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Carbon tetrachloride-induced loss of microsomal messenger-ribonucleic acid activity
Ib; 4
SHORT COMMUN ICATIONS
Briefly, though these resalts are not sufficiently striking to enable one t o conclude that hormonal control of differentiation exists in embryonic blood islands, they encourage further research to discover such mechanisms in the differentiating embryo. This work has been supported by Euratom (Contract E u r a t o m - U L B o16-61-1o ABIB). Erythropoietin was supplied by the National Heart Institute, Bethesda, Md., U.S.A. and by Dr. E. C~OLDWASSER,Argonne Cancer Research Institute, Chicago, to whom we address our thanks. Laboratoire de Morphologie Animale, P. MALPOIX Facultd des Sciences de l'Universitd Libre de Bruxelles, Brussels (Belgium) I 2 3 4 5 6 7 8 9 io ii 12 13
L. O. JACOBSON, C. W. GURNE¥ AND E. GOLDWASSER, Adv. Int. Med., IO (196o) 297. R. J. COLE AND J. PAUL, J. Embryol. Exptl. Morphol., 15 (1966) 245. S. B. KRANTZ, O. GALLIEN LARTIGUE AND E. GOLDWASSER, J. Biol. Chem., 238 (1963) 4085 . E. a . POWSNER AND L. BEERMAN, Blood, 14 (1959) 1213. 8. B. KRANTZ AND E. GOLDWASSER, Biochim. Biophys. Acta, lO 3 (I965) 325 . M. P. PIEBER-PERETTA, R. W. HODGSON AND M. A. PERETTA, Biochim. Biophys. Acta, 95 (1965) 325 . A. KOENER, Bull. SoC. Chim. Biol,, 48 (1966) lO31. L. G. BRITT AND H. HERMANN, J. Embryol. Exptl. Morphol., 7 (1959) 66. V. HAMBURGER AND H. J. HAMILTON, J. Morphol., 88 (1951) 49. A. HELL, J. Embryol, Exptl. Morphol., 12 (1964) 6o9. P. MALPOIX AND S. LIMBOSCH, J. Embryol. Exptl. Morphol., 16 (I966) 439G. A. BRAY, Anal. Biochem., I (196o) 279. V. M. INGRAM AND B. MOSS, Proc. Natl. Acad. Sci. U.S., 54 (1965) 969.
Received April 4th, 1967 Biochim. Biophys. Acta, 145 (1967) i 8 I I84
BBA 93249
Carbon tetrachloride-induced loss of microsomal messenger-ribonucleic acid activity The administration of CC14 to rats results in widespread morphologic and biochemical damage to the liver 1-3. SMUCKLER AND BENDITT4 have shown that CC14 administration causes a decrease in the in vivo incorporation of amino acids into hepatic protein and in the in vitro incorporation of amino acids into protein by microsomes and ribosomes. MAGER, BORNSTEIN AND HALBREICH 5 have reported a "paradoxical" increase in poly (U)-directed phenylalanine incorporation by liver microsomes from CC14-treated animals. This increase accompanies the loss of endogenous microsomal amino acid incorporating activity. These investigators suggested that the destruction of microsomal messenger RNA (mRNA) by CC14 treatment may account for both the decreased endogenous mRNA-directed amino acid incorporation and the increase in poly (U)-directed phenylalanine incorporation. We have found that microsomes from CC14-treated rats function similarly to microsomes made devoid of mRNA activity by a preincubation procedure previously described ~. Abbreviation: m R N A , messenger RNA; poly (U), polyuridylic acid.
Biochin~. Biophys. ,4cta, 145 (1967) 184 187
SHORT COMMUNICATIONS
185
T A B L E I. THE INCORPORATION OF L-[14CIPHENYLALANINE BY MICROSOMES FROM NORMAL AND CCI~-TREATED RATS
2oo-g Female S p r a g u e - D a w l e y r a t s were fasted 16 h prior to the i n t r a p e r i t o n e a l injection of either o.8 ml of mineral oil or 0.8 ml of CC14 in mineral oil (i : i, b y vol.). 2 h later the animals were sacrificed. 2-g p o r t i o n s of liver in io ml of o.25 M sucrose solution containing 6 mM m e r c a p t o e t h a n o l were homogenized in a P o t t e r - E l v e h j e m grinder for 3 ° sec. The h o m o g e n a t e was filtered t h r o u g h a 75/,2 nylon m e s h a n d centrifuged at 15 ooo x g for 15 rain to r e m o v e cell debris, nuclei, and mitochondria, io-ml alJquots of the 15 ooo × g s u p e r n a t a n t were centrifuged for 60 rain at ioo ooo X g. The resulting microsomal pellet was suspended in 2.8 ml of a i mM Tris buffer (pH 7.4), 1.5 mM MgClz, 5 mM KC1 and 6 mM m e r c a p t o e t h a n o l . The s t a n d a r d reaction m i x t u r e s were set u p in duplicate and contained in 2 ml: 12 mM p o t a s s i u m p h o s p h a t e (pH 7.4), 1.5 mM ATP, o. 3 mM GTP, 12 mM MgC1v 4 ° mM creatine p h o s p h a t e , o. 5 m g creatine p h o s p h o k i n a s e , 6 mM m e r c a p t o e t h a n o l , 77 mM sucrose, 43 #M u n i f o r m l y l a b e l e d L-[~4C~phenylalanine (specific a c t i v i t y io/,C//*mole), 0.2 ml ioo ooo x g s u p e r n a t a n t (containing a p p r o x . 2 mg of protein) and o. 4 ml of microsomes (containing approx. 7 mg of protein). W h e r e indicated, p r e i n c u b a t i o n of microsomes was p e r f o r m e d for 12 rain at 37 ° in the absence of L-E~4CJphenylalanine. The preincub a t i o n c o m p o n e n t s and conditions were identical to the conditions described above except for the following changes: I n a v o l u m e of 1.5 ml were p r e s e n t : creatine p h o s p h a t e 27 mM, creatine p h o s p h o kinase o.25 m g a n d MgC12 8 mM. After p r e i n c u b a t i o n s a t u r a t i n g a m o u n t s of poly (U) (4oo/*g), L-p4C]phenylalanine and additional creatine p h o s p h a t e , creatine p h o s p h o k i n a s e , MgC1e and w a t e r were added resulting in a reaction m i x t u r e identical to the s t a n d a r d incubation mix except for the presence of poly (U). I n c u b a t i o n was performed for 15 rain at 37 °. The reaction w a s s t o p p e d w i t h io ~g trichloroacetic acid. The precipitated p r o t e i n s were purified b y a modification ~ of the m e t h o d of Siekevitz and r a d i o a c t i v i t y m e a s u r e d in a t h i n - w i n d o w gas-flow counter. The r e s u l t s are expressed as c o u n t s / m g microsomal p r o t e i n per min.
Microsomes
A (no preincubation )
B (preincubated)
-- P o l y (U)
+ Poly (U)
-- Poly (U)
+ Poly (U)
Normal CC14
312 55
629 1316
3 5
1630 1592
Table IA confirms the findings of MAGER, BORNSTEIN AND HALBREICH5 and shows that non-preincubated microsomes from CC14-treated rats exhibit a reduced capacity to incorporate phenylalanine in the absence of poly (U) and an enhanced capacity to incorporate phenylalanine in the presence of poly (U) when compared to normal non-preincubated microsomes. In other studies 6 we have shown that preineubation of liver microsomes results in a complete loss of endogenous amino acid incorporating activity and an increase in poly (U)-directed phenylalanine incorporation. The result of preincubating microsomes from CC14-treated rats is shown in Table IB and is compared with that obtained when normal microsomes are preincubated. Microsomes from CC14-treated rats after preincubation behave exactly as do normal preincubated mierosomes. Thus, both exhibit no amino acid incorporation in the absence of poly (U) and identical phenylalanine incorporation in the presence of poly (U). This shows that CC14treatment does not alter the responsiveness of the mierosomal protein-synthesizing apparatus to exogenous mRNA (poly (U)), at least not to any greater extent than occurs during the preincubation period. In addition, the behavior of non-preincubated microsomes from CC14-treated rats (Table IA) shouts a great similarity to that of either normal or CC14microsomes which have been made devoid of endogenous mRNA by preincubation. The low level of endogenous mRNA-directed amino acid incorporation by non-preincubated CC14 Biochim. Biophys. dcla, I45 (I967) 184-187
I~6
SHORT COMMUNICATIONS
2500
I
NORMAL MICROSOMES ~#/ (PREINCUE/A TED, ooo_
,soo~
~eINCUaare°Jes
•
I
,,
J
~,vo~v-
d~ ::"
i z""l" L ""~
O/
50
,
NORMALM,CRO,OM. .-O............................... 0 ................................ 0 ............................... (
L~
ioo
200 poLr
L
~oo
400
Ul#gl~il'
Fig. I. I n c o r p o r a t i o n of L-[llC]phenylalanine at different c o n c e n t r a t i o n s of poly (U) by micros o m e s f r o m n o r m a l and CC14-treated rats. E x p e r i m e n t a l conditions are described in t h e l e g e n d to Table I.
microsomes and the somewhat smaller stimulation b y poly (U) is most likely due to a less complete removal of m R N A in vivo. Fig. i shows that microsomes from CC14-treated rats exhibit a poly (U) saturation curve that is considerably different from that of normal non-preincubated microsomes and resembles closely the curve obtained when normal or CC1a microsomes are preincubated in vitro. Thus, phenylalanine incorporating activity of normal non-preincubated microsomes is saturated by IOO#g/ml of poly (U) while preincubated microsomes from either CC14-treated or normal rats are saturated b y from 2oo to 3oo #g/ml of poly (U). Non-preincubated microsomes from CCl~-treated rats show a saturation between 25o and 30o/~g/ml of poly (U) and this suggests an increased T A B L E 1I BINDING
OF 3I-I-LABELED
POLY
(U)
BY MICROSOMES
FROM NORMAL
AND CCI4-TREATED
RATS
Microsomes were p r e p a r e d a n d p r e i n c u b a t e d in the m a n n e r described. To each reaction m i x t u r e 0 . 2 / , m o l e s of 8H-labeled poly (U) (specific activity 3/~C]/zmole) was added. The entire m i x t u r e w a s t h e n layered over 9 ml of a 0.25 M sucrose in io mM Tris buffer (pH 7-4) and 5 mM MgCI, solution and centrifuged at ioo ooo × g for I 11. The m i c r o s o m a l pellet w a s t h e n r e s u s p e n d e d solubilized w i t h H y a m i n e a n d r a d i o a c t i v i t y m e a s u r e d in a P a c k a r d liquid scintillation counter. The results are expressed as c o u n t s / r a g mierosomal p r o t e i n p e r rain.
Microsomes
No preincubation
Preincubated
Normal CC14
678 lO2O
lO8 i 117o
Biochirn. Biophys. Acta, 145 (1967) 184-187
187
SHORT COMMUNICATIONS
ability of such microsomes to interact with poly (U). Non-preincubated microsomes from CC14-treated rats are also more responsive to low levels of poly (U) as are normal preincubated microsomes 6. The binding of all-labeled poly (U) by microsomes from normal and CC14treated rats is shown in Table II. Non-preincubated microsomes from CC14-treated rats bind 40 % more poly (U) than do normal non-preincubated microsomes. In contrast, following preincubation, both microsomal preparations bind comparable amounts of poly (U). In fact, non-preincubated microsomes from CC14-treated rats bind an amount of 3H-labeled poly (U) comparable to that bound by preincubated microsomes from either normal or CC14-treated rats. These binding studies indicate that a likely explanation for the preincubation-induced loss of mRNA activity is that preincubation causes a removal or destruction of microsomal mRNA and thereby increases the number of available poly (U) binding sites on the microsome. SMUCKLER AND BENDITT4 have found that CC14 administration results in a conversion of polyribosomes to monosomes (54-S particles also appear). This may represent the morphological counterpart in a non-membrane bound preparation to the functional changes encountered in the microsomal preparation. In summary we have shown that microsomes free of mRNA activity and microsomes from CC14-treated rats behave similarly exhibiting a very low level of endogenous mRNA-directed amino acid incorporation and a high level of poly (U)-directed phenylalanine incorporation and an increased binding of poly (U). The equal binding of poly (U) and the equal poly (U)-directed phenylalanine incorporation by preincubated microsomes from normal and CCl,-treated rats suggests that CC14treatment removes or destroys microsomal mRNA without destroying the protein-synthetic capacities or poly (U) binding sites of the microsome. Although the result of CCla treatment and preincubation on endogenous microsomal mRNA activity is the same, this does not necessarily imply that this effect is produced in these two cases by the same mechanism.
National Cancer Institute, National Institutes o~ Health, Bethesda, Md. (U.S.A.) I 2 3 4 5 6 7
M. E . W E K S L E R
H. V. GELBOIN
G. R. CAMERON AND W. A. E. KARUNARATNE,J. Pathol. Bacteriol., 42 (1936) I. G. G. VILLELA, Biochem. Pharmacol., 13 (1964) 665. •. l~_. WELDON, B. RUBENSTEIN AND O. ]~UBINSTEIN, Can. J. Biochem., 43 (1965) 647. E. A. SMUCKLER AND E. P. BENDITT, Biochemistry, 4 (1965) 671. J- MAGER, S. t3ORNSTEIN AND A. HALBREICH, Biochim. Biophys. Acta, 95 (1965) 682. M. E. WEKSLER AND H. V. GELBOIN, J. Biol. Chem., 242 (1967) 727 . H. V. GELBOIN AND L. SOKOLOFF, Science, 134 (1961) 611.
Received April 7th, 1967 Biochim. Biophys. /icta, 145 (1967) 184-187
SHORT COMMUN ICATIONS
Briefly, though these resalts are not sufficiently striking to enable one t o conclude that hormonal control of differentiation exists in embryonic blood islands, they encourage further research to discover such mechanisms in the differentiating embryo. This work has been supported by Euratom (Contract E u r a t o m - U L B o16-61-1o ABIB). Erythropoietin was supplied by the National Heart Institute, Bethesda, Md., U.S.A. and by Dr. E. C~OLDWASSER,Argonne Cancer Research Institute, Chicago, to whom we address our thanks. Laboratoire de Morphologie Animale, P. MALPOIX Facultd des Sciences de l'Universitd Libre de Bruxelles, Brussels (Belgium) I 2 3 4 5 6 7 8 9 io ii 12 13
L. O. JACOBSON, C. W. GURNE¥ AND E. GOLDWASSER, Adv. Int. Med., IO (196o) 297. R. J. COLE AND J. PAUL, J. Embryol. Exptl. Morphol., 15 (1966) 245. S. B. KRANTZ, O. GALLIEN LARTIGUE AND E. GOLDWASSER, J. Biol. Chem., 238 (1963) 4085 . E. a . POWSNER AND L. BEERMAN, Blood, 14 (1959) 1213. 8. B. KRANTZ AND E. GOLDWASSER, Biochim. Biophys. Acta, lO 3 (I965) 325 . M. P. PIEBER-PERETTA, R. W. HODGSON AND M. A. PERETTA, Biochim. Biophys. Acta, 95 (1965) 325 . A. KOENER, Bull. SoC. Chim. Biol,, 48 (1966) lO31. L. G. BRITT AND H. HERMANN, J. Embryol. Exptl. Morphol., 7 (1959) 66. V. HAMBURGER AND H. J. HAMILTON, J. Morphol., 88 (1951) 49. A. HELL, J. Embryol, Exptl. Morphol., 12 (1964) 6o9. P. MALPOIX AND S. LIMBOSCH, J. Embryol. Exptl. Morphol., 16 (I966) 439G. A. BRAY, Anal. Biochem., I (196o) 279. V. M. INGRAM AND B. MOSS, Proc. Natl. Acad. Sci. U.S., 54 (1965) 969.
Received April 4th, 1967 Biochim. Biophys. Acta, 145 (1967) i 8 I I84
BBA 93249
Carbon tetrachloride-induced loss of microsomal messenger-ribonucleic acid activity The administration of CC14 to rats results in widespread morphologic and biochemical damage to the liver 1-3. SMUCKLER AND BENDITT4 have shown that CC14 administration causes a decrease in the in vivo incorporation of amino acids into hepatic protein and in the in vitro incorporation of amino acids into protein by microsomes and ribosomes. MAGER, BORNSTEIN AND HALBREICH 5 have reported a "paradoxical" increase in poly (U)-directed phenylalanine incorporation by liver microsomes from CC14-treated animals. This increase accompanies the loss of endogenous microsomal amino acid incorporating activity. These investigators suggested that the destruction of microsomal messenger RNA (mRNA) by CC14 treatment may account for both the decreased endogenous mRNA-directed amino acid incorporation and the increase in poly (U)-directed phenylalanine incorporation. We have found that microsomes from CC14-treated rats function similarly to microsomes made devoid of mRNA activity by a preincubation procedure previously described ~. Abbreviation: m R N A , messenger RNA; poly (U), polyuridylic acid.
Biochin~. Biophys. ,4cta, 145 (1967) 184 187
SHORT COMMUNICATIONS
185
T A B L E I. THE INCORPORATION OF L-[14CIPHENYLALANINE BY MICROSOMES FROM NORMAL AND CCI~-TREATED RATS
2oo-g Female S p r a g u e - D a w l e y r a t s were fasted 16 h prior to the i n t r a p e r i t o n e a l injection of either o.8 ml of mineral oil or 0.8 ml of CC14 in mineral oil (i : i, b y vol.). 2 h later the animals were sacrificed. 2-g p o r t i o n s of liver in io ml of o.25 M sucrose solution containing 6 mM m e r c a p t o e t h a n o l were homogenized in a P o t t e r - E l v e h j e m grinder for 3 ° sec. The h o m o g e n a t e was filtered t h r o u g h a 75/,2 nylon m e s h a n d centrifuged at 15 ooo x g for 15 rain to r e m o v e cell debris, nuclei, and mitochondria, io-ml alJquots of the 15 ooo × g s u p e r n a t a n t were centrifuged for 60 rain at ioo ooo X g. The resulting microsomal pellet was suspended in 2.8 ml of a i mM Tris buffer (pH 7.4), 1.5 mM MgClz, 5 mM KC1 and 6 mM m e r c a p t o e t h a n o l . The s t a n d a r d reaction m i x t u r e s were set u p in duplicate and contained in 2 ml: 12 mM p o t a s s i u m p h o s p h a t e (pH 7.4), 1.5 mM ATP, o. 3 mM GTP, 12 mM MgC1v 4 ° mM creatine p h o s p h a t e , o. 5 m g creatine p h o s p h o k i n a s e , 6 mM m e r c a p t o e t h a n o l , 77 mM sucrose, 43 #M u n i f o r m l y l a b e l e d L-[~4C~phenylalanine (specific a c t i v i t y io/,C//*mole), 0.2 ml ioo ooo x g s u p e r n a t a n t (containing a p p r o x . 2 mg of protein) and o. 4 ml of microsomes (containing approx. 7 mg of protein). W h e r e indicated, p r e i n c u b a t i o n of microsomes was p e r f o r m e d for 12 rain at 37 ° in the absence of L-E~4CJphenylalanine. The preincub a t i o n c o m p o n e n t s and conditions were identical to the conditions described above except for the following changes: I n a v o l u m e of 1.5 ml were p r e s e n t : creatine p h o s p h a t e 27 mM, creatine p h o s p h o kinase o.25 m g a n d MgC12 8 mM. After p r e i n c u b a t i o n s a t u r a t i n g a m o u n t s of poly (U) (4oo/*g), L-p4C]phenylalanine and additional creatine p h o s p h a t e , creatine p h o s p h o k i n a s e , MgC1e and w a t e r were added resulting in a reaction m i x t u r e identical to the s t a n d a r d incubation mix except for the presence of poly (U). I n c u b a t i o n was performed for 15 rain at 37 °. The reaction w a s s t o p p e d w i t h io ~g trichloroacetic acid. The precipitated p r o t e i n s were purified b y a modification ~ of the m e t h o d of Siekevitz and r a d i o a c t i v i t y m e a s u r e d in a t h i n - w i n d o w gas-flow counter. The r e s u l t s are expressed as c o u n t s / m g microsomal p r o t e i n per min.
Microsomes
A (no preincubation )
B (preincubated)
-- P o l y (U)
+ Poly (U)
-- Poly (U)
+ Poly (U)
Normal CC14
312 55
629 1316
3 5
1630 1592
Table IA confirms the findings of MAGER, BORNSTEIN AND HALBREICH5 and shows that non-preincubated microsomes from CC14-treated rats exhibit a reduced capacity to incorporate phenylalanine in the absence of poly (U) and an enhanced capacity to incorporate phenylalanine in the presence of poly (U) when compared to normal non-preincubated microsomes. In other studies 6 we have shown that preineubation of liver microsomes results in a complete loss of endogenous amino acid incorporating activity and an increase in poly (U)-directed phenylalanine incorporation. The result of preincubating microsomes from CC14-treated rats is shown in Table IB and is compared with that obtained when normal microsomes are preincubated. Microsomes from CC14-treated rats after preincubation behave exactly as do normal preincubated mierosomes. Thus, both exhibit no amino acid incorporation in the absence of poly (U) and identical phenylalanine incorporation in the presence of poly (U). This shows that CC14treatment does not alter the responsiveness of the mierosomal protein-synthesizing apparatus to exogenous mRNA (poly (U)), at least not to any greater extent than occurs during the preincubation period. In addition, the behavior of non-preincubated microsomes from CC14-treated rats (Table IA) shouts a great similarity to that of either normal or CC14microsomes which have been made devoid of endogenous mRNA by preincubation. The low level of endogenous mRNA-directed amino acid incorporation by non-preincubated CC14 Biochim. Biophys. dcla, I45 (I967) 184-187
I~6
SHORT COMMUNICATIONS
2500
I
NORMAL MICROSOMES ~#/ (PREINCUE/A TED, ooo_
,soo~
~eINCUaare°Jes
•
I
,,
J
~,vo~v-
d~ ::"
i z""l" L ""~
O/
50
,
NORMALM,CRO,OM. .-O............................... 0 ................................ 0 ............................... (
L~
ioo
200 poLr
L
~oo
400
Ul#gl~il'
Fig. I. I n c o r p o r a t i o n of L-[llC]phenylalanine at different c o n c e n t r a t i o n s of poly (U) by micros o m e s f r o m n o r m a l and CC14-treated rats. E x p e r i m e n t a l conditions are described in t h e l e g e n d to Table I.
microsomes and the somewhat smaller stimulation b y poly (U) is most likely due to a less complete removal of m R N A in vivo. Fig. i shows that microsomes from CC14-treated rats exhibit a poly (U) saturation curve that is considerably different from that of normal non-preincubated microsomes and resembles closely the curve obtained when normal or CC1a microsomes are preincubated in vitro. Thus, phenylalanine incorporating activity of normal non-preincubated microsomes is saturated by IOO#g/ml of poly (U) while preincubated microsomes from either CC14-treated or normal rats are saturated b y from 2oo to 3oo #g/ml of poly (U). Non-preincubated microsomes from CCl~-treated rats show a saturation between 25o and 30o/~g/ml of poly (U) and this suggests an increased T A B L E 1I BINDING
OF 3I-I-LABELED
POLY
(U)
BY MICROSOMES
FROM NORMAL
AND CCI4-TREATED
RATS
Microsomes were p r e p a r e d a n d p r e i n c u b a t e d in the m a n n e r described. To each reaction m i x t u r e 0 . 2 / , m o l e s of 8H-labeled poly (U) (specific activity 3/~C]/zmole) was added. The entire m i x t u r e w a s t h e n layered over 9 ml of a 0.25 M sucrose in io mM Tris buffer (pH 7-4) and 5 mM MgCI, solution and centrifuged at ioo ooo × g for I 11. The m i c r o s o m a l pellet w a s t h e n r e s u s p e n d e d solubilized w i t h H y a m i n e a n d r a d i o a c t i v i t y m e a s u r e d in a P a c k a r d liquid scintillation counter. The results are expressed as c o u n t s / r a g mierosomal p r o t e i n p e r rain.
Microsomes
No preincubation
Preincubated
Normal CC14
678 lO2O
lO8 i 117o
Biochirn. Biophys. Acta, 145 (1967) 184-187
187
SHORT COMMUNICATIONS
ability of such microsomes to interact with poly (U). Non-preincubated microsomes from CC14-treated rats are also more responsive to low levels of poly (U) as are normal preincubated microsomes 6. The binding of all-labeled poly (U) by microsomes from normal and CC14treated rats is shown in Table II. Non-preincubated microsomes from CC14-treated rats bind 40 % more poly (U) than do normal non-preincubated microsomes. In contrast, following preincubation, both microsomal preparations bind comparable amounts of poly (U). In fact, non-preincubated microsomes from CC14-treated rats bind an amount of 3H-labeled poly (U) comparable to that bound by preincubated microsomes from either normal or CC14-treated rats. These binding studies indicate that a likely explanation for the preincubation-induced loss of mRNA activity is that preincubation causes a removal or destruction of microsomal mRNA and thereby increases the number of available poly (U) binding sites on the microsome. SMUCKLER AND BENDITT4 have found that CC14 administration results in a conversion of polyribosomes to monosomes (54-S particles also appear). This may represent the morphological counterpart in a non-membrane bound preparation to the functional changes encountered in the microsomal preparation. In summary we have shown that microsomes free of mRNA activity and microsomes from CC14-treated rats behave similarly exhibiting a very low level of endogenous mRNA-directed amino acid incorporation and a high level of poly (U)-directed phenylalanine incorporation and an increased binding of poly (U). The equal binding of poly (U) and the equal poly (U)-directed phenylalanine incorporation by preincubated microsomes from normal and CCl,-treated rats suggests that CC14treatment removes or destroys microsomal mRNA without destroying the protein-synthetic capacities or poly (U) binding sites of the microsome. Although the result of CCla treatment and preincubation on endogenous microsomal mRNA activity is the same, this does not necessarily imply that this effect is produced in these two cases by the same mechanism.
National Cancer Institute, National Institutes o~ Health, Bethesda, Md. (U.S.A.) I 2 3 4 5 6 7
M. E . W E K S L E R
H. V. GELBOIN
G. R. CAMERON AND W. A. E. KARUNARATNE,J. Pathol. Bacteriol., 42 (1936) I. G. G. VILLELA, Biochem. Pharmacol., 13 (1964) 665. •. l~_. WELDON, B. RUBENSTEIN AND O. ]~UBINSTEIN, Can. J. Biochem., 43 (1965) 647. E. A. SMUCKLER AND E. P. BENDITT, Biochemistry, 4 (1965) 671. J- MAGER, S. t3ORNSTEIN AND A. HALBREICH, Biochim. Biophys. Acta, 95 (1965) 682. M. E. WEKSLER AND H. V. GELBOIN, J. Biol. Chem., 242 (1967) 727 . H. V. GELBOIN AND L. SOKOLOFF, Science, 134 (1961) 611.
Received April 7th, 1967 Biochim. Biophys. /icta, 145 (1967) 184-187