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Rates of protein synthesis—a review
Journal of Neuroscience Methods, 27 (1989) 91-101 Elsevier
91
NSM 00889
Research Papers
Rates of protein synthesis
a review
F.M. S h a h b a z i a n 1, M y r o n S. J a c o b s 2 a n d Abel L a j t h a 3 I Department of Basic Sciences, Cleveland Chiropractic College, Los Angeles, CA 90004-2196 (U.S.A.), 2 Department of Pathobiology and Oral Pathology, New York University Dental School, New York, N Y 10010 (U. S. A.) and 3 Center for Neurochernistry, The Nathan S. Kline Institute for Psychiatric Research, New York, N Y 10035 (U.S.A.) (Received 3 June 1988) (Revised 19 July 1988) (Accepted 19 July 1988)
Key words: Rate of protein synthesis; Rate of protein degradation; Protein synthesis; Protein degradation; Protein turnover; Incorporation of amino acids into protein; Radioisotope administration; Pulse labeling; Infusion; Perfusion; Pellet implantation The rates of protein synthesis can be measured by a variety of methods including pulse labeling, massive precursor administration, Scornik method, continuous feeding of labeled precursor, infusion, and pellet implantation. Each technique has some advantages and disadvantages. Massive precursor administration and infusion are the most widely used. The advantage of massive precursor administration is its simplicity, however, the amino acid concentration used is much higher than physiological levels. Infusion, however, is much more complicated as a technique and requires complicated calculations. The synthesis rates can also be calculated from degradation curves. Some of the above techniques can be used both in vivo and in vitro, and also for different organs (Shahbazian et al. (1987), Int. J. Dev. Neurosci., 5: 39-42). The brain has rapid rates of protein synthesis both in vivo and in vitro, the latter being much lower for adults.
Introduction The structural elements of an organism undergo metabolic turnover when the organism is in a steady state, that is, when it is not experiencing growth or degeneration. The organismal components include proteins, which are synthesized continuously for replenishment or for specific purposes. Protein synthesis occurs at different rates; although protein synthesis to a great extent involves translation from nucleic acids to amino acids, the rate of synthesis is also affected by the
Correspondence: F.M. Shahbazian, Department of Basic Sciences, Cleveland Chiropractic College, 590 North Vermont Avenue, Los Angeles, CA 90004-2196, U.S.A.
process of transcription of RNA (mRNA, tRNA and rRNA), which in turn depends on replication. Some of the proteins synthesized are enzymes or metabolic regulators, whose presence will affect the rate of protein turnover. Finally, the proteins will break down into amino acids, some of which will enter the process of synthesis, and the rest of which will be degraded to end-products (Fig. 1). A stochastic approach makes it possible to determine the magnitude of certain variables without taking into account the structure or arrangement of the system studied. In determining rates of protein synthesis, especially of total proteins, with this approach it is not necessary to know the nature of the proteins studied, how many proteins are present, or what arrangement they have within the system.
0165-0270/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
92
c
~'n"¢
Translation A
~ RNA / tRNA I_ rRNA
FmRNA1
> Metabolicreactions and their regulation
Proteins
I
, Degradation Amino acids I Breakdown
End products Fig. 1. The processes relating to protein metabolism.
Questions might arise about the usefulness of knowing the rate of synthesis of total proteins in a system. The study of synthesis rates of single proteins or of groups of proteins provides different kinds of information; in some instances, the study of total protein synthesis in health or disease is apt to provide more information about the general mechanism of metabolism involved. During the learning process there is a transitory rise in brain protein synthesis possibly affecting all of the proteins of the system involved. Many drugs, chemicals and poisons affect the total protein machinery. In phenylketonuria, many proteins may be altered, and temperature variation, ischemia, and hypoxia can lower the total protein synthesis (Lajtha and Dunlop, 1981). Protein biosynthesis is thought to be greatly affected by a variety of developmental, environmental, and physiological stimuli (Ilan and Singer, 1975). For example, a hormone such as insulin can affect the cells of the liver and other organs, causing physiological changes that in turn can increase the rate of protein synthesis. This stimulation of protein biosynthesis is considered to be one of the factors in cellular response. Ever since the radioisotope method was applied to the study of metabolism, the stochastic approach has been used to determine rates of protein synthesis. In these procedures, a radioisotope precursor such as a labeled amino acid is injected into the animal. The labeled amino acid then passes through a sequence of compartments of the
body (plasma, extracellular environment, intracellular compartment) before being synthesized into protein.
Amino Acids (precursor pools) During the course of its transport, the labeled amino acid becomes associated with a number of specific pools of free amino acids, and also with bound amino acids and proteins (Lajtha ad Dunlop, 1981). The rate at which protein synthesis occurs (as explained in detail below) is often determined by amino acid uptake, as measured by specific radioactivity (SRA; Dunlop et al., 1975b). The concentration and SRA of amino acids in the plasma and extracetlular fluid are generally similar (Dunlop et al., 1977). Since the SRA of amino acids in this pool is higher than that of intracellutar free amino acids, the rates of protein synthesis based on the SRA of free amino acids in the extracellular fluid (k = SRA of protein/SRA of free AA) are lower than those based on intracellular SRA. The intracellular pool of amino, acids consists of precursor amino acids (aminoacyl-tRNA) with higher SRA, and lysosomal amino acids with lower SRA. Since the intracellular amino acid pool measured represents the total of both ribosomal- and lysosomal-associated amino acids, its SRA is diluted, and the rates of protein synthesis calculated would thus be too high.
93
Rates of Protein Synthesis
low et al. (1978a), be able both to describe in simplified form the physiological processes involved, and to provide the chemical framework for quantifying the components of the system. The rules for our model are: (1) The pools of metabolites and end products are in a steady state; i.e. they do not vary in size as they would under conditions of starvation or growth, unless otherwise stated. (2) Materials exchanged between the pools or introduced into them are assumed to mix completely and instantaneously with those already present. (3) A constant fraction of the material in each pool should be transferred or exchanged in unit time. (This is an exponential or first-order process.) A general method for determining rates of protein synthesis is to give a tracer as an single dose and measure SRA of the metabolite at different intervals of time (Waterlow et al., 1975b). If one is
A protein is synthesized through the action of a multitude of enzymes, nucleic acids, and structural moieties such as ribosomes. Several reactions take place simultaneously or consecutively. In considering the total complement of proteins being synthesized in an organ such as the brain, the large number of chemical transformations taking place can be summarized in the following single reaction, which will serve as an all-inclusive model: K~
Precursor amino acids ~ Protein products Kd
where K~ and K a are rate constants of synthesis and degradation, respectively, for that reaction, and they represent the total of the rate constants of hundreds of reactions. For such a complex system, involving a multitude of reactions, we need a well-defined model. Such a model should, as characterized by Water-
k s = synthesis
rate
V = synthesis
rate
constant
AP
Amino CA R E A C T ION :
acid
= total
M A = pool SRA
=
SA
pool
Protein
counts size
= CA/M A
pool
Cp
= total
Mp
= pool
size
= Sp
= Cp/Mp
SRA k d = degradation
counts
rate
C o n s tan t VpA
RATE :
dCp/dt
=
VApS A
-
= degradation
rate
VpAS p
Fig. 2. The reaction and the rate for a two-pool model of protein synthesis.
(z)
94 dealing with a single pool of either a metabolite or its end product, the rate would theoretically be expressed as
d S A / d t = -kS~, SA(t) = X e -kt, X = SA at zero time If, on the other hand, we are dealing with two pools, such as an amino acid metabolite and a protein end product, then the reaction and its rate would appear as in Fig. 2. Under steady-state conditions between the amino acid and protein pools, Mp is constant and K S = K d = k. When both sides of Eqn. 1 (Fig. 2) are divided by Mp, the rate is expressed as
For calculating the rates of protein synthesis, there are several procedures. These procedures can be divided into 4 categories: single isotope administration, multiple isotope administration, continuous isotope administration, and rates of protein degradation, each of which can be divided into several procedures. These are explained below.
Single isotope administration Pulse-labeling (Jones and Mcllwain, 1971).
In view of the fact that the SRA of the protein pool is very small compared to the amino acid pool, after a short time, we have
At zero time, a tracer dose of labeled amino acid is introduced into the intact brain (i.v. or i.p. injection) or into the medium of incubated brain slices. The amino acids present in body fluids and tissues initially reach a high level then decline to an almost constant value after about 30 min. In the case of i.v. injected animals, the level remains constant for only half an hour whereas in the i.p. injected animals, and in incubated slices, the level remains constant for an hour (Oja, 1967). Since turnover of protein in brain is slow, the peak of protein SRA for brain tissue would be low. The following expression can be used to calculate the rates of protein synthesis:
dSp/dt = kSA
d S p / d t = k( S a - Sp)
Under non-steady-state conditions, Mp is changing and Sp = Cp/Mp
If Sp and SA are measured at one point after zero time, the rate of change of SRA of protein with respect to time is approximated as Sp/time (Wannemacher, 1971). From this rate of change and SA, the rate constant ks (Sp/t)/SA can be calculated. This method of measurement, however, is not reliable and assumes that Sp vs time is a straight line. Another method of calculating rate constants of protein synthesis is by integration of
dSp/dt = ( VAp/Mp)SA -- ( VpA/Mp)S o d S p / d t = ksS A -
kdS p
Since K s = K a = k, then
d S p / d t = k ( S A - Sp)
dSp/dt = d ( C p / M p ) / d t = (1/Mp)(dC~,/dt ) - ( Cp/M2 ) ( d M p / d t )
(2)
Growth rate (for young) or degradation rate (for starving) is:
d M p / d t = YAp- VpA dCp/dt
= VApSA -
VpASp
(3) (1)
from before
dSp = k ( S A - Sp) dt
Substituting (3) and (1) into (2), we get
d S p / d t = ( VAp/M p )( SA -- Sp ) = ks( SA -- Sp ) Further, if 3 or more pools of metabolites a n d / o r end products are involved, the formula would be
S(t)=Xle-kat+X2e-k2,+...+X,e
=
kn,
The solution of an equation such as this, which involves three or more rate constants, generally requires the application of computer assistance.
which becomes Sp = SA(1 -- e -ks') SA, Sp, and the time can be measured, and the only unknown, ks, is then estimated by iteration. The result of this method of calculation, however, is not reliable because SA, which is assumed to be constant, actually changes with time. If, on the other hand, SA and Sp are measured at two different points after zero time, there are
95
two ways by which rate constants can be calculated. One way is to estimate the tangent to the curve of Sp vs time (Lajtha et al., 1957) between two points in time, and use the average of SA at those two points Ks-
Sp2 -- Spl
SA2 -~ SA1
12 -- t 1
2
Another way is to estimate the area under the curves described by SA vs t and Sp vs t (Haider and Traver, 1979):
[ area SA VS t
area Sp vs t
The area Sp vs time is negligible because of the small amount of the labeled protein involved. This is the most accurate of the above methods for measuring rate constants.
Massive precursor administration (Dunlop et al., 1974, 1975a, b). In the pulse-labeling approach it is assumed that the true SRA of the precursor is the same as that of the intracellular pool. By injecting a large dose of amino acid (500 mM) for in vivo experiments, by using a high concentration of amino acid (1 raM) for in vitro slice incubation, and by making SRA of precursor very high, the SRA of plasma or incubation medium and organ intracellular fluid (intact brain or brain slice) approach each other. (More concentrated solutions would cause inhibition of protein synthesis.) Other advantages of massive precursor use are that the plateau of constant SRA in the blood and organs would increase to 3 hours, and the speed of equilibration in reaching plateau would increase. Furthermore, all amino acid pools would be flooded uniformly. The calculations for determining the rate constants in this approach can be exactly like those for pulse labeling, There is, however, another way of approximating the rate constants. This consists of determining the change per hour in SRA of protein at any time following zero time and dividing it by SRA of injected precursor in in vivo experiments, or by dividing it by the SRA of the incubation medium in the case of in vitro brain slices.
Scornik method (Scornik, 1974). To do away with the time-course of precursors and their products, Scornik used constant SRA of varying concentrations of injected amino acids, killed the animals after 5 min, removed the liver, and determined the SRA of hepatic proteins. By plotting 1 / d o s e against 1/incorporation and assuming that it is a straight line, by extrapolating to infinite dose ( 1 / d o s e = 0 ) , the rate constants can be calculated as k = Incorporation at infinite dose SRA of injected amino acid The problem is that the line resulting from plotting 1 / d o s e vs 1/incorporation might not be straight.
Multiple isotope administration (Austin, 1972) Several equally spaced injections of isotope precursor are made into the animal. This method approximates the infusion approach (see Section 'Infusion', below).
Continuous isotope administration Continuous feeding of labeled precursor.
To keep SRA of free amino acid constant throughout the experiment, isotopically labeled material was fed to animals (Buchanan, 1961). Within 50 days the SRA of liver approached 95% of that of the food. The rate of protein synthesis, which is calculated by the curve approaching the SRA of food, is only an estimate because the SRA of precursors is only relatively constant. The food taken in is always diluted by material from the breakdown of tissue protein and the SRA of tissue is constantly increasing during the experiment. Also, since the uptake of radioactivity is measured over a long time-course, the turnover rates of mixed protein calculated from protein SRA curves become progressively lower with passing time. For a pure protein this problem is immaterial (Schimke, 1964). Assuming that SA remains constant and is equal to SRA of the precursor in food, the rate constant can be calculated by
S~= S~(1-e ~").
96
lnfusion (Garlick and Marshal, 1972). Labeled amino acid is introduced by venous infusion at a constant rate. Plasma amino acids and the brain free amino acids (70-80% plasma level) reach plateau levels and are maintained for several hours. By multiple sampling of the blood, one can determine when the plateau of constant precursor is reached. The SRA of free amino acid in the plasma (Sp) in rising to that plateau (Spmax) can be expressed as: Sp = Spmax(1 - e -Lpt) where Lp = empirically fitted rate constant (Waterlow and Stephen, 1967, 1968). The rate at which the SRA of tissue reaches the plateau level depends on two factors, pool size of labeled amino acid and rate of protein turnover in the tissue. Assuming that there is only one precursor pool, then the SRA of free amino acid pool in the brain would reach a constant value expressed in the following manner S B = Simax(1
-- e -ks, )
where S B is SRA of protein in brain and Simax is the intracellular SRA of precursor at plateau. The extent of error in evaluating rates of protein synthesis caused by the assumption that the precursor SRA is constant, S = Sims,, rather than having an exponential nature, S = Simax ( 1 e-Zt), can be illustrated as in Fig. 3. The shaded area in the figure indicates the error resulting by not considering the time dependency of precursor rising to a plateau. The error can be corrected in a number of ways: (1) The time course of Si may be determined experimentally. In this case one determines L empirically and uses S~ rather than Simax(2) The experiment can be designed to be long-term. In such a case, the shaded area would be negligible. (3) Animals could be killed at two different times after the plateau is reached. Thus, the following expression may be used to obtain a rate constant: ,A,SB//A l = ( Simax ) k
where S B / t is the rate of increase of SRA of brain proteins between two points in time.
S=SMAX SPECIFOC(s)~ ACTIVITY
4nt S=SMAX (1-e )
TIME Fig. 3. Illustration of the content of the error in determination of protein synthesis rate caused by assuming the precursor spec. act. to be constant when in fact it rises exponentially to a constant level. The amount of label incorporated into protein is approximately proportional to the area under the curve of precursor spec. act. Hence the error is determined by the relative area and the curve for the duration of the experiment. From Watedow et al. (1978b),
(4) A more accurate formula can be derived for the particular organ. In the case of the brain, it would be expressed S B / S ~= k , , / ( 1 - e -RK.¢') - ( l / R ) where S B = SRA of brain proteins Si = SRA of free amino acid in brain at plateau R = protein-bound amino acids free amino acids The value of S B and S i are calculated at 3 or more times during the plateau and their ratios are obtained. R is a constant value and the ratio S u / S i may be plotted against k s for a given value of t. Under the conditions, the value of k S for any experimental ratio of S B / S ~ at time t, can be read directly from the curve. Pellet implantation (Lajtha et al., 1976, 1979). An alternative to infusing amino acid precursor by means of a venous catheter (external infusion) is the implantation of a pellet of the precursor (internal infusion). The pellet should dissolve slowly and at a uniform rate. In this method it is assumed that the SRA of precursor in the brain is constant and that its incorporation into brain protein is a zero order reaction. It is further assumed that the growth rate of protein is the same for all of the different amino acid pools in the brain and that there are, at most, two major protein pools.
97
S
P -
P
S where
-kslt ]
(
1 -
e
P
A
(
1
-
e
P
Sp : SRA of
protein
Sa : S ~
free
of
-ks2t
P2 ) +
in brain
amino
acid
in b r a i n
P =~_Pi
i=l Pi =
Amount
of p r o t e i n
ksl = S y n t h e s i s
rate
in the ith pool
constant
of b r a i n
of the ith pool
of brain.
Fig. 4. A mathematical model for protein synthesis by pellet implantation (Lajtha et al., 1976, 1979).
In determining the rate of protein synthesis by the pellet implantation method, the equation (Fig. 4) may be derived. The two rate constants present in the above equation are illustrated in Fig. 5. From Fig. 5, it can be seen that there are two pools in the brain, which differ in the rates of formation from the precursor amino acid pool. The amount of protein, P2, is measured by extrapolation, whereas P represents the total protein in the homogenate. After all available values are substituted into the equation, the rate constants ks1 and ks2 are usually calculated with the aid of a computer.
Rates of protein degradation In an organism in steady state, the rates of protein synthesis and breakdown should be equal so that the organism would not be gaining or losing protein. So, to calculate the rates of protein synthesis, sometimes the rates of protein degradation are measured in adult animals.
%Unlabeled
100
Protein
~-
*
~
I Adult Mouse 80
I 0
I
i
I
10 TIME
I 20
i
p 30
(hours)
Fig. 5. Percent unlabeled protein calculated on semilog paper vs time from injection of [14C]tyrosine (30 mg/ml) subcutaneously. From Lajtha et al. (1979).
Protein degradation rates are measured in several ways. The main two techniques are singleisotope labeling and double-isotope labeling. Single-isotope technique. In this technique animals are injected with a radioactive precursor, and while the animals are taken care of for a long period such as a month, some animals are killed in the meantime, and SRA of protein is calculated. A plot is prepared from SRA vs time, and from the curve the rate constants are calculated. Since the time is long, the problem of recyclization is important. The extent to which recycling affects the rates of protein degradation depends on several factors, including the precursor used. The result of this recycling is to reduce the rate of decay and prolong its apparent half-life. The most commonly used precursors are [14C]carbonate and [14C]glucose. The former, which is used most often, would end up as the guanidino carbon of the arginine residue as well as the alpha-carboxyl group of glutamate, glycine, alanine, serine, and glutamine, and in both carboxyl groups of aspartate for liver tissue, and only in some of the above amino acids in other tissues examined. However, [14C]glucose would end up in more amino acid residues, causing more recyclization; it is also more costly to use. One of the experiments in which the turnover rate of tubulin (a microtubule protein) was measured is the work by Forgue and Dahl (1978). In
98
this p r o c e d u r e , rats were injected with [14C]carbonate and the experiment was carried out for 15 days. As before, for the rate we have
1800
dSp/dt = KsS A - KdS p
.E O
where Sp and SA are the SRA of protein and amino acid, respectively, and k~ and k a are synthesis and decay rate constants, respectively. Under steady-state conditions we have
1200
\÷ .omo0ena,e
o. 03
¢L
k s ~ k d
so
600
dSp/dt= kd( SA - Sp) After a long time, SA would be very small, so we have:
3
11
15
Days
dSp/dt = -KdS p Integrating the equation above, we have
7
Fig. 6. Decay in the specific radioactivity of tubulin and homogenate proteins labeled by the administration of [U14C]glucose. From Forgue and Dahl (1978).
ln(S o at time t / S p at time 0) = - - k d t And for half-life, we have ln((Sp/2)/2) = --kdq/2 tl/2 = In 2 / k d
For the above experiment, when In SRA of the tubulin, a single protein, is plotted against time, we have a straight line. However, when In SRA of the homogenate is plotted, we no longer observe a straight line, as shown in Fig. 6. As can be seen, the condition for a single protein makes the problem very easy. However, for a mixture of proteins, there are a lot of complications. Garlick et at. (1976) have used the same label and injected rats as above and analyzed the curve for decay of label in the mixture of proteins in liver. The curve obtained from plotting In SRA vs time has been fitted into three exponential expressions. The three-exponential expression for the plot gave a somewhat better fit than a two-exponential one (see Fig. 7). Double isotope technique. Double isotope labeling involves giving a dose of 14C-amino acid, followed by 3H-amino acid a few days later. After a few hours, the animal is killed, the protein(s) in question is isolated, and the ratio of 3H to 14C is
measured. If it is assumed that all the proteins evolved from the same precursor pool, this ratio represents the rate of protein degradation (Arias et al., 1969; Glass and Doyle, 1972). The reason is that 3H represents the measure of initial labeling
105 f
~.
103
kl%d. 1) 9P-
2.28
.o ~
0.274
.>
O
101
4",-" 0.0619
n"
12
24
36
Time (days) Fig. 7. Decay of specific radioactivity of liver proteins labeled by injection of NaH14CO3 . From Garlick et al. (1976).
99 a n d t4C is the r e m a i n d e r of protein(s) d e g r a d e d after a few days. W h e n these values are d i v i d e d , the m o r e the difference b e t w e e n the two, the higher their ratio, a n d the faster the protein(s) is (are) d e g r a d e d . A l t h o u g h the p r o b l e m of recycling still r e m a i n s for the 14C-label, this is a very satisfactory m e t h o d for m e a s u r i n g relative d e c a y rates.
Results and Conclusions T h e rates of p r o t e i n synthesis can b e m e a s u r e d b y a variety of m e t h o d s i n c l u d i n g p u l s e labeling, massive p r e c u r s o r a d m i n i s t r a t i o n , Scornik m e t h o d , c o n t i n u o u s feeding of l a b e l e d precursor, infusion, a n d pellet i m p l a n t a t i o n . The synthesis rates can also b e c a l c u l a t e d f r o m d e g r a d a t i o n curves. E a c h t e c h n i q u e has s o m e a d v a n t a g e s a n d d i s a d vantages. T h e massive p r e c u r s o r technique, infusion (perfusion), a n d m e a s u r e m e n t s f r o m d e g r a d a tion curves are the m o s t w i d e l y used. T h e a d v a n t a g e of massive p r e c u r s o r a d m i n i s t r a t i o n is its
simplicity; however, the a m i n o acid c o n c e n t r a t i o n used is m u c h higher t h a n p h y s i o l o g i c a l levels. Infusion, s o m e t i m e s referred to as perfusion, is m u c h m o r e c o m p l i c a t e d as a technique, as are the calculations. T h e use of d e g r a d a t i o n curves is also simple as a t e c h n i q u e b u t has several d i s a d vantages. I n the single-isotope technique, b e c a u s e of the l o n g time r e q u i r e d (up to weeks), the recycling of the label, l o w e r i n g of the d e c a y rate, increases the half-life. It is also difficult to m e a sure the rate in n e w b o r n or i m m a t u r e a n i m a l s because, b y the e n d of the e x p e r i m e n t , the a n i m a l s have r e a c h e d m a t u r i t y . S o m e o f the techniques, such as pulse labeling a n d massive p r e c u r s o r a d m i n i s t r a t i o n , can be used b o t h in vivo a n d in vitro (slices). The a b o v e techniques can also b e used for a n y tissue or o r g a n ( S h a b h a z i a n et al., 1987), whereas the use of o t h e r techniques is s o m e w h a t limited. T h e Scornik m e t h o d has o n l y b e e n a p p l i e d to in vivo liver b u t it m a y be a p p l i c a b l e to in vitro techniques a n d to a n y tissue. M u l t i p l e i s o t o p e a d m i n i s t r a t i o n , pellet
TABLE I COMPARISON OF AMINO ACID INCORPORATION RATES IN ADULT ORGANS Tissue
Animal
System
Age
Incorporation (% per h)
Reference
Brain Brain Brain Liver Liver Liver Liver Liver Liver Heart Heart Heart (atrium) Heart (vent.) Heart (atrium) Heart (vent.) Heart Diaphragm Diaphragm
Rat Rat Rat Rat Rat Rat Mouse Mouse Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat
6 wk 300-450 g 6 wk 6 wk 90 g 8 wk 7 wk 7 wk 6 wk 6 wk 90 g 300 g 300 g 300 g 300 g 6 wk 90 g
0.62 0.30 0.097 2.15 2.08 2.45 2.62 1.97 0.32 0.66 0.70 0.70 0.40 0.48 0.22 0.043 0.61
Shahbazian et al., (1987) Seta et al. (1973) Shahbazian et al. (1987) Shahbazian et al. (1987) Pain and Garlick (1974) Goldspink and Kelly (1984) Huston and Mortimore (1982) Huston and Mortimore (1982) Shahbazian et al. (1987) Shahbazian et al. (1987) Pain and Garlick (1974) Preedy et al. (1985) Preedy et al. (1985) Preedy et al. (1985) Preedy et al. (1985) Shahbazian et al. (1987) Preedy et al. (1986)
Leg muscle Leg muscle Gastrocnemius muscle
Rat Rat
In vivo Perfusion Slice In vivo In vivo In vivo In vivo Perfusion Slice In vivo In vivo In vivo In vivo Perfusion Perfusion Slice In vivo Whole incubation In vivo Slice
90 g 6 wk 6 wk
0.31 0.46 0.022
Preedy et al. (1986) Shahbazian et al. (1987) Shahbazian et al. (1987)
Rat
In vivo
90 g
0.57
Pain and Garlick (1974)
100 i m p l a n t a t i o n , a n d feeding of labeled precursor can o n l y be used i n vivo. They c a n be applied to a n y tissue. I n f u s i o n c a n o n l y be used for certain organs. F o r brain, the t e c h n i q u e is extremely complex (Seta et al., 1973). The values o b t a i n e d for rates are s o m e w h a t lower t h a n those from the vivo e x p e r i m e n t (See T a b l e I). D e g r a d a t i o n curves can b e used for b o t h i n vivo techniques a n d slices of a n y o r g a n or tissue. M o s t of the research o n t u r n o v e r rates has b e e n d o n e in a d u l t animals. T a b l e I summarizes these results. T h e i n vivo rates give the highest values. A m o n g in vivo techniques, however, p e r f u s i o n / i n f u s i o n give slightly lower values. The tissue slice t e c h n i q u e gives m u c h lower values t h a n in vivo t e c h n i q u e s ( a b o u t 8 0 - 9 0 % lower) for a d u l t animals. I n i m m a t u r e a n i m a l s ( S h a h b a z i a n et al., 1987) i n vitro rates (slices) are close to in vivo rates (10-20% lower). W i t h the exception of kidney, all i m m a t u r e i n vivo rates are higher t h a n a d u l t i n vivo. I n some cases, the i m m a t u r e in vivo rates are d o u b l e to triple the values of the a d u l t in vivo, as i n brain. T h e research i n b r a i n p r o t e i n synthesis shows the ' i n vivo vs i n vitro d e v e l o p m e n t a l difference' to be true for cell types a n d b r a i n regions (Shahb a z i a n et al., 1986c) as well as different subcellular fractions (microsomes are a n e x c e p t i o n ; S h a h b a z i a n et al., 1986a) a n d different m o l e c u l a r weights ( S h a h b a z i a n et al., 1986b). F o r a comprehensive review of p r o t e i n t u r n o v e r i n different organs, cells, subcellular fractions etc., see D u n l o p (1983). I n conclusion, the values for the t u r n o v e r rate d e p e n d o n the t e c h n i q u e used a n d the variance m a y be due to technical artifacts.
References Arias, I.M., Doyle, D. and Sehimke, R.T. (1969) Studies on the synthesis and degradation of the proteins of the endoplasmic reticulum of rat liver, J. Biol. Chem., 244: 3303-3315. Austin, L., Lowry, O.H., Brown, J.G. and Carter, J.G. (1972) The turnover of protein in discrete areas of brain, Biochem. J., 126: 351-359. Buchaman, D.L. (1961) Total carbon turnover measured by
feeding a uniformly labeled diet, Arch. Biochem. Biophys., 94: 500-511. Dunlop, D.S. (1983) Protein turnover in brain: synthesis and degradation. In A. Lajtha (Ed.), Handbook in Neurochemistry 5, Plenum, New York, pp. 25-63. Dunlop, D.S., Van Elden, W. and Lajtha, A. (1974) Measurements of rates of synthesis in rat brain slices. J. Neurochem., 22: 821-830. Dunlop, D.S., Van Elden, W. and Lajtha, A. (1•75a) Optimal conditions for protein synthesis in incubated slices of rat brain, Brain Res., 99: 303-318. Dunlop, D.S., Van Elden, W. and Lajtha, A. (1975b) A method for measuring brain protein synthesis rates in young and adult rats, J. Neurochem., 24: 337-344. Dunlop, D.S., Lajtha, A. and Toth, J. (1977) Measuring brain protein metabolism in young and adult rats. In (EdsA, Roberts et al. Mechanisms, Regulation and Special Function of Protein Synthesis in the Brain, Elsevier. Amsterdam, pp. 79-96. Forgue, S.T. and Dahl, J.L. (1978) The turnover rate of tubulin in rat brain, J. Neurochem., 31: 1289-1297. Garrick, P.J. and Marshal, I. (1972) A technique for measuring protein synthesis, J. Neurochem., 19: 577-583. Garrick, P.J, Waterlow, J.C. and Swick, R.W. (1976) Measurement of protein turnover in rat liver, Biochem. J., 156: 657-663. Glass, R.D. and Doyle, D. (1972) On the measurement of protein turnover in animal cells, J. Biol. Chem, 247: 5234-5242. Goldspink, D.E. and Kelly, F.J. (1984) Protein turnover and growth in the whole body, liver and kidney of the rat from fetus to senility, Biochem. J., 217: 507-516. Haider, M. and Traver, H. (1979) Effect of diet on protein synthesis and nucleic acid levels in rat liver, J. Nutr., 99: 433-445. Hutson, N.J. and Mortimore, G.E. (1982) Suppression of cytoplasmic protein uptake by lysosomes as the mechanism of protein regain in livers of starved-refed mice, J. Biol. Chem., 257: 9548-9554. llan, J. and Singer, M. (1975) Sampling of the leucine pool from the growing peptide chain: differences in leucinespecific activity of peptidyltransfer RNA from free and membrane-bound polysome, J. Mol. Biol., 91: 39-51. Jones, D.A. and Mcllwain, H. (1971) Amino acid distribution and incorporation into proteins in isolated electricallystimulated cerebral tissues, J. Neurochem., 18: 41-58. Lajtha, A. and Dunlop, D. (1981) Turnover of proteins in the nervous system, Life Sci., 29: 755-767. Lajtha, A., Dunlop, D. Patlak, C. and Toth, J. (1979) Compartments of protein metabolism in the developing brain, Biochim. Biophys. Acta, 561: 491-501. Lajtha, A., Furst, S., Gerstein, A. and Waelsch, H. (1957) Amino acid and protein metabolism. I. Turnover of free and protein bound lysine in brain and other organs, J. Neurochem., 1: 289-300. Lajtha, A., Latzkovits, L. and Toth, J. (1976) Comparison of turnover rates of proteins of the brain, liver, and kidney in
101 mouse in vivo following long-term labeling, Biochim. Biophys. Acta, 425: 511-520. Oja, S.S. (1967) Studies on protein metabolism in developing rat brain, Ann. Acad. Sci. Fenn. (Med.), 131: 7-81. Pain, V.M, and Garlick, P.J. (1974) Effect of streptozotocin diabetes and insulin treatment of the rate of protein synthesis in tissues of the rat in vivo, J. Biol. Chem., 249: 4510-4514. Preedy, V.R., Smith, D.M., Kearney, N.F. and Sugden, P.H. (1985) Regional variation and differential sensitivity of rat heart protein synthesis in vivo and in vitro, Biochem. J., 225:487 492. Preedy, V.R., Smith, D.M. and Sugden, P.H. (1986) A comparison of rates of protein turnover in rat diaphragm in vivo and in vitro, Biochem. J., 233: 279-282. Schimke, R.T. (1964) The importance of both synthesis and degradation in the control of arginase levels in rat liver, J. Biol. Chem., 239: 3808-3817. Scornik, O.A. (1974) In vivo rates of translation by ribosomes of normal and regenerating liver, J. Biol. Chem., 249: 3876-3883. Seta, K., Sansur, M. and Lajtha, A. (1973) The rate of incorporation of amino acids into brain proteins during infusion in the rat, Biochim. Biophys. Acta, 294: 472-480. Shahbazian, F.M.. Jacobs, M. and Lajtha, A. (1986a) Rates of amino acid incorporation into particulate proteins in vivo and in slices of young and adult rat brain, J. Neurosci. Res., 15:359 366.
Shahbazian, F.M., Jacobs, M. and Lajtha, A. (1986b) Rates of brain protein turnover as a function of molecular weight. Neurochem. Res. 1 1 : 6 4 7 660. Shahbazian, F.M., Jacobs, M. and Lajtha, A. (1986c) Regional and cellular differences in rat brain protein synthesis, Int. J. Dev. Neurosci., 4: 209-215. Shahbazian, F.M., Jacobs, M. and Lajtha, A. (1987) Rates of protein synthesis in brain and other organs, Int. J, Dev. Neurosci., 5 : 3 9 42. Wannemacher, R.W., Wannemacher, C.F. and Yatvin, M.B. (1971) A m i n o acid regulation of synthesis of ribonucleic acid and protein in the liver of rats, Biochem. J., 124: 385-392. Waterlow, J.C., Garlick, P.J. and Millward, D.J. (1978a) Basic concepts. In Protein Turnover in M a m m a l i a n Tissues and in the Whole Body, North-Holland, New York, pp. 179-223. Waterlow, J.C., Garlick, P.J. and Millward, D.J. (1978b) Measurement of the rate of incorporation of labeled amino acids into tissue proteins. In Protein Turnover in Mammalian Tissues and in the Whole Body, North-Holland, New York, pp. 339-369. Waterlow, J.C. and Stephen, J . M . L (1967) The measurement of total lysine turnover in the rat by IV infusion of k[U-14C]lysine, Clin. Sci., 3 3 : 4 8 9 -506. Waterlow, J,C. and Stephen, J.M.E. (1968) The effect of lob protein diets on the turnover rates of serum, liver and muscle proteins in the rat, measured by continuous infusion of L-[14C]lysine, Clin. Sci., 35:287 305.
91
NSM 00889
Research Papers
Rates of protein synthesis
a review
F.M. S h a h b a z i a n 1, M y r o n S. J a c o b s 2 a n d Abel L a j t h a 3 I Department of Basic Sciences, Cleveland Chiropractic College, Los Angeles, CA 90004-2196 (U.S.A.), 2 Department of Pathobiology and Oral Pathology, New York University Dental School, New York, N Y 10010 (U. S. A.) and 3 Center for Neurochernistry, The Nathan S. Kline Institute for Psychiatric Research, New York, N Y 10035 (U.S.A.) (Received 3 June 1988) (Revised 19 July 1988) (Accepted 19 July 1988)
Key words: Rate of protein synthesis; Rate of protein degradation; Protein synthesis; Protein degradation; Protein turnover; Incorporation of amino acids into protein; Radioisotope administration; Pulse labeling; Infusion; Perfusion; Pellet implantation The rates of protein synthesis can be measured by a variety of methods including pulse labeling, massive precursor administration, Scornik method, continuous feeding of labeled precursor, infusion, and pellet implantation. Each technique has some advantages and disadvantages. Massive precursor administration and infusion are the most widely used. The advantage of massive precursor administration is its simplicity, however, the amino acid concentration used is much higher than physiological levels. Infusion, however, is much more complicated as a technique and requires complicated calculations. The synthesis rates can also be calculated from degradation curves. Some of the above techniques can be used both in vivo and in vitro, and also for different organs (Shahbazian et al. (1987), Int. J. Dev. Neurosci., 5: 39-42). The brain has rapid rates of protein synthesis both in vivo and in vitro, the latter being much lower for adults.
Introduction The structural elements of an organism undergo metabolic turnover when the organism is in a steady state, that is, when it is not experiencing growth or degeneration. The organismal components include proteins, which are synthesized continuously for replenishment or for specific purposes. Protein synthesis occurs at different rates; although protein synthesis to a great extent involves translation from nucleic acids to amino acids, the rate of synthesis is also affected by the
Correspondence: F.M. Shahbazian, Department of Basic Sciences, Cleveland Chiropractic College, 590 North Vermont Avenue, Los Angeles, CA 90004-2196, U.S.A.
process of transcription of RNA (mRNA, tRNA and rRNA), which in turn depends on replication. Some of the proteins synthesized are enzymes or metabolic regulators, whose presence will affect the rate of protein turnover. Finally, the proteins will break down into amino acids, some of which will enter the process of synthesis, and the rest of which will be degraded to end-products (Fig. 1). A stochastic approach makes it possible to determine the magnitude of certain variables without taking into account the structure or arrangement of the system studied. In determining rates of protein synthesis, especially of total proteins, with this approach it is not necessary to know the nature of the proteins studied, how many proteins are present, or what arrangement they have within the system.
0165-0270/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
92
c
~'n"¢
Translation A
~ RNA / tRNA I_ rRNA
FmRNA1
> Metabolicreactions and their regulation
Proteins
I
, Degradation Amino acids I Breakdown
End products Fig. 1. The processes relating to protein metabolism.
Questions might arise about the usefulness of knowing the rate of synthesis of total proteins in a system. The study of synthesis rates of single proteins or of groups of proteins provides different kinds of information; in some instances, the study of total protein synthesis in health or disease is apt to provide more information about the general mechanism of metabolism involved. During the learning process there is a transitory rise in brain protein synthesis possibly affecting all of the proteins of the system involved. Many drugs, chemicals and poisons affect the total protein machinery. In phenylketonuria, many proteins may be altered, and temperature variation, ischemia, and hypoxia can lower the total protein synthesis (Lajtha and Dunlop, 1981). Protein biosynthesis is thought to be greatly affected by a variety of developmental, environmental, and physiological stimuli (Ilan and Singer, 1975). For example, a hormone such as insulin can affect the cells of the liver and other organs, causing physiological changes that in turn can increase the rate of protein synthesis. This stimulation of protein biosynthesis is considered to be one of the factors in cellular response. Ever since the radioisotope method was applied to the study of metabolism, the stochastic approach has been used to determine rates of protein synthesis. In these procedures, a radioisotope precursor such as a labeled amino acid is injected into the animal. The labeled amino acid then passes through a sequence of compartments of the
body (plasma, extracellular environment, intracellular compartment) before being synthesized into protein.
Amino Acids (precursor pools) During the course of its transport, the labeled amino acid becomes associated with a number of specific pools of free amino acids, and also with bound amino acids and proteins (Lajtha ad Dunlop, 1981). The rate at which protein synthesis occurs (as explained in detail below) is often determined by amino acid uptake, as measured by specific radioactivity (SRA; Dunlop et al., 1975b). The concentration and SRA of amino acids in the plasma and extracetlular fluid are generally similar (Dunlop et al., 1977). Since the SRA of amino acids in this pool is higher than that of intracellutar free amino acids, the rates of protein synthesis based on the SRA of free amino acids in the extracellular fluid (k = SRA of protein/SRA of free AA) are lower than those based on intracellular SRA. The intracellular pool of amino, acids consists of precursor amino acids (aminoacyl-tRNA) with higher SRA, and lysosomal amino acids with lower SRA. Since the intracellular amino acid pool measured represents the total of both ribosomal- and lysosomal-associated amino acids, its SRA is diluted, and the rates of protein synthesis calculated would thus be too high.
93
Rates of Protein Synthesis
low et al. (1978a), be able both to describe in simplified form the physiological processes involved, and to provide the chemical framework for quantifying the components of the system. The rules for our model are: (1) The pools of metabolites and end products are in a steady state; i.e. they do not vary in size as they would under conditions of starvation or growth, unless otherwise stated. (2) Materials exchanged between the pools or introduced into them are assumed to mix completely and instantaneously with those already present. (3) A constant fraction of the material in each pool should be transferred or exchanged in unit time. (This is an exponential or first-order process.) A general method for determining rates of protein synthesis is to give a tracer as an single dose and measure SRA of the metabolite at different intervals of time (Waterlow et al., 1975b). If one is
A protein is synthesized through the action of a multitude of enzymes, nucleic acids, and structural moieties such as ribosomes. Several reactions take place simultaneously or consecutively. In considering the total complement of proteins being synthesized in an organ such as the brain, the large number of chemical transformations taking place can be summarized in the following single reaction, which will serve as an all-inclusive model: K~
Precursor amino acids ~ Protein products Kd
where K~ and K a are rate constants of synthesis and degradation, respectively, for that reaction, and they represent the total of the rate constants of hundreds of reactions. For such a complex system, involving a multitude of reactions, we need a well-defined model. Such a model should, as characterized by Water-
k s = synthesis
rate
V = synthesis
rate
constant
AP
Amino CA R E A C T ION :
acid
= total
M A = pool SRA
=
SA
pool
Protein
counts size
= CA/M A
pool
Cp
= total
Mp
= pool
size
= Sp
= Cp/Mp
SRA k d = degradation
counts
rate
C o n s tan t VpA
RATE :
dCp/dt
=
VApS A
-
= degradation
rate
VpAS p
Fig. 2. The reaction and the rate for a two-pool model of protein synthesis.
(z)
94 dealing with a single pool of either a metabolite or its end product, the rate would theoretically be expressed as
d S A / d t = -kS~, SA(t) = X e -kt, X = SA at zero time If, on the other hand, we are dealing with two pools, such as an amino acid metabolite and a protein end product, then the reaction and its rate would appear as in Fig. 2. Under steady-state conditions between the amino acid and protein pools, Mp is constant and K S = K d = k. When both sides of Eqn. 1 (Fig. 2) are divided by Mp, the rate is expressed as
For calculating the rates of protein synthesis, there are several procedures. These procedures can be divided into 4 categories: single isotope administration, multiple isotope administration, continuous isotope administration, and rates of protein degradation, each of which can be divided into several procedures. These are explained below.
Single isotope administration Pulse-labeling (Jones and Mcllwain, 1971).
In view of the fact that the SRA of the protein pool is very small compared to the amino acid pool, after a short time, we have
At zero time, a tracer dose of labeled amino acid is introduced into the intact brain (i.v. or i.p. injection) or into the medium of incubated brain slices. The amino acids present in body fluids and tissues initially reach a high level then decline to an almost constant value after about 30 min. In the case of i.v. injected animals, the level remains constant for only half an hour whereas in the i.p. injected animals, and in incubated slices, the level remains constant for an hour (Oja, 1967). Since turnover of protein in brain is slow, the peak of protein SRA for brain tissue would be low. The following expression can be used to calculate the rates of protein synthesis:
dSp/dt = kSA
d S p / d t = k( S a - Sp)
Under non-steady-state conditions, Mp is changing and Sp = Cp/Mp
If Sp and SA are measured at one point after zero time, the rate of change of SRA of protein with respect to time is approximated as Sp/time (Wannemacher, 1971). From this rate of change and SA, the rate constant ks (Sp/t)/SA can be calculated. This method of measurement, however, is not reliable and assumes that Sp vs time is a straight line. Another method of calculating rate constants of protein synthesis is by integration of
dSp/dt = ( VAp/Mp)SA -- ( VpA/Mp)S o d S p / d t = ksS A -
kdS p
Since K s = K a = k, then
d S p / d t = k ( S A - Sp)
dSp/dt = d ( C p / M p ) / d t = (1/Mp)(dC~,/dt ) - ( Cp/M2 ) ( d M p / d t )
(2)
Growth rate (for young) or degradation rate (for starving) is:
d M p / d t = YAp- VpA dCp/dt
= VApSA -
VpASp
(3) (1)
from before
dSp = k ( S A - Sp) dt
Substituting (3) and (1) into (2), we get
d S p / d t = ( VAp/M p )( SA -- Sp ) = ks( SA -- Sp ) Further, if 3 or more pools of metabolites a n d / o r end products are involved, the formula would be
S(t)=Xle-kat+X2e-k2,+...+X,e
=
kn,
The solution of an equation such as this, which involves three or more rate constants, generally requires the application of computer assistance.
which becomes Sp = SA(1 -- e -ks') SA, Sp, and the time can be measured, and the only unknown, ks, is then estimated by iteration. The result of this method of calculation, however, is not reliable because SA, which is assumed to be constant, actually changes with time. If, on the other hand, SA and Sp are measured at two different points after zero time, there are
95
two ways by which rate constants can be calculated. One way is to estimate the tangent to the curve of Sp vs time (Lajtha et al., 1957) between two points in time, and use the average of SA at those two points Ks-
Sp2 -- Spl
SA2 -~ SA1
12 -- t 1
2
Another way is to estimate the area under the curves described by SA vs t and Sp vs t (Haider and Traver, 1979):
[ area SA VS t
area Sp vs t
The area Sp vs time is negligible because of the small amount of the labeled protein involved. This is the most accurate of the above methods for measuring rate constants.
Massive precursor administration (Dunlop et al., 1974, 1975a, b). In the pulse-labeling approach it is assumed that the true SRA of the precursor is the same as that of the intracellular pool. By injecting a large dose of amino acid (500 mM) for in vivo experiments, by using a high concentration of amino acid (1 raM) for in vitro slice incubation, and by making SRA of precursor very high, the SRA of plasma or incubation medium and organ intracellular fluid (intact brain or brain slice) approach each other. (More concentrated solutions would cause inhibition of protein synthesis.) Other advantages of massive precursor use are that the plateau of constant SRA in the blood and organs would increase to 3 hours, and the speed of equilibration in reaching plateau would increase. Furthermore, all amino acid pools would be flooded uniformly. The calculations for determining the rate constants in this approach can be exactly like those for pulse labeling, There is, however, another way of approximating the rate constants. This consists of determining the change per hour in SRA of protein at any time following zero time and dividing it by SRA of injected precursor in in vivo experiments, or by dividing it by the SRA of the incubation medium in the case of in vitro brain slices.
Scornik method (Scornik, 1974). To do away with the time-course of precursors and their products, Scornik used constant SRA of varying concentrations of injected amino acids, killed the animals after 5 min, removed the liver, and determined the SRA of hepatic proteins. By plotting 1 / d o s e against 1/incorporation and assuming that it is a straight line, by extrapolating to infinite dose ( 1 / d o s e = 0 ) , the rate constants can be calculated as k = Incorporation at infinite dose SRA of injected amino acid The problem is that the line resulting from plotting 1 / d o s e vs 1/incorporation might not be straight.
Multiple isotope administration (Austin, 1972) Several equally spaced injections of isotope precursor are made into the animal. This method approximates the infusion approach (see Section 'Infusion', below).
Continuous isotope administration Continuous feeding of labeled precursor.
To keep SRA of free amino acid constant throughout the experiment, isotopically labeled material was fed to animals (Buchanan, 1961). Within 50 days the SRA of liver approached 95% of that of the food. The rate of protein synthesis, which is calculated by the curve approaching the SRA of food, is only an estimate because the SRA of precursors is only relatively constant. The food taken in is always diluted by material from the breakdown of tissue protein and the SRA of tissue is constantly increasing during the experiment. Also, since the uptake of radioactivity is measured over a long time-course, the turnover rates of mixed protein calculated from protein SRA curves become progressively lower with passing time. For a pure protein this problem is immaterial (Schimke, 1964). Assuming that SA remains constant and is equal to SRA of the precursor in food, the rate constant can be calculated by
S~= S~(1-e ~").
96
lnfusion (Garlick and Marshal, 1972). Labeled amino acid is introduced by venous infusion at a constant rate. Plasma amino acids and the brain free amino acids (70-80% plasma level) reach plateau levels and are maintained for several hours. By multiple sampling of the blood, one can determine when the plateau of constant precursor is reached. The SRA of free amino acid in the plasma (Sp) in rising to that plateau (Spmax) can be expressed as: Sp = Spmax(1 - e -Lpt) where Lp = empirically fitted rate constant (Waterlow and Stephen, 1967, 1968). The rate at which the SRA of tissue reaches the plateau level depends on two factors, pool size of labeled amino acid and rate of protein turnover in the tissue. Assuming that there is only one precursor pool, then the SRA of free amino acid pool in the brain would reach a constant value expressed in the following manner S B = Simax(1
-- e -ks, )
where S B is SRA of protein in brain and Simax is the intracellular SRA of precursor at plateau. The extent of error in evaluating rates of protein synthesis caused by the assumption that the precursor SRA is constant, S = Sims,, rather than having an exponential nature, S = Simax ( 1 e-Zt), can be illustrated as in Fig. 3. The shaded area in the figure indicates the error resulting by not considering the time dependency of precursor rising to a plateau. The error can be corrected in a number of ways: (1) The time course of Si may be determined experimentally. In this case one determines L empirically and uses S~ rather than Simax(2) The experiment can be designed to be long-term. In such a case, the shaded area would be negligible. (3) Animals could be killed at two different times after the plateau is reached. Thus, the following expression may be used to obtain a rate constant: ,A,SB//A l = ( Simax ) k
where S B / t is the rate of increase of SRA of brain proteins between two points in time.
S=SMAX SPECIFOC(s)~ ACTIVITY
4nt S=SMAX (1-e )
TIME Fig. 3. Illustration of the content of the error in determination of protein synthesis rate caused by assuming the precursor spec. act. to be constant when in fact it rises exponentially to a constant level. The amount of label incorporated into protein is approximately proportional to the area under the curve of precursor spec. act. Hence the error is determined by the relative area and the curve for the duration of the experiment. From Watedow et al. (1978b),
(4) A more accurate formula can be derived for the particular organ. In the case of the brain, it would be expressed S B / S ~= k , , / ( 1 - e -RK.¢') - ( l / R ) where S B = SRA of brain proteins Si = SRA of free amino acid in brain at plateau R = protein-bound amino acids free amino acids The value of S B and S i are calculated at 3 or more times during the plateau and their ratios are obtained. R is a constant value and the ratio S u / S i may be plotted against k s for a given value of t. Under the conditions, the value of k S for any experimental ratio of S B / S ~ at time t, can be read directly from the curve. Pellet implantation (Lajtha et al., 1976, 1979). An alternative to infusing amino acid precursor by means of a venous catheter (external infusion) is the implantation of a pellet of the precursor (internal infusion). The pellet should dissolve slowly and at a uniform rate. In this method it is assumed that the SRA of precursor in the brain is constant and that its incorporation into brain protein is a zero order reaction. It is further assumed that the growth rate of protein is the same for all of the different amino acid pools in the brain and that there are, at most, two major protein pools.
97
S
P -
P
S where
-kslt ]
(
1 -
e
P
A
(
1
-
e
P
Sp : SRA of
protein
Sa : S ~
free
of
-ks2t
P2 ) +
in brain
amino
acid
in b r a i n
P =~_Pi
i=l Pi =
Amount
of p r o t e i n
ksl = S y n t h e s i s
rate
in the ith pool
constant
of b r a i n
of the ith pool
of brain.
Fig. 4. A mathematical model for protein synthesis by pellet implantation (Lajtha et al., 1976, 1979).
In determining the rate of protein synthesis by the pellet implantation method, the equation (Fig. 4) may be derived. The two rate constants present in the above equation are illustrated in Fig. 5. From Fig. 5, it can be seen that there are two pools in the brain, which differ in the rates of formation from the precursor amino acid pool. The amount of protein, P2, is measured by extrapolation, whereas P represents the total protein in the homogenate. After all available values are substituted into the equation, the rate constants ks1 and ks2 are usually calculated with the aid of a computer.
Rates of protein degradation In an organism in steady state, the rates of protein synthesis and breakdown should be equal so that the organism would not be gaining or losing protein. So, to calculate the rates of protein synthesis, sometimes the rates of protein degradation are measured in adult animals.
%Unlabeled
100
Protein
~-
*
~
I Adult Mouse 80
I 0
I
i
I
10 TIME
I 20
i
p 30
(hours)
Fig. 5. Percent unlabeled protein calculated on semilog paper vs time from injection of [14C]tyrosine (30 mg/ml) subcutaneously. From Lajtha et al. (1979).
Protein degradation rates are measured in several ways. The main two techniques are singleisotope labeling and double-isotope labeling. Single-isotope technique. In this technique animals are injected with a radioactive precursor, and while the animals are taken care of for a long period such as a month, some animals are killed in the meantime, and SRA of protein is calculated. A plot is prepared from SRA vs time, and from the curve the rate constants are calculated. Since the time is long, the problem of recyclization is important. The extent to which recycling affects the rates of protein degradation depends on several factors, including the precursor used. The result of this recycling is to reduce the rate of decay and prolong its apparent half-life. The most commonly used precursors are [14C]carbonate and [14C]glucose. The former, which is used most often, would end up as the guanidino carbon of the arginine residue as well as the alpha-carboxyl group of glutamate, glycine, alanine, serine, and glutamine, and in both carboxyl groups of aspartate for liver tissue, and only in some of the above amino acids in other tissues examined. However, [14C]glucose would end up in more amino acid residues, causing more recyclization; it is also more costly to use. One of the experiments in which the turnover rate of tubulin (a microtubule protein) was measured is the work by Forgue and Dahl (1978). In
98
this p r o c e d u r e , rats were injected with [14C]carbonate and the experiment was carried out for 15 days. As before, for the rate we have
1800
dSp/dt = KsS A - KdS p
.E O
where Sp and SA are the SRA of protein and amino acid, respectively, and k~ and k a are synthesis and decay rate constants, respectively. Under steady-state conditions we have
1200
\÷ .omo0ena,e
o. 03
¢L
k s ~ k d
so
600
dSp/dt= kd( SA - Sp) After a long time, SA would be very small, so we have:
3
11
15
Days
dSp/dt = -KdS p Integrating the equation above, we have
7
Fig. 6. Decay in the specific radioactivity of tubulin and homogenate proteins labeled by the administration of [U14C]glucose. From Forgue and Dahl (1978).
ln(S o at time t / S p at time 0) = - - k d t And for half-life, we have ln((Sp/2)/2) = --kdq/2 tl/2 = In 2 / k d
For the above experiment, when In SRA of the tubulin, a single protein, is plotted against time, we have a straight line. However, when In SRA of the homogenate is plotted, we no longer observe a straight line, as shown in Fig. 6. As can be seen, the condition for a single protein makes the problem very easy. However, for a mixture of proteins, there are a lot of complications. Garlick et at. (1976) have used the same label and injected rats as above and analyzed the curve for decay of label in the mixture of proteins in liver. The curve obtained from plotting In SRA vs time has been fitted into three exponential expressions. The three-exponential expression for the plot gave a somewhat better fit than a two-exponential one (see Fig. 7). Double isotope technique. Double isotope labeling involves giving a dose of 14C-amino acid, followed by 3H-amino acid a few days later. After a few hours, the animal is killed, the protein(s) in question is isolated, and the ratio of 3H to 14C is
measured. If it is assumed that all the proteins evolved from the same precursor pool, this ratio represents the rate of protein degradation (Arias et al., 1969; Glass and Doyle, 1972). The reason is that 3H represents the measure of initial labeling
105 f
~.
103
kl%d. 1) 9P-
2.28
.o ~
0.274
.>
O
101
4",-" 0.0619
n"
12
24
36
Time (days) Fig. 7. Decay of specific radioactivity of liver proteins labeled by injection of NaH14CO3 . From Garlick et al. (1976).
99 a n d t4C is the r e m a i n d e r of protein(s) d e g r a d e d after a few days. W h e n these values are d i v i d e d , the m o r e the difference b e t w e e n the two, the higher their ratio, a n d the faster the protein(s) is (are) d e g r a d e d . A l t h o u g h the p r o b l e m of recycling still r e m a i n s for the 14C-label, this is a very satisfactory m e t h o d for m e a s u r i n g relative d e c a y rates.
Results and Conclusions T h e rates of p r o t e i n synthesis can b e m e a s u r e d b y a variety of m e t h o d s i n c l u d i n g p u l s e labeling, massive p r e c u r s o r a d m i n i s t r a t i o n , Scornik m e t h o d , c o n t i n u o u s feeding of l a b e l e d precursor, infusion, a n d pellet i m p l a n t a t i o n . The synthesis rates can also b e c a l c u l a t e d f r o m d e g r a d a t i o n curves. E a c h t e c h n i q u e has s o m e a d v a n t a g e s a n d d i s a d vantages. T h e massive p r e c u r s o r technique, infusion (perfusion), a n d m e a s u r e m e n t s f r o m d e g r a d a tion curves are the m o s t w i d e l y used. T h e a d v a n t a g e of massive p r e c u r s o r a d m i n i s t r a t i o n is its
simplicity; however, the a m i n o acid c o n c e n t r a t i o n used is m u c h higher t h a n p h y s i o l o g i c a l levels. Infusion, s o m e t i m e s referred to as perfusion, is m u c h m o r e c o m p l i c a t e d as a technique, as are the calculations. T h e use of d e g r a d a t i o n curves is also simple as a t e c h n i q u e b u t has several d i s a d vantages. I n the single-isotope technique, b e c a u s e of the l o n g time r e q u i r e d (up to weeks), the recycling of the label, l o w e r i n g of the d e c a y rate, increases the half-life. It is also difficult to m e a sure the rate in n e w b o r n or i m m a t u r e a n i m a l s because, b y the e n d of the e x p e r i m e n t , the a n i m a l s have r e a c h e d m a t u r i t y . S o m e o f the techniques, such as pulse labeling a n d massive p r e c u r s o r a d m i n i s t r a t i o n , can be used b o t h in vivo a n d in vitro (slices). The a b o v e techniques can also b e used for a n y tissue or o r g a n ( S h a b h a z i a n et al., 1987), whereas the use of o t h e r techniques is s o m e w h a t limited. T h e Scornik m e t h o d has o n l y b e e n a p p l i e d to in vivo liver b u t it m a y be a p p l i c a b l e to in vitro techniques a n d to a n y tissue. M u l t i p l e i s o t o p e a d m i n i s t r a t i o n , pellet
TABLE I COMPARISON OF AMINO ACID INCORPORATION RATES IN ADULT ORGANS Tissue
Animal
System
Age
Incorporation (% per h)
Reference
Brain Brain Brain Liver Liver Liver Liver Liver Liver Heart Heart Heart (atrium) Heart (vent.) Heart (atrium) Heart (vent.) Heart Diaphragm Diaphragm
Rat Rat Rat Rat Rat Rat Mouse Mouse Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat
6 wk 300-450 g 6 wk 6 wk 90 g 8 wk 7 wk 7 wk 6 wk 6 wk 90 g 300 g 300 g 300 g 300 g 6 wk 90 g
0.62 0.30 0.097 2.15 2.08 2.45 2.62 1.97 0.32 0.66 0.70 0.70 0.40 0.48 0.22 0.043 0.61
Shahbazian et al., (1987) Seta et al. (1973) Shahbazian et al. (1987) Shahbazian et al. (1987) Pain and Garlick (1974) Goldspink and Kelly (1984) Huston and Mortimore (1982) Huston and Mortimore (1982) Shahbazian et al. (1987) Shahbazian et al. (1987) Pain and Garlick (1974) Preedy et al. (1985) Preedy et al. (1985) Preedy et al. (1985) Preedy et al. (1985) Shahbazian et al. (1987) Preedy et al. (1986)
Leg muscle Leg muscle Gastrocnemius muscle
Rat Rat
In vivo Perfusion Slice In vivo In vivo In vivo In vivo Perfusion Slice In vivo In vivo In vivo In vivo Perfusion Perfusion Slice In vivo Whole incubation In vivo Slice
90 g 6 wk 6 wk
0.31 0.46 0.022
Preedy et al. (1986) Shahbazian et al. (1987) Shahbazian et al. (1987)
Rat
In vivo
90 g
0.57
Pain and Garlick (1974)
100 i m p l a n t a t i o n , a n d feeding of labeled precursor can o n l y be used i n vivo. They c a n be applied to a n y tissue. I n f u s i o n c a n o n l y be used for certain organs. F o r brain, the t e c h n i q u e is extremely complex (Seta et al., 1973). The values o b t a i n e d for rates are s o m e w h a t lower t h a n those from the vivo e x p e r i m e n t (See T a b l e I). D e g r a d a t i o n curves can b e used for b o t h i n vivo techniques a n d slices of a n y o r g a n or tissue. M o s t of the research o n t u r n o v e r rates has b e e n d o n e in a d u l t animals. T a b l e I summarizes these results. T h e i n vivo rates give the highest values. A m o n g in vivo techniques, however, p e r f u s i o n / i n f u s i o n give slightly lower values. The tissue slice t e c h n i q u e gives m u c h lower values t h a n in vivo t e c h n i q u e s ( a b o u t 8 0 - 9 0 % lower) for a d u l t animals. I n i m m a t u r e a n i m a l s ( S h a h b a z i a n et al., 1987) i n vitro rates (slices) are close to in vivo rates (10-20% lower). W i t h the exception of kidney, all i m m a t u r e i n vivo rates are higher t h a n a d u l t i n vivo. I n some cases, the i m m a t u r e in vivo rates are d o u b l e to triple the values of the a d u l t in vivo, as i n brain. T h e research i n b r a i n p r o t e i n synthesis shows the ' i n vivo vs i n vitro d e v e l o p m e n t a l difference' to be true for cell types a n d b r a i n regions (Shahb a z i a n et al., 1986c) as well as different subcellular fractions (microsomes are a n e x c e p t i o n ; S h a h b a z i a n et al., 1986a) a n d different m o l e c u l a r weights ( S h a h b a z i a n et al., 1986b). F o r a comprehensive review of p r o t e i n t u r n o v e r i n different organs, cells, subcellular fractions etc., see D u n l o p (1983). I n conclusion, the values for the t u r n o v e r rate d e p e n d o n the t e c h n i q u e used a n d the variance m a y be due to technical artifacts.
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