Molec. Biol. Rep. Vol. 5, 4:251-252, 1979
GLUTAMATE DEHYDROGENASE ACTIVITY IN SUBCELLULAR FRACTIONS OF MOUSE FIBROSARCOMA
Marian HILLAR & Billy LENNOX
Department of Biology, Texas Southern University, Houston, Texas 77004, U.S.A. (Received June 12, 1979)
Abstract
Materials and methods
Glutamate dehydrogenase activity was found to be present at a high level in neoplastic cytosol and microsomes, 3.45 and 9.90 nmoles NADH/min mg protein, respectively. This remains in accordance with the high rate of RNA and protein synthesis in the neoplastic process.
Fibrosarcoma cells were obtained from Department of Radiotherapeutics, M.D. Anderson Hospital, Houston. NADH, a-ketoglutarate were purchased from Sigma Chemical Co., other chemicals - from J.T. Baker Chemical Co.
Culture of Fibrosarcoma in vivo. Introduction
Long biochemical studies seem to clarify somewhat the picture of abnormalities in the cancer cells. General features of such a cell are characterized by increased replication of DNA, increased RNA production, ectopic production of polypeptide hormones and proteins, changes in the antigen composition of the cell, change in the isozyme composition toward higher level of isozymic forms of enzyme typical for fetal tissue, and decrease or increase in some key enzyme activities [8, 9]. Glutamate dehydrogenase (GDH) (EN 1.4.1.3) is a key mitochondrial enzyme responsible for glutamate synthesis and trapping of NH3 in a utilizable form [1, 2], synthesized on ribosomes [3, 4] and induced by glucocorticoids [5]. Because the enzyme is involved in the biosynthesis of amino acids in the cell we were interested in the studies on its level in fast growing tumor tissue.
Trypsinized fibrosarcoma cells were injected subcutaneously into C3H mice and grown for fourteen days before excision [7].
Tissue fractionation Solid fibrosarcoma tumor or rat liver were homogenized (10% homogenate) in 0.074 M Na2HPO4, 0.006 M KH2POa~ pH 7.8, 0.075 M NaC1, and centrifuged at 800 x g for 10 min. Mitochondria were isolated by centrifugation of supernatant at 8 000 x g for 10 min and microsomes were isolated last by centrifugation at 105 000 x g for 30 min. The supernatant from the last centrifugation was used as the cytosol preparation. Isolated fractions were suspended in 0.05 M Na-K phsophate buffer, pH 7.6, and sonified for 2 min in a Heat System sonifier at 80 watts. Fibrosarcoma mitochondria were further centrifuged at 144 000 x g for 20 min to obtain the mitochondrial matrix.
Measurement of" glutamate dehydrogenase activity
Address for correspondence:
Marian HiUar, Department of Biology, Texas Southern University, Houston, Texas 77004.
GDH activity in subcellular fractions was measured by following spectrophotometrically the decrease in absorbance of NADH as a difference 340 nm- 405 nm in the dual wavelength DW-2 Aminco spectrophotometer as described previously [3]. 251
References
Protein determination Protein in subcellular fractions was measured with the Lowry procedure [6].
1. 2. 3.
Results and discussion
Table 1 compares GDH activities in subcellular fractions from mouse fibrosarcoma and rat liver (as an example o f a normal tissue with the highest content o f GDH). GDH activity is not found in normal tissues in the cytosol [2] and only in a
4.
Table. 1. Glutamate dehydrogenase activity in the subcellular fractions of mouse fibrosarcoma
6.
Subcellular fraction
7.
Enzyme activity Fibrosarcoma
Rat liver
Cytosol
3.45•
(5)*
0.00•
(5)*
Microsomes
9.90•
(5)*
0.60•
(4)*
55.67•
(5)*
--
Mitochondrial matrix Mitochondria
76.00•
8. 9.
(7)*
* The figures in the parentheses indicate the number of experiments
very small quantity in microsomes (in rat liver 0.6 nmole NADH/min mg protein). In fibrosarcoma its level is very high in microsomes (9.9 nmole NADH/ min mg protein) and cytosol (3.4 nmole NADH/min mg protein). The mitochondrial matrix seems to contain it in a range comparable to that in normal tissue. The data obtained indicate a highly increased process o f GDH synthesis in carcinogenesis. This remains in accordance with the previous finding o f the decrease in urea cycle activity in liver [8]. GDH is utilized for the reaction o f glutamate formation and shift toward the biosynthetic reactions. This research was supported by a grant from the National Cancer Institute No. RR 08061.
252
5.
Hillar, M. "Regulation of glutamate dehydrogenase activity in isolated rat fiver mitochondria by ADP and progesterone", Biokhimiya 38, 548-551 (1973). Hillar, M. "Glutamate dehydrogenase", J. Bioenergetics 6, 89-126 (1974). Hillar, M., Davis S.A. and Chambers, D., "Studies on subcellular poly(A)-mRNA-rieh RNA fraction with glutamate dehydrogenase template activity", Physiol. Chem. Phys. 9,205-217 (1977). Hillar, M., Davis, S.A., Nikbakht, K.N. and Chambers, D., "Translation of mRNA for glutamate dehydrogenase and spectrophotometric procedure to follow the enzyme biosynthesis", Arch. lnternl. Physiol. Biochim. 85,583-588 (1977). Hillar, M. and Reuben, J., "Hormonal induction of glutamate dehydrogenase in rat fiver", Experientia 32,653-655 (1976). Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R., "Protein measurement with the Foiln phenol reagent", J. Biol. Chem. 193,265-275 (1951). Milas, L., Hunter, N., Basic, I., Mason, K., Grdina, D.J. and Wither, H.R. "Nonspecific immunotherapy of murine solid tumors with Corynebacterium granulosum", J. Natl. Canc. Inst. 54,895-902 (1975). Weber, G., "Enzymatic strategy of the cancer cell", in Cancer Enzymology (Eds. J. Schultz and F. Ahmad), Academic Press, New York 1976, pp. 63-88. Weinhouse, S., Shatton, J.B. and Morris, H.P., "lsozyme composition, gene regulation and metabolism of experimental hepatomas", in Cancer Enzymology (Eds. J. Schultz and F. Ahmad), Academic Press, New York 1976, pp. 41-61.
GLUTAMATE DEHYDROGENASE ACTIVITY IN SUBCELLULAR FRACTIONS OF MOUSE FIBROSARCOMA
Marian HILLAR & Billy LENNOX
Department of Biology, Texas Southern University, Houston, Texas 77004, U.S.A. (Received June 12, 1979)
Abstract
Materials and methods
Glutamate dehydrogenase activity was found to be present at a high level in neoplastic cytosol and microsomes, 3.45 and 9.90 nmoles NADH/min mg protein, respectively. This remains in accordance with the high rate of RNA and protein synthesis in the neoplastic process.
Fibrosarcoma cells were obtained from Department of Radiotherapeutics, M.D. Anderson Hospital, Houston. NADH, a-ketoglutarate were purchased from Sigma Chemical Co., other chemicals - from J.T. Baker Chemical Co.
Culture of Fibrosarcoma in vivo. Introduction
Long biochemical studies seem to clarify somewhat the picture of abnormalities in the cancer cells. General features of such a cell are characterized by increased replication of DNA, increased RNA production, ectopic production of polypeptide hormones and proteins, changes in the antigen composition of the cell, change in the isozyme composition toward higher level of isozymic forms of enzyme typical for fetal tissue, and decrease or increase in some key enzyme activities [8, 9]. Glutamate dehydrogenase (GDH) (EN 1.4.1.3) is a key mitochondrial enzyme responsible for glutamate synthesis and trapping of NH3 in a utilizable form [1, 2], synthesized on ribosomes [3, 4] and induced by glucocorticoids [5]. Because the enzyme is involved in the biosynthesis of amino acids in the cell we were interested in the studies on its level in fast growing tumor tissue.
Trypsinized fibrosarcoma cells were injected subcutaneously into C3H mice and grown for fourteen days before excision [7].
Tissue fractionation Solid fibrosarcoma tumor or rat liver were homogenized (10% homogenate) in 0.074 M Na2HPO4, 0.006 M KH2POa~ pH 7.8, 0.075 M NaC1, and centrifuged at 800 x g for 10 min. Mitochondria were isolated by centrifugation of supernatant at 8 000 x g for 10 min and microsomes were isolated last by centrifugation at 105 000 x g for 30 min. The supernatant from the last centrifugation was used as the cytosol preparation. Isolated fractions were suspended in 0.05 M Na-K phsophate buffer, pH 7.6, and sonified for 2 min in a Heat System sonifier at 80 watts. Fibrosarcoma mitochondria were further centrifuged at 144 000 x g for 20 min to obtain the mitochondrial matrix.
Measurement of" glutamate dehydrogenase activity
Address for correspondence:
Marian HiUar, Department of Biology, Texas Southern University, Houston, Texas 77004.
GDH activity in subcellular fractions was measured by following spectrophotometrically the decrease in absorbance of NADH as a difference 340 nm- 405 nm in the dual wavelength DW-2 Aminco spectrophotometer as described previously [3]. 251
References
Protein determination Protein in subcellular fractions was measured with the Lowry procedure [6].
1. 2. 3.
Results and discussion
Table 1 compares GDH activities in subcellular fractions from mouse fibrosarcoma and rat liver (as an example o f a normal tissue with the highest content o f GDH). GDH activity is not found in normal tissues in the cytosol [2] and only in a
4.
Table. 1. Glutamate dehydrogenase activity in the subcellular fractions of mouse fibrosarcoma
6.
Subcellular fraction
7.
Enzyme activity Fibrosarcoma
Rat liver
Cytosol
3.45•
(5)*
0.00•
(5)*
Microsomes
9.90•
(5)*
0.60•
(4)*
55.67•
(5)*
--
Mitochondrial matrix Mitochondria
76.00•
8. 9.
(7)*
* The figures in the parentheses indicate the number of experiments
very small quantity in microsomes (in rat liver 0.6 nmole NADH/min mg protein). In fibrosarcoma its level is very high in microsomes (9.9 nmole NADH/ min mg protein) and cytosol (3.4 nmole NADH/min mg protein). The mitochondrial matrix seems to contain it in a range comparable to that in normal tissue. The data obtained indicate a highly increased process o f GDH synthesis in carcinogenesis. This remains in accordance with the previous finding o f the decrease in urea cycle activity in liver [8]. GDH is utilized for the reaction o f glutamate formation and shift toward the biosynthetic reactions. This research was supported by a grant from the National Cancer Institute No. RR 08061.
252
5.
Hillar, M. "Regulation of glutamate dehydrogenase activity in isolated rat fiver mitochondria by ADP and progesterone", Biokhimiya 38, 548-551 (1973). Hillar, M. "Glutamate dehydrogenase", J. Bioenergetics 6, 89-126 (1974). Hillar, M., Davis S.A. and Chambers, D., "Studies on subcellular poly(A)-mRNA-rieh RNA fraction with glutamate dehydrogenase template activity", Physiol. Chem. Phys. 9,205-217 (1977). Hillar, M., Davis, S.A., Nikbakht, K.N. and Chambers, D., "Translation of mRNA for glutamate dehydrogenase and spectrophotometric procedure to follow the enzyme biosynthesis", Arch. lnternl. Physiol. Biochim. 85,583-588 (1977). Hillar, M. and Reuben, J., "Hormonal induction of glutamate dehydrogenase in rat fiver", Experientia 32,653-655 (1976). Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R., "Protein measurement with the Foiln phenol reagent", J. Biol. Chem. 193,265-275 (1951). Milas, L., Hunter, N., Basic, I., Mason, K., Grdina, D.J. and Wither, H.R. "Nonspecific immunotherapy of murine solid tumors with Corynebacterium granulosum", J. Natl. Canc. Inst. 54,895-902 (1975). Weber, G., "Enzymatic strategy of the cancer cell", in Cancer Enzymology (Eds. J. Schultz and F. Ahmad), Academic Press, New York 1976, pp. 63-88. Weinhouse, S., Shatton, J.B. and Morris, H.P., "lsozyme composition, gene regulation and metabolism of experimental hepatomas", in Cancer Enzymology (Eds. J. Schultz and F. Ahmad), Academic Press, New York 1976, pp. 41-61.
