thyroid hormone-induced regulation of protein synthesis in tadpole tail muscle

GENERAL AND COMPARATIVE ENLXXRINOLOGY 38,441-450 (199)

Thyroid Hormone-induced Regulation of Protein Synthesis in Tadpole Tail Muscle

MOHAMMEDSALEEM' AND BURRG.ATKINSON~

Cell Science Laboratories, Department of Zoology. University of Western Ontario, London, Ontario, N6A 5B7 Canada

Accepted March 19, 1979

Administration of thyroid hormone to anuran tadpoles markedly depresses the in vivo rate of protein synthesis in tadpole tail muscle (M. Saleem and B. G. Atkinson, 1978, J. Biol. C/tern. 253, 1378-1384). Additional in vitro studies suggested that the decrease in protein synthetic activity was due to an inhibitory factor(s) present in the postribosomal supematant fraction from tail muscle of hormone-treated tadpoles. This research employs the use of postribosomal fractions from tail muscle of control and T,-treated tadpoles in a heterologous, cell-free polypeptide synthesizing system as a means to substantiate the presence of an inhibitor and further elucidate the mechanism by which the thyroid hormone-induced factor(s) depresses protein synthesis. This study establishes that the initial depression in protein synthetic activity is not due to altered ribonuclease activity, enhanced proteolytic enzyme activity, or depleted elongation factors. The depression is partially, if not wholly, attributable to the presence of a thermolabile, trypsin-sensitive inhibitory factor in tail muscle from thyroid hormone-treated tadpoles.

Regression of tadpole tail during metamorphosis is a thyroid hormone- controlled phenomenon (Frieden and Just, 1970; Atkinson, 1971; Cohen et al., 1978). Under the influence of exogenously ad- ministered thyroxine, triiodothyronine (T3), or thyroxine analog, the tadpole tail invo- lutes with consequent degeneration and disappearance of its musculature (Shatfer, 1963; Kaltenbach, 1968; Weber, 1969; Tata, 1966; Tonoue and Frieden, 1970; Fox, 1975; Kistleret al., 1975; Ryffel and Weber, 1973; Saleem and Atkinson, 1978). Current opin- ion regarding the thyroid hormone-induced regression of tadpole tail muscle is that prior to (and concurrent with) detectable increases in proteolytic activity and tail regression, the rate of total protein synthesis in tail muscle declines (Tonoue and Frieden, 1970; Little et al., 1973; Kistler et al., 1975; Merrifield and Atkinson, 1977; Saleem and Atkinson, 1978). Previous

’ M.S. is currently a Postdoctoral Fellow of the Muscular Dystrophy Association of Canada at York University, Downsview, Ontario, M3J lP3 Canada.

* To whom all correspondence should be addressed.

studies in our laboratory have revealed that the T,-induced decrease in the rate of protein synthesis in tadpole tail muscle is partially, if not wholly, due to the presence of a factor in the postribosomal fraction which is inhibitory to ribosomal translational efftciency (Merrifield and Atkinson, 1977; Saleem, 1977; Saleem and Atkinson, 1978). Further studies with tail muscle postribosomal fractions (S,,O) from T,-treated tadpoles established that the ability of this fraction to support poly(U)-directed polyphenylalanine synthesis with tail muscle ribosomal subunits from either control or hormone-treated tadpoles is consistently 40-46% less active than that observed with S,,, fractions from control animals (Saleem, 1977). The heterologous, cell-free studies reported here were undertaken: (1) to clarify whether the decreased rate of protein synthesis in tail muscle of T,-treated tadpoles is due to a hormone-induced inhibitory factor, enhanced hydrolytic enzyme activity, or depletion of elongation factors; (2) to establish if this T,-induced inhibitory factor necessarily requires ribo-

441 0016~6480/79/080441-lOSOl.OO/O Copyright @ 1979 by Afade& Press, Inc. All tights of reproduction in my form reserved.

442 SALEEM AND ATKINSON

somes from a homologous source for its activity; and (3) to further elucidate the level in the translational process at which this inhibitor functions. The results reported in this study strongly support the contention that thyroid hormone treatment of pre- metamorphic tadpoles induces the activation or synthesis of a proteinaceous factor which acts by inhibiting protein synthesis in the tadpole tail muscle. These results also clearly indicate that a thermolabile, trypsin-sensitive inhibitor is present in the pH 5 supernatant of tail muscle postribosomal fractions from T,-treated tadpoles, and that its mode of action does not depend upon ribosomes from a homologous source.

EXPERIMENTAL PROCEDURES Materials

Rana cntesbeiana tadpoles were obtained from the Mogul Ed. Company, Oshkosh, Wisconsin, main- tained at room temperature in water on a diet of canned spinach, and staged according to Taylor and Kollros (1946). Stages IX to XII tadpoles weighing 5 to 8 g were starved for 5 days and subsequently injected intraperitoneally with triiodothyronine at a dose of 30 nmol/g body wt. Five-day starved control animals were injected with a vehicle solution of 200 PM NaOH. After injection tadpoles were not fed and were main- tamed in groups of 25 per 6 liters of water. Various times after vehicle or T, treatment, tadpoles were anesthetized by immersion in ice water and sacrificed by severing the truncus arteriosus. Tails were excised just posterior to the anal pore, the skin removed from the excised tail, and the underlying muscle dissected out.

Unless otherwise stated, all chemicals were reagent grade and purchased from Fisher Scientific (Philadel- phia, Pa.) or Sigma Chemical Company (St. Louis, MO.). Sucrose (density gradient grade, ribonuclease- free) and uniformly labeled [2-r4C]poly(U) (specific

activity was 0.30 Ci/mol polynucleotide P) was obtained from Schwarz/Mann. Uniformly labeled L-

[l*C]phenylalanyl-tRNA (sp. act. 385 Ci/mol), containing 19 other unlabeled aminoacyl-tRNAs from E. co/i strain B, was obtained from New England Nuclear. The potassium salt of poly(U) was obtained from Schwarz/Mann. Sodium fusidate and chartreusin were generously supplied to us by Dr. D. M. Nicholls (York University, Downsview, Ontario, Canada) who re- ceived them as gifts from Squibb Institute for Medical

Research (New Brunswick, N.J.) and Upjohn Com- pany (Kalamazoo, Mich.), respectively.

Methods Preparation of postribosomal and pH 5 supernatant

fractions. Tadpole tail muscle was homogenized in 10 vol of a buffer composed of 0.02 M Tris-HCl (pH 7.6), 0.08 M KCl, 0.01 M MgCl,, 0.25 M sucrose, and 4 mM dithiothreitol. The homogenate was centrifuged at 13,000g for 10 min and the resulting supernatant centrifuged in a Beckman 60 Ti rotor at 150,OOOg for 2 hr at 4” in a Sorvall OTD-2 ultracentrifuge. Occasionally some of the 150,OOOg supernatant fraction was dialyzed overnight at 4” against a buffer composed of 50 mM Tris-HCl (pH 7.6) 80 m&f KCl, 6 m&f MgCl,, and 2 mM dithiothreitol, made to a final concentration of 20% (v/v) glycerol, and stored in small aliquots at -20”. The remaining 150,OOOg supernatant was ad-

justed to pH 5.2 with 1 M acetic acid. After 30 min the resulting precipitate was removed by centrifugation at 15,OOOg for 15 min. The supematant (pH 5 supema- tam) was adjusted to pH 7.6 with 1 M KOH, dialyzed overnight at 4” against a buffer containing 50 m&f Tris-HCl (pH 7.6), 80 n&f KCI, 6 mM MgCl,, and 2 mM dithiothreitol. The dialyzed pH 5 supematant was adjusted to a 20% (v/v) final concentration of glycerol and stored in small aliquots at -20”.

Preparation of preincubnted. salt-washed rat liver ribosomes. Preincubated, salt-washed rat liver ribosomes were prepared as described by Nicholls (1973) and Girgis and Nicholls (1973). Livers from fasted rats were homogenized in 4 vol of a buffer containing 50 rr& Tris-HCl (pH 7.6), 80 mM KCI, 6 m&f MgCl,, 0.25 M sucrose, and 4 mM dithiothreitol. The tissue homogenate was centrifuged in a Spinco Model L2-65 ultracentrifuge at 15,000g for 15 min. Microsomes were sedimented from the 15,000g supernatant by centrifugation for 65 min at 105,OOOg. Microsomes (5 mg protein/ml) were preincubated for 10 min at 37” in a buffered medium containing 5 mg proteiniml equiva- lent of postmicrosomal supernatant, 5 mM ATP, and 0.2 mM GTP. Microsomes were then recovered by centrifuging at 105,OOOg for 65 min. Ribosomes were prepared by resuspending the microsomes in a buffer containing 1% deoxycholate and were recovered by centrifuging for 65 min at 105,OOOg. This pellet was resuspended in a buffered medium containing 50 mit4 Tris-HCl (pH 7.6). 0.5 M KCl, 10 mM M&l,, 0.25 M sucrose, and 4 mM dithiothreitol, and stirred for 12 hr in the cold. Aggregated material was removed by centrifuging for 10 min at 15,OOOg. The clarified supematant was layered over a discontinuous sucrose gradient and the ribosomes sedimented at 105,OOOg for 2.5 hr through 0.5 M sucrose layered over 1 M sucrose prepared in the Tris-KCl-MgCl,-dithiothreitol buffer. The ribosomal pellet was resuspended in a Tris-

Ts REGULATION OF PROTEIN SYNTHESIS 443

KCl-M&l,-dithiothreitol buffer containing 20% (v/v) glycerol and stored in small aliquots at -20”. This preincubated and salt-washed liver ribosome preparation exhibited negligible contamination with EF-1 and EF-2 (Nicholls, 1973). It had an endogenous mRNA-directed [14C]phenylalanyl-tRNA incorporation that was less than 10% of the poly(U)-directed incorporation. This ribosome preparation was used for all investigations of the activity of supematant factors EF-1 and EF-2.

Assay for incorporation of [‘4Clphenylalanyl-tRNA into peptides. The assay was carried out in a volume of 0.4 ml and generally contained 50 mM Tris-HCl (pH 7.6), 80 mM KCl, 6 mM MgCl,, 2 mM dithiothreitol, 0.4 mM GTP, 100 pg poly (U), 33 pg E. coli [14C]phenylalanyl-tRNA (14,000 dpm), and preincubated, salt-washed rat liver ribosomes (50 pg ribosomal RNA). The concentration of postribosomal and pH 5 supematant fractions was varied depending on the experiment, as noted in the figure legends. After incubation at 37” the reaction was stopped by the addi- tion of 2 ml of cold 10% (w/v) trichloracetic acid and the precipitated protein was heated at 90” for 20 min in 5% trichloroacetic acid, washed with ethanol:ether (2:l) saturated with sodium acetate, and washed in ether. The final ether-extracted pellet was air-dried, dissolved in 1 N NaOH, and counted in a Beckmann LS-255 liquid scintillation counter with an efficiency of SO-85%. Incorporation is expressed as disintegrations per minute per milligram of ribosomal RNA and represents corrected values (the counts recovered from tubes lacking either ribosomes or supernatant fractions were subtracted from the initial counts recovered in the complete system).

Ribonuclease assay. The ribonuclease assay was carried out in a volume of 0.4 ml. The reaction mixture contained 50 mM Tris-HCl (pH 7.5 at 25”), 80 mM KCl, 6 mM MgCl,, and 120 pg [2-14C]poly(U). The pH 5 supematant (2.0 mg protein) from the postribosomal fraction of tail muscle from T,-treated or control tadpoles was added as the source of ribonuclease activity. After 10, 20, and 30 min at 37”, 0.4 ml of acidified ethanol (80% ethanol containing 1 M HCl) was added to each tube. The tubes were supplemented with 0.1 ml of yeast RNA (3% (w/v)) and allowed to stand in the cold for at least 1 hr and then 3 ml of 40% ethanol containing 0.5 M HCl was added. After centrifugation, the acid-ethanol-insoluble and acid-ethanol-soluble counts were determined on a Beckmann LS-255 liquid scintillation counter. To ensure ribonuclease was active under these conditions, 25 ng of bovine pancreatic ribonuclease was added to some of the tubes and its effects measured as described above.

Measurement of protein and RNA. RNA was esti- mated from extinction at 260 nm (E,~ = 230) after ap- plying the ferritin correction as described by Wilson

and Hoaglund (1965). The ribosome preparations were used only when the 2601280 ratio gave a value of 1.90- 1.95. Protein concentrations were determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard.

RESULTS The Influence of Endogenous

Aminoacyl-tRNAs, Synthetases, and Elongation Factors on the Zncorpora- tion of [ 14C]Phenylalanyl-tRNA into Polypeptides Previous work in this laboratory estab-

lished that the postribosomal supematant (S,,,) fraction from tail muscle of T,-treated tadpoles is far less efftcient in support- ing the cell-free incorporation of [‘“Cl phenylalanine into polyphenylalanine than a similar fraction obtained from the tail muscle of control tadpoles (Saleem, 1977). Since differences in the endogenous amount of tRNAs capable of accepting phenylalanine could exist in S,,, fractions from control and T,-treated tadpoles, additional experiments were carried out using [ 14C]phenylalanyl-tRNA in place of [14C]phenylalanine. The results indicate that when an S,,, fraction from tail muscle of T,-treated tadpoles is used, and com- pared to the synthetic activity of an SIm fraction from control tadpoles, the marked reduction of the incorporation of [‘“Cl phenylalanine-tRNA into polyphenylalanine (Fig. 1) is similar to that observed when [14C]phenylalanine is the polypeptide pre- cursor (Saleem, 1977).

To completely eliminate the effect of qualitative or quantitative variations in tRNA species and synthetases in this reaction, a pH 5 supernatant fraction (Girgis and Nicholls, 1972; Nicholls et al., 1977) was prepared from the S,,, and used in place of the S15,, fraction. The results establish that although the incorporation of [14C]phenylalanine-tRNA into peptides increases with the amount of pH 5 supematant fraction added to the incubation mixture (Fig. 2), and with time of incubation


P 0.6 i.0

S ,50hgrTubm)

FIG. 1. The incorporation of [W]phenylalanyl- tRNA into polyphenylalanine using the postribosomal supematant (S,,,) fraction from tail muscle of control (0) amd 3-day T,-treated tadpole (0). Varying amounts of postribosomal supematant (S,,) fraction was incubated at 37“ for 10 min in a buffered (see Methods) reaction mixture containing an excess of preincubated salt-washed rat liver ribosomes (50 pg of RNA), excess poly(U) (100 pg), and [Ylphenyl- alanyl-tRNA. Each point represents the mean of three separate experiments; vertical bars signify the standard error of the mean.

6

T

OO.

PHS (mgllube)

FIG. 2. The incorporation of [l*C]phenylalanyl- tRNA into polyphenylalanine using the pH 5 supematant fraction of postribosomal supematants from tail muscle of control ( l ), and l-day (0), 3-day (m), and 5-day a) T,-treated tadpoles. Varying amounts of the pH 5 supematant was incubated at 37’ for 10 min in a reaction mixture as described in Fig. 1. Each point represents the mean of three separate experiments; vertical bars signify the standard error of the mean.

0 0 15 30 46 60

TIME (min.)

FIG. 3. The kinetics of [W]phenylalanyl-tRNA incorporation into polyphenylahutine using the pH 5 supematant of the postribosomal fraction from tail muscle of control ( l ), and l-day (O), 3-day (m), and 5-day (A) T,-treated tadpoles. Incubation conditions are the same as those described in Fig. 2 except that the time of incubation varied from 0 to 60 min and each incubation mixture contained 400 pg of a pH 5 supematant. Each point represents the mean of three separate experiments; vertical bars signify the standard error of the mean.

(Fig. 3), incorporation of [14C]phenylalanine into polyphenylalanine is always markedly lower when the pH 5 supernatant fraction from tail muscle of T,-treated tadpole is used. A comparison of the relative polypeptide synthetic activity of pH 5 supernatants from muscle of l-, 3-, and S-day T,-treated, and control tadpoles (Figs. 2 and 3) establishes that this hormone induces a factor which has an early and sustained effect on polypeptide synthesis (see Saleem and Atkinson, 1978).

The decreased capacity of muscle pH 5 supematant fraction from T,-treated tadpoles to support the incorporation of [ 14C]phenylalanine-tRNA into peptides could also be due to a decreased activity of elongation factors EF-1 and/or EF-2. To account for this possibility, the antibiotics chartreusin [an inhibitor of the EF-1 catalyzed binding of aminoacyl-tRNA to ribosomes (Gregg and Heintz, 1972; Nicholls ef al., 1974)] and fusidic acid [an

T3 REGULATION OF PROTEIN SYNTHESIS 445

inhibitor of the EF-2 catalyzed transloca- tion step (Malkin and Lipman, 1969; Nicholls, 1973)] were employed as probes of elongation factor activity. Chartreusin and fusidic acid were each found to affect the protein synthetic activity of pH 5 supematants from the tail muscle of control animals to the same degree as each affected the activity of pH 5 supematants from tail muscle of T,-treated tadpoles (results not shown). These results would not be ex- pected if the activity of EF-1 and EF-2 were limiting in the pH 5 supernatants from hormone-treated tadpoles; if EF-1 or EF-2 were limiting, less chartreusin or fusidic acid would be required in preparations from T,-treated animals than in control preparations to achieve the same degree of inhibition.

Proteolytic and Ribonuclease Activity in pH 5 Supernatant Fractions Experiments were carried out to test the

possibility that the apparent decrease in peptide synthetic activity observed with the pH 5 supernatant from muscle of T,-treated tadpoles might be due to a high level of proteolytic activity in this fraction. Tail muscle pH 5 supematants from control and T,-treated tadpoles were incubated with the other components of the cell-free protein synthesizing system (see Methods) and the reaction was terminated in some of the tubes after 30 min (protein synthesis was essentially complete by this time under the conditions used; see Fig. 3). To the remaining tubes, additional control or TJ- treated tadpole tail muscle pH 5 supematant, or trypsin was added, and the tubes were incubated for an additional 30 min, after which the reaction was terminated. No loss of acid-precipitable counts could be detected in the tubes supplemented with additional T,-treated or control muscle pH 5 supematant fractions (Table 1). On the other hand, supplementing the reaction mixtures with 10 pg trypsin resulted in over 90% loss of acid-precipitable counts (Table 1).

To test the possibility that enhanced ribonuclease activity might be present in the pH 5 supematant from the tail muscle of T,-treated tadpoles, a direct measurement of poly( U) degradation was performed (Pet- ryshyn and Nicholls, 1976). The data (not shown) indicates that the conversion of acid-ethanol-insoluble counts [i.e., poly(U)] to acid-ethanol-soluble counts after lo-, 20-, or 30-min incubation is the same with pH 5 supematants from control and T,-treated animals.

Inhibitory Activity in the pH 5 Supernatant Fraction from Muscle of T,-Treated Tadpoles Since the decreased peptide synthetic

activity of the pH 5 supematant fraction from tail muscle of T,-treated tadpoles might result from a deficiency in a factor required for translation or be due to the presence of an inhibitor, mixing experiments were designed to test the relevancy of either possibility. The result of experiments in which equal amounts of control and T,-treated muscle pH 5 preparations were mixed and assayed for peptide chain elongation are shown in Table 2. The results in Table 2 indicate that when increas- ing amounts of the pH 5 supernatant from T,-treated tadpoles are added to the pH 5 supematant from control tadpoles an enhanced depression of polypeptide synthesis occurs. These results strongly support the contention that an inhibitor, rather than a factor deficiency, is responsible for the depressed synthetic activity. The sensitivity of the inhibitor to heat (70” for 15 min) and to trypsin digestion (Table 3) suggests that it is of a proteinaceous nature.

DISCUSSION The rate of protein synthesis in tadpole

tail muscle declines following thyroid hormone treatment (Tonoue and Frieden, 1970; Little et al., 1973; Kistler et al., 1975; Saleem and Atkinson, 1978; Merrifield and Atkinson, 1977). The precise mechanism for the decreased rate of protein synthesis

TABL

E 1

ASSE

SSM

ENT

OF

PRO

TEO

LYTI

C EN

ZYM

E AC

TIVI

TY

DU

RIN

G P

OLY

PHEN

YLAL

ANIN

E SY

NTHE

SIS"

Sour

ce

of in

itial

pH 5

sup

emat

ant

Orig

inal

in

cuba

tion

time

(mitt

) So

urce

of

add

ed

pH

5 su

pem

atan

t

Subs

eque

nt

incu

batio

n tim

e (m

in)

Inco

rpor

atio

n of

labe

led

amin

o ac

id i

nto

polyp

heny

lala

nine

b (d

pm/m

g R

NA)

Con

trol

tadp

ole

mus

cle

30

T,-tr

eate

d ta

dpol

e m

uscl

e 30

Con

trol

tadp

ole

mus

cle

30

None

C

ontro

l ta

dpol

e m

uscl

e T,

-trea

ted

tadp

ole

mus

cle

None

C

ontro

l ta

dpol

e m

uscl

e T,

-trea

ted

tadp

ole

mus

cle

10

tryps

in

*g

0 49

,476

k

1446

30

48

,769

+

1154

30

47

,815

k

1200

0

31,2

00

k 16

00

30

33,3

22

k 10

90

30

34,2

76

2 12

77

30

3,14

7 -t

1071

R Th

e pH

5 s

upem

atan

ts

(400

pg)

of p

ostri

boso

mal

ta

il m

uscl

e fra

ctio

ns

from

con

trol

and

3-da

y T,

-trea

ted

tadp

oles

we

re

incu

bate

d fo

r 30

min

in

a co

m-

plet

e bu

ffere

d re

actio

n m

ixtu

re

(see

Met

hods

). Af

ter

the

initia

l 30

min

inc

ubat

ion

the

reac

tion

was

term

inat

ed

in s

ome

of t

he t

ubes

(po

lyphe

nyla

lani

ne

synt

hesi

s wa

s es

sent

ially

co

mpl

ete

by 3

0 m

in (

see

Fig.

3)).

Fre

sh p

H 5

sup

erna

tant

(6

00 K

g) o

r try

psin

(1

0 pg

) wa

s ad

ded

to th

e re

mai

nder

of

the

tube

s, a

nd

thes

e tu

bes

were

in

cuba

ted

at 3

7” f

or a

n ad

ditio

nal

30 m

in.

* Ea

ch v

alue

rep

rese

nts

the

mea

ns a

nd s

tand

ard

devi

atio

n fro

m

the

mea

n of

at l

east

two

ex

perim

ents

.


TABLE 2 EFFECT OF MIXING EQUAL AMOUNTS OF CONTROL AND T3-T~~?~~ TADPOLE TAIL MUSCLE pH 5

SUPERNATANT FRACTION ON THE INCORPORATION OF [14C]PHENyLALANyL-tRNA INTO POLYPHENYLALANINE~

Total amount of pH 5 supematant

(/.a)

Incorporationb (dpm/mg RNA) Percentage

Control Mixture inhibition

200 23,000 18,000 22 k 466 2 1077

400 34,626 25,000 28 2 2300 + 200

800 68,226 29,746 56 k 2100 2 973

n Equal amounts of the pH 5 supernatants of postribosomal tail muscle fractions from control and 3-day TS- treated tadpoles were mixed and incubated for 10 min in a complete, buffered reaction mixture (see Methods).

b Each value represents the mean and standard deviation from the mean of at least two experiments.

is not fully understood. Previous studies of a thyroid hormone-induced inhibitor and from this laboratory (Merrifield and Atkin- elucidate the possible translational site(s) son, 1977; Saleem, 1977; Saleem and Atkin- affected, we studied the inhibitory activity son, 1978) lend support to our contention of the tail muscle postribosomal (S,,,) frac- that one possible mechanism for the de- tion and the pH 5 supernatant from the S,,, creased rate of protein synthesis operates at fraction in a cell-free polypeptide syn- the translational level of protein synthesis thesizing system. The results from this and is mediated through the activity of a study clearly show that the S,,, and pH 5 cytoplasmic factor inhibitory towards pro- supernatant fractions prepared from tail tein synthesis. To substantiate the presence muscle of T,-treated tadpoles are less effl-

TABLE 3 EFFECT OF HEAT ORTRYPSIN TREATMENT ON THE PROTEIN SYNTHESIS INHIBITORY ACTIVITY OF MUSCLE pH 5

SUPERNATANT FRACTIONS FROM ~~~~~~~~~~ TADPOLES

pH 5 supematant Heat-treated trypsinized or from control untreated pH 5 supematant

tadpoles from T,-treated tadpoles* Incorporation (/a) w (dpmlmg RNA)

200 Untreated 23,000 k 466 100 100 18,000 f 1077 100 Untreated 9,614 ” 538 - 100 5,600 k 830 - Heat: 100 0 100 Heat: 100 8,953 f 169 - Trypsin; 100 0 100 Trypsin; 100 9,384 2 154

n Conditions of the incubation were the same as described in Fig. 2 except that pH 5 supematants from only 3-day T,-treated tadpoles were used.

* Heat treatment-pa 5 supematant (2 mg) was heated at 70” for 15 min. Denatured protein was removed by brief centrifugation. Trypsin treatment-pH 5 supematant (2 mg) was treated with 50 pg trypsin at 37” for 15 min, the reaction mixture was diluted and dialyzed overnight against a buffer containing 50 mM Tris-HCl (pH 7.61, 80 mM KCl, 6 mM MgCl*, and 2 mM DDT.


cient in carrying out peptide synthesis than similar fractions from control animals. Moreover, inhibitory activity is detectable in tadpole tail muscle pH 5 supernatants as early as 1 day after T,-treatment of the tadpole (see Saleem and Atkinson (1978) for in vivo comparisons).

enzyme activity. These results suggest that the decrease in the rate of protein synthesis in this cell-free system is not due to a generalized increased degradation of mRNA or protein.

Since the cell-free polypeptide system used for this study was programmed by poly(U) and relied on rat liver ribosomal subunits as the sole source of ribosomes, it seems unlikely that the regulatory factor di- rectly affects initiation factors or is specific for ribosomal subunits from a homologous source. Hormone-induced changes in synthetase activity or in the endogenous amount of free or charged tRNA are also not involved in the depressed synthetic activity reported here, since the pH 5 supernatant used in these studies is devoid of synthetase activity and ribonucleoprotein material (Hoagland et al., 1956). The cell- free methods used in this study provided both a comparative assessment of the protein synthetic regulatory properties of various fractions from tail muscle of control and T,-treated tadpoles and an indication of the sites in the translational machinery which are not affected by this particular regulator.

Chartreusin and fusidic acid were utilized in this cell-free system to assess, in an indi- rect way, the possible depletion or inactiv- ity of translational elongation factors EF-1 and EF-2. The similar degree of sensitivity detected in the pH 5 supematants from tail muscle of control and T,-treated animals to each of these antibiotics suggests, but does not prove, that the depressed synthetic activity of the pH 5 supernatant from T3- treated tadpoles is not due to limited amounts or decreased activity of either one or both elongation factors. These results coupled with pH 5 supernatant mixing experiments demonstrate that an inhibitor, rather than a translational factor deficiency, is responsible for the depressed synthetic activity of the pH 5 supematants from tail muscle of T,-treated tadpoles.

Although studies on whole tail homoge- nates demonstrate little or no detectable in- crease in alkaline ribonuclease (Weber, 1977) or proteolytic enzyme activity (Little et al., 1973) in the early phases of tail involution, enhanced proteolytic enzyme activity in later phases of tail regression is well documented (see reviews by Frieden and Just, 1970; Weber, 1969). We therefore explored the possibility that differential cytoplasmic localization of ribonuclease or proteolytic enzyme activity may vary in situ and result in different levels of activity in the pH 5 supematants from tail muscle of control and T,-treated tadpoles. This study establishes that the pH 5 supematants from T,-treated tadpoles are devoid of any ab- normal alkaline ribonuclease or proteolytic itors, in that it is induced by a hormone.

In recent years naturally occurring protein synthesis inhibitors have been described in brine shrimp embryos (Huang and Warner, 1974; Warner et al., 1977), pea seedlings (Tome et al., 1972), wheat germ (Steward et al., 1977), rabbit reticulocytes and rat liver (Rabinovitz et al., 1969; Hunt et al., 1972; Beuzard et al., 1973; Ranu and London, 1976; Delaunay et al., 1977), and dystrophic mouse muscle (Petryshyn and Nicholls, 1976). Most of the inhibitors mentioned above resemble the inhibitor demonstrated in this study in that they are components of the postribosomal fraction and are heat labile and trypsin sensitive. Although it is of some interest that the protein synthesis inhibitor detected in muscle from dystrophic mice is also only present in muscle tissue undergoing degradation (Pet- ryshyn and Nicholls, 1976), the inhibitor reported in this study is apparently unique and distinct from the dystrophic mouse muscle inhibitor, and other reported inhib-


ACKNOWLEDGMENTS This research was supported by grants from the

Muscular Dystrophy Association of Canada, National Research Council of Canada, and the Richard Ivey Foundation. We are indebted to Dr. D. M. Nicholls of the Department of Biology, York University, Downsview, Ontario, for providing facilities in which some of this work was completed.

REFERENCES Atkinson, B. G. (1971). Patterns of macromolecular

biosynthesis during amphibian metamorphosis. In “Proceedings of 7th Conference on Endocrinol- ogy and Metabolism” (P. B. Breitenbach and A. D. Kenny, eds.), pp. 48-82. Univ. of Missouri Press, Columbia, MO.

Beuzard, Y., Rodvien, R., and London, I. M. (1973). Effect of hemin on the synthesis of hemoglobin and other proteins in mammalian cells. Proc. Nat. Acad. Sci. USA 70, 1022-1026.

Cohen, P. P., Brucker, R. T., and Morris, S. M. (1978). Cellular and molecular aspects of thyroid hormone action during amphibian metamorphosis, In “Hormonal Proteins and Peptides” (C. H. Li, ed.), Vol. 6, pp. 273-381. Academic Press, New York.

Delaunay, J., Ranu, R. S., Levin, D. H., Ernst, V., and London, I. M. (1977). Characterization of a rat liver factor that inhibits initiation of protein synthesis in rabbit reticulocyte lysates. Proc. Nat. Acad. Sci. USA 74, 2264-2268.

Fox, H. (1975). Aspects of tail muscle ultrastructure and its degeneration in Rana remporariu. J. Em- bryo/. Exp. Morphol. 34, 191-207.

Frieden, E., and Just, J. (1970). Hormonal responses in amphibian metamorphosis. In “Biochemical Action of Hormones” (G. Litwack, ed.), Vol. 1, pp. l-52. Academic Press, New York.

Girgis, G. R., and Nicholls, D. M. (1972). Protein synthesis limited by transferase I. Biochim. Biophys. Acta 269, 465-476.

Girgis, G. R., and Nicholls, D. M. (1973). Hypo- physectomy and growth hormone alter the aminoacyl-tRNA binding activity of renal cortical supematant. Endocrinology 93,436-444.

Gregg, R. E., and Heintz, R. L. (1972). The inhibition of eukaryotic aminoacyl transferase I by chartreusin. Arch. Biochem. Biophys. 152, 451-456.

Hoagland, M. B., Keller, E. B., and Zamecnik, P. C. (1956). Enzymatic carboxyl activation of amino acids. J. Biol. Chem. 218, 345-358.

Huang, F. L., and Warner, A. H. (1974). Control of protein synthesis in brine shrimp embryo by re- pression of ribosomal activity. Arch. Biochem. Biophys. 163, 716-727.

Hunt, T., Vanderhoff, G., and London, I. M. (1972).

Control of globin synthesis: The role of heme. J. Mol. Biol. 66, 471-481.

Kaltenbach, J. C. (1971). Histochemical patterns in the tail of the metamorphosing tadpole. In “Hor- mones in Development” (M. Homburgh and E. J. W. Barrington, eds.) pp. 281-297. Ap- pleton-century-crofts, New York.

Kistler, A., Toshizato, K., and Frieden, E. (1975). Changes in amino acid uptake and protein synthesis of bullfrog tadpole liver and tail tissue during triiodothyronine-induced metamorphosis. De- velop. Biol. 46, 151-159.

Little, G. H., Atkinson, B. G., and Frieden, E. (1973). Changes in the rates of protein synthesis and degradation in the tail of Rana catesbeiuna tadpole during normal metamorphosis. Develop. Biol. 30, 366-373.

Lowry, 0. H., Rosebrough, N. T., Farr, A. L., and Randall, R. T. (1951). Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275.

Malkin, M., and Lipman, F. (1969). Fusidic acid: In- hibition of factor T2 in reticulocyte protein synthesis. Science 164, 71-72.

Merrifield, P. A., and Atkinson, B. G. (1977). Differ- ential rates of synthesis of muscle proteins in tail muscle of T,-treated tadpoles. J. Cell Biol. 75, 35la.

Nicholls, D. M. (1973). Increased translation in liver involves a supematant factor following laser irradiation. Biochim. Biophys. Acta 308, 168-179.

Nicholls, D. M., Carey, T., and Sendecki, W. (1977). Growth of liver accompanied by an increased binding of aminocyl-transfer ribonucleic acids. Biochem. J. 166,462-471.

Nicholls, D. M., Petryshyn, R., and Warner, L. (1974). Laser irradiation induces increased activity of liver elongation factor I. Radial. Res. 60, 98- 107.

Petryshyn, R., and Nicholls, D. M. (1976). Protein synthesis in the heart of genetic dystrophic mice. Biochem. Med. 18, 139-151.

Rabinovitz, M., Freedman, M. L., Fisher, J. M., and Maxwell, C. R. (1969). Tanslational control of hemoglobin synthesis. Cold Spring Harbor Symp. Quanr. Biol. 34, 567-578.

Ranu, R. S., and London, I. M. (1976). Regulation of protein synthesis in rabbit reticulocyte lysates: Purification and initial characterization of the cy- clic 3’:5’-AMP independent protein kinase of the hemi-regulated translational inhibitor. Proc. Nat.

Acad. Sci. USA 73, 4349-4353.

Ryffel, G., and Weber, R. (1973). Changes in the patterns of RNA synthesis in different tissues of Xenopus larvae during induced metamorphosis. Exp. Cell Res. 77, 79-88.

Saleem, M. (1977). “Thyroid Hormone-Induced Reg-


ulation of Translational Efficiency.” Ph.D. The- sis, University of Western Ontario, London, On- tario, Canada.

Saleem, M., and Atkinson, B. G. (1978). Thyroid hormone-induced regulation of polyribosomal translational efftciency in tadpole tail muscle. J. Biol. Chem. 253, 1378-1384.

Shaffer, B. M. (1963). The isolatedxenopccs luevis tail: A preparation for studying the central nervous system and metamorphosis in culture. J. Embryo/. Exp. Morphol. 11, 77-90.

Stewart, T. S., Hruby, 0. E., Sharma, 0. K., and Roberts, W. K. (1977). An ATP-dependent inhibition of protein synthesis in ascites cell extracts by wheat germ protein. Biochim. Biophys. Acra 479, 31-38.

Tata, J. R. (1966). Requirement for RNA and protein synthesis for induced regression of the tadpole tail in organ culture. Develop. Biol. 13, 77-94.

Taylor, A. C., and Kollros, J. S. (1946). Stages in the normal development of Rana pipiens larvae. Anal. Rec. 94, 7-24.

Tome, F., Felacetti, K., and Cammarano, P. (1972). Inhibition of polyphenylalanine synthesis by the

supernatant fractions from pea seedlings at various developmental stages. Biochim. Biophys. Actn 277, 198-210.

Tonoue, T., and Frieden, E. (1970). The effect of triiodothyronine on leucine incorporation into tail and other tadpole tissues. J. Biol. Chem. 245, 2359-2362.

Warner, A. H., Shridhou, U., and Finamore, F. J. (1977). Isolation and partial characterization of a protein synthesis inhibitor from brine shrimp embryos. Canad. J. Biochem. 55, 965-974.

Weber, R. (1969). Tissue involution and lysosomal en- zymes during anuran metamorphosis. In “Lyso- somes in Biology and Pathology” (J. T. Dingle and H. B. Fell, eds.), Vol. 2, pp. 437-461. North-Holland, Amsterdam.

Weber, R. (1977). Biochemical characteristics of tail atrophy during anuran metamorphosis. Coll. Int. Centre Rech. Nat. Sci. 266, 137-146.

Wilson, S. H., and Hoagland, M. B. (1965). Studies on the physiology of rat liver polyribosomes: Quan- titative and intracellular distribution of ribosomes. Proc. Nat. Acad. Sci. USA 54, 600-607.

thyroid hormone-induced regulation of protein synthesis in tadpole tail muscle

Documents