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Nitrous oxide alters the pattern of myelin proteins in the nervous system of the fruit bat roussettus aegyptiacus
Neuroscience Letters, 42 (1983) 99-104
99
Elsevier Scientific Publishers Ireland Ltd.
NITROUS OXIDE ALTERS THE PATTERN OF MYELIN PROTEINS IN THE NERVOUS SYSTEM OF THE F R U ~ BAT ROUSSETTUS AEGYPTIACUS
J.L. McLOUGHLIN and R.C. CANFRILL*
MRC Brain Metabolism Research Group, Department of Medical Biochemistry, University of the Witwatersrand, Johannesburg (South Africa) (Received August 30th, 1983; Accepted September 5th, 1983)
Key ",~ords: nitrous oxide - myelin - protein electrophoresis - fruit bat
Proteins of the myelin membrane were separated by SDS-polyacrylamide electrophoresis. The protein patterns obtained from myelin from the forebrain, medulla, cervical and thoracic spinal cord and phrenic nerve of normal animals were compared with those obtained from animals given nitrous oxide or nitrous oxide plus folinic acid. A shift from high molecular weight proteins to lower molecular weight proteins was seen in all regions studied in animals given NzO and folinic acid. These changes were seen also in the medulla and thoracic spinal cord of animals given N20 alone. Peripheral nerve showed a slight increase in the amounts of P0 at the expense of P3 and P2 in NzO and folinic acid-treated animals.
Nutritional and nitrous oxide-induced vitamin Btz deficiency leads to spongiose changes in the spinal cord and peripheral ataxia [2, 8]. Recent studies [6, 12] have highlighted changes in membrane lipid composition and protein distribution in nutritionally deficient fruit bats. While myelin protein distribution on SDS polyacrylamide gels is not affected in nutritional B~2 deficiency [6], the localization of these proteins on a linear sucrose gradient is altered [6]. The electrophoretic distribution of specific myelin proteins derived from the brains of nitrous oxidetreated fruit bats following linear gradient centrifugation is not significantly different from the distribution obtained from normal animals. This is in contrast to the band-spreading phenomenon noted in membranes derived from nutritionally deficient animals [61. Fruit bats exposed to nitrous oxide for 90 min per day for 9 weeks developed severe ataxia and levels of vitamin B~2 were undetectable [11]. It is proposed that v i t a m i n Bt2 deficiency leads to a deficiency of folate because of the inhibition of the methionine synthetase reaction. This in turn causes an increase in the concentration of methylfolate. The resultant low rate of synthesis of methionine may reduce *Author for correspondence 0304-3940/83/$ 03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd.
100 the available pool of S-adenosylmethionine and affect protein and lipid methylation [9]. Folate administered to an animal is metabolized to methylfolate and transported in the circulation as such, therefore, folinic supplementation in nitrous oxideinduced vitamin Bl2 deficiency should exacerbate the condition. In the present study we have investigated the distribution of myelin-specific proteins in a number of discrete areas of the central and peripheral nervous tissue. Myelin proteins were examined because they are necessary for the normal insulatory properties of myelin. This feature may be disturbed in vitamin Bl2 deficiency and give rise to the neurological symptoms seen in the fruit bats. Furthermore, myelin basic protein exists in the dimethyl form in intact myelin and a change in the amount of basic protein could be expected if the rate of protein methylation is impaired in vitamin Bi2 deficient animals. Myelin was prepared from different regions of the brain and spinal cord by purification of the myelin-containing layer from the Hajbs scheme for the preparation of synaptosomes [4]. Crude myelin membranes were washed twice with water and pelleted at 45,000 g for 20 min. The resultant pellet was suspended in water and the protein concentration determined by the method of Lowry et al. [5]. Samples of myelin from each brain area (40 #g protein) were solubilized overnight in 1070 SDS, 10070sucrose, 1°7o mercaptoethanoi and then run on 1207opolyacrylamide gels containing 0.1 07o sodium dodecyi sulphate (SDS) as previously described [7]. Bands were identified after staining with Coomassie blue by comparison with authentic molecular weight standards (Pharmacia). Peripheral nerve myelin proteins were identified and labelled according to the system of Greenfield et al [3]. Proteins were quantitated using a Beckman CDC 200 scanner. Fruit bats were maintained in captivity as decribed previously l l0]. Animals were arbitarily assigned to 3 test groups: (1) bats exposed to 5007o nitrous oxide for 90 min per day for 9 weeks; (2) bats exposed to 50070 nitrous oxide for 90 min per day plus a daily intraperitoneal injection of 150 #g folinic acid; and (3) control (no nitrous oxide or folate injection). Myelin prepared from different regions of the central nervous system of control fruit bats showed the same protein patterns as those described previously [7], and differed from rat myelin only in the occurrence of a single basic protein species (Fig. 1). Ratios of high molecular weight protein (Wolfgram proteins) to low molecular weight protein (basic protein) were variable throughout the brain and spinal cord. This may be related to a regional difference in the apparent distribution of myelin markers previously explored by Waehneldt and Lane [13] in the whole brain particulate preparations. Treatment with nitrous oxide alone was inconclusive, see Tables I and II. In some areas (cervical spinal cord and forebrain) myelin protein distribution was similar to control, whereas in the thoracic spinal cord and medulla there was a decrease in high molecular weight proteins. These changes are highlighted by the basic pro-
101
(o)
M
A
B
C
D
E
M
(b)
94000
94000
67000
67000
A
B
C
D
E
43000 °
43000 ° 30000
e f g h i
b c 20000 d
30000
e f g h i
b c 20000 d
14400 14400
Fig. 1. SDS polyacrylamide gel electrophoresis of myelin proteins derived from 40 ~g each of A, forebrain; B, medulla; C, cervical spinal cord; D, thoracic spinal cord; and E, phrenic nerve. Lane M contains molecular weight marker phosphorylase b 94000; albumin 67000; ovaibumin 43000; carbonic anhydrase 30000; trypsin inhibitor 20000; c~-lactalbumin 14400. CNS myelin proteins are identified as: a, Wolfgram proteins; b, proteolipid protein; c DM-20; d, basic protein. Peripheral nerve myelin proteins are identified as: e, Po; f, P3; g, 1:'4; h, Pt; i, P2. (a) Myelin protein distribution in different samples of nervous tissue from a control bat. (b) Myelin protein distribution in different samples of nervous tissue from a nitrous oxide exposed bat.
TABLE ! REGIONAL DISTRIBUTION OF MYELIN PROTEINS IN BRAIN TISSUE FROM THE FRUIT BAT Regional distribution of myelin proteins in brain tissue from the fruit bat exposed to nitrous oxide and following folinic acid supplementation. Proteins were identified according to their molecular weights and quantitated by area. Values shown are the mean derived from 3 or 4 animals and are taken from the computed results of a Beckman CDC 200 scanner. Statistical significance was determined using Student's ttest; *P< 0.005. B/W was determined as the ratio of low molecular weight protein (basic protein) to high molecular weight protein (Wolfgram proteins). Region
Tubulin
Wolfgram proteins
Proteolipid DM 20 protein
(w)
Basic protein
B/W
(a)
Forebrain Control (3) N20 (4) N20 + folate (3)
25.5 23.3 14.1"
41.1 36.5 26.8*
! 3.9 13.9 23.3*
2.9 3.8 2.8
16.5 22.6 33.0*
0.4 0.6 1.2
Medulla Control (3) NzO (4) N20 + folate (3)
11.2 8.9 5.3"
26.5 20.1" 20.2*
21.6 23~7 29.7
2.7 4.5 3.0
38.0 42.9 41.7
1.5 2.1 2.1
102 TABLE I1 REGIONAL DISTRIBUTION OF MYELIN PROTEINS IN SPINAL CORD AND PERIPHERAL NERVE FROM THE FRUIT BAT
Regional distribution of myelin proteins in spinal cord and peripheral nerve tissue exposed to nitrous oxide and following folinic acid supplementation. Peripheral nerve proteins were identified by the system of Greenfield et al [4]. All other details are contained in the legend to Table I. Difference from control: *P<0.005; **P<0.01. Region
Cervical spinal cord Control (3) NzO (4) NzO + folate (3) Thoracic spinal cord Control (3) N20 (4) N20 + folate (3) Peripheral Nerve Control (3) N20 (4) N20 + folate (3)
Tubulin
5.5 8.7 4.3 ! 1.8 9.2 4.0**
Wolfgram proteins (w)
Proteolipid DM 20 protein
Basic protein
B/W
19.3 ! 9.0 11.8"
30.1 26.0 30.7
5.1 4.8 3.5
40.0 41.5 49.7*
2.2 2.2 4.2
21.7 15.9 12.7"*
26.4 20.7 32.5
5.0 4.5 3.2
35. ! 49.8 47.4**
1.6 3.8 3.8
Po
P3
P4
P~
Pz
43.0 44.1 51.5"*
35.6 37.1 28.9**
I 1.3 10.1 12.7
3.3 3.3 2.1
6.7 5.4 4.9**
tein/Wolfgiam proteins ratio (B/W) which was unchanged in the cervical spinal cord and forebrain. The addition of folinic acid to the regime caused a significant decrease in the high molecular weight Wolfgram proteins, and in all areas except the medulla there was a concomitant rise in the proportion of basic protein. These changes are reflected in an increased B/W ratio. Peripheral nerve myelin protein composition was altered by the combined nitrous oxide and folinic acid treatment, but was unchanged in preparations from animals treated with nitrous oxide alone. Protein Po was increased at the expense of proteins P2 and P3 (Table II), in contrast to the loss of high molecular weight proteins and their replacement with lower molecular weight basic protein seen in the central nervous system myelin samples. In myelin prepared from fruit bats made deficient in vitamin BI2 by dietary deprivation, changes in the myelin protein composition were not detectable when symptoms of neurological disturbance were apparent [7]. Nitrous oxide administration, on the other hand, caused a rearrangement of the relative protein composition which was exacerbated by folinic acid administration. The dramatic effect of nitrous oxide plus folinic acid may be ascribed to a combination of mechanisms which increase methylfolate concentrations.
103 Two mechanisms are proposed to explain the observed effect of nitrous oxide on myelin membrane proteins. (1) Vitamin Bn depletion by nitrous oxide causes a depletion of the Sadenosylmethionine pool for protein methylation by inhibiting methionine synthetase. Since this process is very rapid, gross changes in membrane structure may become apparent whereas there were no detectable changes i n membrane composition in vitamin B~2 deficiency induced by dietary manipulation because of its slow onset. (2) Nitrous oxide is a potent anaesthetic and analgesic agent. Since its anaesthetic actions are classically described as interference with the lipid bilayer, changes in membrane protein components would be necessary to stabilise the structure of the membrane. Myelin basic protein has previously been ~ ~cribed an important role in stabilising the myelin membrane, and these results may be a direct demonstration of this property. Furthermore, nitrous oxide has recently been shown to interfere with the function of membrane receptor proteins in vitro, and a specific interaction of nitrous oxide with the opiate binding site has been published recently [1], providing further evidenced for a direct membrane interaction. The authors are grateful to the Medical Research Council of South Africa for its generous support, and to Dr. J. van der Westhuyzen for experimental animals.
1 Daras, C., CantriU, R.C. and Gillman, M.A., ['HlNaloxone displacement: evidence for nitrous oxide as opiod receptor agonist, Europ. J. Pharmacol., 89 (1983) 177-178. 2 Green R., von Tonder, S.V., Oettle, G.J., Cole, G. and Metz, J., Neurological changes in fruit bats deficient in vitamin Btz, Nature (Lond.), 254, (1975) 14P-150. 3 Greenfield, S., Brostoff, S., Eylar, E.H. and Moreil, P., Protein composition of myelin of the peripheral nervous system, J. Neurochem., 20 (1973) 1207-1216. 4 Haj6s, F., An improved method for the preparation of synaptosomal fractions in high purity, Brain Res., 93 (1975) 485-489. 5 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 165-175. 6 McLoughlin, J.L. and Cantrill, R.C., Vitamin Bn deficiency alters the distribution of membrane proteins in the fruit bat nervous sytem, Brain Res., in press. 70ldfield, M., Van der Westhuyzen, J., McLoughlin, J.L. and Cantrill, R.C., Protein profile of the myelin membrane of the fruit bat Roussettus aep~J,pticus, Comp. Biochem. Physiol., in press. 8 Scott, J.M. and Weir, D.G., The methyl folate trap, Lancet, 2 (1981) 337-340. 9 Small, D.A. and Carnegie, P.R., Myelopathy associated with vitamin Bt2 deficiency, Trends Neuro Sci., 4 (1981) x-xi. 10 Van der Westhuyzen, J., Cantrill, R.C., Fernandez-Costa, F. and Metz, J., The lipid composition of the brain in the vitamin B~z deficient fruit bat (Roussettus Aegyptiacus) with neurological impairment, J. Neurochem., 37 (1981) 543-549. 11 Van der Westhuyzen, J., Fernandez-Costa, F., Metz, J. Kanazawa, S., Drivas, G. and Herbert, V., Cobalamin (vitamin BI,) analogs are absent in plasma from fruit bats exposed to nitrous oxide, Proc. Soc. exp. Biol. Med., 171 (1982) 88-91.
104 12 Van der Westhuyzen, J., CantriH, R.C., Fernandez-Costa, F. and Metz, J., Effect o f a vitamin BRz deficient diet on lip and fatty acid composition of spinal cord myelin in the fruit bat, J. Nutr., 113 (1983) 531-537. ! 3 Waehneldt, T.V. and Lane, J.D., Dissociation of myelin from its 'enzyme markers' during ontogeny, J. Neurc
99
Elsevier Scientific Publishers Ireland Ltd.
NITROUS OXIDE ALTERS THE PATTERN OF MYELIN PROTEINS IN THE NERVOUS SYSTEM OF THE F R U ~ BAT ROUSSETTUS AEGYPTIACUS
J.L. McLOUGHLIN and R.C. CANFRILL*
MRC Brain Metabolism Research Group, Department of Medical Biochemistry, University of the Witwatersrand, Johannesburg (South Africa) (Received August 30th, 1983; Accepted September 5th, 1983)
Key ",~ords: nitrous oxide - myelin - protein electrophoresis - fruit bat
Proteins of the myelin membrane were separated by SDS-polyacrylamide electrophoresis. The protein patterns obtained from myelin from the forebrain, medulla, cervical and thoracic spinal cord and phrenic nerve of normal animals were compared with those obtained from animals given nitrous oxide or nitrous oxide plus folinic acid. A shift from high molecular weight proteins to lower molecular weight proteins was seen in all regions studied in animals given NzO and folinic acid. These changes were seen also in the medulla and thoracic spinal cord of animals given N20 alone. Peripheral nerve showed a slight increase in the amounts of P0 at the expense of P3 and P2 in NzO and folinic acid-treated animals.
Nutritional and nitrous oxide-induced vitamin Btz deficiency leads to spongiose changes in the spinal cord and peripheral ataxia [2, 8]. Recent studies [6, 12] have highlighted changes in membrane lipid composition and protein distribution in nutritionally deficient fruit bats. While myelin protein distribution on SDS polyacrylamide gels is not affected in nutritional B~2 deficiency [6], the localization of these proteins on a linear sucrose gradient is altered [6]. The electrophoretic distribution of specific myelin proteins derived from the brains of nitrous oxidetreated fruit bats following linear gradient centrifugation is not significantly different from the distribution obtained from normal animals. This is in contrast to the band-spreading phenomenon noted in membranes derived from nutritionally deficient animals [61. Fruit bats exposed to nitrous oxide for 90 min per day for 9 weeks developed severe ataxia and levels of vitamin B~2 were undetectable [11]. It is proposed that v i t a m i n Bt2 deficiency leads to a deficiency of folate because of the inhibition of the methionine synthetase reaction. This in turn causes an increase in the concentration of methylfolate. The resultant low rate of synthesis of methionine may reduce *Author for correspondence 0304-3940/83/$ 03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd.
100 the available pool of S-adenosylmethionine and affect protein and lipid methylation [9]. Folate administered to an animal is metabolized to methylfolate and transported in the circulation as such, therefore, folinic supplementation in nitrous oxideinduced vitamin Bl2 deficiency should exacerbate the condition. In the present study we have investigated the distribution of myelin-specific proteins in a number of discrete areas of the central and peripheral nervous tissue. Myelin proteins were examined because they are necessary for the normal insulatory properties of myelin. This feature may be disturbed in vitamin Bl2 deficiency and give rise to the neurological symptoms seen in the fruit bats. Furthermore, myelin basic protein exists in the dimethyl form in intact myelin and a change in the amount of basic protein could be expected if the rate of protein methylation is impaired in vitamin Bi2 deficient animals. Myelin was prepared from different regions of the brain and spinal cord by purification of the myelin-containing layer from the Hajbs scheme for the preparation of synaptosomes [4]. Crude myelin membranes were washed twice with water and pelleted at 45,000 g for 20 min. The resultant pellet was suspended in water and the protein concentration determined by the method of Lowry et al. [5]. Samples of myelin from each brain area (40 #g protein) were solubilized overnight in 1070 SDS, 10070sucrose, 1°7o mercaptoethanoi and then run on 1207opolyacrylamide gels containing 0.1 07o sodium dodecyi sulphate (SDS) as previously described [7]. Bands were identified after staining with Coomassie blue by comparison with authentic molecular weight standards (Pharmacia). Peripheral nerve myelin proteins were identified and labelled according to the system of Greenfield et al [3]. Proteins were quantitated using a Beckman CDC 200 scanner. Fruit bats were maintained in captivity as decribed previously l l0]. Animals were arbitarily assigned to 3 test groups: (1) bats exposed to 5007o nitrous oxide for 90 min per day for 9 weeks; (2) bats exposed to 50070 nitrous oxide for 90 min per day plus a daily intraperitoneal injection of 150 #g folinic acid; and (3) control (no nitrous oxide or folate injection). Myelin prepared from different regions of the central nervous system of control fruit bats showed the same protein patterns as those described previously [7], and differed from rat myelin only in the occurrence of a single basic protein species (Fig. 1). Ratios of high molecular weight protein (Wolfgram proteins) to low molecular weight protein (basic protein) were variable throughout the brain and spinal cord. This may be related to a regional difference in the apparent distribution of myelin markers previously explored by Waehneldt and Lane [13] in the whole brain particulate preparations. Treatment with nitrous oxide alone was inconclusive, see Tables I and II. In some areas (cervical spinal cord and forebrain) myelin protein distribution was similar to control, whereas in the thoracic spinal cord and medulla there was a decrease in high molecular weight proteins. These changes are highlighted by the basic pro-
101
(o)
M
A
B
C
D
E
M
(b)
94000
94000
67000
67000
A
B
C
D
E
43000 °
43000 ° 30000
e f g h i
b c 20000 d
30000
e f g h i
b c 20000 d
14400 14400
Fig. 1. SDS polyacrylamide gel electrophoresis of myelin proteins derived from 40 ~g each of A, forebrain; B, medulla; C, cervical spinal cord; D, thoracic spinal cord; and E, phrenic nerve. Lane M contains molecular weight marker phosphorylase b 94000; albumin 67000; ovaibumin 43000; carbonic anhydrase 30000; trypsin inhibitor 20000; c~-lactalbumin 14400. CNS myelin proteins are identified as: a, Wolfgram proteins; b, proteolipid protein; c DM-20; d, basic protein. Peripheral nerve myelin proteins are identified as: e, Po; f, P3; g, 1:'4; h, Pt; i, P2. (a) Myelin protein distribution in different samples of nervous tissue from a control bat. (b) Myelin protein distribution in different samples of nervous tissue from a nitrous oxide exposed bat.
TABLE ! REGIONAL DISTRIBUTION OF MYELIN PROTEINS IN BRAIN TISSUE FROM THE FRUIT BAT Regional distribution of myelin proteins in brain tissue from the fruit bat exposed to nitrous oxide and following folinic acid supplementation. Proteins were identified according to their molecular weights and quantitated by area. Values shown are the mean derived from 3 or 4 animals and are taken from the computed results of a Beckman CDC 200 scanner. Statistical significance was determined using Student's ttest; *P< 0.005. B/W was determined as the ratio of low molecular weight protein (basic protein) to high molecular weight protein (Wolfgram proteins). Region
Tubulin
Wolfgram proteins
Proteolipid DM 20 protein
(w)
Basic protein
B/W
(a)
Forebrain Control (3) N20 (4) N20 + folate (3)
25.5 23.3 14.1"
41.1 36.5 26.8*
! 3.9 13.9 23.3*
2.9 3.8 2.8
16.5 22.6 33.0*
0.4 0.6 1.2
Medulla Control (3) NzO (4) N20 + folate (3)
11.2 8.9 5.3"
26.5 20.1" 20.2*
21.6 23~7 29.7
2.7 4.5 3.0
38.0 42.9 41.7
1.5 2.1 2.1
102 TABLE I1 REGIONAL DISTRIBUTION OF MYELIN PROTEINS IN SPINAL CORD AND PERIPHERAL NERVE FROM THE FRUIT BAT
Regional distribution of myelin proteins in spinal cord and peripheral nerve tissue exposed to nitrous oxide and following folinic acid supplementation. Peripheral nerve proteins were identified by the system of Greenfield et al [4]. All other details are contained in the legend to Table I. Difference from control: *P<0.005; **P<0.01. Region
Cervical spinal cord Control (3) NzO (4) NzO + folate (3) Thoracic spinal cord Control (3) N20 (4) N20 + folate (3) Peripheral Nerve Control (3) N20 (4) N20 + folate (3)
Tubulin
5.5 8.7 4.3 ! 1.8 9.2 4.0**
Wolfgram proteins (w)
Proteolipid DM 20 protein
Basic protein
B/W
19.3 ! 9.0 11.8"
30.1 26.0 30.7
5.1 4.8 3.5
40.0 41.5 49.7*
2.2 2.2 4.2
21.7 15.9 12.7"*
26.4 20.7 32.5
5.0 4.5 3.2
35. ! 49.8 47.4**
1.6 3.8 3.8
Po
P3
P4
P~
Pz
43.0 44.1 51.5"*
35.6 37.1 28.9**
I 1.3 10.1 12.7
3.3 3.3 2.1
6.7 5.4 4.9**
tein/Wolfgiam proteins ratio (B/W) which was unchanged in the cervical spinal cord and forebrain. The addition of folinic acid to the regime caused a significant decrease in the high molecular weight Wolfgram proteins, and in all areas except the medulla there was a concomitant rise in the proportion of basic protein. These changes are reflected in an increased B/W ratio. Peripheral nerve myelin protein composition was altered by the combined nitrous oxide and folinic acid treatment, but was unchanged in preparations from animals treated with nitrous oxide alone. Protein Po was increased at the expense of proteins P2 and P3 (Table II), in contrast to the loss of high molecular weight proteins and their replacement with lower molecular weight basic protein seen in the central nervous system myelin samples. In myelin prepared from fruit bats made deficient in vitamin BI2 by dietary deprivation, changes in the myelin protein composition were not detectable when symptoms of neurological disturbance were apparent [7]. Nitrous oxide administration, on the other hand, caused a rearrangement of the relative protein composition which was exacerbated by folinic acid administration. The dramatic effect of nitrous oxide plus folinic acid may be ascribed to a combination of mechanisms which increase methylfolate concentrations.
103 Two mechanisms are proposed to explain the observed effect of nitrous oxide on myelin membrane proteins. (1) Vitamin Bn depletion by nitrous oxide causes a depletion of the Sadenosylmethionine pool for protein methylation by inhibiting methionine synthetase. Since this process is very rapid, gross changes in membrane structure may become apparent whereas there were no detectable changes i n membrane composition in vitamin B~2 deficiency induced by dietary manipulation because of its slow onset. (2) Nitrous oxide is a potent anaesthetic and analgesic agent. Since its anaesthetic actions are classically described as interference with the lipid bilayer, changes in membrane protein components would be necessary to stabilise the structure of the membrane. Myelin basic protein has previously been ~ ~cribed an important role in stabilising the myelin membrane, and these results may be a direct demonstration of this property. Furthermore, nitrous oxide has recently been shown to interfere with the function of membrane receptor proteins in vitro, and a specific interaction of nitrous oxide with the opiate binding site has been published recently [1], providing further evidenced for a direct membrane interaction. The authors are grateful to the Medical Research Council of South Africa for its generous support, and to Dr. J. van der Westhuyzen for experimental animals.
1 Daras, C., CantriU, R.C. and Gillman, M.A., ['HlNaloxone displacement: evidence for nitrous oxide as opiod receptor agonist, Europ. J. Pharmacol., 89 (1983) 177-178. 2 Green R., von Tonder, S.V., Oettle, G.J., Cole, G. and Metz, J., Neurological changes in fruit bats deficient in vitamin Btz, Nature (Lond.), 254, (1975) 14P-150. 3 Greenfield, S., Brostoff, S., Eylar, E.H. and Moreil, P., Protein composition of myelin of the peripheral nervous system, J. Neurochem., 20 (1973) 1207-1216. 4 Haj6s, F., An improved method for the preparation of synaptosomal fractions in high purity, Brain Res., 93 (1975) 485-489. 5 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 165-175. 6 McLoughlin, J.L. and Cantrill, R.C., Vitamin Bn deficiency alters the distribution of membrane proteins in the fruit bat nervous sytem, Brain Res., in press. 70ldfield, M., Van der Westhuyzen, J., McLoughlin, J.L. and Cantrill, R.C., Protein profile of the myelin membrane of the fruit bat Roussettus aep~J,pticus, Comp. Biochem. Physiol., in press. 8 Scott, J.M. and Weir, D.G., The methyl folate trap, Lancet, 2 (1981) 337-340. 9 Small, D.A. and Carnegie, P.R., Myelopathy associated with vitamin Bt2 deficiency, Trends Neuro Sci., 4 (1981) x-xi. 10 Van der Westhuyzen, J., Cantrill, R.C., Fernandez-Costa, F. and Metz, J., The lipid composition of the brain in the vitamin B~z deficient fruit bat (Roussettus Aegyptiacus) with neurological impairment, J. Neurochem., 37 (1981) 543-549. 11 Van der Westhuyzen, J., Fernandez-Costa, F., Metz, J. Kanazawa, S., Drivas, G. and Herbert, V., Cobalamin (vitamin BI,) analogs are absent in plasma from fruit bats exposed to nitrous oxide, Proc. Soc. exp. Biol. Med., 171 (1982) 88-91.
104 12 Van der Westhuyzen, J., CantriH, R.C., Fernandez-Costa, F. and Metz, J., Effect o f a vitamin BRz deficient diet on lip and fatty acid composition of spinal cord myelin in the fruit bat, J. Nutr., 113 (1983) 531-537. ! 3 Waehneldt, T.V. and Lane, J.D., Dissociation of myelin from its 'enzyme markers' during ontogeny, J. Neurc