Effect of chronic administration of morphine on mouse brain aminoacyl-tRNA synthetase and tRNA-amino acid binding

Brain Research, 53 (1973) 373-386

373

© Elsevier Scientific Publishing Company Amsterdam - Printed in The Netherlands

EFFECT OF CHRONIC ADMINISTRATION OF MORPHINE ON MOUSE BRAIN AMINOACYL-tRNA SYNTHETASE AND tRNA-AMINO ACID BINDING

R A N A J I T K U M A R D A T T A AND (LATE) W I L L I A M A N T O P O L

Division of Laboratories, Beth Israel Medical Center, New York, N. Y. 10003 and Department of Pathology, Mount Sinai School of Medicine of The City University of New York, New York, N. Y. 10029 (U.S.A.) (Accepted October 15th, 1972)

SUMMARY

Effects of the chronic administration of morphine to mice on the activity of aminoacyl-tRNA synthetase and the amino acid-tRNA binding were investigated. Aminoacyl-tRNA synthetases and tRNAs were purified partially from brains of control mice and mice chronically treated with increasing doses of morphine (up to 100 mg/kg). The partially purified brain synthetases from the control and morphinetreated mice catalyzed the in vitro binding of [14C]labeled arginine, leucine and phenylalanine with unfractionated tRNAs obtained from E. coli, mouse brains and mouse liver. However, the synthetase purified from the brains of the morphine-treated mice had lower specific activities than that purified from the brains of control mice. The decreased specific activities of synthetase of brains of morphine-treated mice were fairly proportional to the doses of morphine administered and persisted for 5 days after withdrawal of morphine. Administration of a single dose of morphine produced no significant decrease in synthetase activity. Moreover, chronic administration of morphine did not significantly alter physico-chemical properties and amino acid-binding capacities of tRNAs from the brains of mice chronically treated with morphine. Morphine did, however, moderately inhibit the binding of amino acids with E. coli tRNA in the presence of synthetase isolated from brains of either control mice or morphinetreated mice. The pretreatment of purified synthetase with morphine caused slight decreases of synthetase activity. Morphine did not bind in vivo with synthetase or tRNA in the presence of synthetases isolated from brains of either control mice or morphinetreated mice. The pretreatment of purified synthetase with morphine caused slight decreases of synthetase activity. Morphine did not bind in vivo with synthetase or tRNA during chronic administration of morphine nor with purified synthetase or tRNA in vitro.

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DATTA AND W. ANTOPOL

INTRODUCTION

The administration of a single dose of 10-60 mg/kg morphine sulfate in rats causes significant inhibition of the protein-synthesizing capacity of brain ribosomes in vitro6. A similar inhibition of protein synthesis in rat brain in vivo occurs following chronic administration of morphine 5. In HeLa cells, morphine congeners, levorphanol and levallorphan, inhibit protein synthesislTAs. Since the mechanism of this inhibition is not clear (reviewed by Clouet4) the present investigators are interested in determining the level at which the inhibition occurs in the multi-stage protein synthesis process. The activation of an amino acid and the binding of the activated form to its specific transfer RNA (tRNA) is the initial step of protein synthesis. This step is: Amino acid q- ATP + enzyme in soluble supernatant -+aminoacyladenylate-enzyme q- pyrophosphate. The aminoacyladenylate-enzymecomplex then combines with tRNA to yield aminoacyl-tRNA + AMP + enzyme. Since this integrated step of activation and binding, which is catalyzed by the enzyme aminoacyl-tRNA synthetase (called synthetase hereafter) can be followed independently of the other steps of protein synthesis, the effort is concentrated on this activation and binding process in the presence of morphine. In order to work with a comparatively clean cell-free system, attempts are made to purify partially the aminoacyl-tRNA synthetase from brains of control mice and those treated chronically with increasing doses of morphine. Results presented in this communication indicate that morphine in vitro inhibits moderately the tRNA-amino acid binding, and decreases, during its chronic administration, the specific activities of synthetase of brain tissue of mice so treated. METHODS AND MATERIALS

Animals Inbred C57BL male mice weighing 25-30 g were kept at 22-25 °C with food and water ad libitum throughout the experiments. Acute administration of morphine Unless stated otherwise, in acute experiments, a single dose (30 mg/kg body weight) of morphine sulfate dissolved in 0.25 ml of normal saline (pH adjusted to 7.0) was administered intraperitoneally to the experimental group. The control group received an equal volume of normal saline. Three hours later, mice were sacrificed and brains were immediately removed. Chronic administration of morphine In chronic administration, mice received morphine (dissolved in normal saline, pH adjusted to 7.0) intraperitoneally. Initially, a dose of 5 mg/kg body weight was administered once daily for the first 2 days. The dose was increased by 5 mg/kg every second day until 100 mg/kg was reached. Mice tolerated the increasing doses of morphine with no mortality at the highest dose of morphine. Mice were sacrificed at 22-26

MORPHINE ON AMINOACYL-tRNA SYNTHETASE

375

h after the final dose and the brains were immediately removed. Unless stated otherwise, the brains from mice treated chronically with morphine are called 'morphinized brains' and brains of the control mice 'control brains' in the text.

Purification of aminoacyl-tRNA synthetase Brains (cerebrum, cerebellum, cerebral peduncle and pons included) from 4-6 mice were homogenized in 50 ml of sucrose-phosphate buffer (0.25 M sucrose, 0.1 M potassium phosphate, pH 6.5). The homogenate (H) was centrifuged at 3,500 rev./min (approximately 1640 x g) for 10 min, and cell debris and nuclei were discarded as the residue. The post-nuclear fraction (PN) was then centrifuged at 105,000 x g for 45 min. The supernatant was collected and centrifuged once more at 105,000 x g for 45 min. The supernatant (soluble supernatant, SS) was adjusted to pH 5.0 by dropwise addition of 1 N acetic acid when a flocculant precipitate was obtained. This was centrifuged at 3,500 rev./min for 10 min and the precipitate (5P) was collected. This was then solubilized in 5 ml of sucrose-phosphate buffer (pH 6.5) and centrifuged at 3,500 rev./min for 10 min. The supernatant (5S) was collected, mixed with DEAEcellulose in suspension (100 mg of cellulose/20 mg protein in 5S) and occasionally stirred for 15 min. The suspension was centrifuged at 15,000 x g for 20 min and the clear supernatant collected. This supernatant (5SD) contained partially purified aminoacyl-tRNA synthetase and was found to be free from amino acids, nucleotides and tRNA. No attempt was made to fractionate this into specific synthetases. The enzyme similarly purified from the morphinized brains did not contain any detectable morphine by gas chromatographic analysis. The purified enzymes from the control brains as well as from the morphinized brains did not bind with [14C]morphine added during the purification procedure or during their assays.

Preparation of tRNA About 5 g of brain or liver tissue was homogenized in a Waring blender with 20 ml of water-saturated phenol and 150 ml of 1.0 M NaCI, 5 mM EDTA in 0.1 M Tris-HCl buffer, pH 7.5. The homogenate was centrifuged at 3,500 rev./min (approximately 1640 x g) for 15 min. The upper aqueous layer was carefully collected and mixed with 3 vol. of 95 ~o ethanol. The precipitate which formed when the mixture was cooled in ice for 30 min was separated by centrifugation and resuspended as above in 25 ml of 0.1 M Tris-HC1 buffer, pH 7.5. The suspension was passed through a column (2 cm × 12 cm) of 2.5 g of DEAE-cellulose previously equilibrated with cold 0.1 MTrisHC1 buffer, pH 7.5. The column was washed with 100 ml of Tris-HC1 buffer, pH 7.5 and the tRNA was eluted with 1.0 M NaC1 in 0.I M Tris-HCl buffer, pH 7.5. The first 5 ml of NaC1 solution was discarded as hold-up. Sufficient salt solution (50-100 ml) was then collected until the optical density (at 260 nm) of the eluates was less than one. The combined eluates were extracted once with an equal volume of water-saturated phenol and once with an equal volume of ether. To the aqueous solution containing the tRNA 3 vol. of 95 ~ ethanol were added and the mixture was allowed to stand overnight at 0--4 °C when a precipitate was formed. This was then collected by centrifugation, washed with 80 ~o ethanol and then dried in vacuum. The procedure

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R.K. DATTA AND W. ANTOPOL

was basically the same as that of Brunngraber z. The purified tRNA from the morphinized brains contained no detectable morphine. The purified tRNA from the control and the morphinized brains did not bind in vitro with [14C]morphine either during the purification procedure or during the assays of aminoacyl-tRNA synthetase.

Chromatographic elution profiles of tRNA tRNA was chromatographically fractionated on DEAE-cellulose at pH 4.2 in the presence of 4.0 M urea as described by Cherayil and BockL Partially purified tRNA as described in the previous section was applied to a column (1.2 cm × 90 cm) equilibrated with 0.33 M NaCI in 4.0 M urea and 0.02 M sodium acetate buffer, pH 4.2. Elution was accomplished at 22-25 °C with a gradient linear in NaC1, 0.35-0.60 M, in a total volume of 400 ml containing 4.0M urea and buffer. Fraction size was 5 ml collected every 30 min. The optical density of the fractions was monitored at 260 nm. Assay of aminoacyl-tRNA synthetase The acylation of tRNA with [14C]amino acids was carried out according to the procedure of Zubay 24. The acylation medium contained 37.5 pmoles cacodylate buffer, pH 7.0, 7.5/zmoles magnesium acetate, 30/zmoles ammonium chloride, 1.25 #moles ATP, 0.1/zmole [14C]amino acid, 0.05 mg tRNA, 0.5-1.5 mg partially purified aminoacyl-tRNA synthetase in a total volume of 0.5 ml. After 30 min of incubation at 37 °C 0.1 ml aliquots were withdrawn for assay of acid-precipitable radioactivity with the filter paper technique of Hoskinson and Khorana 1~. As a check, the incubation was stopped by adding an equal volume of freshly distilled phenol and shaken for 20 min at 3-5 °C. Following centrifugation at 3,500 rev./min (approximately 1640 × g) for 10 min the aqueous layer was collected. This was treated with 3 vol. of 95 ~ ethanol containing 2 ~ potassium acetate and kept at 0 °C overnight when a very minute precipitate was formed. This was collected by centrifugation, washed once with ethanol, solubilized in 0.5 ml hyamine hydroxide for 12 h in the cold and transferred to the vials for measuring radioactivity. Results of this and the filter paper technique for the assay of synthetase activity were within ~- 5 ~ . Because of the ease of operation, the filter paper technique was then routinely used. All assays were carried out in duplicate, and the mean specific activities were expressed as pmoles of amino acid bound by 0.05 mg tRNA/mg synthetase protein/30 min. Chemical determinations Protein was measured with the Folin phenol reagent with crystalline bovine serum albumin as a standard. RNA was determined by Mejbaum's orcinol method and also by the UV-spectrophotometric procedure. D N A was determined by Dische's diphenylamine method. Amino acids were measured by the colorimetric method of Rosen 2°. Total lipids were extracted and determined by the method of Folch et aLL Polysaccharides were determined by the method of Hess and Slade 11. Nucleotides, obtained after hydrolysis of tRNA with 0.3 N K O H solution at 37 °C for 18 h, were separated by paper chromatography, using the method of Magasanik et al. 16. The paper strips were scanned with a UV lamp to locate and identify the nucleotide spots.

MORPHINEON AMINOACYL-tRNA SYNTHETASE

377

Each spot was cut out and eluted overnight in 0.066 M phosphate buffer, pH 7.0. The UV absorption spectrum for each spot was obtained in the range of 245-290 nm and the amounts of the respective nucleotides were calculated using the factors of Elson et aL s. Chemicals L-[U-14C]Arginine (210 mCi/mmole), L-[U-14C]leucine (248 mCi/mmole) and L-[U-14C]phenylalanine (345 mCi/mmole) were purchased from Tracer Lab., Waltham, Mass., U.S.A. Morphine [N-methyl-14C]hydrochloride (53 mCi/mmole) was purchased from Amersham/Searle, Arlington, Ill., U.S.A. t R N A (sodium salt, stripped and free from ribonuclease) isolated from Escherichia coli cells strain B was purchased from General Biochemicals, Chagrin Falls, Ohio, U.S.A. RESULTS

Partial purification and activity of aminoacyl-tRN.4 synthetase The procedure for partial purification of aminoacyl-tRNA synthetase from mouse brain by pH 5.0 precipitation of the 105,000 × g soluble supernatant followed by stripping with DEAE-cellulose was described above in the Methods and Materials section. As assayed with purified tRNA from E. coli, this enzyme preparation from control brains (5SD) catalyzed the binding of [14C]arginine, [14C]leucine and [14C]phenylalanine. The enzyme was purified 29-fold for binding arginine, 81-fold for leucine and 24-fold for phenylalanine (Table I). No attempt was made to further fractionate this enzyme preparation into specific synthetases. When the same purification procedure was followed with morphinized brains the same degrees of purification and yields were obtained (Table II). The enzyme (5SD) was purified 31-fold for binding arginine, 70-fold for leucine and 28-fold for phenylalanine. TABLE I PURIFICATION OF A M I N O A C Y L - t R N A SYNTHETASE FROM NORMAL MOUSE BRAIN

Purification procedures and assay conditions were as described in the Methods and Materials and the Results sections. Results are based on the binding of E. coli tRNA with [14C]arginine. Fraction

Specific activity (pmoles of arginine bound/mg protein~50 t~gtRNA/30 rain)

Homogenate (H) 0.7 Post-nuclear supernatant (PN) 3.1 Soluble supernatant (SS) 12.4 Solubilized pH 5 precipitate (5S) 20.3 DEAE-treated purified enzyme (5SD)* 20.4

Purification (X)

YieM(%)

4.4 17.8 29.1

100 106 97 27

29.2

28

1.0

* The enzyme (5SD) was 81-fold purified with respect to [14C]leucinebinding, and 24-fold purified with respect to [14Clphenylalaninebinding.

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R. K. DATTA AND W. ANTOPOL

TABLE II PURIFICATION OF AMINOACYL-tRNA SYNTHETASEFROMTHE MORPHINIZEDMOUSEBRAIN Purification procedures and assay conditions were as described in the Methods and Materials and the Results sections. Results are based on the binding of E. coli tRNA with [laC]arginine.

Fraction

Specific activity Purification (X) (pmoles of arginine bound/mg protein/50 t~g t RN,4 / 30 min

Homogenate (H) 0.4 Post-nuclear supernatant (PN) 2.0 Soluble supernatant (SS) 8.7 Solubilized pH 5 precipitate (5S) 9.4 DEAE-treated purified enzyme (5SD)* 12.3

Yield (%)

5.0 21.9 23.5

100 100 100 45

31.0

33

1.0

* The enzyme (5SD) was 70-fold purified with respect to [14C]leucine binding, and 28-fold purified with respect to [14C]phenylalanine binding. TABLE III PHYS1CO-CHEMICAL PROPERTIES OF tRNAs ISOLATED FROM THE CONTROL AND THE CHRONICALLY MORPHINE-TREATED MICE Mean values of 3 preparations are given.

Physico-chemical property

t RNA isolated from

R~ T-- ~TCA-insoluble (%) 1"~ ~ TCA-soluble (%) D N A (%) Protein (%) Amino acids ) Lipids PolysaccharidesJ Adenylate (moles/100 moles) Cytidylate (moles/100 moles) Guanylate (moles/100 moles) Uridylate (moles] 100 moles) AMP + CMP/GMP + UMP E ] ~m 260 n m

Control brains

Morphinized brains

Control liver

95.2 < 1 < 1 < 1

94.1 <1 <1 <1

96.0 <1 <:1 <1

Not detectable

Not detectable

Not detectable

19.0 23.3 30.9 18.0 0.86 203

260 nm Ratio - 280 nm

18.7 22.7 31.5 17.5 0.84 205

1.70

1.72

203 1.70

Partial purification o f t R N A f r o m brains In order to compare activities of partially purified enzymes in binding amino a c i d s w i t h t R N A s f r o m t h e c o n t r o l a n d t h e m o r p h i n i z e d b r a i n s as well as t h e p h y s i c o chemical properties of brain tRNAs purification of tRNA

from these two sources, isolation and partial

by the method described in the Methods and Materials section

MORPHINEON AMINOACYL-tRNA SYNTHETASE

379

were attempted. For comparison, t R N A from liver of control mice was similarly purified. These t R N A s from brain and liver were relatively free from D N A , protein, amino acids, lipids and polysaccharides and had characteristic UV absorption (Table III). The chromatographic elution profiles of the t R N A s were almost identical. The t R N A from control brains was not significantly different from t R N A from the morphinized brains in physico-chemical properties studied (Table III). Amino acid-binding capacities o f brain t R N A s from the control and the morphinized brains As shown in Table IV, synthetase (5SD) from control brains catalyzed the binding of [14C] labeled arginine, leucine and phenylalanine with mouse brain and mouse liver t R N A as well as with E. coli t R N A . Though there are some differences in relative activities of the synthetase from control brains in binding amino acids with t R N A s from 4 different sources, there was no significant difference in relative activities of the enzyme from control brains in binding amino acids with t R N A s from the control and the morphinized brains. Similarly, synthetase (5SD) isolated from the morphinized brains also catalyzed binding of amino acids with mouse brain and mouse liver t R N A as well as with E. coli t R N A . Here too, there were no significant differences in relative activities of the enzyme from the morphinized brains in binding amino acids with t R N A s from the control and the morphinized brains. It was presumed, therefore, that the amino acid-binding capacities of t R N A s isolated from the control and the morphinized brains did not alter significantly with chronic morphine administration. Specific activities o f synthetase f r o m control and morphinized brains Based on the binding of E. coli t R N A and [14C] labeled arginine, leucine and phenylalanine, the purified enzyme (5SD) from the morphinized brains had decreased specific activities as compared with those of the enzyme (5SD) from the control TABLE IV In vitro BINDING

OF [14C]AMINO ACIDS WITH

tRNAs

FROM DIFFERENT SOURCES BY SYNTHETASES FROM

THE CONTROL AND THE MORPHINIZED BRAINS

Mean values of two experiments using different enzyme preparations are given. Source of synthetase

Control brains:

Morphinized brains:

Source of tRNA

E. coli Control brains Morphinized brains Control liver E. coli Control brains Morphinized brains Control liver

Relative activity (%); in binding with Arginine

Leucine

Phenylalanine

100 78 80 86 100 81 85 86

100 112 102 98 100 112 110 100

100 114 105 114 100 117 106 100

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R . K . DATTA AND W. ANTOPOL

TABLE V SPECIFIC ACTIVITIES OF SYNTHETASES FROM THE CONTROL AND THE MORPHINIZED BRAINS

Twenty control and 20 morphinized mice were used in the experiment. Brains from 4 mice were pooled for enzyme purification and assays. Results are based on binding of E. coil tRNA with [z4C]amino acids listed in the Table.

Enzyme (5SD) from Fold purified over Binding with homogenate ( H) with respect to binding with the amino acid concerned

Specific activity pmoles amino acid Mean relative bound/mg enzyme activity (%) protein~50 #g tRNA /30 min (mean S.E.M.)

Control mice Morphinized mice

29-32 31-33

Arginine Arginine

20.3 5- 1.4 12.3 ± 0.9

100 61

Control mice Morphinized mice

78-82 70-78

Leucine Leucine

14.0 ± 1.3 7.8 ± 0.5

100 56

Control mice Morphinized mice

24-27 28-30

Phenylalanine Phenylalanine

9.4 ± 0.7 6.2 ± 0.3

100 65

I

t

!

I

o~ >.

.40 u

o_ c

o

"20

Last dose (mg/Kg)

20

40

60

80

100

Day

8

16

24

32

40

45

Fig. 1. Effect of chronic administration of morphine on the activity of aminoacyl-tRNA synthetase. Gradually increasing doses of morphine were administered as outlined in the Methods and Materials section. Brains from 4 mice were pooled for enzyme purification and assays. Results are based on the activities of purified synthetases in binding E. coli tRNA with leucine. The decrease was calculated as percentage decrease of specific activity of synthetase of the morphinized brains from that of synthetase of the control brains. The specific activities of synthetase of the control brains did not change significantly during the entire period of the experiment. The 'day' in the abscissa indicates the day in which the indicated last dose of morphine was administered and enzyme activities measured, and does not convey, in any way, that the decrease of specific activities of synthetase is proportional to the time of exposure to morphine. Dotted line indicates decrease of synthetase activity when morphine was withdrawn after the final dose of 100 mg/kg was administered.

MORPHINEON AMINOACYL-tRNA SYNTHETASE

381

brains, when enzymes from both the sources were purified to a comparable extent (Table V). This was true of all fractions during the course of purification of the enzyme in binding E. coli t R N A and [14C]arginine (Tables I and II). The decreases of synthetase activities of the morphinized brains (compared to that of the control brains) were fairly proportional to the doses of morphine administered during chronic morphinization (Fig. 1). In one experiment, brain synthetases were partially purified and their activities determined 5 days after the final injection of morphine or saline. The enzyme from the morphinized brains still had decreased activity as compared to that from the control (Fig. 1).

Effect of acute administration of morphine on the specific activities of synthetase Administration of a single dose of 30 mg/kg body weight of morphine did not significantly decrease the specific activities of synthetase (5SD) of the morphinized brain as compared to the synthetase (5SD) of the control brains, when assayed with E. coli t R N A and [14C]arginine. In vitro effect of morphine on purified synthetases from control and morphinized brains In vitro additions of morphine (up to 0.2 m M ) decreased the specific activities of the purified synthetase (5SD) from the control as well as from the morphinized brains when assayed with E. coli t R N A and [14C]arginine and [aac]leucine (Fig. 2). In the presence of morphine, the purified synthetases from the morphinized brains was inhibited slightly more than the enzyme from the control brains. The dose-response relationship was evident up to 0.05 m M concentrations of morphine. When the purified synthetase from the control brains was pretreated with 0.2 m M morphine for 2 h ,

I

3O

u 20 ARGININE

c

LEUCINE Cl

i

0,05

,,

,

~l

0.1

t

i

i

I

O. 15

0.05

0. I0

0.15

CONCENTRATION OF MORPHINE (raM)

Fig. 2. In vitroeffect of morphine on the activities of synthetases from the control and the morphinized brains. Regults (means with ranges) are based on 3 assays of activities of synthetases from two sources in binding E. coli tRNA with amino acids indicated in the figure. The decreases in activities of synthetases in the presence of morphine were calculated as percentage decreases of the activities of synthetases in the absence of morphine. --©--C)--, Synthetase from the control brains; ~ , Synthetase from the morphinizcd brains.

0.2

382

R.K. DATTA AND W. ANTOPOL

TABLE VI EFFECT OF PRETREATMENT W I T H MORPHINE ON THE ACTIVITy OF PURIFIED SYNTHETASE

Results are based on two different assays with synthetase from the control brains and E. coli tRNA. Pretreatment of synthetase with

Binding with

None Morphine None Morphine

Activity pmoles of amino acid bound/ mg enzyme protein/ 50 pg tRNA/30 min (mean)

Relative activity (%)

[laC]arginine [14C]arginine

20.7 18.6

100 90

[laC]leucin e [14C]leucine

13.7 11.6

100 85

in the assay m e d i u m w i t h o u t t R N A and a m i n o acid a n d m o r p h i n e then was r e m o v e d by dialysis for 4 h the p r e t r e a t e d synthetase h a d a specific activity for binding E, coli t R N A a n d arginine a n d leucine 10-15 ~ lower t h a n a similar synthetase p r e p a r a t i o n to which m o r p h i n e was n o t a d d e d d u r i n g p r e t r e a t m e n t ~Tabl~ v I ) . . In vitro effect o f morphine on the binding property o f t R N A s I n o r d e r to examine whether m o r p h i n e altered the a m i n o acid-binding p r o p e r t y o f t R N A , t R N A s were p r e t r e a t e d with 1 m M m o r p h i n e for 2 h in the assay m e d i u m without enzyme a n d a m i n o acid. t R N A s were purified a n d freed o f m o r p h i n e as described in the M e t h o d s a n d M a t e r i a l s section. The c h r o m a t o g r a p h i c profile on D E A E cellulose o f the p r e t r e a t e d E. coli t R N A was similar to that o f the u n t r e a t e d E. coli t R N A . The p r e t r e a t e d t R N A s f r o m E. eoli, the c o n t r o l brains a n d the m o r p h i n i z e d TABLE VII EFFECTOFPRETREATMENTWITHMORPHINEONTHEBINDINGPROPERTYOFtRNAs Results are based on two different assays with synthetase from the control brains. tRNA

Pretreatment

Binding with

Binding of amino acids pmoles amino acid bound/mg enzyme protein~50 i~g tRNA/30 mitt (mean)

E. coli

None Morphine

[14C]arginine 114C]arginine

20.6 20.1

E. coil

None Morphine

[14C]leucine [14C]leucine

13.8 13.6

Control brains

None Morphine

[14C]arginine [x4C]arginine

20.0 19.9

Morphinized brains

None Morphine

[a4C]arginine [laC]arginine

19.6 19.9

MORPHINE ON A M I N O A C Y L - t R N A SYNTHETASE

383

brains, all bound amino acids at the same rate as the untreated tRNAs (Table VII), suggesting that morphine had no effect on the binding property of tRNA. It was further found that, under the conditions employed in synthetase assays, tRNAs from E. coli, the control brains and the morphinized brains did not bind [14C]labeled morphine. DISCUSSION

The activation of amino acid and the binding to its specific tRNA involve amino acid, tRNA and the enzyme aminoacyl-tRNA synthetase. This integrated step can be followed independently of the other steps of protein synthesis so that any changes which occur in the components as a result of chronic administration of morphin~ can be studied. Of the components of this step of protein synthesis, amino acid is least expected to be altered by morphine. Therefore, our efforts were concentrated on studies of possible alterations in the functions of tRNA as well as the activities of synthetases. In the present study, physico-chemical properties (Table III) and chromatographic elution profiles of tRNA from the morphinized brains were not significantly different from those of tRNA from the control brains. Further, the amino acid-binding capacities of tRNAs isolated from the control brains and from the morphinized brains were not altered significantly by chronic administration of morphine (Table IV). When tRNAs from E. coli, control brains and morphinized brains were pretreated with morphine, these tRNAs bound amino acids almost at the same rate as the untreated tRNAs, suggesting that morphine had no effect on the amino acid-binding properties of tRNAs (Table VII). Further, under the conditions employed in synthetase assays, tRNAs from E. coli, the control brains and the morphinized brains did not bind [14C]morphine and the tRNA purified from the morphinized brains did not contain detectable morphine. In view of these findings it is presumed that the chronic administration of morphine to mice does not change the chromatographic elution profiles, physico-chemical properties and amino acid-binding properties of tRNAs. There are several reports of changes in the tRNA pattern under different physiological and pathological conditions( such as viral infection14,21, hormonal stimulation1,1~, embryogenesis and carcinogenesis12, 2a. In most cases, the changes were the altered chromatographic elution profiles. Though, there is, to our knowledge, no reported case of altered tRNA patterns as a result of administration of analgesic or narcotic drugs, it is hard to rule out, as we used unfractionated tRNAs in our study, whether the chronic administration of morphine alters structures and functions of specific tRNAs. It is qtfitepossible that the physico-chemical properties of some of the tR,NAs are altered to some minor extent so that it is not included in the material obtained by the preparation procedure or is not revealed in the chromatographic elution profiles. In the present study, the minor bases of tRNAs, particularly the.methylated bases, were not investigated. Since the methylated bases of tRNAs play an important role in amino acid-binding properties of tRNAI~ and since morphine is partly demethylated in the body (reviewed by Scrafani and Clouet22), it is of interest to investigate whether the chronic administration of morphine causes transmethylation of tRNA,

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and, if so, whether it affects the amino acid-binding properties of tRNA so methylated. This study indicates that there is no significant in vivo effect of a single acuge dose of morphine on specific activities of synthetase in binding tRNA with amino acids. However, after chronic administration of morphine the specific activities of synthetase decreased significantly (Table V). This was observed in the purified synthetase as well as in fractions during the course of purification of the enzyme (Tables I and |I). The decreases in the activity of purified synthetase were dose-dependent and persisted for at least 5 days after the final dose of morphine (Fig. 1). In view of these findings, the chronic administration of morphine appears to be responsible for the decreases in specific activities of synthetase. The sustained decreases in activities of synthetase during a period of 5 days following the last dose of morphine suggest the desirability of a longer duration of morphine withdrawal to determine whether the morphine-induced decreases in synthetase activities are irreversible. Further, it is possible that the chronic administration of morphine may decrease the production of synthetases in the brain. An experiment is desirable to check this possibility. The absence of morphine (determined by gas chromatographic analysis) in the purified preparation of synthetase from the morphinized brains and the inability of the purified synthetase to bind [14C]morphine in the assay conditions suggest that the decreases in specific activities of synthetase apparently were not related to the binding of morphine with synthetase. However, some direct effect was noted in vitro, where morphine moderately decreased the specific activities of purified synthetase from the control, as well as from the morphinized brains (Fig. 2). In addition, the in vitro pretreatment of the purified synthetase with morphine caused some decrease in the activity of the enzyme in binding amino acids with tRNA. We found that nalorphine, a prototype narcotic antagonist, did not decrease in vitro specific activities of purified synthetase nor reverse the morphine-induced decrease of specific activities of the purified synthetase. Work is under way to determine the mechanism by which morphine decreases these specific activities. It is possible that the morphine-induced decreases of synthetase activity may be mediated through structural changes of the synthetase molecule during chronic administration of morphine. Alternatively, it may be that the metabolites of morphine bind with synthetase and interfere with the binding of amino acid to tRNA. Grollman 1° attributed inhibition of protein synthesis in HeLa cells, resulting from exposure to ipecac alkaloids, to interference at aminoacyl-tRNA transfer step. It has yet to be found whether the chronic administration of morphine decreases the amounts of aminoacyl-tRNA making it rate limiting to inhibit protein synthesis. Whatever be the mechanism, the present study detected, at least, one step in the protein synthesis where morphine exerts an inhibitory influence. Previous studies in our laboratory have indicated that the chronic administration of morphine produces dose-dependent decreases in uridine incorporating abilities of brain tissues of mice 7 and causes some inhibition of RNA polymerase activities of brain nuclei 7a. It will be interesting, therefore, to determine whether the inhibition of protein synthesis is a selective effect of the chronic administration of morphine or is secondary to the inhibition of RNA synthesis. While the decreases of protein and

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R N A synthesis appear to be due to biochemical responses to chronic a d m i n i s t r a t i o n of m o r p h i n e , the pharmacological significance of these biochemical changes in m a m mals is yet to be determined. ACKNOWLEDGEMENTS Portions o f this study were presented at the First N a t i o n a l Meeting of The A m e r i c a n Society for N e u r o c h e m i s t r y in A l b u q u e r q u e , New Mexico, in 1970. The study was supported by the N a t i o n a l Institute of Health, U.S. Public Health Service G r a n t No. F R 05492, The Saul Singer F o u n d a t i o n , Charles H. Silver F u n d a n d The Lenore W e i n s t e i n F u n d , New York. The a u t h o r s t h a n k Mrs. A m m i n i M o o r t h y , Miss Shirley M a r r i n , Mr. Richard Seelig, Mr. Steven Friedfeld, Mrs. Victoria D i x o n a n d Mrs. Esther M c M a n u s for their able assistance. R. K. D. expresses gratefulness to Dr. R. J. Stenger, Director of Pathology a n d L a b o r a t o r i e s of Beth Israel Medical Center, for his valuable suggestions in writing the manuscript.

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