282

Biochimica et Biophysica Acta, 383 (1975) 282--289

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 98239 E F F E C T O F B I O T I N ON R I B O N U C L E I C ACID S Y N T H E S I S

R.L. BOECKX and K. DAKSHINAMURTI Department of Biochemistry, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba (Canada)

(Received October 7th, 1974)

Summary A single injection o f biotin to b i o t i n - d e f i c i e n t rats p r o d u c e s a t w o - f o l d increase in t h e i n c o r p o r a t i o n , b o t h in vivo and in vitro o f precursors into nucleic acids as early as 2 h a f t e r the biotin t r e a t m e n t . T h e specific activity o f the p r e c u r s o r p o o l is n o t a f f e c t e d b y biotin. Analysis o f t h e p o l y s o m e profile at various times following biotin t r e a t m e n t and a kinetic s t u d y of the e f f e c t of excess p o l y ( U ) o n the i n c o r p o r a t i o n o f p h e n y l a l a n i n e b y cell-free a m i n o acid i n c o r p o r a t i o n e x p e r i m e n t s indicate a m a r k e d decrease in messenger-free ribosomes in rat liver a f t e r biotin a d m i n i s t r a t i o n .

Introduction T h e i n c o r p o r a t i o n o f a m i n o acids into proteins of liver, intestinal m u c o s a , pancreas and skin in vivo and in vitro is m a r k e d l y decreased in b i o t i n d e f i c i e n c y and a single injection o f biotin t o t h e biotin d e f i c i e n t rats s t i m u l a t e d a m i n o acid i n c o r p o r a t i o n b o t h in vivo and in vitro m o r e t h a n t w o fold ( D a k s h i n a m u r t i and Litvak [ 1 ] , B o e c k x and D a k s h i n a m u r t i [2,3] ). Analysis o f the p r o d u c t s o f a m i n o acid i n c o r p o r a t i o n into liver p r o t e i n s in vivo and in vitro i n d i c a t e d t h a t the syntheses of s o m e proteins were s t i m u l a t e d several fold b u t o t h e r s were n o t s t i m u l a t e d at all ( B o e c k x and D a k s h i n a m u r t i [ 2 ] ) . Such a specificity in the s t i m u l a t i o n o f p r o t e i n synthesis m e d i a t e d b y b i o t i n has earlier been r e f e r r e d to ( D a k s h i n a m u r t i and C h e a h - T a n [ 4 ] , D a k s h i n a m u r t i et al. [5] ). In all o f these cases, the b i o t i n - m e d i a t e d s t i m u l a t i o n was abolished b y t r e a t m e n t o f t h e animals with i n h i b i t o r s o f p r o t e i n or R N A synthesis like p u r o m y c i n , e t h i o n i n e or a c t i n o m y c i n D. T h e e f f e c t o f b i o t i n on p r o t e i n synthesis was p r e c e d e d b y a s t i m u l a t i o n in t h e i n c o r p o r a t i o n o f o r o t i c acid into nuclear and r i b o s o m a l R N A seen as early as 2--4 h a f t e r b i o t i n t r e a t m e n t . Nuclear R N A f r o m b i o t i n - t r e a t e d rats s u p p o r t e d higher levels o f a m i n o acid i n c o r p o r a t i o n b y n o r m a l rat liver r i b o s o m e s , as c o m p a r e d w i t h similar R N A isolated f r o m b i o t i n - d e f i c i e n t rats ( D a k s h i n a m u r t i and Litvak [1] ).

283 We present here further evidence to suggest t h a t the synthesis of certain kinds of RNA is stimulated by biotin.

Experimental Materials ATP, GTP, UTP, phosphoenolpyruvate and pyruvate kinase were obtained from the Sigma Chemical Corp. L-[4,5-3H]leucine (50 C i / m m o l ) w a s obtained from the International Chemical and Nuclear Corp. L-[U-14 C] phenylalanine (380 Ci/mol) and [6 -14C]orotate (50 Ci/mol) were purchased from the New England Corp. Polyuridylic acid (ammonium salt) was obtained from Miles Laboratories. a-Amylase (EC 3.2.1.1) from swine pancreas was purchased from the Worthington Biochemical Corporation. Polyethyleneimine~ellulose thin-layer chromatography plates (20 × 20 cm) were purchased from Brinkman Instruments (Canada) Ltd. Preparation of animals Biotin deficiency was produced in weanling (45--50 g) male Holtzman rats by feeding t h e m for 5--6 weeks an avidin diet (Dakshinamurti and Cheah-Tan [6] ). In all experiments reported the effect of a single injection of biotin into biotin-deficient rats was examined. In addition, since the experimental (biotininjected) and control (biotin-deficient) rats were starved for the duration of the experiment (from 0 to 18 h as indicated), complications owing to differences in food intake between the two groups were eliminated. All animals used in an experiment were of the same age and weight. The animals received either a single intraperitoneal injection of 1 ml of 0.82 mM D(+)-biotin in 0.15 M NaC1 or 1 ml of 0.15 M NaC1. [6-14 C] Orotate incorporation into nuclear R N A in vivo The incorporation of orotate in vivo was done as described previously (Dakshinamurti and Litvak [1] ). Biotin-deficient rats were injected at various times before sacrifice with a single intraperitoneal dose of biotin. In some experiments the rats were injected with actinomycin D 1 h before the biotin injection. A dose of 100 pg of actinomycin D per 100 g body weight was used (Dakshinamurti and Litvak [1] ). 30 min before sacrifice, all animals were injected with 100 pCi of [6-14C]orotate. Animals were sacrificed and liver nuclear RNA was prepared as described by Hiatt [7]. RNA was dissolved in 0.01 M sodium acetate buffer (pH 5.1) containing 0.1 M NaC1 and aliquots were used for RNA assay according to Cerriotti [8] and for determination of radioactivity. The effect of biotin injection on the specific radioactivities of the acidsoluble nucleotide pool as well as of UTP itself following [6-14 C] orotate injection was studied. UTP was separated from the mixed nucleotides using thin layer chromatography according to Neuhard [9]. The liver homogenate was treated with an equal volume of 0.5 M perchloric acid and centrifuged. The perchloric acid supernatant was neutralized by the addition of concentrated KOH and centrifuged. An aliquot of the supernatant was mixed with a Norite A suspension (one volume of a 1% suspension). The mixture was centrifuged,

284 and the supernatant discarded. The charcoal pellet was treated with small volumes (2 ml) of 0.1 M NH4OH in 50% EtOH for 20 min at 37°C. The suspension was centrifuged and the supernatant was evaporated to dryness in a stream of nitrogen. The residue was dissolved in 0.1 ml of distilled water containing 100 nmoles of UTP. This was applied to 20 $ 20 cm polyethyleneimine-cellulose thin-layer chromatography plates and developed as described by Neuhard [9]. The solvent systems used were 2 M LiC1/2 M acetic acid(1 : 1, v/v) (3 cm) followed by 2.5 M LiC1/2 M acetic acid (1 : 1, v/v) (15 cm) in the first dimension and 2.5 M a m m o n i u m acetate/3.6% boric acid (pH 7.0) (5 cm) followed by 3.5 M a m m o n i u m acetate/5% boric acid (pH 7.0) (15 cm) in the second dimension. Plates containing only 100 nmol of UTP were also run. Following development, the plates were air-dried and the UTP spots were located under ultraviolet light. The UTP spots were cut out and eluted with minimal volumes of 4 M NH4OH. Absorbance at 260 nm and radioactivity were determined in aliquots of the eluate. The absorbance of eluates from plates containing only unlabelled UTP was subtracted and the difference in absorbance was assumed to be due to labelled UTP.

[5- 3H] UTP incorporation into nuclear R N A in vitro At various time intervals before sacrifice, animals were given a single intraperitoneal dose of biotin (200 pg in one ml of saline). Three animals were used for each time interval studied, and following sacrifice, the livers of rats in that group were pooled. Nuclei were prepared by the m e t h o d of Widnell and Tata [10] and were gently dispersed in 0.25 M sucrose/1 mM MgC12. DNA was assayed as described by Widnell and Tata [10] and the nuclear suspension was diluted to a concentration of 4 mg DNA/ml. The incorporation of UTP into nuclei was studied as previously described (Dakshinamurti and Litvak [1]) except that 2 pCi of [5-3H] UTP per 1.5 ml incubation was used. Final UTP concentration was 1.3 mM.

Analysis o f polysome profiles At various times before sacrifice, animals were given a single intraperitoneal dose of biotin (200 gg in 1 ml of saline). Animals were sacrificed and livers were immediately removed and chilled in ice-cold homogenizing buffer (0.25 M sucrose/20 mM Tris/4 mM MgC12/10 mM KC1/1 mM 2-mercaptoethanol, pH 7.5). Livers were finely minced and gently homogenized in 2.5 vol. of this buffer using 8 strokes of a teflon-glass Potter-Elvejhem homogenizer rotating at 1500 rev./min. The homogenate was centrifuged at 12 000 × g for 10 min. The upper half of the supernatant was siphoned off taking care to avoid contamination by the lipid layer floating on the top. a-Amylase (900 units/mg) was added to this post-mitochondrial supernatant to a final concentration of 50 U/ml and the solution was incubated at 4°C for 30 min to remove glycogen (Gamulin et al. [11] ). Sodium deoxycholate (10% w/v) was added to a final concentration of 1.3% (w/v), 20 A2 s 4 units were carefully layered over a 0.15% to 0.27% (percentage by weight) sucrose isokinetic gradient (Noll [12] ). The gradients contained KC1 (50 mM)/MgC12 (5 mM)/Tris (20 mM), pH 7.5. Gradients were spun for 2 h at 180 000 X g in an SW 41 swinging bucket

285 rotor using the Beckman Model L3-40 ultracentrifuge. Following centrifugation the gradients were analyzed using an Isco Model 183 Density Gradient Fractionator. O u t p u t from the centrifuge tube was monitored for ultravioletabsorbing material at 254 nm. In order to identify the various peaks observed in this procedure, the isokinetic sucrose gradients were calibrated as described by Noll [12]. Normal laboratory chow fed rat liver polysomes were used. These were prepared by the m e t h o d of Wettstein et al. [13]. Ribosomal RNA was isolated by the m e t h o d of Hill and Bresnick [14] and analyzed on 5% to 40% sucrose density gradients. Centrifugation was carried out for 15 h in an SW 25.1 rotor using the Beckman L3-40 ultra-centrifuge. Gradients were analyzed using a gradient fractionator as described above.

Determination of messenger-free ribosomes The dual-label technique of Loeb [15] was used to compare the effect of polyuridylic acid on cell-free amino acid incorporating systems from biotin treated and control (biotin-deficient) rats. Biotin deficient animals were injected with biotin (200 pg in 1 ml of saline) 18 h prior to sacrifice. Control animals received saline only. Microsomes and pH 5 fraction were prepared as described previously (Boeckx and Dakshinamurti [3]). pH 5 fraction was prepared from normal (laboratory chow fed) rat livers. Protein was assayed by the biuret method according to Rendina [16]. Incubation mixtures contained the following in an initial volume of 10 ml: 35 mM Tris (pH 7.4); 50 mM KC1; 5 mM MgCI: ; 0.25 M sucrose; 4.5 mM 2-mercaptoethanol; 1 mM ATP; 0.15 mM GTP; 5 mM phosphoenolpyruvate; 0.25 mg pyruvate kinase; 60 pM each of 20 amino acids; 0 or 4 mg polyuridylic acid; 3 pCi each of L-[4,5 -3 H] leucine and L-[U -14 C] phenylalanine; 30 mg pH 5.0 fraction protein and 50 mg of microsomal protein. The reaction was carried out at 31°C. 6 1-ml samples of the reaction mixture taken at various time intervals were pipetted into precooled (0 ° C) test tubes containing one ml 10% trichloroacetic acid. The trichloroacetic acid precipitates were prepared for determination of 14C and 3H radioactivities as described previously (Boeckx and Dakshinamurti [3] ). All scintillation counting was performed using a Beckman LS-250 liquid scintillation system. Data was collected on paper punch tape and analyzed using a CDC 1700 computer and program SCINT (Boeckx et al. [17]). Data are usually expressed as picomoles or nanomoles of the original isotopically labelled c o m p o u n d incorporated. Results The effect of a single injection of biotin 16 hours before sacrifice on the incorporation in vivo of [6 -14 C ] o r o t a t e into nuclear RNA was examined. The results presented in Table I show a 2.5-fold increase in incorporation. Actinomycin D injected 1 h prior to biotin t r e a t m e n t abolished the stimulation in [6 -14C]orotate incorporation. Biotin treatment did n o t have any effect on the extent of labelling of the liver acid soluble fraction (Table II) at any of the time intervals studied. The effect of biotin injection to biotin-deficient rats on the specific acitivity of the liver UTP pool following a 30 min pulse of

286

TABLE

I

INCORPORATION

OF [6-14C]OROTATE

INTO NUCLEAR

Animal status

Biotin deficient Biotin deficient Biotin deficient

+ biotin + actinomycin

D + biotin

RNA

[ 6 . 1 4 C] O r o t a t e incorporated per mg of nuclear RNA* (pmol)

Relative incorporate

18 ÷ 21 200 ÷ 35 7 0 ,+ 1 7

1.0 2.5 0.9

* Means ± standard deviations of 6 experiments.

[6-1 4 C] o r o t a t e was also studied. Results given in Table III show no significant change in the specific radioactivity of liver UTP due to biotin injection. Since no significant alterations are seen in the e x t e n t of labelling of the acid soluble fraction or in the specific activity of the precursor nucleotide pool following biotin injection, one could assume that the increase in orotate incorporation observed following biotin t r e a t m e n t is related to a stimulation of RNA synthesis. This is, in fact, c o r r o b o r a t e d by in vitro experiments. As early as 5 h following biotin administration, the stimulation of UTP i n c o r p o r atio n into nuclear RNA in vitro had reached a plateau (Fig. 1). Biotin injection resulted in a two-fold stimulation of b o t h the total as well as the rate of the incorporation reaction. Thus, the activity of RNA polymerase reaction is increased two-fold. A significant effect of biotin is seen as early as 2 h following biotin t r e a t m e n t . Analysis of the polysome profiles at various times following biotin administration indicates a marked increase in heavier aggregates accompanied by a c o n c o m i t a n t decrease in the p r o p o r t i o n of m onom ers (Fig. 2). By measuring the areas under the profiles in Fig. 2, we calculated t hat the p r o p o r t i o n of m o n o s o mes d r o p p e d f r om 38% (time 0) to 20% (after 8 h). Examination of the sucrose density gradient profiles of ribosomal RNA isolated f r o m biotin treated and control animals showed no apparent differences in this RNA fraction. The increase in larger polysomes suggests t hat there is an increase in m R NA c o n t e n t with a c o n c o m i t a n t decrease in messenger-free ribosomes. TABLE EFFECT

II OF BIOTIN

INJECTION

ON THE LABELLING

Time after biotin (h)

[6.14 ] Orotate incorporated per g fresh liver* (nmol)

0 2 4 8 16

6.49 8.06 7.75 8.03 7.40 * M e a n s ,+ s t a n d a r d

+ 0.76 -+ 0 . 6 9 -+ 0 . 2 9 +- 2 . 0 5 ,+ 0 . 7 5

deviations of 3 experiments.

OF THE ACID-SOLUBLE

FRACTION

287

TABLE III EFFECT OF BIOTIN INJECTION ON THE SPECIFIC ACTIVITY OF THE ACID-SOLUBLE UTP POOL Means + standard deviations of 3 experiments. Time after biotin

p C i o f 14 C f r o m [6-14C]Orotate p e r tool o f U T P

0 4

1 7 9 3 . 2 +- 8 5 . 8 1 5 2 1 . 7 +- 2 9 8 . 1

.< Z o

Z f~

20

.00

0

z_© ._1 0

0 t,.) a_

1(? i

i

,

a

5

10

15

20

TIME AFTER BI©TIN ~HOURS) F i g . 1. T h e e f f e c t o f b i o t i n i n j e c t i o n o n [ 5 - 3 H ] U T P i n c o r p o r a t i o n i n t o n u c l e a r R N A in v i t r o b y i s o l a t e d nuclei. Both the extent of i n c o r p o r a t i o n (e) and the rate of i n c o r p o r a t i o n (expressed as p m o l of [ 5 -3 H] U T P i n c o r p o r a t e d p e r r a i n p e r m g D N A ) (o) are s h o w n . D e t a i l s of t h e p r o c e d u r e axe d e s c r i b e d in the text. 0 HRS, 1.O 0.5

6 HRS

1.5

4.0

1.0 Z

~

0°5

3.0

8 HRS,

1,5 1.0 0.5

5

10

15

20

FRACTION NUMBER

i

i

i

i

10

20

30

40

x o,

Fig. 2. P o l y s o m e p r o f i l e s o f b i o t i n d e f i c i e n t r a t liver at v a r i o u s t i m e s f o l l o w i n g t h e i n j e c t i o n o f b i o t i n . 20 A 2 5 4 u n i t s o f d e t e r g e n t t r e a t e d p o s t - m i t o e h o n d r i a l s u p e r n a t a n t w e r e l a y e r e d o n t o a 0 . 1 5 % to 0 . 2 7 % i s o k i n e t i c s u c r o s e g r a d i e n t a n d c e n t r i f u g e d f o r 2 h a t 1 8 0 0 0 0 X g. C o m p a r i s o n w i t h a c a l i b r a t i o n c u r v e i n d i c a t e s t h a t t h e p e a k e l u t i n g in f r a c t i o n 3 is t h e 80 S m o n o m e r . F i g . 3. A p l o t o f t h e r a t e o f p o l y ( U ) d i r e c t e d p h e n y l a l a n i n e i n c o r p o r a t i o n (qbu) v e r s u s t h e c u m m u l a t i v e l e u c i n e i n c o r p o r a t i o n (SOt) as o b s e r v e d i n cell-free p r o t e i n s y n t h e s i z i n g s y s t e m s f r o m b i o t i n d e f i c i e n t (o) a n d b i o t i n t r e a t e d b i o t i n d e f i c i e n t ( e ) r a t liver. D e t a i l s o f t h e p r o c e d u x e a r e p r e s e n t e d in t h e t e x t .

288 Evidence to support this hypothesis comes f r o m the kinetic study of the effect o f polyuridylic acid on the incorporation of phenylalanine in deficient and biotin-treated cell-free amino acid i ncorporat i on systems. Fig. 3 shows a plot of the rate o f poly(U) directed phenylalanine i ncorporat i on (¢u) versus cumulative leucine i ncor por at i on (?t0 t). As described by Loeb [ 1 5 ] , the number of ribosomes losing their endogenous m R N A is p r o p o r t i o n a l to the cumulative leucine incorporation. Also, in the presence of excess poly(U), the rate of poly(U) directed phenylalanine incorporation (q5u) will be proportional to the n u m b e r of messenger-free ribosomes. The relationship between these two factors is described by ~bu = k' (No + k ?t0 t) where t = time o f incubation No = n u m b e r of messenger-free ribosomes at t = 0 ?to = rate of leueine incorporation due to natural template RNA ~bu = rate of phenylalanine incorporation due to polyuridylic acid k',k = constants. A plot of Cu versus )to t should give a straight line with a y intercept (at ?to t = 0) at k'No. The intercept, therefore, is a measure of the n u m b e r of messenger-free ribosomes. Fig. 3 indicates that following biotin t r e a t m e n t , the value of k'N0 changes from 2.3 to 1.4 suggesting a marked decrease in the n u m b e r of messenger-free ribosomes in these microsomal preparations.

Discussion The role of biotin as the prosthetic group of the carbon dioxide fixation enzymes has been discussed by Knappe [ 1 8 ] . Apart from this funct i on, we have described the involvement of biotin in the synthesis of several hepatic glycolytic enzymes (Dakshinamurti and Cheah-Tan [ 4 ] , Dakshinamurti et al. [ 5 ] , Dakshinamurti and Hong [19] ). In addition, we have shown t hat amino acid in co r p o r atio n in liver, intestinal mucosa and skin both in vivo and in vitro is stimulated by a single dose of biotin (Boeckx and Dakshinamurti [ 3 ] ) . However, the pattern of the biotin-mediated stimulation of amino acid incorporation is complex. The synthesis of different proteins is stimulated from eightfold to none at all. We have also presented evidence t o indicate that the biotin-mediated effect on amino acid incorporation into protein is preceded by a stimulation in the incorporation of orotic acid or UTP into RNA. Nuclear RNA isolated f r o m biotin-treated rats is more efficient in supporting the i ncorporat i on of amino acids by the cell-free ribosomal - pH 5 fraction incubation system when compared with similar RNA preparations f r om biotin