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Comparative properties of glutamic-alanine transaminase from several sources
ARCHIVES
OF
BIOCHEMISTRY
AND
Comparative
Properties
SARAH the Department
of Pharmacology,
(1964)
of Glutamic-Alanine
from
From
106, 501-505
BIOPHYSICS
Several
HOPPER’
Sources AND H. L. SEGAL’
St. Louis Received
Transaminase
University July
School
of Medicine,
St. Louis,
Missouri
25, 1963
Rat liver mitochondrial glutamic-alanine transaminase can be distinguished from the supernatant enzyme on the basis of its markedly greater lability and its nonresponsiveness to glucocorticoid administration. The activity of this enzyme in rat skeletal muscle, heart, and kidney are considerably less than that in liver and are also nonresponsive to glucocorticoids. Enzyme from some of these tissues and from liver of some other species cross reacts with antiserum to the rat liver supernatant enzyme. Both glutamic-alanine and glutamic-aspartic transaminase from several sources are inhibited by aminooxyacetic acid. Previous reports from this laboratory have dealt with the glutamic-alanine transaminase of rat liver supernatant fraction (l-3). In this paper is presented a comparison of some of the properties of this enzyme from the mitochondrial and supernatant fractions of rat liver, as well as from several other tissues of the rat and from the livers of other species. EXPERIMENTAL
PREPARATIOK
OF TISSUE
FRACTIONS
Liver and skeletal muscle homogenates were made in 9 volumes of 0.25 M sucrose, and heart and kidney homogenates in 4 volumes of 0.25 M sucrose. Blending was in a Servall Omni-mixer for 30 seconds at 0°C. The usual tissue extract was the supernatant fraction of a 15 minute, 10,OOOg centrifugation of the homogenate. For preparation of subcellular fractions, the homogenate of rat liver was centrifuged successively at 6009 for 6 minutes, 10,000g for 15 minutes, and 100,000g for 90 minutes. The sediments, respectively referred to as nuclei, mitochondria, and microsomes, were washed twice in the original volume of 0.25 M sucrose and suspended in the original volume of 1 Present address: National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda 14, Maryland. 2 Present address: Biology Department, State University of New York, Buffalo 14, New York. 501
0.01 M KPOa buffer, pH 7.3, by gentle enization with a Teflon pestle.
ENZYME
hand
homog-
ASSAYS
Assay of glutamic-alanine transaminase in postmitochondrial fractions was by Assay Method II, as previously described (1), involving the coupling of pyruvate formation to DPNH oxidation in the presence of lactic dehydrogenase. Assay of the enzyme in mitochondrial fractions was usually performed by a slightly different method to minimize nonspecific DPNH oxidation. A suspension of mitochondria was incubated at 37°C. in a volume of 1 ml. containing 37 pmoles of nn-alanine, 4.6 pmoles of a-ketoglutarate, and 100 pmoles of sodium pyrophosphate buffer, pH 7.8. The reaction was stopped by the addition of 0.5 ml. of 1.0 N trichloroacetic acid. Denatured protein was removed by centrifugation, and 0.5 ml. of the supernatant solution was transferred to a cuvette, neutralized with 0.5 ml. of 0.33 ,V sodium hydroxide, and assayed spectrophotometrically for pyruvate in the presence of 0.6 rmole of DPNH, 300 kmoles of KPOd buffer, pH 7.3, and 3 units of lactic dehydrogenase. Small corrections for endogenous pyruvate formation were made with control tubes lacking either alanine or ol-ketoglutarate. For glutamic-aspartic transaminase assays, a coupled assay was used in which aspartate replaced alanine and malic dehydrogenase replaced lactic dehydrogenase (1). Units are on a pmole basis, as previously defined (l), and specific activity is units per milligram of
502
HOPPER
AND SEGAL
protein. Protein was determined by the method of Lowry et al. (4).
FmoIe of DPNH, 3 units of lactic dehydrogenase, and 100 pmoles of KPO4 buffer, pH 7.3. Another aliquot of the supernatant soluRESULTS tion from the incubation mixture was asPROPERTIES OF THE ENZYME OF RAT sayed semi-quantitatively for glutamate by LIVER MITOCHONDRIA paper electrophoresis, by comparison on the paper after ninhydrin staining with known The presence of glutamic-alanine transamounts of a standard glutamate solution. aminase activity in rat liver mitochondria has been reported previously (5, 6). The Electrophoresis was performed at pH 4.7 in intention of the present studies was to corn- pyridine-acetic acid buffer (33.5 ml. of each per liter) at 1200 V. for 135 hours. pare the properties of the mitochondrial Two pmoles of pyruvate and approxienzyme with that in the supernatant fracmately 2 pmoles of glutamate were formed in tion, and in particular to determine whether the activity of the former, like that of the 30 minutes per milliliter of the incubation mixture. Neither pyruvate nor glutamate latter (7), is responsive to glucocorticoid was formed in two control tubes from which administration. either alanine or a-ketoglutarate was omitThe distribution of activity in a rat liver ted. homogenate is shown in Table I. Essentially Unlike the enzyme in the supernatant all of the activity was recovered in the 100, fraction, whose activity is increased 3- to 5000s supernatant and the mitochondrial of 2 mg. per day fractions. The greater portion (up to 90 % fold after administration in some experiments) is present in the per rat of prednisolone acetate for 5 days (l), the activity of the mitochondrial enzyme was soluble fraction of rat liver. To confirm that pyruvate formation in the unaffected by this treatment. Thus the average specific activity from four untreated mitochondrial preparations was via a transamination reaction, the stoichiometry of animals was 0.209 (range: 0.189+.225), and animals was pyruvate and glutamate formation was from 5 prednisolone-treated 0.216 (range: 0.198dI.234). measured. One-tenth ml. of a mitochondrial The pH profile of the mitochondrial acsuspension was incubated at 37°C. for 30 tivity resembled that of the supernatant minutes with 12.5 pmoles of cr-ketoglutarate, in the 100 pmoles of m-alanine, and 100 pmoles of enzyme, with maximum activity KP04 buffer, pH 7.3, in a final volume of 1.0 range pH 7.5-8.0 in 0.1 M sodium pyrophosphate buffer. ml. The reaction was stopped by immersing Attempts to solubilize the mitochondrial the tubes in boiling water for 5 minutes, and denatured protein was removed by cen- activity met with meager success. Twice washed mitochondria were resuspended in trifugation. An aliquot of the supernatant deionized water, 0.1% Triton X-100, 0.01 Ad solution was assayed spectrophotometrically for pyruvate by DPISH oxidation in the KP04 buffer, pH 7.3, or 0.25 il8 sucrose. presence of lactic dehydrogenase. The assay With the exception of Triton, which inhibited approximately 50$X, the activity of medium contained 0.1 ml. of the supernatant solution from the incubation mixture, 0.75 the enzyme was the same regardIess of the suspension medium. Subsequent freezing and thawing of all of these mitochondrial susTABLE I pensions resulted in a marked loss of activINTRACELLULAR DISTRIBUTION OF RAT LIVER ity. Mitochondrial suspensions in 0.01 M GLUTAMIC-ALANJNE TRXVSAMINASE ACTIVITY pH 7.3, which were sonicated KPOd, Fraction 70 total units Specific activity (Raytheon 10kc) for 5 minutes, contained variable levels of activity. In some cases the Homogenate 100 0.26 Nuclei Mitochondria Microsomes Supernatant
0 32
Nil
0
0.19 Nil
65
0.34
activity was approxilnately and after sonic treatment.
the same before
In other cases, sonication resulted in varying degrees of activity loss. When mitochondrial sonicates
GLUTAMIC-ALANINE
were centrifuged at 100,OOOg for 40 minutes, low and variable activity remained in the supernatant layer. Similar results were obtained when the mitochondrial suspensions were sonicated in the same buffer containing 1O-3 M ethylenediaminetetraacetate or 1O-3 M glutathione. The relatively small amounts of activity solubilized by sonication as described above were used to test the heat and acid stability of the mitochondrial enzyme. The sonicate was heated to 55°C. for 5 minutes, cooled in ice, the pH lowered to 5.0 by dropwise addition of 1.0 N acetic acid, and precipitated material centrifuged off. This procedure is a useful step in the purification of the supernatant enzyme (1) and gives yields of up to 96 5%.In contrast, only 5 % of the activity from the mitochondrial extracts was recovered in the soluble phase after such treatment and none among the precipitated material. Tests of cross-reactivity of the solubilized mitochondrial enzyme with antiserum to the supernatant enzyme (3) were not feasible because of the instability of the activity under the conditions of the incubation. DISTRIBUTION OF GLUTAMIC-ALANINE TRANSAMINASE ACTIVITY
The activity present in the 10,OOOgsupernatant fraction of several tissuesis shown in Table II. Of the tissues tested, all exhibited considerably less activity than rat liver, except dog liver, which was several-fold higher. The responseof the activity in rat tissues to glucocorticoid administration was tested. With the exception of liver, none were significantly altered. Rosen et al. have reported that the activity in brain is also not responsive to glucocorticoid administration (8). The extracts of dog, human, pork, and beef liver were carried through the first two steps of the purification developed for the rat liver enzyme (1)) involving heat and acid precipitation (as described in the previous section), followed by precipitation with ammonium sulfate (25 g. per 100 ml. of solution at pH 5.0). The enzyme from beef liver was unstable to the heat and acid treatment, whereas the recoveries in this step with the
503
TRANSAMISASE TABLE
II
DISTRIBUTION 9ND 1MMUN0L0121c.4~ CROSS-REACTIVITYOF SOLUBLE GLI= T.\MIC-AL~NINE TR.WSAMIN.IHE The antiserum was prepared using the soluble enzyme purified from rat liver as antigen. The values in the last column are the volumes of antiserum required to neutralize one unit of enzyme from the source shown. SOUICe
Rat liver Rat skeletal Rat heart Rat kidney Human liver Dog liver Pork liver Beef liver * Not
Units/g.
Specific activity
A;tis;;um
36.4 4.2 2.2 0.6 14.2 133.0 1.7 1.5
0.28
0.77
tissue
muscle
m
0.13
0.97
0.07 0.01 0.28 1.57 -,I -
1.50 2.35 2.51 3.69 -
measured.
other liver extracts was comparable to that from the rat (60-95 %). The dog, human, and pork enzymes were also recovered in high yield in the ammonium sulfate precipitate in a lo- to 20-fold overall purification, as with the rat liver enzyme. Enzyme from the several sources was tested for cross-reactivity with rabbit antiserum prepared to the rat liver enzyme (3). These results are presented in the last column of Table II. A high degree of crossreactivity was observed between antiserum to rat liver supernatant enzyme and the enzyme from rat skeletal muscle, with lesser degrees of cross-reactivity with the enzyme from other rat tissues or from livers of other species.None of the enzyme activities failed to give somedegree of cross-reactivity. INHIBITION
BY
AMINOOXYACETIC
Acrn
Aminooxyacetic acid has been found to be a potent inhibitor of glutamic-alanine transaminase of rat liver supernatant fraction, competitive with the amino acid substrates and uncompetitive with the keto acid substrates (2). It has also been reported to inhibit glutamic-r-aminobutyric transaminase of brain and Escherichia coli (9). A characteristic of the inhibition of the rat liver supernatant enzyme was a striking pH dependence in the range 7 to 8, with a
504
HOPPER
AND
TABLE INHIBITION
OF GLUTAMIC-ALANINE AMINOOXYACETATE
SEGAL III
(GALT) AND GLUTAMIC-ASPARTIC TRANSAMINASE (GAsT) (AOA) AND DEPENDENCE OF THE INHIBITION UPON pH
BY
y0 Inhibition Enzyme and source
GAlT Liver Liver
supernatant mitochondria
Heart supernatant GAsT Liver supernatant Liver Heart
mitochondria supernatant
AOA concentration (W
PH 7.8
a.3
80
18
-a
0
90 36
86 23
65 -
34 -
6.8
7.3
10-T
80
3 x 10-G
95 63
10-T
9.0
10-T
14
0
0
-
-
10-h 10-5 10-S
83
15 6
15 0
-
-
94
-
10-s 10-s
15 97
27
-
8 85
-
0
-
-
a Not measured.
diminishing degree of inhibition as the pH was increased (2). The effect of this compound on glutamic-alanine and glutamicaspartic transaminases of rat liver mitochondria and rat liver and rat heart supernatant fraction was tested at several pH values. The results are presented in Table III. There was marked inhibition in all cases, although the alanine enzyme was more sensitive to the inhibitor than the aspartate enzyme. A qualitatively similar pH dependence was also present in all cases. DISCUSSION
As would be expected from the ubiquity of the substrates, glutamic-alanine transaminase activity is of extremely wide tissue and species distribution (lo), although the relative activity from tissue to tissue varies widely. With the exception of beef liver, the liver supernatant enzymes from all species studied exhibited similar properties with respect to heat and acid stability and solubility in ammonium sulfate solutions, as well as cross-reactivity with anti-rat enzyme serum. The glutamic-alanine transaminase activities of the supernatant and mitochondrial fractions of rat liver were distinguishable on the basis of their relative stabilities, which differed markedly, and of their response to glucocorticoids. The presence of distinct isozymes of glutamic-alanine transaminase in
the soluble and mitochondrial fractions places this enzyme in the same category with malic dehydrogenase (11,12), glutamicaspartic transaminase (13), and isocitric dehydrogenase (14), which are also present in distinguishable forms in the soluble and mitochondrial fractions of tissues. It is of interest that the rat liver mitochondrial glutamic-alanine transaminase activity is not under glucocorticoid control, in contrast to the supernatant enzyme, thus demonstrating a distinction in the synthetic machinery of the two enzymes. A number of transaminases have now been shown to exhibit marked sensitivity to the inhibitor, aminooxyacetic acid (2,9). Several additional compounds containing aminooxy groups or potential aminooxy groups, including cycloserine, have also recently been demonstrated to be inhibitors of transaminases (15, 16). Cycloserine also inhibits the pyridoxal-linked amino acid decarboxylation (17), tryptophanase (17), and alanine racemase (18) reactions, as well as o-alanyln-alanine synthetase (18). The kinetics of the inhibition by the several aminooxy compounds studied (15, 16) appear to conform closely to the kinetic mechanism reported for aminooxyacetate inhibition of rat liver supernatant glutamic-alanine transaminase, including the pH dependency (2). This class of compounds merits examination as possible inhibitors of pyridoxal enzymes in general.
GLUTAMIC-ALANINE ACKNOWLEDGMENTS This material was taken from a thesis submitted by Sarah Hopper to the Graduate School of St. Louis University in partial fulfilment of the requirements for the Ph.D. degree. One of us (HLS) is a Research Career Awardee of the United States Public Health Service. This work was supported by a grant (A-3642) from the same agency. REFERENCES 1. SEGAL, H. L., BEATTIE, D. S., AND HOPPER, S., J. Biol. Chem. 237, 1914 (1962). 2. HOPPER, S., AND SEGAL, H. L., J. Biol. Chem. 237, 3189 (1962). 3. SEGAL, H. L., Rosso, R. G., HOPPER, S., AND WEBER, M. M., J. Biol. Chem. 237, 3303 (1962). 4. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 5. ROWSELL, E. V., Biochem. J. 64, 235 (1956). 6. KAFER, E., AND POLLAK, J. K., Exptl. Cell Res. 22, 120 (1961).
TRANSAMINASE
505
7. ROSEN, F., ROBERTS, N. R., AND NICHOL, C. A., J. Biol. Chem. 234, 476 (1959). 8. ROSEN, F., ROBERTS, N. R., BUDNICK, L. E., AND NICHOL, C. A., Science 127, 287 (1958). 9. WALLACH, D. P., Biochem. Pharmacol. 6, 323 (1961). 10. MEISTER, A., Advan. Enzymol. 16, 185 (1955). 11. DELBR~CK, A., SCHIMASSEK, H., BARTSCH, K., AND BUTCHER, TH., Biochem. Z. 331, 297 (1959). 12. WIELAND, TH., PFLEIDERER, G., HAUPT, I., AND W~~RNER, W., Biochem. 2. 332, 1 (1959). 13. BOYD, J. W., Biochem. J. 61, 434 (1961). 14. LOWENSTEIN, J. M., AND SMITH, S. R., Biochim. Biophys. Acta 66, 385 (1962). 15. BRAWNSTEIN, A. E., AZARKH, R. M., AND TIN-SEN’, S., Biokhimiya 26, 882 (1962). 16. POLYANOVSKII, 0. L., AND TORCHINSKII, Yu. M., Doklady Akad. Nauk, S.S.S.R. 141, 488 (1961). 17. YAMADA, K., SAWAKI, S., AND HAYAMI, S., J. Vitaminol. 3, 68 (1957). 18. STROMINGER, J. L., ITO, E., AND THRENN, R. H., J. Am. Chem. Sot. 62, 998 (1960).
OF
BIOCHEMISTRY
AND
Comparative
Properties
SARAH the Department
of Pharmacology,
(1964)
of Glutamic-Alanine
from
From
106, 501-505
BIOPHYSICS
Several
HOPPER’
Sources AND H. L. SEGAL’
St. Louis Received
Transaminase
University July
School
of Medicine,
St. Louis,
Missouri
25, 1963
Rat liver mitochondrial glutamic-alanine transaminase can be distinguished from the supernatant enzyme on the basis of its markedly greater lability and its nonresponsiveness to glucocorticoid administration. The activity of this enzyme in rat skeletal muscle, heart, and kidney are considerably less than that in liver and are also nonresponsive to glucocorticoids. Enzyme from some of these tissues and from liver of some other species cross reacts with antiserum to the rat liver supernatant enzyme. Both glutamic-alanine and glutamic-aspartic transaminase from several sources are inhibited by aminooxyacetic acid. Previous reports from this laboratory have dealt with the glutamic-alanine transaminase of rat liver supernatant fraction (l-3). In this paper is presented a comparison of some of the properties of this enzyme from the mitochondrial and supernatant fractions of rat liver, as well as from several other tissues of the rat and from the livers of other species. EXPERIMENTAL
PREPARATIOK
OF TISSUE
FRACTIONS
Liver and skeletal muscle homogenates were made in 9 volumes of 0.25 M sucrose, and heart and kidney homogenates in 4 volumes of 0.25 M sucrose. Blending was in a Servall Omni-mixer for 30 seconds at 0°C. The usual tissue extract was the supernatant fraction of a 15 minute, 10,OOOg centrifugation of the homogenate. For preparation of subcellular fractions, the homogenate of rat liver was centrifuged successively at 6009 for 6 minutes, 10,000g for 15 minutes, and 100,000g for 90 minutes. The sediments, respectively referred to as nuclei, mitochondria, and microsomes, were washed twice in the original volume of 0.25 M sucrose and suspended in the original volume of 1 Present address: National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda 14, Maryland. 2 Present address: Biology Department, State University of New York, Buffalo 14, New York. 501
0.01 M KPOa buffer, pH 7.3, by gentle enization with a Teflon pestle.
ENZYME
hand
homog-
ASSAYS
Assay of glutamic-alanine transaminase in postmitochondrial fractions was by Assay Method II, as previously described (1), involving the coupling of pyruvate formation to DPNH oxidation in the presence of lactic dehydrogenase. Assay of the enzyme in mitochondrial fractions was usually performed by a slightly different method to minimize nonspecific DPNH oxidation. A suspension of mitochondria was incubated at 37°C. in a volume of 1 ml. containing 37 pmoles of nn-alanine, 4.6 pmoles of a-ketoglutarate, and 100 pmoles of sodium pyrophosphate buffer, pH 7.8. The reaction was stopped by the addition of 0.5 ml. of 1.0 N trichloroacetic acid. Denatured protein was removed by centrifugation, and 0.5 ml. of the supernatant solution was transferred to a cuvette, neutralized with 0.5 ml. of 0.33 ,V sodium hydroxide, and assayed spectrophotometrically for pyruvate in the presence of 0.6 rmole of DPNH, 300 kmoles of KPOd buffer, pH 7.3, and 3 units of lactic dehydrogenase. Small corrections for endogenous pyruvate formation were made with control tubes lacking either alanine or ol-ketoglutarate. For glutamic-aspartic transaminase assays, a coupled assay was used in which aspartate replaced alanine and malic dehydrogenase replaced lactic dehydrogenase (1). Units are on a pmole basis, as previously defined (l), and specific activity is units per milligram of
502
HOPPER
AND SEGAL
protein. Protein was determined by the method of Lowry et al. (4).
FmoIe of DPNH, 3 units of lactic dehydrogenase, and 100 pmoles of KPO4 buffer, pH 7.3. Another aliquot of the supernatant soluRESULTS tion from the incubation mixture was asPROPERTIES OF THE ENZYME OF RAT sayed semi-quantitatively for glutamate by LIVER MITOCHONDRIA paper electrophoresis, by comparison on the paper after ninhydrin staining with known The presence of glutamic-alanine transamounts of a standard glutamate solution. aminase activity in rat liver mitochondria has been reported previously (5, 6). The Electrophoresis was performed at pH 4.7 in intention of the present studies was to corn- pyridine-acetic acid buffer (33.5 ml. of each per liter) at 1200 V. for 135 hours. pare the properties of the mitochondrial Two pmoles of pyruvate and approxienzyme with that in the supernatant fracmately 2 pmoles of glutamate were formed in tion, and in particular to determine whether the activity of the former, like that of the 30 minutes per milliliter of the incubation mixture. Neither pyruvate nor glutamate latter (7), is responsive to glucocorticoid was formed in two control tubes from which administration. either alanine or a-ketoglutarate was omitThe distribution of activity in a rat liver ted. homogenate is shown in Table I. Essentially Unlike the enzyme in the supernatant all of the activity was recovered in the 100, fraction, whose activity is increased 3- to 5000s supernatant and the mitochondrial of 2 mg. per day fractions. The greater portion (up to 90 % fold after administration in some experiments) is present in the per rat of prednisolone acetate for 5 days (l), the activity of the mitochondrial enzyme was soluble fraction of rat liver. To confirm that pyruvate formation in the unaffected by this treatment. Thus the average specific activity from four untreated mitochondrial preparations was via a transamination reaction, the stoichiometry of animals was 0.209 (range: 0.189+.225), and animals was pyruvate and glutamate formation was from 5 prednisolone-treated 0.216 (range: 0.198dI.234). measured. One-tenth ml. of a mitochondrial The pH profile of the mitochondrial acsuspension was incubated at 37°C. for 30 tivity resembled that of the supernatant minutes with 12.5 pmoles of cr-ketoglutarate, in the 100 pmoles of m-alanine, and 100 pmoles of enzyme, with maximum activity KP04 buffer, pH 7.3, in a final volume of 1.0 range pH 7.5-8.0 in 0.1 M sodium pyrophosphate buffer. ml. The reaction was stopped by immersing Attempts to solubilize the mitochondrial the tubes in boiling water for 5 minutes, and denatured protein was removed by cen- activity met with meager success. Twice washed mitochondria were resuspended in trifugation. An aliquot of the supernatant deionized water, 0.1% Triton X-100, 0.01 Ad solution was assayed spectrophotometrically for pyruvate by DPISH oxidation in the KP04 buffer, pH 7.3, or 0.25 il8 sucrose. presence of lactic dehydrogenase. The assay With the exception of Triton, which inhibited approximately 50$X, the activity of medium contained 0.1 ml. of the supernatant solution from the incubation mixture, 0.75 the enzyme was the same regardIess of the suspension medium. Subsequent freezing and thawing of all of these mitochondrial susTABLE I pensions resulted in a marked loss of activINTRACELLULAR DISTRIBUTION OF RAT LIVER ity. Mitochondrial suspensions in 0.01 M GLUTAMIC-ALANJNE TRXVSAMINASE ACTIVITY pH 7.3, which were sonicated KPOd, Fraction 70 total units Specific activity (Raytheon 10kc) for 5 minutes, contained variable levels of activity. In some cases the Homogenate 100 0.26 Nuclei Mitochondria Microsomes Supernatant
0 32
Nil
0
0.19 Nil
65
0.34
activity was approxilnately and after sonic treatment.
the same before
In other cases, sonication resulted in varying degrees of activity loss. When mitochondrial sonicates
GLUTAMIC-ALANINE
were centrifuged at 100,OOOg for 40 minutes, low and variable activity remained in the supernatant layer. Similar results were obtained when the mitochondrial suspensions were sonicated in the same buffer containing 1O-3 M ethylenediaminetetraacetate or 1O-3 M glutathione. The relatively small amounts of activity solubilized by sonication as described above were used to test the heat and acid stability of the mitochondrial enzyme. The sonicate was heated to 55°C. for 5 minutes, cooled in ice, the pH lowered to 5.0 by dropwise addition of 1.0 N acetic acid, and precipitated material centrifuged off. This procedure is a useful step in the purification of the supernatant enzyme (1) and gives yields of up to 96 5%.In contrast, only 5 % of the activity from the mitochondrial extracts was recovered in the soluble phase after such treatment and none among the precipitated material. Tests of cross-reactivity of the solubilized mitochondrial enzyme with antiserum to the supernatant enzyme (3) were not feasible because of the instability of the activity under the conditions of the incubation. DISTRIBUTION OF GLUTAMIC-ALANINE TRANSAMINASE ACTIVITY
The activity present in the 10,OOOgsupernatant fraction of several tissuesis shown in Table II. Of the tissues tested, all exhibited considerably less activity than rat liver, except dog liver, which was several-fold higher. The responseof the activity in rat tissues to glucocorticoid administration was tested. With the exception of liver, none were significantly altered. Rosen et al. have reported that the activity in brain is also not responsive to glucocorticoid administration (8). The extracts of dog, human, pork, and beef liver were carried through the first two steps of the purification developed for the rat liver enzyme (1)) involving heat and acid precipitation (as described in the previous section), followed by precipitation with ammonium sulfate (25 g. per 100 ml. of solution at pH 5.0). The enzyme from beef liver was unstable to the heat and acid treatment, whereas the recoveries in this step with the
503
TRANSAMISASE TABLE
II
DISTRIBUTION 9ND 1MMUN0L0121c.4~ CROSS-REACTIVITYOF SOLUBLE GLI= T.\MIC-AL~NINE TR.WSAMIN.IHE The antiserum was prepared using the soluble enzyme purified from rat liver as antigen. The values in the last column are the volumes of antiserum required to neutralize one unit of enzyme from the source shown. SOUICe
Rat liver Rat skeletal Rat heart Rat kidney Human liver Dog liver Pork liver Beef liver * Not
Units/g.
Specific activity
A;tis;;um
36.4 4.2 2.2 0.6 14.2 133.0 1.7 1.5
0.28
0.77
tissue
muscle
m
0.13
0.97
0.07 0.01 0.28 1.57 -,I -
1.50 2.35 2.51 3.69 -
measured.
other liver extracts was comparable to that from the rat (60-95 %). The dog, human, and pork enzymes were also recovered in high yield in the ammonium sulfate precipitate in a lo- to 20-fold overall purification, as with the rat liver enzyme. Enzyme from the several sources was tested for cross-reactivity with rabbit antiserum prepared to the rat liver enzyme (3). These results are presented in the last column of Table II. A high degree of crossreactivity was observed between antiserum to rat liver supernatant enzyme and the enzyme from rat skeletal muscle, with lesser degrees of cross-reactivity with the enzyme from other rat tissues or from livers of other species.None of the enzyme activities failed to give somedegree of cross-reactivity. INHIBITION
BY
AMINOOXYACETIC
Acrn
Aminooxyacetic acid has been found to be a potent inhibitor of glutamic-alanine transaminase of rat liver supernatant fraction, competitive with the amino acid substrates and uncompetitive with the keto acid substrates (2). It has also been reported to inhibit glutamic-r-aminobutyric transaminase of brain and Escherichia coli (9). A characteristic of the inhibition of the rat liver supernatant enzyme was a striking pH dependence in the range 7 to 8, with a
504
HOPPER
AND
TABLE INHIBITION
OF GLUTAMIC-ALANINE AMINOOXYACETATE
SEGAL III
(GALT) AND GLUTAMIC-ASPARTIC TRANSAMINASE (GAsT) (AOA) AND DEPENDENCE OF THE INHIBITION UPON pH
BY
y0 Inhibition Enzyme and source
GAlT Liver Liver
supernatant mitochondria
Heart supernatant GAsT Liver supernatant Liver Heart
mitochondria supernatant
AOA concentration (W
PH 7.8
a.3
80
18
-a
0
90 36
86 23
65 -
34 -
6.8
7.3
10-T
80
3 x 10-G
95 63
10-T
9.0
10-T
14
0
0
-
-
10-h 10-5 10-S
83
15 6
15 0
-
-
94
-
10-s 10-s
15 97
27
-
8 85
-
0
-
-
a Not measured.
diminishing degree of inhibition as the pH was increased (2). The effect of this compound on glutamic-alanine and glutamicaspartic transaminases of rat liver mitochondria and rat liver and rat heart supernatant fraction was tested at several pH values. The results are presented in Table III. There was marked inhibition in all cases, although the alanine enzyme was more sensitive to the inhibitor than the aspartate enzyme. A qualitatively similar pH dependence was also present in all cases. DISCUSSION
As would be expected from the ubiquity of the substrates, glutamic-alanine transaminase activity is of extremely wide tissue and species distribution (lo), although the relative activity from tissue to tissue varies widely. With the exception of beef liver, the liver supernatant enzymes from all species studied exhibited similar properties with respect to heat and acid stability and solubility in ammonium sulfate solutions, as well as cross-reactivity with anti-rat enzyme serum. The glutamic-alanine transaminase activities of the supernatant and mitochondrial fractions of rat liver were distinguishable on the basis of their relative stabilities, which differed markedly, and of their response to glucocorticoids. The presence of distinct isozymes of glutamic-alanine transaminase in
the soluble and mitochondrial fractions places this enzyme in the same category with malic dehydrogenase (11,12), glutamicaspartic transaminase (13), and isocitric dehydrogenase (14), which are also present in distinguishable forms in the soluble and mitochondrial fractions of tissues. It is of interest that the rat liver mitochondrial glutamic-alanine transaminase activity is not under glucocorticoid control, in contrast to the supernatant enzyme, thus demonstrating a distinction in the synthetic machinery of the two enzymes. A number of transaminases have now been shown to exhibit marked sensitivity to the inhibitor, aminooxyacetic acid (2,9). Several additional compounds containing aminooxy groups or potential aminooxy groups, including cycloserine, have also recently been demonstrated to be inhibitors of transaminases (15, 16). Cycloserine also inhibits the pyridoxal-linked amino acid decarboxylation (17), tryptophanase (17), and alanine racemase (18) reactions, as well as o-alanyln-alanine synthetase (18). The kinetics of the inhibition by the several aminooxy compounds studied (15, 16) appear to conform closely to the kinetic mechanism reported for aminooxyacetate inhibition of rat liver supernatant glutamic-alanine transaminase, including the pH dependency (2). This class of compounds merits examination as possible inhibitors of pyridoxal enzymes in general.
GLUTAMIC-ALANINE ACKNOWLEDGMENTS This material was taken from a thesis submitted by Sarah Hopper to the Graduate School of St. Louis University in partial fulfilment of the requirements for the Ph.D. degree. One of us (HLS) is a Research Career Awardee of the United States Public Health Service. This work was supported by a grant (A-3642) from the same agency. REFERENCES 1. SEGAL, H. L., BEATTIE, D. S., AND HOPPER, S., J. Biol. Chem. 237, 1914 (1962). 2. HOPPER, S., AND SEGAL, H. L., J. Biol. Chem. 237, 3189 (1962). 3. SEGAL, H. L., Rosso, R. G., HOPPER, S., AND WEBER, M. M., J. Biol. Chem. 237, 3303 (1962). 4. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 5. ROWSELL, E. V., Biochem. J. 64, 235 (1956). 6. KAFER, E., AND POLLAK, J. K., Exptl. Cell Res. 22, 120 (1961).
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