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Amino acids and the maintenance of osmotic equilibrium in brain tissue
Vol . 5, pp . 2321-2329, 1966 " Life Sciences Printed in Great Britain .
Pergamon Press Ltd.
AMINO ACIDS AND THE MAINTENANCE OF OSMOTIC EQUILIBRIUM IN BRAIN TISSUES Claude F . Baxter and C . Leo Ortiz Neurochemistry Laboratories Veterans Administration Hospital, Sepulveda, California, Department of Biochemistry, City of Hope Medical Center, Duarte, and Department oP Physiology, UCLA School of Medicine, Los Angeles, California (Received
5 October 1966)
It is well established that in marine invertebrates, intracellular amino acid concentrations play a role in establishing osmotic equilibrium between different tissues and their environment (1) .
Changes in "free" amino acid levels which
parallel alterations in environmental salinity have been described also in muscle tissue and plasma oP amphibians (2,3) . The functions of non-protein bound amino acids in the vertebrate central nervous system (CNS) are not fully understood . Levels of these nitrogenous compounds have been shown to remain extremely constant under a large variety of physiological stress conditions (4) .
On the basis of several lines oP investigation,
it would appear that amino acids such as glutamic acid (GA) and gamma-aminobutyric acid (GABA), may play a role in modü~ring or mediating excitation or inhibition in the nervous system (for reviews see 5,6) .
Since close osmotic regulation in the verte-
brate CNS is of great physiological importance, it seemed oP interest to determine whether GA, GABA and amino acids in general were involved in the maintenance of an osmotic equilibrium in sThis résearch wás sûpported by Grant BNB-03g43 from the National Institute oP Neurological Diseases and Blindness, U . S . Public Health Service . 2321
Yol. 5, No . 24
BRAIN AMINO ACIDS
2322 this tissue .
The experiments reported here show that amino acid levels in the brains of the western toad (Bufo boreas ) are altered when these amphibians are adapted to an environment of brackish water .
The changes observed in amino acid levels of brain tissue
are greater than any reported for vertebrates in response to a normal physiological stress situation . Methods Adult western toads ( Bufo boreas ) ranging in weight from 25 to 100 grams and of both sexes were collected in Ventura County, California, by a local supplier .
In the laboratory, they
were housed in separate groups, 10 to 15 toads to each 30 gallon aquarium .
The aquariums were stacked on adjustable shelving
inclined at an angle of approximately 20° from the horizontal . This arrangement permitted the addition of water to one half of each aquarium, while leaving the other half dry . changed daily .
The water was
Control groups of toads were kept in aquariums
with distilled water throughout the experimental period .
Experi-
mental groups of toads were first conditioned to the laboratory environment for at least one week in distilled water .
They were
then exposed for two days to a mixture of 20~ ocean water and 80z distilled water .
Subsequently, they were adapted for 2 to 3 days
to an environment containing 40z ocean water and 60x distilled water .
The final osmolality of the brackish water varied from
400 to 420 m.Osm/1, as determined by freezing point depression with an Advanced Osmometer; . For the biochemical assay of brain tissue, both adapted and control toads were decapitated .
Brains were removed rapidly,
lightly blotted, weighed and homogenized in 80~ Ethyl alcohol . Advanced
nstruments, Inc ., Newton Highlands, Mass .
BRAIN AMINO ACIDS
Yol . 5, No . 24
2323
Tissue extracts were prepared as previously described (7) .
For
some experiments the extracts of several toad brains were pooled . Ninhydrin positive compounds in these extracts were separated and measured with a Technicon amino acid analyzer system connected to s Gilford Model 2000 spectrophotometer and recorder .
In one
experiment, total brain extracts were prepared according to the methods developed by Stein and Moors (8) with the exception that BioRad Ag2 X8 resin was used to remove excess picric acid .
These
particular extracts were analyzed on a Beckman-Spinco amino acid analyzer .
GA and glycine were measured enzymatically according
to methods described recently (9,10) except that alcoholic rather than trichloracetic acid extracts of brain tissue were prepared for these assays .
The enzymatic methods used to determine OABA
and urea have álso been described (11,12) . The relative water content of the toad brains was measured using either sectioned whole brain, or alcoholic homogenates representing known amounts oP brain tissue . Samples were dried in plastic dishes at 60°C under reduced pressure until constant weight was attained . Results The behavior of toads adapted to brackish water was indistinguishable Prom controls maintained in the distilled water environment, except that the Former spent more time in the aqueous half oP the aquarium and occasionally appeared a little sluggish . Amino acid levels in the brain of toads of any single shipment by the supplier were quite uniform, as was the biochemical response to oamotlc stress .
There appeared to be, however, seasonal varia-
tions, both in normal amino acid levels and in the amount of change produced by a brackish water environment .
Thus, GABA
levels in osmotically stressed toads were elevated in some
2324
BRAIN AMINO ACIDS
Vol . 5, No . 24
experiments by only 30X while in others as much as 80~ . These observations are consistent with the numerous reports by investigators who found large seasonal variations in metabolism and metabolites of amphibians (13) .
Such variations
did not relate directly to the nutritional state of the animals or to the environmental temperature (14,15) . TABLE I Effect of Environmental Salinity Upon GABA Levels in Brains of Toad Bufo boreas
Condition
Sexy
Animal #
Control H2O
M F H
8 8 8
Ave .
4ox Ocean H2O
M F H
8 8 8
Ave .
body wt . gm+~
GABA ug/gmt
Increase
38 46 50
80 86 82
26~t 264 275
45
83
268
38 54 38
77 82 72
358 341 365
35 29 33
43
77
348
32
Average body weight of group . by more than * 10 gm : }a Average brain weight . than : 8 mg .
brain wt . mgr*
No toad within group differed
No brain within group differed by more
t
2 brains were pooled . Each result, expressed in ug/gram brain tissue (wet weight), is the avérage of 4 analyses . 1do assay differed by more than * 15 ug/gm from the average .
A
M = male ; F = female ; H ~ hermaphrodite . There was no significant difference in the response to
osmotic stress by males, females and hermaphrodites (Table I) . effort was made in subsequent experiments to separate the sexes .
No
Vol . 5, No . 24
BRAIN AàdINO ACIDS
232 5
TABLE II Compositional Changes in Major Nitrogenous Constituents of Toad Brain as a Result of Adaptation to Brackish Water CONTROL 40f OCEAN H2O umoles/gm . tissue (wet weight)
AMINO ACID
CHANGE
x
Aspartic
0 .9
2~1
+149
Alanine
0 .37
0 .87
+135
Glycine
0 .87
1 .6
+ 84
Glutamic
5 .5
9 .1
+ 65
2 .7
4 .0
+ 48
3 .5
4 "8
+ 3T
15 .8
37 .8
+139
1 .7
0 .8
- 55
Y
-aminobutyric
Glutamine + Asparagine* Urea Ethanolamine*
Results obtained with Beckman-Spinco amino acid analyzer only . Some amino acid changes in toad brain are shown in Table II .
A check of these results for GA, GABA, glycine and urea
by enzymatic assay, yielded values which agreed quite well with those obtained Prom the amino acid analyzer .
Amino acids not
listed in Table II varied little in the brains of control and adapted toads, or were present in such low concentrations that changes could be only of little consequence in maintaining the . osmotic equilibrium of brain tissues . The possibility that dehydration of brain tissue might account for the increased levels of amino acids in the brains of ocean water adapted toads was explored . Dry weight increased by a corresponding 6 to 12x (Table III) which is far less than the smallest increase recorded in Table II .
2326
Vol . 5, No . 24
BRAIN AMINO ACIDS TABLE III Changes in Brain Dry Weight in Toads Adapted to Brackish Water
Expt . ~ A*
B~f
CONTROL z
No . of Animals
40~ OCEAN H2O
x
z
15
H2O content Dry weight
85 .3 14 .7
84 .4 15 .6
-l .l +6 .1
8
H2O content Dry weight
84 .8 15 .2
83 .0 17 .0
-2 .1 +12 .0
Corresponding to results presented in Table I . sf Corresponding to results presented in Table II . Discussion The results reported here indicate that vertebrate brain tissue resembles invertebrate nervous tissue (16) in that amino acids appear to have functional significance in osmotic regulation Primarily those amino acids which are related to the tricarboxylic acid cycle responded to osmotic stress .
As a percentage change,
the increase in aspartic acid levels was the most dramatic . However, on a quantitative (molar) basis the increase of GA was of greater significance and the elevation of OABA equal to that of aspartic acid .
Overall, the intracellular amino acids and urea
levels were increased in excess of 30 umoles/gm of tissue .
Since
Curtis and co-workers have shown that both aA and GABA have profound effects upon the electrical activity of neuronal tissue of toads (17), it is noteworthy that large elevations of both of these amino acids in the brain of osmotically adapted toads were not accompanied by marked behavioral changes .
These observations
do not necessarily conflict with the hypothesis that ascribes transmitter functions to some amino acids in the nervous system .
Yol . 5, No . 24
BRAIN AMINO ACIDS
2327
It must be assumed, however, that the osmotically active amino acid pool is spatially differentiated from amino acids concerned directly with neuronal activity . The increased cerebral glycine concentration in osmotically stressed toads may be related to the decrease observed in ethanolamine (18) .
The possible function of urea in osmotic
regulation of amphibian muscle has been mentioned (19) and the osmotic mechanism by which urea reduces cerebral edema has been studied (20) .
The results here suggest that brain tissue is
similar to muscle tissue in that urea may be of importance in the osmotic response to a euryhaline environment .
Also, the changes
of amino acids in amphibian brain were somewhat similar to those reported for muscle tissue (2) .
One notable difference was that
the taurine level in brain tissue did not show the large elevation which was found in muscle tissue . The overall mechanisms by xhich intracellular amino acid levels are altered in response to a saline environment are not clearly understood .
Some studies in aquatic invertebrates
suggest activation of enzyme systems by cationic constituents (21) while other studies favor a mechanism involving anions (22) . There is some evidence which suggests that in amphibian muscle tissue, amino acids and urea are formed by an enhanced degradation of larger molecules (3) . Preliminary studies regarding these mechanisms in amphibian nervous tissue have been made (23) and will be reported in detail in a subsequent publication .
Our observations are in
agreement with the concept that inorganic ions mediate the mechanism responsible for the accumulation of amino acids in vertebrate nervous tissues .
2328
BRAIN AMINO ACIDS
Vol . 5, No . 24
Summary A selective elevation of amino acid levels in the vertebrate central nervous system was produced by adapting male, female, or hermaphrodite toads of the species Bufo boreas to brackish xater .
The largest quantitative changes observed were
in levels of glutamic acid, aspartic acid, Y -aminobutyric acid and urea .
Tissue dehydration accounts for only a very small
portion of these changes . Acknow ledgment We are indebted to Dr . John Pierce, Department of Biological Chemistry, UCLA, for making available to us the use of his Beckman-Spinco amino acid analyzer for a confirmatory run . References 1.
M . Florkin and E . Schoffeniels, In Studies in Comparative Biochemistry (edited by K . A . Munday) . p6 . Pergamon Press, London (1965) .
2.
M . S . Gordon, Biol . Bull ., 12
3.
R . R . Tercafs and E . Schoffeniels, Life Sci ., 1
4.
E . Roberts and D . G . Simonsen, In Amino Acid Pools (edited by J . T . Holden) . p348 .
5.
218
(1965) . (1962) .
19
Elsevier, Amsterdam (1962) .
D . R . Curtis, In Studies in Physiology . p34 "
Springer Verlag,
Berlin (1965) . 6.
D . R . Curtis and J . C . Watkins, J . Neurochem ., 6
7.
C . F . Baxter, Methods in Medical Research , 9
8.
W . H . Stein and S . Moore, J . Biol . Chem ., 211
9.
L . T . Graham, Jr ., R . Werman and M . H . Aprison, Life Sci ., 4
117
192
(1960) .
(1961) .
915
(1954) .
1085 (1965) .
10 . M . H . Aprison and R . Werman, Life Sci ., 4
2075
(1965) .
11 . C . F . Baxter and E . Roberts, Proc . Soc . Exptl . Biol . Med ., 101
811
(1959) .
BRAIN AMINO ACIDS
Vol . 5, No . 24
2329
12 . E . Bent and H . U . Bergmeyer, In Methode of Enzymatic Analysis , (edited by H . U . Bergmeyer) . p401 .
Academic Prees, New York
(1963) . 13 . S . Mizell, J . Cellular Comp . Physiol ., 66 14 . P . A . Wright, Endocrinology , 64
551
15 . C . L . Smith, J . Exptl . Biol ., 26
251
(1965) .
(1959) .
412
(1950) .
16 . E . Schoffeniela, Arch . Int . Physiol . Biochim ., 68
696 (1960) .
17 . D . R . Curtis, J . W . Phillis and J . C . Watkina, Brit . J . Pharmacol . Chemotherapy , 16
262
(1961) .
18 . A . Weissbach and D . B . Sprinson, J . Biol . Chem ., 203
1031
(1953) . 19 . M . S . oordon, K . Schmidt- Nielaen and H . M . Kelly, J . Exptl . Biol ., ~8
(1961) .
659
20 . D . J . Reed and D . M . Woodbury, J . Physiol ., 164 21 . E . Schoffeniela and R . aillea, Life Sci ., 1
834
252
(1962) .
(1963) .
22 . A . E . Chaplin, A . K . Huggins and K . A . Munday, Comp . Biochem . Physiol ., 16
49
(1965) .
23 . C . F . Baxter, Federation Proc ., 25
713
(1966) .
Pergamon Press Ltd.
AMINO ACIDS AND THE MAINTENANCE OF OSMOTIC EQUILIBRIUM IN BRAIN TISSUES Claude F . Baxter and C . Leo Ortiz Neurochemistry Laboratories Veterans Administration Hospital, Sepulveda, California, Department of Biochemistry, City of Hope Medical Center, Duarte, and Department oP Physiology, UCLA School of Medicine, Los Angeles, California (Received
5 October 1966)
It is well established that in marine invertebrates, intracellular amino acid concentrations play a role in establishing osmotic equilibrium between different tissues and their environment (1) .
Changes in "free" amino acid levels which
parallel alterations in environmental salinity have been described also in muscle tissue and plasma oP amphibians (2,3) . The functions of non-protein bound amino acids in the vertebrate central nervous system (CNS) are not fully understood . Levels of these nitrogenous compounds have been shown to remain extremely constant under a large variety of physiological stress conditions (4) .
On the basis of several lines oP investigation,
it would appear that amino acids such as glutamic acid (GA) and gamma-aminobutyric acid (GABA), may play a role in modü~ring or mediating excitation or inhibition in the nervous system (for reviews see 5,6) .
Since close osmotic regulation in the verte-
brate CNS is of great physiological importance, it seemed oP interest to determine whether GA, GABA and amino acids in general were involved in the maintenance of an osmotic equilibrium in sThis résearch wás sûpported by Grant BNB-03g43 from the National Institute oP Neurological Diseases and Blindness, U . S . Public Health Service . 2321
Yol. 5, No . 24
BRAIN AMINO ACIDS
2322 this tissue .
The experiments reported here show that amino acid levels in the brains of the western toad (Bufo boreas ) are altered when these amphibians are adapted to an environment of brackish water .
The changes observed in amino acid levels of brain tissue
are greater than any reported for vertebrates in response to a normal physiological stress situation . Methods Adult western toads ( Bufo boreas ) ranging in weight from 25 to 100 grams and of both sexes were collected in Ventura County, California, by a local supplier .
In the laboratory, they
were housed in separate groups, 10 to 15 toads to each 30 gallon aquarium .
The aquariums were stacked on adjustable shelving
inclined at an angle of approximately 20° from the horizontal . This arrangement permitted the addition of water to one half of each aquarium, while leaving the other half dry . changed daily .
The water was
Control groups of toads were kept in aquariums
with distilled water throughout the experimental period .
Experi-
mental groups of toads were first conditioned to the laboratory environment for at least one week in distilled water .
They were
then exposed for two days to a mixture of 20~ ocean water and 80z distilled water .
Subsequently, they were adapted for 2 to 3 days
to an environment containing 40z ocean water and 60x distilled water .
The final osmolality of the brackish water varied from
400 to 420 m.Osm/1, as determined by freezing point depression with an Advanced Osmometer; . For the biochemical assay of brain tissue, both adapted and control toads were decapitated .
Brains were removed rapidly,
lightly blotted, weighed and homogenized in 80~ Ethyl alcohol . Advanced
nstruments, Inc ., Newton Highlands, Mass .
BRAIN AMINO ACIDS
Yol . 5, No . 24
2323
Tissue extracts were prepared as previously described (7) .
For
some experiments the extracts of several toad brains were pooled . Ninhydrin positive compounds in these extracts were separated and measured with a Technicon amino acid analyzer system connected to s Gilford Model 2000 spectrophotometer and recorder .
In one
experiment, total brain extracts were prepared according to the methods developed by Stein and Moors (8) with the exception that BioRad Ag2 X8 resin was used to remove excess picric acid .
These
particular extracts were analyzed on a Beckman-Spinco amino acid analyzer .
GA and glycine were measured enzymatically according
to methods described recently (9,10) except that alcoholic rather than trichloracetic acid extracts of brain tissue were prepared for these assays .
The enzymatic methods used to determine OABA
and urea have álso been described (11,12) . The relative water content of the toad brains was measured using either sectioned whole brain, or alcoholic homogenates representing known amounts oP brain tissue . Samples were dried in plastic dishes at 60°C under reduced pressure until constant weight was attained . Results The behavior of toads adapted to brackish water was indistinguishable Prom controls maintained in the distilled water environment, except that the Former spent more time in the aqueous half oP the aquarium and occasionally appeared a little sluggish . Amino acid levels in the brain of toads of any single shipment by the supplier were quite uniform, as was the biochemical response to oamotlc stress .
There appeared to be, however, seasonal varia-
tions, both in normal amino acid levels and in the amount of change produced by a brackish water environment .
Thus, GABA
levels in osmotically stressed toads were elevated in some
2324
BRAIN AMINO ACIDS
Vol . 5, No . 24
experiments by only 30X while in others as much as 80~ . These observations are consistent with the numerous reports by investigators who found large seasonal variations in metabolism and metabolites of amphibians (13) .
Such variations
did not relate directly to the nutritional state of the animals or to the environmental temperature (14,15) . TABLE I Effect of Environmental Salinity Upon GABA Levels in Brains of Toad Bufo boreas
Condition
Sexy
Animal #
Control H2O
M F H
8 8 8
Ave .
4ox Ocean H2O
M F H
8 8 8
Ave .
body wt . gm+~
GABA ug/gmt
Increase
38 46 50
80 86 82
26~t 264 275
45
83
268
38 54 38
77 82 72
358 341 365
35 29 33
43
77
348
32
Average body weight of group . by more than * 10 gm : }a Average brain weight . than : 8 mg .
brain wt . mgr*
No toad within group differed
No brain within group differed by more
t
2 brains were pooled . Each result, expressed in ug/gram brain tissue (wet weight), is the avérage of 4 analyses . 1do assay differed by more than * 15 ug/gm from the average .
A
M = male ; F = female ; H ~ hermaphrodite . There was no significant difference in the response to
osmotic stress by males, females and hermaphrodites (Table I) . effort was made in subsequent experiments to separate the sexes .
No
Vol . 5, No . 24
BRAIN AàdINO ACIDS
232 5
TABLE II Compositional Changes in Major Nitrogenous Constituents of Toad Brain as a Result of Adaptation to Brackish Water CONTROL 40f OCEAN H2O umoles/gm . tissue (wet weight)
AMINO ACID
CHANGE
x
Aspartic
0 .9
2~1
+149
Alanine
0 .37
0 .87
+135
Glycine
0 .87
1 .6
+ 84
Glutamic
5 .5
9 .1
+ 65
2 .7
4 .0
+ 48
3 .5
4 "8
+ 3T
15 .8
37 .8
+139
1 .7
0 .8
- 55
Y
-aminobutyric
Glutamine + Asparagine* Urea Ethanolamine*
Results obtained with Beckman-Spinco amino acid analyzer only . Some amino acid changes in toad brain are shown in Table II .
A check of these results for GA, GABA, glycine and urea
by enzymatic assay, yielded values which agreed quite well with those obtained Prom the amino acid analyzer .
Amino acids not
listed in Table II varied little in the brains of control and adapted toads, or were present in such low concentrations that changes could be only of little consequence in maintaining the . osmotic equilibrium of brain tissues . The possibility that dehydration of brain tissue might account for the increased levels of amino acids in the brains of ocean water adapted toads was explored . Dry weight increased by a corresponding 6 to 12x (Table III) which is far less than the smallest increase recorded in Table II .
2326
Vol . 5, No . 24
BRAIN AMINO ACIDS TABLE III Changes in Brain Dry Weight in Toads Adapted to Brackish Water
Expt . ~ A*
B~f
CONTROL z
No . of Animals
40~ OCEAN H2O
x
z
15
H2O content Dry weight
85 .3 14 .7
84 .4 15 .6
-l .l +6 .1
8
H2O content Dry weight
84 .8 15 .2
83 .0 17 .0
-2 .1 +12 .0
Corresponding to results presented in Table I . sf Corresponding to results presented in Table II . Discussion The results reported here indicate that vertebrate brain tissue resembles invertebrate nervous tissue (16) in that amino acids appear to have functional significance in osmotic regulation Primarily those amino acids which are related to the tricarboxylic acid cycle responded to osmotic stress .
As a percentage change,
the increase in aspartic acid levels was the most dramatic . However, on a quantitative (molar) basis the increase of GA was of greater significance and the elevation of OABA equal to that of aspartic acid .
Overall, the intracellular amino acids and urea
levels were increased in excess of 30 umoles/gm of tissue .
Since
Curtis and co-workers have shown that both aA and GABA have profound effects upon the electrical activity of neuronal tissue of toads (17), it is noteworthy that large elevations of both of these amino acids in the brain of osmotically adapted toads were not accompanied by marked behavioral changes .
These observations
do not necessarily conflict with the hypothesis that ascribes transmitter functions to some amino acids in the nervous system .
Yol . 5, No . 24
BRAIN AMINO ACIDS
2327
It must be assumed, however, that the osmotically active amino acid pool is spatially differentiated from amino acids concerned directly with neuronal activity . The increased cerebral glycine concentration in osmotically stressed toads may be related to the decrease observed in ethanolamine (18) .
The possible function of urea in osmotic
regulation of amphibian muscle has been mentioned (19) and the osmotic mechanism by which urea reduces cerebral edema has been studied (20) .
The results here suggest that brain tissue is
similar to muscle tissue in that urea may be of importance in the osmotic response to a euryhaline environment .
Also, the changes
of amino acids in amphibian brain were somewhat similar to those reported for muscle tissue (2) .
One notable difference was that
the taurine level in brain tissue did not show the large elevation which was found in muscle tissue . The overall mechanisms by xhich intracellular amino acid levels are altered in response to a saline environment are not clearly understood .
Some studies in aquatic invertebrates
suggest activation of enzyme systems by cationic constituents (21) while other studies favor a mechanism involving anions (22) . There is some evidence which suggests that in amphibian muscle tissue, amino acids and urea are formed by an enhanced degradation of larger molecules (3) . Preliminary studies regarding these mechanisms in amphibian nervous tissue have been made (23) and will be reported in detail in a subsequent publication .
Our observations are in
agreement with the concept that inorganic ions mediate the mechanism responsible for the accumulation of amino acids in vertebrate nervous tissues .
2328
BRAIN AMINO ACIDS
Vol . 5, No . 24
Summary A selective elevation of amino acid levels in the vertebrate central nervous system was produced by adapting male, female, or hermaphrodite toads of the species Bufo boreas to brackish xater .
The largest quantitative changes observed were
in levels of glutamic acid, aspartic acid, Y -aminobutyric acid and urea .
Tissue dehydration accounts for only a very small
portion of these changes . Acknow ledgment We are indebted to Dr . John Pierce, Department of Biological Chemistry, UCLA, for making available to us the use of his Beckman-Spinco amino acid analyzer for a confirmatory run . References 1.
M . Florkin and E . Schoffeniels, In Studies in Comparative Biochemistry (edited by K . A . Munday) . p6 . Pergamon Press, London (1965) .
2.
M . S . Gordon, Biol . Bull ., 12
3.
R . R . Tercafs and E . Schoffeniels, Life Sci ., 1
4.
E . Roberts and D . G . Simonsen, In Amino Acid Pools (edited by J . T . Holden) . p348 .
5.
218
(1965) . (1962) .
19
Elsevier, Amsterdam (1962) .
D . R . Curtis, In Studies in Physiology . p34 "
Springer Verlag,
Berlin (1965) . 6.
D . R . Curtis and J . C . Watkins, J . Neurochem ., 6
7.
C . F . Baxter, Methods in Medical Research , 9
8.
W . H . Stein and S . Moore, J . Biol . Chem ., 211
9.
L . T . Graham, Jr ., R . Werman and M . H . Aprison, Life Sci ., 4
117
192
(1960) .
(1961) .
915
(1954) .
1085 (1965) .
10 . M . H . Aprison and R . Werman, Life Sci ., 4
2075
(1965) .
11 . C . F . Baxter and E . Roberts, Proc . Soc . Exptl . Biol . Med ., 101
811
(1959) .
BRAIN AMINO ACIDS
Vol . 5, No . 24
2329
12 . E . Bent and H . U . Bergmeyer, In Methode of Enzymatic Analysis , (edited by H . U . Bergmeyer) . p401 .
Academic Prees, New York
(1963) . 13 . S . Mizell, J . Cellular Comp . Physiol ., 66 14 . P . A . Wright, Endocrinology , 64
551
15 . C . L . Smith, J . Exptl . Biol ., 26
251
(1965) .
(1959) .
412
(1950) .
16 . E . Schoffeniela, Arch . Int . Physiol . Biochim ., 68
696 (1960) .
17 . D . R . Curtis, J . W . Phillis and J . C . Watkina, Brit . J . Pharmacol . Chemotherapy , 16
262
(1961) .
18 . A . Weissbach and D . B . Sprinson, J . Biol . Chem ., 203
1031
(1953) . 19 . M . S . oordon, K . Schmidt- Nielaen and H . M . Kelly, J . Exptl . Biol ., ~8
(1961) .
659
20 . D . J . Reed and D . M . Woodbury, J . Physiol ., 164 21 . E . Schoffeniela and R . aillea, Life Sci ., 1
834
252
(1962) .
(1963) .
22 . A . E . Chaplin, A . K . Huggins and K . A . Munday, Comp . Biochem . Physiol ., 16
49
(1965) .
23 . C . F . Baxter, Federation Proc ., 25
713
(1966) .