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Antigenic differences associated with conformational changes in glutamate dehydrogenase
226
BIOCHIMICAET BIOPHYSICAACTA
BBA 65052 A N T I G E N I C D I F F E R E N C E S ASSOCIATED W I T H C O N F O R M A T I O N A L C H A N G E S IN G L U T A M A T E D E H Y D R O G E N A S E NORMAN TALAL AND GORDON M. TOMKINS National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Md. (U.S.A.)
Received February 24th, 1964)
SUMMARY
The reactions between glutamate dehydrogenase (L-giutamate:DPN(TPN)oxidoreductase (deaminating), EC 1.4.1.3) crystallized from bovine liver and specific rabbit anti-glutamate dehydrogenase immune sera have been studied. This immunologic system can be resolved into three precipitin lines (a, b, and c) by immunoelectrophoresis and double diffusion. Quantitative precipitin experiments demonstrate that the immunologic reactivity of the enzyme varies in relation to changes induced by regulator molecules such as ADP or diethylstilbestrol. Studies performed with supernatant fractions from the precipitin experiments reveal a selective absorption of specific antibodies and sequential disappearance of precipitin lines starting with a. These results are explained on the basis of antigenic differences associated with different conformational states of glutamate dehydrogenase. INTRODUCTION Glutamate dehydrogenase (L-glutamate:DPN(TPN)oxidoreductase (deaminating), EC 1.4.1.3) crystallized from bovine liver is an equilibrium system composed of different molecular conformations of the enzyme which have different relative catalytic activities towards amino acid substrates, e.g. glutamate and alaninel, z. Immunoelectrophoretic studies recently reported 3 demonstrated three enzymically active glutamate dehydrogenase-anti glutamate dehydrogenase precipitin lines (a, b, and c) and thus showed that at least some of these molecular forms of glutamate dehydrogenase could be separated immunologically. The relative prbportions of these immunologic forms were influenced by reagents such as ADP, GTP and diethylstilbestrol which had been shown previously to affect the structure of glutamate dehydrogenasel,4, ~. In this report additional quantitative evidence is presented to show that the immunologic behavior of glutamate dehydrogenase varies as a result of antigenic differences arising from conformational changes induced by these "regulator molecules". An explanation is also proposed to account for the resolution into immunologically distinct components of this system which is in rapid, reversible equihbrium. Biochirn. Biophys. Aeta,
89 (1964) 226-231
ANTIGENIC DIFFERENCESIN GLUTAMICDEHYDROGENASE
227
MATERIALSAND METHODS Glutamate dehydrogenase (as a suspension of crystals in ammonium sulfate), ADP, DPN, DPNH~, diethylstilbestrol and amino acid substrates were obtained from the Sigma Chemical Company, St. Louis, Missouri. GTP was purchased from Pabst Laboratories, Milwaukee, Wisconsin. Antisera against native and denatured glutamate dehydrogenase was prepared in rabbits by methods previously described 3. The enzyme was examined for contamination and found to be homogeneous by the methods described earlier ~. Quantitative precipitin experiments were performed by adding increasing amounts of glutamate dehydrogenase (in 0.05 M Tris buffer, pH 8.0) to o.5-ml aliquots of rabbit antiserum prepared against glutamate dehydrogenase denatured with sodium dodecyl sulfate. Final volumes were 0. 7 ml. Precipitation was carried out at 37 ° for I h and then at 4 ° for 48 h. Antigen-antibody precipitates were centrifuged at 2500 rev./min for 45 min and washed twice with chilled o.15 M NaC1 buffered with o.15 M phosphate at pH 7.2. They were dissolved in 0.5 N NaOH and made up to 2 ml in the buffered NaC1. Protein concentrations were determined by the method of LOWRY et a l : . The supernatant fractions from quantitative precipitin experiments were examined for residual enzyme and antibody activities. Glutamate and alanine dehydrogenase activities were assayed by methods previously described 7 and antibody was detected by double diffusion in 1% agar gel 8.
Fig. I. Immunoelectrophoresis 9 of glutamate dehydrogenase in 1% agar gel buffered with 0.05 M veronal at pH 8.2. Electrophoresis was carried out in o.o25 M veronal buffer (pH 8.2) at I4O V and ioo mA for 4 h at room temperature. Precipitin lines a, b and c were revealed with rabbit anti-glutamate dehydrogenase antiserum 6 and lines a and b with antiserum 2. Line a, (illustrated schematically), did not appear in this experiment until glutamate dehydrogenase activity was identified by tetrazolium reduction employing o.o6 M L-glutamate and o.75 mM DPN as substrates 8. RESULTS Fig. I shows a typical immunoelectrophoretic experiment in which the glutamate dehydrogenase system has been resolved by reaction with anti-glutamate dehydrogenase antisera into 3 separate precipitin lines, a, b and c. It was previously reported 3 that these three immunologic forms of glutamate dehydrogenase have different relative catalytic activities towards L-glutamate and L-alanine and are affected by regulator molecules. Form a, for example, shows only glutamate dehydrogenase activity and is most apparent when experiments are performed in the Biochim. Biophys. Acta, 89 (1964) 226-23I
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N. TALAL, G. M. TOMKINS
presence of ADP. Conversely, form c has predominantly alanine dehydrogenase activity and is most readily observed when GTP or diethylstilbestrol are incorporated in the agar. Form b has both enzyme activities and is present under all conditions. It was shown s by specific absorption and double diffusion experiments that forms b and c are antigenically different. The results of three quantitative precipitin experiments employing the same antiserum are shown in Fig. 2. In each instance, increasing amounts of glutamate dehydrogenase were added to a constant amount of antiserum and the total precipitable protein measured. In two experiments the precipitates were allowed to form in the presence of D P N H , and either ADP or diethylstilbestrol. In the third experiment (control) no attempt was made to influence the equilibrium between the glutamateactive and alanine-active forms of the enzyme. 420 360 300
yo
/
_...J~Es ,o-,~ b-#u@o-*
o- 240
DPNH210"4 z_ 180
g
I~'0
I
125
.....
f
250 GDI:'I ADDED (p.g)
f
375
I
500
Fig. 2. Quantitative precipitin curves obtained by adding increasing amounts of glutamate dehydrogenase ( G D H ) t o constant arnouzlts of antiserum. P~cipit~.~on w a s ~ e d out in the absence of regulator molecules (control) or in the presence of DPNt-Is z.o. Io'-* M plus either ADP, 2.2 5 , IO-a M or diethylstilbestrol (DES) x.o. Io-4 M.
As illustrated, the three curves have the same general contour. The control and diethylstilbestrol curves are the same in the range of antibody excess (low glutamate dehydrogenase concentrations) and vary slightly at higher antigen (glutamate dehydrogenase) concentrations. However, the control and ADP curves are strikingly different in two respects: I, the equivalence point in the ADP system is reached at approx. 3o-4o% of the glutamate dehydrogenase concentration required to reach equivalence in the control system, and 2, under the influence of ADP, the enzyme precipitates only about 6o% of the potentially precipitable anti-glntamate dehydrogenase antibody; i.e. approx. 4o% of the antibody is directed against antigenic forms of glutamate dehydrogenase not present in the system containing ADP. In immunodiffusion experiments, the proximity of precipitin lines to the antibody reservoir and their position relative to each other is a complex function involving reactant diffusion rates, relative concentrations and immunologic equivalence. Assuming uniform diffusion rates for both antibody and glutamate dehydrogenase, the precipitin line closest to the antiserum reservoir (i.e., a in Fig. I) represents the antigen-antibody system attaining equivalence at lowest reactant concentration (i.e., the ADP system in Fig. 2). The following points demonstrate that line a is in Biochim. Biophys. Acta, 89 (1964) 226-23I
ANTIGENIC DIFFERENCES IN GLUTAMIC DEHYDROGENASE
i/~i:il:
229
if!i/¸
Fig. 3. Double diffusion experiments performed in 1 9'o agar gel (buffered at p H 8.2 with o.o 5 M veronal) containing either 2.25 . lO -8 M ADP (left) or i.o. lO -4 M diethylstilbestrol (DES) (right). Antiserum 6 (Ab) was placed in the center wells and the peripheral wells contained the following amounts of glutamate dehydrogenase (GDH): (I) Io/2g, (2) 5o/~g, (3) IOO/*g, (4) 2oo#g, (5) 50o/~g, (6) I mg. Precipitin lines were stained for glutamate dehydrogenase (GDH) activity by the tetrazolium method prior to photography.
fact related to ADP and does form at lowest antigen concentration: I, the appearance of line a is favored by ADP and it shows only glutamate dehydrogenase activity s; 2, in immunodiffusion experiments (Fig. 3) employing small amounts of glutamate dehydrogenase (IO/~g), weak or no precipitin lines formed unless ADP was. added to the agar in which case a strong line (a) appeared; 3, for a given glutamate dehydrogenase
Fig. 4. Successive supernatants (S1 through $5) from the control quantitative precipitin experiment (see Fig. 2) studied for residual antibody activity by double diffusion against I mg of glutamate dehydrogenase (GDH) (dissolved in o.o 5 M Tris, pH 8.0). The experiment is performed in i % agar gel buffered at p H 8.2 with 0.0 5 veronal. The original antiserum (Orig. AB.) used in the quantitative precipitin experiment is included for comparison.
Biochim. Biophys. Acla, 89 (I964) 226-231
23 °
N. TALAL,G. M. TOMKINS
concentration above IO ~tg, precipitin lines began to form at lower antigen concentration (i.e., closer to the antiserum reservoir) in "ADP agar" than in "plain" or "diethylstilbestrol agar" (Fig. 3). The supernatants from the quantitative precipitin experiments were studied for residual antibody activity to determine whether antibodies to the 3 immunologic forms were absorbed simultaneously or one at a time. A stepwise disappearance of a single precipitin line at a time (starting with a) was observed and indicated strongly the existence of separate, distinct antibodies directed against different antigenic forms of the glutamate dehydrogenase molecule. Thus, as expected from the equivalence data, anti-a antibodies were removed first, i.e., at the lowest glutamate dehydrogenase concentrations, so that the first supernatant (Fig. 4, $1) reacted very weakly with a 320
.| az O "; o o
240
ontrol
f60
4O
ADp 2.sXIO-$ ~ I D P N ~
30 ~:~
20
~o
"~0- ' ~ -60' - ~
90
,~ ,~
,~o 2',o do 2
120 150 180 GDH ADDED (k~g)
2,v
Fig. 5. Supernatants from the three quantitative precipitin experiments (see Fig. 2) studied for residual glutamate dehydrogenase (GDH) and alanine dehydrogenase (AlaDH) activities. DES stands for diethylstilbestrol. and the second (S,) not at all. Anti-b antibodies were completely absorbed at slightly higher glutamate dehydrogenase concentrations so that line b was revealed with $1 and S 2 but not with S3. Anti-c antibodies were present up to S 5 but not beyond. This demonstration of specific antibodies for a, b and c is further evidence that these three forms of glutamate dehydrogenase are antigenicaUy different, as previously established in other experiments for b and c (ref. 3). The supernatants were also examined for glutamate dehydrogenase and alanine dehydrogenase activities (Fig. 5)- No enzyme activity was detected in the area of antibody excess since all of the added glutamate dehydrogenase was removed in the precipitate. Beginning with S3 there was a progressive and parallel increase in both glutamate and alanine dehydrogenase activities, again indicating that (different forms of) the same protein catalyze the two reactions. DISCUSSION
These experiments and those reported elsewhere 3 demonstrate the existence of three different immunologic forms of glutamate dehydrogenase. Since different forms of the enzyme are apparently in rapid equilibrium1, 2, their resolution by immunoelectro-
Biochim. Biophys. Acta, 89 (I964) 226-231
ANTIGENIC DIFFERENCES IN GLUTAMIC DEHYDROGENASE
231
phoresis into distinct precipitin lines was at first surprising. Indeed, electrophoresis of glutamate dehydrogenase in agar gel shows only one band of protein or enzyme activity and the precipitin lines in immunoelectrophoresis all have the same electrophoretic mobility. This suggests, therefore, that primarily the immunochemical properties of the system are responsible for its resolution. The presence of multiple precipitin lines in immunodiffusion experiments generally implies more than one antigen-antibody system with the antigens distributed on different molecules. Different antigens on a single molecule produce only one precipitin line TM. A single antigen on molecules of vastly different size (and, therefore, rates of diffusion) could produce more than one line only if the smaller (rapidly diffusing) molecule failed to precipitate all the antibody n. In such a situation the precipitin line farthest from the antiserum reservoir would disappear first as antibody was progressively absorbed. However, as shown in Fig. 4, it is precisely this line (c) that is the last to disappear in the glutamate dehydrogenase system, suggesting that the 3 precipitin lines represent different antigen-antibody systems. The resolution of the glutamate dehydrogenase system into different precipitin lines depends, therefore, on reaction between different antigenic forms of glutamate dehydrogenase and their respective specific antibodies. Both immunoelectrophoretic and quantitative experiments suggest that these antigenic forms are related to conformational changes induced in the catalytically active molecule by regulator substances such as ADP, GTP, and diethylstilbestrol. The resolution of the different forms into discrete precipitin lines p~obably depends, therefore, on different equivalent concentrations for these different antigen-antibody systems. Thus, as the antigens, consisting of the different antigenic forms of glutamate dehydrogenase in equilibrium, react with the antiserum, the first precipitin line to form (a) removes all of the available anti-a antibody. The remaining antibodies then diffuse past line a and precipitate, in turn, forms b and c producing the other two precipitin lines. In this way, the selective sequential exhaustion of the antisera resolves the glutamate dehydrogenase equilibrium mixture into discrete precipitin lines. REFERENCES 1 G. M. TOMKINS AND K. L. YIELDING, Cold Spring Harb. Syrup. Quant. Biol., 26 (1961) 331 z G. M. TOMKINS, K. L. YIELDING, N. TALAL AND J. CURRAN, Cold Spring Harb. Syrup. Quant. Biol., 28 (1963) 461. s N. TALAL, G. 1~. TOMKINS, J. F. MUSHINSKI AND K. L. YIELDING, J. Mol. Biol., 8 (1964) 46. 4 C. FRIEDEN, J. Biol. Chem., 234 (1959) 8o9. 5 K. L. YIELDING AND G. M. TOMKINS, Proc. Natl. Acad. Sci. U.S., 46 (196o) 1483. 6 O. H. LOWRY, N. J. ROSEBROUGH, A. C. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. G. M. TOMKINS, K. L. YIELDING AND J. CURRAN, Proc. Natl. Acad. Sci. U.S., 47 (1961) 27o. 8 0 . OUCHTERLONY, Acta Pathol. Microbiol. Scand., 32 (1953) 231. P. GRABAR AND C. A. WILLIAMS, Biochim. Biophys. Acta, IO (1953) 193. 10 C. LAPRESLE, Ann. Inst. Pasteur, 89 (1955) 654. Xl I. FINGER, E. A. I~ABAT, A. E. BEZER AND A. KIDD, J. Immunol., 84 (196o) 227.
Biochim. Biophys. Acta, 89 (1964) 226-23I
BIOCHIMICAET BIOPHYSICAACTA
BBA 65052 A N T I G E N I C D I F F E R E N C E S ASSOCIATED W I T H C O N F O R M A T I O N A L C H A N G E S IN G L U T A M A T E D E H Y D R O G E N A S E NORMAN TALAL AND GORDON M. TOMKINS National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Md. (U.S.A.)
Received February 24th, 1964)
SUMMARY
The reactions between glutamate dehydrogenase (L-giutamate:DPN(TPN)oxidoreductase (deaminating), EC 1.4.1.3) crystallized from bovine liver and specific rabbit anti-glutamate dehydrogenase immune sera have been studied. This immunologic system can be resolved into three precipitin lines (a, b, and c) by immunoelectrophoresis and double diffusion. Quantitative precipitin experiments demonstrate that the immunologic reactivity of the enzyme varies in relation to changes induced by regulator molecules such as ADP or diethylstilbestrol. Studies performed with supernatant fractions from the precipitin experiments reveal a selective absorption of specific antibodies and sequential disappearance of precipitin lines starting with a. These results are explained on the basis of antigenic differences associated with different conformational states of glutamate dehydrogenase. INTRODUCTION Glutamate dehydrogenase (L-glutamate:DPN(TPN)oxidoreductase (deaminating), EC 1.4.1.3) crystallized from bovine liver is an equilibrium system composed of different molecular conformations of the enzyme which have different relative catalytic activities towards amino acid substrates, e.g. glutamate and alaninel, z. Immunoelectrophoretic studies recently reported 3 demonstrated three enzymically active glutamate dehydrogenase-anti glutamate dehydrogenase precipitin lines (a, b, and c) and thus showed that at least some of these molecular forms of glutamate dehydrogenase could be separated immunologically. The relative prbportions of these immunologic forms were influenced by reagents such as ADP, GTP and diethylstilbestrol which had been shown previously to affect the structure of glutamate dehydrogenasel,4, ~. In this report additional quantitative evidence is presented to show that the immunologic behavior of glutamate dehydrogenase varies as a result of antigenic differences arising from conformational changes induced by these "regulator molecules". An explanation is also proposed to account for the resolution into immunologically distinct components of this system which is in rapid, reversible equihbrium. Biochirn. Biophys. Aeta,
89 (1964) 226-231
ANTIGENIC DIFFERENCESIN GLUTAMICDEHYDROGENASE
227
MATERIALSAND METHODS Glutamate dehydrogenase (as a suspension of crystals in ammonium sulfate), ADP, DPN, DPNH~, diethylstilbestrol and amino acid substrates were obtained from the Sigma Chemical Company, St. Louis, Missouri. GTP was purchased from Pabst Laboratories, Milwaukee, Wisconsin. Antisera against native and denatured glutamate dehydrogenase was prepared in rabbits by methods previously described 3. The enzyme was examined for contamination and found to be homogeneous by the methods described earlier ~. Quantitative precipitin experiments were performed by adding increasing amounts of glutamate dehydrogenase (in 0.05 M Tris buffer, pH 8.0) to o.5-ml aliquots of rabbit antiserum prepared against glutamate dehydrogenase denatured with sodium dodecyl sulfate. Final volumes were 0. 7 ml. Precipitation was carried out at 37 ° for I h and then at 4 ° for 48 h. Antigen-antibody precipitates were centrifuged at 2500 rev./min for 45 min and washed twice with chilled o.15 M NaC1 buffered with o.15 M phosphate at pH 7.2. They were dissolved in 0.5 N NaOH and made up to 2 ml in the buffered NaC1. Protein concentrations were determined by the method of LOWRY et a l : . The supernatant fractions from quantitative precipitin experiments were examined for residual enzyme and antibody activities. Glutamate and alanine dehydrogenase activities were assayed by methods previously described 7 and antibody was detected by double diffusion in 1% agar gel 8.
Fig. I. Immunoelectrophoresis 9 of glutamate dehydrogenase in 1% agar gel buffered with 0.05 M veronal at pH 8.2. Electrophoresis was carried out in o.o25 M veronal buffer (pH 8.2) at I4O V and ioo mA for 4 h at room temperature. Precipitin lines a, b and c were revealed with rabbit anti-glutamate dehydrogenase antiserum 6 and lines a and b with antiserum 2. Line a, (illustrated schematically), did not appear in this experiment until glutamate dehydrogenase activity was identified by tetrazolium reduction employing o.o6 M L-glutamate and o.75 mM DPN as substrates 8. RESULTS Fig. I shows a typical immunoelectrophoretic experiment in which the glutamate dehydrogenase system has been resolved by reaction with anti-glutamate dehydrogenase antisera into 3 separate precipitin lines, a, b and c. It was previously reported 3 that these three immunologic forms of glutamate dehydrogenase have different relative catalytic activities towards L-glutamate and L-alanine and are affected by regulator molecules. Form a, for example, shows only glutamate dehydrogenase activity and is most apparent when experiments are performed in the Biochim. Biophys. Acta, 89 (1964) 226-23I
228
N. TALAL, G. M. TOMKINS
presence of ADP. Conversely, form c has predominantly alanine dehydrogenase activity and is most readily observed when GTP or diethylstilbestrol are incorporated in the agar. Form b has both enzyme activities and is present under all conditions. It was shown s by specific absorption and double diffusion experiments that forms b and c are antigenically different. The results of three quantitative precipitin experiments employing the same antiserum are shown in Fig. 2. In each instance, increasing amounts of glutamate dehydrogenase were added to a constant amount of antiserum and the total precipitable protein measured. In two experiments the precipitates were allowed to form in the presence of D P N H , and either ADP or diethylstilbestrol. In the third experiment (control) no attempt was made to influence the equilibrium between the glutamateactive and alanine-active forms of the enzyme. 420 360 300
yo
/
_...J~Es ,o-,~ b-#u@o-*
o- 240
DPNH210"4 z_ 180
g
I~'0
I
125
.....
f
250 GDI:'I ADDED (p.g)
f
375
I
500
Fig. 2. Quantitative precipitin curves obtained by adding increasing amounts of glutamate dehydrogenase ( G D H ) t o constant arnouzlts of antiserum. P~cipit~.~on w a s ~ e d out in the absence of regulator molecules (control) or in the presence of DPNt-Is z.o. Io'-* M plus either ADP, 2.2 5 , IO-a M or diethylstilbestrol (DES) x.o. Io-4 M.
As illustrated, the three curves have the same general contour. The control and diethylstilbestrol curves are the same in the range of antibody excess (low glutamate dehydrogenase concentrations) and vary slightly at higher antigen (glutamate dehydrogenase) concentrations. However, the control and ADP curves are strikingly different in two respects: I, the equivalence point in the ADP system is reached at approx. 3o-4o% of the glutamate dehydrogenase concentration required to reach equivalence in the control system, and 2, under the influence of ADP, the enzyme precipitates only about 6o% of the potentially precipitable anti-glntamate dehydrogenase antibody; i.e. approx. 4o% of the antibody is directed against antigenic forms of glutamate dehydrogenase not present in the system containing ADP. In immunodiffusion experiments, the proximity of precipitin lines to the antibody reservoir and their position relative to each other is a complex function involving reactant diffusion rates, relative concentrations and immunologic equivalence. Assuming uniform diffusion rates for both antibody and glutamate dehydrogenase, the precipitin line closest to the antiserum reservoir (i.e., a in Fig. I) represents the antigen-antibody system attaining equivalence at lowest reactant concentration (i.e., the ADP system in Fig. 2). The following points demonstrate that line a is in Biochim. Biophys. Acta, 89 (1964) 226-23I
ANTIGENIC DIFFERENCES IN GLUTAMIC DEHYDROGENASE
i/~i:il:
229
if!i/¸
Fig. 3. Double diffusion experiments performed in 1 9'o agar gel (buffered at p H 8.2 with o.o 5 M veronal) containing either 2.25 . lO -8 M ADP (left) or i.o. lO -4 M diethylstilbestrol (DES) (right). Antiserum 6 (Ab) was placed in the center wells and the peripheral wells contained the following amounts of glutamate dehydrogenase (GDH): (I) Io/2g, (2) 5o/~g, (3) IOO/*g, (4) 2oo#g, (5) 50o/~g, (6) I mg. Precipitin lines were stained for glutamate dehydrogenase (GDH) activity by the tetrazolium method prior to photography.
fact related to ADP and does form at lowest antigen concentration: I, the appearance of line a is favored by ADP and it shows only glutamate dehydrogenase activity s; 2, in immunodiffusion experiments (Fig. 3) employing small amounts of glutamate dehydrogenase (IO/~g), weak or no precipitin lines formed unless ADP was. added to the agar in which case a strong line (a) appeared; 3, for a given glutamate dehydrogenase
Fig. 4. Successive supernatants (S1 through $5) from the control quantitative precipitin experiment (see Fig. 2) studied for residual antibody activity by double diffusion against I mg of glutamate dehydrogenase (GDH) (dissolved in o.o 5 M Tris, pH 8.0). The experiment is performed in i % agar gel buffered at p H 8.2 with 0.0 5 veronal. The original antiserum (Orig. AB.) used in the quantitative precipitin experiment is included for comparison.
Biochim. Biophys. Acla, 89 (I964) 226-231
23 °
N. TALAL,G. M. TOMKINS
concentration above IO ~tg, precipitin lines began to form at lower antigen concentration (i.e., closer to the antiserum reservoir) in "ADP agar" than in "plain" or "diethylstilbestrol agar" (Fig. 3). The supernatants from the quantitative precipitin experiments were studied for residual antibody activity to determine whether antibodies to the 3 immunologic forms were absorbed simultaneously or one at a time. A stepwise disappearance of a single precipitin line at a time (starting with a) was observed and indicated strongly the existence of separate, distinct antibodies directed against different antigenic forms of the glutamate dehydrogenase molecule. Thus, as expected from the equivalence data, anti-a antibodies were removed first, i.e., at the lowest glutamate dehydrogenase concentrations, so that the first supernatant (Fig. 4, $1) reacted very weakly with a 320
.| az O "; o o
240
ontrol
f60
4O
ADp 2.sXIO-$ ~ I D P N ~
30 ~:~
20
~o
"~0- ' ~ -60' - ~
90
,~ ,~
,~o 2',o do 2
120 150 180 GDH ADDED (k~g)
2,v
Fig. 5. Supernatants from the three quantitative precipitin experiments (see Fig. 2) studied for residual glutamate dehydrogenase (GDH) and alanine dehydrogenase (AlaDH) activities. DES stands for diethylstilbestrol. and the second (S,) not at all. Anti-b antibodies were completely absorbed at slightly higher glutamate dehydrogenase concentrations so that line b was revealed with $1 and S 2 but not with S3. Anti-c antibodies were present up to S 5 but not beyond. This demonstration of specific antibodies for a, b and c is further evidence that these three forms of glutamate dehydrogenase are antigenicaUy different, as previously established in other experiments for b and c (ref. 3). The supernatants were also examined for glutamate dehydrogenase and alanine dehydrogenase activities (Fig. 5)- No enzyme activity was detected in the area of antibody excess since all of the added glutamate dehydrogenase was removed in the precipitate. Beginning with S3 there was a progressive and parallel increase in both glutamate and alanine dehydrogenase activities, again indicating that (different forms of) the same protein catalyze the two reactions. DISCUSSION
These experiments and those reported elsewhere 3 demonstrate the existence of three different immunologic forms of glutamate dehydrogenase. Since different forms of the enzyme are apparently in rapid equilibrium1, 2, their resolution by immunoelectro-
Biochim. Biophys. Acta, 89 (I964) 226-231
ANTIGENIC DIFFERENCES IN GLUTAMIC DEHYDROGENASE
231
phoresis into distinct precipitin lines was at first surprising. Indeed, electrophoresis of glutamate dehydrogenase in agar gel shows only one band of protein or enzyme activity and the precipitin lines in immunoelectrophoresis all have the same electrophoretic mobility. This suggests, therefore, that primarily the immunochemical properties of the system are responsible for its resolution. The presence of multiple precipitin lines in immunodiffusion experiments generally implies more than one antigen-antibody system with the antigens distributed on different molecules. Different antigens on a single molecule produce only one precipitin line TM. A single antigen on molecules of vastly different size (and, therefore, rates of diffusion) could produce more than one line only if the smaller (rapidly diffusing) molecule failed to precipitate all the antibody n. In such a situation the precipitin line farthest from the antiserum reservoir would disappear first as antibody was progressively absorbed. However, as shown in Fig. 4, it is precisely this line (c) that is the last to disappear in the glutamate dehydrogenase system, suggesting that the 3 precipitin lines represent different antigen-antibody systems. The resolution of the glutamate dehydrogenase system into different precipitin lines depends, therefore, on reaction between different antigenic forms of glutamate dehydrogenase and their respective specific antibodies. Both immunoelectrophoretic and quantitative experiments suggest that these antigenic forms are related to conformational changes induced in the catalytically active molecule by regulator substances such as ADP, GTP, and diethylstilbestrol. The resolution of the different forms into discrete precipitin lines p~obably depends, therefore, on different equivalent concentrations for these different antigen-antibody systems. Thus, as the antigens, consisting of the different antigenic forms of glutamate dehydrogenase in equilibrium, react with the antiserum, the first precipitin line to form (a) removes all of the available anti-a antibody. The remaining antibodies then diffuse past line a and precipitate, in turn, forms b and c producing the other two precipitin lines. In this way, the selective sequential exhaustion of the antisera resolves the glutamate dehydrogenase equilibrium mixture into discrete precipitin lines. REFERENCES 1 G. M. TOMKINS AND K. L. YIELDING, Cold Spring Harb. Syrup. Quant. Biol., 26 (1961) 331 z G. M. TOMKINS, K. L. YIELDING, N. TALAL AND J. CURRAN, Cold Spring Harb. Syrup. Quant. Biol., 28 (1963) 461. s N. TALAL, G. 1~. TOMKINS, J. F. MUSHINSKI AND K. L. YIELDING, J. Mol. Biol., 8 (1964) 46. 4 C. FRIEDEN, J. Biol. Chem., 234 (1959) 8o9. 5 K. L. YIELDING AND G. M. TOMKINS, Proc. Natl. Acad. Sci. U.S., 46 (196o) 1483. 6 O. H. LOWRY, N. J. ROSEBROUGH, A. C. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. G. M. TOMKINS, K. L. YIELDING AND J. CURRAN, Proc. Natl. Acad. Sci. U.S., 47 (1961) 27o. 8 0 . OUCHTERLONY, Acta Pathol. Microbiol. Scand., 32 (1953) 231. P. GRABAR AND C. A. WILLIAMS, Biochim. Biophys. Acta, IO (1953) 193. 10 C. LAPRESLE, Ann. Inst. Pasteur, 89 (1955) 654. Xl I. FINGER, E. A. I~ABAT, A. E. BEZER AND A. KIDD, J. Immunol., 84 (196o) 227.
Biochim. Biophys. Acta, 89 (1964) 226-23I