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Effect of the ATP level on the overall protein biosynthesis rate in a wheat germ cell-free system
BB ELSEVIER
etBiochi~ic~a BiophysicaA~ta Biochimica et Biophysica Acta 1293 (1996) 207-212
Effect of the: ATP level on the overall protein biosynthesis rate in a wheat germ cell-free system S.V. Matveev a,,, L.M. Vinokurov ~, L.A. Shaloiko a, C. Davies a, E.A. Matveeva b, Yu.B. Alakhov a a Branch of the Shemyakin and Ot,chinnikot" Institute ofBioorganic Chemistr3", Russian Academy of Sciences, 142292 Pushchino, Moscow Region, Russia b Institute of Cell Biophysics, Russian Academy of Sciences, 142292 Pushchino, Moscow Region, Russia Received 26 October 1995; accepted 23 November 1995
Abstract
A sensitive assay which examines the effects of ATP level on the overall activity of a cell-free translation system in a protein synthesis is described. The translational activity of cell-free system was measured in terms of a rate of protein synthesis directed by the 'test' template. The test template encodes a photoluminescent protein, obelin. The rate of obelin accumulation was determined from the kinetic curves of obelin mRNA tran:dation. Time-dependent nucleotide level measurements were conducted throughout the translation processes. It has been shown that the rate of translation decreases exponentially with the decrease of the ATP level. This fall in the overall translation rate is due in pa~: to the mRNA becoming inactive in the translation process. This is not caused by degradation, this mRNA can be restored for translatio:a in a fresh cell-free system by phenol treatment. The reported results provide evidence that the level of ATP unambiguously determines the translational activity of the system. Keywords." ATP regulation; Cell-free translation; Obelin mRNA; Luminescence
1. Introduction
According to Atkinson's formulation [1], protein synthesis depends strongly on small changes in the energy charge of a cell. Evidence of the existing correlation between small changes in the ATP abundance and relatively large changes in tt':e rate of protein synthesis have been obtained in many sy:~tems: (i) in isolated fat cells [2], (ii) in rat hepatocytes and rabbit reticulocytes [3,4] and, (iii) in isolated rat muscle cells [5]. The number and size cf polyribosomes decrease during incubation of rat thymus cytoplasm cells in conditions of a lower ATP content in c2~toplasm [6]. These results were interpreted as the inhibition of initiation of translation by ADP and AMP. This conclusion confirms the ADP concentration effect on the degree of the triple initiation complex formation in the cell-free translation system from
Abbreviation~: CP, creatine phosphate; CK, creatine kinase. • Corresponding author. Fax: + 7 095 9252342. 0167-4838/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 4 8 3 8 ( 9 5 ) 0 0 2 4 4 - 8
rabbit reticulocytes [7]. On the other hand, the rate of initiation of translation depends on the A D P / A T P relationship and does not depend on the absolute ATP, ADP and AMP concentrations in the cell-free translation system from Ehrlich tumor cells [8]. Generally the proven fact is that biosynthesis decreases with the decrease of the ATP content [1-5]. The changes in the rate of the initiation step, which is subjected by the ATP content [6-8], was assumed as a real mechanism of that dependence. This is the reason why numerous authors have suggested that the ATP level can play the role of a protein synthesis regulator both in vitro and in vivo. To substantiate this assumption it is necessary, in the first place to show a definite dependence of the protein synthesis rate on the ATP-dependent parameter and, in the second to show in what way this variation of the biosynthesis rate can be achievable. Thanks to the unique sensitivity of the obelin testing method and the designed technique for measurements of the ATP concentration in the translation system we obtained data which enabled us to state the mechanism of such regulation.
208
S.V. Matt eet et al. / Biochimica et Biophysica Acta 1293 (1996) 207-212
2. Materials and methods
2.6. Assay o f obelin
2.1. Materials
Five microliters of the activation buffer containing obelin were added to 0.5 ml of 100 mM Tris, 10 mM EDTA (pH 8.8). An 0.2 ml aliquot of the solution (100 mM CaCI~, 100 mM Tris, pH 8.8) was injected into this volume after the sample was inserted into a luminometer photomultiplier (Model BLM 8802, Nauka, Krasnoyarsk). The luminometcr was calibrated using a light standard [13]. One luminescent unit was equal to 108 quanta/sec. The rate constant of the obelin luminescent reaction was 6.9 sec- i.
Creatine-phosphate kinase, Hepes, potassium acetate, magnesium acetate, spermidine, dithiothreitol, gelatin, 2mercaptoethanol and L-amino acids were from Sigma; ATP, GTP, creatine phosphate were from ICN Biomedicals; Tris, EDTA were from Serva; other chemical reagents of analytical grade were from Reachim (Russia). 2.2. Obelin mRNA
Obelin mRNA was obtained from the Pcull linearized pNOV plasmid [9,10] with the aid of sp6 polymerase [11]. The transcripts were isolated as previously described [11]. The mRNA was dissolved in water to a 0.063 m g / m l concentration. 2.3. Preparation o f the wheat germ extract
The wheat germ extract was prepared according to the procedure of Erickson and Blobel [12] and stored until use at - 70°C. 2.4. Conditions f o r in vitro obelin synthesis
Translation of the obelin mRNA was carried out in the wheat germ cell-free system. The translation mixture (25 /zl) included 8 /zl of a wheat germ extract with an absorbance of A260 = 85 O.U. Other components of the mixture were 40 mM Hepes (pH 7.6), 80 mM potassium acetate, 3.2 mM magnesium acetate, 0.2 mM spermidine, 6 mM dithiothreitol, 2 mM ATP, 50 /xM GTP and 100 /zM each of the 20 amino acids. Creatine phosphokinase (1.12 ~g) with a specific activity of 350 U / m g and creatine phosphate up to 8 mM were added to 25 ~1 of the mixture if the synthesis of obelin was performed in the presence of the ATP-regenerating system. The translation mixture (25 /xl) was incubated for a specific time at 24°C before addition of (0.063 /zg) obelin mRNA. The kinetics of obelin accumulation in the process of translation was measured by luminescence of the activated apo-obelin. 2.5. Activation o f apo-obelin
2.7. Nucleotide measurements
Nucleotide levels throughout protein synthesis were assessed by HPLC analysis on a reverse-phase column in an ion-pair exchange regime. A 5 ,ttl aliquot was withdrawn from the translation system at the times indicated in each experiment and blended with an equal volume of 20 mM tetrabutylammonium, 200 mM NaH2PO ~ (pH 6.0). The sample was loaded onto a Separon C18 TESSEK (Czech Republic) column and eluted with 20 mM tetrabutylammonium, 200 mM NaH2PO 4 (pH 6.0) for 10 min at a flow rate of 0.8 m l / m i n . Absorbance at 254 nm was monitored by a model 210 Hitachi flow spectrophotometer. Individual nucleotide peaks were identified by analysis of known standards. Measurements of the relative nucleotide levels were performed using a Shimadzu CR5A integrator.
3. Results Fig. 1 shows the kinetics of obelin synthesis in a cell-free wheat germ translation system. The addition of a new portion of the template elevates the rate of protein synthesis (Fig. 1). However, the increase in the level of
~,
1200
i
.~ ~ 900 e-.
"~ 600 e..
E=~ --g.~
300 o
0 Activation of apo-obelin was done in 100 ~1 of the reactivation buffer 10 mM Tris-HCl, 0.5 M NaCI, 1 mM EDTA, 5 mM mercaptoethanol, 0.1% gelatin (pH 7.0) containing 5 /zl aliquot from the translation system and 1 /xl of 40 g M coelenterazine solution in methanol. The activation mixture was incubated for 4 h at room temperature in the dark.
I
e-,
120
240
360
Time of translation (min) Fig. 1. Kinetics of protein synthesis. Accumulationof obelin in the wheat germ cell-free translation system in the presence of CP and CK is shown by the luminescence of activated apoobelin, solid circles. Arrows show at which time a new [x)nion of mRNA was added to an aliquot of the translating system. Curves designated with triangles (1), diamonds (2) and hexagons (3) represent the result of this translation.
S. V. Matveev et al. / Biochimica et Biophysica Acta 1293 (1996) 207-212
synthesized protein deperds on the time of addition of a fresh portion of the template. After the system reaches a plateau, the addition of mRNA does not lead to the recovery in protein synthesis (Fig. I, curve 3). Consequently, the system loses its capability for synthesis during the incubation process. The data in Fig. 2 show the incubation time dependence of nucleotide levels in the translation mixtures without mRNA at a different composition of the mixtures. The control experiments in th.: presence of [14C]Leu show no incorporation of radiolabelled Leu in the hot TCA insoluble fraction of the translation mixture. Thus, the decrease in the ATP level is induced by the non-cotranslation ATPase of the wheat germ extract. We used obelin mRNA as the 'test' template to examine the properties of the translation mixture depending on the incubation time. A ~lefined concentration of obelin mRNA was added at a specific time into the translation mixture after its incubation in the absence of exogenous template. The mRNA cortcentration was chosen in such a way that the cotranslational consumption of ATP remained
1
0
0
~
II'~
< 0
o
A
ATP
~ 0
~
]12
209
low (not exceeding the experimental error of ATP determination) in comparison with ATP hydrolysis in processes not connected with translation. Fig. 3 shows the kinetic curves of apo-obelin accumulation in these experiments. When the translation begins in conditions with a low ATP level (Fig. 3, panels B, C, D, curves 4, 5, 6), the curves exhibit a distinctly expressed non-linearity at the first third of the kinetic curve. The system appears to accelerate, reaching a maximal rate (Vm) of translation somewhere in the middle of the kinetic curve (Fig. 3). This feature of the kinetic curves can be evidence that the rate of initiation is a limiting one for the starting of protein synthesis but differs from the rate of reinitiation which defines the efficiency of the translation process as a whole. The values of maximal rate of translation (Vm) achievable under these conditions are shown in Fig. 2 as columns. The decrease of Vm proceeds concurrently with the percentage decrease of the ATP in the solution. The only deviation from this tendency is the Vm value obtained when the obelin mRNA was added to CP containing translation mixture at zero time of incubation, Figs. 2 and
100
B
6
i
o 120
o 240
o 0
100
60
120 6
100
C 75
< Z
D
E O
75
50
25
0
0
60
120
0
Illu 60
0 120
Time of incubation of the translation mixture (min) Fig. 2. Nucleotide levels in a translation mixture incubated without obelin mRNA. Levels of ATP (circles), ADP (.squares) and AMP (triangles) were measured in the translation mixtures with the different composition at the initial moment of incubation: panel (A), 8 mM CP and 0.045% CK; panel (B), 8 mM CP and no CK; panel (C), no CP and 0.045% CK; and panel (D), no CP and no CK. Maximal rates of obelin synthesis followed by an addition of mRNA to the translation mixture were determined from the kinetic curves in Fig. 2 and are plotted as columns at the time of mRNA addition.
S. V. Matveet" et al. / Biochimica et Biophysica Acta 1293 (1996) 207-212
210
I
Table 1 Correlation coefficients calculated for different dependences between Vm and the A(n)P level in the system from data given in Fig. 2
I
600
g
2
120
3
8o
B
Vm (LU/min) In V,,,
ATP
ADP
AMP
ADP/ATP
ATPo / A T P
-~ 0.72
0~ 0.58
b-~ 0.7
0.71 0.89
0.73 0.96
40 ADP(t) / ATP(t) "~
O~ 0
~
120
- '" ~ 60 120
1 180
C
O0 0
120
D
2
4
I
8
-3 4
40
40
4
O 5
5 e-
0wv 0
6 60
120
0 0
60
12(1 0
Time of the obelin mRNA translation (min) Fig. 3. Kinetics of obelin synthesis. Accumulation of obelin in translation systems with the same content as for Fig. 2 are shown as the luminescence of activated apoobelin. The curves differed with the time of incubation of the translation mixture before the mRNA addition. At the times shown below, 0.06 /zg of the obelin mRNA was added to 25 /,tl aliquots of the translation mixture. Panel A, 0' (1), 30' (2), 60' (3), 90' (4), 120' (5), 180' (6), and 240' (7); panels B, C and D, 0' (1), 10' (2), 20' (3), 30' (4), 40' (5), and 60' (6).
3, panels A and C. The reason for this could be explained by a time delay in renaturation of the translation system components in the presence of CP. We cannot exclude that storage of the wheat germ extract at - 7 0 ° C leads to the effect of renaturation kinetics on the translation activity of the system. The best linear correlations are observed between In Vm and the ratio A T P 0 / A T P ( t ) on the one hand, and In Vm and the ratio ADP(t)/ATP(t)on the other hand, (Table 1), (ATP 0 represents the initial ATP concentration in thc translation mixture, or ATP 0 = A T P ( t ) + A D P ( t ) + A M P ( t ) at any moment of time of the incubation process). These dependences are shown in Fig. 4. Panel A shows
-3
I 4
-6
0
I 8
I 12
16
ATP 0 / ATP(t) Fig. 4. Correlation of In Vm with the ATP levels expressed in terms of A D P ( t ) / A T P ( t ) in panel A and ATP0/ATP(t) in panel B. The data designated with the different signs correspond to the data in the different panels of Fig. 2: downward triangles (panel A), circles (panel B), squares (panel C), upward triangles (panel D).
a different tilting of the curves about the axis. However, we have to assume the overall dependences for the same wheat germ extract and m R N A in varying translation conditions. The dependences in panel B satisfy this condition and can be represented as In Vm
=
- -
b ATPo/ATP (t) + a
(1)
Table 2 Obelin mRNA translation in the system with initial conditions as for the control experiment in Fig. 1. The ATP level in the system was restored by the CP addition at 150 rain of the translation process Fig. 1, control data, no addition Time of translation (min): ATP (% ATPo) ADP (% ATP0) AMP (% ATPo) LU per/a.I of translation system ( L U / ~ I )
+ C P up to 16 mM
+CP up to 16 mM + fresh mRNA (0.06/zg/25 ,u,I)
150
180
210
155
180
210
155
180
210
28 37 27 620
21 32 36 625
17 29 42 622
82 10 < 1 619
91 2
90 3
91 3
620
622
79 11 < 1 620
89 3 992
840
S. V. Matveec et al. / Biochimica et Biophysica Acta 1293 (1996) 207-212
600
1
I
~.-~ 400
~-
0
l
,3 60
120
180
Time of translation (min) Fig. 5. Kinetics of the translation of obelin mRNA, which remained active after the desired time Ihrough the biosynthesis reaction with condition as for Fig. 1. The obelin mRNA was prepared by phenol treatment of 25/xl aliquot withdrawn from the translation system at 0 min for curve 1, 60 min for curve 2 and 150 rain for curve 3. The total RNA was added to 25 /zl of fresh trartslation mixture and the luminescence of activated apo-obelin are shown ~.s the result of translation.
where b is the tangent of the experimental curve angle of inclination to the ATPo/ATP axis while a is the segment on the In Vm axis intercepted by the experimental curve. This does not mean that coefficients a and b are constants.
800
i
I
T-
800
i
i I
A 600
", 7,'. I
2 40o
600
2
400
3
"t 4 .~ /
E 2oo
'~..J 60
120
200
-
-
4 5
0
180
0
60
16
i 120
180
e.~
.~ 12o
C
.
I
o= ~ so
Indeed, coefficient b can depend on the activity of the enzymes and differs for different wheat germ extracts. The influence of temperature can be expressed by the dependence of coefficient a on temperature. Let us return for a moment to Fig. I. In accordance with Eq. l, the translation activity of the system could be resumed by the return of the ATP level to the initial one. The data represented in Table 2 show that the ATP level is restored after some CP was added to the translation system at 150 min of the translation process in Fig. I. Simultaneously, the translational activity of the cell-free system is also resumed after the addition of a new mRNA portion (Table 2). However, there is practically no de novo synthesis of the obelin followed by the ATP level recovering without new mRNA addition, (Table 2). This means that there is not any translatable mRNA in this translation system after the ATP level was restored at 150 min of translation process, as in Fig. I. It is well known (for example, [14]) that the template degradation takes place in cell extract solutions. It is also known that the translation in a batch system from wheat germ can be directed for six hours or more, [15]. Hence, it would be expected that the mRNA does not degrade fully during the first 150 min of translation process. The data in Fig. 5 show that the obelin mRNA presents in the translation system in time of its incubation because some mRNA activity can be detected in a fresh translation mixture after deproteinization by phenol treatment. This facts signify that the decrease in the ATP level during the incubation of the cell-free translation system leads to the irreversible masking of a part of the active mRNA.
4. Discussion
5 O0
211
12o
1
80
c-
The main conclusion which follows from the presented results is that the maximal achieved translation rate of test templates with the defined concentration in the wheat germ cell-free system is a function only of the ATP level at a constant temperature. On the grounds of the revealed regularities we can simulate theoretical kinetics of protein synthesis when the protein accumulation rate at any moment of time depends only on the relationship of ATPo/ATP, V(t) = A exp( - b ATP0/ATP ( t ) )
e-
4
.~ 4o
40
~
-
4
© 0
o 60
120
0
60
120
Time of the obelin m R N A translation (min) Fig. 6. Experimental and theoretical kinetics of obclin synthesis. Experimental parts (panels A and C) correspond to the conditions of panels A and D in Fig. 3. Theoretical panels B and D was constructed in accordance with the model of translation described in the text.
where A = e". Coefficients A and b were determined from data in Fig. 4. ATP0 equals 2 mM as in real experiments. The ATP(t) functional dependence for each case was obtained by approximating the real experimental points of the ATP level by polynomials. Fig. 6, panels B and D show the model curves calculated from the ATP level data presented in Fig. 2, panels A and D, respectivcly, and shifted for l0 rain from the beginning of translation as the luminescent activity of the newly synthesized obelin is exhibited only after this time,
212
S. V. Matceec et al. / Biochimica et Biophysica Acta 1293 (1996) 207-212
Fig. 3. With the exception of curve I in Fig. 6A, the other curves coincide well with the respective of experimental kinetic curves. The theoretical curves for panels B and C in Fig. 2 are not shown as they negligibly differ from the curves of Fig. 6D, due to the virtually same ATP(t) dependence in all three cases. The dependence of In Vm on ATP0/ATI~t) in Fig. 4 is built on the basis of data on the rate of translation of the newly added template, hence the state of the mRNA and the degree of its degradation do not affect the results. The model kinetic curves in no way take into account the state of mRNA in the process of translation. None the less, a close agreement with the experiment is observed. Consequently, the decrease of the rate of translation is defined by the decreasing in the activity of the translation system followed by the ATP exhaustion. The mechanism of this phenomenon can be assumed as either the inhibition of stages of the translational cycle a n d / o r the restriction of the number of mRNA particles, which could be take place in translation process. We cannot discuss the first possibility because there are no experimental data except that the ATP level affects on the rate of the initiation of translation [8]. But the data presented in this study permit us to consider that the translation system has the mechanism for the masking of that mRNA which could not be translated throughout because of the lack of ATP energy.
References [1] Atkinson. D.E. (1968) Biochemistry 7, 4030-4034. [2] I,yons, R.T., Nordeen. S.K. and Young, D.A. (1980) J. Biol. Chem. 255, 6330-6334. [3] Gjerde, H. and Helgeland, L. (1984) Acta PhatTaacol. Toxicol. 54, 385-388. [4] Rupniak, H.T.R. and Quincey, R.V. (1975) FEBS Left. 58, 99-101. [5] Bylund-Fellenius. A.G., Ojamaa. K.M., Li, J.B., Wassner, S.J. and Jefferson, L.S. (1984) Am. J. Physiol. 246. E297-E305. [6] Mendelsohn. S.L., Nordeen, S.K. and Young, D.A. (1977) Biochem. Biophys. Res. Commun. 79. 53-60. [7] Walton. G.M. and Gill, G.N. (1975) Biochim. Biophys. Acta 390, 231-245. [8] Hucul, J.A.. Henshaw, E.C. and Young. D.A. (1985) J. Biol. Chem. 260, 15585-15591. [9] Bondar'. V.S., Trofimov, K.P. and Vysotski, E.S. (1992) Biochimiya (Moscow) 57. 1481-1490. [10] lllarionov, B.A.. Markova, S.V.. Bondar', V.S., Vysotski, E.S. and Gitelson, J.l. (1992) Proc. Russian Acad. Sci. 326, 911-913 (in Russian). [1 I] Gurevich, V.V., Pokrovskaya, I.D., Obukhova, T.A. and Zozulya, S.A. (1991) Anal. Biochem. 195. 207-213. [12] Erickson, A.H. and Blobel, G. (1983) in Methods in Enzymology (Fleischer, S and Fleischer B., eds.), Vol. 96, pp. 38-50, Academic Press, New York. [13] Hastings, J.W. and Weber. G. (1963) J. Opt. Soc. Am. 53. 14101415. [14] Decker. C.J. and Parker, R. (1994) Trends Biochem. Sci. 19, 336-34015. [15] Kawarasaki, Y., Kawai, T., Nakano, H. and Yamane, T. (1995) Anal. Biochem. 226, 320-324.
etBiochi~ic~a BiophysicaA~ta Biochimica et Biophysica Acta 1293 (1996) 207-212
Effect of the: ATP level on the overall protein biosynthesis rate in a wheat germ cell-free system S.V. Matveev a,,, L.M. Vinokurov ~, L.A. Shaloiko a, C. Davies a, E.A. Matveeva b, Yu.B. Alakhov a a Branch of the Shemyakin and Ot,chinnikot" Institute ofBioorganic Chemistr3", Russian Academy of Sciences, 142292 Pushchino, Moscow Region, Russia b Institute of Cell Biophysics, Russian Academy of Sciences, 142292 Pushchino, Moscow Region, Russia Received 26 October 1995; accepted 23 November 1995
Abstract
A sensitive assay which examines the effects of ATP level on the overall activity of a cell-free translation system in a protein synthesis is described. The translational activity of cell-free system was measured in terms of a rate of protein synthesis directed by the 'test' template. The test template encodes a photoluminescent protein, obelin. The rate of obelin accumulation was determined from the kinetic curves of obelin mRNA tran:dation. Time-dependent nucleotide level measurements were conducted throughout the translation processes. It has been shown that the rate of translation decreases exponentially with the decrease of the ATP level. This fall in the overall translation rate is due in pa~: to the mRNA becoming inactive in the translation process. This is not caused by degradation, this mRNA can be restored for translatio:a in a fresh cell-free system by phenol treatment. The reported results provide evidence that the level of ATP unambiguously determines the translational activity of the system. Keywords." ATP regulation; Cell-free translation; Obelin mRNA; Luminescence
1. Introduction
According to Atkinson's formulation [1], protein synthesis depends strongly on small changes in the energy charge of a cell. Evidence of the existing correlation between small changes in the ATP abundance and relatively large changes in tt':e rate of protein synthesis have been obtained in many sy:~tems: (i) in isolated fat cells [2], (ii) in rat hepatocytes and rabbit reticulocytes [3,4] and, (iii) in isolated rat muscle cells [5]. The number and size cf polyribosomes decrease during incubation of rat thymus cytoplasm cells in conditions of a lower ATP content in c2~toplasm [6]. These results were interpreted as the inhibition of initiation of translation by ADP and AMP. This conclusion confirms the ADP concentration effect on the degree of the triple initiation complex formation in the cell-free translation system from
Abbreviation~: CP, creatine phosphate; CK, creatine kinase. • Corresponding author. Fax: + 7 095 9252342. 0167-4838/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 4 8 3 8 ( 9 5 ) 0 0 2 4 4 - 8
rabbit reticulocytes [7]. On the other hand, the rate of initiation of translation depends on the A D P / A T P relationship and does not depend on the absolute ATP, ADP and AMP concentrations in the cell-free translation system from Ehrlich tumor cells [8]. Generally the proven fact is that biosynthesis decreases with the decrease of the ATP content [1-5]. The changes in the rate of the initiation step, which is subjected by the ATP content [6-8], was assumed as a real mechanism of that dependence. This is the reason why numerous authors have suggested that the ATP level can play the role of a protein synthesis regulator both in vitro and in vivo. To substantiate this assumption it is necessary, in the first place to show a definite dependence of the protein synthesis rate on the ATP-dependent parameter and, in the second to show in what way this variation of the biosynthesis rate can be achievable. Thanks to the unique sensitivity of the obelin testing method and the designed technique for measurements of the ATP concentration in the translation system we obtained data which enabled us to state the mechanism of such regulation.
208
S.V. Matt eet et al. / Biochimica et Biophysica Acta 1293 (1996) 207-212
2. Materials and methods
2.6. Assay o f obelin
2.1. Materials
Five microliters of the activation buffer containing obelin were added to 0.5 ml of 100 mM Tris, 10 mM EDTA (pH 8.8). An 0.2 ml aliquot of the solution (100 mM CaCI~, 100 mM Tris, pH 8.8) was injected into this volume after the sample was inserted into a luminometer photomultiplier (Model BLM 8802, Nauka, Krasnoyarsk). The luminometcr was calibrated using a light standard [13]. One luminescent unit was equal to 108 quanta/sec. The rate constant of the obelin luminescent reaction was 6.9 sec- i.
Creatine-phosphate kinase, Hepes, potassium acetate, magnesium acetate, spermidine, dithiothreitol, gelatin, 2mercaptoethanol and L-amino acids were from Sigma; ATP, GTP, creatine phosphate were from ICN Biomedicals; Tris, EDTA were from Serva; other chemical reagents of analytical grade were from Reachim (Russia). 2.2. Obelin mRNA
Obelin mRNA was obtained from the Pcull linearized pNOV plasmid [9,10] with the aid of sp6 polymerase [11]. The transcripts were isolated as previously described [11]. The mRNA was dissolved in water to a 0.063 m g / m l concentration. 2.3. Preparation o f the wheat germ extract
The wheat germ extract was prepared according to the procedure of Erickson and Blobel [12] and stored until use at - 70°C. 2.4. Conditions f o r in vitro obelin synthesis
Translation of the obelin mRNA was carried out in the wheat germ cell-free system. The translation mixture (25 /zl) included 8 /zl of a wheat germ extract with an absorbance of A260 = 85 O.U. Other components of the mixture were 40 mM Hepes (pH 7.6), 80 mM potassium acetate, 3.2 mM magnesium acetate, 0.2 mM spermidine, 6 mM dithiothreitol, 2 mM ATP, 50 /xM GTP and 100 /zM each of the 20 amino acids. Creatine phosphokinase (1.12 ~g) with a specific activity of 350 U / m g and creatine phosphate up to 8 mM were added to 25 ~1 of the mixture if the synthesis of obelin was performed in the presence of the ATP-regenerating system. The translation mixture (25 /xl) was incubated for a specific time at 24°C before addition of (0.063 /zg) obelin mRNA. The kinetics of obelin accumulation in the process of translation was measured by luminescence of the activated apo-obelin. 2.5. Activation o f apo-obelin
2.7. Nucleotide measurements
Nucleotide levels throughout protein synthesis were assessed by HPLC analysis on a reverse-phase column in an ion-pair exchange regime. A 5 ,ttl aliquot was withdrawn from the translation system at the times indicated in each experiment and blended with an equal volume of 20 mM tetrabutylammonium, 200 mM NaH2PO ~ (pH 6.0). The sample was loaded onto a Separon C18 TESSEK (Czech Republic) column and eluted with 20 mM tetrabutylammonium, 200 mM NaH2PO 4 (pH 6.0) for 10 min at a flow rate of 0.8 m l / m i n . Absorbance at 254 nm was monitored by a model 210 Hitachi flow spectrophotometer. Individual nucleotide peaks were identified by analysis of known standards. Measurements of the relative nucleotide levels were performed using a Shimadzu CR5A integrator.
3. Results Fig. 1 shows the kinetics of obelin synthesis in a cell-free wheat germ translation system. The addition of a new portion of the template elevates the rate of protein synthesis (Fig. 1). However, the increase in the level of
~,
1200
i
.~ ~ 900 e-.
"~ 600 e..
E=~ --g.~
300 o
0 Activation of apo-obelin was done in 100 ~1 of the reactivation buffer 10 mM Tris-HCl, 0.5 M NaCI, 1 mM EDTA, 5 mM mercaptoethanol, 0.1% gelatin (pH 7.0) containing 5 /zl aliquot from the translation system and 1 /xl of 40 g M coelenterazine solution in methanol. The activation mixture was incubated for 4 h at room temperature in the dark.
I
e-,
120
240
360
Time of translation (min) Fig. 1. Kinetics of protein synthesis. Accumulationof obelin in the wheat germ cell-free translation system in the presence of CP and CK is shown by the luminescence of activated apoobelin, solid circles. Arrows show at which time a new [x)nion of mRNA was added to an aliquot of the translating system. Curves designated with triangles (1), diamonds (2) and hexagons (3) represent the result of this translation.
S. V. Matveev et al. / Biochimica et Biophysica Acta 1293 (1996) 207-212
synthesized protein deperds on the time of addition of a fresh portion of the template. After the system reaches a plateau, the addition of mRNA does not lead to the recovery in protein synthesis (Fig. I, curve 3). Consequently, the system loses its capability for synthesis during the incubation process. The data in Fig. 2 show the incubation time dependence of nucleotide levels in the translation mixtures without mRNA at a different composition of the mixtures. The control experiments in th.: presence of [14C]Leu show no incorporation of radiolabelled Leu in the hot TCA insoluble fraction of the translation mixture. Thus, the decrease in the ATP level is induced by the non-cotranslation ATPase of the wheat germ extract. We used obelin mRNA as the 'test' template to examine the properties of the translation mixture depending on the incubation time. A ~lefined concentration of obelin mRNA was added at a specific time into the translation mixture after its incubation in the absence of exogenous template. The mRNA cortcentration was chosen in such a way that the cotranslational consumption of ATP remained
1
0
0
~
II'~
< 0
o
A
ATP
~ 0
~
]12
209
low (not exceeding the experimental error of ATP determination) in comparison with ATP hydrolysis in processes not connected with translation. Fig. 3 shows the kinetic curves of apo-obelin accumulation in these experiments. When the translation begins in conditions with a low ATP level (Fig. 3, panels B, C, D, curves 4, 5, 6), the curves exhibit a distinctly expressed non-linearity at the first third of the kinetic curve. The system appears to accelerate, reaching a maximal rate (Vm) of translation somewhere in the middle of the kinetic curve (Fig. 3). This feature of the kinetic curves can be evidence that the rate of initiation is a limiting one for the starting of protein synthesis but differs from the rate of reinitiation which defines the efficiency of the translation process as a whole. The values of maximal rate of translation (Vm) achievable under these conditions are shown in Fig. 2 as columns. The decrease of Vm proceeds concurrently with the percentage decrease of the ATP in the solution. The only deviation from this tendency is the Vm value obtained when the obelin mRNA was added to CP containing translation mixture at zero time of incubation, Figs. 2 and
100
B
6
i
o 120
o 240
o 0
100
60
120 6
100
C 75
< Z
D
E O
75
50
25
0
0
60
120
0
Illu 60
0 120
Time of incubation of the translation mixture (min) Fig. 2. Nucleotide levels in a translation mixture incubated without obelin mRNA. Levels of ATP (circles), ADP (.squares) and AMP (triangles) were measured in the translation mixtures with the different composition at the initial moment of incubation: panel (A), 8 mM CP and 0.045% CK; panel (B), 8 mM CP and no CK; panel (C), no CP and 0.045% CK; and panel (D), no CP and no CK. Maximal rates of obelin synthesis followed by an addition of mRNA to the translation mixture were determined from the kinetic curves in Fig. 2 and are plotted as columns at the time of mRNA addition.
S. V. Matveet" et al. / Biochimica et Biophysica Acta 1293 (1996) 207-212
210
I
Table 1 Correlation coefficients calculated for different dependences between Vm and the A(n)P level in the system from data given in Fig. 2
I
600
g
2
120
3
8o
B
Vm (LU/min) In V,,,
ATP
ADP
AMP
ADP/ATP
ATPo / A T P
-~ 0.72
0~ 0.58
b-~ 0.7
0.71 0.89
0.73 0.96
40 ADP(t) / ATP(t) "~
O~ 0
~
120
- '" ~ 60 120
1 180
C
O0 0
120
D
2
4
I
8
-3 4
40
40
4
O 5
5 e-
0wv 0
6 60
120
0 0
60
12(1 0
Time of the obelin mRNA translation (min) Fig. 3. Kinetics of obelin synthesis. Accumulation of obelin in translation systems with the same content as for Fig. 2 are shown as the luminescence of activated apoobelin. The curves differed with the time of incubation of the translation mixture before the mRNA addition. At the times shown below, 0.06 /zg of the obelin mRNA was added to 25 /,tl aliquots of the translation mixture. Panel A, 0' (1), 30' (2), 60' (3), 90' (4), 120' (5), 180' (6), and 240' (7); panels B, C and D, 0' (1), 10' (2), 20' (3), 30' (4), 40' (5), and 60' (6).
3, panels A and C. The reason for this could be explained by a time delay in renaturation of the translation system components in the presence of CP. We cannot exclude that storage of the wheat germ extract at - 7 0 ° C leads to the effect of renaturation kinetics on the translation activity of the system. The best linear correlations are observed between In Vm and the ratio A T P 0 / A T P ( t ) on the one hand, and In Vm and the ratio ADP(t)/ATP(t)on the other hand, (Table 1), (ATP 0 represents the initial ATP concentration in thc translation mixture, or ATP 0 = A T P ( t ) + A D P ( t ) + A M P ( t ) at any moment of time of the incubation process). These dependences are shown in Fig. 4. Panel A shows
-3
I 4
-6
0
I 8
I 12
16
ATP 0 / ATP(t) Fig. 4. Correlation of In Vm with the ATP levels expressed in terms of A D P ( t ) / A T P ( t ) in panel A and ATP0/ATP(t) in panel B. The data designated with the different signs correspond to the data in the different panels of Fig. 2: downward triangles (panel A), circles (panel B), squares (panel C), upward triangles (panel D).
a different tilting of the curves about the axis. However, we have to assume the overall dependences for the same wheat germ extract and m R N A in varying translation conditions. The dependences in panel B satisfy this condition and can be represented as In Vm
=
- -
b ATPo/ATP (t) + a
(1)
Table 2 Obelin mRNA translation in the system with initial conditions as for the control experiment in Fig. 1. The ATP level in the system was restored by the CP addition at 150 rain of the translation process Fig. 1, control data, no addition Time of translation (min): ATP (% ATPo) ADP (% ATP0) AMP (% ATPo) LU per/a.I of translation system ( L U / ~ I )
+ C P up to 16 mM
+CP up to 16 mM + fresh mRNA (0.06/zg/25 ,u,I)
150
180
210
155
180
210
155
180
210
28 37 27 620
21 32 36 625
17 29 42 622
82 10 < 1 619
91 2
90 3
91 3
620
622
79 11 < 1 620
89 3 992
840
S. V. Matveec et al. / Biochimica et Biophysica Acta 1293 (1996) 207-212
600
1
I
~.-~ 400
~-
0
l
,3 60
120
180
Time of translation (min) Fig. 5. Kinetics of the translation of obelin mRNA, which remained active after the desired time Ihrough the biosynthesis reaction with condition as for Fig. 1. The obelin mRNA was prepared by phenol treatment of 25/xl aliquot withdrawn from the translation system at 0 min for curve 1, 60 min for curve 2 and 150 rain for curve 3. The total RNA was added to 25 /zl of fresh trartslation mixture and the luminescence of activated apo-obelin are shown ~.s the result of translation.
where b is the tangent of the experimental curve angle of inclination to the ATPo/ATP axis while a is the segment on the In Vm axis intercepted by the experimental curve. This does not mean that coefficients a and b are constants.
800
i
I
T-
800
i
i I
A 600
", 7,'. I
2 40o
600
2
400
3
"t 4 .~ /
E 2oo
'~..J 60
120
200
-
-
4 5
0
180
0
60
16
i 120
180
e.~
.~ 12o
C
.
I
o= ~ so
Indeed, coefficient b can depend on the activity of the enzymes and differs for different wheat germ extracts. The influence of temperature can be expressed by the dependence of coefficient a on temperature. Let us return for a moment to Fig. I. In accordance with Eq. l, the translation activity of the system could be resumed by the return of the ATP level to the initial one. The data represented in Table 2 show that the ATP level is restored after some CP was added to the translation system at 150 min of the translation process in Fig. I. Simultaneously, the translational activity of the cell-free system is also resumed after the addition of a new mRNA portion (Table 2). However, there is practically no de novo synthesis of the obelin followed by the ATP level recovering without new mRNA addition, (Table 2). This means that there is not any translatable mRNA in this translation system after the ATP level was restored at 150 min of translation process, as in Fig. I. It is well known (for example, [14]) that the template degradation takes place in cell extract solutions. It is also known that the translation in a batch system from wheat germ can be directed for six hours or more, [15]. Hence, it would be expected that the mRNA does not degrade fully during the first 150 min of translation process. The data in Fig. 5 show that the obelin mRNA presents in the translation system in time of its incubation because some mRNA activity can be detected in a fresh translation mixture after deproteinization by phenol treatment. This facts signify that the decrease in the ATP level during the incubation of the cell-free translation system leads to the irreversible masking of a part of the active mRNA.
4. Discussion
5 O0
211
12o
1
80
c-
The main conclusion which follows from the presented results is that the maximal achieved translation rate of test templates with the defined concentration in the wheat germ cell-free system is a function only of the ATP level at a constant temperature. On the grounds of the revealed regularities we can simulate theoretical kinetics of protein synthesis when the protein accumulation rate at any moment of time depends only on the relationship of ATPo/ATP, V(t) = A exp( - b ATP0/ATP ( t ) )
e-
4
.~ 4o
40
~
-
4
© 0
o 60
120
0
60
120
Time of the obelin m R N A translation (min) Fig. 6. Experimental and theoretical kinetics of obclin synthesis. Experimental parts (panels A and C) correspond to the conditions of panels A and D in Fig. 3. Theoretical panels B and D was constructed in accordance with the model of translation described in the text.
where A = e". Coefficients A and b were determined from data in Fig. 4. ATP0 equals 2 mM as in real experiments. The ATP(t) functional dependence for each case was obtained by approximating the real experimental points of the ATP level by polynomials. Fig. 6, panels B and D show the model curves calculated from the ATP level data presented in Fig. 2, panels A and D, respectivcly, and shifted for l0 rain from the beginning of translation as the luminescent activity of the newly synthesized obelin is exhibited only after this time,
212
S. V. Matceec et al. / Biochimica et Biophysica Acta 1293 (1996) 207-212
Fig. 3. With the exception of curve I in Fig. 6A, the other curves coincide well with the respective of experimental kinetic curves. The theoretical curves for panels B and C in Fig. 2 are not shown as they negligibly differ from the curves of Fig. 6D, due to the virtually same ATP(t) dependence in all three cases. The dependence of In Vm on ATP0/ATI~t) in Fig. 4 is built on the basis of data on the rate of translation of the newly added template, hence the state of the mRNA and the degree of its degradation do not affect the results. The model kinetic curves in no way take into account the state of mRNA in the process of translation. None the less, a close agreement with the experiment is observed. Consequently, the decrease of the rate of translation is defined by the decreasing in the activity of the translation system followed by the ATP exhaustion. The mechanism of this phenomenon can be assumed as either the inhibition of stages of the translational cycle a n d / o r the restriction of the number of mRNA particles, which could be take place in translation process. We cannot discuss the first possibility because there are no experimental data except that the ATP level affects on the rate of the initiation of translation [8]. But the data presented in this study permit us to consider that the translation system has the mechanism for the masking of that mRNA which could not be translated throughout because of the lack of ATP energy.
References [1] Atkinson. D.E. (1968) Biochemistry 7, 4030-4034. [2] I,yons, R.T., Nordeen. S.K. and Young, D.A. (1980) J. Biol. Chem. 255, 6330-6334. [3] Gjerde, H. and Helgeland, L. (1984) Acta PhatTaacol. Toxicol. 54, 385-388. [4] Rupniak, H.T.R. and Quincey, R.V. (1975) FEBS Left. 58, 99-101. [5] Bylund-Fellenius. A.G., Ojamaa. K.M., Li, J.B., Wassner, S.J. and Jefferson, L.S. (1984) Am. J. Physiol. 246. E297-E305. [6] Mendelsohn. S.L., Nordeen, S.K. and Young, D.A. (1977) Biochem. Biophys. Res. Commun. 79. 53-60. [7] Walton. G.M. and Gill, G.N. (1975) Biochim. Biophys. Acta 390, 231-245. [8] Hucul, J.A.. Henshaw, E.C. and Young. D.A. (1985) J. Biol. Chem. 260, 15585-15591. [9] Bondar'. V.S., Trofimov, K.P. and Vysotski, E.S. (1992) Biochimiya (Moscow) 57. 1481-1490. [10] lllarionov, B.A.. Markova, S.V.. Bondar', V.S., Vysotski, E.S. and Gitelson, J.l. (1992) Proc. Russian Acad. Sci. 326, 911-913 (in Russian). [1 I] Gurevich, V.V., Pokrovskaya, I.D., Obukhova, T.A. and Zozulya, S.A. (1991) Anal. Biochem. 195. 207-213. [12] Erickson, A.H. and Blobel, G. (1983) in Methods in Enzymology (Fleischer, S and Fleischer B., eds.), Vol. 96, pp. 38-50, Academic Press, New York. [13] Hastings, J.W. and Weber. G. (1963) J. Opt. Soc. Am. 53. 14101415. [14] Decker. C.J. and Parker, R. (1994) Trends Biochem. Sci. 19, 336-34015. [15] Kawarasaki, Y., Kawai, T., Nakano, H. and Yamane, T. (1995) Anal. Biochem. 226, 320-324.