Journal of Neuroscience Research 3:375-387 (1978)
Studies on the Effects of Chronic Ethanol Ingestion on the Properties of Rat Brain Ribosomes Sujata Tewari, Sandra Murray, and Ernest I?Noble Department of Psychiatry and Human Behavior, College o f Medicine, University o f California, lrvine
Previous observations have demonstrated decreased in vivo and in vitro protein synthesis by brain ribosomal systems following long-term ethanol ingestion. For further investigation of the properties of brain ribosomes, the 40s and 60s ribosomal subunits were successfully isolated from control and chronic 10% ethanol-drinking rats. For a successful dissociation of ribosomes into subunits NH4C1, puromycin and a high-salt treatment at 10°C were essential with a critical concentration of Mg2+ since ribosomes could not be resolved at less than 7 mM Mg2+. Analysis of the A23, profile of the subunits o n t h e sucrose gradients showed no significant differences between the control and ethanol-ingesting groups. Studies on H-labeled ribosomes following in vivo RNA labeling showed correspondence of the radioactive profiles from t h e incorporation of [ S 3 H ] orotic acid into RNA with the sucrose gradient absorbance profile of 6 0 s and 40s ribosomal subunits. Furthermore, active reassociation of both subunits occurred at 37OC as demonstrated b y t h e increased [ l4c1phenylalanine incorporation in the presence of poly(U). Results further showed that the poly(U)-dependent [ 14C]phenylalanine incorporation was significantly reduced by t h e subunits from the ethanol-ingesting animals. These findings suggest that long-term ingestion of ethanol caused functional changes in the properties of brain ribosomes, specifically o n the reassociation process of the two subunits. Key words: brain, protein synthesis, alcohol, ethanol, ribosomal subunits INTRODUCTION
The inhibition of in vivo and in vitro RNA and protein synthesis in brain tissue following long-term ethanol ingestion has been demonstrated repeatedly in recent years [Tewari and Noble, 1971; Jarlstedt and Hamberger, 1972; Noble and Tewari, 1973; Renis et al, 19751 . Subsequently, studies of effects on chronic ethanol ingestion were reported on Abbreviations: mRNA - Messenger RNA; rRNA polyuridylic acid; S - Svedberg coefficient.
-
Ribosomal RNA; tRNA
-
Transfer RNA; poly (U)
Dr Noble is now at the National Institute on Alcohol Abuse and Alcoholism, 5600 Fishers Lane, Rockville, MD 20825. Address reprint requests to Dr Sujata Tewari, Department of Psychiatry and Human Behavior, College of Medicine, University of California, lrvine CA 92717. 0360-4012/78/0305/6-0375$02.60 @ 1978 Alan R . Liss, Inc
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the specific reactions of protein biosynthesis [Tewari and Noble, 1971; Fleming et al, 19751. Some of these findings have established the primary effects of ethanol at the ribosomal level where marked inhibition of [ l4 C] polyphenylalanine synthesis occurred independent of the source of the pH 5 precipitable enzymes fraction [Noble and Tewari, 19751. It was suggested that reduced association of mRNA with the ribosomes due to functional alterations of one or both subunits could be the primary cause for the observed ethanol-induced inhibition. In recent years extensive studies have established the ribosomes as the site for protein synthesis with a number of studies available on the structure and properties of these particles [Weinberg and Penman, 1970; Mejbaum and Pritchard, 1971; Tanaka and Ogata, 1972; Stoffler et al, 1974; Shires et al, 19751. These reports have shown the eukaryotic ribosomes to be composed of two subunits, containing three types of RNA and some 70 proteins. The sedimentation coefficient values, although variable from species to species, are thought to be 6 0 s and 4 0 s for the two respective subunits [Petermann, 19641. No studies are available on the effects of ethanol on ribosomal subunits and the present study was undertaken to isolate the dissociated 4 0 s and 6 0 s ribosomal subunits from brain tissue and to investigate the chronic effects of ethanol on the dissociation and reassociation properties of brain ribosomes. EXPERIMENTAL PROCEDURES Chemicals
All chemicals were reagent A grade and solutions were prepared with glass doubledistilled deionized water. Whatman glass filter disks (GF/A 2.4 cm) were purchased from Quickfit Reeve Angle, Inc (Clifton, New Jersey). Bovine serum albumin and Trizma [tris(hydroxymethyl)aminomethane] were products of Sigma Chemical Co (St. Louis, Missouri). The chemicals for scintillation fluid, PPO (2,s-diphenyloxazole) and POPOP {1,4-bis [2-(5-phenyloxazolyl)] -benzene}were purchased from Packard Instruments Co (Donners Grove, Illinois). Standard RNA (yeast) was from Calbiochem (San Diego, California). Labeled [5-3H] orotic acid (specific activity 17 Ci/mmole) and [' C] phenylalanine (41 0 mCi/mmole) were purchased from Schwartz/Mann Bioresearch (Orangeburg, New York). Administration of Ethanol
Male four-week-old Sprague-Dawley rats from Simonsen Laboratory were housed as described by Tewari and Noble (1970). The control group received water while the .ethanol group was given a 10%(v/v) ethanol-water solution ad libitum for a period of at least six weeks. Ethanol was routinely replaced with water 24 hours prior to experimentation. Both groups received laboratory chow ad libitum. Administration of RNA Precursor
Intraventricular injection of [5-3HI orotic acid into lateral brain ventricles was performed according to the techniques of Noble et a1 [1967]. Composition of Medium M
The medium contained 0.25 M sucrose, 0.02 M Trizma, pH 7.6,O.l M KCl, 0.04 M NaCl, 0.007 M magnesium acetate, and 0.006 M 2-mercaptoethanol [Tewari and Noble, 19701.
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Preparation of Ribosomal Subunits
1) Isolation and purification of ribosomes. Brains from both control and ethanol groups were treated identically. Ribosomes (RI) and pH 5 precipitable enzymes fraction were isolated by differential centrifugation of brain homogenates in medium M according to the procedures of Tewari and Baxter [ 19691 and Tewari and Noble [ 19701 . Ribosomes were purified by centrifuging 9 ml of the suspension through a discontinuous 0.5 M and 1 .O M sucrose gradient (9 ml each) containing 0.5 M NH4C1, 0.1 M Tris-HC1 (pH 7.6), 0.01 M MgClz, and 0.006 M 2-mercaptoethanol [Skogerson and Moldave, 1968; Martin and Wool, 19681. 2) Stripping of peptidyl-tRNA. Purified ribosomes (RII) obtained by the above procedures were stripped of peptidyl-tRNA by using a modification of the procedures of Skogerson and Moldave [1967] and Gasior and Moldave [1972]. At a concentration of approximately 2.5 mg of ribosomal protein/ml of 75 mM Tris-HC1, pH 7.6 (1 m1/4 gm brain), 0.6 ml of the suspension was incubated at 37°C for 25 minutes with 1 mM puromycin dihydrochloride, 80 mM NH4 C1, 75 mM Tris (pH 7.6), 12 mM MgClz, and 6 mM 2-mercaptoethanol in a final volume of 24 ml. When necessary, lml of the 105,OOOg supernatant fraction was added as a source of RNase inhibitor and transfer factors. Following the incubation, the reaction mixture was cooled in ice briefly and centrifuged at 105,OOOg for 30 minutes at 4" C. The reisolated puromycin-treated pellet (RIII) was suspended at a concentration of 5 mg/ml (1 m1/10 gm brain) in 80 mM KC1, 12 mM MgC12, 50 mM Tris-HC1 (pH 7.6), and 6 mM 2-mercaptoethanol, and was clarified briefly at 1,500g for 10 minutes at 4°C. 3) Separation and isolation of 60s and 40s subunits following high-salt treatments. The 1,500g supernatant (R Sup) obtained above was mixed with an equal volume of a 2.5 M KC1 and 10 mM MgClz mixture, and 1-ml samples were layered onto 34 ml of a 15-3576 (w/v) linear sucrose gradient containing 30 mM Tris-HC1(pH 7.6), 6 mM 2-mercaptoethanol, and either 0.6 M KC1 with 15 mM MgClz or 0.4 M KCl with 7 mM MgC12 [Martin and Wool, 1969; Mechler and Mach, 1971; Gasior and Moldave, 19721 . Gradients were centrifuged for 12-14 hours at 10°C and 37,OOOg in a SW 27 rotor. Cushions of 0.5 ml of 2% sucrose and 1.5 ml of 60% sucrose on the top and bottom of the gradient were included for convenience in monitoring gradient absorbance. One-milliliter fractions were collected and absorbance at 260 nm was monitored in a Gilford recording spectrophotometer, model 2400, fitted with a flow-through cell. Resulting subunit peak fractions corresponding to 60s and 40s were pooled separately and isolated by centrifugation for four hours at 10°C and 105,OOOg. The subunit pellets were frozen at -70°C after rehomogenizing in a storage medium containing 0.35 M sucrose, 50 mM Tris (pH 7.6), 7 mM MgClZ, and 6 mM 2-mercaptoethanol. Determination of Radioactivity
The incorporation of ["C] phenylalanine into protein was measured by determining the activity in hot TCA-extractable residue according to Tewari and Noble [1970]. The incorporation of [5-jH] orotic acid into RNA of the ribosomal subunit was determined by cold TCA and ethanol ether extractions on Millipore filter disks (2.4 cm, Whatman) [Tewari et al, 19751 . The radioactivity was determined using a PPO-POPOP mixture and a liquid scintillation counter, Beckman model LS 250 [Tewari and Noble, 19701. The protein and RNA content of the subcellular fractions were determined by the methods of Lowry et a1 [1951] and Mejbaum [1967] respectively.
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RESULTS Dissociation of Ribosomes Into the Large and Small Subunits
To determine the effects of long-term ethanol ingestion on the dissociation properties, brain ribosomes (RI fraction) from control and ethanol-ingesting groups were prepared and purified by centrifugation through a discontinuous sucrose gradient containing NH4C1to yield an RII fraction prior to dissociation into 6 0 s and 40s subunits (Fig 1). The NH4C1 treatment of RI fraction was thought to be essential since possible protein factors are removed by this process [Skogerson and Moldave, 1967; Borgese et al, 19731 . Figure 1 outlines a brief fractionation scheme. Details of the procedure have been described under Materials and Methods. The purified ribosomes (RII fraction) of both control and ethanol-ingesting groups could be completely dissociated into 6 0 s and 40s subunits by NH4C1, puromycin, and high-salt treatments, followed by continuous gradient centrifugation at 10°C. No dissociation occurred at 4°C where aggregation of ribosomes prevented dissociation and is similar to observations reported by Borgese et a1 [1973] and Martin and Wool [1969]. As with other mammalian ribosomes, brain ribosomes were more resistant t o dissociation than bacterial ribosomes and required a specific ratio of K+ and Mg2+ concentrations. Under present conditions, the presence of puromycin and the combination of high K+ with specified Mgz+ concentration were found to be crucial for successful dissociation.
RIBOSOMES
(RI Fraction)
layered on 0.5H and 1H sucrose buffer containing 0.5MNn C1. 44,000 g, 16-20 brs,
Supernatant
Ilibosomal Pellet
(RII Fraction)
I'uromycin incubation at 37012 for 25 min. 105.000 g, 150 min, C'4
I
Supernatant
Ribosomal 'Pellet (RIII Fraction) 1500 g, 10 min,
4.C
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Effects of Varying Magnesium Concentrations
Figures 2A and 2B show the A260 profiles of the ribosomal fraction on a sucrose density gradient before (RII fraction) and after puromycin treatment (R Sup fraction) to release the nascent polypeptide chains as described in Figure 1. In this experiment, 1 ml of RII fraction was layered onto a 15-35% gradient (Fig 2A) while another aliquot was incubated with puromycin, isolated, treated with high K+ (0.6 M) and 15 mM Mgz+ , and the resultant (R Sup) fraction layered on a similar gradient (Fig 2B). The profile obtained in Figure 2A shows several distinct peaks from both control and ethanol-treated brain showing the general distribution of polysomes in the NH4C1-treated RII fraction, and. some of these peaks were distinctly heavier than the larger subunit. The reisolation of RII fraction into ribosomal supernatant fraction gave two very distinct absorbance peaks following puromycin and high-salt treatment (Fig 2B). There was no significant difference observed between the absorbance profiles of the two subunits from control or ethanoltreated groups and both groups behaved similarly toward dissociation treatments. Dissocia tion characteristics were again examined by reducing the Mg2+ concentration from 15 to 7 mM. In this experiment fractionation of the ribosomal supernatant fraction on a gradien containing 0.6 M KC1 and 7 mM Mg2+ also gave excellent dissociation into two ribosomal subunits (Fig 3). No significant differences were again observed between the AZM)profiles of control and alcohol-drinking animals.
Control Ethanol
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Fig 2. Sucrose gradient analysis of cerebral ribosomes. Samples with approximately 2 mg of protein were layered onto 34 ml of 15-35% (w/v) sucrose gradients containing 0.6 M KC1 and 15 mM MgCl2. A) Before puromycin treatment. B) After successive puromycin and high-salt treatments. Control (-). Ethanol (- - -).
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Fig 3. Separation of ribosomes into subunits. After puromycin treatment of the NH4Cl-washed ribosomes and further treatment with 1.3 M KCl 11 mM MgClz approximately 1.46 mg of the R Sup fraction was layered onto gradients containing 0.6 M KC1/7 mM MgCl2. Gradients were monitored at 260 Ethanol (- - - - - -). nm continuously and collected as previously described. Control (-).
Although excellent dissociation of ribosomal subunits could be obtained in the presence of 15 mM Mg2+ and 7 mM Mg2+, further lowering of Mg2+ was proven unsuccessful. To illustrate this crucial requirement of MgZ+,AZm profiles of the control and ethanol-treated ribosomes in a sucrose gradient were examined after treatment of RII fraction with puromycin and centrifuging in the presence of varying concentrations of Mgz+ and high K+. No resolution was found to occur in either group when Mgz+ concentrations were lowered to 1.5 or 0.1 mM with a constant K+ level of 0.6 M (Figs 4A and 4B). Protein Recovery
To ensure similar experimental processing, the recovery of ribosomal protein between the control and ethanol groups were compared during the entire fractionation procedure (Table I). TABLE I. Protein Recovery During the Fractionation of Ribosomes
Fractions Ribosomes Purified ribosomes (RII) Postpuromycin-treated supernatant fraction (R Sup) Large ribosomal subunits (60s) Small ribosomal subunits (40s) ~~
Control (% of RI protein)
Ethanol-treated (%of RI protein)
100" 51
100 53
11 4 4
13 I 6
~~~~~~~~~~~~~~~~~~~
alOO% = approximately 2.47 mg of ribosomal protein/gm wet weight of brain tissue. Preparation of the various fractions is described in Figure 1. Subunits were separately pooled peaks from gradients after puromycin and high-salt treatments, sedimented at 105,OOOg for four hours, and resuspended in buffer. protein content of each fraction was assayed according to Lowry et a1 [ 221.
Ethanol and Brain Ribosomes
38 1
3.0 2.6
2.0
1.6
1.0
0.6
Bottom
Raetion h b s r
Fig 4. Effect of Mgz+ level in sucrose gradients on ribosomal subunit separation. Following puromycin treatment, ribosomal suspensions with approximately 0.8 mg of protein were layered onto 15-35% (v/w) sucrose gradients containing either A) 0.6 M KCI and 1.5 mM MgClz, or B) 0.6 M KCI and 0.1 mM Ethanol (- - -). MgClz. Control (-).
Following the discontinuous sucrose gradient centrifugation of equal amounts of control and ethanol RI protein fractions with NH4C1, approximately 57 and 53% of the ribosomal protein were recovered in the two RII fractions, respectively. Further incubation of the RII fraction with puromycin removed most of the proteins leaving only 11 and 13% of the total protein in the control and ethanol (R Sup) fractions containing the partially dissociated ribosomes. Most of this protein could be recovered in the final pooled fractions containing the 60s and 40s subunits after resedimentation at 105,OOOg. The final recoveries for both control subunits were 4%, while those of ethanol-treated subunits were 7 and 6% for the larger and smaller subunits, respectively. The distribution of the protein content of the two ribosomal subunits was similar between the two groups with 40s values slightly lower in each group. The total yield of the isolated subunit protein was slightly higher in the 60s and 40s particles of brain from ethanol-drinking animals when compared to the control, while individually the protein content of the larger subunit was also higher than the 40s fraction in the ethanol-treated brains. Since complete dissociation of ribosomes could be obtained in the presence of both 15 and 7 mM Mg2+, the recovery of protein content was also compared under these conditions and the protein values for the recovery into the 60s and 40s subunits are given in Table 11. Studies show no significant differences in the total yield of subunit protein from the gradients with 15 or 7 mM Mg2+. If at all, the 15 mM Mg2+ gradient gave a slightly
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TABLE 11. Effects of Mg2+ Concentration on the Recovery of Ribosomal Protein in the 60s and 40s Fractions* Treatment
1. Pre-high-salt treatment 2. Post-high-salt treatment a) 15 mM Mg2+ b) 7 mM Mg2+
Fraction
mg protein/gm brain weight
R Sup
0.351
6 0 s unit 40s unit 60s unit 4 0 s unit
0.119 0.072 0.096 0.068
%recovery of R Sup protein fraction 100 34 21 27.4 19.4
*Experimental conditions were similar to those described in Figure 2. Each of the two separate aliquots of the puromycin-treated R Sup fractions was individually fractionated on separate gradients containing 15 mM Mgz+ and 7 mM Mg2+ with 0.6 M K+. Total recovery of subunit fractions was 5 5 and 47% with 15 and 7 mM Mg2+, respectively.
higher yield than the 7 mM Mg2+ gradient, with 55% recovery of total protein at the higher Mg2+ concentration. The total protein recovered under these conditions in the larger subunit and smaller subunit were 34 and 21%, respectively. Lowering of Mg2+ to 7 mM resulted in 27% of ribosomal protein recovered in 60s and 19%in 4 0 s fraction. These studies were carried out with subunits from control brains only. Effects of lntraventricular Administration of [5-3 HI Orotic Acid
The subunits were further characterized by labeling their rRNA content with a single intraventricular pulse of [5-3H]orotic acid (an RNA precursor) for 24 hours. The one-day period was chosen to differentiate the labeling of rRNA from mRNA since the half-life of ribosomal RNA in brain tissue has been found to be 11 days [Khan and Wilson, 1965; Bondy, 19661 . The labeled ribosomes were next dissociated into subunits by the procedures described and radioactivity was measured into cold TCA-insoluble residue (Fig 5 ) . Findings given in Figure 5 demonstrated that the AZm sucrose gradient profiles of labeled control ribosomal subunits could be measured only at the peaks for 4 0 s and 60s (dotted line). The radioactivity was determined in each fraction and the solid line represents the amount of radioactivity recovered in fractions following absorbance measurement at AZm.Data clearly show that the incorporation of [5-3HI orotic acid into rRNA as measured by cold TCA-precipitable counts were only in the fractions corresponding to the 60s and 4 0 s peaks of control brains. There were no detectable amounts of radioactivity present in the initial heavier fractions or subsequent fractions collected after the shoulder of the 40s peak. The incorporation of [5-jH] orotic acid into subunit RNA fractions was further determined by pooling the individual peaks and sedimenting them at 105,OOOg for four hours at 10°C. Results given in Table I11 show that both subunits had similar specific activities and 50% of the radioactivity initially present in the ribosomal supernatant fraction could be recovered as labeled RNA in each RNA fraction of the 60s and 40s fractions. In Vitro Reassociation of Subunits
Following the labeling of rRNA other functional properties of the subunit were examined by studying the poly(U)-dependent incorporation of ["C] phenylalanine into
Et h an o l an d Brain Ribosomes
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15
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Fig 5. In vivo labeling of rRNA. Twenty-four hours after intrdventricukar injection of 30 pCi [3H] orotic acid (specific activity 17 Ci/mmole) into each rat brain, ribosomal subunits were isolated by methods described, following treatment by puromycin and high salts (1.3 M KC1/11 mM MgC12). Indicated amounts (control ribosomal sup) were layered onto the gradients listed, and after centrifugation (see Methods), gradients were monitored continuously at 260 nm and collected in approximately 30 1.1-ml fractions. Aliquots (0.05 ml) were removed from selected fractions for determining radioactivity. TABLE 111. In vivo Incorporation of [S3H] Orotic Acid Into RNA of Brain Ribosomal Fractions*
Fractions Ribosomal supernatant prior to fractionation Pool 60s 40s
Control ( l o 3 x counts/min/mg RNA 199.0
% Activity
100 50 50
102.0 102.0 ~~
*Radioactivity was determined by cold TCA precipitation. Ribosomal supernatant was postpuromycin and post-low-speed-soluble fraction before high-KC1 treatment. The subunits were separately pooled peaks from fractions obtained from centrifugation of ribosomal supernatant after high-salt treatment.
protein under appropriate in vitro reassociating conditions. Results obtained in Table IV show that the subunits were able to recombine under in vitro conditions and incorporate [ 14C]phenylalanine into protein. The stimulation of [ 14C]phenylalanine incorporation by poly(U) was negligible in the presence of a single subunit fraction. Increased activity was obtained only when Cm and C ~ O were present together (137%). This increased activity was RNase sensitive as demonstrated by a large decrease in the hot TCA-precipitable [I4C] phenylalanine activity. Addition of RNase was without effect on the activity of single subunits.
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TABLE IV. Effects of Poly(U) on [ 14C] Phenylalanine Incorporation Into Protein by Ribosomal Subunits* Endogenous activity (%)
Experimental conditions
100.0 137.0 54.0 101.6 100.0 101.0 100.0
*loo% = 7,500 counts/min/mg ribosomal protein.
Incubation medium consisted of 75 mM Tris-HC1, pH 7.6,40 mM NH4CI, 7 mM MgCl?, 6 mM 2-mercaptoethanol, 100 pg poly(U), 200 pg bovine serum albumin, approximately 30 pg protein of 6 0 s fraction and 19 pg of 40s fraction. Incubation period was 30 minutes at 37°C. RNase, when added, was at a concentration of 20 pg/ml of incubation medium. The source of the pH 5 enzyme fraction was from control brain only and was added at a concentration of 200 pg/ml in incubation medium.
Effects of Ethanol Ingestion on I n Vitro [14C1Polyphenylalanine Synthesis
The reassociation of 60s and 40s subunits was again examined to determine the possible effects of chronic ethanol ingestion. Table V compares the activity of the subunits between the control and ethanol-ingesting groups both in the presence and absence of poly(U) (ie, under endogenous conditions). Data show that under either experimental conditions the subunits prepared from the control brain demonstrated a higher activity than those isolated from the ethanol-treated brain. The addition of polyuridylic acid, resultedin increased activity in the ethanol group, with reduced l 4 C activity when comparedto the control. These results indicate that the in vitro recombination of 60s and 40s ribosomal subunits was significantly inhibited following chronic ethanol ingestion.
DISCUSSION
In this paper we have examined the effects of chronic ethanol ingestion on the separation of cerebral ribosomes into the two subunits, 60s and 40S, by puromycin and high-salt treatments. In addition, the subunits were characterized by in vivo labeling of their rRNA content with [!G3H]orotic acid and by their ability to synthesize [14C] polyphenylalanine under in vitro conditions. Our findings show that, similar to rat liver TABLE V. Effects of Ethanol o n the Poly(U)-Dependent Phenylalanine Incorporation ~
~~
Experimental conditions C 6 0 s + C 40s E 6 0 s + E 40s
Endogenous, % control activity 100 78
Endogenous + poly(U) % control activity 140 104
Incubation conditions were similar to those described in Table IV. Approximately 30 pg of 60s subunit fraction and 19 pg of 40s subunit fraction were used from each of the control (C) and ethanolingesting (E) groups. 100% = 5,547 counts/min.
Ethanol and Brain Ribosomes
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ribosomes [Lawford, 19691, brain ribosomes from both control and ethanol-ingesting animals could be dissociated into subunits by releasing the peptidyl transfer RNA and nascent polypeptide chains by treating first with puromycin (Figs 2A and 2B) and then immediately increasing and decreasing the K+ and Mgz+ concentrations, respectively, during sucrose gradient centrifugation. Our observations indicate that brain ribosomes from both control and ethanol-ingesting groups were not only highly sensitive to changes in the K+/MgZ+ratio, but, in addition, were highly resistant to dissociation into subunits. In contrast to liver, where lowering of Mg2+ concentrations have been reported successful, brain ribosomes from both groups required a higher Mg2+ concentration (7-1 5 mM) and had to be sequentially subjected to puromycin and high-KC1 treatment. Somewhat similar data are available from other laboratories showing differences in acrylamide gel electrophoresis of proteins and high Mgz+ requirement between brain and liver ribsomal subunits. Furthermore, the lowering of Mg2+ concentrations from 1.5 to 0.1 mM in the gradient (Figs 4A and 4B) or in an incubation medium containing puromycin and 0.5 M KC1 at the same time was also found to be unsuccessful for dissociation of ribosomes into subunits (unpublished data), although this treatment has been used successfully with rat liver ribosomes [Lawford et al, 1966; Lawford, 19691 . Under the present experimental conditions, no ethanol-induced changes in the K+ or Mgz+ requirement could be observed. Among other properties, the dissociation process of control and ethanol-ingesting ribosomes after the high-salt treatment was temperature sensitive and was only effective at 10°C since further lowering of temperature did not result in subunit resolution for both groups. The present studies have further examined the nature of labeling of RNA contained in the subunits with [5-jH] orotic acid. For the production of a functional ribosomal subunit, studies on ribosomal assembly have shown interaction of RNA and protein. These subunits are known to contain three species of RNA [Weinberg and Penman, 19701 . In this report we have presented evidence of substantial incorporation of [5-3H] orotic acid into rRNA of both subunit fractions after 24 hours of pulse labeling. Somewhat similar observations have previously reported the labeling of cytoplasmic rRNA after 2 and 24 hours of initial pulse [Noble and Tewari, 1973, Tewari, and Noble, 1975al. These studies showed the incorporation of [5-jH] uridine or [5-jH] orotic acid specifically into tRNA and rRNA in rodent brain. The subunits from both control and ethanol-ingesting groups were further characterized by in vitro reassociation studies which demonstrated a definite high poly(U) response in the presence of both subunits. However, while the ingestion of ethanol had not changed the dissociation characteristics, the reassociation process as determined by increased [I4C] phenylalanine incorporation by poly(U) was found to be adversely affected. It is of interest to note that some detectable I4C activity was always present in the absence of poly(U) under incubation conditions containing one or both subunits (Table IV). However, the RNase-sensitive poly(U) stimulation was present only when the incubation media contained both subunits (Tables IV and V). Thus, at this moment, the nature of this endogenous activity is not clearly understood. The decreased incorporation of ["C] phenylalanine into protein by ethanol subunits is particularly signficant since the incorporative ability was determined in the presence of the control pH 5 enzyme fraction which served as the source of aminoacyltransfer RNA synthetases, tRNA, and other transfer factors. Furthermore, the observed effects of ethanol were not attributable to technical procedures since ribosomes from both groups were processed identically, which is reflected in comparable protein recovery
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during the entire fractionation procedure (Table I). Similar inhibition of [ 14C] polyphenylalanine synthesis by ethanol-treated ribosomes has been reported previously and it was suggested that functional alterations of these particles might be responsible for possible reduced reduced mRNA association resulting in decreased availability [Tewari and Noble, 1975a, b] . These ideas are consistent with the present data on decreased activity in the presence of the 40s and 60s subunits of the ethanol group and previous findings on the labeling pattern of ribosomal RNA and subsequent inhibition of protein synthesis [Tewari and Noble 1975a; Noble and Tewari, 19751. However, more experiments are required to understand fully the chronic effects of ethanol on the reassociation properties of the 60s and 40s ribosomal subunits. In conclusion, our findings suggest that ethanol ingestion definitely affected the in vitro recombination of ribosomal subunits involved in carrying out effective protein synthesis. The eukaryotic ribosomes are heterogeneous in nature and even to this date the functional significance of this heterogeneity is unclear. Since it is believed that the functional characteristics of ribosomes are highly dependent on a variety of factors [MacInnes 1972, 19731, it is extremely suggestive that ribosomes exert regulatory roles in cellular metabolic processes. This is of particular importance in brain tissue which is a highly specialized, diversified organ and is itself heterogeneous in nature. The present findings on decreased activity in the presence of ethanol ribosomes are thus highly provocative in postulating that ethanol interferes with normal functioning of brain tissue at the ribosomal level. The present observation could therefore be a function of instability in one or both of the 40s subunit tFWA complexes and a functional change in the 60s subunits engendered by ethanol. The exact nature of this deficiency is currently under investigation. ACKNOWLEDGMENTS
This work was supported in part by research grant AA00252 from the National Institute on Alcohol Abuse and Alcoholism (NIAAA), ADAMNA. One of us (ST) is a recipient of a Research Scientist Development Award, Type 11, from the NIAAA (AA 0899). REFERENCES Bondy SC (1966). The ribonucleic acid metabolism of the brain. J Neurochem 13:955-959. Borgese D, Blobel G, Sabatini DD (1973). In vitro exchange of ribosomal subunits between free and membrane bound ribosomes. J Mol Biol 74:415-438. Fleming EW, Tewari S, Noble EP (1975) “Effects of chronic ethanol ingestion on brain aminoacyltRNA synthetic and tRNA.” J Neurochem 24:553-560. Gasior E, Moldave K (1972). Evidence for a soluble protein fraction specific for the interaction between aminoacylate transfer RNA’s and 4 0 s subunit of mammalian ribosomes. J Mol Biol66:391-402. Jarlstedt J , Hamberger A (1972). Experimental alcoholism in rats, effect of acute ethanol intoxication on the in vitro incorporation of [3H]leucine into neuronal and glial proteins. J Neurochem 19:2299-2306. Khan AA, Wilson JE (1965). Studies of turnover in mammalian subcellular particles: Brain nuclei, mitochondria and microsomes. J Neurochem 12:81-86. Lawford GR (1969). The effect of incubation with puromycin on the dissociation of rat liver ribosomes into active subunits. Biochem Biophys Res Commun 37:143-150. Lawford GR, Langford P, Schachter H (1966). The inhibition of rat liver polyribosome breakdown in the presence of liver supernatant. J Biol Chem 241:1835-1839. Lowry OH, Rosebrough JJ, Farr AL, Randall RJ (1951). Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275.
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MacInnes JW (1972). Differences between ribosomal subunits from brain and those from other tissue. J Mol Biol 65:157-161. MacInnes JW (1973). Mammalian brain ribosomes are behaviorally and structurally heterogeneous. Nature New Biol 241:244-246. Martin TE, Wool 1G (1968). Formation of active hybrids from subunits of muscle ribosomes from normal and diabetic rats. Proc Natl Acad Sci 60:569-574. Martin TE, Wool IG (1969). Active hybrid 8 0 s particles formed from subunits of a rat, rabbit and protozoan (Tetrahymena pyriformis) ribosomes. J Mol Biol43:151-161. Mechler B, Mach B (1971). Preparation and properties of ribosomal subunits from mouse plasmocytoma tumors. J Biochem 21:552-564. Mejbaum W (1967). In Campbell PN, Sargent JR (eds): “Techniques in Protein Biosynthesis.” London: Academic Press, vol 1, pp 301-303. Mejbaum NH, Pritchard MG (1971). Interaction of informational macromolecules with ribosomes. IV. Dissociation of RNA-binding ability from ribosomes by treatment with salts. Biochim Biophys Acta 246:269-279. Noble EP, Wurtman RJ, Axelrod J (1967). A simple and rapid method for injecting [H3] norepinephrine into the lateral ventricules of the rat brain. Life Sci 6:281-291. Noble EP, Tewari S (1973). Protein and ribonucleic acid metabolism in brains of mice following chronic alcohol consumption. Ann NY Acad Sci 215: 333-345. Noble EP, Tewari S (1975). Ethanol and brain ribosomes. Fed Proc 34(10): 1942-1947. Petermann ML (1964). “The Physical and Chemical Properties of Ribosomes.” New York: Elsevier. Renis M, Giovine A, Bertolino A (1975). Protein synthesis in mitochondria1 and microsomal fractions from rat brain and liver after acute or chronic ethanol administration. Life Sci 16: 1447-1458. Shires TK, McLaughlin CM, Pitot HC (1975). The selectivity and stoichiometry of membrane binding sites for polyribosomes, ribosomes and ribosomal subunits in vitro. Biochem J 146:513-526. Skogerson L, Moldave K (1967). The binding of aminoacyl transferase I1 to ribosomes. Biochem Biophys Res Commun 27:568-572. Skogerson L, Moldave K (1968). Evidence for aminoacyl-tRNA binding, peptide bond synthesis and translocase activities in the aminoacyl transfer reaction. Arch Biochem Biophys 125:497-505. Etoffler G, Wool 1G I in A. Rak K (1974). The identification of the eukaryotic ribosomal proteins homologous with Escherichia coli proteins L7 and L12. Proc Natl Acad Sci USA 71(12):47234726. Tanaka T, Ogata K (1972). Two classes of membrane-bound ribosomes in rat liver cells and their albumin synthesizing activity. Biochem Biophys Res Commun 49(4): 1069- 1074. Tewari S , Baxter CF (1969). Stimulatory effect of yaminobutyric acid upon amino acid incorporation into protein by a ribosomal system from immature rat brain. J Neurochem 16: 171-180. Tewari S, Fleming EW, Noble EP (1975). Alterations in brain RNA metabolism following chronic ethanol ingestion. J Neurochem 24:561-569. Tewari S, Noble EP (1971). Ethanol and brain protein synthesis. Brain Res 26:469-474. Tewari S, Noble EP (1975a). In Rothschild MA, Oratz M, Schreiber SS (eds): “Alcohol and Abnormal Protein Biosynthesis: Biochemical and Clinical.” Pergamon, pp 421 -428. Tewari S, Noble EP (1975b). In Gross MM (ed): “Advances in Experimental Biology and Medicine (Alcohol Intoxication and Withdrawal 11): New York: Plenum Press, vol59, p p 37-53. Weinberg RA, Penman S (1970). Processing of 4 5 s nucleolar RNA. J Mol Biol 47:169-178.
Studies on the Effects of Chronic Ethanol Ingestion on the Properties of Rat Brain Ribosomes Sujata Tewari, Sandra Murray, and Ernest I?Noble Department of Psychiatry and Human Behavior, College o f Medicine, University o f California, lrvine
Previous observations have demonstrated decreased in vivo and in vitro protein synthesis by brain ribosomal systems following long-term ethanol ingestion. For further investigation of the properties of brain ribosomes, the 40s and 60s ribosomal subunits were successfully isolated from control and chronic 10% ethanol-drinking rats. For a successful dissociation of ribosomes into subunits NH4C1, puromycin and a high-salt treatment at 10°C were essential with a critical concentration of Mg2+ since ribosomes could not be resolved at less than 7 mM Mg2+. Analysis of the A23, profile of the subunits o n t h e sucrose gradients showed no significant differences between the control and ethanol-ingesting groups. Studies on H-labeled ribosomes following in vivo RNA labeling showed correspondence of the radioactive profiles from t h e incorporation of [ S 3 H ] orotic acid into RNA with the sucrose gradient absorbance profile of 6 0 s and 40s ribosomal subunits. Furthermore, active reassociation of both subunits occurred at 37OC as demonstrated b y t h e increased [ l4c1phenylalanine incorporation in the presence of poly(U). Results further showed that the poly(U)-dependent [ 14C]phenylalanine incorporation was significantly reduced by t h e subunits from the ethanol-ingesting animals. These findings suggest that long-term ingestion of ethanol caused functional changes in the properties of brain ribosomes, specifically o n the reassociation process of the two subunits. Key words: brain, protein synthesis, alcohol, ethanol, ribosomal subunits INTRODUCTION
The inhibition of in vivo and in vitro RNA and protein synthesis in brain tissue following long-term ethanol ingestion has been demonstrated repeatedly in recent years [Tewari and Noble, 1971; Jarlstedt and Hamberger, 1972; Noble and Tewari, 1973; Renis et al, 19751 . Subsequently, studies of effects on chronic ethanol ingestion were reported on Abbreviations: mRNA - Messenger RNA; rRNA polyuridylic acid; S - Svedberg coefficient.
-
Ribosomal RNA; tRNA
-
Transfer RNA; poly (U)
Dr Noble is now at the National Institute on Alcohol Abuse and Alcoholism, 5600 Fishers Lane, Rockville, MD 20825. Address reprint requests to Dr Sujata Tewari, Department of Psychiatry and Human Behavior, College of Medicine, University of California, lrvine CA 92717. 0360-4012/78/0305/6-0375$02.60 @ 1978 Alan R . Liss, Inc
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the specific reactions of protein biosynthesis [Tewari and Noble, 1971; Fleming et al, 19751. Some of these findings have established the primary effects of ethanol at the ribosomal level where marked inhibition of [ l4 C] polyphenylalanine synthesis occurred independent of the source of the pH 5 precipitable enzymes fraction [Noble and Tewari, 19751. It was suggested that reduced association of mRNA with the ribosomes due to functional alterations of one or both subunits could be the primary cause for the observed ethanol-induced inhibition. In recent years extensive studies have established the ribosomes as the site for protein synthesis with a number of studies available on the structure and properties of these particles [Weinberg and Penman, 1970; Mejbaum and Pritchard, 1971; Tanaka and Ogata, 1972; Stoffler et al, 1974; Shires et al, 19751. These reports have shown the eukaryotic ribosomes to be composed of two subunits, containing three types of RNA and some 70 proteins. The sedimentation coefficient values, although variable from species to species, are thought to be 6 0 s and 4 0 s for the two respective subunits [Petermann, 19641. No studies are available on the effects of ethanol on ribosomal subunits and the present study was undertaken to isolate the dissociated 4 0 s and 6 0 s ribosomal subunits from brain tissue and to investigate the chronic effects of ethanol on the dissociation and reassociation properties of brain ribosomes. EXPERIMENTAL PROCEDURES Chemicals
All chemicals were reagent A grade and solutions were prepared with glass doubledistilled deionized water. Whatman glass filter disks (GF/A 2.4 cm) were purchased from Quickfit Reeve Angle, Inc (Clifton, New Jersey). Bovine serum albumin and Trizma [tris(hydroxymethyl)aminomethane] were products of Sigma Chemical Co (St. Louis, Missouri). The chemicals for scintillation fluid, PPO (2,s-diphenyloxazole) and POPOP {1,4-bis [2-(5-phenyloxazolyl)] -benzene}were purchased from Packard Instruments Co (Donners Grove, Illinois). Standard RNA (yeast) was from Calbiochem (San Diego, California). Labeled [5-3H] orotic acid (specific activity 17 Ci/mmole) and [' C] phenylalanine (41 0 mCi/mmole) were purchased from Schwartz/Mann Bioresearch (Orangeburg, New York). Administration of Ethanol
Male four-week-old Sprague-Dawley rats from Simonsen Laboratory were housed as described by Tewari and Noble (1970). The control group received water while the .ethanol group was given a 10%(v/v) ethanol-water solution ad libitum for a period of at least six weeks. Ethanol was routinely replaced with water 24 hours prior to experimentation. Both groups received laboratory chow ad libitum. Administration of RNA Precursor
Intraventricular injection of [5-3HI orotic acid into lateral brain ventricles was performed according to the techniques of Noble et a1 [1967]. Composition of Medium M
The medium contained 0.25 M sucrose, 0.02 M Trizma, pH 7.6,O.l M KCl, 0.04 M NaCl, 0.007 M magnesium acetate, and 0.006 M 2-mercaptoethanol [Tewari and Noble, 19701.
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Preparation of Ribosomal Subunits
1) Isolation and purification of ribosomes. Brains from both control and ethanol groups were treated identically. Ribosomes (RI) and pH 5 precipitable enzymes fraction were isolated by differential centrifugation of brain homogenates in medium M according to the procedures of Tewari and Baxter [ 19691 and Tewari and Noble [ 19701 . Ribosomes were purified by centrifuging 9 ml of the suspension through a discontinuous 0.5 M and 1 .O M sucrose gradient (9 ml each) containing 0.5 M NH4C1, 0.1 M Tris-HC1 (pH 7.6), 0.01 M MgClz, and 0.006 M 2-mercaptoethanol [Skogerson and Moldave, 1968; Martin and Wool, 19681. 2) Stripping of peptidyl-tRNA. Purified ribosomes (RII) obtained by the above procedures were stripped of peptidyl-tRNA by using a modification of the procedures of Skogerson and Moldave [1967] and Gasior and Moldave [1972]. At a concentration of approximately 2.5 mg of ribosomal protein/ml of 75 mM Tris-HC1, pH 7.6 (1 m1/4 gm brain), 0.6 ml of the suspension was incubated at 37°C for 25 minutes with 1 mM puromycin dihydrochloride, 80 mM NH4 C1, 75 mM Tris (pH 7.6), 12 mM MgClz, and 6 mM 2-mercaptoethanol in a final volume of 24 ml. When necessary, lml of the 105,OOOg supernatant fraction was added as a source of RNase inhibitor and transfer factors. Following the incubation, the reaction mixture was cooled in ice briefly and centrifuged at 105,OOOg for 30 minutes at 4" C. The reisolated puromycin-treated pellet (RIII) was suspended at a concentration of 5 mg/ml (1 m1/10 gm brain) in 80 mM KC1, 12 mM MgC12, 50 mM Tris-HC1 (pH 7.6), and 6 mM 2-mercaptoethanol, and was clarified briefly at 1,500g for 10 minutes at 4°C. 3) Separation and isolation of 60s and 40s subunits following high-salt treatments. The 1,500g supernatant (R Sup) obtained above was mixed with an equal volume of a 2.5 M KC1 and 10 mM MgClz mixture, and 1-ml samples were layered onto 34 ml of a 15-3576 (w/v) linear sucrose gradient containing 30 mM Tris-HC1(pH 7.6), 6 mM 2-mercaptoethanol, and either 0.6 M KC1 with 15 mM MgClz or 0.4 M KCl with 7 mM MgC12 [Martin and Wool, 1969; Mechler and Mach, 1971; Gasior and Moldave, 19721 . Gradients were centrifuged for 12-14 hours at 10°C and 37,OOOg in a SW 27 rotor. Cushions of 0.5 ml of 2% sucrose and 1.5 ml of 60% sucrose on the top and bottom of the gradient were included for convenience in monitoring gradient absorbance. One-milliliter fractions were collected and absorbance at 260 nm was monitored in a Gilford recording spectrophotometer, model 2400, fitted with a flow-through cell. Resulting subunit peak fractions corresponding to 60s and 40s were pooled separately and isolated by centrifugation for four hours at 10°C and 105,OOOg. The subunit pellets were frozen at -70°C after rehomogenizing in a storage medium containing 0.35 M sucrose, 50 mM Tris (pH 7.6), 7 mM MgClZ, and 6 mM 2-mercaptoethanol. Determination of Radioactivity
The incorporation of ["C] phenylalanine into protein was measured by determining the activity in hot TCA-extractable residue according to Tewari and Noble [1970]. The incorporation of [5-jH] orotic acid into RNA of the ribosomal subunit was determined by cold TCA and ethanol ether extractions on Millipore filter disks (2.4 cm, Whatman) [Tewari et al, 19751 . The radioactivity was determined using a PPO-POPOP mixture and a liquid scintillation counter, Beckman model LS 250 [Tewari and Noble, 19701. The protein and RNA content of the subcellular fractions were determined by the methods of Lowry et a1 [1951] and Mejbaum [1967] respectively.
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RESULTS Dissociation of Ribosomes Into the Large and Small Subunits
To determine the effects of long-term ethanol ingestion on the dissociation properties, brain ribosomes (RI fraction) from control and ethanol-ingesting groups were prepared and purified by centrifugation through a discontinuous sucrose gradient containing NH4C1to yield an RII fraction prior to dissociation into 6 0 s and 40s subunits (Fig 1). The NH4C1 treatment of RI fraction was thought to be essential since possible protein factors are removed by this process [Skogerson and Moldave, 1967; Borgese et al, 19731 . Figure 1 outlines a brief fractionation scheme. Details of the procedure have been described under Materials and Methods. The purified ribosomes (RII fraction) of both control and ethanol-ingesting groups could be completely dissociated into 6 0 s and 40s subunits by NH4C1, puromycin, and high-salt treatments, followed by continuous gradient centrifugation at 10°C. No dissociation occurred at 4°C where aggregation of ribosomes prevented dissociation and is similar to observations reported by Borgese et a1 [1973] and Martin and Wool [1969]. As with other mammalian ribosomes, brain ribosomes were more resistant t o dissociation than bacterial ribosomes and required a specific ratio of K+ and Mg2+ concentrations. Under present conditions, the presence of puromycin and the combination of high K+ with specified Mgz+ concentration were found to be crucial for successful dissociation.
RIBOSOMES
(RI Fraction)
layered on 0.5H and 1H sucrose buffer containing 0.5MNn C1. 44,000 g, 16-20 brs,
Supernatant
Ilibosomal Pellet
(RII Fraction)
I'uromycin incubation at 37012 for 25 min. 105.000 g, 150 min, C'4
I
Supernatant
Ribosomal 'Pellet (RIII Fraction) 1500 g, 10 min,
4.C
Ethanol a n d Brain Ribosomes
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Effects of Varying Magnesium Concentrations
Figures 2A and 2B show the A260 profiles of the ribosomal fraction on a sucrose density gradient before (RII fraction) and after puromycin treatment (R Sup fraction) to release the nascent polypeptide chains as described in Figure 1. In this experiment, 1 ml of RII fraction was layered onto a 15-35% gradient (Fig 2A) while another aliquot was incubated with puromycin, isolated, treated with high K+ (0.6 M) and 15 mM Mgz+ , and the resultant (R Sup) fraction layered on a similar gradient (Fig 2B). The profile obtained in Figure 2A shows several distinct peaks from both control and ethanol-treated brain showing the general distribution of polysomes in the NH4C1-treated RII fraction, and. some of these peaks were distinctly heavier than the larger subunit. The reisolation of RII fraction into ribosomal supernatant fraction gave two very distinct absorbance peaks following puromycin and high-salt treatment (Fig 2B). There was no significant difference observed between the absorbance profiles of the two subunits from control or ethanoltreated groups and both groups behaved similarly toward dissociation treatments. Dissocia tion characteristics were again examined by reducing the Mg2+ concentration from 15 to 7 mM. In this experiment fractionation of the ribosomal supernatant fraction on a gradien containing 0.6 M KC1 and 7 mM Mg2+ also gave excellent dissociation into two ribosomal subunits (Fig 3). No significant differences were again observed between the AZM)profiles of control and alcohol-drinking animals.
Control Ethanol
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Fig 2. Sucrose gradient analysis of cerebral ribosomes. Samples with approximately 2 mg of protein were layered onto 34 ml of 15-35% (w/v) sucrose gradients containing 0.6 M KC1 and 15 mM MgCl2. A) Before puromycin treatment. B) After successive puromycin and high-salt treatments. Control (-). Ethanol (- - -).
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Fig 3. Separation of ribosomes into subunits. After puromycin treatment of the NH4Cl-washed ribosomes and further treatment with 1.3 M KCl 11 mM MgClz approximately 1.46 mg of the R Sup fraction was layered onto gradients containing 0.6 M KC1/7 mM MgCl2. Gradients were monitored at 260 Ethanol (- - - - - -). nm continuously and collected as previously described. Control (-).
Although excellent dissociation of ribosomal subunits could be obtained in the presence of 15 mM Mg2+ and 7 mM Mg2+, further lowering of Mg2+ was proven unsuccessful. To illustrate this crucial requirement of MgZ+,AZm profiles of the control and ethanol-treated ribosomes in a sucrose gradient were examined after treatment of RII fraction with puromycin and centrifuging in the presence of varying concentrations of Mgz+ and high K+. No resolution was found to occur in either group when Mgz+ concentrations were lowered to 1.5 or 0.1 mM with a constant K+ level of 0.6 M (Figs 4A and 4B). Protein Recovery
To ensure similar experimental processing, the recovery of ribosomal protein between the control and ethanol groups were compared during the entire fractionation procedure (Table I). TABLE I. Protein Recovery During the Fractionation of Ribosomes
Fractions Ribosomes Purified ribosomes (RII) Postpuromycin-treated supernatant fraction (R Sup) Large ribosomal subunits (60s) Small ribosomal subunits (40s) ~~
Control (% of RI protein)
Ethanol-treated (%of RI protein)
100" 51
100 53
11 4 4
13 I 6
~~~~~~~~~~~~~~~~~~~
alOO% = approximately 2.47 mg of ribosomal protein/gm wet weight of brain tissue. Preparation of the various fractions is described in Figure 1. Subunits were separately pooled peaks from gradients after puromycin and high-salt treatments, sedimented at 105,OOOg for four hours, and resuspended in buffer. protein content of each fraction was assayed according to Lowry et a1 [ 221.
Ethanol and Brain Ribosomes
38 1
3.0 2.6
2.0
1.6
1.0
0.6
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Raetion h b s r
Fig 4. Effect of Mgz+ level in sucrose gradients on ribosomal subunit separation. Following puromycin treatment, ribosomal suspensions with approximately 0.8 mg of protein were layered onto 15-35% (v/w) sucrose gradients containing either A) 0.6 M KCI and 1.5 mM MgClz, or B) 0.6 M KCI and 0.1 mM Ethanol (- - -). MgClz. Control (-).
Following the discontinuous sucrose gradient centrifugation of equal amounts of control and ethanol RI protein fractions with NH4C1, approximately 57 and 53% of the ribosomal protein were recovered in the two RII fractions, respectively. Further incubation of the RII fraction with puromycin removed most of the proteins leaving only 11 and 13% of the total protein in the control and ethanol (R Sup) fractions containing the partially dissociated ribosomes. Most of this protein could be recovered in the final pooled fractions containing the 60s and 40s subunits after resedimentation at 105,OOOg. The final recoveries for both control subunits were 4%, while those of ethanol-treated subunits were 7 and 6% for the larger and smaller subunits, respectively. The distribution of the protein content of the two ribosomal subunits was similar between the two groups with 40s values slightly lower in each group. The total yield of the isolated subunit protein was slightly higher in the 60s and 40s particles of brain from ethanol-drinking animals when compared to the control, while individually the protein content of the larger subunit was also higher than the 40s fraction in the ethanol-treated brains. Since complete dissociation of ribosomes could be obtained in the presence of both 15 and 7 mM Mg2+, the recovery of protein content was also compared under these conditions and the protein values for the recovery into the 60s and 40s subunits are given in Table 11. Studies show no significant differences in the total yield of subunit protein from the gradients with 15 or 7 mM Mg2+. If at all, the 15 mM Mg2+ gradient gave a slightly
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TABLE 11. Effects of Mg2+ Concentration on the Recovery of Ribosomal Protein in the 60s and 40s Fractions* Treatment
1. Pre-high-salt treatment 2. Post-high-salt treatment a) 15 mM Mg2+ b) 7 mM Mg2+
Fraction
mg protein/gm brain weight
R Sup
0.351
6 0 s unit 40s unit 60s unit 4 0 s unit
0.119 0.072 0.096 0.068
%recovery of R Sup protein fraction 100 34 21 27.4 19.4
*Experimental conditions were similar to those described in Figure 2. Each of the two separate aliquots of the puromycin-treated R Sup fractions was individually fractionated on separate gradients containing 15 mM Mgz+ and 7 mM Mg2+ with 0.6 M K+. Total recovery of subunit fractions was 5 5 and 47% with 15 and 7 mM Mg2+, respectively.
higher yield than the 7 mM Mg2+ gradient, with 55% recovery of total protein at the higher Mg2+ concentration. The total protein recovered under these conditions in the larger subunit and smaller subunit were 34 and 21%, respectively. Lowering of Mg2+ to 7 mM resulted in 27% of ribosomal protein recovered in 60s and 19%in 4 0 s fraction. These studies were carried out with subunits from control brains only. Effects of lntraventricular Administration of [5-3 HI Orotic Acid
The subunits were further characterized by labeling their rRNA content with a single intraventricular pulse of [5-3H]orotic acid (an RNA precursor) for 24 hours. The one-day period was chosen to differentiate the labeling of rRNA from mRNA since the half-life of ribosomal RNA in brain tissue has been found to be 11 days [Khan and Wilson, 1965; Bondy, 19661 . The labeled ribosomes were next dissociated into subunits by the procedures described and radioactivity was measured into cold TCA-insoluble residue (Fig 5 ) . Findings given in Figure 5 demonstrated that the AZm sucrose gradient profiles of labeled control ribosomal subunits could be measured only at the peaks for 4 0 s and 60s (dotted line). The radioactivity was determined in each fraction and the solid line represents the amount of radioactivity recovered in fractions following absorbance measurement at AZm.Data clearly show that the incorporation of [5-3HI orotic acid into rRNA as measured by cold TCA-precipitable counts were only in the fractions corresponding to the 60s and 4 0 s peaks of control brains. There were no detectable amounts of radioactivity present in the initial heavier fractions or subsequent fractions collected after the shoulder of the 40s peak. The incorporation of [5-jH] orotic acid into subunit RNA fractions was further determined by pooling the individual peaks and sedimenting them at 105,OOOg for four hours at 10°C. Results given in Table I11 show that both subunits had similar specific activities and 50% of the radioactivity initially present in the ribosomal supernatant fraction could be recovered as labeled RNA in each RNA fraction of the 60s and 40s fractions. In Vitro Reassociation of Subunits
Following the labeling of rRNA other functional properties of the subunit were examined by studying the poly(U)-dependent incorporation of ["C] phenylalanine into
Et h an o l an d Brain Ribosomes
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Fig 5. In vivo labeling of rRNA. Twenty-four hours after intrdventricukar injection of 30 pCi [3H] orotic acid (specific activity 17 Ci/mmole) into each rat brain, ribosomal subunits were isolated by methods described, following treatment by puromycin and high salts (1.3 M KC1/11 mM MgC12). Indicated amounts (control ribosomal sup) were layered onto the gradients listed, and after centrifugation (see Methods), gradients were monitored continuously at 260 nm and collected in approximately 30 1.1-ml fractions. Aliquots (0.05 ml) were removed from selected fractions for determining radioactivity. TABLE 111. In vivo Incorporation of [S3H] Orotic Acid Into RNA of Brain Ribosomal Fractions*
Fractions Ribosomal supernatant prior to fractionation Pool 60s 40s
Control ( l o 3 x counts/min/mg RNA 199.0
% Activity
100 50 50
102.0 102.0 ~~
*Radioactivity was determined by cold TCA precipitation. Ribosomal supernatant was postpuromycin and post-low-speed-soluble fraction before high-KC1 treatment. The subunits were separately pooled peaks from fractions obtained from centrifugation of ribosomal supernatant after high-salt treatment.
protein under appropriate in vitro reassociating conditions. Results obtained in Table IV show that the subunits were able to recombine under in vitro conditions and incorporate [ 14C]phenylalanine into protein. The stimulation of [ 14C]phenylalanine incorporation by poly(U) was negligible in the presence of a single subunit fraction. Increased activity was obtained only when Cm and C ~ O were present together (137%). This increased activity was RNase sensitive as demonstrated by a large decrease in the hot TCA-precipitable [I4C] phenylalanine activity. Addition of RNase was without effect on the activity of single subunits.
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TABLE IV. Effects of Poly(U) on [ 14C] Phenylalanine Incorporation Into Protein by Ribosomal Subunits* Endogenous activity (%)
Experimental conditions
100.0 137.0 54.0 101.6 100.0 101.0 100.0
*loo% = 7,500 counts/min/mg ribosomal protein.
Incubation medium consisted of 75 mM Tris-HC1, pH 7.6,40 mM NH4CI, 7 mM MgCl?, 6 mM 2-mercaptoethanol, 100 pg poly(U), 200 pg bovine serum albumin, approximately 30 pg protein of 6 0 s fraction and 19 pg of 40s fraction. Incubation period was 30 minutes at 37°C. RNase, when added, was at a concentration of 20 pg/ml of incubation medium. The source of the pH 5 enzyme fraction was from control brain only and was added at a concentration of 200 pg/ml in incubation medium.
Effects of Ethanol Ingestion on I n Vitro [14C1Polyphenylalanine Synthesis
The reassociation of 60s and 40s subunits was again examined to determine the possible effects of chronic ethanol ingestion. Table V compares the activity of the subunits between the control and ethanol-ingesting groups both in the presence and absence of poly(U) (ie, under endogenous conditions). Data show that under either experimental conditions the subunits prepared from the control brain demonstrated a higher activity than those isolated from the ethanol-treated brain. The addition of polyuridylic acid, resultedin increased activity in the ethanol group, with reduced l 4 C activity when comparedto the control. These results indicate that the in vitro recombination of 60s and 40s ribosomal subunits was significantly inhibited following chronic ethanol ingestion.
DISCUSSION
In this paper we have examined the effects of chronic ethanol ingestion on the separation of cerebral ribosomes into the two subunits, 60s and 40S, by puromycin and high-salt treatments. In addition, the subunits were characterized by in vivo labeling of their rRNA content with [!G3H]orotic acid and by their ability to synthesize [14C] polyphenylalanine under in vitro conditions. Our findings show that, similar to rat liver TABLE V. Effects of Ethanol o n the Poly(U)-Dependent Phenylalanine Incorporation ~
~~
Experimental conditions C 6 0 s + C 40s E 6 0 s + E 40s
Endogenous, % control activity 100 78
Endogenous + poly(U) % control activity 140 104
Incubation conditions were similar to those described in Table IV. Approximately 30 pg of 60s subunit fraction and 19 pg of 40s subunit fraction were used from each of the control (C) and ethanolingesting (E) groups. 100% = 5,547 counts/min.
Ethanol and Brain Ribosomes
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ribosomes [Lawford, 19691, brain ribosomes from both control and ethanol-ingesting animals could be dissociated into subunits by releasing the peptidyl transfer RNA and nascent polypeptide chains by treating first with puromycin (Figs 2A and 2B) and then immediately increasing and decreasing the K+ and Mgz+ concentrations, respectively, during sucrose gradient centrifugation. Our observations indicate that brain ribosomes from both control and ethanol-ingesting groups were not only highly sensitive to changes in the K+/MgZ+ratio, but, in addition, were highly resistant to dissociation into subunits. In contrast to liver, where lowering of Mg2+ concentrations have been reported successful, brain ribosomes from both groups required a higher Mg2+ concentration (7-1 5 mM) and had to be sequentially subjected to puromycin and high-KC1 treatment. Somewhat similar data are available from other laboratories showing differences in acrylamide gel electrophoresis of proteins and high Mgz+ requirement between brain and liver ribsomal subunits. Furthermore, the lowering of Mg2+ concentrations from 1.5 to 0.1 mM in the gradient (Figs 4A and 4B) or in an incubation medium containing puromycin and 0.5 M KC1 at the same time was also found to be unsuccessful for dissociation of ribosomes into subunits (unpublished data), although this treatment has been used successfully with rat liver ribosomes [Lawford et al, 1966; Lawford, 19691 . Under the present experimental conditions, no ethanol-induced changes in the K+ or Mgz+ requirement could be observed. Among other properties, the dissociation process of control and ethanol-ingesting ribosomes after the high-salt treatment was temperature sensitive and was only effective at 10°C since further lowering of temperature did not result in subunit resolution for both groups. The present studies have further examined the nature of labeling of RNA contained in the subunits with [5-jH] orotic acid. For the production of a functional ribosomal subunit, studies on ribosomal assembly have shown interaction of RNA and protein. These subunits are known to contain three species of RNA [Weinberg and Penman, 19701 . In this report we have presented evidence of substantial incorporation of [5-3H] orotic acid into rRNA of both subunit fractions after 24 hours of pulse labeling. Somewhat similar observations have previously reported the labeling of cytoplasmic rRNA after 2 and 24 hours of initial pulse [Noble and Tewari, 1973, Tewari, and Noble, 1975al. These studies showed the incorporation of [5-jH] uridine or [5-jH] orotic acid specifically into tRNA and rRNA in rodent brain. The subunits from both control and ethanol-ingesting groups were further characterized by in vitro reassociation studies which demonstrated a definite high poly(U) response in the presence of both subunits. However, while the ingestion of ethanol had not changed the dissociation characteristics, the reassociation process as determined by increased [I4C] phenylalanine incorporation by poly(U) was found to be adversely affected. It is of interest to note that some detectable I4C activity was always present in the absence of poly(U) under incubation conditions containing one or both subunits (Table IV). However, the RNase-sensitive poly(U) stimulation was present only when the incubation media contained both subunits (Tables IV and V). Thus, at this moment, the nature of this endogenous activity is not clearly understood. The decreased incorporation of ["C] phenylalanine into protein by ethanol subunits is particularly signficant since the incorporative ability was determined in the presence of the control pH 5 enzyme fraction which served as the source of aminoacyltransfer RNA synthetases, tRNA, and other transfer factors. Furthermore, the observed effects of ethanol were not attributable to technical procedures since ribosomes from both groups were processed identically, which is reflected in comparable protein recovery
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Tewari, Murray, and Noble
during the entire fractionation procedure (Table I). Similar inhibition of [ 14C] polyphenylalanine synthesis by ethanol-treated ribosomes has been reported previously and it was suggested that functional alterations of these particles might be responsible for possible reduced reduced mRNA association resulting in decreased availability [Tewari and Noble, 1975a, b] . These ideas are consistent with the present data on decreased activity in the presence of the 40s and 60s subunits of the ethanol group and previous findings on the labeling pattern of ribosomal RNA and subsequent inhibition of protein synthesis [Tewari and Noble 1975a; Noble and Tewari, 19751. However, more experiments are required to understand fully the chronic effects of ethanol on the reassociation properties of the 60s and 40s ribosomal subunits. In conclusion, our findings suggest that ethanol ingestion definitely affected the in vitro recombination of ribosomal subunits involved in carrying out effective protein synthesis. The eukaryotic ribosomes are heterogeneous in nature and even to this date the functional significance of this heterogeneity is unclear. Since it is believed that the functional characteristics of ribosomes are highly dependent on a variety of factors [MacInnes 1972, 19731, it is extremely suggestive that ribosomes exert regulatory roles in cellular metabolic processes. This is of particular importance in brain tissue which is a highly specialized, diversified organ and is itself heterogeneous in nature. The present findings on decreased activity in the presence of ethanol ribosomes are thus highly provocative in postulating that ethanol interferes with normal functioning of brain tissue at the ribosomal level. The present observation could therefore be a function of instability in one or both of the 40s subunit tFWA complexes and a functional change in the 60s subunits engendered by ethanol. The exact nature of this deficiency is currently under investigation. ACKNOWLEDGMENTS
This work was supported in part by research grant AA00252 from the National Institute on Alcohol Abuse and Alcoholism (NIAAA), ADAMNA. One of us (ST) is a recipient of a Research Scientist Development Award, Type 11, from the NIAAA (AA 0899). REFERENCES Bondy SC (1966). The ribonucleic acid metabolism of the brain. J Neurochem 13:955-959. Borgese D, Blobel G, Sabatini DD (1973). In vitro exchange of ribosomal subunits between free and membrane bound ribosomes. J Mol Biol 74:415-438. Fleming EW, Tewari S, Noble EP (1975) “Effects of chronic ethanol ingestion on brain aminoacyltRNA synthetic and tRNA.” J Neurochem 24:553-560. Gasior E, Moldave K (1972). Evidence for a soluble protein fraction specific for the interaction between aminoacylate transfer RNA’s and 4 0 s subunit of mammalian ribosomes. J Mol Biol66:391-402. Jarlstedt J , Hamberger A (1972). Experimental alcoholism in rats, effect of acute ethanol intoxication on the in vitro incorporation of [3H]leucine into neuronal and glial proteins. J Neurochem 19:2299-2306. Khan AA, Wilson JE (1965). Studies of turnover in mammalian subcellular particles: Brain nuclei, mitochondria and microsomes. J Neurochem 12:81-86. Lawford GR (1969). The effect of incubation with puromycin on the dissociation of rat liver ribosomes into active subunits. Biochem Biophys Res Commun 37:143-150. Lawford GR, Langford P, Schachter H (1966). The inhibition of rat liver polyribosome breakdown in the presence of liver supernatant. J Biol Chem 241:1835-1839. Lowry OH, Rosebrough JJ, Farr AL, Randall RJ (1951). Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275.
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MacInnes JW (1972). Differences between ribosomal subunits from brain and those from other tissue. J Mol Biol 65:157-161. MacInnes JW (1973). Mammalian brain ribosomes are behaviorally and structurally heterogeneous. Nature New Biol 241:244-246. Martin TE, Wool 1G (1968). Formation of active hybrids from subunits of muscle ribosomes from normal and diabetic rats. Proc Natl Acad Sci 60:569-574. Martin TE, Wool IG (1969). Active hybrid 8 0 s particles formed from subunits of a rat, rabbit and protozoan (Tetrahymena pyriformis) ribosomes. J Mol Biol43:151-161. Mechler B, Mach B (1971). Preparation and properties of ribosomal subunits from mouse plasmocytoma tumors. J Biochem 21:552-564. Mejbaum W (1967). In Campbell PN, Sargent JR (eds): “Techniques in Protein Biosynthesis.” London: Academic Press, vol 1, pp 301-303. Mejbaum NH, Pritchard MG (1971). Interaction of informational macromolecules with ribosomes. IV. Dissociation of RNA-binding ability from ribosomes by treatment with salts. Biochim Biophys Acta 246:269-279. Noble EP, Wurtman RJ, Axelrod J (1967). A simple and rapid method for injecting [H3] norepinephrine into the lateral ventricules of the rat brain. Life Sci 6:281-291. Noble EP, Tewari S (1973). Protein and ribonucleic acid metabolism in brains of mice following chronic alcohol consumption. Ann NY Acad Sci 215: 333-345. Noble EP, Tewari S (1975). Ethanol and brain ribosomes. Fed Proc 34(10): 1942-1947. Petermann ML (1964). “The Physical and Chemical Properties of Ribosomes.” New York: Elsevier. Renis M, Giovine A, Bertolino A (1975). Protein synthesis in mitochondria1 and microsomal fractions from rat brain and liver after acute or chronic ethanol administration. Life Sci 16: 1447-1458. Shires TK, McLaughlin CM, Pitot HC (1975). The selectivity and stoichiometry of membrane binding sites for polyribosomes, ribosomes and ribosomal subunits in vitro. Biochem J 146:513-526. Skogerson L, Moldave K (1967). The binding of aminoacyl transferase I1 to ribosomes. Biochem Biophys Res Commun 27:568-572. Skogerson L, Moldave K (1968). Evidence for aminoacyl-tRNA binding, peptide bond synthesis and translocase activities in the aminoacyl transfer reaction. Arch Biochem Biophys 125:497-505. Etoffler G, Wool 1G I in A. Rak K (1974). The identification of the eukaryotic ribosomal proteins homologous with Escherichia coli proteins L7 and L12. Proc Natl Acad Sci USA 71(12):47234726. Tanaka T, Ogata K (1972). Two classes of membrane-bound ribosomes in rat liver cells and their albumin synthesizing activity. Biochem Biophys Res Commun 49(4): 1069- 1074. Tewari S , Baxter CF (1969). Stimulatory effect of yaminobutyric acid upon amino acid incorporation into protein by a ribosomal system from immature rat brain. J Neurochem 16: 171-180. Tewari S, Fleming EW, Noble EP (1975). Alterations in brain RNA metabolism following chronic ethanol ingestion. J Neurochem 24:561-569. Tewari S, Noble EP (1971). Ethanol and brain protein synthesis. Brain Res 26:469-474. Tewari S, Noble EP (1975a). In Rothschild MA, Oratz M, Schreiber SS (eds): “Alcohol and Abnormal Protein Biosynthesis: Biochemical and Clinical.” Pergamon, pp 421 -428. Tewari S, Noble EP (1975b). In Gross MM (ed): “Advances in Experimental Biology and Medicine (Alcohol Intoxication and Withdrawal 11): New York: Plenum Press, vol59, p p 37-53. Weinberg RA, Penman S (1970). Processing of 4 5 s nucleolar RNA. J Mol Biol 47:169-178.
