F. J. & GRANT C. W. M. (1978) A model for ganglioside behaviour in cell membranes. Biochim. bio- ture; potentiometry; Gallus domesticus; Salmo gairdneri; brain. phys. Acta 507, 280-293.