Effects of Chronic Ethanol Treatment on Rat Liver M itochondrial Protein Synthesis Jerome D. Bernstein a n d Ralph Penniall. Ph.D.

Chronic ethanol treatment of male SpragueDawley rats resulted in a 50% decrease in the rate of incorporation of precursor leucine into isolated mitochondria. This decrease is manifest in a decreased labeling of three polypeptides of inner mitochondrial membranes that are the major products of in vitro mitochondrial protein synthesis under the conditions employed. Immunoprecipitation of cytochrome-c oxidase revealed that these three polypeptides are subunits 1, 2, and 3 of cytochrome-c oxidase and have apparent molecular weights of 33,000, 25,000, and 20,000. Sixty percent of the total incorporated radioactivity is associated with these polypeptides. A decrease in the contents of subunit 2 and of a second polypeptide with an apparent molecular weight of 22,000 was also noted as an effect of chronic ethanol treatment.

M

UCH evidence links chronic ethanol ingestion to respiratory aberrations in liver mit~chondria.'-~Previous work of this laboratory6 and others7.' has established that a decrease occurs in both content and activity of cytochrome-c oxidase (ferrocytochrome-c: oxygen oxidoreductase EC 1.9.3.1) in liver mitochondria of ethanol-treated rats. We have also noted aberrations in other mitochondrial components-a decreased content of ubiquinone and of N A D H dehydrogenase (NADH:K,FE(CN), reductase).6 Mitochondria1 protein synthetic activity appears to be limited to the production of 10-15 polypeptides of the inner membrane,9-" which are associated with enzyme complexes involved in electron transport and phosphorylation."-" Chronic ethanol treatment has been shown to reduce the rate of leucine incorporation into isolated liver mitochondria of both rats7 and pigs.I7 To our knowledge, there is only one report1' on changes in the mitochondrial synthesis of particular polypeptides as affected by ethanol treatment. In this article, we present evidence that establishes that chronic ethanol treatment depresses the synthesis of three subunits of cytochrome-c oxidase.

MATERIALS AND METHODS

Diet

.

Male Sprague-Dawley rats, 500-600 g, obtained from Charles River Breeding Labs, Inc., were fed a liquid diet in which carbohydrate, protein, and fat contributed 69.946, 23.3%. and 4.6% of total calories, respectively. Ethanol, when used, isocalorically replaced dextrose for 35.5% of the total calorie^.'^'^ Rats were divided by weight into groups of 4 animals: 2 received dietary ethanol and 2 control animals were pair-fed the average preceding day's caloric intake of the 2 animals receiving ethanol. Animals were introduced to the ethanol diet by stages, receiving 18% of total calories as ethanol for 3 days, 27% the next 4 days, and 35.5% on day 8. Animals were sacrificed after 4 mo on the diet. Essentially identical rates of weight gain were sustained by animals on control (0.9 f 0.13 g/day), ethanol (0.9 f 0. I I g/day), and standard rat chow (0.9 & 0.02 g/day) diets.

Mitochondria Mitochondria were prepared according to Chappell and Hansford,19using a medium containing 250 mM sucrose, 10 mM THAM (tris [hydroxymethyl] aminomethane), and I m M EDTA (ethylenediamine tetra-acetic acid), pH 7.8.'" Sterile reagents and conditions were maintained throughout the isolation.*' Final pellets were drained well; homogenized by hand in 2-3-ml isolation medium; and adjusted to a content of 10 mg protein/ml, following determinations of protein by the method of Lowry et aLZ2Mitochondria isolated from both groups of animals were similarly contaminated to the extent of 5 % with microsomal protein.

Amino Acid Incorporation into Total Proteins Amino acid incorporation by mitochondria was measured in the sterile incubation system described by Beattie and Ibrahim.20 In all experiments, 10-mg samples of mitochondrial protein were incubated in duplicate with uniformly labeled L-"C leucine in a total volume of 10 ml at 30'C in a metabolic shaker. Aliquots of the system were delivered at ~~

~

From the Department of Biochemistry and Nutrition. School of Medicine. University of North Carolina, chapel Hill, N . C . Supported in part by Grant 7506from the North Carolina Alcoholism Research Authority and Grant AGO0256 from the National Institutes ofHealth. Reprint requests should be addressed to Ralph Penniall. Ph.D.. Department of Biochemistry and Nutririon. School of Medicine, University of North Carolina, Chapel Hill, N.C. 27514. 0 1978 by Grune & Stratton. Inc. Ol45-6O08/78/0203-O042~02.O0/0

Alcoholism: Clinical and Experimental Research, Vol. 2 . No. 3 (July). 1978

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intervals onto strips of Whatman No. 3 filter paper and processed by the procedure of Mans and N o ~ e l l iutilizing ,~~ cold 10% TCA (trichloroacetic acid) containing 50 mM Lleucine in the initial washes. Strips were counted to 1.5% error in scintillation vials, containing 10 ml of Scintiverse cocktail (Fisher Scientific Co.) and 1 ml distilled water, in a Beckman CPM-100 Liquid Scintillation Counter with an efficiency for "C of 90%.

Fractionation of Incubated Mitochondria When the number and/or character of the labeled components was to be determined, incorporation was stopped by mixing the flask contents with an equal volume of ice-cold 20 mM L-leucine, 250 mM sucrose, pH 7.8. Mitochondria were then reisolated and washed according to Burke and Beattie.l' Mitochondria1 membranes were prepared using dilute acetic acid and lubrol." The lubrol pellet was washed by suspension and recentrifugation with 5% TCA and 3 times with distilled water; it was then dissolved in 0.5 ml I % SDS (sodium dodecyl sulfate). Aliquots were used for the following: protein determination according to the method of Lowry et al;" determination of specific radioactivity; or electrophoresis as described below. In experiments in which enzyme activities were to be measured, lubrol pellets were brought directly into homogeneous suspension in a system containing 2% Triton X-100,0.2 M potassium phosphate, pH 7.

l m m unoprecipitation Rabbits were immunized with beef heart cytochrome-c oxidase,z6 and immune sera were harvested according to Campbell et aI.%Crude y-Gpreparations were precipitated by use of ammonium sulfate." Such precipitates were dissolved in 50 mM potassium phosphate, pH 7; dialyzed for 48 hr at 4°C versus 200 volumes of buffer; and finally rendered sterile by subsequent passage through an appropriate Millipore filter. The derived y-G globulin preparations were titrated with a constant amount of mitochondria as indicated below. Well washed mitochondria were suspended in a system containing 2% (v/v) Triton X-100,200 mM potassium phosphate (pH 7). and centrifuged for 10 min at 5000 g. Cytochrome contents in the supernatant were determined from difference spectra using the 605-630 nm absorbance peak of cytochrome-aa, ( t m M = 12) and the 550-540 nm absorbance peak (tmM = 19.1) for cytochrome-c + c,.*" Aliquots of the supernatant were then mixed with varying amounts of immune y-G globulin in a final volume of 7 ml containing 0.29% Triton X-100and 50 mM potassium phosphate, pH 7. The tubes were incubated I hr at 38°C and at O'C for 12 hr and then centrifuged for 20 min at 10,OOOg. The pellets were resuspended once in 50 mM potassium phosphate, pH 7, and reisolated. Two points should be emphasized at this juncture. Titered y-G globulin preparations were employed in immunoprecipitation experiments at the antigen-antibody ratio determined to yield quantitative precipitation of rat liver cytochrome-c oxidase. Secondly, control experiments, employing nonimmune y-G globulin (derived prior to immunization) gave no evidence of nonspecific precipitation of rat liver enzyme.

Electrophoresis Samples were dissolved in the dissociation medium of Swank and Munkres,19 heated 20 min at 70'C, and then electrophoresed in 8 % (unless otherwise indicated) polyacrylamide gels (15 cm) containing 0.1% SDS and 8 M urea. Electrophoresis was conducted at 2.5 mA per gel tube for about 12 hr. Horse liver alcohol dehydrogenase, lactic dehydrogcnase, sperm whale myoglobin, lysozyme. and cortrosyn were used to calculate molecular weights as described by Weber and Osborn.,O Immediately after electrophoresis, gels were either sliced for radioactive counting (as indicated below) or they were stained in Coomassie blue R-250 according to Weber and Osborn and subjected to densitometry in an lsco Gel Scanner.

Counfingof Gels Gels were sliced into 1 .O-mm slices with a Hoefer Scientific S L 280 Electronic Gel Slicer. Samples of 2 consecutive slices were digested in scintillation vials in I ml of 30% hydrogen peroxide for 12 hr at 55'C. Twenty milliliters of scintillation cocktail containing 3 parts toluene solution (6 g PPO [2.5-diphenyloxazole] and 0.1 g POPOP [ 1,4-bis(2-[5phenyloxazoyl]) benzene] per liter) and I part Triton X-100 were then added. Samples were counted as described above.

Enzyme Assays Triplicate assays were performed spectrophotometrically at 25'C, using freeze-thawed mitochondria to obviate permeability problem^.^ Cytochrome-c oxidase activity was determined according to Elliot et aL3I

Chemicals Uniformly labeled L-"C leucine (290 Ci/mole) was o b tained from ICN Chemical and Radioisotope Division. All protein standards (except cortrosyn [Organon]) plus amino acids, bicine (N,N-bis[2-hydroxyethyl] glycine), ATP, phosphoenol pyruvate, pyruvate kinase. chloramphenicol, and cycloheximide were obtained from Sigma Chemical Company. Sodium dithionite and Triton X-100 were o b tained from J. T. Baker Chemical Company. PPO and POPOP were obtained from New England Nuclear, and acrylamide and N.N'-methylene-bisacrylamide were obtained from Eastman Chemical Company and from Aldrich Chemical Company, respectively. Lubrox W X was a gift from ICI United States, Inc.

RESULTS

As can be seen in Fig. 1, the rates of precursor incorporation into protein by isolated mitochondria were depressed approximately 50% as a result of ethanol treatment. With mitochondria from animals on both control and ethanol-containing diets, incorporation rates were approximately linear for 15 min and then decreased slightly for the following 15 min. As expected, the uptake of leucine was completely inhibited by chloramphenicol and insensitive to

ETHANOL AND LIVER MITOCHONDRIAL PROTEIN SYNTHESIS

Time (min) Fig. 1. Effects of chronic ethanol treatment on rate of incorporation of L-"C leucine by isolated mitochondria. Figures represent mean f SE derived from duplicate incubations of mitochondria of each of 3 alcohol-treated (---I or control (-1 animals with 0.26 pCi/rnl L-"C leucine et 30"C in the complete system for protein synthesis as described in Materials and Methods.

cycloheximide (not shown). It should be noted that the results demonstrate that mitochondria derived from animals fed the control liquid diet are fully competent insofar as amino acid incorporation is concerned. The incorporation rates observed for control animals compare very well to previous report^^^.^^ in which mitochondria from animals fed standard laboratory diets were incubated under identical conditions insofar as medium, protein concentration, time and the specific radioactivity of precursor are concerned. To assess the character of the labeled products, mitochondria were fractionated with dilute acetic acid and lubrol after an incubation with L-I4C leucine. The results of three separate experiments are shown in Table I ; in each experiment, the mitochondria from a pair of animals (one control and one ethanol-treated) were

303

simultaneously fractionated. As can be seen, with both control and ethanol-treated animals, derivation of the lubrol pellet accomplished a similar enrichment of the incorporated radioactivity. It is also apparent that the respective specific radioactivities of the lubrol pellets reflect the lesser incorporation of leucine by mitochondria from ethanol-treated animals. At both the stage of intact mitochondria and of the lubrol pellet, the specific radioactivity of the material derived from ethanol-treated animals was 46%, and 4496, respectively, of that of control animals. Technical complications in the initial experiments obviated construction of a balance sheet for incorporated radioactivity. Thus, no data of this nature are given in Table 1. However, in one experiment, it was possible to determine that the recoveries of protein in the lubrol pellet were 11.6% and 13.6% of the total mitochondrial protein for the ethanol-treated and control animals, respectively. With both types of mitochondria, the recoveries of radioactivity were 50%. Such recoveries of protein and radioactivity in the lubrol pellet compare well with those reported by Burke and Beattie.24 When scanned densitometrically, electrophoretic separations of lubrol pellets revealed approximately 16 distinct polypeptides with apparent molecular weights. between 10,000 and 60,000. Thus, the electrophoretic fractionation of this preparation in the presence of 8 M urea indicates it to be substantially more complex than realized heretofore. A set of comparative electrophoretic separations of lubrol pellet components from mitochondria of ethanol-treated and control animals is presented in Fig. 2. Despite the complexity of the patterns, it is readily apparent that the relative content of two polypeptides with apparent molecular weights of 25,000 and 22,000 are

Table 1. Purification of the Lubrol-Insoluble Components of Mitochondria1 Membranes

-

Fraction

Ethanol-Treated (DPM/mg Protein)

Control (DPM/rng Protein)

Change (%)

Whole mitochondria Lubrol-insoluble Ratio'

3,808 f 739 18,535 f 3.41 6 4.9 f 0 . 5

7.116 f 1.199 33,016 f 1.578 4.6 f 0.9

46 44 NS

Isolated mitochondria of 3 control and 3 ethanol-treated animals were labeled in vitro with 1 pCi/ml L-I'C leucine for 30 min. washed, reisolated. and treated with dilute acetic acid and lubrol as described in Materials and Methods. Figures represent mean *SE at a level of significance of 0.1 > p > 0.05. 'Specific radioactivity of the lubrol pellet divided by that of whole mitochondria.

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/\/I

[

u, 60

*

I

40

30

x Molecular Weight

greatly diminished (60%-80%) in mitochondria from ethanol-treated animals. Similar differences were noted in the two other pairs of animals. It should be added that no differences in the staining intensity were found in the region of the gel containing polypeptides with apparent molecular weights less than 20,000 (not included in Fig. 2).

20

I Fig. 2. Polypeptide profile of lubrol pellets derived from mitochondria of a control and an ethanol-treated animal. Lubrol pellets derived from labeled mitochondria (1 pCi/ml L-"C leucine) of a control and ethanol-treated animal were prepared, electrophoresed. and stained as described in Materials and Methods. Approximately 100 pg protein were applied to gels. Asterisks indicate polypeptides that correspond in apparent molecular weight to those that acquired incorporated L-"C leucine (see Fig. 4).

When duplicate gels were sliced and counted to determine the number and character of the products of in vitro labeling, it was revealed that 3 polypeptides with apparent molecular weights of 33,000, 25,000, and 20,000 were substantially labeled (Fig. 3). While there were indications of minor labeling of additional components, none of these were labeled to a significant degree

Fig. 3. Electrophoretic profile of radioactivity incorporated by mitochondria of an ethanol-treated and a control animal. Isolated mitochondria of an ethanol-treated (---I and control (-) animal were labeled with 1 pCi/ml L-"C leucine for 30 min at 30"C in the complete system for protein synthesis and fractionated with acetic acid and lubrol. Lubrol pellets containing approximately 160 pg of protein were electrophoresed followed by gel slicing and scintillation counting as described in Materials and Methods. The specific activities of the lubrol pellets ara 31.438 and 15,119 DPM/mg protein for the control and ethanoltreated animals, respectively. The apparent molecular weights of the labeled polypeptides were calculated to be 33.000.26.000. and 20,000. respectively.

ETHANOL AND LIVER MITOCHONDRIAL PROTEIN SYNTHESIS

under the conditions of these experiments. The comparison of the lubrol pellets from ethanoltreated and control animals (Fig. 3) indicates that the extent of incorporation of precursor into each of the three polypeptides is greatly diminished in the ethanol-treated animals. These differences were similarly pronounced in the two other pairs of anim’als tested. It was apparent from the distribution of radioactivity in the gels that the three aforementioned components, on the basis of apparent molecular weight, correspond precisely to three of the components of the lubrol pellet that were asterisked previously (Fig. 2). It is pertinent to add that Burke and BeattieZ4similarly detected the predominant labeling of three polypeptides in lubrol pellets from rat liver mitochondria. T h e apparent molecular weights of their polypeptides were 40,000, 27,000, and 20,000. While the difference between 25,000 and 27,000 is within the error of the method, the difference in apparent molecular weight determined for the largest product of mitochondrial synthesis (i.e., 40,000 versus 33,000) could well stem from the incorporation of urea into the electrophoretic system. It should be noted that subunit 1 of beef heart cytochrome-c oxidase displays an apparent molecular weight of 35,300 in the presence of urea, as opposed to an apparent molecular weight of 38,000 in the absence of urea.33Thus, inasmuch as w e submit evidence below that the largest of the three labeled products of the lubrol pellet is most likely the largest subunit of rat liver cytochrome-c oxidase, we believe that a similar effect of urea is responsible for the difference in apparent molecular weights observed by Burke and Beattie and ourselves. Proof that the three main products of in vitro

305

mitochondria1 protein synthesis a r e cytochrome-c oxidase subunits and that their labeling is depressed in ethanol-treated animals was obtained by use of rabbit immune r - G globulin preparations. The results of a representative experiment are shown in Table 2. It can be noted that, with mitochondria from both control and ethanol-treated animals, immune precipitation rendered essentially all the cytochrome-c oxidase insoluble with either no or only minimal simultaneous precipitation of cytochromes c + c I . Under these conditions, 60% of the total radioactivity was also precipitated from both mitochondrial preparations (Table 2). On the other hand, when mitochondria were treated with r - G globulin derived from normal rabbit sera, neither cytochrome-c oxidase activity nor radioactivity was removed from the supernatant (not shown). While the coprecipitation of radioactivity and cytochrome-c oxidase of Table 2 gave good indication that these two parameters might bear a relationship to one another, the point needed additional proof. This was acquired when immunoprecipitates were subjected to separation by SDS-urea electrophoresis. The results obtained with the preparations of the experiment of Table 2 are presented in Fig. 4. It can be seen that the immunoprecipitates from the mitochondria of both control and ethanoltreated animals displayed t h r e e labeled polypeptides, which again contained the bulk of the label. These three entities are identical in apparent molecular weight to the three principal labeled products in lubrol pellets of earlier experiments (Fig. 3). Again, pronounced differences were noted between control and ethanol-treated animals, regarding the extent of labeling of these three entities by isolated

Table 2. lmmunoprecipitation of Cytochrome-c Oxidase from Mitochondria Labeled in Vitro Cytochrome-c DPM (Totall

Mitochondria1 lysate Immune precipitation supernatant Percent removed bv antibodv

Oxidase (units)

Cytochrome+ c, (nmolel

c

Ethanol

Control

Ethanol

Control

Ethanol

Control

4.032 1.645 59.2

10.425 3,903 62.6

1.280 0.09 1 93

1.370 0.069 95

0.98’ 0.9 1 7

0.64

0.59 0

Mitochondria of a control and an ethanol-treated animal were incubated in vitro with 1 pCi/ml L-”C leucine for 3 0 min under conditions indicated in Materials and Methods; they were then washed, reisolated. and lysed in 2% Triton X- 100:0.2 M potassium phosphate. pH 7. An aliquot of each supernatant. containing 0.7 nmole cytochrome-aas.was treated with an optimal level of 7-G globulin. which had been derived as described in Materials and Methods. Cytochrome-c oxidase activity (pmole cytochrome-c oxidized/minl and cytochrome-c cI content were determined as described in Materials and Methods. Larger aliquots of ethanol-treated mitochondria were required to provide the indicated level of cytochrome-c oxidase.

.

+

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300

250

200

150

100

50

0

10

5

15

20

25 30 Vial Number

35

40

45

50

55

Fig. 4. The electrophoretic resolution of the products of in vitro mitochondria1 protein synthesis in immunoprecipitates of rat liver cytochrome-c oxidase. Immunoprecipitates (containing 0.7 nmole cytochrome-aa,), which had been derived in the exmeriment of TaMe 2 from a control (approximately 6000 DPM) (-) and an ethanol-treated (approximately 2400 DPM) (---I animal, were subjected to electrophoresis, gel slicing, and liquid scintillation counting as described in Materials and Methods. The apparent molecular weights of the labeled polypeptides were calculated to be 33,000.25.000. and 20.000 respectively.

‘

4

2

1

30

I

5

I

20 10 Molecular Weight

I

5

Fig. 6. The polypeptide composition of an immunoprecipitate of rat liver cytochrome-c oxidase. The upper trace shows the electrophoretic profile of an immunoprecipitate of approximately 0.5 nmole cytochromeaa3. precipitated from isolated control rat liver mitochondria as described in Materials and Methods. The lower trace is en electrophoretic profile of the y-G globulin (100 pg protein) alone. Gels (12.6%)were stained with Cootpassie blue. Proteins are numbered for reference.

mitochondria. Interestingly, the incorporation of precursor into all three polypeptides is similarly diminished. When all of t h e proteins of an immunoprecipitate of rat liver cytochrome-c oxidase are visualized by staining with Coomassie blue, profiles such as that shown in Fig. 5 are obtained. Denser (12.5%) gels were employed in these separations to enhance the separation of -y-G globulin subunits and cytochrome-c oxidase components. As a consequence, the latter entities are more sharply resolved (Fig. 5 ) than in previous separations (e.g., in Fig. 4). Despite this difference, it can be seen that the three largest polypeptides of the separation shown in Fig. 5 are identical in molecular weight to those labeled components previously demonstrated in the lubrol pellets (Fig. 3) and in immunoprecipitates derived from lysed mitochondria (Fig. 4). It should be noted that the numbering of t h e polypeptides of t h e liver cytochrome-c oxidase immunoprecipitate of Fig. 5 was performed according to the pattern adopted by Downer et al.34 for the beef heart enzyme, excluding certain entities from consideration as component subunits (e.g., the peak migrating between subunits 4 and 5 of Fig. 3) on

ETHANOL AND LIVER MITOCHONDRIAL PROTEIN SYNTHESIS

the basis of rationales used by Downer et al. We have reported the striking similarities in the polypeptide composition patterns of purified beef heart cytochrome-c oxidase versus the rat liver enzyme when this is done.35 Thus, we submit that the evidence is strong that the principal products of in vitro protein synthesis by liver mitochondria are the three largest subunits of cytochrome-c oxidase. DISCUSSION

Our results indicate that chronic ethanol treatment effects a 50% decrease in the incorporation of precursor leucine into protein by rat liver mitochondria in vitro (Fig. 1, Table 1). Rubin et al.7 noted a 30% decrease in this parameter in rats maintained on a chronic ethanol diet. Our results show also that at least 50% of the incorporated leucine (Fig. 3) appears in three polypeptides of the lubrol pellet. Similar findings were made by Burke and Beattie2' and Ibrahim et al.,32 showing that these three polypeptides were labeled to similar extents in vivo or in vitro. It would thus appear that the observed effects of chronic ethanol treatment on mitochondrial protein synthesis in vitro may well reflect changes that obtain in vivo. However, inasmuch as it has been demonstrated2' that isolated rat liver mitochondria can synthesize as many as eight polypeptides in vitro, an effect of ethanol on mitochondrial synthesis of polypeptides other than the three noted in the lubrol pellet cannot be overruled. Future experiments involving longer incorporation times and/or increased precursor activities will be addressed to this point. Burke et a1.I7 studied protein synthesis in isolated liver mitochondria from pigs chronically treated with ethanol and noted a 35% decrease in the amount of L-3H leucine incorporated. The electrophoretic profiles of Burke et al. resemble closely those noted by Burke and Beattiez4for rat liver. Of the total of seven or eight polypeptides that were synthesized by pig liver mitochondria, a decrease in incorporation of precursor occurred in only one, a proteolipid with an apparent molecular weight of 40,000. This entity may well correspond to the largest polypeptide in the lubrol pellet, which shows an identical apparent molecular weight when electrophoresed without urea. Thus, it would appear that different species may

307

respond differently to chronic ethanol treatment in terms of which specific products of mitochondrial protein synthesis are affected. In pigs, the labeling of only one polypeptide is diminished, while in rats, the labeling of three major products is diminished. While striking decreases in the extent of labeling of all three polypeptides of the lubrol pellet were noted as a consequence of ethanol ingestion, the relative content of only one of these components, that with an apparent molecular weight of 25,000, was decreased. In regard to this point, Costantino and Attardi36.37 have resolved ten distinct polypeptides of mitochondrial origin in HeLa cells labeled in vivo in the presence of emetine, an inhibitor of cytoplasmic protein synthesis. Three of the polypeptides (designated 2, 5, and 8) exhibit apparent molecular weights of 39,000, 27,500 and 19,500, respectively, in the absence of urea.37 These values are in exact agreement with those of Burke and BeattieZ4and, as we have indicated above, may be considered analogous to the entities we designate as subunits 1, 2, and 3 of rat liver cytochrome-c oxidase. In pulse-chase studies in the presence of cycloheximide, Costantino and Attardi36 noted a relatively rapid decline in radioactivity of component 5 (to 25% its initial radioactivity after 4 hr) and a much slower decline in the radioactivity of components 2 and 8. In fact, these components retained most of their incorporated radioactivity during a 12-hr period. If component 5 (of Costantino and Attardi) may be considered to be analogous to the polypeptide with an apparent molecular weight of 25,000 in SDS-8 M ureazQ(i.e., subunit 2), the shorter half-life of this component of HeLa cells could be the basis for the decreased content of its apparent counterpart in ethanol-treated animals (Fig. 2). By similar reasoning, the polypeptides of lubrol pellets of rat liver mitochondria with apparent molecular weights of 33,000 and 20,000 may be considered to correspond to components 2 and 8 of Costantino and Attardi.37Thus, while the labeling of these components is depressed (Figs. 3 and 4), the fact that levels of these entities are not demonstrably altered by chronic ethanol (Fig. 2) may be due to their longer relative halflives analogous to components 2 and 8 of Costantino and AttardL3' We believe it is important to emphasize that our experiments provide strong evidence that

308

the labeled polypeptides of the lubrol pellet are subunits 1, 2, and 3 of cytochrome-c oxidase. The immunochemical coprecipitation of more than 90% of the cytochrome-c oxidase and the larger part of incorporated radioactivity (60%) of mitochondria (Table 2). together with the observations that the 3 labeled components of lubrol pellets (Fig. 3) are identical in apparent molecular weight to those precipitated by immune y-Gglobulin preparations (Fig. 4), lead us to this view. This view is bolstered by the striking similarities in the apparent polypeptide compositions of rat liver and beef heart cytochrome-c oxidase.g5 The finding that these entities are components of cytochrome-c oxidase corroborates in essence the findings of other investigators with nonmammalian cells. In all cases, the three largest polypeptides of the enzyme complex a r e of mitochondrial origin I I. l4.:lK:i!I and are the major products of mitochondrial protein synthesis both in vivo and in vitro.4".4' It is evident that, in the derivations of both lubrol pellets (Table 1) and cytochrome-c oxidase immunoprecipitates (Table 2), recoveries of radioactivity are not quantitative. While the exact character of the lubrol pellet is unclear at present, we find that its derivation yields essentially identical enrichments of both specific radioactivity and NADH-K,Fe(CN), reductase specific activity.,' On the basis of these and other finding^,'^ we believe it will eventually be determined that lubrol pellet is a derived inner mitochondrial membrane preparation. Whether this should prove to be so or not, the extractions involved in derivation of the lubrol pellet could be expected to remove soluble components and to select for intact insoluble enzyme complexes of the mitochondrion. Thus, we believe the losses of radioactivity stem from the loss of unintegrated components of insoluble complexes. On the other hand, the immune y-G globulin used in the derivation of cytochrome-c oxidase immunoprecipitates selects for components of that entity. Consequently, extraneous mitochondrial translation products unrelated to cytochrome-c oxidase, as well as unintegrated oxidase components for which there was insufficient specific antibody(ies), would have remained unprecipitated and contributed to those losses in radioactivity.

BERNSTEIN AND PENNIALL

A t present, the underlying mechanism(s) of ethanol's actions on mitochondrial protein synthesis remain obscure. Our experiments confirm previous observation^^*".^^ of the diminished capacity of isolated liver mitochondria to incorporate precursors into protein after chronic ethanol treatment. However, like Rubin et al.,7 we find that ethanol's effects on protein synthesis are not accompanied by any profound change in the efficiency of ATP generation by isolated mitochondria. In addition, preliminary studies of mitochondria derived from isolated parenchymal cells from control and ethanoltreated rats, following an in vitro incubation of the cells, indicate no differences in either their total adenine nucleotide content o r in their content of neutral protease, as manifest with either endogenous protein or ATEE (n-acetyl tyrosine ethyl ester) as substrates (Penniall et al., unpublished observations). M ~ r l a n d ' ~and . ~ Mmland ~ and Sjetnan4' have investigated extensively the inhibitory effects of chronic ethanol administration on protein synthesis by the intact rat liver. They conclude that ethanol's actions are dependent on both the concentration and length of exposure to ethanol and involve neither detectable changes in liver amino acid pool sizes nor changes in rates of protein catabolism. At this time we cannot make similar conclusions concerning liver mitochondria per se, but we intend to investigate these parameters in future experiments. The effects of chronic ethanol ingestion, as manifested with isolated mitochondria, might well be mediated at the level of cytoribosomal and/or mitoribosomal protein synthesis. Poyton and K a ~ a n a g hhave ~ ~ shown t h a t an intramitochondrial pool of subunits of yeast cytochrome-c oxidase of cytoribosomal origin is necessary for sustained mitochondria1 protein synthesis in vitro and that the synthesis of subunits 1, 2, and 3 fell sharply on depletion of this pool. The addition of cell sap preparations could restore synthesis of subunits 1, 2, and 3. This work provides a direct explanation for the frequently observed dependence of mitochondrial protein synthesis on prior or concomitant cytoribosomal protein ~ynthesis.'~It is possible that ethanol's effects on synthesis of subunits 1, 2, and 3 are mediated by an effect on intramitochondrial and/or cell sap pools of low molecular weight components of cytoribosomal

ETHANOL AND LIVER MITOCHONDRIAL PROTEIN SYNTHESIS

origin. The 22,000 dalton component of the lubrol pellet, which is diminished in content in mitochondria of the ethanol-treated animals and apparently a cytoribosomal translation product, could also play a regulatory role in mitochondrial protein synthesis, either alone or in conjunction with the necessary oxidase

309

subunits of cytoribosomal origin. Whether depleted levels of such polypeptides are responsible for the depressions in mitochondrial protein synthetic capacity and the depressed levels of cytochrome-c oxidases-’ remains to be determined.

REFERENCES I . Videla L, Bernstein J, Israel Y:Metabolic alterations produced in the liver by chronic ethanol administration. Biochem J 134507-514, 1973 2. Cederbaum AI, Lieber CS, Rubin E: Effects of chronic ethanol treatment on mitochondrial functions: Damage to coupling site I. Arch Biochem Biophys 165:560-569, 1974 3. Gordon ER: Mitochondria1 functions in an ethanolinduced fatty liver. J Biol Chem 248:8271-8280, 1973 4. Thompson JA, Reitz RC: Mechanism for effect of chronic ethanol ingestion on rat liver mitochondrial ATPlinked functions. Presented before the Second International Symposium on Ethanol and Aldehyde Metabolizing Systems, University of Pennsylvania, Philadelphia, October 1976, p 31 5. Koch OR, Bedetti CD. Gamboni M, et al: Functional alterations in liver mitochondria in chronic experimental alcoholism. Exp Mol Pathol25:253-262, 1976 6. Bernstein JD, Penniall R: Effects of chronic ethanol treatment upon rat liver mitochondria. Biochem Pharmacol (in press) 7. Rubin E, Beattie DS, Lieber C: Effects of ethanol on the biogenesis of mitochondrial membranes and associated mitochondrial functions. Lab Invest 23:62&627, 1970 8. Cederbaum Al, Lieber CS, Toth A, et al: Effects of ethanol and fat on the transport of reducing equivalents into rat liver mitochondria. J Biol Chem 248:4977-4986, 1973 9. Tzagaloff A. Rubin MS, Sierra MF: Biosynthesis of mitochondrial enzymes. Biochim Biophys Acta 301:71- 104. 1973 10. Poyton RO, Schatz G: Cytochrome-c oxidase from baker’s yeast. 111. Physical characterization of isolated subunits and chemical evidence for two different classes of polypeptides. J Biol Chem 250:752-761, 1975 1 I . Sebald W, Machleidt W, Otto J: Products of mitochondrial protein synthesis of Neurosporu crussu. Determination of equimolar amounts of three products in cytochrome c oxidase on the basis of amino acid analysis. Eur J Biochem 38:311-324, 1973 12. Tzagaloff A. Meagher P: Assembly of the mitochondrial membrane system. VI. Mitochondrial synthesis of the subunit proteins of the rutamycinsensitive adenosine triphosphatase. J Biol Chem 247:594-603, 1972 13. Weiss H: Cytochrome b in Neurosporu crussu mitochondria. A membrane protein containing subunits of cytoplasmic and mitochondrial origin. Eur J Biochem 30:469-478, 1972 14. Mason TL, Schatz G: Cytochrome c oxidase from baker’s yeast. 11. Site of translation of the protein components. J Biol Chem 248:1355-1360, 1973 15. Katan MB, Groot GSP: Isolation, partial characterization, and mode of biosynthesis of cytochrome bc, (com-

plex 111)of yeast, in Quaglioriello E, Papa S, Palmieri F, et al ( 4 s ) : Electron Transfer Chains and Oxidative Phosphorylation. Amsterdam, North Holland, 1975, p 127 16. Jack1 G, Sebald W: Identification of two products of mitochondrial protein synthesis associated with mitochondrial adenosine triphosphatase from Neurosporu crussu. Eur J Biochem 54:97-106, 1975 17. Burke JP, Tumbleson ME, Hicklin KW, et al: Effect of chronic ethanol ingestion on mitochondrial protein synthesis in Sinclair (S-I) miniature swine (38687). Proc SOC Exp Biol Med 148:1051-1056. 1975 18. Thompson JA. Reitz RC: Studies on the acute and chronic effects of ethanol ingestion on choline oxidation. Ann NY Acad Sci 273:194-204, 1976 19. Chappell JB, Hansford RG: Preparation of mitochondria from animal tissues and yeasts, in Birnie GD (ed): Subcellular Components: Preparation and Fractionation. Baltimore, University Park Press, 1972, p 77 20. Beattie DS, lbrahim NG: Optimal conditions for amino acid incorporation by isolated rat liver mitochondria. Stimulation by valinomycin and other agents. Biochemistry 12:176- 180, 1973 21. Beattie DS. Basford RE, Koritz SB: Bacterial contamination and amino acid incorporation by isolated mitochondria. J Biol Chem 242:3366-3368, 1967 22. Lowry OH, Rosebrough NJ. Farr AL, et al: Protein measurement with the folin phenol reagent. J Biol Chem 193~265-275,1951 23. Mans RJ, Novelli GD: Measurement of the incorporation of radioactive amino acids into protein by a filterpaper disk method. Arch Biochem Biophys 94:48-53, 1961 24. Burke JP, Beattie DS: Products of rat liver mitochondrial protein synthesis: Electrophoretic analysis of the number and size of these proteins and their solubility in chloroform: methanol. Arch Biochem Biophys 164:1-11, 1974 25. Van Buuren KJH, Van Gelder BF, Eggelte TA: Biochemical and biophysical studies on cytochrome aa3 I. Steady-state kinetics of cytochrome aa3. Biochim Biophys Acta 234:468-480. 1971 26. Campbell DH, Garrey JS, Cremer NE, et al: Separation and preservation of serum, in: Methods in Immunology. New York, W.A. Benjamin, 1964. p 22 27. Heide K, Schwick HG: Salt fractionation of immunoglobulins, in Weir DM ( 4 ) : Handbook of Experimental Immunology. Oxford, Blackwell Scientific. 1973, P61 28. Wilson DF: The stoichiometry and site specificity of the uncoupling of mitochondrial oxidative phosphorylation by salicylanilide derivatives. Biochemistry 8:2475-248 1 , 1969

310

29. Swank RT, Munkres KD: Molecular weight analysis of oligopeptides by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate. Anal Biochem 39:462-477, 1971 30. Weber K, Osborn M: The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem 244:4406-4412, 1969 31. Elliott WB, Holbrook JP, Penniall R: Studies on a cytochrome oxidase antibody Ill. Cross reactivity. Biochim Biophys Acta 251:277-280,1971 32. lbrahim NG, Burke JP, Beattie DS: Mitochondrial protein synthesis in vitro is not an artifact. FEBS Lett 29:73-76, 1973 33. Capaldi RA, Bell RL, Branchek T: Changes in the order of migration of polypeptides in complex 111 and cytochrome c oxidase under different conditions of SDS polyacrylamide gel electrophoresis. Biochem Biophys Res Commun 74:425-433, 1977 34. Downer NW, Robinson NC, Capaldi RA: Characterization of a seventh subunit of beef heart cytochrome c oxidase. Similarities between the beef heart enzyme and that from other species. Biochemistry 15:2930-2936, 1976 35. Bernstein JD, Bucher JR, Penniall R: Origin of mitochondrial enzymes. V. The polypeptide character and biosynthesis of rat liver cytochrome c oxidase. J Bioenerg Biomembr (submitted for publication) 36. Costantino P, Attardi G: Metabolic properties of the products of mitochondrial protein synthesis in HeLa cells. J Biol Chem 252:1702-171 I , 1977 37. Costantino P, Attardi G: Identification of discrete electrophoretic components among the products of mitochondrial protein synthesis in HeLa cells. J Mol Biol 96~291-306,1975 38. Rubin MS, Tzagaloff A: Assembly of the mitochondrial membrane systems X. Mitochondrial synthesis of

BERNSTEIN AND PENNIALL

three of the subunit proteins of yeast cytochrome oxidase. J Biol Chem 248:4275-4279,1973 39. Koch G: Synthesis of the mitochondrial inner membrane in cultured Xenopur laevis oocyres. J Biol Chem 251 :6O97-6107. 1976 40. Von Ruecker A, Werner S, Neupert W: Synthesis of cytochrome oxidase components in isolated mitochondria of Neurospora crassa. FEBS Lett 47:290-294,1974 41. Poyton RO, Groot GSP: Biosynthesis of polypeptides of cytochrome c oxidase by isolated mitochondria. Proc Natl Acad Sci USA 72:172-176, 1975 42. Schnaitman C, Greenawalt JW: Enzymatic p r o p erties of the inner and outer membranes of rat liver mitochondria. J Cell Biol38:158-175, 1968 43. Koch OR, Boveris A, De Favelukes S S , et al: Biochemical lesion of liver mitochondria from rats after chronic ethanol consumption. Exp Mol Pathol 27:2 13-220, 1977 44. Msrland J: Effects of chronic ethanol treatment on tryptophan oxygenase, tyrosine aminotransferase and general protein metabolism in the intact and perfused rat liver. Biochem Pharmacol23:21-35, 1974 45. Mgrland J: Hepatic tryptophan oxygenase activity as a marker of changes in protein metabolism during chronic ethanol treatment. Acta Pharmacol Toxicol 35:155- 168, 1974 46. M~rlandJ, Sjetnan AE: Effect of ethanol intake on the incorporation of labelled amino acids into liver protein. Biochem Pharmacol25:2125-2130, 1976 47. Poyton RO, Kavanagh J: Regulation of mitochondrial protein synthesis by cytoplasmic proteins. Proc Natl Acad Sci USA 73:3947-395 I. 1976 48. Schatz G, Mason TL: The biosynthesis of mitochondrial proteins, in Snell EE, Boyer PD, Meister A, et al(eds): Annual Review of Biochemistry, (vol43). Palo Alto, Annual Reviews, 1974, p. 70