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In vitro conversion of radioglucose to free and protein-bound amino acids by virus-infected mouse brain
In vitro Conversion of Radioglucose to Free and Protein-
Bound Amino Acids by Virus-Infected Kivie Moldave,2
Mouse Brain’
Max E.Rafelson. Jr.: Dorothy Lagerborg, Pearson and Richard J. Wmleti
Harold
E.
Prom the Departments of Biochemistry and Nutrition and of Microbiology, University of Southern California, School of Medicine; and the Laboratory Division, Los Angeles County Hospital; Los Angeles, California
Received October 19, 1953 INTRODUCTION
When minced l-day-old mouse brain is incubated with uniformly labeled glucose-O*, radioactivity is found in all of the protein-bound amino acids with the exceptions of proline and threonine (l-3). The propagation of Theiler’s GD VII mouse encephalomyelitis virus in this system stimulates the incorporation of glucose-Cl4fragments into all of the labeled amino acids except lysine and histidine, these being unlabeled in virus-infected cultures (1, 3). It appeared probable that free amino acids are intermediates between radioglucose and labeled protein-bound amino acids (2). Such a situation would require that the free amino acids have higher specific activities than the corresponding protein-bound amino acids. Examination of the free amino acids might also yield information to explain the inactivity of proline and threonine and the viral inhibition of the incorporation of carbon from glucose into lysine and histidine. The present experiments were carried out to determine the extent of in vitro incorporation of Cl4 from uniformly labeled glucose into the free amino acids of mouse brain, and to investigate the possible relationships between the turnover of the free and the protein-bound amino acids. 1 Supported by a grant from the National Advisory Council of the National Institutes of Health. 2 Present address: McArdle Memorial Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin. 3 Present address : Department of Biological Chemistry, University of Illinois College of Medicine, Chicago 12, Illinois. 383
384
MOLDAVE
ET
AL.
METHODS
Preparation
and Incubation
of Tissues
Fifty to 70 mg. of minced l-day-old mouse brain was incubated aseptically for 24 hr. at pH 9 in 3 ml. of Simms’ solution containing 3 mg. of uniformly labeled glucose-Cl4 with an activity of approximately 436,000 counts/min./mg. (1,4). Free amino acids from 125 to 150 flasks were pooled in each of the four in vitro experiments. Tests for sterility were made on all flasks, and virus titer was determined on the virus-infected cultures at the end of the incubation period (5). As unincubated controls, 10 g. of fresh l-day-old mouse brain was fractionated immediately upon sacrifice of the mice and removal of the brain. Completely independent pools were used for each of the four in vilro and the two control experiments.
Fractionation
of Tissue
Isolation of the free amino acids was carried out essentially by the procedures of Roberts et al. (6, 7). The contents of 20 fla,sks (or 20 fresh mouse brains) were added to anhydrous ethanol in amounts to give a final alcohol concentration of 79730/ The tissue, in 70% alcohol, was immediately homogenized in a Waring blendor and centrifuged, and the supernatant fluid was carefully filtered. This filtrate was evaporated to dryness at room temperature with a stream of air, and the resulting residue was extracted three times with 1 ml. water/100 mg. of fresh tissue at 60°C. In the series of experiments in which the amino acids were chromatographed on Dowex 50 resin columns, a further extraction was made with 0.01 N HCl at room temperature. The combined aqueous and acid extracts obtained from approximately 10 g. of brain tissue incubated in 125-150 flasks were pooled and concentrated with a stream of air at 50°C. to a volume of 10 ml. The free amino acids were “purified” by adsorption at pH 2 on acid-cycle Dowex 50 resin columns 1.2 cm. in diameter and 5 cm. in length, washing with 0.01 N HCl followed by distilled water and elution with 1 N NH,OH. Recovery experiments showed that no loss of aspartic acid, alanine, or lysine resulted from this procedure. Acidic substances, such as cysteic acid or taurine; or neutral substances, such as monosaccharides or urea; should be eliminated in the effluent. The NHdOH eluate was evaporated to dryness, taken up in the appropriate solvents, and chromatographed on starch or Dowex 50 resin columns. The “protein” fraction was isolated from the alcohol-insoluble residues by shaking with ethanol-ether and refluxing with chloroform-ethanol mixtures as described previously (4, 8). Portions of the “protein” fractions were hydrolyzed and prepared for chromatography by the procedure previously used (I, 4).
Analysis
of Amino Acids
Two methods were employed to separate the amino acids. In Expts. I, II, and III, the “purified” free amino acid preparations and the hydrolyzed “protein” fractions were separated on starch columns with 2:l propanol-0.5 N HCl according to the procedures of Moore and Stein (9-12) as previously described (1). The free and protein-bound amino acids from Expts. IV, V, and VI were separated on 15 and loo-cm. Dowex 50 resin columns (13) and were pooled as previously described
(2).
TABLE Concenlrations
and Specific
dctivities
of dmino
I Acids
Separated
Free amino acid3
fiimolesa
Aspartic
Controld Infectede Freshf
acid
by Starch
Colwm~
‘rotein-bound acids
amino
pXU01E%=
33.9 32.2 27.6
1
-
S.A.6
19.1 19.2
5350 7350
11.9 20.0
2800 3750
Control Infected Fresh
0 0 1.4
GIycine
Control Infected Fresh
30.0 31.4 32.0
5050 6200 -
19.9 19.8
2700 3100
Histidine
Control Infected FPA
45.8 51.9 42.0
1900 2100 -
16.2 11.0
1350 0
Lysine
cuntro1 infected Fresh
24.0 0 23.3
750 -
32.8 12.0
900 0
Phenylalanine
Control Infected Fresh
1.3 1.7 2.0
4800 9800
5.9 5.7
3050 3600
Proline
Control Infected Fresh
0 0 0
-
35.3 31.8
0 0
Serine
Control Infected Fresh
2.5 2.2 1.6
18,Ooa 34,ioo -
20.7 20.8
2600 5150
Threonine
Control Infected Fresh
9.3 9.5 11.4
0 0 -.
21.3 21.3
0 0
Tyrosine
Control Infected Fresh
1.2 1.5 1.2
19,300 33,400 -
5.8 5.9
2480 3660
Control Infected Fresh
35.4 37.5 35.7
12,900 14,200
30.6 31.0
3700 54uo
Cystiuc
Glutamic
acid0
+ alanine
385
-
-
386
MOLDAVE
TABLE
ET
AL.
I-Continued Free amino
acids
rmcdee
SAP
1Proteinhgd
amino
__
plldC3C
_-
--
S.A?
Valineo + methionine
Control Infected Fresh
3.3 3.1 4.6
19,600 30,300 -
30.3 30.6
1700 3200
Arginine
Control Infected Fresh
31.0”
2200
2350 3450
29.6
-
17.7 18.0
Control Infected Fresh
2.7 6.3 3.9
17,500 19,700 -
31.0 30.8
3350 4650
Leucine
+ ammonia
-I- isoleucine
-
-
-
-
0 Micromoles of free amino acid/100 g fresh tissue. b Specific activity expressed as counts/mm ./micromole amino acid. c Micromoles of amino acid for 100 mg. of hydrolyzed “protein.” d Experiment I: 24-hr. incubation without virus. 0 Experiment II: 24-hr. incubation with Theiler’s GD VII virus. f Experiment III : Fresh l-day-old mouse brain. 0 Values are for the combined amino acids. b No arginine was recovered from the starch column in this experiment. Free amino acid concentrations are expressed as micromoles of amino acid/100 g. of fresh tissue. Protein-bound amino acid concentrations are expressed as micromoles of amino acid/100 mg. of hydrolyzed “protein.” Radioactivity associated with the amino acids is expressed as the specific activity (counts/min./micromole amino acid). In all experiments the identity and purity of the effluent amino acids were checked by ascending paper chromatography in one dimension using watersaturated phenol as developer. RESULTS
Table I presents the data for Expts. I, II, and III in which the free and the protein-bound amino acids were separated using starch-column chromatography. Table II gives the results of Expts. IV, V, and VI in which the separations were carried out using Dowex 50 columns. The values for the amounts and specific activities of the proteinbound amino acids (alcohol-insoluble fraction) from control and virusinfected brain are similar to those previously reported for the trichloroacetic acid-insoluble residues (l-3). As was then pointed out, all of the
TABLE Amounts
and Specific
Activities
II
of Amino
Acids
Separated Free amino
by Dowex
60 Columns
acids %A?
lmloles”
S.A?
Alanine
ControP Infected8 Fresh/
6.3 5.4 5.8
3100 7300 -
11.6 11.6 12.6
1850 4100 -
Arginine
Control Infected Fresh
18.9 16.8 17.8
1800 3550 -
11.8 12.8 13.2
1400 3100 -
Control Infected Fresh
29.7 28.6 24.6
6900 10,800 -
22.0 22.8 22.4
4850 6900 -
Control Infected Fresh
0.9 0.7 1.8
2200 5ooo -
8.7 15.2 12.1
1000 2750 -
Control Infected Fresh
28.1 29.7 30.3
8700 13,300 -
23.1 23.3 20.8
4550 7200 -
Glycine
Control Infected Fresh
33.3 28.6 29.4
4200 5450 -
20.2 19.8 20.7
2450 3600 -
Histidine
Control Infected Fresh
43.1 40.6 39.9
850 950 -
16.4 11.6 17.1
800 0 -
Isoleucine
Control Infected Fresh
2.0 1.9 1.8
5ooo 8700 -
10.9 11.0 10.9
1800 2650 -
I,eucine
Control Infected Fresh
3.3 2.8 2.7
7100 11,500 -
20.4 21.1 20.3
2100 3200 -
Lysine
Control Infected Fresh
19.9 0 21.7
-
32.1 12.9 32 2
Control Infected Fresh
1.9 2.3 2.5
10,400 25,800 -
10.7 11.3 18.6
Aspartic
acid
Cystine
Glutantic
Methionine
acid
387
900
-
400 0 2450 3350 -
388
MOLDAVE
TABLE
ET
AL.
II-Continued
= Free amino
iI
‘rot&-bound acids
acids
I LltlOl‘2S~
S.A.)
pmolesC
amino
S.A.b
--
Proline
-
Control Infected Fresh
0 0 0
Serine
Control Infected Fresh
1.6 1.9 1.1
12,300 24,800 -
19.4 18.8 20.7
1500 3600 -
Threonine
Control Infected Fresh
9.9 10.4 12.1
0 0
19.6 19.3 19.5
-
0 0
Control Infected Fresh
2.4 2.1 3.0
650 1050 -
21.1 20.9 15.0
200 700 -
Control Infected Fresh
4.7 5.1 6.1
11,900 16,500 -
13.8 14.6 13.9
1300 2850 -
Valine
Phenylalanine
+ tyrosine
34.4 35.8 33.7
-
-
-
0 0 -
-
0 Micromoles of free amino acid/100 g. fresh tissue. a Specific activity expressed as counts/min./micromole amino acid. c Micromoles of amino acid for 100 mg. of hydrolyzed “protein.” d Experiment IV: 24-hr. incubation without virus. e Experiment V: 24-hr. incubation with Theiler’s GD VII virus. f Fresh l-day-old mouse brain.
protein-bound amino acids except proline and threonine incorporate radioactivity from glucose. Infection with Theiler’s GD VII virus increasesthe appearance of CA into all of the labeled amino acids except histidine and lysine, which are reduced in amount and which do not incorporate carbon from glucose in the virus-infected cultures. Tables I and II show that some of the observations on the proteinbound amino acids may be correlated with the amounts and specific activities of the free amino acids. With the exception of free cystine, which was not found in the starch columns, the agreement in the amounts and activities of the free amino acids by the two methods was reasonably
CONVERSION
OF RADIOGLUCOSE
389
good, In most casesthe specific activities of the free amino acids were significantly higher than those for the corresponding protein-bound acids. The amounts of most of the free amino acids from fresh l-day old mouse brain were found to be similar to those found in the unincubated control tissues. Three ninhydrin-reacting substances which were recovered from the free amino acid preparations in unincubated mouse brain, did not correspond in position to any of the known amino acids. The possibility that one of these substancesmay be y-aminobutyric acid, whose presence in mouse brain has been demonstrated by Roberts and Frankel (14), is now being investigated. Other possibilities include glutathione or ethanolamine. Unknown ninhydrin-reacting material was not recovered from any of the incubated mouse brain preparations whether analyzed by starch or by resin chromatography. Separation of the free amino acids from fresh brain by starch columns also failed to reveal the presence of any ninhydrin-reacting substances other than the amino acids listed in Table I. The data in Tables I and II reveal that the concentrations of most of the free amino acids were similar in the control and virus-infected tissues.An important exception, however, was free lysine which was present in the uninfected tissue but not in the infected tissues. Free proline was absent from fresh or incubated brain. The data in Tables I and II show that radioactivity was associated with all of the free amino acids except threonine. The specific activities of most of the free amino acids were appreciably higher in the infected than in the uninfected cultures. DISCUSSION
The data presented have demonstrated that radioactive carbon from glucose is incorporated into certain of the free amino acids of minced l-day-old mouse brain during a period of 24 hr. incubation. The propagation of Theiler’s GD VII virus in this system is associated with increased incorporation of glucose-Cl4 fragments into most of the free amino acids. This effect would appear to be related to the previously noted stimulation by virus propagation of the incorporation of glucase-Cl4fragments into the protein-bound amino acids in this system. A number of the observations in Tables I and Ii are in accord with the suggestion that free amino acids are intermediates in the incorporation of radioactive carbon from glucose into the protein-bound amino acids.
390
MOLDAVE
ET AL.
1. The free amino acids had specific activities that were appreciably higher than their protein-bound counterparts. 2. Free threonine, though present in relatively large amounts, was, like the bound threonine, devoid of radioactivity. 3. Inbound proline was absent from fresh or incubated brain, and no radioactive carbon was present in the protein-bound proline. 4. Free radioactive lysine was present in the incubated control tissues but was inactive and reduced in amount in the infected cultures. Not in accord with the suggestion of the intermediate role of free amino acids is the observation that free radioactive histidine is present in both control and infected cultures. However the significant reduction in the protein-bound histidine in the infected cultures as previously noted may explain the absence of radioactivity in histidine in the virus-infected cultures. The present experiments suggest that one of the effects of the virus may be on the reaction sequences leading from glucose and its metabolites to free amino acids. A rough approximation of the “turnover” of the protein-bound amino acids may be obtained by the following relationship : relative specific Specific activity of protein-bound amino acid = activity of proteinSpecific’ activity of free amino acid bound amino acids Calculations of this type for all of the protein-bound amino acids in both virus-infected and control mouse brains indicate that the “relative turnover” of most of the protein-bound amino acids is of the same order of magnitude in control and virus-infected brain. Exceptions to this are methionine, lysine, histidine, and valine. The “relative turnover” of the first three is lower in the virus-infected brain, while the “turnover” of valine appeared to be increased in the virus-infected brain. SUMMARY
The amounts and specific activities of the free amino acids of minced l-day-old mouse brain incubated 24 hr. with glucose-U4 in the presence and absence of Theiler’s GD VII virus were determined and compared with the corresponding values for the protein-bound amino acids. Virus propagation stimulated the incorporation of radioactive carbon from glucose into most of the free amino acids as well as the proteinbound amino acids.
CONVERSION
OF
391
RADIOGLUCOSE
The specific activities of the labeled, free amino acids were higher than those of their protein-bound counterparts, suggesting that free ammo acids were intermediates in the conversion of metabolites derived from glucose into protein-bound amino acids. The data suggest that the effect of the virus was primarily on the reactions leading to the formation of labeled amino acids rather than on the subsequent incorporation of the labeled amino acids. REFERENCES
M. E., JR., WINZLER, 193, ‘205 (1951).
1. RAFELSON,
2. WINZLER, J. Biol. 3. MOLDAVE,
R. J., AND PEARSON,
R. J., MOLDAVE, K., RAFELSON, M. Chem. 199, 485 (1952). K., WINZLER, R. J., ‘AND PEARSON,
E.,
H. E., J. Biol.
JR., AND PEARSON,
H. E., J. Biol.
Chem.
Chem. H. E., 209, 357
(1953). 4. RAFELSON,
M.
E.,
JR.,
WINZLER,
R. J., AND PEARSON,
H.
E., J.
Biol.
Chem.
447 (1950). H., Science 109, 14 (1949). Cancer Research 9, 645 (1949). R. J., AND PEARSON, H. E., J. BioZ.
Chem.
181, 595 (1949). 5. 6. 7. 8.
PEARSON, ROBERTS, ROBERTS, RAFELSON,
H. E., J. Immunol. 84, E., AND TISHKOFF, G. E., AND FRANKEL, S., M. E., JR., WINZLER,
181, 583 (1949). 9. STEIN, W. H., AND MOORE,
S. J., J. BioZ. Chem. 178, 337 (1948). 10. MOORE, S., AND STEIN, W. H., J. BioZ. Chem. 178, 367 (1948). 11. MOORE, S., AND STEIN, W. H., J. BioZ. 178,53 (1949). 12. STEIN, W. H., AND MOORE, S., J. BioZ. Chem. 178.79 (1949). 13. MOORE, S., AND STEIN, W. H., J. BioZ. Chem. 192, 663 (1951). 14. ROBERTS, E., AND FRANKEL, S., J. BioZ. Chem. 187, 55 (1950).
Bound Amino Acids by Virus-Infected Kivie Moldave,2
Mouse Brain’
Max E.Rafelson. Jr.: Dorothy Lagerborg, Pearson and Richard J. Wmleti
Harold
E.
Prom the Departments of Biochemistry and Nutrition and of Microbiology, University of Southern California, School of Medicine; and the Laboratory Division, Los Angeles County Hospital; Los Angeles, California
Received October 19, 1953 INTRODUCTION
When minced l-day-old mouse brain is incubated with uniformly labeled glucose-O*, radioactivity is found in all of the protein-bound amino acids with the exceptions of proline and threonine (l-3). The propagation of Theiler’s GD VII mouse encephalomyelitis virus in this system stimulates the incorporation of glucose-Cl4fragments into all of the labeled amino acids except lysine and histidine, these being unlabeled in virus-infected cultures (1, 3). It appeared probable that free amino acids are intermediates between radioglucose and labeled protein-bound amino acids (2). Such a situation would require that the free amino acids have higher specific activities than the corresponding protein-bound amino acids. Examination of the free amino acids might also yield information to explain the inactivity of proline and threonine and the viral inhibition of the incorporation of carbon from glucose into lysine and histidine. The present experiments were carried out to determine the extent of in vitro incorporation of Cl4 from uniformly labeled glucose into the free amino acids of mouse brain, and to investigate the possible relationships between the turnover of the free and the protein-bound amino acids. 1 Supported by a grant from the National Advisory Council of the National Institutes of Health. 2 Present address: McArdle Memorial Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin. 3 Present address : Department of Biological Chemistry, University of Illinois College of Medicine, Chicago 12, Illinois. 383
384
MOLDAVE
ET
AL.
METHODS
Preparation
and Incubation
of Tissues
Fifty to 70 mg. of minced l-day-old mouse brain was incubated aseptically for 24 hr. at pH 9 in 3 ml. of Simms’ solution containing 3 mg. of uniformly labeled glucose-Cl4 with an activity of approximately 436,000 counts/min./mg. (1,4). Free amino acids from 125 to 150 flasks were pooled in each of the four in vitro experiments. Tests for sterility were made on all flasks, and virus titer was determined on the virus-infected cultures at the end of the incubation period (5). As unincubated controls, 10 g. of fresh l-day-old mouse brain was fractionated immediately upon sacrifice of the mice and removal of the brain. Completely independent pools were used for each of the four in vilro and the two control experiments.
Fractionation
of Tissue
Isolation of the free amino acids was carried out essentially by the procedures of Roberts et al. (6, 7). The contents of 20 fla,sks (or 20 fresh mouse brains) were added to anhydrous ethanol in amounts to give a final alcohol concentration of 79730/ The tissue, in 70% alcohol, was immediately homogenized in a Waring blendor and centrifuged, and the supernatant fluid was carefully filtered. This filtrate was evaporated to dryness at room temperature with a stream of air, and the resulting residue was extracted three times with 1 ml. water/100 mg. of fresh tissue at 60°C. In the series of experiments in which the amino acids were chromatographed on Dowex 50 resin columns, a further extraction was made with 0.01 N HCl at room temperature. The combined aqueous and acid extracts obtained from approximately 10 g. of brain tissue incubated in 125-150 flasks were pooled and concentrated with a stream of air at 50°C. to a volume of 10 ml. The free amino acids were “purified” by adsorption at pH 2 on acid-cycle Dowex 50 resin columns 1.2 cm. in diameter and 5 cm. in length, washing with 0.01 N HCl followed by distilled water and elution with 1 N NH,OH. Recovery experiments showed that no loss of aspartic acid, alanine, or lysine resulted from this procedure. Acidic substances, such as cysteic acid or taurine; or neutral substances, such as monosaccharides or urea; should be eliminated in the effluent. The NHdOH eluate was evaporated to dryness, taken up in the appropriate solvents, and chromatographed on starch or Dowex 50 resin columns. The “protein” fraction was isolated from the alcohol-insoluble residues by shaking with ethanol-ether and refluxing with chloroform-ethanol mixtures as described previously (4, 8). Portions of the “protein” fractions were hydrolyzed and prepared for chromatography by the procedure previously used (I, 4).
Analysis
of Amino Acids
Two methods were employed to separate the amino acids. In Expts. I, II, and III, the “purified” free amino acid preparations and the hydrolyzed “protein” fractions were separated on starch columns with 2:l propanol-0.5 N HCl according to the procedures of Moore and Stein (9-12) as previously described (1). The free and protein-bound amino acids from Expts. IV, V, and VI were separated on 15 and loo-cm. Dowex 50 resin columns (13) and were pooled as previously described
(2).
TABLE Concenlrations
and Specific
dctivities
of dmino
I Acids
Separated
Free amino acid3
fiimolesa
Aspartic
Controld Infectede Freshf
acid
by Starch
Colwm~
‘rotein-bound acids
amino
pXU01E%=
33.9 32.2 27.6
1
-
S.A.6
19.1 19.2
5350 7350
11.9 20.0
2800 3750
Control Infected Fresh
0 0 1.4
GIycine
Control Infected Fresh
30.0 31.4 32.0
5050 6200 -
19.9 19.8
2700 3100
Histidine
Control Infected FPA
45.8 51.9 42.0
1900 2100 -
16.2 11.0
1350 0
Lysine
cuntro1 infected Fresh
24.0 0 23.3
750 -
32.8 12.0
900 0
Phenylalanine
Control Infected Fresh
1.3 1.7 2.0
4800 9800
5.9 5.7
3050 3600
Proline
Control Infected Fresh
0 0 0
-
35.3 31.8
0 0
Serine
Control Infected Fresh
2.5 2.2 1.6
18,Ooa 34,ioo -
20.7 20.8
2600 5150
Threonine
Control Infected Fresh
9.3 9.5 11.4
0 0 -.
21.3 21.3
0 0
Tyrosine
Control Infected Fresh
1.2 1.5 1.2
19,300 33,400 -
5.8 5.9
2480 3660
Control Infected Fresh
35.4 37.5 35.7
12,900 14,200
30.6 31.0
3700 54uo
Cystiuc
Glutamic
acid0
+ alanine
385
-
-
386
MOLDAVE
TABLE
ET
AL.
I-Continued Free amino
acids
rmcdee
SAP
1Proteinhgd
amino
__
plldC3C
_-
--
S.A?
Valineo + methionine
Control Infected Fresh
3.3 3.1 4.6
19,600 30,300 -
30.3 30.6
1700 3200
Arginine
Control Infected Fresh
31.0”
2200
2350 3450
29.6
-
17.7 18.0
Control Infected Fresh
2.7 6.3 3.9
17,500 19,700 -
31.0 30.8
3350 4650
Leucine
+ ammonia
-I- isoleucine
-
-
-
-
0 Micromoles of free amino acid/100 g fresh tissue. b Specific activity expressed as counts/mm ./micromole amino acid. c Micromoles of amino acid for 100 mg. of hydrolyzed “protein.” d Experiment I: 24-hr. incubation without virus. 0 Experiment II: 24-hr. incubation with Theiler’s GD VII virus. f Experiment III : Fresh l-day-old mouse brain. 0 Values are for the combined amino acids. b No arginine was recovered from the starch column in this experiment. Free amino acid concentrations are expressed as micromoles of amino acid/100 g. of fresh tissue. Protein-bound amino acid concentrations are expressed as micromoles of amino acid/100 mg. of hydrolyzed “protein.” Radioactivity associated with the amino acids is expressed as the specific activity (counts/min./micromole amino acid). In all experiments the identity and purity of the effluent amino acids were checked by ascending paper chromatography in one dimension using watersaturated phenol as developer. RESULTS
Table I presents the data for Expts. I, II, and III in which the free and the protein-bound amino acids were separated using starch-column chromatography. Table II gives the results of Expts. IV, V, and VI in which the separations were carried out using Dowex 50 columns. The values for the amounts and specific activities of the proteinbound amino acids (alcohol-insoluble fraction) from control and virusinfected brain are similar to those previously reported for the trichloroacetic acid-insoluble residues (l-3). As was then pointed out, all of the
TABLE Amounts
and Specific
Activities
II
of Amino
Acids
Separated Free amino
by Dowex
60 Columns
acids %A?
lmloles”
S.A?
Alanine
ControP Infected8 Fresh/
6.3 5.4 5.8
3100 7300 -
11.6 11.6 12.6
1850 4100 -
Arginine
Control Infected Fresh
18.9 16.8 17.8
1800 3550 -
11.8 12.8 13.2
1400 3100 -
Control Infected Fresh
29.7 28.6 24.6
6900 10,800 -
22.0 22.8 22.4
4850 6900 -
Control Infected Fresh
0.9 0.7 1.8
2200 5ooo -
8.7 15.2 12.1
1000 2750 -
Control Infected Fresh
28.1 29.7 30.3
8700 13,300 -
23.1 23.3 20.8
4550 7200 -
Glycine
Control Infected Fresh
33.3 28.6 29.4
4200 5450 -
20.2 19.8 20.7
2450 3600 -
Histidine
Control Infected Fresh
43.1 40.6 39.9
850 950 -
16.4 11.6 17.1
800 0 -
Isoleucine
Control Infected Fresh
2.0 1.9 1.8
5ooo 8700 -
10.9 11.0 10.9
1800 2650 -
I,eucine
Control Infected Fresh
3.3 2.8 2.7
7100 11,500 -
20.4 21.1 20.3
2100 3200 -
Lysine
Control Infected Fresh
19.9 0 21.7
-
32.1 12.9 32 2
Control Infected Fresh
1.9 2.3 2.5
10,400 25,800 -
10.7 11.3 18.6
Aspartic
acid
Cystine
Glutantic
Methionine
acid
387
900
-
400 0 2450 3350 -
388
MOLDAVE
TABLE
ET
AL.
II-Continued
= Free amino
iI
‘rot&-bound acids
acids
I LltlOl‘2S~
S.A.)
pmolesC
amino
S.A.b
--
Proline
-
Control Infected Fresh
0 0 0
Serine
Control Infected Fresh
1.6 1.9 1.1
12,300 24,800 -
19.4 18.8 20.7
1500 3600 -
Threonine
Control Infected Fresh
9.9 10.4 12.1
0 0
19.6 19.3 19.5
-
0 0
Control Infected Fresh
2.4 2.1 3.0
650 1050 -
21.1 20.9 15.0
200 700 -
Control Infected Fresh
4.7 5.1 6.1
11,900 16,500 -
13.8 14.6 13.9
1300 2850 -
Valine
Phenylalanine
+ tyrosine
34.4 35.8 33.7
-
-
-
0 0 -
-
0 Micromoles of free amino acid/100 g. fresh tissue. a Specific activity expressed as counts/min./micromole amino acid. c Micromoles of amino acid for 100 mg. of hydrolyzed “protein.” d Experiment IV: 24-hr. incubation without virus. e Experiment V: 24-hr. incubation with Theiler’s GD VII virus. f Fresh l-day-old mouse brain.
protein-bound amino acids except proline and threonine incorporate radioactivity from glucose. Infection with Theiler’s GD VII virus increasesthe appearance of CA into all of the labeled amino acids except histidine and lysine, which are reduced in amount and which do not incorporate carbon from glucose in the virus-infected cultures. Tables I and II show that some of the observations on the proteinbound amino acids may be correlated with the amounts and specific activities of the free amino acids. With the exception of free cystine, which was not found in the starch columns, the agreement in the amounts and activities of the free amino acids by the two methods was reasonably
CONVERSION
OF RADIOGLUCOSE
389
good, In most casesthe specific activities of the free amino acids were significantly higher than those for the corresponding protein-bound acids. The amounts of most of the free amino acids from fresh l-day old mouse brain were found to be similar to those found in the unincubated control tissues. Three ninhydrin-reacting substances which were recovered from the free amino acid preparations in unincubated mouse brain, did not correspond in position to any of the known amino acids. The possibility that one of these substancesmay be y-aminobutyric acid, whose presence in mouse brain has been demonstrated by Roberts and Frankel (14), is now being investigated. Other possibilities include glutathione or ethanolamine. Unknown ninhydrin-reacting material was not recovered from any of the incubated mouse brain preparations whether analyzed by starch or by resin chromatography. Separation of the free amino acids from fresh brain by starch columns also failed to reveal the presence of any ninhydrin-reacting substances other than the amino acids listed in Table I. The data in Tables I and II reveal that the concentrations of most of the free amino acids were similar in the control and virus-infected tissues.An important exception, however, was free lysine which was present in the uninfected tissue but not in the infected tissues. Free proline was absent from fresh or incubated brain. The data in Tables I and II show that radioactivity was associated with all of the free amino acids except threonine. The specific activities of most of the free amino acids were appreciably higher in the infected than in the uninfected cultures. DISCUSSION
The data presented have demonstrated that radioactive carbon from glucose is incorporated into certain of the free amino acids of minced l-day-old mouse brain during a period of 24 hr. incubation. The propagation of Theiler’s GD VII virus in this system is associated with increased incorporation of glucose-Cl4 fragments into most of the free amino acids. This effect would appear to be related to the previously noted stimulation by virus propagation of the incorporation of glucase-Cl4fragments into the protein-bound amino acids in this system. A number of the observations in Tables I and Ii are in accord with the suggestion that free amino acids are intermediates in the incorporation of radioactive carbon from glucose into the protein-bound amino acids.
390
MOLDAVE
ET AL.
1. The free amino acids had specific activities that were appreciably higher than their protein-bound counterparts. 2. Free threonine, though present in relatively large amounts, was, like the bound threonine, devoid of radioactivity. 3. Inbound proline was absent from fresh or incubated brain, and no radioactive carbon was present in the protein-bound proline. 4. Free radioactive lysine was present in the incubated control tissues but was inactive and reduced in amount in the infected cultures. Not in accord with the suggestion of the intermediate role of free amino acids is the observation that free radioactive histidine is present in both control and infected cultures. However the significant reduction in the protein-bound histidine in the infected cultures as previously noted may explain the absence of radioactivity in histidine in the virus-infected cultures. The present experiments suggest that one of the effects of the virus may be on the reaction sequences leading from glucose and its metabolites to free amino acids. A rough approximation of the “turnover” of the protein-bound amino acids may be obtained by the following relationship : relative specific Specific activity of protein-bound amino acid = activity of proteinSpecific’ activity of free amino acid bound amino acids Calculations of this type for all of the protein-bound amino acids in both virus-infected and control mouse brains indicate that the “relative turnover” of most of the protein-bound amino acids is of the same order of magnitude in control and virus-infected brain. Exceptions to this are methionine, lysine, histidine, and valine. The “relative turnover” of the first three is lower in the virus-infected brain, while the “turnover” of valine appeared to be increased in the virus-infected brain. SUMMARY
The amounts and specific activities of the free amino acids of minced l-day-old mouse brain incubated 24 hr. with glucose-U4 in the presence and absence of Theiler’s GD VII virus were determined and compared with the corresponding values for the protein-bound amino acids. Virus propagation stimulated the incorporation of radioactive carbon from glucose into most of the free amino acids as well as the proteinbound amino acids.
CONVERSION
OF
391
RADIOGLUCOSE
The specific activities of the labeled, free amino acids were higher than those of their protein-bound counterparts, suggesting that free ammo acids were intermediates in the conversion of metabolites derived from glucose into protein-bound amino acids. The data suggest that the effect of the virus was primarily on the reactions leading to the formation of labeled amino acids rather than on the subsequent incorporation of the labeled amino acids. REFERENCES
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