439
Biochimica et Biophysica Acta, 407 (1975) 439--441
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 98433 STIMULATION OF MITOCHONDRIAL PROTEIN SYNTHESIS BY METAL IONS
MARSHALL
P. P R I M A C K
Department of Medicine, Columbia University,College of Physicians and Surgeons, N e w York, N.Y. (U.S.A.) (Received June 23rd, 1975)
Summary 1--10 pM Cu 2÷, Ag ÷, and Au 3÷ were found to stimulate rat liver mitochondrial protein synthesis in vitro. Cu 2÷ and Ag ÷ also produced an increase in mitochondrial volume ("swelling"). Thus, thyroid hormones and their analogs are not unique, as suggested previously (Buchanan, J.L., Primack, M.P. and Tapley, D.F. (1970) Endocrinology 87, 993--999), in stimulating both mitochondrial protein synthesis and swelling. Furthermore, the data suggest a role for Cu z÷ in the regulation of mitochondrial protein synthesis.
Introduction In previous experiments substances that induce mitochondrial swelling were examined for their effects on mitochondrial protein synthesis [1]. Thyroid hormones and their analogs were the only swelling agents that increased protein synthesis. In particular, metal ions had no effect on protein synthesis at 1 #M, were usually inhibitory at 10 #M and were markedly inhibitory at 100 pM. Other concentrations were not tested at that time. However, in more recent experiments concentrations of swelling agents from 1 to 10 pM have been examined for their effects on protein synthesis and swelling. While steroids, fatty acids and most metals produced no effect or inhibition, this report demonstrates that Cu 2÷, Ag+ and Au 3÷, chemically related in the periodic table of the elements, markedly enhanced protein synthesis. Materials and Methods Mitochondria from 150 g male Sherman rats were prepared in 0.25 M sucrose and incubated for studies on protein synthesis and swelling in a medium of 50 mM Tris • HC1 at pH 7.4, 100 mM sucrose, 50 mM KC1, 10 mM
440 MgC12, 10 mM sodium succinate, 10 mM potassium phosphate at pH 7.4, 1 pM [3 H] leucine (900 Ci/mol) and 19 other amino acids at 5 pM each as previously described [ 1]. Results and Discussion The effects of CuC12, AgNOs, and HAuCI4 on leucine incorporation and swelling are presented in Table I. All three stimulated leucine incorporation at 2 pM and by 6 pM stimulation of incorporation was maximal. Effects on swelling were variable. AgNO3 and CuC12 produced statistically significant swelling by 6 and 10 pM, respectively, whereas HAuC14 appeared to inhibit to a small extent the swelling normally observed in the medium employed. Since CuSO4 was as affective as CuC12 in stimulating leucine incorporation and swelling and since neither NaC1 nor NaNO3 produced swelling or increased leucine incorporation, the anions were n o t responsible for the observed effects. In other experiments it was shown that the increase in leucine incorporation reflected a true stimulation of protein synthesis since it was suppressed by inhibitors of protein synthesis (chloramphenicol and puromycin) and inhibitors of ATP formation (dinitrophenol and oligomycin) b u t was unaffected by cycloheximide, an inhibitor of cytoplasmic protein synthesis [1], and since the increase in incorporation was n o t the result of a change in the specific activity of the endogenous pool of leucine [2]. The ability to stimulate protein synthesis was also observed when succinate was replaced by other oxidizable substrates including pyruvate, malate, fumarate, isocitrate, a-ketoglutarate, citrate, ~-glycerophosphate and ~-hydroxybutyrate. When succinate and phosphate were replaced by ATP plus a regenerating system of phosphoenolpyruvate and pyruvate kinase [3] 4 pM CuC12, AgNO3, and HAuC14 increased leucine incorporation 85 + 25, 140 -+ 40 and 115 -+ 30% (mean + S.E.), respectively, even TABLE I THE EFFECT SWELLING
OF CuC12, AgNO 3 AND HAuC14 ON MITOCHONDRIAL
PROTEIN
SYNTHESIS
AND
T h e r e s u l t s are e x p r e s s e d as m e a n ± S . E . T h e n u m b e r o f e x p e r i m e n t s is g i v e n in p a r e n t h e s e s , n.s. = n o t statistically s i g n i f i c a n t . Metal
~uM
P e r c e n t c h a n g e in leucine incorporation
P
Percent change in pellet weights
P
CuC12
2 4 6 10
33 81 127 55
+- 1 3 ± 11 ± 22 +_ 2 4
(8) (31) (14) (9)
Biochimica et Biophysica Acta, 407 (1975) 439--441
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 98433 STIMULATION OF MITOCHONDRIAL PROTEIN SYNTHESIS BY METAL IONS
MARSHALL
P. P R I M A C K
Department of Medicine, Columbia University,College of Physicians and Surgeons, N e w York, N.Y. (U.S.A.) (Received June 23rd, 1975)
Summary 1--10 pM Cu 2÷, Ag ÷, and Au 3÷ were found to stimulate rat liver mitochondrial protein synthesis in vitro. Cu 2÷ and Ag ÷ also produced an increase in mitochondrial volume ("swelling"). Thus, thyroid hormones and their analogs are not unique, as suggested previously (Buchanan, J.L., Primack, M.P. and Tapley, D.F. (1970) Endocrinology 87, 993--999), in stimulating both mitochondrial protein synthesis and swelling. Furthermore, the data suggest a role for Cu z÷ in the regulation of mitochondrial protein synthesis.
Introduction In previous experiments substances that induce mitochondrial swelling were examined for their effects on mitochondrial protein synthesis [1]. Thyroid hormones and their analogs were the only swelling agents that increased protein synthesis. In particular, metal ions had no effect on protein synthesis at 1 #M, were usually inhibitory at 10 #M and were markedly inhibitory at 100 pM. Other concentrations were not tested at that time. However, in more recent experiments concentrations of swelling agents from 1 to 10 pM have been examined for their effects on protein synthesis and swelling. While steroids, fatty acids and most metals produced no effect or inhibition, this report demonstrates that Cu 2÷, Ag+ and Au 3÷, chemically related in the periodic table of the elements, markedly enhanced protein synthesis. Materials and Methods Mitochondria from 150 g male Sherman rats were prepared in 0.25 M sucrose and incubated for studies on protein synthesis and swelling in a medium of 50 mM Tris • HC1 at pH 7.4, 100 mM sucrose, 50 mM KC1, 10 mM
440 MgC12, 10 mM sodium succinate, 10 mM potassium phosphate at pH 7.4, 1 pM [3 H] leucine (900 Ci/mol) and 19 other amino acids at 5 pM each as previously described [ 1]. Results and Discussion The effects of CuC12, AgNOs, and HAuCI4 on leucine incorporation and swelling are presented in Table I. All three stimulated leucine incorporation at 2 pM and by 6 pM stimulation of incorporation was maximal. Effects on swelling were variable. AgNO3 and CuC12 produced statistically significant swelling by 6 and 10 pM, respectively, whereas HAuC14 appeared to inhibit to a small extent the swelling normally observed in the medium employed. Since CuSO4 was as affective as CuC12 in stimulating leucine incorporation and swelling and since neither NaC1 nor NaNO3 produced swelling or increased leucine incorporation, the anions were n o t responsible for the observed effects. In other experiments it was shown that the increase in leucine incorporation reflected a true stimulation of protein synthesis since it was suppressed by inhibitors of protein synthesis (chloramphenicol and puromycin) and inhibitors of ATP formation (dinitrophenol and oligomycin) b u t was unaffected by cycloheximide, an inhibitor of cytoplasmic protein synthesis [1], and since the increase in incorporation was n o t the result of a change in the specific activity of the endogenous pool of leucine [2]. The ability to stimulate protein synthesis was also observed when succinate was replaced by other oxidizable substrates including pyruvate, malate, fumarate, isocitrate, a-ketoglutarate, citrate, ~-glycerophosphate and ~-hydroxybutyrate. When succinate and phosphate were replaced by ATP plus a regenerating system of phosphoenolpyruvate and pyruvate kinase [3] 4 pM CuC12, AgNO3, and HAuC14 increased leucine incorporation 85 + 25, 140 -+ 40 and 115 -+ 30% (mean + S.E.), respectively, even TABLE I THE EFFECT SWELLING
OF CuC12, AgNO 3 AND HAuC14 ON MITOCHONDRIAL
PROTEIN
SYNTHESIS
AND
T h e r e s u l t s are e x p r e s s e d as m e a n ± S . E . T h e n u m b e r o f e x p e r i m e n t s is g i v e n in p a r e n t h e s e s , n.s. = n o t statistically s i g n i f i c a n t . Metal
~uM
P e r c e n t c h a n g e in leucine incorporation
P
Percent change in pellet weights
P
CuC12
2 4 6 10
33 81 127 55
+- 1 3 ± 11 ± 22 +_ 2 4
(8) (31) (14) (9)
