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The effect of monovalent cations on the incorporation of various amino acids in protein synthesis by isolated rat spleen nuclei
580
SHORT COMMUNICATIONS
BBA 93427
The effect of monovolent cations on the incorporotion of vorious amino acids in protein synthesis by isoloted rat spleen nuclei The previous paper reported that cell nuclei retaining the ability to synthesize proteins in vitro could be isolated from rat spleen cells using isotonic sucrose solution containing 3.0 mM MgC12 as the isolation medium 1. In the course of the experiments, the effect of monovalent cations on the incorporation of various amino acids was studied, since special note has been taken of the effect of monovalent cations on the protein synthesis by isolated nuclei. ALLFREY et al3 presented a detailed report on this problem, using an isolated calf thymus nuclei system, and concluded that incorporation of amino acids into proteins by isolated nucleus is dependent on Na ÷. This result has sometimes been used as a criterion for nuclear protein synthesis 3. However, the results of other incorporation profiles effected by monovalent cations have been described 4,5. Studies on this subject should be performed in more detail. Along this line, the author studied the effect of monovalent cations on the incorporation of several amino acids in the protein synthesis by isolated nuclei from rat spleen cells. Figs. i and 2 depict the experimental results. Regarding the enhancement of the incorporation by Na +, the seven amino acids examined in the present experiment fell into two separate groups which may be designated Group l, comprising glycine, L-leucine, L-methionine and r.-phenylalanine, and Group II, comprising L-alanine, L-proline and L-threonine (Fig. i). The incorporation of amino acids of Group I was not notably enhanced by Na +. The maximal increase obtained in this case was limited to less than 30 % of the controls (no monovalent cations added). On the other hand, 3C
15
2C
I0
~o
'5
"5
.~_ 0
NQ÷ (mM)
0~ :
5 15 K~-(rnM)
4~ 90
Fig. I. E f f e c t of N a + on t h e i n c o r p o r a t i o n of [14C]amino acids into p r o t e i n s b y isolated r a t spleen nuclei. Nuclei were isolated from r a t spleen cells according to t h e p r o c e d u r e described p r e v i o u s l y 1. T h e c o m p l e t e m i x t u r e ( I . o m l ) c o n t a i n e d t h e following c o m p o n e n t s a t t h e final c o n c e n t r a t i o n s t a t e d : 19o m M sucrose, 20 m M glucose, 6. 5 m M MgCI~, 25 m M Tris-HC1 (pH 7.o), a p p r o x . I m g (dry wt.) of nuclei, o. I/~C of [14C]amino acid a n d NaC1 ( c o n c e n t r a t i o n specified in abscissa). I n c u b a t i o n w a s carried o u t a t 37 ° for 3 ° mill w i t h g e n t l e s h a k i n g , a n d m a t e r i a l s precipitable w i t h 5 % trichloroacetic acid were u s e d for [14C]amino acid incorporation. I - I , L-[1*C]alanine; A-LX, [I*C]glycine; D-U], L-[14C]leucine; O - O , L-[14C]methionine; ® - ® , L-[14C]phenylalanine; 0 - - 0 , L[ltC]proline; A - • , L-piC]threonine. Fig. 2. E f f e c t of K + o n t h e i n c o r p o r a t i o n of [i*Clamino acids into p r o t e i n s b y isolated r a t spleen nuclei. E x p e r i m e n t a l c o n d i t i o n s a n d s y m b o l s as in Fig. I.
Biochim. Biophys. Acta, 182 (1969) 58o-582
I IN
~-Ala L-Pro L-Thr
Group II
GUY L-Leu L-Met L-Phe
Group I
acid
Amino
123 98 119
240 6.2 I40
82
Specific activity (mC/mmole)
241 ‘7 32
2258
925 3%
I.2
9 0.8
5’ 63 79 73
1008
255
1218
‘035 385’ 108 2952
Naf Counts/min
None
pmoles
38
I2
45”
57 72 79 95
pmoles
cations in the incubation medium
Countslmin
Monovalent
PRESENCE
OR IN
128 418
592
106 2610
3941
813
Counts/min
16
6
22’
87;
45 74
pmoles
of each monovalent
THE
K’
Incubation conditions as described in the legend to Fig. I. Concentration experiments marked with an asterisk in which it was adjwted to 5 mM.
MOLAR AMOUNTOF AMINO ACIDS INCORPORATEDBY ISOLATED NUCLEI
TABLE THE
ABSENCE
OF MONOVALENT
CATIONS
224 II 27
640 3104 75 1643
8 0.5 I.0
35 59 55 53
219 IO 30
3344 76 1763
940
Rb+ Countslmin
Li+
Countslmin
pmoles
8 0.5 I.1
57
56
i;
pmoles
cation was adjusted to 15 mM except in the
582
SHORT COMMUNICATIONS
the incorporation of the amino acids of Group I I was greatly enhanced by Na +. The enhancement of L-threonine incorporation reached 30 times the control values at 15 mM Na +. The corresponding values for L-alanine and for L-proline were 5 times at 5 mM and 15 times at 15 raM, respectively. When Na + was replaced with K +, these seven amino acids fell into the same two separate groups (Fig. 2). The incorporation of the amino acids of Group I was again not notably enhanced by K+, while the amino acid of Group II was incorporated at a much greater rate under the same conditions. This was similar to the results obtained in the experiments on the effect of Na +, though the extent of enhancement by K + was approximately half that produced by Na +. Effects of Li+ and Rb + were also examined in a similar fashion, but no marked differences in the incorporation of these seven amino acids was observed. Table I gives the molar amounts of each amino acid that were incorporated into proteins during 30 min incubation. It is evident that the amounts of Group I amino acids incorporated were independent of monovalent cations, while the amino acids comprising Group II were incorporated to a comparable extent only if Na + or K + was present. It can be mentioned, therefore, that the strict requirement for Na + is true only for the incorporation of a certain group of amino acids and that Na + can be replaced by K+, although the latter is only half as effective. A simple correlation between the chemical structure of amino acids and the requirement for a monovalent cation has not yet been observed by the author. Results obtained with other isolated nuclear systems in which slight enhancement by Na + and some inhibition by K+ were shown in the incorporation of glycine, L-leucine and L-lysinO-~ may mean that these amino acids would also fall into Group I in those isolated nuclear systems. The author would like to thank Dr. H. Naora, Dr. M. Amano, Dr. M. Izawa, Dr. T. Mita and Dr. H. Mitsui for providing facilities and for useful discussions. The author is also indebted to Dr. T. Shiroya and Dr. Y. Akita for their interest and encouragement.
Biology Division, National Cancer Center, Research Institute, Chuo-ku, Tokyo (Japan)
JuNjI TsuzuKI*
I J. TsUzuKI AND H. NAORA, Biochim. Biophys. Acta, 169 (1968) 55 o. 2 V. G. ALLFREY, R. MEUDT, J. w . HOPKINS AND A. E. I~IRSKY, Proc. Natl. Acad. Sci. U.S., 47 (1961) 907. 3 R. RENDI, Exptl. Cell Res., 19 (196o) 489. 4 J. MALEC, L. KORNACKA AND M. WOJNAROWSKA,Exptl. Cell Res., 34 (1964) 188. 5 K. R. REES AND C*. F. ROWLAND, Biochem. J., 78 (1961) 89. 6 W. G. FLAMM, M. L. BIRNSTIEL AND P. FILNER, Biochim. Biophys. Acta, 76 (1963) i i o . 7 M. L. BIRNSTIEL, M. I. H. CHIPCHASE AND R. J. HAYES, Biochim. Biophys. Acta, 55 (1962) 728.
Received March 24th, 1969 * P r e s e n t address: L a b o r a t o r y of R a d i a t i o n Biology, F a c u l t y of Science, U n i v e r s i t y of Tokyo, H o n g o , Tokyo, J a p a n .
Biochim. Biophys. Acta, i82 (I969) 580-582
SHORT COMMUNICATIONS
BBA 93427
The effect of monovolent cations on the incorporotion of vorious amino acids in protein synthesis by isoloted rat spleen nuclei The previous paper reported that cell nuclei retaining the ability to synthesize proteins in vitro could be isolated from rat spleen cells using isotonic sucrose solution containing 3.0 mM MgC12 as the isolation medium 1. In the course of the experiments, the effect of monovalent cations on the incorporation of various amino acids was studied, since special note has been taken of the effect of monovalent cations on the protein synthesis by isolated nuclei. ALLFREY et al3 presented a detailed report on this problem, using an isolated calf thymus nuclei system, and concluded that incorporation of amino acids into proteins by isolated nucleus is dependent on Na ÷. This result has sometimes been used as a criterion for nuclear protein synthesis 3. However, the results of other incorporation profiles effected by monovalent cations have been described 4,5. Studies on this subject should be performed in more detail. Along this line, the author studied the effect of monovalent cations on the incorporation of several amino acids in the protein synthesis by isolated nuclei from rat spleen cells. Figs. i and 2 depict the experimental results. Regarding the enhancement of the incorporation by Na +, the seven amino acids examined in the present experiment fell into two separate groups which may be designated Group l, comprising glycine, L-leucine, L-methionine and r.-phenylalanine, and Group II, comprising L-alanine, L-proline and L-threonine (Fig. i). The incorporation of amino acids of Group I was not notably enhanced by Na +. The maximal increase obtained in this case was limited to less than 30 % of the controls (no monovalent cations added). On the other hand, 3C
15
2C
I0
~o
'5
"5
.~_ 0
NQ÷ (mM)
0~ :
5 15 K~-(rnM)
4~ 90
Fig. I. E f f e c t of N a + on t h e i n c o r p o r a t i o n of [14C]amino acids into p r o t e i n s b y isolated r a t spleen nuclei. Nuclei were isolated from r a t spleen cells according to t h e p r o c e d u r e described p r e v i o u s l y 1. T h e c o m p l e t e m i x t u r e ( I . o m l ) c o n t a i n e d t h e following c o m p o n e n t s a t t h e final c o n c e n t r a t i o n s t a t e d : 19o m M sucrose, 20 m M glucose, 6. 5 m M MgCI~, 25 m M Tris-HC1 (pH 7.o), a p p r o x . I m g (dry wt.) of nuclei, o. I/~C of [14C]amino acid a n d NaC1 ( c o n c e n t r a t i o n specified in abscissa). I n c u b a t i o n w a s carried o u t a t 37 ° for 3 ° mill w i t h g e n t l e s h a k i n g , a n d m a t e r i a l s precipitable w i t h 5 % trichloroacetic acid were u s e d for [14C]amino acid incorporation. I - I , L-[1*C]alanine; A-LX, [I*C]glycine; D-U], L-[14C]leucine; O - O , L-[14C]methionine; ® - ® , L-[14C]phenylalanine; 0 - - 0 , L[ltC]proline; A - • , L-piC]threonine. Fig. 2. E f f e c t of K + o n t h e i n c o r p o r a t i o n of [i*Clamino acids into p r o t e i n s b y isolated r a t spleen nuclei. E x p e r i m e n t a l c o n d i t i o n s a n d s y m b o l s as in Fig. I.
Biochim. Biophys. Acta, 182 (1969) 58o-582
I IN
~-Ala L-Pro L-Thr
Group II
GUY L-Leu L-Met L-Phe
Group I
acid
Amino
123 98 119
240 6.2 I40
82
Specific activity (mC/mmole)
241 ‘7 32
2258
925 3%
I.2
9 0.8
5’ 63 79 73
1008
255
1218
‘035 385’ 108 2952
Naf Counts/min
None
pmoles
38
I2
45”
57 72 79 95
pmoles
cations in the incubation medium
Countslmin
Monovalent
PRESENCE
OR IN
128 418
592
106 2610
3941
813
Counts/min
16
6
22’
87;
45 74
pmoles
of each monovalent
THE
K’
Incubation conditions as described in the legend to Fig. I. Concentration experiments marked with an asterisk in which it was adjwted to 5 mM.
MOLAR AMOUNTOF AMINO ACIDS INCORPORATEDBY ISOLATED NUCLEI
TABLE THE
ABSENCE
OF MONOVALENT
CATIONS
224 II 27
640 3104 75 1643
8 0.5 I.0
35 59 55 53
219 IO 30
3344 76 1763
940
Rb+ Countslmin
Li+
Countslmin
pmoles
8 0.5 I.1
57
56
i;
pmoles
cation was adjusted to 15 mM except in the
582
SHORT COMMUNICATIONS
the incorporation of the amino acids of Group I I was greatly enhanced by Na +. The enhancement of L-threonine incorporation reached 30 times the control values at 15 mM Na +. The corresponding values for L-alanine and for L-proline were 5 times at 5 mM and 15 times at 15 raM, respectively. When Na + was replaced with K +, these seven amino acids fell into the same two separate groups (Fig. 2). The incorporation of the amino acids of Group I was again not notably enhanced by K+, while the amino acid of Group II was incorporated at a much greater rate under the same conditions. This was similar to the results obtained in the experiments on the effect of Na +, though the extent of enhancement by K + was approximately half that produced by Na +. Effects of Li+ and Rb + were also examined in a similar fashion, but no marked differences in the incorporation of these seven amino acids was observed. Table I gives the molar amounts of each amino acid that were incorporated into proteins during 30 min incubation. It is evident that the amounts of Group I amino acids incorporated were independent of monovalent cations, while the amino acids comprising Group II were incorporated to a comparable extent only if Na + or K + was present. It can be mentioned, therefore, that the strict requirement for Na + is true only for the incorporation of a certain group of amino acids and that Na + can be replaced by K+, although the latter is only half as effective. A simple correlation between the chemical structure of amino acids and the requirement for a monovalent cation has not yet been observed by the author. Results obtained with other isolated nuclear systems in which slight enhancement by Na + and some inhibition by K+ were shown in the incorporation of glycine, L-leucine and L-lysinO-~ may mean that these amino acids would also fall into Group I in those isolated nuclear systems. The author would like to thank Dr. H. Naora, Dr. M. Amano, Dr. M. Izawa, Dr. T. Mita and Dr. H. Mitsui for providing facilities and for useful discussions. The author is also indebted to Dr. T. Shiroya and Dr. Y. Akita for their interest and encouragement.
Biology Division, National Cancer Center, Research Institute, Chuo-ku, Tokyo (Japan)
JuNjI TsuzuKI*
I J. TsUzuKI AND H. NAORA, Biochim. Biophys. Acta, 169 (1968) 55 o. 2 V. G. ALLFREY, R. MEUDT, J. w . HOPKINS AND A. E. I~IRSKY, Proc. Natl. Acad. Sci. U.S., 47 (1961) 907. 3 R. RENDI, Exptl. Cell Res., 19 (196o) 489. 4 J. MALEC, L. KORNACKA AND M. WOJNAROWSKA,Exptl. Cell Res., 34 (1964) 188. 5 K. R. REES AND C*. F. ROWLAND, Biochem. J., 78 (1961) 89. 6 W. G. FLAMM, M. L. BIRNSTIEL AND P. FILNER, Biochim. Biophys. Acta, 76 (1963) i i o . 7 M. L. BIRNSTIEL, M. I. H. CHIPCHASE AND R. J. HAYES, Biochim. Biophys. Acta, 55 (1962) 728.
Received March 24th, 1969 * P r e s e n t address: L a b o r a t o r y of R a d i a t i o n Biology, F a c u l t y of Science, U n i v e r s i t y of Tokyo, H o n g o , Tokyo, J a p a n .
Biochim. Biophys. Acta, i82 (I969) 580-582