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Tyrosine transaminase degradation in perfused liver after inhibition of protein synthesis by cycloheximide
398
SHORT COMMUNICATIONS
EBA 93381
Tyrosine transaminase degradation in perfused liver after inhibition of protein synthesis by cycloheximide The tyrosine transaminase (L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) of rat liver is characterized by its rapid turnover, being degraded with a half-life of 2-3 h (refs. I, 2). It was recently shown by KENNEYa that cyc]oheximide, a well-established inhibitor of mammalian protein synthesis, upon administration to rats in vivo blocked the degradation of tyrosine transaminase as well as synthesis of the enzyme. Other reports indicate that the paradoxical effect of cycloheximide on tyrosine transaminase may be rather variable 4-~. Since the activity of this enzyme is known to be affected by a large number of humoral agents in vivo 1,2,7-11, we found it desirable to study the effect of cycloheximide in the isolated, perfused rat liver. In this system, we have previously shown that tryptophan oxygenase (EC I.I3.I.I2), another liver enzyme with rapid turnover, is degraded with a half-life of 2 h in the presence of cycloheximide12. A similar result has now been obtained for tyrosine transaminase, indicating that most of the effects of cycloheximide on the enzyme in vivo may be indirect. Livers, as well as blood for the perfusion, were obtained from adult male Wistar rats, weighing about 300 g. The animals were fasted overnight prior to the experimental procedures. The livers were perfused at 37 ° in a small-volume perfusator with 25 ml of perfusate composed of heparinized blood diluted with two parts of an albumin-containing balanced salt solution z3. Details of the perfusion technique will be described in a separate communication z4. Liver samples, removed at intervals by tying off distal parts of the lobes, were frozen at --20 ° and used for enzyme assay on the following day. Tyrosine transaminase activity was assayed in homogenates by the procedure of ROSEN eb al. ~5. Addition of the synthetic glucocorticoid dexamethasone (0.I mg/ml) to the perfusate produced a marked increase in tyrosine transaminase activity (Fig. i). Without hormone, the basal enzyme level fell continuously during perfusion. This is probably due to a lack of amino acids, as indicated by the perfusion experiments of BARNABEI AND SEREN116.
Addition of cycloheximide at a dose level (20/~g/ml) which inhibits protein synthesis b y 95 % (refs. 12, 14) blocks the induction of tyrosine transaminase, and leads to a rapid fall in enzyme activity (Fig. I). The basal (non-induced) enzyme level is also lowered by cycloheximide treatment. In both cases the half-life of the enzyme is approx. 2-3 h. This is in agreement with previous measurements 1,2, and indicates that degradation of tyrosine transaminase is unaffected by cycloheximide in the perfused liver. Very little is known about the mechanism of enzyme degradation. Oxygen is apparently required, since no breakdown of tryptophan oxygenase or tyrosine transaminase is seen in excised livers or in liver slices under anaerobic conditions ~7. This suggests that enzyme degradation is an energy-dependent process which may conceivably be blocked by interference with energy metabolism. To investigate this possibility, we tested the effect of 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation, on the degradation of tyrosine transaminase in the presence of cycloheximide. Fig. 2 summarizes the results obtained with dinitrophenol in concentraBioc~im. Biophvs. Acta, 174 (1969) 398-4oo
399
SHORT COMMUNICATIONS
300[ •~, :.,u 2oc
",,
IOQ
o g 100
5o
.u'l'N ~ Perfusion t i m e (h)
Time afteraddition of inhibitor (h)
Fig. i. Effect of dexamethasone and cycloheximide on tyrosine transaminase activity in perfused rat liver. Dexamethasone was added at zero time, cycloheximide at arrow. O - O , dexamethasone only; & - - - A, dexamethasone plus cycloheximide; O - O , no additions; A - - . A cycloheximide only. Enzyme activities are expressed in per cent of the activity at the beginning of perfusion. Each curve represents a perfusion of a single liver. Several perfusions of each type have been performed. Fig. 2. Effect of dinitrophenol and cycloheximide on the activity of tyrosine transaminase in perfused rat liver. Livers were perfused with dexamethasone for various time periods (2.o-3. 5 h) prior to the first addition of inhibitor (defined as zero time on abscissa). E], cycloheximide at o h; O, cycloheximide at o h, dinitrophenol (io-4M) at 0.5 h; A, dinitrophenol (1.6.io-4M) at o h, cycloheximide at I h; 0 , dinitrophenol (4.io-4M) at o h, cycloheximide at I h; &, dinitrophenol (2.io -~ M) at o h, cycloheximide at I h. Enzyme activities are expressed in per cent of the activity at the time of addition of the first inhibitor ( + ) . Each kind of symbol represents a single liver perfusion.
tions previously shown to inhibit energy-requiring processes in perfused livers is. Cycloheximide was used in a standard concentration of 20 ffg/ml in all cases. Clearly, the rate of tyrosine transaminase degradation is unaffected even by rather large doses of dinitrophenol, indicating that enzyme degradation does not depend on continuous mitochondrial ATP generation. Since inhibition of enzyme synthesis is the only observed effect of cycloheximide on tyrosine transaminase in the perfused liver, indirect mechanisms may account for the diverse effects seen in vivo. Cycloheximide seems to stimulate the secretion of cathecholamines, adrenal corticoids and pituitary hormones 19,2°, and a recent report suggested that the induction of tyrosine transaminase by cycloheximidea,5 m a y be due to secretion of glucagon 21. Our own preliminary results indicate that insulin may be responsible for inhibition of tyrosine transaminase degradation 22. It is thus apparent that hormones may play a part in the paradoxical responses elicited by cycloheximide in vivo. Although other mechanisms are possible, the interference by hormones will make an exact analysis in vivo difficult. The complexity of this situation illustrates the utility of organ perfusion in studies involving metabolic regulation. This work has been supported by grants from the Norwegian Council for Science and the Humanities. The technical assistance of Mrs. TURID GANGN~ES and Miss LILLY SKUTLE is gratefully acknowledged. Biochim. t~iophys. Acta, 174 (1969) 398-400
400
Department ol Physiology and Biochemistry Dental Faculty, University o10slo, Oslo (Norway) Department o[ Pharmacology Medical Faculty, University o/Oslo, Oslo (Norway)
SHORT COMMUNICATIONS KRISTIAN F . J E R V E L L
P E R O . SEGLEN
1 E. C. C. LIN AND W. E. KNOX, J. Biol. Chem., 233 (1958 ) 1186. 2 F. T. KENNEY, J. Biol. Chem., 237 (1962) 3495. 3 F. T. KENNEY, Science, 156 (1967) 525. 4 S. FIALA AND E. FIALA, Nature, 21o (1966) 53o. 5 P. F. BENSON AND P. M. YOUNG, Biochem. J., lO6 (1968) 54 P. 6 C. MAVRIDES AND E. A. LANE, Science, 156 (1967) 1376. 7 0 . GREENGARD AND G. T. BAKER, Science, 154 (1966) 1461. 8 D. HOLTEN AND F. T. KENNE'¢, J. Biol. Chem., 242 (1967) 4372. 9 W. D. WICKS, Science, 16o (1968) 997. io F. T. KENNEY, J. Biol. Chem., 242 (1967) 4367 . I i I. B. BLACK AND J. AXELROD, Proc. Natl. Acad. Sci. U.S., 59 (1968) 1231. 12 P. O. SEGLEN AND K. F. JERVELL, Abstr. Federation European Biochem. Soc. 4th Meeting, Oslo, 1967, p. 121. 13 H. SCHIMASSEK, Li[e Sci., I I (1962) 629. 14 P. O. SEGLEN AND K. F. JERVELL,Z. Physiol. Chem., in t h e press. 15 F. ROSEN, H. R. HARDING, R. J. MILI-IOLLAND AND C. A. NICHOL, J. Biol. Chem., 238 (1963) 3725 • 16 O. BARNABEI AND F. SERENI, Boll. Soc. Ital. Biol. Sper., 36 (196o) 1656. 17 R. T. SCHIMKE, Natl. Cancer Inst. Monograph, 27 (1967) 3Ol. i8 E. TRIA AND O. BARNABEI, Protoplasma, 63 (1967) 3 o. 19 A. TOTHILL, Brit. y. Pharmacol. Chemotherap., 32 (1968) 322. 20 S. FIALA AND E. FIALA, Biochim. Biophys. ,4eta, lO 3 (1965) 699. 21 S. FIALA AND E. S. FIALA, Federation Proc., 27 (1968) 837. 22 P. O. SEGLEN, Z. Physiol. Chem., 349 (1968) 1229.
Received August 29th, 1968 Biochim. Biophys. dcta, 174 (1959) 398-4oo
BBA 93382
Some effects of various tetracyclines on incorporation of amino acids into polypeptides by Escherichia coli extracts The specific reaction in the protein biosynthesis pathway that is sensitive to the tetracycline antibiotics is thought to be the binding of an aminoacyl-tRNA to the messenger RNA-ribosome complex1,2. The availability of a series of tetracycline analogues prompted a comparative study of the inhibitory activity of these compounds against Escherichia coli. The present paper reports on the relative inhibitory potency of these analogues against bacterial growth, peptide synthesis by cell-free extracts, and binding of aminoacyl-tRNA to ribosomes. When the inhibition of growth of logarithmic phase E. coli cells was measured, oxytetracycline was the most active of the analogues tested (Table I). The 5° % inhibitory doses of chlortetracycline and tetracycline (not shown) were approximately equal to the value for oxytetracycline. When the same series of analogues was tested in an in vitro valine-incorporating system (S-3o)3 (Fig. Ia), oxytetracycline again was the most active inhibitor. Tetracycline and chlortetracycline (not shown) gave results similar to oxytetracyBiochim. Biophys. Acta, 174 (1969) 4oo-402
SHORT COMMUNICATIONS
EBA 93381
Tyrosine transaminase degradation in perfused liver after inhibition of protein synthesis by cycloheximide The tyrosine transaminase (L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) of rat liver is characterized by its rapid turnover, being degraded with a half-life of 2-3 h (refs. I, 2). It was recently shown by KENNEYa that cyc]oheximide, a well-established inhibitor of mammalian protein synthesis, upon administration to rats in vivo blocked the degradation of tyrosine transaminase as well as synthesis of the enzyme. Other reports indicate that the paradoxical effect of cycloheximide on tyrosine transaminase may be rather variable 4-~. Since the activity of this enzyme is known to be affected by a large number of humoral agents in vivo 1,2,7-11, we found it desirable to study the effect of cycloheximide in the isolated, perfused rat liver. In this system, we have previously shown that tryptophan oxygenase (EC I.I3.I.I2), another liver enzyme with rapid turnover, is degraded with a half-life of 2 h in the presence of cycloheximide12. A similar result has now been obtained for tyrosine transaminase, indicating that most of the effects of cycloheximide on the enzyme in vivo may be indirect. Livers, as well as blood for the perfusion, were obtained from adult male Wistar rats, weighing about 300 g. The animals were fasted overnight prior to the experimental procedures. The livers were perfused at 37 ° in a small-volume perfusator with 25 ml of perfusate composed of heparinized blood diluted with two parts of an albumin-containing balanced salt solution z3. Details of the perfusion technique will be described in a separate communication z4. Liver samples, removed at intervals by tying off distal parts of the lobes, were frozen at --20 ° and used for enzyme assay on the following day. Tyrosine transaminase activity was assayed in homogenates by the procedure of ROSEN eb al. ~5. Addition of the synthetic glucocorticoid dexamethasone (0.I mg/ml) to the perfusate produced a marked increase in tyrosine transaminase activity (Fig. i). Without hormone, the basal enzyme level fell continuously during perfusion. This is probably due to a lack of amino acids, as indicated by the perfusion experiments of BARNABEI AND SEREN116.
Addition of cycloheximide at a dose level (20/~g/ml) which inhibits protein synthesis b y 95 % (refs. 12, 14) blocks the induction of tyrosine transaminase, and leads to a rapid fall in enzyme activity (Fig. I). The basal (non-induced) enzyme level is also lowered by cycloheximide treatment. In both cases the half-life of the enzyme is approx. 2-3 h. This is in agreement with previous measurements 1,2, and indicates that degradation of tyrosine transaminase is unaffected by cycloheximide in the perfused liver. Very little is known about the mechanism of enzyme degradation. Oxygen is apparently required, since no breakdown of tryptophan oxygenase or tyrosine transaminase is seen in excised livers or in liver slices under anaerobic conditions ~7. This suggests that enzyme degradation is an energy-dependent process which may conceivably be blocked by interference with energy metabolism. To investigate this possibility, we tested the effect of 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation, on the degradation of tyrosine transaminase in the presence of cycloheximide. Fig. 2 summarizes the results obtained with dinitrophenol in concentraBioc~im. Biophvs. Acta, 174 (1969) 398-4oo
399
SHORT COMMUNICATIONS
300[ •~, :.,u 2oc
",,
IOQ
o g 100
5o
.u'l'N ~ Perfusion t i m e (h)
Time afteraddition of inhibitor (h)
Fig. i. Effect of dexamethasone and cycloheximide on tyrosine transaminase activity in perfused rat liver. Dexamethasone was added at zero time, cycloheximide at arrow. O - O , dexamethasone only; & - - - A, dexamethasone plus cycloheximide; O - O , no additions; A - - . A cycloheximide only. Enzyme activities are expressed in per cent of the activity at the beginning of perfusion. Each curve represents a perfusion of a single liver. Several perfusions of each type have been performed. Fig. 2. Effect of dinitrophenol and cycloheximide on the activity of tyrosine transaminase in perfused rat liver. Livers were perfused with dexamethasone for various time periods (2.o-3. 5 h) prior to the first addition of inhibitor (defined as zero time on abscissa). E], cycloheximide at o h; O, cycloheximide at o h, dinitrophenol (io-4M) at 0.5 h; A, dinitrophenol (1.6.io-4M) at o h, cycloheximide at I h; 0 , dinitrophenol (4.io-4M) at o h, cycloheximide at I h; &, dinitrophenol (2.io -~ M) at o h, cycloheximide at I h. Enzyme activities are expressed in per cent of the activity at the time of addition of the first inhibitor ( + ) . Each kind of symbol represents a single liver perfusion.
tions previously shown to inhibit energy-requiring processes in perfused livers is. Cycloheximide was used in a standard concentration of 20 ffg/ml in all cases. Clearly, the rate of tyrosine transaminase degradation is unaffected even by rather large doses of dinitrophenol, indicating that enzyme degradation does not depend on continuous mitochondrial ATP generation. Since inhibition of enzyme synthesis is the only observed effect of cycloheximide on tyrosine transaminase in the perfused liver, indirect mechanisms may account for the diverse effects seen in vivo. Cycloheximide seems to stimulate the secretion of cathecholamines, adrenal corticoids and pituitary hormones 19,2°, and a recent report suggested that the induction of tyrosine transaminase by cycloheximidea,5 m a y be due to secretion of glucagon 21. Our own preliminary results indicate that insulin may be responsible for inhibition of tyrosine transaminase degradation 22. It is thus apparent that hormones may play a part in the paradoxical responses elicited by cycloheximide in vivo. Although other mechanisms are possible, the interference by hormones will make an exact analysis in vivo difficult. The complexity of this situation illustrates the utility of organ perfusion in studies involving metabolic regulation. This work has been supported by grants from the Norwegian Council for Science and the Humanities. The technical assistance of Mrs. TURID GANGN~ES and Miss LILLY SKUTLE is gratefully acknowledged. Biochim. t~iophys. Acta, 174 (1969) 398-400
400
Department ol Physiology and Biochemistry Dental Faculty, University o10slo, Oslo (Norway) Department o[ Pharmacology Medical Faculty, University o/Oslo, Oslo (Norway)
SHORT COMMUNICATIONS KRISTIAN F . J E R V E L L
P E R O . SEGLEN
1 E. C. C. LIN AND W. E. KNOX, J. Biol. Chem., 233 (1958 ) 1186. 2 F. T. KENNEY, J. Biol. Chem., 237 (1962) 3495. 3 F. T. KENNEY, Science, 156 (1967) 525. 4 S. FIALA AND E. FIALA, Nature, 21o (1966) 53o. 5 P. F. BENSON AND P. M. YOUNG, Biochem. J., lO6 (1968) 54 P. 6 C. MAVRIDES AND E. A. LANE, Science, 156 (1967) 1376. 7 0 . GREENGARD AND G. T. BAKER, Science, 154 (1966) 1461. 8 D. HOLTEN AND F. T. KENNE'¢, J. Biol. Chem., 242 (1967) 4372. 9 W. D. WICKS, Science, 16o (1968) 997. io F. T. KENNEY, J. Biol. Chem., 242 (1967) 4367 . I i I. B. BLACK AND J. AXELROD, Proc. Natl. Acad. Sci. U.S., 59 (1968) 1231. 12 P. O. SEGLEN AND K. F. JERVELL, Abstr. Federation European Biochem. Soc. 4th Meeting, Oslo, 1967, p. 121. 13 H. SCHIMASSEK, Li[e Sci., I I (1962) 629. 14 P. O. SEGLEN AND K. F. JERVELL,Z. Physiol. Chem., in t h e press. 15 F. ROSEN, H. R. HARDING, R. J. MILI-IOLLAND AND C. A. NICHOL, J. Biol. Chem., 238 (1963) 3725 • 16 O. BARNABEI AND F. SERENI, Boll. Soc. Ital. Biol. Sper., 36 (196o) 1656. 17 R. T. SCHIMKE, Natl. Cancer Inst. Monograph, 27 (1967) 3Ol. i8 E. TRIA AND O. BARNABEI, Protoplasma, 63 (1967) 3 o. 19 A. TOTHILL, Brit. y. Pharmacol. Chemotherap., 32 (1968) 322. 20 S. FIALA AND E. FIALA, Biochim. Biophys. ,4eta, lO 3 (1965) 699. 21 S. FIALA AND E. S. FIALA, Federation Proc., 27 (1968) 837. 22 P. O. SEGLEN, Z. Physiol. Chem., 349 (1968) 1229.
Received August 29th, 1968 Biochim. Biophys. dcta, 174 (1959) 398-4oo
BBA 93382
Some effects of various tetracyclines on incorporation of amino acids into polypeptides by Escherichia coli extracts The specific reaction in the protein biosynthesis pathway that is sensitive to the tetracycline antibiotics is thought to be the binding of an aminoacyl-tRNA to the messenger RNA-ribosome complex1,2. The availability of a series of tetracycline analogues prompted a comparative study of the inhibitory activity of these compounds against Escherichia coli. The present paper reports on the relative inhibitory potency of these analogues against bacterial growth, peptide synthesis by cell-free extracts, and binding of aminoacyl-tRNA to ribosomes. When the inhibition of growth of logarithmic phase E. coli cells was measured, oxytetracycline was the most active of the analogues tested (Table I). The 5° % inhibitory doses of chlortetracycline and tetracycline (not shown) were approximately equal to the value for oxytetracycline. When the same series of analogues was tested in an in vitro valine-incorporating system (S-3o)3 (Fig. Ia), oxytetracycline again was the most active inhibitor. Tetracycline and chlortetracycline (not shown) gave results similar to oxytetracyBiochim. Biophys. Acta, 174 (1969) 4oo-402