Preferential synthesis of extracellular ribonuclease and some intracellular proteins in actinomycin D-treated Bacillus subtilis

BIOCHIMICA ET BIOPHYSICA ACTA

221

BBA 96143

PREFERENTIAL SYNTHESIS OF EXTRACELLULAR RIBONUCLEASE AND SOME INTRACELLULAR PROTEINS IN ACTINOMYCIN D-TREATED BA C I L L U S S U B T I L I S

K A Z U O Y A M A G U C H I , K I Y O S H I K A D O W A K I AND

BUNJI

MARUO

Division of Enzymology, Institute o/Applied Microbiology, University o] Tokyo, Tokyo (Japan) (Received October 22nd, 1968)

SUMMARY

I. Actinomycin D, at a concentration of I/~g/ml, inhibited almost completely the formation of extracellular ~-amylase (EC 3.2.1.1) by Bacillus subtilis K, while the accumulation of ribonuclease (EC class 2.7.7) continued at a decreased, but constant, rate in the same culture. The latter was, however, inhibited immediately and completely by the addition of 5 #g/ml of the antibiotic. 2. Actinomycin D (I/,g/ml) did not completely inhibit the incorporation of labeled leucine into cellular proteins; a decreased rate of incorporation (lO-2O °/o of the control) remained for at least 60 rain in the actinomycin D-treated cells. On the other hand, 5 #g/ml of this drug resulted in the inhibition of leucine incorporation (more than 97 ~o inhibition) within 30 rain. 3. Purification of individual enzymes demonstrated that the actinomycin Dtreated cells incorporated labeled leucine well into the ribonuclease protein but insignificantly into the ~-amylase protein. The inhibition ratio, as judged by the enzyme formation, was in good agreement with that calculated from the leucine incorporation. 4. Fractionation by DEAE-cellulose column chromatography and gel filtration on Sephadex G-75 indicated that labeled leucine was also incorporated preferentially into limited species of intracellular proteins in the presence of 1/~g/ml of actinomycin D, as in the case of the syntheses of extracellular enzymes. The results clearly indicate that actinomycin D at low concentrations has differential effects upon the syntheses de novo of intracellular proteins, as well as extracellular enzymes in B. subtilis K cells.

INTRODUCTION

Although it has generally been accepted that the antibiotic actinomycin D, at low concentration, inhibits DNA-dependent RNA synthesis both in vivo and in vitro, resulting in inhibition of protein synthesis, the precise mechanism for its inhibition has not been elucidated. We have demonstrated with the Gram-positive bacterium, Bacillus subtilis K that the formation of extracellular ribonuclease (EC class 2.7.7) is inhibited to the extent of only 50 ~o by I #g/ml of actinomycin D, whereas this antibiotic inhibited Biochim. Biophys. Acta, 179 (1969) 221-231

222

g. YAMAGUCHIet al.

the formation of c~-amylase (EC 3.2.1.1), proteinase (EC class 3.4.4) and alkaline phosphatase (EC 3.1.3.1) by more than 9 ° °/o under the same conditions 1,~. Some other bacterial enzymes have also been observed to be affected non-uniformly by this drug ~-5. If the residual formation of ribonuclease in the actinomycin D-treated cells is definitely due to the continuation of synthesis de novo, then the investigation of such a differential inhibition will be both interesting and important, since these anomalous phenomena should reflect both the unique mechanism of action of this particular antibiotic and the possible difference between the biosynthetic processes for the highly susceptible and moderately susceptible enzymes. Our previous finding 1 that the residual formation of ribonuclease was strongly inhibited b y chloramphenicol and 5-fluorouracil, which are both specific inhibitors for protein synthesis, strongly supports the notion of synthesis de novo of this enzyme in the presence of a low concentration of actinomycin D. However, the observation by SWEATON et al. 6 of a specific inhibitor for extracellular ribonuclease inside the B. subtilis cells suggests another possible explanation for the differential effect: actinomycin D would have inhibited the net synthesis of ribonuclease protein as strongly as that of x-amylase but the liberation of latent ribonuclease into the medium or the activation of a precursor protein of ribonuclease would have continued even in the presence of the antibiotic. The present study with the double-labeling technique has demonstrated that the latter possibility is not the case: isolation and analysis of labeled ribonuclease and x-amylase proved that preferential synthesis of ribonuclease de novo in the actinomycin D-treated cells took'place. The present paper further shows that the relative resistance to actinomycin D is not specific for the synthesis of extracellular ribonuclease: differential inhibition of biosynthesis is also observed among the groups of intracellular proteins.

MATERIALS AND METHODS

The organism and culture conditions B. subtilis K (IAM 1523) was used throughout. Bacterial cells were grown under

aeration in a bouillon-yeast extract medium 1 at 3 °o for 20-22 h, and the culture was diluted 5o-fold with a glutamate-citrate medium 7. The organism was further grown for 9 h to reach the early stationary phase (absorbance at 550 nm about 3.o), at which the activities of s-amylase and ribonuclease were 50-60 units/ml and 1.5-2.o units/ml, respectively. In some experiments the cells grown in the bouillon yeast extract medium were harvested and resuspended in a lactate medium as described previously 1. A s s a y of enzyme activity

x-Amylase was assayed by the method of HAGIHARA8. For the assay of ribonuclease, i ml of the reaction mixture which contained the enzyme, 2.5 mg of purified yeast RNA, 50/,moles of Tris (pH 7.5), and 5 #moles of EDTA was incubated at 3 °o for io rain. After the addition of 0.5 ml of IO % trichloroacetic acid containing 0. 4 °/o uranyl acetate and keeping in an ice-bath for 60 rain, the precipitate was removed by centrifugation. A portion (0.2 ml) of the clear supernatant was diluted with 5 ml of deionized water and the absorbance at 260 nm was measured with a spectrophotoBiochim. Biophys. Acta, 179 (1969) 221-231

223

ACTINOMYCIN I ) AND PROTEIN SYNTHESIS

meter (Shimadzu, OR-5o ). The complete hydrolysis of I mg of RNA in IO rain was defined as I unit of enzyme activity.

Measurement o] protein synthesis Actinomycin D was added to a culture in the early stationary phase of growth at 3 o°. At various times before and after the addition of the dlug, a I-ml sample of the culture was withdrawn and mixed with 0.2 #C of [x4C]leucine. After exactly I rain, I ml of IO °/o trichloroacetic acid containing 0.5 mg unlabeled L-leucine was added to terminate the incorporation of radioactivity. The precipitate was heated at 9°o for 20 rain, collected on a Millipore filter HA, washed with 15 ml of 5 % trichloroacetic acid and dried, and the radioactivity was measured with an automatic Geiger-Miiller gas-flow counter (nihon Musen, Tokyo).

Double-labeling experiment The culture grown in the glutamate-citrate medium for 9 h was divided into two portions: actinomycin D (I/~g/ml) was added to one portion. After 30 rain of incubation with or without the antibiotic, 50 #C (1. 5/*moles) of L-[14C]leucine was added to the actinomycin D-containing culture and 3oo/*C (1. 5/*moles) of L-[3H]leucine to the other. Both cultures were incubated for I h with aeration. Each culture was mixed with 15o/,moles of unlabeled L-leucine, incubated for a further 5 min and then chilled at o °.

Preparation o] S-I4o The two labeled cultures were mixed together and centrifuged at 7000 x g for IO rain. Harvested cells were once washed with o.oi M Tris buffer (pH 7.5) containing 5 mM MgC12, resuspended in the same buffer and disrupted by sonication (IO kcycles/ see for IO rain) at o °. The sonicate was centrifuged at 15 ooo x g for 20 rain to remove cell debris. The 15 ooo × g supernatant fraction was centrifuged at 14o ooo ×g for 9° rain. The 14o ooo x g sediment was resuspended in Tris-MgC12 buffer and was designated as the ribosomal fraction. The 14o ooo x g supernatant fraction was dialyzed overnight against Tris-MgC12 buffer at 4 °, and designated as S-I4O.

DEA E-cellulose and Amberlite IRP-64 (XE-64) column chromatography DEAE-cellulose (from Bio-Rad laboratories) was equilibrated with o.oi M Tris buffer (pH 7.6). A I-ml sample was applied to a column (0. 9 cm × 35 cm), eluted first with 4 ° ml of the same buffer and then with 200 ml of NaC1 in o.oi M Tris buffer (pH 7.6) (linear gradient of NaC1 from o to 0.6 M). Amberlite IRP-64 (XE-64) (from Rohm and Haas) was equilibrated with o.I M sodium phosphate buffer (pH 5.9) according to the method of HIRS 9. A 1.5-ml sample was applied to the column (0. 9 c m x 33 cm) and eluted with the same buffer.

Gel ]iltration Sephadex G-75 (from Pharmacia Fine Chemicals) was washed with o.oi M Tris buffer (pH 7.6). A 2-ml sample was put on a column (0. 9 cm × 115 cm) and eluted with the same buffer.

Preparation o] unlabeled carrier enzymes To prepare the unlabeled carrier enzymes, 1.o6 kg of (NH4)~SO 4 was added to 2 1 of the glutamate-citrate medium prepared from a culture grown for 18 h at 3 o°. The precipitate dissolved in o.oi M Tris buffer (pH 7.6) was extensively dialyzed against the same buffer and put on a DEAE-cellulose column (2 cm ×30 cm) to separate m-amylase from ribonuclease. Chromatography was carried out as described above. Biochim. Biophys. Acta, 179 (1969) 221-231

K. YAMAGUCHIet al.

224

Chemicals L-[x4C]Leucine (250 mC/mmole) and L-ESH]leucine (200 mC/mmole) were purchased from New England Nuclear and Daiichi Pure Chemicals, respectively. Actinomycin D was a gift of Merck, Sharp and Dohme Research Laboratory.

Scintillation counting A i-ml sample was added to 0.25 ml of I M hyamine in methanol and incubated at 45 ° for I or 2 days, and then IO ml of dioxane scintillator 10 was added. Radioactivity was measured with a scintillation spectrometer Packard Model 314 A. RESULTS

E//ect o/actinomycin D on enzyme/ormation and leucine incorporation As shown in Fig. I, ~-amylase formation was almost completely suppressed within IO min after the addition of I/*g/ml of actinomycin D, whereas ribonuclease

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formation was not reduced for at least 2o rain and thereafter its formation continued at a slower rate for more than I h. It should be noted that o.25/*g/ml of the drug rather stimulated the ribonuclease formation after about 30 min as previously reported 1. However, a high concentration of actinomycin D (5 #g/ml) immediately stopped the ribonuclease formation (Fig. 2). Fig. 3 shows that the leucine incorporation rapidly declined after the addition of actinomycin D to the culttlre. At a concentration of I/,g/m], however, actinomycin D did not completely inhibit protein synthesis but about 15 % of normal extent of incorporation remained after 20 rain incubation in the presence of the drug. On the other hand, by the addition of 5 #g/ml of actlnomycin D protein synthesis was almost completely suppressed. These results were confirmed b y the fact that the incorporation of leucine continued at a decreased but constant rate for at least 30 rain when Biochim. Biophys. Acta, 179 (1969) 221-z3I

225

ACTINOMYCIN D AND PROTEIN SYNTHESIS

Actinomycin D

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Fig. 2. T h e effect of 5/~g/ml of a c t i n o m y c i n D o n t h e f o r m a t i o n of r i b o n u c l e a s e . A f t e r 9.5 h of i n c u b a t i o n in g l u t a m a t e - c i t r a t e m e d i u m , a c t i n o m y c i n D (5/~g/ml) w a s a d d e d to a c u l t u r e (o time). 0 - 0 , control; O - - - O , a c t i n o m y c i n D. Fig. 3. T h e effect of a c t i n o m y c i n D on t h e i n c o r p o r a t i o n of [14C]leucine for i min. A f t e r 9 h of i n c u b a t i o n in g l u t a m a t e - c i t r a t e m e d i u m , a c t i n o m y c i n D w a s a d d e d (o time). T h e IOO % v a l u e s a x e 33 67o c o u n t s / m i n for i # g / m l a n d 26 7oo c o u n t s / m i n for 5 # g / m l of a c t i n o m y c i n D. O - O , i.o # g / m l ; 0 - 0 , 5 / , g / m l of a c t i n o m y c i n D.

[14C]leucine was added to the culture after actinomycin D-treatment for 3o min (Fig. 4)- As described above, extracellular ribonuclease continued to be synthesized at a decreased rate but x-amylase did not under the same conditions. The incorporation of [14C]uracil into RNA was not completely suppressed by actinomycin D at a concentration of I pg/ml, while no incorporation was detected in the presence of 5 #g/ml of actinomycin D. Therefore, it is not likely that the incorporation of leucine at a concentration of I/~g/ml of actinomycin D was due to the presence of stable messenger RNA, although the RNA synthesis could not be measured exactly by the incorporation of [14C]uracil in B. subtilis, since actinomycin D seemed to bring about a change of the specific radioactivity of UTP pool in the cells (unpublished data).

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Biochim. Biophys. Mcta, 179 (1969) 221-231

226

K. YAMAGUCHIet al.

The incorporation o/labeled leucine into e-amylase and ribonuclease In order to investigate whether actinomycin D inhibits enzyme syntheses de novo differentially, the enzymes being synthesized in the actinomycin D-treated cells were labeled with [14C]leucine and the enzymes synthesized in untreated cells with [aH]leucine as described in MATERIALSAND METHODS. TABLE I THE INCREASE OF ENZYME ACTIVITY DURING THE 1-h LABELING PERIOD + A c t i n o m y c i n D designates the addition of i / , g / m l of actinomycin D 3 ° rain prior to the zero time. =-Amylase

o time (units/ml) i h after (units/ml) Increase (units/ml) I n h i b i t i o n (%)

Ribonuclease

-- d ctinomycin D

+ A ctinomycin D

-- A ctinomycin D

+ A ctinornycin D

69.3 114.o 44.7 o

53-3 57.1 3 .8 92

1.682 2.268 °.586 o

1.514 1.794 0.280 52

Table I shows the increase in enzyme activities during the labeled-leucine incorporation. Ribonuclease formation was apparently less sensitive to actinomycin D than e-amylase formation. 0~-Amylase formation occurred only slowly (8 % of control) under the present conditions of I #g/ml of the drug. To purify both e-amylase and ribonuclease, the labeled cultures were mixed and centrifuged at 7000 × g for IO min to remove the bacterial ceils. After the supernatant was concentrated b y lyophilization, it was dissolved in a small amount of o.oi M Tris buffer (pH 7.6) and dialyzed overnight against the same buffer. The sample was subjected to DEAE-cellulose column chromatography (see MATERIALS AND METHODS). Under these conditions, ribonuclease was not adsorbed on DEAE-cellulose, while eamylase was adsorbed and eluted with o.2-o.3 M N a t l . This double-labeled ribonuclease fraction was added to the unlabeled carrier ribonuclease fraction obtained by DEAE-cellulose column chromatography (see MATERIALS AND MEXHODS) and the mixture was purified according to the method of NISHIMURAn modified as follows: the ribonuclease fraction was acidified to p H 2 with 2 M H~S04 and after standing overnight at 4 ° the precipitate was removed b y centrifugation. The supernatant was dialyzed against o.oi M Tris buffer (pH 7.6) containing o.I M NaC1, concentrated with polyethylene glycol and put on a Sephadex G-75 column. After being concentrated by lyophylization, the ribonuclease fraction from the gel filtration was adsorbed on Amberlite IRP-64 and eluted with o.I M sodium phosphate buffer (pI-I 5.9) (Fig. 5). The ribonuclease preparation was thus purified about 6o-fold and its specific activity was almost as high as a commercial preparation of crystalline p~ncreatic ribonuclease A (Sigma). Since NISHIMURA11 reported that the specific activity of crystalline extracellular ribonuclease of B. subtilis was almost the same as that of crystalline pancreatic ribonuclease A, the present preparation, though not crystallized, could be considered as pure as a crystalline one. The e-amylase fraction from DEAE-cellulose column chromatography was mixed with carrier Biochim. Biophys. ,4cta, 179 (1969) 221-231

ACTINOMYCIN

D

AND PROTEIN SYNTHESIS

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a-amylase and purified in accordance with the method of HAGIHARATM. Finally, aamylase precipitated in 4 ° °/o acetone solution was applied to a DEAE-cellulose column and eluted as described in MATERIALSAND METHODS (Fig. 5). Table II shows the specific activity and molar ratio of [14C]- to [~H]leucine incorporated in both enzyme fractions at each purification step. Ratios of 14C to ~H in a-amylase fractions decreased to 0.07 as its purification proceeded. On the contrary, those of ribonuclease fractions increased to 0. 9. In Fig. 5, it is clearly seen that the elution pattern of enzyme activity coincided well with that of radioactivity in both chromatograms and that [14C]leucine was incorporated only to a small extent as compared with [3H]leucine, in the case of a-amylase. As shown in Table II and Fig. 5, TABLE

II

PURIFICATION

OF k-AMYLASE

AND RIBONUCLEASE

Step of purification

AND THEIR

M O L A R R A T I O S O F 14C T O 3I-I

Unit[l~g Tyr equiv

14C/3H

Inhibition (%)

i -9 13 16

780 IOO 7o0

-o.24 0.06 0.07 --

-76 94 93 --

3° 60 270 700

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-62 64 28

I 700 i 5oo

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12 --

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16o

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Biochim. Biophys. Acta, 179 (1969) 221-231

K. YAMAGUCHI et al.

228

the incorporated radioactivity behaved identically with enzyme proteins and this indicated that ~-amylase and ribonuclease proteins were actually labeled. Therefore, the above results must have reflected the preferential synthesis de novo of ribonuclease protein in the presence of a low concentration of actinomycin D. Proteins synthesized in the presence o/actinomycin D The following experiment was done to decide whether there were some proteins, which, like extracellular ribonuclease, were preferentially synthesized in the organism

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Biochim. Biophys. Acta, 179 (1969) 221-231

ACTINOMYCIN D AND PROTEIN SYNTHESIS

229

pre-treated with I #g/ml of actinomycin D for 3o min. Actinomycin D-treated cells were labeled with L-EJ4C]leucine and untreated cells with L-I3H]leucine. After the incubation for I h, both cultures were chased with unlabeled leucine and mixed, and double-labeled S-I4O was prepared (see MATERIALSAND METHODS). The chromatogram from a DEAE-cellulose column of S-I4O (Fig. 6a) demonstrated that the incorporation of [14C]leucine was strongly inhibited b y actinomycin D and, at the same time, their molar ratios of 1~C to 3H were very similar in all fractions except for Ic. Fraction Ic appeared to be low molecular weight compound(s) (tool. wt. about IOOO) containing leucine residues since Fraction Ic was not hot trichloroacetic acid-precipitable, could not be eluted with the void volume of the buffer from a Sephadex G-Io column, and when hydrolyzed with 6 M HC1 at IiO ° for 20 h, all radioactivity of Fraction Ic was recovered as free leucine on a paper chromatogram. When compared in detail the distribution of 14C with that of 3H in Fig. 6a, it was noted that there was a small but definite discrepancy in Fraction V (Fig. 6b). The molar ratio of 14C to 3H of Fraction Vb was 0.27 and higher than that of Fraction Vc (o.16) and those of the hot trichloroacetic acid-insoluble fractions from whole cell, S-I4O and ribosomal fraction were 0.22, o.21 and o.18, respectively. In order to ascertain this discrepancy, both Fractions Vb and Vc were further fractionated by gel filtration of Sephadex G-75. As shown in Figs. 7 a and b, the e]ution pattern of 1~C was very different from that of 3H. Peaks 1- 4 in both Fractions Vb and Vc were hot trichloroacetic acid-insoluble and estimated to consist of proteins of molecular weight higher than IO ooo, as judged from the elution volumes in Figs. 7 a and b, and Peak 5 of Fractions Vb and Vc were hot trichloroacetic acid-soluble. These results suggested that I #g/ml of actinomycin D had a differential effect upon the incorporation of leucine into proteins. S.A.

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DISCUSSION

Assuming that the effect of actinomycin D on the specific radioactivity of the leucine pool is negligible, it is expected that the molar ratio of 14C to 3H in each enzyme Biochim. Biophys. Acta, 179 (1969) 221-231

23 °

K. YAMAGUCHIet al.

fraction reflects the ratio of synthesis de novo rates for each enzyme with and without the antibiotic. The good agreement of inhibition ratios measured b y enzyme activity, with those b y leucine incorporation indicated a differential effect of the drug on synthesis de novo of the enzymes (Tables I and II). For ribonuclease, the inhibition measured b y the enzyme activity was not exactly the same as that of leucine incorporation. This m a y be due to a small change of specific radioactivity in the leucine pool after the addition of the drug or due to the presence of actinomycin-sensitive ribonuclease, which was separated as the fast moving component from the main of the ribonuclease in Amberlite IRP-64 column chromatography (Fig. 5b). Since the differential effect of actinomycin D, which had been found in extracellular enzyme synthesis, was also observed in the syntheses of intracellular proteins (Figs. 6 and 7), this effect was not specific to extracellular ribonuclease synthesis. A possibility that the increase of ribonuclease activity in the presence of the drug might be due to the activation of a precursor protein for extracellular ribonuclease or the accelerated liberation of latent ribonuclease is therefore not likely. LEVINTHAL et al. 1~ suspected that the smaller fragments of proteins might be synthesized in actinomycin D-treated cells. The molecular weight of this ribonuclease is about IO 700 (ref. I4) and as shown in Fig. 7, those of some proteins (Peaks 2 and 4 of Fraction Vb and Peaks 2 and 3 of Fraction Vc), the syntheses of which were more resistant to the drug, were more than IO ooo. Furthermore, each peak of 14C radioactivity agreed well with that of SH and no such abnormal fraction that had incorporated only leucine, in the presence of the drug, was detected. Therefore it is unlikely that all of the proteins labeled with [14Clleucine are the unusual fragments of proteins. HAYWOOD AND SINSHEIMERls also reported that there was a protein fraction in protoplasts of Escherichia coli which is synthesized even in the presence of actinomycin D (IO ~g/ml). The fraction was heterogeneous and contained some components of molecular weight greater than IO ooo. The following two explanations are possible for the differential effect of actinomycin D on synthesis de novo of protein; (i) the different susceptibility of the genes to the drug, that is, differential synthesis of messenger RNA, (ii) the different stability of messenger RNA. The presence of the long-lived messenger RNA in bacteria has been suggested by several authors 16-19. We cannot, however, employ this speculation as an explanation of the phenomenon on the following experimental bases, (i) ribonuclease formation was strongly inhibited by 5-fluorouracil 1, which probably was incorporated into messenger RNA as a base analogue, (ii) the formation of ribonuclease and the leucine incorporation into proteins continued at a decreased but constant rate at least for i h in the presence of I ~g/ml of the antibiotic (Figs. i and 3) but were instantly and completely inhibited b y 5/~g/ml of the drug (Figs. 2 and 3), (iii) as previously reported ~°, actinomycin D-inhibited enzyme formation could be recovered by the addition of DNA and the formation of ribonuclease was recovered specifically at low concentrations of DNA whereas the recovery of ~-amylase formation was only slight. In the previous paper 21, we reported that a careful determination of the composition of pulse-labeled RNA showed small but definite differences in guaninecytosine contents when its formation was partially inhibited. This suggests that actinomycin D has a differential effect upon RNA synthesis. PERRY22 and YAMADA AND KAWAMATA23 have reported that rapidly labeled RNA, with a larger sedimentation Biochim. Biophys. Acta, 179 (1969) 221-231

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231

coefficient was preferentially inhibited at a low concentration of actinomycin D. It is plausible that the frequency of binding of actinomycin D to the guanine moiety of DNA is parallel with the size of the cistron. Actually, the incorporation of leucine into higher molecular weight protein (Peak I of Fractions Vb and Vc) was very sensitive to actinomycin D as shown in Fig. 7. However, the higher molecular weight protein was not usually more sensitive to the antibiotic as seen in Peaks 2 and 3 of Fraction Vb. It must be considered that the sensitivity of the cistron to actinomycin D is dependent not only on its guanine content but also on the extent of affinity of the guanine moiety to the drug. We previously presented 2° the following possibility as an explanation of this phenomenon; the syntheses of some kinds of messenger RNA's are more resistant to actinomycin D than other messenger RNA's: messenger RNA which codes ribonuclease protein is preferentially synthesized in the presence of the drug, or the regulator gene which represses the ribonuclease synthesis is highly susceptible to the drug. Considering results of binding studies of actinomycin D to DNA, we suspect that specific sites having higher affinity to actinomycin D are absent in the gene for ribonuclease, therefore actinomycin D attaches to this gene only after the specific sites in other genes have been saturated with the drug.

ACKNOWLEDGMENTS

The authors express their thanks to Drs. G. DENKEWALTER and H. B. WooDRUET, Merck, Sharp and Dohme Research Laboratory, for their generous gift of the actinomycin D. This work was supported in part by a grant from the Japan Foundation for Applied Enzymology.

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Biochim. Biophys. Acta, 179 (1969) 221-231