The stimulatory effect of 3-methylcholanthrene on microsomal amino acid incorporation and benzpyrene hydroxylase activity and its inhibition by actinomycin D

PRELIMINARY NOTES

657

PN 6111 The stimulatory effect of 3-methylcholonthrene on microsomal amino acid incorporation and benzpyrene hydroxylose activity and its inhibition by octinomycin D The administration in vivo of 3-methylcholanthrene increases rat-liver microsomal benzpyrene hydroxylase activity and a number of other microsomal enzymes 1~. 3-Methylcholanthrene stimulation is specific. Thus certain N-demethylases, 0demethylases, and ring hydroxylases are stimulated while other microsomal enzymes e.g. meperidine demethylase, glucose-6-phosphatase (EC3.1.3.9) and D P N H cytochrome c reductase (EC 1.6.2.1) remain unchanged or are depressed. It has been shown that 3-methylcholanthrene pretreatment increases microsomal amino acid incorporation into protein when either freO, s or soluble RNA bound amino acids 5 are used as precursors. The 3-methylcholanthrene stimulation of benzpyrene hydroxylase e and arninoazo dye demethylase (N-methyl aminoazo dye oxidase) 7 is prevented by puromycin. These results suggest that the 3-methylcholanthrene stimulation of benzpyrene hydroxylase is due to enzyme synthesis. Current evidence indicates that "messenger RNA" is synthesized by a DNAdirected RNA-polymerase (EC 2.7.7.6 ) and that messenger RNA specifies the amino acid sequence on a microsomal incorporation site during protein synthesis 8. Actinomycin D inhibits DNA-directed RNA synthesis in mammalian tissue cultures 9 and the hormonal induction of rat-liver tryptophan pyrrolase (triptophan peroxidase, EC 1. I I. 1.4)1°. This investigation deals with the relationship between 3-methylcholanthrene-induced changes in the amounts of a microsomal enzyme, microsomal amino acid incorporation, and the RNA metabolism of the cell. Table I shows the increased amino acid incorporation in microsomes derived from 3-methylcholanthrene-treated rats. The degree of stimulation varied in each experiment, but was always within 15 to 70 %. The increased incorporation was observed with saturating amounts of each cofactor, over a 4-fold range of labeled amino acid concentration, and further addition of soluble RNA or supernatant fluid did not alter total counts incorporated in either preparation. Incorporation in both preparations was equally inhibited by puromycin and completely prevented by RNAase (EC 2.7.7.16). The protein-synthesizing apparatus of the microsomes can be described as consisting of two parts, the messenger RNA serving as information and the "incorporation site", that is, the microsomal RNA-protein complex with which messenger RNA functions to synthesize protein. In order to investigate the effect of 3methylcholanthrene on amino acid incorporation we utilized the NIRENBERG--I~ATTHEI incorporating system as applied to mammalian liver n. Table I shows that the addition of polynridylic acid (poly-U) stimulates [14C]phenylalanine incorporation in the microsomes from either normal or 3-methylcholanthrene-treated rats. In both preparations addition of lOO Fg of poly-U saturates the phenylalanine incorporation sites and gives 24 % more incorporation in the microsomes from 3-methylcholanthrenetreated rats. Preincubated microsomes lose their capacity for L-phenylalanine incorporation. The addition of poly-U restores their ability to incorporate this amino Biochira. Biophys. Acla, 72 (I963) 657--660

658

PRELIMINARY NOTES TABLE I

THE EFFECT OF POLYURIDYLIC ACID ON" AMINO ACID INCORPORATION IN LIVER MICROSOMES FROM N O R M A L A N D 3-METHYLCI-IOLANTHRENE-TREATED RATS G r o u p s of 3 - 4 m a l e r a t s w e i g h i n g 40-5 ° g were injected i n t r a p e r i t o n e a l l y w i t h I m g 3 - m e t h y l c h o l a n t h r e n e in o.25 m l corn oil a n d killed i8 h t h e r e a f t e r . Controls were g i v e n corn oil. E a c h flask c o n t a i n e d 2 o # m o l e s p o t a s s i u m p h o s p h a t e (pH 7.4), o . 5 # m o l e e a c h of A T P a n d G T P , i o # m o l e s MgClz, 4 o # m o l e s p h o s p h o c r e a t i n e , o.25 m g creatine p h o s p h o k i n a s e (EC 2.7.3.2), i o o # m o l e s G S H , o.o85 # m o l e of u n i f o r m l y labeled L-[14Clphenylalanine (specific a c t i v i t y 9.8 # C / # m o l e ) a n d m i c r o s o m e s a n d s u p e r n a t a n t e q u i v a l e n t to 2oo a n d 67 m g of r a t liver, respectively. All flasks were in duplicate. I n c u b a t i o n s were m a d e a t 37 ° for 15 rain as described 5. P r e i n c u b a t i o n s were a t 37 ° for 2o rain in t h e a b s e n c e of t h e r a d i o a c t i v e p h e n y l a l a n i n e a n d in t h e presenoe of all t h e c o f a c t o r s listed above. A f t e r p r e i n c u b a t i o n t h e [14C]amino acid, a d d i t i o n a l o.25 m g c r e a t i n e k i n a s e a n d 4 ° # m o l e s c r e a t i n e p h o s p h a t e were a d d e d a n d t h e i n c u b a t i o n s m a d e as d e s c r i b e d s. Normal rats Conditions

A dditions

8-Methylcholanthrenetreated rats

(counts/rain/rag protein)

No p r e i n c u b a t i o n s No p r e i n c u b a t i o n s No p r e i n c u b a t i o n s Preincubated Preincubated Preincubated

None P o l y u r i d y l i c acid P o l y u r i d y l i c acid None P o l y u r i d y l i c acid P o l y u r i d y l i c acid

(ioo#g) (2oo#g) (ioo#g) (2oo#g)

380 774

587 955 963 Io 1238 1541

737

13 lO55 lO59

acid. This effect is apparently due to a release of messenger RNJ" tror~ the microsomes by preincubation and its replacement by poly-U. When endogenoJls messenger RNA activity was removed and [14CJphenylalanine incorporation measured in the presence of IOO and 200 #g of poly-U, there was 17 and 45 % greater incorporation in the microsomes from 3-methylcholanthrene-treated rats. With this system, the increased rate of incorporation observed with microscmes from 3-methylcholanthrenetreated rats suggests that one effect of 3-methylcholanthrene is to increase the number of available microsomal amino acid incorporation sites. Table II shows that administration of 3-methylcholanthrene causes an I8-fold rise in benzpyrene hydroxylase activity. Actinomycin D given alone has no effect. When 3-methylcholanthrene and actinomycin D are given simultaneously there is a 70 % inhibition of the 3-methylcholanthrene stimulation. This suggests that 3-methylcholanthrene induction of enzyme activity depends on the synthesis of RNA and when this process is blocked by actinomycin D, enzyme synthesis is inhibited. The failure of actinomycin D to inhibit completely the 3-methylcholanthrene effect may be due either to a second pathway for enzyme synthesis independent of actinomycin I) inhibition of RNA synthesis, or to incompleteness of the actinomycin D block of RNA synthesis. Table II also shows the effect of actinomycin D and 3-methylcholanthrene on [I4Clphenylalanine incorporation in the non-preincubated microsomes and in the preincubated system with poly-U added. The inhibition by actinomycin D in the non-preincubated system suggests that the maintenance of normal mierosomal incorporating activity depends on continued messenger RNA synthesis. When this process is blocked by actinomycin D in vivo over a I6-h pericd the amino acid incorporating activity of the microsomes is lowered by 30 %. In ~ fis system, 3-methylcholanthrene given alone caused a 5° % increase in incorpoi,~ting activity. When 3-methylcholanthrene and actinomycin D were both given the 3-methylcholanthrene Biochirn.

Biophys.

Acta,

72 (1963) 657-660

PRELIMINARY NOTES

659

TABLE II THE INHIBITION BY ACTINOMYCIND o F METHYLCHOLANTHRENESTIMULATIONOF BENZPYRENE HYDROXYLASE ACTIVITYAND MICROSOMALAMINO ACID INCORPORATION Groups of five male rates weighing 4o-5 ° g were injected intraperitoneally with o.o3o mg actinomycin D in 0.25 ml of o.154 M NaC1 in 0.04 M NaHIPO4-Na2HPO, (pH 7.4) at o, 4, 8 and 12 h. i mg 3-methylcholanthrene in o.25 corn oil was injected at i h. Controls were given buffer and/or corn oil. The rats were killed at 18 h. The benzpyrene hydroxylase assay was by a modified procedure of V~ATTENBERC.AND LEONGTM. Activity refers to/~g formed in 12 min at 37° by homogenate equivalent to IOO mg wet liver. Similar results were obtained with isolated microsomes. Enzyme activity Hydroxybenzpyrene

Pretreatment 3-Methyleholanthrene

Actinomycin D*

formed (as 3-OHbenzpyrene

--

--

--

+

+ +

-+

o.8o 1.o8 14.45 3.8o

(ag)

~-['4C]PkenylaIanine incorporation (counts/rain/rag protein) No preincubation

(%)

165 113 248 132

-68 15° 80

Preincubation, then zoo ~g poly-U

(%)

314 133 412 145

--

42 131 46

" i33/zg actinomycin D added in vitro had no effect on phenylalanine incorporation. effect was p r e v e n t e d a n d the a c t i v i t y was similar to t h a t of microsomes from rats given a c t i n o m y c i n D only. This suggests t h a t the 3 - m e t h y l c h o l a n t h r e n e effect on a m i n o acid i n c o l p o r a t i o n is m e d i a t e d t h r o u g h a s t i m u l a t i o n of messenger R N A synthesis. W h e n the p r e i n c u b a t e d microsomes with poly-U added were used as a n assay system, the effects of 3 - m e t h y l c h o l a n t h r e n e a n d a c t i n o m y c i n D were similar to those observed with the n o n - p r e i n c u b a t e d system. The p r e i n c u b a t e d , poly-U a d d e d system m a y be considered as an assay of available microsomal incorporation sites i n d e p e n d e n t of the a m o u n t of messenger R N A present. Our results suggest t h a t the n u m b e r of these sites is increased b y 3 - m e t h y l c h o l a n t h r e n e t r e a t m e n t a n d the formation of these sites is a function of a n a c t i n o m y c i n D sensitive reaction. YANKOFSKY AND SPIEGELMANTM have shown the presence in E. coli of a sequence in D N A c o m p l e m e n t a r y to ribosomal RNA. P r e s u m a b l y microsomal R N A is synthesized t h r o u g h a R N A polymerase reaction using this D N A sequence as information. If this is so, a c t i n o m y c i n D should inhibit microsomal R N A synthesis as well as messenger R N A synthesis. Our results support the hypothesis t h a t 3 - m e t h y l c h o l a n t h r e n e induces microsomal e n z y m e synthesis b y s t i m u l a t i n g b o t h the production of messenger R N A a n d microsomal a m i n o acid incorporation sites engaged in enzyme synthesis. The authors wish to t h a n k Dr. E. MAXWELL for valuable suggestions for r e m o v a l of messenger R N A a c t i v i t y from microsomes a n d Dr. H. FALK for a generous gift of 3 - h y d r o x y benz-(a)pyrene. Mr. H. WATERS gave valuable technical assistance.

Diagnostic Research Branch, National Cancer Institute, Bethesda, M d . (U.S.A.)

HARRY V. GELBOIN NORMA R. BLACKBURN

1 A. H. CONNEY, E. C. MILLER AND J. A. MILLER, J. Biol. Chem., 228 (I957) 753. t A. H. CONNEY, J. R. GILLETTE,S. K. INSCOE, E. R. TRAMSAND H. S. POSNER, Science, 13o (1959) 1478. a H. V. GELBOIN, J. A. MILLER AND E. C. MILLER, Cancer Res., 19 (1959) 975. , A. VON D~R DECKEN AND T. HULTIN, Arch. Biochem. Biophys., 90 (196o) 2oi.

Biochim. Biophys. Acta, 72 (1963) 657-660

660 5 6 7 s 0 10

H. H. A. F. E. O. 1 1 E. 12 S. ls L.

PRELIMINARY NOTES V. GELBOII'¢ AND L. SOKOLOFF,Science, 134 (1961) 611. V. GELBOIN, unpublished results. H. CONNEY, personal communication. JAcoB AND J. MONOD~ J. Mol. Biol., 3 (1961) 318. REICH, E. M. FRANKLIN, A. J. SHATKIN AND E. L. TATUM, Science, 134 (1961) 356. GREENGARD AND G. Acs, BiocMm. Biophys. Acta, 61 (1962) 652. S. MAXWELL, Proc. Natl. Acad. Sci. U.S., 48 (1962). A. YANKOFSKY AND S. SPIEGELMAN, Proc. Natl. Acad. Sci. U.S., 48 (1962) 1466. W. WATTENBERG AND J. L. LEONG, Histochem. Cytochem., IO (1962) 412.

Received March 25th, 1963 Biochim. Biophys. Acta, 72 (1963) 657-660

PN 6113 Ribonucleic acid metabolism in unfertilized and fertilized sea-urchin eggs It is known, from the work of HULTIN1 that the ribosomes of unfertilized sea-urchin eggs have a low capacity for amino acid incorporation into their proteins; these ribosomes undergo a rapid increase in activity soon after fertilization or treatment with parthenogenetic agents. It is also known that protein synthesis is negligible in living unfertilized sea-urchin eggs and that it begins soon after fertilization S. More recently, NEMER3 and W I L T AND HULTIN4 reported the fact that addition of polyuridylic acid (poly-U) induces a considerable increase in the incorporation of phenylalanine by the ribosomes isolated from unfertilized sea-urchin eggs; the activation of this process by poly-U is much less in the case of ribosomes obtained from fertilized eggs. The most likely conclusion is that the inactivity of the ribosomes, in unfertilized eggs, is due to the fact that they are devoid of messenger RNA's (m-RNA's); the latter would be produced by the nucleus very soon after fertilization or activation. However, other hypotheses, as pointed out by one of us 5, can be proposed in order to explain the low activity of protein synthesis in eggs at very early stages of their development: for instance, the ribosomes, if their surface is blocked by an inhibitor, might be unable to accept m-RNA's; alternately, the latter might be synthesized during ovogenesis and remain in an inactive form during the egg maturation. Fertilization or activation, according to such hypotheses (which are well in keeping with our general ideas about the metabolic changes induced by fertilization in sea-urchin eggs6, 7) would only set free pre-existing processes which were inhibited or repressed in the unfertilized egg. The present note describes a few experiments designed for testing the two alternatives (synthesis of new m-RNA's, or removal of an inhibitory factor by fertilization). (a) First of all, the experiments of NEMER3 and of WILT AND HULTIN4 have been repeated and confirmed (on the 12 000 ×g supernatant of homogenized unfertilized Arbacia eggs). In agreement with WILT AND HULTIN 4, it was found that considerable amounts of poly-U (800/~g/ml) must be added in order to obtain a large (5 times) stimulation of phenylalanine incorporation in this system in vi to. This stimulating effect of poly-U could not be replaced by the addition of 400/zg of RNA isolated from unfertilized sea-urchin eggs by the phenol method of GEORGIEVAND MANTIEVA s. Biochim. Biophys. Acta, 72 (1963) 660-662