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In vivo stimulation of 14C-amino acid incorporation into nonhistone proteins in rat liver chromatin induced by insulin and cortisol
644
SHORT COMMUNICATIONS
BBA 93560
In vivo stimulation of 14C-amino acid incorporation into nonhistone proteins in rat liver chromatin induced by insulin and cortisol
A considerable number of hormones (somatotropic hormone, testosterone, thyroxin 1, insulin 2 and cortisol a,4) have been found to stimulate the nuclear protein biosynthesis in rat liver in vivo. Furthermore, isolated liver nuclei from cortisolpretreated rats display an increased incorporation rate of amino acids into the nuclear protein in vitro 4. Studies in vivo on the specificity of amino acid incorporation into the nuclear protein of insulin or eortisol-pretreated rats revealed a much higher uptake of leucine or glutamate when compared to lysine 2,4. It was presumed that these hormones are able to stimulate a specific fraction of nuclear proteins. This theory was supported by TENG AND HAMILTON5 who demonstrated an estrogen-induced stimulation of nonhistone acidic proteins in the uterus of ovariectomized rats. Hormonal induction by cortisol caused an increase in the diffuse chromatin content of the hepatic nuclei, as a result of a corresponding decrease in the compact chromatin content 8. It seemed to be highly interesting to elucidate whether or not a specific protein fraction of isolated chromatin was stimulated if insulin or cortisol was employed. Sprague Dawley rats (I5O g) maintained on a normal diet were used throughout this study. All insulin and cortisol treatments were carried out by means of intraperitoneal injections. Bovine insulin (25 U/mg) was obtained from NOVO, cortisol from Merck. [1-14CT-DL-leucine (55.2 C/mole) and reconstituted uniformly 14C-labeled protein hydrolysate (54 C/carbon atom) were purchased from the Radiochemical Centre, Amersham; hyamine from Packard; sucrose special enzyme grade from Mann, N.Y. All other reagents were of analytical purity and delivered by iquka, Merck or Riedel de Haen. Protein was determined by the method of LOWRY et al. ~, RNA with orcinol ~ and DNA with diphenylamin 9. Labeling of nuclear protein in rat liver was performed by injecting intraperitoneally 2 #C ]I-14C]-DL-leucine or 2 #C 14C-labeled protein hydrolysate. Liver nuclei (protein: DNA ratio, 4.9±o.25; RNA : DNA ratio, o . 2 4 ± o . o i ) were isolated in sucrose solutions of different densities l°,n. Chromatin and nonhistone acidic proteins were prepared by modified procedures of refs. I2, 13. Nuclear pellet obtained from three rat livers was suspended in 4 o m l of o.o75 M NaCl-o.o24 M EDTA buffer (pH 8.0) and centrifuged at I 5 o o × g for 15 rain. This step was repeated three times. The pellet was suspended twice in o.5 M Tris-HC1 buffer, pH 8.0, and centrifuged as described above. The pellet was homogenized in 8 ml of o.o 5 M Tris-HC1, pH 8.o, in a glass potter with a Teflon pestle and centrifuged at IO o o o X g for 15 rain. This pellet was resuspended in 6 ml of o.o5 M Tris-HC1 buffer, pH 8.0, and carefully layered on 25 ml of 1. 7 M suerose-o.o5 M Tris HC1 buffer, pH 8.o. After centrifugation at 55 ooo x g for 3 h (Spinco L2 centrifuge; SW 25.1 rotor) the crude chromatin was suspended twice in io ml H20 and centrifuged at IO ooo ×g for IO rain. The isolated chromatin was suspended in 4.8 M CsC1 and subjected to a 13o ooo ×g centrifugation (SW 5° rotor) at 4 ° for 24 h. This treatment caused the sedimentation of DNA while the chromosomal proteins were floating to the top la. Further treatment of the top pellicule was performed as already described 5. 2-ml protein samples were precipitated with 2 ml 4 ° °/o (w/v) trichloroacetic acid and washed with 2 ml IO o/ .o trichloroacetic acid (w/v). The precipitate was exBiochim. ]giophys. Acta, 224 (196o) 644 646
645
SHORT COMMUNICATIONS
tracted three times with absolute ether and dried overnight in a dessicator. The residue was dissolved in I ml hyamine at 37 ° for 12 h and subjected to radioactive counting (Packard Tricarb 3o03). 18 rats were divided into 2 cortisol, 2 insulin and 2 control groups. 6 h before decapitation each rat of the cortisol group was injected with 5 mg cortisol (suspended in I ml o.14 M NaC1), while each rat from the insulin group received a 5-h pretreatment with 5 I.U. bovine insulin (dissolved in I ml o.14 M NaC1). The control rats were treated with physiological saline solution. Exactly one hour before decapitation each rat of experiment A was injected intraperitoneally with 2 #C [I-14C]I)L-leucine, and alternatively, the rats of experiment B with 2/zC uniformly ~4Clabeled reconstituted protein hydrolysate. The results are presented in Table I. TABLE I EFFECT OF CORTISOL AND INSULIN ON 14C-AMINO ACID INCORPOR&TION INTO RAT LIVER CHROMATIN
in vivo Effect of p r e t r e a t m e n t w i t h eortisol and insulin on the u p t a k e in vivo of (a) [i-14C]-DL-leucine (2 /,C/per rat) and (b) uniformly l'C-labeled protein h y d r o l y s a t e (2 E¢C/per rat) into proteins of r a t liver chromatin. H o r m o n e - t r e a t e d rats received intraperitoneal injections of 5 mg cortisol 6 h and 5 I,U. insulin 5 h before sacrifice. Control rats were treated with physiological saline solution. The 14C-amino acids were administered to the rats intraperitoneally exactly i h before decapitation.
Protein [faction
Counts~rain per mg protein Control rats
Cortisol-treated rats
Insulin-treated rats
Histones: (a) (b)
28 60
41 87
32 65
N o n h i s t o n e Proteins: (a) (b)
47 9°
128 245
lO6 189
Both hormones had almost no affect on the biosynthesis of histones, and there was no significant change in the chemical composition of rat liver chromatin (RNA/ DNA ratio, o.o65~:o.oI; total protein/DNA ratio, 1.9±o.15). This is contrasted b y the marked stimulation of 14C-amino acid incorporation into nonhistone acidic proteins. These results strongly support our previous theory on the nature of cortisolor insulinqnduced stimulation of nuclear protein, and they agree well with the results of TENG AND HAMILTON 5 who demonstrated the increased synthesis of nonhistone acidic protein caused b y estrogen. The authors are grateful to Dr. H. Probst and Priv. Doz. Dr. U. Weser (Physiologisch-Chemisches Institut Tfibingen) for untiring support and helpful criticism. This work was supported by a grant from the Deutsche Forschungsgemeinschaft, Bad Godesberg. Kreiskrankenhaus,
744 Niirtingen (Germany)
MAX D I E T E R BUCK
PETER SCHAUDER*
* P r e s e n t address: D e p a r t m e n t of Medicine, University of W a s h i n g t o n , Seattle, W a s h i n g t o n 98105 .
Biochim. Biophys. Acta, 224 (197 o) 644-646
646
SHORT COMMUNICATIONS
J. R. TATA, Nature, 212 (1966) 1312. M. D. BUCK, P. SCHAUDER AND U. WESER, Z. Natur[orsch., 25b (197 o) 276. A. DEISSEROTH, Biochim. Biophys. Acta, 186 (1969) 392. P. SCHAUDER AND M. D. BUCK, in preparation. C. S. TENG AND T. I~I. HAMILTON, Proc. Natl. Acad. Sci. U.S., 63 fi969) 465 . G. S. ARBUZOVA, I. M. GRYAZNOVA,T. ~¥[. MOROZOVA AND R. I. SALGANIK, Mol. Biol., 2 (1968) 249. 7 0 . H. LOWRY, N. J. ROSEBROUGtt, A. L. FARR AND •. J. RANDALL, J. Biol. Chem., 193 (I95I) 265. 8 ]{. B. HURLBERT, H. SCHMITZ, A. F. BRUMM AND V. R. POTTER, d. Biol. Chem., 209 (1954) 23. 9 I~. BURTON, Biochem. J., 62 (1956) 315 . Io C. C. WIDNELL AND J. t~. TATA, Biochem. J., 92 (1964) 313 . 1I R. R. MCGREGOR AND H. R. MAHLER, Arch. Biochem. Biophys., 12o (1967) 136. i2 I{. D. SMITH, R. B. CHURCH AND B. J. MCCARTHY, Biochemistry, 8 (1969) 4271. 13 J. BONNER ~_ND J. WIDHOLM, Proc. Natl. Acad. Sei. U.S., 57 (1967) 1379I 2 3 4 5 6
Received August
I S t h , 1970
Biochim. Biophys. Acta, 224 (197 o) 644-646
SHORT COMMUNICATIONS
BBA 93560
In vivo stimulation of 14C-amino acid incorporation into nonhistone proteins in rat liver chromatin induced by insulin and cortisol
A considerable number of hormones (somatotropic hormone, testosterone, thyroxin 1, insulin 2 and cortisol a,4) have been found to stimulate the nuclear protein biosynthesis in rat liver in vivo. Furthermore, isolated liver nuclei from cortisolpretreated rats display an increased incorporation rate of amino acids into the nuclear protein in vitro 4. Studies in vivo on the specificity of amino acid incorporation into the nuclear protein of insulin or eortisol-pretreated rats revealed a much higher uptake of leucine or glutamate when compared to lysine 2,4. It was presumed that these hormones are able to stimulate a specific fraction of nuclear proteins. This theory was supported by TENG AND HAMILTON5 who demonstrated an estrogen-induced stimulation of nonhistone acidic proteins in the uterus of ovariectomized rats. Hormonal induction by cortisol caused an increase in the diffuse chromatin content of the hepatic nuclei, as a result of a corresponding decrease in the compact chromatin content 8. It seemed to be highly interesting to elucidate whether or not a specific protein fraction of isolated chromatin was stimulated if insulin or cortisol was employed. Sprague Dawley rats (I5O g) maintained on a normal diet were used throughout this study. All insulin and cortisol treatments were carried out by means of intraperitoneal injections. Bovine insulin (25 U/mg) was obtained from NOVO, cortisol from Merck. [1-14CT-DL-leucine (55.2 C/mole) and reconstituted uniformly 14C-labeled protein hydrolysate (54 C/carbon atom) were purchased from the Radiochemical Centre, Amersham; hyamine from Packard; sucrose special enzyme grade from Mann, N.Y. All other reagents were of analytical purity and delivered by iquka, Merck or Riedel de Haen. Protein was determined by the method of LOWRY et al. ~, RNA with orcinol ~ and DNA with diphenylamin 9. Labeling of nuclear protein in rat liver was performed by injecting intraperitoneally 2 #C ]I-14C]-DL-leucine or 2 #C 14C-labeled protein hydrolysate. Liver nuclei (protein: DNA ratio, 4.9±o.25; RNA : DNA ratio, o . 2 4 ± o . o i ) were isolated in sucrose solutions of different densities l°,n. Chromatin and nonhistone acidic proteins were prepared by modified procedures of refs. I2, 13. Nuclear pellet obtained from three rat livers was suspended in 4 o m l of o.o75 M NaCl-o.o24 M EDTA buffer (pH 8.0) and centrifuged at I 5 o o × g for 15 rain. This step was repeated three times. The pellet was suspended twice in o.5 M Tris-HC1 buffer, pH 8.0, and centrifuged as described above. The pellet was homogenized in 8 ml of o.o 5 M Tris-HC1, pH 8.o, in a glass potter with a Teflon pestle and centrifuged at IO o o o X g for 15 rain. This pellet was resuspended in 6 ml of o.o5 M Tris-HC1 buffer, pH 8.0, and carefully layered on 25 ml of 1. 7 M suerose-o.o5 M Tris HC1 buffer, pH 8.o. After centrifugation at 55 ooo x g for 3 h (Spinco L2 centrifuge; SW 25.1 rotor) the crude chromatin was suspended twice in io ml H20 and centrifuged at IO ooo ×g for IO rain. The isolated chromatin was suspended in 4.8 M CsC1 and subjected to a 13o ooo ×g centrifugation (SW 5° rotor) at 4 ° for 24 h. This treatment caused the sedimentation of DNA while the chromosomal proteins were floating to the top la. Further treatment of the top pellicule was performed as already described 5. 2-ml protein samples were precipitated with 2 ml 4 ° °/o (w/v) trichloroacetic acid and washed with 2 ml IO o/ .o trichloroacetic acid (w/v). The precipitate was exBiochim. ]giophys. Acta, 224 (196o) 644 646
645
SHORT COMMUNICATIONS
tracted three times with absolute ether and dried overnight in a dessicator. The residue was dissolved in I ml hyamine at 37 ° for 12 h and subjected to radioactive counting (Packard Tricarb 3o03). 18 rats were divided into 2 cortisol, 2 insulin and 2 control groups. 6 h before decapitation each rat of the cortisol group was injected with 5 mg cortisol (suspended in I ml o.14 M NaC1), while each rat from the insulin group received a 5-h pretreatment with 5 I.U. bovine insulin (dissolved in I ml o.14 M NaC1). The control rats were treated with physiological saline solution. Exactly one hour before decapitation each rat of experiment A was injected intraperitoneally with 2 #C [I-14C]I)L-leucine, and alternatively, the rats of experiment B with 2/zC uniformly ~4Clabeled reconstituted protein hydrolysate. The results are presented in Table I. TABLE I EFFECT OF CORTISOL AND INSULIN ON 14C-AMINO ACID INCORPOR&TION INTO RAT LIVER CHROMATIN
in vivo Effect of p r e t r e a t m e n t w i t h eortisol and insulin on the u p t a k e in vivo of (a) [i-14C]-DL-leucine (2 /,C/per rat) and (b) uniformly l'C-labeled protein h y d r o l y s a t e (2 E¢C/per rat) into proteins of r a t liver chromatin. H o r m o n e - t r e a t e d rats received intraperitoneal injections of 5 mg cortisol 6 h and 5 I,U. insulin 5 h before sacrifice. Control rats were treated with physiological saline solution. The 14C-amino acids were administered to the rats intraperitoneally exactly i h before decapitation.
Protein [faction
Counts~rain per mg protein Control rats
Cortisol-treated rats
Insulin-treated rats
Histones: (a) (b)
28 60
41 87
32 65
N o n h i s t o n e Proteins: (a) (b)
47 9°
128 245
lO6 189
Both hormones had almost no affect on the biosynthesis of histones, and there was no significant change in the chemical composition of rat liver chromatin (RNA/ DNA ratio, o.o65~:o.oI; total protein/DNA ratio, 1.9±o.15). This is contrasted b y the marked stimulation of 14C-amino acid incorporation into nonhistone acidic proteins. These results strongly support our previous theory on the nature of cortisolor insulinqnduced stimulation of nuclear protein, and they agree well with the results of TENG AND HAMILTON 5 who demonstrated the increased synthesis of nonhistone acidic protein caused b y estrogen. The authors are grateful to Dr. H. Probst and Priv. Doz. Dr. U. Weser (Physiologisch-Chemisches Institut Tfibingen) for untiring support and helpful criticism. This work was supported by a grant from the Deutsche Forschungsgemeinschaft, Bad Godesberg. Kreiskrankenhaus,
744 Niirtingen (Germany)
MAX D I E T E R BUCK
PETER SCHAUDER*
* P r e s e n t address: D e p a r t m e n t of Medicine, University of W a s h i n g t o n , Seattle, W a s h i n g t o n 98105 .
Biochim. Biophys. Acta, 224 (197 o) 644-646
646
SHORT COMMUNICATIONS
J. R. TATA, Nature, 212 (1966) 1312. M. D. BUCK, P. SCHAUDER AND U. WESER, Z. Natur[orsch., 25b (197 o) 276. A. DEISSEROTH, Biochim. Biophys. Acta, 186 (1969) 392. P. SCHAUDER AND M. D. BUCK, in preparation. C. S. TENG AND T. I~I. HAMILTON, Proc. Natl. Acad. Sci. U.S., 63 fi969) 465 . G. S. ARBUZOVA, I. M. GRYAZNOVA,T. ~¥[. MOROZOVA AND R. I. SALGANIK, Mol. Biol., 2 (1968) 249. 7 0 . H. LOWRY, N. J. ROSEBROUGtt, A. L. FARR AND •. J. RANDALL, J. Biol. Chem., 193 (I95I) 265. 8 ]{. B. HURLBERT, H. SCHMITZ, A. F. BRUMM AND V. R. POTTER, d. Biol. Chem., 209 (1954) 23. 9 I~. BURTON, Biochem. J., 62 (1956) 315 . Io C. C. WIDNELL AND J. t~. TATA, Biochem. J., 92 (1964) 313 . 1I R. R. MCGREGOR AND H. R. MAHLER, Arch. Biochem. Biophys., 12o (1967) 136. i2 I{. D. SMITH, R. B. CHURCH AND B. J. MCCARTHY, Biochemistry, 8 (1969) 4271. 13 J. BONNER ~_ND J. WIDHOLM, Proc. Natl. Acad. Sei. U.S., 57 (1967) 1379I 2 3 4 5 6
Received August
I S t h , 1970
Biochim. Biophys. Acta, 224 (197 o) 644-646