186 BIOCHIMICA ET BIOPHYSICA ACrA

BBA 95455

THE KINETICS OF PROTEIN SYNTHESIS IN EARLY AMPHIBIAN

DEVELOPMENT

R. E. E C K E R AND L. D E N N I S S M I T H

Division of Biological and Medical Research, Argonne National Laboratory, Argonne, Ill. (U.S.A.)

(Rece ived J a n u a r y i7 th , 1966)

SUMMARY

The kinetics of incorporation of tritiated leucine were used to study protein ssmthesis in activated eggs and early embryos of Rana pipiens. The results support the conclusion that yolk proteins are degraded to provide steady-state amino acid pools intermediate in the synthesis of cytoplasmic proteins de novo. The kinetic data can be used to calculate the rate of protein synthesis.

INTRODUCTION

In an earlier report 1 we demonstrated significant incorporation of injected tritiated leucine into the proteins of Rana pipiens embryos during the early cleavage stages. This has suggested that protein synthesis de novo plays some part in the events of early embryogenesis. However, since these earlier experiments demonstrated only total levels of activity after 6 h of incorporation, they gave no information relative to the kinetics of protein synthesis in this period.

The present investigation is a study of amino acid pool kinetics during early development in R. pipiens. The kinetic data are used to draw conclusions concerning the mechanism and rate of protein synthesis.

MATERIALS AND METHODS

Measurement of protein synthesis in eggs or embryos of the leopard frog, R. pipiens, was accomplished by microinjection of tritiated D,L-leucine (I.O mC/ml; 4.0 C/mmole) into the eggs, followed by assay of the activity found in saline-soluble proteins. The details of these methods have been described previously 1. In the present study three-egg samples, rather than ten-egg samples, were used to allov¢ homogenization at early times after injection for kinetic analysis.

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PROTEIN SYNTHESIS IN AMPHIBIANS I87

In addition to measurements of incorporation into saline-soluble proteins, kinetics of loss of act ivi ty from the leucine pool were also determined. Injected eggs were homogenized in 2 ml of 5 % trichloroacetic acid and the trichloroacetic acid- insoluble material was removed by centrifugation. The supernatant liquid was extracted three times with 5 vol. of ether, transferred to scintillation vials, evaporated overnight at 55 ° and counted as previously described 1.

To determine the size of the leucine pool at various stages, 300 eggs were ho- mogenized in a dialysis tube for each determination. The homogenate was subjected to pressure filtration at one atmosphere of nitrogen. The residue was washed twice with a small volume of normal saline and all of the filtrates were combined. The final volume was about 1.5 ml. Leucine content in the filtrate was determined by amino acid analysis, kindly performed for us by Dr. A. B. EDMUNDSON.

RESULTS

When eggs of R. pipiens are activated by the injection of tr i t iated leucine, incorporation of the isotope into saline-soluble proteins begins immediately. Fig. I shows the incorporation kinetics observed, and also those obtained with eggs injected 13o and 260 min after activation with a glass needle. In all cases the relationships are essentially the same, that is, incorporation is initially very rapid but soon decays to a constant maximum level.

150C

c 5CE

iio . / 7 . " .

~ o 2o0 3oo ~ o 5& Time a f te r activation (rain)

4 0 0 0

3occ

c

8

6 0 0 8 ~o ~cb 1go 280 go ~o s~o a l o Time after activation (ram)

Fig. i. Kinetics of incorporation of tri t iated leucine into saline-soluble proteins of a~tivated eggs. • - • , activated by injection of the isotope; O - O , activated by needle and injected with isotope i3o min later; A - A , activated by needle and injected with isotope 26o min later. Volume in- jected was 18 m/~l.

Fig. 2. Kinetics of loss of tr i t iated leucine from the cold acid-soluble pool after activation by injection of the isotope. Volume injected was 8.2 m/~l.

The kinetics of incorporation, indicated as loss of act ivi ty from the leucine pool, are shown in Fig. 2. This curve is essentially a mirror image of those in Fig. I . I t appears from this graph that not all of the available leucine is incorporated by the eggs. However, the act ivi ty tha t remains unincorporated is not utilized by an L-leucine-requiring strain of Escherichia coli, while about half of the act ivi ty from the original isotope solution is incorporated by the same strain. In addition, recent

Biochim. Biophys. Acta, I29 (1966) i86-I92

1 8 8 R. E. E C K E R , L. D. S M I T H

experiments with tr i t iated L-leucine show that all of the counts are taken up by the act ivated eggs, with the same kinetics as shown in Fig. 2. Apparent ly the eggs will not utilize the D-isomer from a racemic mixture.

Thus, the very rapid at ta inment of a maximum level of incorporation shown in Fig. I is due to a rapid exhaustion of the isotope from the" leucine pool. This conclusion is further supported by the data in Fig. 3 and in Table I. In the experiment shown in Fig. 3 eggs were injected either once, twice, or three times, with 9o-min intervals between injections. With each subsequent injection, total incorporation increased to about the same extent and with about the same kinetics.

T A B L E I

EFFECT ON TRITIATED LEUCINE INCORPORATION OF PRIOR OR SUBSEQUENT EXPOSURE TO UNLABELED LEUCINE

Al l i n j e c t i o n s w e r e m a d e w i t h a p i p e t t e c a l i b r a t e d t o d e l i v e r 18 m/~l o f s o l u t i o n . C o n c e n t r a t i o n of t h e u n l a b e l e d l e u c i n e w a s 2 m g / m l ; p H w a s 7.o.

Activated by Time alter activation (min) Total time o/ Counts/rain injection o/: incorporation incorporated

Tritiated leucine Unlabeled leucine (rain) per 3 eggs* infected injected

U n l a b e l e d l e u c i n e 2o - - 3 ° 67 U n l a b e l e d l e u c i n e 2o - - 9 ° 246 STEINBERG'S s o l u t i o n 18 - - 3 ° 461 STEINBERG'S s o l u t i o n 18 - - 9 ° 746 T r i t i a t e d l e u c i n e - - 9o 12o 711 T r i t i a t e d l e u c i n e - - 90 i 8 o 714 U n l a b e l e d l e u c i n e i o o - - 3 ° 147 U n l a b e l e d l e u c i n e i o o - - 9 ° 6Ol T r i t i a t e d l e u c i n e - - - - 3 ° 742 T r i t i a t e d l e u c i n e - - - - 9 ° 834

" A v e r a g e o f t w o g r o u p s i n j e c t e d .

Table I shows what happens to the kinetics of incorporation when the size of the leucine pool is increased by pre-injection of an excess of the unlabeled amino acid. A 4-fold increase in the size of the leucine pool markedly diminishes the rate of incorporation of the tr i t iated leucine injected immediately thereafter. An equal volume of STEINBERG'S solution s injected into the eggs just prior to the tri t iated leucine has little or no effect on incorporation. If Ioo min is allowed between the time the excess unlabeled leucine is injected and the time of injection of the labeled leucine, incorporation rate is increased, but still is significantly less than the control. On the other hand, when the tri t iated leucine is injected first and allowed to incorporate for 9 ° rain before the size of the leucine pool is increased, there was no change in the amount of incorporated activity. That is, once the label has been incorporated, it cannot be "chased" out by the unlabeled leucine.

The very early kinetics of protein synthesis cannot be determined in fertilized eggs, since one cannot practicably begin injections until about 20-30 min after fertilization. However, at later times, it appears that the kinetics in fertilized eggs and in act ivated eggs are the same. Fig. 4 shows the kinetics of incorporation in 4-cell embryos. This experiment was done on the sam~ day and with the same batch of eggs as the experiments shown in Fig. I. The kinetic,~ of incorporation by activated

Biochim. Biophys. Acta, 129 (1966) 186 -192

PROTEIN SYNTHESIS IN AMPHIBIANS 189

~, ~ ,5 9

~ i ~ r ~ t ° '-

o so

o

,oo ,~ l ~o ~ ~ 3~o 4oo Time after ad.ivation (rnin)

1500

--c 50( / 26o !'~o 460 5~o 660

Time after fet~.itizaticm ( rain )

Fig. 3- Kinetics of incorporation into eggs after repeated successive injections. O - O , injected once; A - A , injected twice; O-C), injected three times. Volume injected was 14 m/~l.

Fig. 4. Kinetics of incorporation into 4-cell embryos. Volume injected was 18 m/zl.

eggs, injected 26o min after activation, and of 4-cell embryos, injected 25o min after fertilization, are almost identical.

Estimates of quenching by the protein-hyamine system 1 indicate that perhaps as little as 20 % of the incorporated leucine is extracted in saline. The remainder of the activity is presumably incorporated into saline-insoluble proteins and discarded in the preparative procedure used. Efforts to recover these counts have been un- successful. Although the majority of the protein of the egg can be rendered soluble in aqueous buffers, we have been unable to increase either the total activity recovered or the specific activity over that obtained with the saline extraction.

DISCUSSION

I t has been suggested that incorporation studies of the kind presented here may not be a true indication of net protein synthesis 3,4. However, several fines of evidence support the assumption that there is net incorporation of the injected leucine into newly synthesized proteins. The possibility of protein turnover is excluded by the data in Table I. These data show that the isotope, once incorporated, cannot be "chased" out by adding to the pool large excesses of unlabeled leucine. In addition, Table II shows that puromycin, a drug known to inhibit a specific step iu protein biosynthesis 5, inhibits leucine incorporation in direct proportion to the amount of drug injected. Thus, it appears reasonable to conclude that leucine incorporation, as determined in this system, is a valid measure of the conventional synthesis of new proteins.

I t has been proposede, ~ that, in the amphibian, cytoplasmic proteins are synthesized from amino acid and peptide pools derived from hydrolysis of yolk proteins. We shall make the further assumption, as suggested by DEUCHAR 7, that the primary products of yolk degradation are amino acids rather than peptides; that is, the activated egg and the developing embryo contain a steady-state amino acid pool, intermediate in the process leading from yolk degradation to the synthesis of cytoplasmic proteins.

yolk protein > amino acid pools > new protein

Biochim. Biophys. Acta, 129 (1966) I86-I92

1 9 0 R . E . E C K E R , L. D. SMITH

T A B L E I I

EFFECT OF PRE-INJECTED POROMYCIN ON INCORPORATION OF TRITIATED LEUCINE

E g g s w e r e a c t i v a t e d b y i n j e c t i o n o f t h e p u r o m y c i n a n d , a f t e r t h e p e r i o d o f e x p o s u r e i n d i c a t e d , w e r e i n j e c t e d w i t h t r i t i a t e d l e u c i n e . V o l u m e of t h e p u r o m y c i n s o l u t i o n s i n j e c t e d w a s i i m / , l . 7 .1 m/~l o f t h e t r i t i a t e d l e u c i n e w a s u s e d . E g g s w e r e i n j e c t e d a n d h o m o g e n i z e d i n g r o u p s o f i o . W h e r e m o r e t h a n i o e g g s w e r e i n j e c t e d , r e s u l t s s h o w n a r e a v e r a g e s o f t h e s e v e r a l g r o u p s . C o n t r o l s w e r e a c t i v a t e d c o n v e n t i o n a l l y a n d i n j e c t e d w i t h t h e i s o t o p e a t t h e s a m e t i m e a f t e r a c t i v a t i o n a s t h e e x p e r i m e n t a l g r o u p s , a n d a l l o w e d t h e s a m e t i m e f o r i n c o r p o r a t i o n .

Female Dose Time o/ Time o/ Total number Incorporation ~ug per egg) pre-exposure incorporation o[ eggs (% control)

to drug (min) (min) iniected

II

o . o 2 2 9 o 2 1 o 2o 58.1 o . o 5 5 9 0 2 1 o 20 35 .2 o . o 5 5 12o 12o 3 ° 32 .8 o . i i o 9 ° 2 1 o i o 24 .6 o . i i o 12o 12o i o 17. 4 0 . 2 2 0 12o 12o 20 8. 5

o.o22 12o 12o i o 9 3 . 6 o . i i o 12o 12o 2 0 29 .6 o . 2 2 o 12o 12o 2 o 13. 5

If this assumption is valid, a number of experimental data could be predicted. For example, a small amount of isotopically labeled amino acid, injected into the amino acid pool, would be expected to dilute out as protein synthesis occurs, as shown in Fig. 2. Furthermore, it should dilute out with essentially the same kinetics irrespective of the time of injection, as shown in Fig. I. One would also expect that a large increase in the size of the leucine pool would have a marked effect on the kinetics of dilution, as shown in Table I.

Using this model, it should be possible to predict the exact kinetics of in- corporation of the injected leucine. To do this, several subordinate assumptions must be made. First, it must be assumed that the size of the leucine pool does not normally change over the period of time under study. Amino acid analysis of the leucine pool shows this to be a valid assumption, for unactivated eggs, 2-cell embryos and i6-ceU embryos all have the same size leucine pool, that is, about 0.12 #g per individual.

The second subordinate assumption is that the rate of dilution of the tritiated leucine in the pool, - - d L / d t , is equal to the rate of flow of leucine through the pool, [, times the proportion of the total leucine pool, V, that is labeled, L. That is,

--dL/dt =/L/V. (1)

This assumption also implies that the amount of leucine injected as labeled amino acid is an insignificant fraction of the total pool. This can be shown to be a fact. The largest volume injected (18 m/,1) increased the size of the pool by less than 3 %.

The third assumption is that the amount of tritiated leucine in the pool is equal to the amount injected, L 0, less the amount incorporated. That is,

L = L o - P*/k, (2)

where P* is the amount of the isotope incorporated into the protein measured and k is the proportion of the injected leucine detectable in the protein measured. Then,

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P R O T E I N S Y N T H E S I S I N A M P H I B I A N S I9I

integrating Eqn. I and substituting the result into Eqn. 2, the following kinet~ic equation is obtained:

P* ~ kLo(I--e-~/v) '(3)

A graph of this equation, with P* plotted as a function of t, shows a rapid increase in P* at early times with a slow approach to a maximum value at later times, similar to the graphs in Fig. I. The initial slope in this relationship is KLo]/V. kL o represents the maximum level of incorporation attainable and can be estimated from the value of P* when t is relatively large. Thus, the value of ]/V can be determined as the quotient of the initial slope and the maximum value of P*. Since the value of V is known, the rate of flow,/, of leucine through the pool can be calculated.

By this method, the value of the parameter / has been determined for each of the experiments represented in Figs. I, 2 and 4. These results are shown in Table III. The average value of / from these calculations is 2.0-IO -4/~g/min. Assuming a value

T A B L E I I I

CALCULATIONS OF THE CONSTANT, f , FROM THE DATA IN FIGS. I , 2 AND 4

Type of eggs Time injected Value of the constants in Eqn. 3 used (rain after

activation or kLo f /V (min -t) /~ug/min × IO -~) fertilization) (counts/min)

A c t i v a t e d * o 53 ° o . o i 2 i .4 A c t i v a t e d " 13 o 62 o o . o 17 2. o A c t i v a t e d " 2 6 o i o o o o . o 2 1 2 . 5 A c t i v a t e d " o 2 1 o o O.Ol 7 2 . 0 F e r t i l i z e d * * " 2 5 0 1 1 9 o o . o 1 9 2. 3

* D a t a f r o m F i g . i . "* D a t a f r o m F i g . 2.

* ' * D a t a f r o m F i g . 4-

of IO ~ for the leucine content of the proteins made by the egg during this period, the rate of protein synthesis during the early cleavage stages of R. pipiens devel- opment is calculated to be 2. IO -a/~g per individual per min. I t should be emphasized that this is the total rate of protein synthesis, because the kinetics of leucine in- corporation are used to determine the total rate of flow of leucine through the amino acid pool, and not the rate of incorporation of the amino acid into one specific class of proteins.

The parameter k in Eqn. 3 represents the proportion of the injected isotope that is detected in the saline-soluble proteins and would therefore reflect any changes in this proportion. The curves in Fig. I show that the maximum attainable level of incorporation, kLo, was higher in the eggs injected at later times after activation. A similar pattern is observed in Fig. 3. Since, in a given experiment, each egg was injected with the same amount of tritiated leucine, L0, the increase in maximum incorporation must be due to an increase in the value of k. This would suggest either that a greater proportion of the total protein, synthesized at the later stages studied,

Biochim. Biophys. Acta, I 2 9 ( I 9 6 6 ) I 8 6 - I 9 2

192 R . E . ECKER, L. D. SMITH

is saline-soluble or that the leucine content of newly synthesized saline-soluble proteins is greater at later times. Either suggestion implies a change in the nature of protein synthesis over the period of time studied.

Although we have presented a simplified model in which amino acids are the important intermediates in the conversion of yolk to cytoplasmic proteins, the experimental data do not exclude the possibility that peptides may be involved in this intermediate role; nor do they eliminate the possibility that some cytoplasmic proteins may appear directly from storage in the yolk. However, it is clear that a significant amount of conventional synthesis of protein occurs and that the rate of this synthesis is essentially constant over at least the first 6 h of development.

ACKNOWLEDGEMENTS

This work was supported by the U. S. Atomic Energy Commission. We grate- fully acknowledge the technical assistance of Miss JOAN STACHURA.

R E F E R E N C E S

i L. D. SMITH AND R. E. ECKER, Science, 15o (1965) 777. 2 M. STEINBERG, Carnegie Inst. Wash. Yearbook, 56 (1957) 347. 3 J- BRACI-IET, The Biochemistry o/ Development, Pergamon, New York, I96O, p. 59. 4 E. M. DEUCI-IAR, Biol. Rev., 37 (1962) 378. 5 D. •ATHANS, Federation Proc., 23 (1964) 984. 6 R. A. FLICKINGER, Syrup. Germ Cells and Development, Inst. Intern. d'Embryol, et Fond., Fon-

dazione A. Baselli, Milan, 1961, p. 29. 7 E. M. DEUCHAR, in R. WEBER, The Biochemistry o/ Animal Develcpment, Vol. i, Academic

Press, New York, 1965, p. 271.

Biochim. Biophys. Acta, 129 (1966) 186-192