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Basal and induced levels of tryptophan pyrrolase and tyrosine transaminase in the chick
DEVELOPMENTAL
Basal
BIOLOGY
13,
and Induced and Tyrosine
182198
(1966)
Levels of Tryptophan Pyrrolase Transaminase in the Chick1
W. EUGENE KNOX AND HANS M. EPPENBERCER” Department Cancer
of Biological Chemistry, Harvard Research Institute, New England Boston, Massachusetts Accepted
November
15,
Medical School and Deaconess Hospital,
the
1965
INTRODUCTION
The present studies were designed to compare the regulation of the tryptophan pyrrolase (EC 1.11.1.4 L-tryptophan: HzO, oxidoreductase) and tyrosine transaminase (EC 2.6.1.5 L-tyrosine: 2-oxoglutarate aminotransferase) in the liver of the chick embryos and the hatched chicks with the known behavior of these enzymes in the livers of rats. The levels of both enzymes in the liver of chick embryos have been increased by treatment with tryptophan (Gordon, 1956), and the level of the liver tyrosine transaminase in the young hatched chick was elevated by hydrocortisone treatment (Chan and Cohen, 1964; Lerner, 1959). Our results show that both enzymes are present in more or less constant specific activity in the Iiver from early embryonic life, and that there is a striking difference in the behavior of these two enzymes after treatment with inducers. Hydrocortisone elevates the tyrosine transaminase during the embryonic period, but not the tryptophan pyrrolase, This induction was not prevented by actinomycin. Tryptophan elevates the tryptophan pyrrolase, and it induces the tyrosine transaminase to a much smaller amount throughout the period studied. Formylase (EC 3.5.1.9: aryl-formylamineamidohydrolase) the second ‘This investigation was supported by United States Public Health Service Grant AM 00567-12, by Research Career Award AM-K6261803 from the National Institute of Arthritis and Metabolic Diseases of the National Institutes of Health, United States Public Health Service, and by United States Atomic Energy Commission Contract At( 30-l )-901 with the New England Deaconess Hospital (NYO-901-40). ‘Present address: Department of Biochemistry, Brandeis University, Waltham, Massachusetts. 182
INDUCED
ENZYMES
IN
CHICK
EMBRYOS
183
enzyme in the pathway of tryptophan metabolism, was low or absent in the embryonic liver. It increased dramatically after hatching, but its level was unaffected by treatment with the inducers. METHODS
White Leghorn SPAFAS chicken eggs were obtained from a commercial supplier and incubated in the laboratory. Preliminary results with Rhode Island Red eggs were entirely similar. The viability of untreated control eggs was consistently over 90%, and their age was uniform as judged by hatching within a 12-hour period on the scheduled 21st day. The ages given for the embryos are the day of incubation in the laboratory. The age of the hatched chicks was counted from the time of hatching within 2-hour periods during the last 12 hours of incubation. The injected solutions were sterile (autoclaved) solutions in 0.9% NaCI. Injections were made with sterile precautions into the air space of the egg through a hole drilled with forceps and sealed with Scotch tape. The hatched chicks received the doses intraperitoneally, with care taken to avoid the yolk sac. All controls received similar injections of saline alone. The mortality in embryos was determined by daily candling during the treatment period and inspection at the time of killing. More of the younger embryos died after injections than the older ones, but less than one-third at any age died with the doses used. The mortality was significantly higher with larger doses of the inducers. At harvest the living embryos were weighed, chilled on ice, and the livers were transferred to preweighed beakers containing the cold solution for homogenization. These were subsequently reweighed to determine the weight of single livers or the average liver weight per embryo in the pool. Pools of about twenty H-day-old, eight 16-day-old, five 19-day-old embryo livers and individual hatched chick livers were used. Experiments in chick embryos younger than 11 days were prevented by the insensitivity of the assay for tryptophan pyrrolase and the greatly increased toxicity of tryptophan. However, it was possible to measure the tyrosine transaminase in homogenates of whole 5-day-old embryos, and to induce this enzyme with cortisol. Injections of 0.075 mg of cortisol in 0.25 ml of saline were made into the chorioallantois just beside the embryo, and the embryos were removed 5 hours later. A 20% homogenate of the total embryo in pools of about 24 embryos
184
KNOX
AND
EPPENBERGER
each was assayed as usual for the tyrosine transaminase. The activities observed were referred entirely to the liver weight (determined separately) for comparative purposes. Enzyme Assays. The livers were promptly homogenized in a glass homogenizer with enough cold 0.15 A4 KC1 containing 0.005 A4 Ltryptophan and 0.02 M a-ketoglutarate to make a 20% homogenate, and centrifuged in the cold at 100,000 g for 1 hour in the Spinco model L preparative ultracentrifuge. The clear supernatant solution was carefully withdrawn with a syringe to avoid the sedimented particulate layer and the floating lipid layer. Assays were done immediately, about 2 hours after death of the chicks, using the supernatant fractions. The tryptophan pyrrolase was determined at 37°C by readings of the optical density at 360 rnp in a Gilford spectrophotometer of a reaction system against a water blank (Knox and Ogata, 1965). Additions, in this order, to the reaction mixture were: 0.5 ml of 20% liver supernatant solution, 1.2 ml of 0.2 M phosphate, pH 7.0, 0.2 ml of 20 pM hematin, 0.3 ml of formylase (429 pmoles/hr/ml at 37°C) (Knox, 1955), and 1 ml of water. After temperature equilibration, 0.2 ml of 0.05 M L-tryptophan and 0.1 ml of 0.3 M freshly neutralized ascorbic acid solution were added to start the reaction, making the total volume 3.5 ml. Activities are expressed as units (micromoles of kynurenine formed per hour) per gram of wet liver. Formylase was assayed with formylkynurenine as substrate as described by Knox (1955). The tyrosine transaminase was determined at 25°C by the enol borate method (Lin et al., 1958), with the components, which included pyridoxal phosphate and a-ketoglutarate, proportionately reduced to a total volume of 1 ml containing 0.1 ml of the 20% liver supernatant solution. The total activity (see below) was expressed as units (micromoles of p-hydroxyphenylpyruvate formed per hour) per gram wet liver. Actinomycin D (obtained through the courtesy of Merck, Sharp and Dohme Research Laboratories, Rahway, New Jersey) was dissolved in a minute amount of 95% ethanol and diluted with 0.9% NaCl to about 50 pg/ml. The concentration was determined from its absorption at 445 rnp (C = 19,600, pH 455.0 (Waksman et al., 1958) ). a-Methyl-nn-tryptophan was obtained from the Regis Chemical Co., Chicago, and also through the courtesy of Merck, Sharp and Dohme Research Laboratories, Rahway, New. Jersey.
INDUCED
ENZYMES
IN
CHICK
185
EMBRYOS
RESULTS
Preliminary surveys were made of the assay conditions required for maximum activities of the tryptophan pyrrolase and tyrosine transaminase from chick and chick embryo livers (Table 1). The tryptophan pyrrolase activity of infant rats was not increased by addition of hematin (Greengard and Feigelson, 1963) as was that of older rats ( Greengard and Feigelson, 1961) . The enzyme in chick embryos and chicks was similar in that hematin [and ascorbic acid (Knox and Ogata, 1965)] addition did not increase the activity in embryo livers, but it doubled that in 40-day-old chick livers. Preincubation of the concentrated liver extract from embryos or chicks with methemoglobin and tryptophan did not increase the activity, as has been observed to occur with rat liver extracts (Knox et al., 1965). The routine conditions of assay that were adopted therefore included addition of hematin and ascorbic acid, as well as formylase, but without the TABLE EFFECT OF PREINCUBATION TYROSINE TRANSAMINASE
TREATMENTS ACTIVITIES
1 UPON THE OF CHICK
Untreated Preparation
Tryptophan pyrrolase Embryo (17 days) (37”) Chick (40 days) (25”) Tyrosine transaminase (25”) Embryo (16 days) Chick (1 day)
Basal
TRYPTOPHAX PYRROLASE LIVER PREPARATIONS~~*
chicks
Cortisol-treated
Total
(2)c
2.6
(6)
0.54
(6)c
0.50
(6)
3.7
(6) (2)
18.6 11.9
chicks
Banal
2.5
40
AND
(6) (3)
2.20
15.4
Total
(6)
2.44
(6)
(5)
76.0
(3)
(2)
52.1
(7)
a The “basal” activities of tryptophan pyrrolasc were measured as described in the text, and the “total” activities were measured after preincubation at 37” for 30 minutes of the 20% supernatant fraction of liver with an equal volume of the KCltryptophan solution containing 1.0 mg horse methemoglobin per milliliter. These tryptophan pyrrolase assays in 40-day-old chicks were at 25”, and not at 37”. The “basal” activities of tyrosine transaminase were measured by omitting the 20 mM a-ketoglutarate and 5 mM tryptophan usually added during homogenization, and the “total” activities were measured with these additions, as described in the text. Parentheses give the number of separate livers or pools of livers measured. b Values are stated in units per gram of liver. c Omission of hematin and ascorbic acid from the basal assay of tryptophan pyrrolase did not affect the activity from embryonic livers, but halved that from 40-dayold chicks.
186
KNOX
AND
EPPENBERGER
preincubation treatment. Tryptophan was present during homogenization of the livers for its possible stabilizing effect ( Greengard and Feigelson, 1962). A preliminary survey of the properties of the tyrosine transaminase from chick liver revealed that its activity was notably higher in livers homogenized in the presence of a-ketoglutarate (Table 1). .cu-Ketoglutarate was for this reason added routinely to all homogenates for measurement of the tyrosine transaminase levels. In other experiments, addition of a-ketoglutarate was found to activate the enzyme, not simply to stabilize the enzyme, during the period between homogenization and assay. The particle-free supernatant fraction prepared without a-ketoglutarate had about 20% of the total demonstrable activity, but when the undiluted fraction was allowed to stand for an additional 2 hours at 0°C with added a-ketoglutarate the full activity appeared (Table 1). The activation by added a-ketoglutarate was demonstrable in older livers and after hydrocortisone treatment. The coenzyme, pyridoxal phosphate (tested at 78 pg/ml, 7.4 times the concentration used in the assay), and also 0.005 M L-tryptophan, a substrate of the transaminase (Jacoby and La Du, 1964), activated the soluble enzyme in the same way as a-ketoglutarate, but to a lesser extent. The combination of a-ketoglutarate and pyridoxal phosphate was somewhat more effective than a-ketoglutarate alone. The routine assay conditions adopted included addition of #a-ketoglutarate and tryptophan to the homogenate in order to measure the maximal activity. The average and range of the control and induced levels of tryptophan pyrrolase in livers at different ages, from ll-day-old embryos until they were hatched, are given in Fig. 1. The numerical values with the standard deviations and with the body and liver weights are given in Table 2. Tryptophan pyrrolase was present at a nearly constant activity per gram of liver throughout the period examined. The total activity per liver therefore increased with liver growth. Before the 11th day tryptophan injections killed the embryos, but after this age tryptophan injections elevated the enzyme level. This induction was especially marked in the 11-day-old embryos and in the chick immediately upon hatching, but the effect was less between these ages. Additional experiments to be described (Fig. 2) showed that the smaller increases in the 18day-old embryos were significant. Hydrocortisone injections, on the other hand, did not significantly
AND
km)
0.81 0.92 2.31 8.27
39.7 41.7 74.5 375
Liver
0.004 0.08 0.39 0.60
weight
LEVELS
0.097 3.7 19.0 30.8
Body
Average
INDUCED
1.54 0.99
2.1
1.6 1.7 2.4
rt 0.23 zk 0.34
IL- 1.1
xk 0.3 z!T 0.4 * 0.6
-
Control
TABLE AND
f 1.2* -
5 1.1* + 0.5* f 0.6
3.00*1.14* -
6.6
8.4 3.3 3.3
+ Tryptophan
pyrrolaw
HATCHED
PYRROLASE
Tryptophan
OF TRYPTOPHAN
2 TYROSINE
1.17 2.97
2.6
2.5 2.6 2.3
+ 0.74 rt 0.86*
+ 1.3
*.0.65 + 1.2 k 0.5
+ Cortisol
CHICKS~J
AND
12.3 12.6
f 5.1 + 1.8
6.‘id 24.0 f 4.0 17.7 2~ 5.2 13.2 ii 4.1
Control
TRAXSAMINASE
transaminasec
LIVERS
19.6
38.5 25.8 30.0
-
+ 8.1
+ 4.0* + 3.7 + 7.2*
+ Tryptophan
Tyrosine
IS
35.3 49.5
f 20.6 + 6.3*
37.3d 122.3 f 25.1* 118.8 f 15.4* 81.7 k 32.4*
+ Cortisol
OF EMBRYO
Q The 5-day-old embryos were given 0.075 or 0.15 mg cortisol in 0.25 ml saline at -5 hours; the older embryos were given 0.15 mg cortisol in 0.5 ml saline at -6 hours, or 4 mg L-tryptophan in 0.2 ml saline at -72, -48, and -24 hours; and all were killed at sero time. The hatched chicks were given 1 ml of saline containing 19 mg L-tryptophan per 100 gm body weight, or 2.4 mg hydrocortisone acetate per 100 gm body weight 6 hours before death. b Activities are the means of 3-6 livers or pools of livers, k standard deviations, expressed as micromoles per hour per gram liver. c Asterisk (*): significantly different from controls (P < 0.05), with c calculated from the small number of pools (3-6). The means, each consisting of many animals per pool, are probably less variable than indicated in this way. d Five-day-old embryos: assayed in total body homogenate and expressed as though all activity was in the liver.
Embryos 5 days II days 16 days 19 days Chicks 12 hours 24-30 hours 10 days 40 davs
Age
BASAL
2
if4
g 2g
i
%
188
KNOX
AND
EPPENBERGER
IO Hatch
Tr
‘\
\
/ / _--- Tt1 kT
1 \ r*
T/
*’..‘
._
Days (embryo) FIG.
treated hatching. different
1. Liver tryptophan pyrrolase activities in control (-()-), cortisol(- - - x - - -), and L-tryptophan-treated (- -@- -) chicks before and after Ranges of the values are marked by vertical lines. Those significantly (P < 0.05) from the control values are marked by asterisks.
elevate the tryptophan pyrrolase in the chick livers until several weeks after hatching. The responsiveness of the tryptophan pyrrolase level in 16-day-old embryos was studied in more detail, because the increase after tryptophan treatment at this age was less than in livers at earlier or later ages. The time course was determined of the response to L-tryptophan and to its nonmetabolizable analog, a-methyl-nL-tryptophan ( Fig. 2). The small elevation 24 hours after tryptophan treatment was confirmed, and was found to be statistically significant (P < 0.01). A similar elevation was present as early as 6 hours after the tryptophan treatment. There was no evanescent rise to the higher levels seen in younger or older livers. a-Methyl-m-tryptophan, which induces the tryptophan pyrrolase in rats (Sourkes and Townsend, 1955; Greengard, 1964), was much more effective as an inducer than L-tryptophan in the 16-day-old chick embryo livers. The enzyme level rose progressively during the 24-hour period following a single dose of a-methylnr,-tryptophan to a level (8.9 units per gram) comparable to that produced in the younger embryos by L-tryptophan (8.4 units per
INDUCED
ENZYMES
IN
CHICK
189
EMBRYOS
6
24 ROUti: AFTER TREATMENT
FIG. 2. Induction of tryptophan pyrrolase in l&day-old chick embryo livers after single doses of 20 pmoles of L-tryptophan (--@--) or a-methyl-nLtryptophan (-x-). Saline-injected controls (-O-). 12 standard errors of the means are indicated by arrows for the means of four and six pools 24 hours after inductions, and by the shaded zone for the mean of 13 pools of control livers. Other points represent a single pool of five or more livers.
gram in 11-day-old embryos, Table 2). Three daily injections of a-methyl-nL-tryptophan did not further elevate the enzyme. The omission of hematin from the enzyme assays of both the control and a-methyl-nL-tryptophan-treated livers did not lower their tryptophan pyrrolase in pyrrolase activities. This indicated that the tryptophan both kinds of preparations from Is-day-old embryos was fully conjugated with its prosthetic group, a state of the enzyme characteristic of young rats (Greengard and Feigelson, 1963) and those induced by tryptophan (Greengard and Feigelson, 1961) or a-methyl-nn-tryptophan ( Greengard, 1964). Formylase, which hydrolyzes the formylkynurenine formed by tryptophan pyrrolase to kynurenine (Knox, 1955), was low or absent in
196
KNOX
AND
EPPENBERGER
the embryonic chick livers. Formylkynurenine accumulated during the tryptophan pyrrolase reaction in preparations from these young livers. For this reason it was necessary to add formylase to the tryptophan pyrrolase assays in embryo and l-day-old chick livers in order to measure kynurenine production. Assays of formylase activity at different ages showed that a pronounced developmental formation of the enzyme occurred at the time of hatching (Table 3). When significant activity had appeared (14 days after hatching), treatment of the chicks with tryptophan or hydrocortisone 6 hours earlier did not affect the developing formylase level. This treatment with tryptophan increased the level of the tryptophan pyrrolase whose reaction provides the substrate for the formyIase (Table 2). TABLE KYNURENINE
FORMYLASE
ACTIVITY
IN
3 LIVERS
OF
CHICKS
AT
Controla Age (days)
Embryo: 37” assays 16 20 Chick: 25” assays 1 1 4 7
Number of assays
2 1 4 4 4 4
Average
DIFFERENT
AGES
1nducedo.b Range
Average
RaIlgl2
33-120 -
47* 45t 1991
30-70 30-60 15%229
3.9 11.1 49 153 356
125-171 318-388
a Activities are expressed as micromoles per hour per gram liver. * Induction treatments were: * hydrocortisone acetate, t hydrocortisone acetate plus tryptophan, and $ tryptophan, all given 6 hours before death at the same dosages recorded for hatched chicks in Table 2.
The average and range of the control and induced levels of tyrosine transaminase in livers at different ages from 11-day-old embryos to hatched chicks are given in Fig. 3. Additional levels from 5-day-old embryos, and from chicks after hatching until 2 days old, along with the standard deviations and with body and liver weights for all determinations, are given in Table 2. Tyrosine transaminase was present from the earliest period examined (measured in whole-body homogenates of the 5-day-old embryos and expressed per weight of liver). From the 11th embryonic day the activity per gram of liver decreased. The total activity per liver therefore remained approxi-
INDUCED
ENZYMES
1N
CHICK
191
EMBRYOS
Hatch i
-----_______ I -xi WI
‘\
‘\
‘\
‘.
*.
-
* 60r’ . 4 930c: t’
x 1
O
I
II
I
I
I
I,
Days (embryo)
I6
I
I
1 II
I9
<;;g “
I
12
J&2 Hours
I, (chick)
tyrosine-a-ketoglutarate transaminase activities in control FIG. 3. Liver (-O-), cortisol-treated ( - - - x - - -), and L-tryptophan-treated (--a-) chicks before and after hatching. Ranges of the values are marked by vertical lines. Those significantly different (P < 0.05) from the control values are marked by asterisks.
mately constant as the liver grew. Tryptophan, which elevates this enzyme level in adrenalectomized rats (Kenney and Flora, 1961; Rosen and Milholland, 1963) and in chick embryos (Gordon, 1956), produced no more than 2-fold elevations at any age. These elevations appeared to be statistically significant in 11- and 19-day-old embryos. n-Tyrosine had a similar effect, but was tested less extensively because of its insolubility. Hydrocortisone injections, on the other hand, elevated the tyrosine transaminase level unambiguously at all ages tested. Its effect declined with age, producing relatively smaller elevations in chicks older than the 11-day-old embryos. Additional experiments to be described in l-day-old chicks (Table 5) showed that this smaller effect was significant. A large and significant elevation with hydrocortisone has been reported in 2-week-old chicks (Chan and Cohen, 1964). The fraction of the tyrosine transaminase activity demonstrable in homogenates without preincubation with a-ketoglutarate increased
I92
KNOX
AND
EPPENBERGER
slightly, from 20 to 33% with increasing age, and was not higher after hydrocortisone treatment ( Table 1) . The inhibition by actinomycin of the inductions of the two enzymes was tested. Actinomycin did not inhibit the substrate-type induction of the tryptophan pyrrolase with a-methyl-ix-tryptophan in the 16day-old embryos, or the smaller elevation with L-tryptophan in the lo-day-old chicks. In the older chicks, actinomycin plus tryptophan tripled the already elevated tryptophan pyrrolase level that was produced by tryptophan alone. Significant elevation of the tryptophan pyrrolase by hydrocortisone was observed in still older chicks (40-60 days), though it had not been found in younger ones (Table l), and this elevation by hydrocortisone in the older chicks was prevented by actinomycin (Table 4). The elevations of tyrosine transaminase by hydrocortisone were not inhibited by actinomycin in either embryos or l-day-old chicks (Table 5). Actinomycin was equally ineffective if given to the embryos before the hydrocortisone, as shown in the table, or if given together with the hydrocortisone. Doses of actinomycin from one-third to five times those shown in the table and given 2-18 TABLE EFFECT
OF ACTINOMYCIN
4
ON THE EI~EVATION CHICK LIVERS~,*
OF TRYPTOPHAN
PYRROLASE
IN
(Inducer) Age (days)
Control
Actinomyoin
(a-Methyl-nn-tryptophan)
Embryos 16 (3 pools)
2..5 * 0.05
8.3
* 2.0
pools)
1.5
* 0.23
1.18
k 0.65
3.0
+ 0.34
1.27
f 0.23
2.97
*
1.14
(Hydrocortisone 40-60
8.5
(2 pools)
(L-Tryptophan)
Chicks 10 (4-5
PlUS actinomycin
AlOllt?
(5-9
pools)
0.99
+ 0.86
8.9
f
1.24
phosphate) 1.15
+ 0.12
a Embryos received 4.4 mg a-methyl-nn-tryptophan and 23 rg actinomycin D 24 hours before death. Ten-day-old chicks received 20 mg L-tryptophan and 57 rg actinomycin D 6 hours before death. Older chicks (40-60 days) received 2.5 mg hydrocortisone phosphate per 100 gm body weight and 100 pg actinomycin D 4.5 hours before death. b Activities are given as the mean units of tryptophan pyrrolase per gram liver + c, for the number of determinations given in parentheses.
INDUCED
ENZYMES
IN
CHICK
193
EMBRYOS
hours before the hydrocortisone also failed to prevent the elevation of the transaminase. The elevation by hydrocortisone of the tyrosine transaminase in embryo or very young chicks therefore appears to be insensitive to actinomycin, in contrast to the results in other animals. DISCUSSION
The behavior of the three enzymes examined here in the chick contrasts with their known behavior in rodents. Neither the tryptophan pyrrolase nor the tyrosine transaminase has been detected in the livers of fetal rodents (Auerbach and Waisman, 1959; Nemeth, 1959; Sereni et al., 1959). These enzymes appear after birth. In the chick embryos the tryptophan pyrrolase was present as early as the 11th day. The tyrosine transaminase activity, for which detection is more sensitive, was found in the 5-day-old whole-embryo homogenates. The specific activities of these enzymes did not change strikingly during the developental period studied. On the other hand, the dramatic developmental formation of the formylase at about the time of hatching provides another instance of the evocation of apparently de novo enzyme formation in a tissue during development. In mammalian livers which have been examined, this enzyme is present in the fetal livers before the appearance of the tryptophan pyrrolase (Nemeth, 1951)) and is always present in great excess (Knox and Mehler, 1951). The nature of two of the enzymes was also different in the chick. The character of the tryptophan pyrrolase, with regard to the apparent degree of conjugation with its hematin prosthetic group, was TABLE15 EFFECT
OF ACTINOMYCIN IN
LIVERS
ON INDUCTION OF EMBRYO
Preparation
Embryos, 16 days (6 pools of 3-8 each) Chicks, 24-30 hours (5 or 6 chicks)
Control
OF TYROSINE AXD
HATCHED
TRANSAMINASE
BY CORTISOL
CHICKS~~*
Actillomycin
Cortisol + aotinomycin
Cortisol
17.6
+ 4.65
24.6
f 12.9
89.0
f 38.0
82.3
+ 28.8
12.6
k 1.8
19.2
* 6.8
49.3
f 6.1
48.9
+ 21.8
u Embryos received 20 pg actinomycin D 18 hours hours before death. Chicks received 23 rg actinomycin acetate together 6 hours before death. h Activities are expressed as means f (r in micromoles
before and 0.15 mg cortisol D and 1 .O mg hydrocortisone per hour
per gram
of liver.
6
194
KNOX
AND
EPPENBEBGER
not different in younger and older chick livers from that reported in younger and older rats. The enzyme activity from chick embryos and young rats was not increased upon addition of hematin, but was increased in enzymes from 40-day-old chicks and from normal adult rat livers (Greengard and Feigelson, 1961). These findings in the rat have been interpreted as indicating that the enzyme in the developing rat and the tryptophan-induced adult rats is fully conjugated with its prosthetic group, while that of the normal and hydrocortisone-induced rats is partially unconjugated (Greengard and Feigelson, 1963). The same is apparently so for the enzyme in younger and older chicks. On the other hand, no indication could be found in chicks at any age for a;precursor of the tryptophan pyrrolase which becomes activated by incubation of the concentrated liver extract with tryptophan and a source of hematin, as has been found in rats (Knox et al., 1965). The form of the tyrosine transaminase in chick livers was decidedly different from the enzyme found in rat liver extracts. A large proportion of the total activity was present in a form that was catalytically inactive until a concentrated solution of the liver extract was incubated in the cold with a-ketoglutarate. L-Tryptophan and pyridoxal phosphate, compounds with which the enzyme specifically combines, also activated the enzyme in the same way as a-ketoglutarate. The proportion of the inactive form decreased somewhat with the age of the chicks and also with hydrocortisone treatment. It is possible that this inactive form represents a physiological precursor of the enzyme. A smaller increase in activity of the tyrosine transaminase after incubation of extracts from rat livers in a complex mixture has been reported (Tryfiates and Litwack, 1964). The effect of the inducers on the several enzymes at different ages revealed unexpected patterns. Both tryptophan and hydrocortisone were demonstrably effective as enzyme inducers during the same age period in the young chick livers, but the response to these inducers was enzyme specific. The tryptophan pyrrolase was elevated significantly only by the substrate-type inducers until the chicks were over one month old, when a 3-fold increase by hydrocortisone was first detected. The tyrosine transaminase was elevated markedly by hydrocortisone from the 5th embryonic day throughout the period examined. The much smaller, though significant, elevation of the tyrosine transaminase activity by treatment with tryptophan, may not represent a real increase in enzyme amount. Tryptophan activated the enzyme in uitro, and it is also a substrate (Jacoby and La Du, 1964). The ele-
INDUCED
ENZYMES
IN
CHICK
EMBRYOS
195
vated activity might represent more efficient conversion in vivo of the inactive to the active form of the enzyme by tryptophan than was produced in vitro by the preincubation of the enzyme with its substrates. The formylase during the period of its first formation was not affected by either type of inducer. Thus in the same organ during the tryptophan pyrrolase increased with same period of development, tryptophan and a-methyl-m-tryptophan treatment, tyrosine transaminase increased with hydrocortisone, and formylase was not changed by either type of inducer. The presence of tryptophan pyrrolase in the embryonic livers before the appearance of formylase, the next enzyme in the metabolic pathway, coupled with the elevation of the tryptophan pyrrolase by tryptophan, provided an opportunity to test the occurrence of sequential induction of enzymes in animal tissues. When tryptophan was administered, the amount of tryptophan oxidized was presumably increased and extra formylkynurenine was formed. But the level of formylase, which acts on the formylkynurenine was not affected, even though the experiments were done in chicks that had already begun to make this enzyme and had not yet formed the maximum amount. The relative ineffectiveness of administered tryptophan in 16- and 19-day-old embryos, where it did not increase the tryptophan pyrrolase level as much as it did in younger or older livers, cannot be attributed to unresponsiveness of the enzyme level at this time. It was elevated maximally during this period by treatment with the nonmetabolizable analog, a-methyl-nr,-tryptophan. Possibly tryptophan was rapidly utilized in other reactions, such as protein synthesis, during this period, and was not available for the induction. At 11 days of age, the embryo is small (3.7 gm) and increases relatively slowly in absolute weight per day. Tryptophan then induces maximally. By 16 days, the absolute weight gain per day has markedly increased. Then tryptophan induces very little. The large absolute increase in liver size during this period is associated with increase of a number of enzymes (Eppenberger et al., 1962/1963). The failure of actinomycin to inhibit the substrate-type induction of tryptophan pyrrolase and its inhibition of hydrocortisone induction of the same enzyme (in older chicks) duplicates the results observed in rats (Greengard et al., 1963a). These could be interpreted in support of the mechanisms that have been postulated: that hormone-type induction, but not substrate-type induction, requires new RNA synthesis (Greengard, 1963), and that substrate-type induction occurs by
196
KNOX
AND
EPPENBEBGER
slowing the degradation of the enzyme ( Schimke et al., 1965). However, the action of actinomycin appears to be more complicated than originally assumed. Although it did not increase the tryptophan pyrrolase when given alone, when given with tryptophan it markedly heightened the induction obtained with tryptophan alone. Other paradoxical effects of this inhibitor have been reported. They include its elevation of the tryptophan pyrrolase and tyrosine transaminase levels when given alone for several days to intact or adrenalectomized rats (Rosen et al., 1964), and its intensification of the hydrocortisone inductions of the same enzymes when given 5 hours after the hormone ( Garren et al., 1964). The two actinomycin effects observed here, its failure to prevent substrate-type induction and its inhibition of the hormone-type induction of tryptophan pyrrolase, were necessarily of the two types of determined at different ages. The sensitivities inductions to inhibition by actinomycin have therefore not been compared in exactly similar situations. The complete failure of actinomycin to inhibit the hydrocortisoneinduced elevations of tyrosine transaminase in chicks under 1 day of age is different from its effect on the hydrocortisone induction of tyrosine transaminase in rats (Greengard et al., 1963a,b; Garren et al., 1964), and different from its effect on tryptophan pyrrolase in the older chicks examined here. Without a comparison of its effect on the tyrosine transaminase in older chicks, no conclusions can be drawn; but the possibilities exist that induction of tyrosine transaminase in chicks at any age is not sensitive to actinomycin inhibition, or that hydrocortisone induces this enzyme in the young chick liver by a mechanism not requiring new RNA synthesis. Note added in proof: Preliminary results by Dr. N. S. Constantsas indicate that actinomycin D in 5-week-old chicks ( 100 pg per chick) also does not prevent the induction of tyrosine transaminase by hydrocortisone phosphate (2.4 mg per 100 gm body weight) as it does in similar experiments reported for tryptophan pyrrolase (Table 4). Groups of four controls and those treated 4.5 hours earlier with actinomycin, hydrocortisone phosphate, and hydrocortisone phosphate plus actinomycin average 4.5, 15.3, 72.4, and 66.2 units of tyrosine transaminase per gram of liver, respectively. SUMMARY
In the liver of chick embryos tyrosine transaminase was present from the 5th day, tryptophan pyrrolase from the 11th day, and form-
INDUCED
ENZYMES
IN
CHICK
EMBRYOS
197
ylase appeared at the time of hatching. The tyrosine transaminase level was elevated 5-fold by treatment with hydrocortisone but not with tryptophan, and the tryptophan pyrrolase was elevated a similar amount by treatment with tryptophan but not with hydrocortisone. The dramatic developmental elevation of formylase was not affected by either treatment. The behavior of the enzymes is therefore markedly different in the chick embryo than in the fetal rat. This difference was emphasized by the finding that the hydrocortisone-induced elevation of tyrosine transaminase in the chick embryo was not prevented by actinomycin. We are grateful to Mrs. Marta M. Piras for doing the formylase determinations of Table 3, and to Dr. N. Hasegawa for the determination of the actinomycin inhibition of the tryptophan pyrrolase inductions by hydrocortisone phosphate in the 40- to 60-day-old chicks of Table 4. REFERENCES V. H., and WAISMAN, H. A. ( 1959). Tryptophan peroxidase-oxidase, histidase, and transaminase activity in the liver of the developing rat. .I. Biol. Chem. 234, 304-306. CHAN, S., and COHEN, P. P. (1964). A comparative study of the effect of hydrocortisone injection on tryosine transaminase activity of different vertebrates. Arch. Biochem. Biophys. 164, 335-337. EPPENBERGEH, H. M., VON FELLENBEHG, R., RICHTERICH, R., and AEBI, H. ( 1962/1963). Die Ontogenese von zytoplasmatischen Enzymen beim Hiihnerembryo. Enzymol. Biol. Clin. 2, 139-174. GARREN, L. D., HOWELL, R. R., TOMKINS, G. M., and CROCCO, R. M. (1964). A paradoxical effect of actinomycin D: the mechanism of regulation of enzyme synthesis by hydrocortisone Proc. Natl. Acd. Sci. U.S. 52, 1121-1129. GORDON, M. W. (1956). Role of adaptive enzyme formation in morphogenesis. In “Progress in Neurobiology” (S. R. Korey and J. I. Nurnberger, eds.), Vol. 1, pp. 83-100. Harper (Hoeber), New York. GREENGAHD, 0. ( 1963). The role of coenzymes, cortisone and RNA in the control of liver enzyme levels. In “Advances in Enzyme Regulation” (G. Weber, ed.), Vol. 1, pp. 61-76. Macmillan (Pergamon), New York. GREENCARD, 0. ( 1964). Tryptophan analogues and the mechanism of induction of rat-liver tryptophan pyrrolase, in viuo. Biochim. Biophys. Acta 85, 492-494. GREENGARD, O., and FEIGELSON, P. ( 1961). The activation and induction of rat liver tryptophan pyrrolase in uivo by its substrate. J. Biol. Chem. 236, 158-161. GREENGARD, O., and FEIGELSON, P. ( 1962). The purification and properties of liver tryptophan pyrrolase. J. BioZ. Chem. 237, 1903-1967. GREENGARD, O., and FEIGELSON, P. ( 1963). Relationships of the apoenzyme and coenzyme of tryptophan pyrrolase in developing and regenerating rat liver. Ann. N.Y. Acad. Sci. 111, 227-232. GREENGARD, O., SMITH, M. A., and ACS, G. (1963a). Relation of cortisone and AUEHBACH,
198
KNOX
AND
EPPENBERGER
synthesis of ribonucleic acid to induced and developmental enzyme formation. 1. Biol. them. 238, 1548-1551. GREENGARD, O., WEBER, G., and SINGHAL, R. L. ( 1963b). Glycogen deposition in the liver induced by cortisone: dependence on enzyme synthesis. Science 141, 160-161. JACOBY, G. A., and LA Du, B. N. (1964). Studies on the specificity of tyrosinecY-ketoglutarate transaminase. J. Biol. Chem. 239, 419-424. KENNEY, F. T., and FLORA, R. M. (1961). Induction of tyrosine-cu-ketoglutarate transaminase in rat liver. I. Hormonal nature. J. Biol. C&m. 236, 2699-2702. KNOX, W. E. (1955). Tryptophan oxidation. In “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. II, pp. 242-253. Academic Press, New York. KNOX, W. E., and MEHLER, A. H. (1951). The adaptive increase of the tryptophan peroxidase-oxidase system of liver. Science 113, 237-238. KNOX, W. E., and OGATA, M. (1965). Effects of peroxide, catalase, and hematin in the assay of liver tryptophan pyrrolase. J. Biol. Chem. 240, 2216-2221. KNOX, W. E., PIRAS, M., and TOKUYAMA, K. ( 1965). Activation of tryptophan pyrrolase after substrate and hormone induction. Federation Proc. 24, 474. LERNER, S. A. ( 1959). The existence and control of six transaminases in the vertebrate liver. Biochemical Sciences Honors Thesis, Harvard College, Cambridge, Massachusetts. LIN, E. C. C., PITT, B. M., OVEN, M., and KNOX, W. E. (1958). The assay of aromatic amino acid transaminations and keto acid oxidation by the enol borate-tautomerase method. J. Bill. Chem. 233, 668-673. NEMETH, A. M. (1959). Mechanisms controlling changes in tryptophan peroxidase activity in developing mammalian liver. J. Biol. Chem. 234, 2921-2924. NEMETH, A. M. ( 1961). Enzyme formation in developing mammalian liver. Biochim. Biophys. Acta 48, 189-191. ROSEN, F., and MILHOLLAND, R. J. ( 1963). Glucocorticoids and transaminase activity. VII. Studies on the nature and specificity of substrate induction of tyrosine-a-ketoglutarate transaminase and tryptophan pyrrolase. J. Biol. Chem. 238, 3730-3735. ROSEN, F., RAINA, P. N., MILHOLLAND, R. J., and NICHOL, C. A. (1964). Induction of several adaptive enzymes by actinomycin D. Science 146, 661-663. SCHIMKE, R. T., SWEENEY, E. W., and BERLIN, C. M. (1965). The roles of synthesis and degradation in the control of rat liver tryptophan pyrrolase. J. Biol. Chem. 24Q, 322-331. SERENI, F., KENNEY, F. T., and KRETCHMER, N. (1959). Factors infhiencing the development of tyrosine-cu-ketoglutarate transaminase activity in rat liver. J. Biol. Chem. 234, 609-612. SOURKES, T. L., and TOWNSEND, E. (1955). Effects of e-methyl-nn-tryptophan on the oxidation of tryptophan. Can. J. Biochem. Physiol. 33, 73~740. TRYFIATES, G. P., and LITWACK, G. (1964). Appearance of an increment of tyrosine aminotransferase activity in a cell-free system. Biochktry 3, 14&J1487. WAICSMAN, S. A., KATZ, E., and VINING, L. C. (1958). Nomenclature of the actinomycins. Proc. Natl. Acad. Sci. U.S. 44, 602-612.
Basal
BIOLOGY
13,
and Induced and Tyrosine
182198
(1966)
Levels of Tryptophan Pyrrolase Transaminase in the Chick1
W. EUGENE KNOX AND HANS M. EPPENBERCER” Department Cancer
of Biological Chemistry, Harvard Research Institute, New England Boston, Massachusetts Accepted
November
15,
Medical School and Deaconess Hospital,
the
1965
INTRODUCTION
The present studies were designed to compare the regulation of the tryptophan pyrrolase (EC 1.11.1.4 L-tryptophan: HzO, oxidoreductase) and tyrosine transaminase (EC 2.6.1.5 L-tyrosine: 2-oxoglutarate aminotransferase) in the liver of the chick embryos and the hatched chicks with the known behavior of these enzymes in the livers of rats. The levels of both enzymes in the liver of chick embryos have been increased by treatment with tryptophan (Gordon, 1956), and the level of the liver tyrosine transaminase in the young hatched chick was elevated by hydrocortisone treatment (Chan and Cohen, 1964; Lerner, 1959). Our results show that both enzymes are present in more or less constant specific activity in the Iiver from early embryonic life, and that there is a striking difference in the behavior of these two enzymes after treatment with inducers. Hydrocortisone elevates the tyrosine transaminase during the embryonic period, but not the tryptophan pyrrolase, This induction was not prevented by actinomycin. Tryptophan elevates the tryptophan pyrrolase, and it induces the tyrosine transaminase to a much smaller amount throughout the period studied. Formylase (EC 3.5.1.9: aryl-formylamineamidohydrolase) the second ‘This investigation was supported by United States Public Health Service Grant AM 00567-12, by Research Career Award AM-K6261803 from the National Institute of Arthritis and Metabolic Diseases of the National Institutes of Health, United States Public Health Service, and by United States Atomic Energy Commission Contract At( 30-l )-901 with the New England Deaconess Hospital (NYO-901-40). ‘Present address: Department of Biochemistry, Brandeis University, Waltham, Massachusetts. 182
INDUCED
ENZYMES
IN
CHICK
EMBRYOS
183
enzyme in the pathway of tryptophan metabolism, was low or absent in the embryonic liver. It increased dramatically after hatching, but its level was unaffected by treatment with the inducers. METHODS
White Leghorn SPAFAS chicken eggs were obtained from a commercial supplier and incubated in the laboratory. Preliminary results with Rhode Island Red eggs were entirely similar. The viability of untreated control eggs was consistently over 90%, and their age was uniform as judged by hatching within a 12-hour period on the scheduled 21st day. The ages given for the embryos are the day of incubation in the laboratory. The age of the hatched chicks was counted from the time of hatching within 2-hour periods during the last 12 hours of incubation. The injected solutions were sterile (autoclaved) solutions in 0.9% NaCI. Injections were made with sterile precautions into the air space of the egg through a hole drilled with forceps and sealed with Scotch tape. The hatched chicks received the doses intraperitoneally, with care taken to avoid the yolk sac. All controls received similar injections of saline alone. The mortality in embryos was determined by daily candling during the treatment period and inspection at the time of killing. More of the younger embryos died after injections than the older ones, but less than one-third at any age died with the doses used. The mortality was significantly higher with larger doses of the inducers. At harvest the living embryos were weighed, chilled on ice, and the livers were transferred to preweighed beakers containing the cold solution for homogenization. These were subsequently reweighed to determine the weight of single livers or the average liver weight per embryo in the pool. Pools of about twenty H-day-old, eight 16-day-old, five 19-day-old embryo livers and individual hatched chick livers were used. Experiments in chick embryos younger than 11 days were prevented by the insensitivity of the assay for tryptophan pyrrolase and the greatly increased toxicity of tryptophan. However, it was possible to measure the tyrosine transaminase in homogenates of whole 5-day-old embryos, and to induce this enzyme with cortisol. Injections of 0.075 mg of cortisol in 0.25 ml of saline were made into the chorioallantois just beside the embryo, and the embryos were removed 5 hours later. A 20% homogenate of the total embryo in pools of about 24 embryos
184
KNOX
AND
EPPENBERGER
each was assayed as usual for the tyrosine transaminase. The activities observed were referred entirely to the liver weight (determined separately) for comparative purposes. Enzyme Assays. The livers were promptly homogenized in a glass homogenizer with enough cold 0.15 A4 KC1 containing 0.005 A4 Ltryptophan and 0.02 M a-ketoglutarate to make a 20% homogenate, and centrifuged in the cold at 100,000 g for 1 hour in the Spinco model L preparative ultracentrifuge. The clear supernatant solution was carefully withdrawn with a syringe to avoid the sedimented particulate layer and the floating lipid layer. Assays were done immediately, about 2 hours after death of the chicks, using the supernatant fractions. The tryptophan pyrrolase was determined at 37°C by readings of the optical density at 360 rnp in a Gilford spectrophotometer of a reaction system against a water blank (Knox and Ogata, 1965). Additions, in this order, to the reaction mixture were: 0.5 ml of 20% liver supernatant solution, 1.2 ml of 0.2 M phosphate, pH 7.0, 0.2 ml of 20 pM hematin, 0.3 ml of formylase (429 pmoles/hr/ml at 37°C) (Knox, 1955), and 1 ml of water. After temperature equilibration, 0.2 ml of 0.05 M L-tryptophan and 0.1 ml of 0.3 M freshly neutralized ascorbic acid solution were added to start the reaction, making the total volume 3.5 ml. Activities are expressed as units (micromoles of kynurenine formed per hour) per gram of wet liver. Formylase was assayed with formylkynurenine as substrate as described by Knox (1955). The tyrosine transaminase was determined at 25°C by the enol borate method (Lin et al., 1958), with the components, which included pyridoxal phosphate and a-ketoglutarate, proportionately reduced to a total volume of 1 ml containing 0.1 ml of the 20% liver supernatant solution. The total activity (see below) was expressed as units (micromoles of p-hydroxyphenylpyruvate formed per hour) per gram wet liver. Actinomycin D (obtained through the courtesy of Merck, Sharp and Dohme Research Laboratories, Rahway, New Jersey) was dissolved in a minute amount of 95% ethanol and diluted with 0.9% NaCl to about 50 pg/ml. The concentration was determined from its absorption at 445 rnp (C = 19,600, pH 455.0 (Waksman et al., 1958) ). a-Methyl-nn-tryptophan was obtained from the Regis Chemical Co., Chicago, and also through the courtesy of Merck, Sharp and Dohme Research Laboratories, Rahway, New. Jersey.
INDUCED
ENZYMES
IN
CHICK
185
EMBRYOS
RESULTS
Preliminary surveys were made of the assay conditions required for maximum activities of the tryptophan pyrrolase and tyrosine transaminase from chick and chick embryo livers (Table 1). The tryptophan pyrrolase activity of infant rats was not increased by addition of hematin (Greengard and Feigelson, 1963) as was that of older rats ( Greengard and Feigelson, 1961) . The enzyme in chick embryos and chicks was similar in that hematin [and ascorbic acid (Knox and Ogata, 1965)] addition did not increase the activity in embryo livers, but it doubled that in 40-day-old chick livers. Preincubation of the concentrated liver extract from embryos or chicks with methemoglobin and tryptophan did not increase the activity, as has been observed to occur with rat liver extracts (Knox et al., 1965). The routine conditions of assay that were adopted therefore included addition of hematin and ascorbic acid, as well as formylase, but without the TABLE EFFECT OF PREINCUBATION TYROSINE TRANSAMINASE
TREATMENTS ACTIVITIES
1 UPON THE OF CHICK
Untreated Preparation
Tryptophan pyrrolase Embryo (17 days) (37”) Chick (40 days) (25”) Tyrosine transaminase (25”) Embryo (16 days) Chick (1 day)
Basal
TRYPTOPHAX PYRROLASE LIVER PREPARATIONS~~*
chicks
Cortisol-treated
Total
(2)c
2.6
(6)
0.54
(6)c
0.50
(6)
3.7
(6) (2)
18.6 11.9
chicks
Banal
2.5
40
AND
(6) (3)
2.20
15.4
Total
(6)
2.44
(6)
(5)
76.0
(3)
(2)
52.1
(7)
a The “basal” activities of tryptophan pyrrolasc were measured as described in the text, and the “total” activities were measured after preincubation at 37” for 30 minutes of the 20% supernatant fraction of liver with an equal volume of the KCltryptophan solution containing 1.0 mg horse methemoglobin per milliliter. These tryptophan pyrrolase assays in 40-day-old chicks were at 25”, and not at 37”. The “basal” activities of tyrosine transaminase were measured by omitting the 20 mM a-ketoglutarate and 5 mM tryptophan usually added during homogenization, and the “total” activities were measured with these additions, as described in the text. Parentheses give the number of separate livers or pools of livers measured. b Values are stated in units per gram of liver. c Omission of hematin and ascorbic acid from the basal assay of tryptophan pyrrolase did not affect the activity from embryonic livers, but halved that from 40-dayold chicks.
186
KNOX
AND
EPPENBERGER
preincubation treatment. Tryptophan was present during homogenization of the livers for its possible stabilizing effect ( Greengard and Feigelson, 1962). A preliminary survey of the properties of the tyrosine transaminase from chick liver revealed that its activity was notably higher in livers homogenized in the presence of a-ketoglutarate (Table 1). .cu-Ketoglutarate was for this reason added routinely to all homogenates for measurement of the tyrosine transaminase levels. In other experiments, addition of a-ketoglutarate was found to activate the enzyme, not simply to stabilize the enzyme, during the period between homogenization and assay. The particle-free supernatant fraction prepared without a-ketoglutarate had about 20% of the total demonstrable activity, but when the undiluted fraction was allowed to stand for an additional 2 hours at 0°C with added a-ketoglutarate the full activity appeared (Table 1). The activation by added a-ketoglutarate was demonstrable in older livers and after hydrocortisone treatment. The coenzyme, pyridoxal phosphate (tested at 78 pg/ml, 7.4 times the concentration used in the assay), and also 0.005 M L-tryptophan, a substrate of the transaminase (Jacoby and La Du, 1964), activated the soluble enzyme in the same way as a-ketoglutarate, but to a lesser extent. The combination of a-ketoglutarate and pyridoxal phosphate was somewhat more effective than a-ketoglutarate alone. The routine assay conditions adopted included addition of #a-ketoglutarate and tryptophan to the homogenate in order to measure the maximal activity. The average and range of the control and induced levels of tryptophan pyrrolase in livers at different ages, from ll-day-old embryos until they were hatched, are given in Fig. 1. The numerical values with the standard deviations and with the body and liver weights are given in Table 2. Tryptophan pyrrolase was present at a nearly constant activity per gram of liver throughout the period examined. The total activity per liver therefore increased with liver growth. Before the 11th day tryptophan injections killed the embryos, but after this age tryptophan injections elevated the enzyme level. This induction was especially marked in the 11-day-old embryos and in the chick immediately upon hatching, but the effect was less between these ages. Additional experiments to be described (Fig. 2) showed that the smaller increases in the 18day-old embryos were significant. Hydrocortisone injections, on the other hand, did not significantly
AND
km)
0.81 0.92 2.31 8.27
39.7 41.7 74.5 375
Liver
0.004 0.08 0.39 0.60
weight
LEVELS
0.097 3.7 19.0 30.8
Body
Average
INDUCED
1.54 0.99
2.1
1.6 1.7 2.4
rt 0.23 zk 0.34
IL- 1.1
xk 0.3 z!T 0.4 * 0.6
-
Control
TABLE AND
f 1.2* -
5 1.1* + 0.5* f 0.6
3.00*1.14* -
6.6
8.4 3.3 3.3
+ Tryptophan
pyrrolaw
HATCHED
PYRROLASE
Tryptophan
OF TRYPTOPHAN
2 TYROSINE
1.17 2.97
2.6
2.5 2.6 2.3
+ 0.74 rt 0.86*
+ 1.3
*.0.65 + 1.2 k 0.5
+ Cortisol
CHICKS~J
AND
12.3 12.6
f 5.1 + 1.8
6.‘id 24.0 f 4.0 17.7 2~ 5.2 13.2 ii 4.1
Control
TRAXSAMINASE
transaminasec
LIVERS
19.6
38.5 25.8 30.0
-
+ 8.1
+ 4.0* + 3.7 + 7.2*
+ Tryptophan
Tyrosine
IS
35.3 49.5
f 20.6 + 6.3*
37.3d 122.3 f 25.1* 118.8 f 15.4* 81.7 k 32.4*
+ Cortisol
OF EMBRYO
Q The 5-day-old embryos were given 0.075 or 0.15 mg cortisol in 0.25 ml saline at -5 hours; the older embryos were given 0.15 mg cortisol in 0.5 ml saline at -6 hours, or 4 mg L-tryptophan in 0.2 ml saline at -72, -48, and -24 hours; and all were killed at sero time. The hatched chicks were given 1 ml of saline containing 19 mg L-tryptophan per 100 gm body weight, or 2.4 mg hydrocortisone acetate per 100 gm body weight 6 hours before death. b Activities are the means of 3-6 livers or pools of livers, k standard deviations, expressed as micromoles per hour per gram liver. c Asterisk (*): significantly different from controls (P < 0.05), with c calculated from the small number of pools (3-6). The means, each consisting of many animals per pool, are probably less variable than indicated in this way. d Five-day-old embryos: assayed in total body homogenate and expressed as though all activity was in the liver.
Embryos 5 days II days 16 days 19 days Chicks 12 hours 24-30 hours 10 days 40 davs
Age
BASAL
2
if4
g 2g
i
%
188
KNOX
AND
EPPENBERGER
IO Hatch
Tr
‘\
\
/ / _--- Tt1 kT
1 \ r*
T/
*’..‘
._
Days (embryo) FIG.
treated hatching. different
1. Liver tryptophan pyrrolase activities in control (-()-), cortisol(- - - x - - -), and L-tryptophan-treated (- -@- -) chicks before and after Ranges of the values are marked by vertical lines. Those significantly (P < 0.05) from the control values are marked by asterisks.
elevate the tryptophan pyrrolase in the chick livers until several weeks after hatching. The responsiveness of the tryptophan pyrrolase level in 16-day-old embryos was studied in more detail, because the increase after tryptophan treatment at this age was less than in livers at earlier or later ages. The time course was determined of the response to L-tryptophan and to its nonmetabolizable analog, a-methyl-nL-tryptophan ( Fig. 2). The small elevation 24 hours after tryptophan treatment was confirmed, and was found to be statistically significant (P < 0.01). A similar elevation was present as early as 6 hours after the tryptophan treatment. There was no evanescent rise to the higher levels seen in younger or older livers. a-Methyl-m-tryptophan, which induces the tryptophan pyrrolase in rats (Sourkes and Townsend, 1955; Greengard, 1964), was much more effective as an inducer than L-tryptophan in the 16-day-old chick embryo livers. The enzyme level rose progressively during the 24-hour period following a single dose of a-methylnr,-tryptophan to a level (8.9 units per gram) comparable to that produced in the younger embryos by L-tryptophan (8.4 units per
INDUCED
ENZYMES
IN
CHICK
189
EMBRYOS
6
24 ROUti: AFTER TREATMENT
FIG. 2. Induction of tryptophan pyrrolase in l&day-old chick embryo livers after single doses of 20 pmoles of L-tryptophan (--@--) or a-methyl-nLtryptophan (-x-). Saline-injected controls (-O-). 12 standard errors of the means are indicated by arrows for the means of four and six pools 24 hours after inductions, and by the shaded zone for the mean of 13 pools of control livers. Other points represent a single pool of five or more livers.
gram in 11-day-old embryos, Table 2). Three daily injections of a-methyl-nL-tryptophan did not further elevate the enzyme. The omission of hematin from the enzyme assays of both the control and a-methyl-nL-tryptophan-treated livers did not lower their tryptophan pyrrolase in pyrrolase activities. This indicated that the tryptophan both kinds of preparations from Is-day-old embryos was fully conjugated with its prosthetic group, a state of the enzyme characteristic of young rats (Greengard and Feigelson, 1963) and those induced by tryptophan (Greengard and Feigelson, 1961) or a-methyl-nn-tryptophan ( Greengard, 1964). Formylase, which hydrolyzes the formylkynurenine formed by tryptophan pyrrolase to kynurenine (Knox, 1955), was low or absent in
196
KNOX
AND
EPPENBERGER
the embryonic chick livers. Formylkynurenine accumulated during the tryptophan pyrrolase reaction in preparations from these young livers. For this reason it was necessary to add formylase to the tryptophan pyrrolase assays in embryo and l-day-old chick livers in order to measure kynurenine production. Assays of formylase activity at different ages showed that a pronounced developmental formation of the enzyme occurred at the time of hatching (Table 3). When significant activity had appeared (14 days after hatching), treatment of the chicks with tryptophan or hydrocortisone 6 hours earlier did not affect the developing formylase level. This treatment with tryptophan increased the level of the tryptophan pyrrolase whose reaction provides the substrate for the formyIase (Table 2). TABLE KYNURENINE
FORMYLASE
ACTIVITY
IN
3 LIVERS
OF
CHICKS
AT
Controla Age (days)
Embryo: 37” assays 16 20 Chick: 25” assays 1 1 4 7
Number of assays
2 1 4 4 4 4
Average
DIFFERENT
AGES
1nducedo.b Range
Average
RaIlgl2
33-120 -
47* 45t 1991
30-70 30-60 15%229
3.9 11.1 49 153 356
125-171 318-388
a Activities are expressed as micromoles per hour per gram liver. * Induction treatments were: * hydrocortisone acetate, t hydrocortisone acetate plus tryptophan, and $ tryptophan, all given 6 hours before death at the same dosages recorded for hatched chicks in Table 2.
The average and range of the control and induced levels of tyrosine transaminase in livers at different ages from 11-day-old embryos to hatched chicks are given in Fig. 3. Additional levels from 5-day-old embryos, and from chicks after hatching until 2 days old, along with the standard deviations and with body and liver weights for all determinations, are given in Table 2. Tyrosine transaminase was present from the earliest period examined (measured in whole-body homogenates of the 5-day-old embryos and expressed per weight of liver). From the 11th embryonic day the activity per gram of liver decreased. The total activity per liver therefore remained approxi-
INDUCED
ENZYMES
1N
CHICK
191
EMBRYOS
Hatch i
-----_______ I -xi WI
‘\
‘\
‘\
‘.
*.
-
* 60r’ . 4 930c: t’
x 1
O
I
II
I
I
I
I,
Days (embryo)
I6
I
I
1 II
I9
<;;g “
I
12
J&2 Hours
I, (chick)
tyrosine-a-ketoglutarate transaminase activities in control FIG. 3. Liver (-O-), cortisol-treated ( - - - x - - -), and L-tryptophan-treated (--a-) chicks before and after hatching. Ranges of the values are marked by vertical lines. Those significantly different (P < 0.05) from the control values are marked by asterisks.
mately constant as the liver grew. Tryptophan, which elevates this enzyme level in adrenalectomized rats (Kenney and Flora, 1961; Rosen and Milholland, 1963) and in chick embryos (Gordon, 1956), produced no more than 2-fold elevations at any age. These elevations appeared to be statistically significant in 11- and 19-day-old embryos. n-Tyrosine had a similar effect, but was tested less extensively because of its insolubility. Hydrocortisone injections, on the other hand, elevated the tyrosine transaminase level unambiguously at all ages tested. Its effect declined with age, producing relatively smaller elevations in chicks older than the 11-day-old embryos. Additional experiments to be described in l-day-old chicks (Table 5) showed that this smaller effect was significant. A large and significant elevation with hydrocortisone has been reported in 2-week-old chicks (Chan and Cohen, 1964). The fraction of the tyrosine transaminase activity demonstrable in homogenates without preincubation with a-ketoglutarate increased
I92
KNOX
AND
EPPENBERGER
slightly, from 20 to 33% with increasing age, and was not higher after hydrocortisone treatment ( Table 1) . The inhibition by actinomycin of the inductions of the two enzymes was tested. Actinomycin did not inhibit the substrate-type induction of the tryptophan pyrrolase with a-methyl-ix-tryptophan in the 16day-old embryos, or the smaller elevation with L-tryptophan in the lo-day-old chicks. In the older chicks, actinomycin plus tryptophan tripled the already elevated tryptophan pyrrolase level that was produced by tryptophan alone. Significant elevation of the tryptophan pyrrolase by hydrocortisone was observed in still older chicks (40-60 days), though it had not been found in younger ones (Table l), and this elevation by hydrocortisone in the older chicks was prevented by actinomycin (Table 4). The elevations of tyrosine transaminase by hydrocortisone were not inhibited by actinomycin in either embryos or l-day-old chicks (Table 5). Actinomycin was equally ineffective if given to the embryos before the hydrocortisone, as shown in the table, or if given together with the hydrocortisone. Doses of actinomycin from one-third to five times those shown in the table and given 2-18 TABLE EFFECT
OF ACTINOMYCIN
4
ON THE EI~EVATION CHICK LIVERS~,*
OF TRYPTOPHAN
PYRROLASE
IN
(Inducer) Age (days)
Control
Actinomyoin
(a-Methyl-nn-tryptophan)
Embryos 16 (3 pools)
2..5 * 0.05
8.3
* 2.0
pools)
1.5
* 0.23
1.18
k 0.65
3.0
+ 0.34
1.27
f 0.23
2.97
*
1.14
(Hydrocortisone 40-60
8.5
(2 pools)
(L-Tryptophan)
Chicks 10 (4-5
PlUS actinomycin
AlOllt?
(5-9
pools)
0.99
+ 0.86
8.9
f
1.24
phosphate) 1.15
+ 0.12
a Embryos received 4.4 mg a-methyl-nn-tryptophan and 23 rg actinomycin D 24 hours before death. Ten-day-old chicks received 20 mg L-tryptophan and 57 rg actinomycin D 6 hours before death. Older chicks (40-60 days) received 2.5 mg hydrocortisone phosphate per 100 gm body weight and 100 pg actinomycin D 4.5 hours before death. b Activities are given as the mean units of tryptophan pyrrolase per gram liver + c, for the number of determinations given in parentheses.
INDUCED
ENZYMES
IN
CHICK
193
EMBRYOS
hours before the hydrocortisone also failed to prevent the elevation of the transaminase. The elevation by hydrocortisone of the tyrosine transaminase in embryo or very young chicks therefore appears to be insensitive to actinomycin, in contrast to the results in other animals. DISCUSSION
The behavior of the three enzymes examined here in the chick contrasts with their known behavior in rodents. Neither the tryptophan pyrrolase nor the tyrosine transaminase has been detected in the livers of fetal rodents (Auerbach and Waisman, 1959; Nemeth, 1959; Sereni et al., 1959). These enzymes appear after birth. In the chick embryos the tryptophan pyrrolase was present as early as the 11th day. The tyrosine transaminase activity, for which detection is more sensitive, was found in the 5-day-old whole-embryo homogenates. The specific activities of these enzymes did not change strikingly during the developental period studied. On the other hand, the dramatic developmental formation of the formylase at about the time of hatching provides another instance of the evocation of apparently de novo enzyme formation in a tissue during development. In mammalian livers which have been examined, this enzyme is present in the fetal livers before the appearance of the tryptophan pyrrolase (Nemeth, 1951)) and is always present in great excess (Knox and Mehler, 1951). The nature of two of the enzymes was also different in the chick. The character of the tryptophan pyrrolase, with regard to the apparent degree of conjugation with its hematin prosthetic group, was TABLE15 EFFECT
OF ACTINOMYCIN IN
LIVERS
ON INDUCTION OF EMBRYO
Preparation
Embryos, 16 days (6 pools of 3-8 each) Chicks, 24-30 hours (5 or 6 chicks)
Control
OF TYROSINE AXD
HATCHED
TRANSAMINASE
BY CORTISOL
CHICKS~~*
Actillomycin
Cortisol + aotinomycin
Cortisol
17.6
+ 4.65
24.6
f 12.9
89.0
f 38.0
82.3
+ 28.8
12.6
k 1.8
19.2
* 6.8
49.3
f 6.1
48.9
+ 21.8
u Embryos received 20 pg actinomycin D 18 hours hours before death. Chicks received 23 rg actinomycin acetate together 6 hours before death. h Activities are expressed as means f (r in micromoles
before and 0.15 mg cortisol D and 1 .O mg hydrocortisone per hour
per gram
of liver.
6
194
KNOX
AND
EPPENBEBGER
not different in younger and older chick livers from that reported in younger and older rats. The enzyme activity from chick embryos and young rats was not increased upon addition of hematin, but was increased in enzymes from 40-day-old chicks and from normal adult rat livers (Greengard and Feigelson, 1961). These findings in the rat have been interpreted as indicating that the enzyme in the developing rat and the tryptophan-induced adult rats is fully conjugated with its prosthetic group, while that of the normal and hydrocortisone-induced rats is partially unconjugated (Greengard and Feigelson, 1963). The same is apparently so for the enzyme in younger and older chicks. On the other hand, no indication could be found in chicks at any age for a;precursor of the tryptophan pyrrolase which becomes activated by incubation of the concentrated liver extract with tryptophan and a source of hematin, as has been found in rats (Knox et al., 1965). The form of the tyrosine transaminase in chick livers was decidedly different from the enzyme found in rat liver extracts. A large proportion of the total activity was present in a form that was catalytically inactive until a concentrated solution of the liver extract was incubated in the cold with a-ketoglutarate. L-Tryptophan and pyridoxal phosphate, compounds with which the enzyme specifically combines, also activated the enzyme in the same way as a-ketoglutarate. The proportion of the inactive form decreased somewhat with the age of the chicks and also with hydrocortisone treatment. It is possible that this inactive form represents a physiological precursor of the enzyme. A smaller increase in activity of the tyrosine transaminase after incubation of extracts from rat livers in a complex mixture has been reported (Tryfiates and Litwack, 1964). The effect of the inducers on the several enzymes at different ages revealed unexpected patterns. Both tryptophan and hydrocortisone were demonstrably effective as enzyme inducers during the same age period in the young chick livers, but the response to these inducers was enzyme specific. The tryptophan pyrrolase was elevated significantly only by the substrate-type inducers until the chicks were over one month old, when a 3-fold increase by hydrocortisone was first detected. The tyrosine transaminase was elevated markedly by hydrocortisone from the 5th embryonic day throughout the period examined. The much smaller, though significant, elevation of the tyrosine transaminase activity by treatment with tryptophan, may not represent a real increase in enzyme amount. Tryptophan activated the enzyme in uitro, and it is also a substrate (Jacoby and La Du, 1964). The ele-
INDUCED
ENZYMES
IN
CHICK
EMBRYOS
195
vated activity might represent more efficient conversion in vivo of the inactive to the active form of the enzyme by tryptophan than was produced in vitro by the preincubation of the enzyme with its substrates. The formylase during the period of its first formation was not affected by either type of inducer. Thus in the same organ during the tryptophan pyrrolase increased with same period of development, tryptophan and a-methyl-m-tryptophan treatment, tyrosine transaminase increased with hydrocortisone, and formylase was not changed by either type of inducer. The presence of tryptophan pyrrolase in the embryonic livers before the appearance of formylase, the next enzyme in the metabolic pathway, coupled with the elevation of the tryptophan pyrrolase by tryptophan, provided an opportunity to test the occurrence of sequential induction of enzymes in animal tissues. When tryptophan was administered, the amount of tryptophan oxidized was presumably increased and extra formylkynurenine was formed. But the level of formylase, which acts on the formylkynurenine was not affected, even though the experiments were done in chicks that had already begun to make this enzyme and had not yet formed the maximum amount. The relative ineffectiveness of administered tryptophan in 16- and 19-day-old embryos, where it did not increase the tryptophan pyrrolase level as much as it did in younger or older livers, cannot be attributed to unresponsiveness of the enzyme level at this time. It was elevated maximally during this period by treatment with the nonmetabolizable analog, a-methyl-nr,-tryptophan. Possibly tryptophan was rapidly utilized in other reactions, such as protein synthesis, during this period, and was not available for the induction. At 11 days of age, the embryo is small (3.7 gm) and increases relatively slowly in absolute weight per day. Tryptophan then induces maximally. By 16 days, the absolute weight gain per day has markedly increased. Then tryptophan induces very little. The large absolute increase in liver size during this period is associated with increase of a number of enzymes (Eppenberger et al., 1962/1963). The failure of actinomycin to inhibit the substrate-type induction of tryptophan pyrrolase and its inhibition of hydrocortisone induction of the same enzyme (in older chicks) duplicates the results observed in rats (Greengard et al., 1963a). These could be interpreted in support of the mechanisms that have been postulated: that hormone-type induction, but not substrate-type induction, requires new RNA synthesis (Greengard, 1963), and that substrate-type induction occurs by
196
KNOX
AND
EPPENBEBGER
slowing the degradation of the enzyme ( Schimke et al., 1965). However, the action of actinomycin appears to be more complicated than originally assumed. Although it did not increase the tryptophan pyrrolase when given alone, when given with tryptophan it markedly heightened the induction obtained with tryptophan alone. Other paradoxical effects of this inhibitor have been reported. They include its elevation of the tryptophan pyrrolase and tyrosine transaminase levels when given alone for several days to intact or adrenalectomized rats (Rosen et al., 1964), and its intensification of the hydrocortisone inductions of the same enzymes when given 5 hours after the hormone ( Garren et al., 1964). The two actinomycin effects observed here, its failure to prevent substrate-type induction and its inhibition of the hormone-type induction of tryptophan pyrrolase, were necessarily of the two types of determined at different ages. The sensitivities inductions to inhibition by actinomycin have therefore not been compared in exactly similar situations. The complete failure of actinomycin to inhibit the hydrocortisoneinduced elevations of tyrosine transaminase in chicks under 1 day of age is different from its effect on the hydrocortisone induction of tyrosine transaminase in rats (Greengard et al., 1963a,b; Garren et al., 1964), and different from its effect on tryptophan pyrrolase in the older chicks examined here. Without a comparison of its effect on the tyrosine transaminase in older chicks, no conclusions can be drawn; but the possibilities exist that induction of tyrosine transaminase in chicks at any age is not sensitive to actinomycin inhibition, or that hydrocortisone induces this enzyme in the young chick liver by a mechanism not requiring new RNA synthesis. Note added in proof: Preliminary results by Dr. N. S. Constantsas indicate that actinomycin D in 5-week-old chicks ( 100 pg per chick) also does not prevent the induction of tyrosine transaminase by hydrocortisone phosphate (2.4 mg per 100 gm body weight) as it does in similar experiments reported for tryptophan pyrrolase (Table 4). Groups of four controls and those treated 4.5 hours earlier with actinomycin, hydrocortisone phosphate, and hydrocortisone phosphate plus actinomycin average 4.5, 15.3, 72.4, and 66.2 units of tyrosine transaminase per gram of liver, respectively. SUMMARY
In the liver of chick embryos tyrosine transaminase was present from the 5th day, tryptophan pyrrolase from the 11th day, and form-
INDUCED
ENZYMES
IN
CHICK
EMBRYOS
197
ylase appeared at the time of hatching. The tyrosine transaminase level was elevated 5-fold by treatment with hydrocortisone but not with tryptophan, and the tryptophan pyrrolase was elevated a similar amount by treatment with tryptophan but not with hydrocortisone. The dramatic developmental elevation of formylase was not affected by either treatment. The behavior of the enzymes is therefore markedly different in the chick embryo than in the fetal rat. This difference was emphasized by the finding that the hydrocortisone-induced elevation of tyrosine transaminase in the chick embryo was not prevented by actinomycin. We are grateful to Mrs. Marta M. Piras for doing the formylase determinations of Table 3, and to Dr. N. Hasegawa for the determination of the actinomycin inhibition of the tryptophan pyrrolase inductions by hydrocortisone phosphate in the 40- to 60-day-old chicks of Table 4. REFERENCES V. H., and WAISMAN, H. A. ( 1959). Tryptophan peroxidase-oxidase, histidase, and transaminase activity in the liver of the developing rat. .I. Biol. Chem. 234, 304-306. CHAN, S., and COHEN, P. P. (1964). A comparative study of the effect of hydrocortisone injection on tryosine transaminase activity of different vertebrates. Arch. Biochem. Biophys. 164, 335-337. EPPENBERGEH, H. M., VON FELLENBEHG, R., RICHTERICH, R., and AEBI, H. ( 1962/1963). Die Ontogenese von zytoplasmatischen Enzymen beim Hiihnerembryo. Enzymol. Biol. Clin. 2, 139-174. GARREN, L. D., HOWELL, R. R., TOMKINS, G. M., and CROCCO, R. M. (1964). A paradoxical effect of actinomycin D: the mechanism of regulation of enzyme synthesis by hydrocortisone Proc. Natl. Acd. Sci. U.S. 52, 1121-1129. GORDON, M. W. (1956). Role of adaptive enzyme formation in morphogenesis. In “Progress in Neurobiology” (S. R. Korey and J. I. Nurnberger, eds.), Vol. 1, pp. 83-100. Harper (Hoeber), New York. GREENGAHD, 0. ( 1963). The role of coenzymes, cortisone and RNA in the control of liver enzyme levels. In “Advances in Enzyme Regulation” (G. Weber, ed.), Vol. 1, pp. 61-76. Macmillan (Pergamon), New York. GREENCARD, 0. ( 1964). Tryptophan analogues and the mechanism of induction of rat-liver tryptophan pyrrolase, in viuo. Biochim. Biophys. Acta 85, 492-494. GREENGARD, O., and FEIGELSON, P. ( 1961). The activation and induction of rat liver tryptophan pyrrolase in uivo by its substrate. J. Biol. Chem. 236, 158-161. GREENGARD, O., and FEIGELSON, P. ( 1962). The purification and properties of liver tryptophan pyrrolase. J. BioZ. Chem. 237, 1903-1967. GREENGARD, O., and FEIGELSON, P. ( 1963). Relationships of the apoenzyme and coenzyme of tryptophan pyrrolase in developing and regenerating rat liver. Ann. N.Y. Acad. Sci. 111, 227-232. GREENGARD, O., SMITH, M. A., and ACS, G. (1963a). Relation of cortisone and AUEHBACH,
198
KNOX
AND
EPPENBERGER
synthesis of ribonucleic acid to induced and developmental enzyme formation. 1. Biol. them. 238, 1548-1551. GREENGARD, O., WEBER, G., and SINGHAL, R. L. ( 1963b). Glycogen deposition in the liver induced by cortisone: dependence on enzyme synthesis. Science 141, 160-161. JACOBY, G. A., and LA Du, B. N. (1964). Studies on the specificity of tyrosinecY-ketoglutarate transaminase. J. Biol. Chem. 239, 419-424. KENNEY, F. T., and FLORA, R. M. (1961). Induction of tyrosine-cu-ketoglutarate transaminase in rat liver. I. Hormonal nature. J. Biol. C&m. 236, 2699-2702. KNOX, W. E. (1955). Tryptophan oxidation. In “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. II, pp. 242-253. Academic Press, New York. KNOX, W. E., and MEHLER, A. H. (1951). The adaptive increase of the tryptophan peroxidase-oxidase system of liver. Science 113, 237-238. KNOX, W. E., and OGATA, M. (1965). Effects of peroxide, catalase, and hematin in the assay of liver tryptophan pyrrolase. J. Biol. Chem. 240, 2216-2221. KNOX, W. E., PIRAS, M., and TOKUYAMA, K. ( 1965). Activation of tryptophan pyrrolase after substrate and hormone induction. Federation Proc. 24, 474. LERNER, S. A. ( 1959). The existence and control of six transaminases in the vertebrate liver. Biochemical Sciences Honors Thesis, Harvard College, Cambridge, Massachusetts. LIN, E. C. C., PITT, B. M., OVEN, M., and KNOX, W. E. (1958). The assay of aromatic amino acid transaminations and keto acid oxidation by the enol borate-tautomerase method. J. Bill. Chem. 233, 668-673. NEMETH, A. M. (1959). Mechanisms controlling changes in tryptophan peroxidase activity in developing mammalian liver. J. Biol. Chem. 234, 2921-2924. NEMETH, A. M. ( 1961). Enzyme formation in developing mammalian liver. Biochim. Biophys. Acta 48, 189-191. ROSEN, F., and MILHOLLAND, R. J. ( 1963). Glucocorticoids and transaminase activity. VII. Studies on the nature and specificity of substrate induction of tyrosine-a-ketoglutarate transaminase and tryptophan pyrrolase. J. Biol. Chem. 238, 3730-3735. ROSEN, F., RAINA, P. N., MILHOLLAND, R. J., and NICHOL, C. A. (1964). Induction of several adaptive enzymes by actinomycin D. Science 146, 661-663. SCHIMKE, R. T., SWEENEY, E. W., and BERLIN, C. M. (1965). The roles of synthesis and degradation in the control of rat liver tryptophan pyrrolase. J. Biol. Chem. 24Q, 322-331. SERENI, F., KENNEY, F. T., and KRETCHMER, N. (1959). Factors infhiencing the development of tyrosine-cu-ketoglutarate transaminase activity in rat liver. J. Biol. Chem. 234, 609-612. SOURKES, T. L., and TOWNSEND, E. (1955). Effects of e-methyl-nn-tryptophan on the oxidation of tryptophan. Can. J. Biochem. Physiol. 33, 73~740. TRYFIATES, G. P., and LITWACK, G. (1964). Appearance of an increment of tyrosine aminotransferase activity in a cell-free system. Biochktry 3, 14&J1487. WAICSMAN, S. A., KATZ, E., and VINING, L. C. (1958). Nomenclature of the actinomycins. Proc. Natl. Acad. Sci. U.S. 44, 602-612.