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1H-NMR spectra of rat synaptic plasma membranes: effect of temperature and comparison with fluorescence polarization
Brain Research, 344 ( 1985 ) 1~2-- 1~)6 ~Zl~evic~
162
BRE 21046
1
H,~
Spectra of ~ ilynaptl¢ ~ ~ll,m~: effect of t ~ M u r e c ~ ~ ~ fluorescence polarization
and
GEORGE P. KREISHMAN1and ROBERT J. HITZEMANN2 1Department of Chemistry and 2Departments of Psychiatry, Pharmacology and Cell Biophysics, University of Cincinnati, Cincinnati, OH (U.S.A.) (Accepted April 30th. t985) Key words: nuclear magnetic resonance (NMR) spectrum
synaptic membrane
fluorescence polarization
1H-NMR spectra of rat synaptic plasma membranes obtained over the temperature range of 24-46 °C are presented. The data illustrate that a transition occurs from a more ordered to less ordered state at approximately 37 °C. This phenomenon was not related to using D20 as the solvent and was replicated, although with less sensitivity, using fluorescence polarization methodology. The physical properties of synaptic plasma membranes are important elements in our understanding of such diverse p h e n o m e n a as neuronal differentiationS,6,15 and the mechanism(s) of drug action 1.3,7-1°. For the most part. two techniques have been used to examine the physical properties of synaptic membranes, fluorescence polarization (FPZ) and electron spin resonance (ESR) spectroscopy. F P Z has had especially wide application, due to its sensitivity, to the diversity of available membrane probes and to the relatively inexpensive equipment required. Furthermore. some recent studies show that measures of the steady-state fluorescence anisotropy (rs) for at least one probe, 1.6-diphenyl-l,3,5 hexatriene ( D P H ) can be converted to a measure of m e m b r a n e order without the necessity of determining the fluorescence lifetime14, 20. However. despite their advantages. FPZ and E S R are perturbing techniques which require the introduction of a probe molecule Thus, one is precisely studying probe-membrane interactions. A non-perturbing alternative to F P Z or E S R is nuclear magnetic resonance ( N M R ) spectroscopy. N M R can measure membrane motions within the slower, potentially more important time domains (104-106 s-ll than are possible with F P Z and E S R (time d o m a i n / > 108 s-l) (ref. 2). N M R can be used to probe lipid mobility at various depths within the membrane 19 or to
examine the lipid packing arrangement 4. The spectra obtained by N M R in natural membranes often reveal a broad gel to liquid-crystalline phase transition which is seen by more direct techniques e.g. differential scanning calorimetry ~3. In contrast. FPZ and ESR may under identical conditions reveal a sharp phase transition and not report the peripheral boundaries of the gel to liquid crystalline transition 13. Furthermore. N M R experiments report that protein does not perturb lipid order, suggesting a rapid exchange of the bulk and protein boundary lipidsg, 18. Differently, E S R and F P Z report that proteins immobilize membrane lipids and that the exchange between the bulk and boundary lipids is slow in reference to the time scale being used tl In the present study we present 1H-NMR Delayed Fourier Transform (DFT) 17 spectra (in D 2 0 ) of rat synaptic plasma membranes obtained at temperatures over the range of 2 4 - 4 6 °C. With the appropriate choice of spectral parameters, large spectral changes can be observed for small changes in membrane molecular dynamics. In addition, various portions of the membranes can be monitored simultaneously. These data are then compared with the results obtained by F P Z techniques over the same temperature range. Synaptic plasma membranes (SPMs) were pre-
Correspondence: R.J. Hitzemann. Department of Psychiatry, University of Cincinnati. College of Medicine. 231 Bethesda Ave.. Cincinnati. OH 45267-0559. U.S.A. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
163 pared from the rat forebrain as described previouslyS. Membrane purity, as assessed by both morphological and chemical criteria was estimated to be greater than 85%. The membranes were washed 3 times in D20-phosphate buffered saline (PBS) -pH (pD) = 8.2 and then resuspended in this buffer at a concentration of approximately 10 mg/ml. A small aliquot of this suspension was used for the protein determination essentially as described by Lowry et al. ~2. On the basis of the protein determination, the sample was adjusted to 5 mg/ml and 0.7 ml was transferred to a 5 mm N M R tube. The sample was purged with dry N~ and then sealed using epoxy glue. The sample was then frozen at - 7 0 °C until analysis. The I H - N M R spectra were obtained using a Nicolet NMC 300 MHz F T - N M R spectrometer. The delay between the excitation pulse and the start of acquisition was set at 240 Bs; (in non-sonicated lipid bilayers, the relaxation time(s) for the majority of the protons is ~ 100 ~//S) 17. Under these conditions, less than 5% of the total spectral intensity is observed when the receiver is turned on. A n y increase in the relaxation rates of the protons will lead to a marked increase in the observed spectral intensity. Spectra were obtained over a temperature range of 22-46 °(2 utilizing a NTC temperature control unit. The samples were allowed to equilibrate at each new temperature for 40 rain prior to spectral acquisition. The spectra were processed with the same scaling factor so that direct comparisons of the intensities could be made. Chemical shifts are reported relative to H O - D at 4.5 ppm for each temperature. (What appear to be chemical shifts in the lipid resonances are actually a result of the temperature dependence of the H O - D resonance. This 'shift' was compensated for in the subtraction routine.) Resonance identification was made on the basis of comparison with published spectra and by comparison with the spectra of model membranes composed of dimyrsitylphosphatidylcholine, cholesterol and gangliosides (1:0.3:0.1, molar ratio). It should be noted that under the spectral conditions employed, the protein protons, because of their fast relaxation, will not contribute significantly to the spectra. FPZ measurements were made essentially as described elsewhere s. Synaptic membranes were suspended in both D 2 0 - P B S and normal PBS in order to check for potential isotope effects. The final mem-
brane concentration was 10 j~g/ml; the probe ( D P H ) concentration was 0.025/2g/ml. Fluorescence measurements were made in a T-format fluorometer (HH-2 fluorometer, H and L Instruments, Bulingame, CA). Under the conditions employed, samples could be diluted 8-fold with no change in polarization suggesting that light-scattering did not contribute appreciably to the observed results. The temperature variation of the ln(r~) was fit to either one or two straight lines with a minimum chi square residual taken as the best fit. A significantly (P < 0.05) better correlation was achieved with two lines than one for both the D~O and H 2 0 measurements. The 1H-NMR D F T spectra of rat brain synaptic plasma membranes as a function of temperature are shown in Fig. 1. Identifiable resonances are the terminal methyl resonance at 0.6-0.7 ppm, the broad methylene resonance at 0.6-2.0 ppm, and the choline resonance at 3.0 ppm. The sharp resonances at 3.0 and 3.6 ppm are probably derived from carbohydrate protons, perhaps sialic acid. Since these resonances exhibited little temperature dependence, they were used as the internal references for the spectral subtraction routine. The temperature effects are most easily seen in the difference spectra obtained by subtracting the 24 °C spectra from the spectra at higher temperatures. The data show that there are no significant temperature effects over the range of 24-32 °C. As temperature is increased above 32 °C, the methyl group resonance (0.6 ppm), the broad methylene resonance ({1.6-2.(I ppm) and the choline resonance (3 ppm) increase in intensity with increasing temperature, indicating greater Ill-mobility. Since the difference spectra are characteristic of a phospholipid and not of a protein or saccharide, the increase in intensity must be attributed to spectral changes in the lipids. The increase in intensity cannot be attributed to chemical shift or changes of resonances. If this were the case, then peak(s) of negative intensity would be observed in the difference spectra. No negative peaks were observed over the spectral region studied of 11 to - 2 ppm. The increase in spectral intensity can, therefore, be only attributed to an increase in the T 2 of the lipid protons which yields a larger free induction decay after the preacquisition delay time and hence greater observable spectral intensity. The increase in spectral intensity with temperature for the methylene resonance is sigmoidal in
164
nature suggesting a cooperative change in the lipid
p r o x i m a t e l y 38 °C. T h e increase in the spectral inten-
bilayer from a m o r e ordered to less ordered state
sity over the entire t e m p e r a t u r e range is approxi-
(Fig. 2). The midpoint for this temperature is ap-
mately a factor of 2. At this time. it is not possible to ~nULT RAT 5 P M VER3USTEMPERATURE ~JH,561-,577
! DIFFERENCE SPECTR6OF ADULT ~T 5PP RJH.SeI-,572
r
=
qLtC
:
3qC
=
28C
I, ,
l~
,
!
~
A
~"~
-
Lt2C
-
qOC
- 2~C
38C
- 2qC
/,% " '
, ~ ~ ~ I ~ I ~ @ ~ 4 ~ U ~
30C
-
2½C
\
~ .~.~u~1 ~u\ ,
-,,.,. " , v . . _
.
.
.
.
.
.
. 3
-
= 24r
i 2
1
"
"
¢~
--
PPM
Fig. 1. The ~H-NMR-DFT spectra of adult rat cortex SPM as a function of temperature (right). The difference spectra of a given'temperature minus that at 24 °C (left). This experiment was repeated on 3 different membrane preparations with essentially identical resuits.
165
50
4(3
>" 30
C
20
10
i 26
30
34
38
42
T e m p e r a t u r e 'C
Fig. 2. The intensity in arbitrary units of the -0.5 to 2 ppm spectral region of the difference spectrum v e r s u s temperature.
ascertain whether the spectral intensity increase is due to a small change in T 2 of the majority of lipids or whether isolated domains have 'melted' since both would elicit the same spectral response. However, to our knowledge, this is the first demonstration of an apparent cooperative transition of the lipids in synaptic membranes which occurs in the physiological range. The question arises as to why this transition has not been observed in previous experiments with F P Z or ESR (see e.g. ref. 7). Two possible answers were investigated. One, the transition is an artifact of using D 2 0 as the solvent. Two, the high temperature 'arm' of the transition is normally not investigated, especially above 40 °C, and thus its presence would not be reliably detected. To examine both of these questions, the FPZ experiments shown in Fig. 3 were conducted. The fluorescence data are presented in the form of Arrhenius plots (ln r Svs l/T) for synaptic membranes in D 2 0 and H 2 0 PBS. No significant difference was found between the D 2 0 and H 2 0 data, suggesting there was no significant isotope effect on membrane physical properties. However, for membranes suspended in both solvents, a significant 'break' in the Arrhenius plot was found at approximately 37 °C. Using classic thermodynamic analyses, such breaks have been interpreted as a change in the membrane flow activation energy ( d E ) (see e.g. ref. 3). A
more pedestrian explanation is that the membrane in the immediate probe environment undergoes a transition from a more ordered to a less ordered state. Some studies have reported no break in the F P Z Arrhenius plot for synaptic membranes, even at high temperatures. The reason(s) for this discrepancy are not clear, given the similarities in SPM preparation and fluorescence methodology. However, the presence of a transition in our m e m b r a n e preparations seems assured, given the qualitative agreement between both the I H - N M R and F P Z data. The fact that the two methods agree is perhaps somewhat surprising given the marked difference(s) in the actual parameters measured and given the abundant literature showing the sharp contrast between N M R and F P Z data (e.g. ref. 16). The agreement of the two methods, in our opinion, illustrates the robust nature of the findings. The data presented here clearly illustrate the usefulness and advantages of I H - N M R over other techniques in analyzing the lipid matrix of SPMs. While
5 3C
Adult Ral Cortex
SP/v~ b I o~9 '~
H20
•
D?Q
b
1340
0
51<
5 O:
3~5
320
325
1/T x IO 3
330
335
K
Fig. 3. Plot of in (r~) vs 1/T in H20 and D,O buffers. Lines shown are those which give the least chi-square residuals to the experimental data. Data are the mean of 4 experiments, each with a different membrane preparation.
166 F P Z is clearly a m o r e sensitive t e c h n i q u e t h a n ~HN M R in t e r m s of tissue r e q u i r e m e n t s , with the ap-
In conclusion, the increase in r e s o n a n c e intensity associated with i n c r e a s i n g t e m p e r a t u r e a b o v e 34 ~'("
p r o p r i a t e selection of spectral p a r a m e t e r s (e.g. the
illustrates that at physiological t e m p e r a t u r e s synaptic
p r e a c q u i s i t i o n d e l a y times), the I H - N M R m e t h o d is
m e m b r a n e s are especially sensitive to small changes
m o r e sensitive than F P Z to small p e r t u r b a t i o n s in
in t e m p e r a t u r e . T h e i m p o r t a n c e of this sensitivity in
m e m b r a n e o r d e r . A l t h o u g h the d a t a p r e s e n t e d are
the r e g u l a t i o n of n e r v o u s activity is u n c l e a r but m a y
precisely a m e a s u r e
help to explain the sensitivity of n e r v e m e m b r a n e s to
of spin-spin r e l a x a t i o n t i m e s
(T2), the data can be i n t e r p r e t e d in t e r m s of m e m -
agents which h a v e t e m p e r a t u r e - l i k e effects such as
brane o r g a n i z a t i o n . T h e q u e s t i o n arises as to w h e t h er or not o t h e r N M R t e c h n i q u e s , e.g. ~3C- 2H- or t~F-
e t h a n o l and the gaseous anesthetics.
labelled m o l e c u l e s , w o u l d d e t e c t similar m e m b r a n e
T h e a u t h o r s wish to t h a n k R u t h R u p l e y far editori-
b e h a v i o r to that r e p o r t e d here. T h e a n s w e r is p r o b a -
al and typing assistance. This study was s u p p o r t e d in
bly not since the sensitivity of the ~ H - N M R m e t h o d is
part by U S P H S G r a n t M H - 3 7 3 7 7 and a g r a n t from
d e p e n d e n t u p o n the short T 2 r e l a x a t i o n t i m e of pro-
the Scottish R i t e S c h i z o p h r e n i a F o u n d a t i o n . Partial
tons in relation to the p r e - a c q u i s i t i o n delay time. Nu-
funding of the N M R s p e c t r o m e t e r was p r o v i d e d by a
clei such as~3C, 2H and 19F h a v e m u c h l o n g e r T , relaxation times 2t.
grant from the N a t i o n a l S c i e n c e F o u n d a t i o n I C H E 8102974).
1 Chin, J.H. and Goldstein, D.B., Effects of low concentrations of ethanol on the fluidity of spin-labelled erythrocyte and brain membranes, Mol. Pharmacol.. 13 (1977) 435-441. 2 Cornell. B.A., Heller, R.G., Raison, J., Separovic, F., Smith, R., Vary, J.C. and Morris, C.. Biological membranes are rich in low frequency motion, Biochim. Biophys. Acta, 732 (1983) 473-478. 3 Crews, F.T.. Majchrowicz, E. and Meeks, R., Changes in cortical synaptosomal plasma membrane fluidity and composition in ethanol-dependent rats, Psychopharmacology, 81 (1983) 208-213. 4 Cullis, P.R. and DeKruijff, B, Lipid polymorphism and the functional roles of lipids in biological membranes, Biochim. Biophys. Acta, 559 (1979) 399-420. 5 De Laat, S.W., Van der Saag, P.T., Nelemans, A.S. and Shinitzky, M., Microviscosity changes during differentiation of neuroblastoma cells, Biochim. Biophys. Acta, 509 (1978) 188-193, 6 De laat, S. and Van der Saag, P.T., The plasma membrane as a regulatory site in growth and differentiation of neuroblastoma ceils, Int. Rev. Cytol., 74 (1982) t-54. 7 Harris, R.A. and Schroeder, F., Effects of barbiturates and ethanol on the physical properties of brain membranes, J. Pharmacol. Exp. Ther., 223 (1982) 424"431. 8 Hitzemann, R.J. and Johnson, D.A., Developmental changes in synaptic membrane lipid composition and fluidity, Neurochem. Res., 8 (1983) 121-131. 9 Jarrell, H.C.. Butler, K.W., Byrd, R.A., Deslauriers, R., Ekiel, I. and Smith, I.C.P., A 2H-NMR study of Acholeplasma laidlawii membranes highly enriched in myristic acid, Biochim. Biophys. Acta 688 (1982) 622"636. 10 Johnson, D.A., Lee, N.M.. Cooke, R. and Loh, H.H., Ethanol-induced fluidization of the brain lipid bitayers: required presence of cholesterol in membranes for the expression of tolerance, Mol. Pharmacol., 15 (1979) 739-746. 11 Jost, P.C., Griffith, O.H., Capaldi, R.A. and VanderKooi, G., Evidence for boundary lipid in membranes, Proc. Natl. Acad. Sci. U.S.A.. 70 (1973) 480-486.
12 Lowry. O.H., Rosebrough. N.J., Farr. A.L. and Randall. R.J . Protein measurement with the Folin phenol reagent. I. Biol Chem,. 193 (1951) 265-275. 13 McElhaney, R.N.. The structure and function of the Acholeplasma laidlawii plasma membrane. Biochim. Biophys. Acta. 779 (19841 1-42. 14 Pottel. H.. Van der Meer. W. and Herreman, W., Correlation between the order parameter and the steady-state fluorescence anisotropy of 1,6-diphenyt- 1.3,5-hexatriene and an evaluation of membrane fluidity, Biochim. Biophys. Acta. 730(1983) 181-186. 15 Sandra. A.. Paltzer. W.B and Thomas. M.J . Morphological differentiation of murine neuroblastoma induced bv liposomes. Lipid specificity and pathway of liposome uptake, Exp. CellRes.. 132 (1981) 473-477 16 Seelig. J.. Tamm. L.. Hymol. L. andFleischer. S.. Duetenum and phosphorus nuclear magnetic resonance and fluorescence depolarization studies of functional reconstituted sarcoplasmic reticulum membrane vesicles. Biochemistry, 20 ~1981~ 3922-3932. 17 Seiter. C.H.A., Feigenson. G . W , Chan. S.t. and Hsu. M.C.. Delayed fourier transform proton magnetic resonance spectroscopy, J. Am. Chem. Soc, 94 cl972t 2535-2537. 18 Smith I.C.P.. Butler. K.W.. Tullock. A.P. Davis. J.H. and Bloom. M.. The properties of gel state lipid in membranes of Acholeplasma laidlawii as observed by 2H NMR. FEBS Lett.. 100 (1979] 57-61. 19 Stockton. G.W.. Johnson. K.G.. Butler, K.W.. Tullock, A.P.. Boulanger, Y.. Smith. I.C.P., Davis. J.H and Bloom. M.. Deuterium NMR study of lipid organization on Acholeplasma laidlawii membranes. Nature. (London, 269 (1977) 267-268. 20 Van Blitterswijk, W.J.. Van Hoeven. R.P. and Van tier Meer, B.W.. Lipid structural order parameters treciprocal of fluidity) in biomembranes derived from steady-state fluorescence polarization measurements. Biochim Biophys. Acta. 644 (1981) 323-332. 21 James. T.L., Nuclear Magnetic Resonance in Biochemtstrv. Academic Press. New York. 1975 pp. 298-347.
162
BRE 21046
1
H,~
Spectra of ~ ilynaptl¢ ~ ~ll,m~: effect of t ~ M u r e c ~ ~ ~ fluorescence polarization
and
GEORGE P. KREISHMAN1and ROBERT J. HITZEMANN2 1Department of Chemistry and 2Departments of Psychiatry, Pharmacology and Cell Biophysics, University of Cincinnati, Cincinnati, OH (U.S.A.) (Accepted April 30th. t985) Key words: nuclear magnetic resonance (NMR) spectrum
synaptic membrane
fluorescence polarization
1H-NMR spectra of rat synaptic plasma membranes obtained over the temperature range of 24-46 °C are presented. The data illustrate that a transition occurs from a more ordered to less ordered state at approximately 37 °C. This phenomenon was not related to using D20 as the solvent and was replicated, although with less sensitivity, using fluorescence polarization methodology. The physical properties of synaptic plasma membranes are important elements in our understanding of such diverse p h e n o m e n a as neuronal differentiationS,6,15 and the mechanism(s) of drug action 1.3,7-1°. For the most part. two techniques have been used to examine the physical properties of synaptic membranes, fluorescence polarization (FPZ) and electron spin resonance (ESR) spectroscopy. F P Z has had especially wide application, due to its sensitivity, to the diversity of available membrane probes and to the relatively inexpensive equipment required. Furthermore. some recent studies show that measures of the steady-state fluorescence anisotropy (rs) for at least one probe, 1.6-diphenyl-l,3,5 hexatriene ( D P H ) can be converted to a measure of m e m b r a n e order without the necessity of determining the fluorescence lifetime14, 20. However. despite their advantages. FPZ and E S R are perturbing techniques which require the introduction of a probe molecule Thus, one is precisely studying probe-membrane interactions. A non-perturbing alternative to F P Z or E S R is nuclear magnetic resonance ( N M R ) spectroscopy. N M R can measure membrane motions within the slower, potentially more important time domains (104-106 s-ll than are possible with F P Z and E S R (time d o m a i n / > 108 s-l) (ref. 2). N M R can be used to probe lipid mobility at various depths within the membrane 19 or to
examine the lipid packing arrangement 4. The spectra obtained by N M R in natural membranes often reveal a broad gel to liquid-crystalline phase transition which is seen by more direct techniques e.g. differential scanning calorimetry ~3. In contrast. FPZ and ESR may under identical conditions reveal a sharp phase transition and not report the peripheral boundaries of the gel to liquid crystalline transition 13. Furthermore. N M R experiments report that protein does not perturb lipid order, suggesting a rapid exchange of the bulk and protein boundary lipidsg, 18. Differently, E S R and F P Z report that proteins immobilize membrane lipids and that the exchange between the bulk and boundary lipids is slow in reference to the time scale being used tl In the present study we present 1H-NMR Delayed Fourier Transform (DFT) 17 spectra (in D 2 0 ) of rat synaptic plasma membranes obtained at temperatures over the range of 2 4 - 4 6 °C. With the appropriate choice of spectral parameters, large spectral changes can be observed for small changes in membrane molecular dynamics. In addition, various portions of the membranes can be monitored simultaneously. These data are then compared with the results obtained by F P Z techniques over the same temperature range. Synaptic plasma membranes (SPMs) were pre-
Correspondence: R.J. Hitzemann. Department of Psychiatry, University of Cincinnati. College of Medicine. 231 Bethesda Ave.. Cincinnati. OH 45267-0559. U.S.A. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
163 pared from the rat forebrain as described previouslyS. Membrane purity, as assessed by both morphological and chemical criteria was estimated to be greater than 85%. The membranes were washed 3 times in D20-phosphate buffered saline (PBS) -pH (pD) = 8.2 and then resuspended in this buffer at a concentration of approximately 10 mg/ml. A small aliquot of this suspension was used for the protein determination essentially as described by Lowry et al. ~2. On the basis of the protein determination, the sample was adjusted to 5 mg/ml and 0.7 ml was transferred to a 5 mm N M R tube. The sample was purged with dry N~ and then sealed using epoxy glue. The sample was then frozen at - 7 0 °C until analysis. The I H - N M R spectra were obtained using a Nicolet NMC 300 MHz F T - N M R spectrometer. The delay between the excitation pulse and the start of acquisition was set at 240 Bs; (in non-sonicated lipid bilayers, the relaxation time(s) for the majority of the protons is ~ 100 ~//S) 17. Under these conditions, less than 5% of the total spectral intensity is observed when the receiver is turned on. A n y increase in the relaxation rates of the protons will lead to a marked increase in the observed spectral intensity. Spectra were obtained over a temperature range of 22-46 °(2 utilizing a NTC temperature control unit. The samples were allowed to equilibrate at each new temperature for 40 rain prior to spectral acquisition. The spectra were processed with the same scaling factor so that direct comparisons of the intensities could be made. Chemical shifts are reported relative to H O - D at 4.5 ppm for each temperature. (What appear to be chemical shifts in the lipid resonances are actually a result of the temperature dependence of the H O - D resonance. This 'shift' was compensated for in the subtraction routine.) Resonance identification was made on the basis of comparison with published spectra and by comparison with the spectra of model membranes composed of dimyrsitylphosphatidylcholine, cholesterol and gangliosides (1:0.3:0.1, molar ratio). It should be noted that under the spectral conditions employed, the protein protons, because of their fast relaxation, will not contribute significantly to the spectra. FPZ measurements were made essentially as described elsewhere s. Synaptic membranes were suspended in both D 2 0 - P B S and normal PBS in order to check for potential isotope effects. The final mem-
brane concentration was 10 j~g/ml; the probe ( D P H ) concentration was 0.025/2g/ml. Fluorescence measurements were made in a T-format fluorometer (HH-2 fluorometer, H and L Instruments, Bulingame, CA). Under the conditions employed, samples could be diluted 8-fold with no change in polarization suggesting that light-scattering did not contribute appreciably to the observed results. The temperature variation of the ln(r~) was fit to either one or two straight lines with a minimum chi square residual taken as the best fit. A significantly (P < 0.05) better correlation was achieved with two lines than one for both the D~O and H 2 0 measurements. The 1H-NMR D F T spectra of rat brain synaptic plasma membranes as a function of temperature are shown in Fig. 1. Identifiable resonances are the terminal methyl resonance at 0.6-0.7 ppm, the broad methylene resonance at 0.6-2.0 ppm, and the choline resonance at 3.0 ppm. The sharp resonances at 3.0 and 3.6 ppm are probably derived from carbohydrate protons, perhaps sialic acid. Since these resonances exhibited little temperature dependence, they were used as the internal references for the spectral subtraction routine. The temperature effects are most easily seen in the difference spectra obtained by subtracting the 24 °C spectra from the spectra at higher temperatures. The data show that there are no significant temperature effects over the range of 24-32 °C. As temperature is increased above 32 °C, the methyl group resonance (0.6 ppm), the broad methylene resonance ({1.6-2.(I ppm) and the choline resonance (3 ppm) increase in intensity with increasing temperature, indicating greater Ill-mobility. Since the difference spectra are characteristic of a phospholipid and not of a protein or saccharide, the increase in intensity must be attributed to spectral changes in the lipids. The increase in intensity cannot be attributed to chemical shift or changes of resonances. If this were the case, then peak(s) of negative intensity would be observed in the difference spectra. No negative peaks were observed over the spectral region studied of 11 to - 2 ppm. The increase in spectral intensity can, therefore, be only attributed to an increase in the T 2 of the lipid protons which yields a larger free induction decay after the preacquisition delay time and hence greater observable spectral intensity. The increase in spectral intensity with temperature for the methylene resonance is sigmoidal in
164
nature suggesting a cooperative change in the lipid
p r o x i m a t e l y 38 °C. T h e increase in the spectral inten-
bilayer from a m o r e ordered to less ordered state
sity over the entire t e m p e r a t u r e range is approxi-
(Fig. 2). The midpoint for this temperature is ap-
mately a factor of 2. At this time. it is not possible to ~nULT RAT 5 P M VER3USTEMPERATURE ~JH,561-,577
! DIFFERENCE SPECTR6OF ADULT ~T 5PP RJH.SeI-,572
r
=
qLtC
:
3qC
=
28C
I, ,
l~
,
!
~
A
~"~
-
Lt2C
-
qOC
- 2~C
38C
- 2qC
/,% " '
, ~ ~ ~ I ~ I ~ @ ~ 4 ~ U ~
30C
-
2½C
\
~ .~.~u~1 ~u\ ,
-,,.,. " , v . . _
.
.
.
.
.
.
. 3
-
= 24r
i 2
1
"
"
¢~
--
PPM
Fig. 1. The ~H-NMR-DFT spectra of adult rat cortex SPM as a function of temperature (right). The difference spectra of a given'temperature minus that at 24 °C (left). This experiment was repeated on 3 different membrane preparations with essentially identical resuits.
165
50
4(3
>" 30
C
20
10
i 26
30
34
38
42
T e m p e r a t u r e 'C
Fig. 2. The intensity in arbitrary units of the -0.5 to 2 ppm spectral region of the difference spectrum v e r s u s temperature.
ascertain whether the spectral intensity increase is due to a small change in T 2 of the majority of lipids or whether isolated domains have 'melted' since both would elicit the same spectral response. However, to our knowledge, this is the first demonstration of an apparent cooperative transition of the lipids in synaptic membranes which occurs in the physiological range. The question arises as to why this transition has not been observed in previous experiments with F P Z or ESR (see e.g. ref. 7). Two possible answers were investigated. One, the transition is an artifact of using D 2 0 as the solvent. Two, the high temperature 'arm' of the transition is normally not investigated, especially above 40 °C, and thus its presence would not be reliably detected. To examine both of these questions, the FPZ experiments shown in Fig. 3 were conducted. The fluorescence data are presented in the form of Arrhenius plots (ln r Svs l/T) for synaptic membranes in D 2 0 and H 2 0 PBS. No significant difference was found between the D 2 0 and H 2 0 data, suggesting there was no significant isotope effect on membrane physical properties. However, for membranes suspended in both solvents, a significant 'break' in the Arrhenius plot was found at approximately 37 °C. Using classic thermodynamic analyses, such breaks have been interpreted as a change in the membrane flow activation energy ( d E ) (see e.g. ref. 3). A
more pedestrian explanation is that the membrane in the immediate probe environment undergoes a transition from a more ordered to a less ordered state. Some studies have reported no break in the F P Z Arrhenius plot for synaptic membranes, even at high temperatures. The reason(s) for this discrepancy are not clear, given the similarities in SPM preparation and fluorescence methodology. However, the presence of a transition in our m e m b r a n e preparations seems assured, given the qualitative agreement between both the I H - N M R and F P Z data. The fact that the two methods agree is perhaps somewhat surprising given the marked difference(s) in the actual parameters measured and given the abundant literature showing the sharp contrast between N M R and F P Z data (e.g. ref. 16). The agreement of the two methods, in our opinion, illustrates the robust nature of the findings. The data presented here clearly illustrate the usefulness and advantages of I H - N M R over other techniques in analyzing the lipid matrix of SPMs. While
5 3C
Adult Ral Cortex
SP/v~ b I o~9 '~
H20
•
D?Q
b
1340
0
51<
5 O:
3~5
320
325
1/T x IO 3
330
335
K
Fig. 3. Plot of in (r~) vs 1/T in H20 and D,O buffers. Lines shown are those which give the least chi-square residuals to the experimental data. Data are the mean of 4 experiments, each with a different membrane preparation.
166 F P Z is clearly a m o r e sensitive t e c h n i q u e t h a n ~HN M R in t e r m s of tissue r e q u i r e m e n t s , with the ap-
In conclusion, the increase in r e s o n a n c e intensity associated with i n c r e a s i n g t e m p e r a t u r e a b o v e 34 ~'("
p r o p r i a t e selection of spectral p a r a m e t e r s (e.g. the
illustrates that at physiological t e m p e r a t u r e s synaptic
p r e a c q u i s i t i o n d e l a y times), the I H - N M R m e t h o d is
m e m b r a n e s are especially sensitive to small changes
m o r e sensitive than F P Z to small p e r t u r b a t i o n s in
in t e m p e r a t u r e . T h e i m p o r t a n c e of this sensitivity in
m e m b r a n e o r d e r . A l t h o u g h the d a t a p r e s e n t e d are
the r e g u l a t i o n of n e r v o u s activity is u n c l e a r but m a y
precisely a m e a s u r e
help to explain the sensitivity of n e r v e m e m b r a n e s to
of spin-spin r e l a x a t i o n t i m e s
(T2), the data can be i n t e r p r e t e d in t e r m s of m e m -
agents which h a v e t e m p e r a t u r e - l i k e effects such as
brane o r g a n i z a t i o n . T h e q u e s t i o n arises as to w h e t h er or not o t h e r N M R t e c h n i q u e s , e.g. ~3C- 2H- or t~F-
e t h a n o l and the gaseous anesthetics.
labelled m o l e c u l e s , w o u l d d e t e c t similar m e m b r a n e
T h e a u t h o r s wish to t h a n k R u t h R u p l e y far editori-
b e h a v i o r to that r e p o r t e d here. T h e a n s w e r is p r o b a -
al and typing assistance. This study was s u p p o r t e d in
bly not since the sensitivity of the ~ H - N M R m e t h o d is
part by U S P H S G r a n t M H - 3 7 3 7 7 and a g r a n t from
d e p e n d e n t u p o n the short T 2 r e l a x a t i o n t i m e of pro-
the Scottish R i t e S c h i z o p h r e n i a F o u n d a t i o n . Partial
tons in relation to the p r e - a c q u i s i t i o n delay time. Nu-
funding of the N M R s p e c t r o m e t e r was p r o v i d e d by a
clei such as~3C, 2H and 19F h a v e m u c h l o n g e r T , relaxation times 2t.
grant from the N a t i o n a l S c i e n c e F o u n d a t i o n I C H E 8102974).
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