Inhibition by lipoperoxidation of amino acid incorporation by rough microsomal membranes in vitro and its partial reversibility

ARCHIVES

OF

BIOCHEMISTRY

AND

BIOPHYSICS

Inhibition by Lipoperoxidation Microsomal Membranes

171,

695-707

(1975)

of Amino Acid Incorporation by Rough in Vitro and Its Partial Reversibility T. K. SHIRES

The Toxicology

Center,

Departments

of Pharmacology Medicine, Iowa Received

and Pathology, City, Iowa 52242 April

The

University

of Zowa,

College

of

28, 1975

Amino acid incorporation in vitro by rough microsomes is inhibited by concurrent lipoperoxidation of the membranes. Disjunction of polysomes from their binding sites was not demonstrable. During the lipoperoxidative inhibition, membrane alteration was indicated by (i) a decline in activity of glucose-6-phosphatase and N-ethylmorphine demethylase, (ii) increased permeability, gauged by the release of radioactively labeled intravesicular protein and glycoprotein, and (iii) the generation of malonyldialdehyde. Despite these effects on the membrane, the lipoperoxidation-associated inhibition of incorporation was found to be transient and restricted to the early minutes of incubation. An accumulation of unidentified lipoperoxidative intermediates early in incubation is indicated as the source of inhibition. Apparently, inhibitory intermediates can escape the membrane since incorporation by free as well as bound polysomes was demonstrated.

In differentiated eukaryotic cells, the membranous component of the rough endoplasmic reticulum has been viewed as perhaps having an active role in translation by the attached polysomes (1). The need for postulating membrane involvement has been advanced by reports of mRNA attachment to membranes of the rough reticulum (l-3). Aside from the stabilizing effect of the membrane on template (11, little is known of specific forms of membrane influence. An experimental approach toward demonstrating such a role for membranes is to alter the structure of the membrane component of rough membranes in vitro and correlate changes in the synthetic functions of the attached polysomes with those in the underlying membrane . The course reported here has employed lipoperoxidation as the method of structural alteration. The choice was influenced by studies with the hepatetoxic agent carbon tetrachloride whose toxic mechanism in vivo involves lipoperoxidation of hepatic cell membranes (4) in addition to an inhibition of protein synthesis (5-7). Carbon tet695

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rights

0 1975 by Academic of reproduction

Press, in any form

Inc. reserved.

rachloride is thought to require metabolic cleavage into its active form by the membranes in order for lipoperoxidation to occur (41, and, in order to avoid this kind of complication, the study reported here used the well-studied and simpler system of lipoperoxidation stimulated by reduced pyridine nucleotides. In NADPHor NADHstimulated lipoperoxidation of microsomal membranes, active NADPH-cytochrome c reductase (8-10) or NADPH-cytochrome b, reductase (11) is involved. Lipoperoxidative events in the membranes include a transient formation of various phospholipid peroxides, destruction of polyunsaturated fatty acids primarily from the p position, and formation of a variety of oxidation products, terminal among which is malonyldialdehyde which is measured as an assay of lipoperoxidative activity (8, 9, 12-14). By using mild incubation conditions (25°C) for simultaneous NADPHstimulated lipoperoxidation and amino acid incorporation, incorporation was inhibited. If peroxidation and incorporation were carried out separately and sequentially on the same set of membranes, the

696

T. K. SHIRES

incorporative unperoxidized

activity approached controls.

MATERIALS Isolation microsomnl

and labeling membranes.

that of

AND METHODS of polysomes

and

rough

rough microsomal membranes, 13Hlleucine (New England Nuclear, 34-36 Cilmmol) was injected (40 mCi/lOO g body weight) into the portal vein. After 30 s, the liver was perfused with saline and the membranes isolated (15). For labeling of intravesicular protein, [3H]leucine (New England Nuclear, 35-36 Ci/mmol) was injected intraperitoneally (100 mCi/lOO g body weight) 30 min before sacrifice of the rat and its rough membranes isolated (221. For labeling of intravesicular glycoprotein, n-t6-3H(N)1glucosaminehydrochloride (New England Nuclear, 10.1 Ci/mmol) was injected intraperitoneally (200 mCillO0 g) 30 min before sacrifice. Where study of intravesicular contents was involved the rough membrane preparations were not given the routine washes in 0.44 M sucrose/T,,K,,M,,,.

Male Holtzman rata, lOO150 g, were housed on wire in a light-cycled room (6:OOPM-6:OO AM, dark), with food (Wayne Lablox or Teklabl and water available ad libitum. Rats in all experiments were fasted for 16-18 h before decapitation. The livers were perfused with saline via the inferior vena cava (with the portal vein severed), rinsed in cold 0.44 M sucrose/T,,K,,M,,, (50 mM TrisHCl, pH 7.4, 25 mM KCl, 1.0 mM MgCl,),’ weighed, and homogenized in two volumes of cold 0.44 M sucrose/T,,K,,M,,, in a motor-driven Potter-ElMeasurement of the release of labeled polysomes vehjem homogenizer with Teflon pestle (10 strokes). An incubation mixture, 1.9 The homogenate was centrifuged at 2O,OOOg,,, for 10 from rough membranes. ml, containing membranes, was made 1.8 M with min in a Sorvall SS34 rotor at 4°C and the postmitorespect to sucrose in a final volume of 7 ml. Rough chondrial supernatant fluid taken from its upper membranes and the polysomes dissociated from two-thirds after removal of lipid from the surface. These and all subsequent steps were carried out in them were fractionated according to the flotation centrifugation procedure described by Shires et al. an ice bath or a cold room at 4°C. Rough and smooth microsomal membranes were (171, centrifuging the mixture in 1.8 M sucrose for 18 h in a Beckman 50 Ti rotor at 50,000 rpm. Polysomes separated from the postmitochondrial supernatant fraction by a modification (15-17) of the method of no longer attached to the membrane collected at the Moyer et al. (181. Separation was carried out in a bottom of the tube, while the membranes and stillattached polysomes collected centripetally in a layer Beckman 50 Ti rotor spun 3 h. Both membrane fractions were washed three times in 0.44 M su- atop the 1.8 M sucrose. The tube is fractionated as described (17) so that the polysome pellet, the memcroseiT,K,,M,,, by homogenizing with 20 strokes and sedimenting at 105,OOOg,,, for 45 min. Poly- branes, and soluble portions of the sucrose can be individually counted in a liquid scintillation specsomes were also isolated from the postmitochondrial trometer. The radioactivity (disintegrations per minsupernatant fraction. The method used was that ute) for each fraction is determined, and expressed described by Shires et al. (19-20). Radioactive labeling of various components of the as a percentage of the total recovered disintegrarough microsomal preparations was carried out as tions (17). RNA extraction and analysis on polyacrylamide follows: For labeling of the polysomes attached to gels. RNA was extracted with phenol-sodium dodethe membranes of the rough microsomal preparacyl sulfate, as previously described (19). The phenol tion, [5-3H]orotic acid (AmershamSearle, Arlington (Mallinckrodt) was 2 x redistilled. Sodium dodecyl Heights, Ill., 15-23 Ci/mmol) was injected intraperisulfate was obtained from the British Drug Co. toneally 18-20 h before sacrifice of the animal and isolation of membranes (19). For labeling of mem- Acrylamide gels were cast with 2.8% acrylamide (19) brane phosphatidylcholine, 10 mg of Casamino acids in g-cm quartz tubes, loaded with 20-150 pg of RNA, (Difco) was injected intraperitoneally followed by 50 and run about 4 h at room temperature at 6 mA and &i/l00 g of [methyl-3H1choline chloride (New Eng- with a methylene blue marker. The gels were land Nuclear, 2.3-3.0 Ci/mmol) 30 min before sacri- scanned at 259 nm in a Gilford scanner attachment on a Model 240 spectrophotomer with recorder. fice (21). Approximately 16% of the radioactivity Amino acid incorporation. The incubation mixpresent in the rough microsomal preparation immeture in 1 ml of buffer A (20 mM Tris-HCl, pH 7.4 at diately after its separation from the smooth appeared in the supernatant fluid after one wash. The 25”C, 100 mM NH,Cl, 5 mM Mg&l, 1 mM dithiothreiloss of radioactivity with subsequent washes tel) included 1 Fmol of ATP, 0.5 pmol of GTP, 10 (tricyclohexylamine dropped to 6% after the third wash and could not be pm01 of phospho(enol)pyruvat salt), 20 pg of pyruvate kinase, 1 pmol each of 20 further reduced with more washes. For labeling of nacent polypeptide chains on the polysomes of the amino acids except for 0.7 pmol of leucine, 0.2 ml of freshly prepared pH 5 fraction (about 9 mg of protein) and 0.27-0.3 pmol of [4,5JH(N)lleucine (35.4’ Abbreviations used: T,&K,M,,o, 50 mM Tris53.2 Cilmmol, New England Nuclear). All nonradioHCl, pH 7.4, 25 mM KCl, 1.0 mM MgCl,; Cl,AcOH, active compounds were obtained from Sigma Chemitrichloroacetic acid. cal Co. The pH 5 fraction was prepared by homoge-

AMINO

ACID

INCORPORATION

BY

nizing rat liver in 2.5 volumes of cold buffered sucrose (0.3 M sucrose, 10 mhf Tris-HCl, pH 7.6 at 25°C 5 mM MgCl,, and 1 mM dithiothreitol; buffer B). The homogenate was centrifuged in the Sorvall SS34 rotor at 15,000 rpm for 15 min, and the supernatant fraction was centrifuged for 5 h at 50,000 rpm in the Beckman 50 Ti rotor. The upper two-thirds of the high speed supernatant fraction (S31 was diluted with two volumes of cold double-distilled H,O containing 1 mM dithiothreitol. The pH of the diluted mixture was adjusted to 5.1 with 1 N acetic acid in an ice bath, the precipitate collected by centrifugation, and resuspended (1 ml per 6 ml of original S3) in buffer A. For incubation, each tube contained 1 mg of rough membranes measured as protein, or 180-200 pg of RNA. All incubations were carried out at 25°C. Incorporation was stopped by addition of 1 ml of cold 10% Cl,AcOH containing 10 mM leucine (unlabeled). The precipitates were washed five or six times with 5% Cl,AcOH (with leucine), incubated at 90°C for 30 min in 5% Cl,AcOH, and extracted twice with acetone:ether (l:l, v/v). After the solvent had thoroughly evaporated, 0.5 ml of formic acid (99%, Eastman) was used to dissolve the pellets in sealed tubes at 37”C, followed by mixture with PCS scintillation cocktail (AmershamSearle). Each tube was rinsed with additional PCS, and the washings and solubilized pellets combined for scintillation counting. Incorporation was measured as the amount of hot acid- and solvent-resistant radioactivity (disintegrations per minute) from tubes incubated with the complete amino acid incorporation system minus the radioactivity in tubes from which the ATP-generating system, ATP and GTP were omitted during incubation (15). With a membrane suspension containing 180-200 pg of RNA, use of more than 0.2 ml of either the pH 5 fraction or the ATP-generating system (plus ATP) did not enhance incorporation, while use of less than 0.2 ml of either diminished it. Similarly, 0.5 pm01 of GTP and 1 pmol of each amino acid was found to give maximal incorporation with buffer A. In subsequent descriptions, the term “energy” or “energy system” refers to the combination GTP, ATP, and ATP-generating system. The term “incorporation system” refers to the energy system, plus pH 5 fraction and amino acids dissolved in buffer A. Analytical procedures. Glucose-6-phosphatase (Dglucose-6-phosphate phosphohydrolase, EC 3.1.3.91 was determined by the method of Nordlie and Arion (23). NADPH-cytochrome c reductase activity (NADPH-cytochrome c oxidoreductase, EC 1.6.2.3) was measured as detailed by Masters et al. (24). Ethylmorphine N-demethylase activity was determined by measuring formaldehyde production by the method of Nash (25) as modified by Cochin and Axelrod (26). Assays were performed in 5 or 10 ml in a 25°C water bath over a magnetic stirrer using 15-

ROUGH

MICROSOMAL

MEMBRANES

697

25-ml Ehrlenmeyer flasks containing 50 mM TrisHCl, pH 7.4 at 25”C, 150 mM KCl, 10 mM MgCl,, 8 mM ethylmorphine (Dionine, Merck), 0.2 mM NADPH, and l-2 mg of rough membranes per ml. The reaction was started by addition of NADPH, and l-ml aliquots were removed at 1-min intervals and transferred to vials containing 1 ml of 15% Cl,AcOH in an ice bath. Lipid peroxidation was measured by the amount of malonyldialdehyde produced using the thiobarbituric acid assay of Ottolenghi (27) as modified by Poyer (14). The only modification involved incubation of the reaction mixture for 90 min at 60°C. For purposes of standardization or experimental use, malonyldialdehyde was made from malonaldehyde bis(dimethyl)acetal (Pfaltz and Bauerl with 1 N HCl and measured by using a molar absorption of 23,000 at 270 nm at pH 7.5 (28). Scintillation counting was done with a Beckman LS-250 counter equipped with automatic quench compensation and automatic external standard counting. Disintegrations per minute were calculated with a Wang 600 calculator placed on-line with the scintillation counter and teletype. All chemicals, unless otherwise specified, were obtained from the Sigma Chemical Company. RESULTS

Lipoperoxidation ration

and Amino Acid Incorpo-

The incorporation of labeled amino acid precursor into acid-precipitable polypeptide in vitro by rough microsomal membranes at 25°C is approximately linear for 15-20 min (Fig. 1A). Inclusion of NADPH in the incubation mixture reduces the rate of incorporation and retards its onset during the first 5 min of incubation (Fig. 1A). NADP, when incubated in the complete incorporating system, had little effect on the amount of labeled precursor entering new polypeptide compared with the control (Fig. 1A). The presence of hydroxybutyl toluene with NADPH in the incorporating system effectively prevented the depressive effect of the reduced pyridine nucleotide on incorporation (Fig. 1). As shown in Fig. 2, the relative inhibition of incorporation caused by incubation with NADPH was found to correlate well with the extent of NADPH-promoted lipoperoxidation measured by malonyldialdehyde production. Incubation with NADH instead of NADPH also inhibited incorporation and stimulated peroxidation in a

698

T. K. SHIRES

INCUBATION

TIME

FIG. 1. Time course of amino acid incorporation by rough microsomal membranes during lipoperoxidation. (A) Rough membranes (1 mg) were incubated at 25°C in a series of tubes in lml final volume of a complete amino acid incorporating system containing 1 mM NADPH (O), 1 mM NADPH plus 50 PM hydroxybutyltoluene (A), or 1 mM NADP (a). Control tubes (0) contained only the incorporation mixture and membranes. Incubation was interrupted at intervals by addition of 1 ml of cold 10% Cl,AcOH (with 10 mM unlabeled leucine). (B) Rough membranes (1 mg) were incubated for 30 min at 25°C in the presence of 1 mM NADPH CO), 1 mM NADPH plus hydroxybutyltoluene (A), or 1 mM NADP (A). A complete amino acid incorporating system (at 25°C) was added directly to each tube and the incubation continued with interruption at each time interval by addition of cold 10% Cl,AcOH. For control tubes, the initial incubation was carried out in the absence of NADPH (0). Final volume during the first incubation was 0.75 ml, and addition of the incorporating system for the second incubation brought the volume to 1 ml. The extent of lipoperoxidation using 1 mg of membrane and 1 mM NADPH was unaffected by volume change over a range of 0.6-1.25 ml. A single preparation of freshly isolated rough membranes was used for all results shown.

manner dependent on its concentration (Fig. 2). Equimolar combinations of the two reduced pyridine nucleotides in increasing concentrations heightened both lipoperoxidation and the inhibition of incorporation over that obtained when either pro-oxidant was used by itself. As a lipoperoxidative agent, NADPH was slightly more effective than NADH, as was the case with the inhibition of incorporation. The results with NADH are at variance with previous reports (e.g., Ref. (2911,but differences in the incubation system employed from those used elsewhere should be noted. Both the generation of malonyldialdehyde and the inhibition of incorporation was prevented by incubation with hydroxybutyl toluene (Fig. 2). Measurement of the lipoperoxidation of rough membranes occurring concurrently in the same incubation mixture with an

active amino acid incorporating system revealed some diminution in the extent of peroxidation compared with levels attained with incubations containing only NADPH and membranes. Equimolar amounts of NADPH and NADH stimulated lipoperoxidation in the presence of incorporating system over that with NADPH alone (Fig. 3). Whether incorporation, as a process of the rough membrane, is the source of this attenuation of lipoperoxidation or whether elements used in the incorporating system were responsible was tested as shown in Table I. The NADPHstimulated production of malonyldialdehyde by rough membranes was noticeably reduced in the presence of the pH 5 fraction. Unlike several other sulfhydryl reagents (291, dithiothreitol included in the peroxidizing mixture did not effect the generation of malonyldialdehyde. Other ele-

AMINO

ACID

INCORPORATION

BY

ROUGH

MICROSOMAL

MEMBRANES

699

Concentration(mM)

FIG. 2. Correlation between lipoperoxidation and amino acid incorporation with increasing concentration of reduced pyridine nucleotides. Rough microsomal membranes (1 mg) were incubated with NADPH CO,-), NADH CO,---), or equimolar amounts of NADPH and NADH CO,-) in the presence of a complete amino acid incorporating system. Also shown are incubations with NADPH (A,-) or NADPH + NADH (A,-) in the presence of hydroxybutyl toluene. After incubation for 30 min at 25°C those tubes with 13H11eucine were measured for incorporation of the label (left panel) and the remainder for lipoperoxidation (right panel). All, experiments shown were carried out in a single rough membrane preparation immediately after its isolation.

ments of the amino acid incorporating syscase-8phosphate activity was reduced tem had no influence on the extent of memfrom a mean 22 pmol P mg-’ h-l in the brane lipoperoxidation (Table II). presence of NADPH, and ethylmorphine Evidence that lipoperoxidation occurdemethylase activity was decreased from a ring with concurrent incubation of mean 4.6 to 1.9 nmol/min-’ mg-‘. DemethNADPH and the amino acid incorporation ylase assays were made on membranes isosystem caused significant changes in the lated from livers of rats injected with 60 rough membrane was seen when memmg/kg of phenobarbital daily for 4 days. brane enzymatic activity was considered.. No difference in the amount of malonyldiLipoperoxidation of microsomal memaldehyde was observed between membrane preparations has been reported to branes isolated from induced and noninlower N-ethylmorphine demethylase activduced animals. ity (29-31, 33) and glucose-E-phosphatase Partial Reversal of Inhibition of Incorporaactivity (31, 32) but not that of NADPHtion cytochrome c reductase (31). All of these findings could be significantly reproduced As was shown in Fig. lA, concurrent with actively incorporating rough memincubation of rough membranes with rebranes incubated with NADPH at 25°C. duced pyridine nucleotides and an amino Membranes (1 mg/ml) were incubated 30 acid incorporating system caused a decline min with or without 1 mM NADPH in the in the incorporative performance. Howpresence of a complete incorporating sysever, if, instead of using this concurrent tem. NADPH-cytochrome c reductase ac- scheme (Scheme 1, below), membranes are tivity was unchanged in the lipoperoxiincubated first with NADPH alone foldized rough membranes, remaining about lowed by a second incubation with the in125 nmol of cytochrome c reduced corporating system (Scheme 21, no decline min-lrng-’ with or without NADPH. Glucould be found, even though the mem-

700

T.

K. SHIRES TABLE

20

I

EFFECT OF ELEMENTS OF THE AMINO ACID INCORPORATING SYSTEM ON LIPOPEROXIDATION ROUGH MICROKBMAL MEMBRANE@

I

Portion

5

10 lncuballon

15 tome

20 (minutes)

25

30

FIG. 3. The effect of the amino acid incorporation system on lipoperoxidation of rough microsomal membranes by reduced pyridine nucleotides. Rough membranes (1 mg) in a final volume of 1 ml were incubated with 1 mM NADPH CO), 1 mM each NADPH and NADH (0) at 25°C in the presence (- - - -) or absence (--) of a complete amino acid incorporating system. Lipoperoxidation was arrested by addition of 50 pM hydroxybutyl toluene and the malonyldialdehyde produced for each time period assayed immediately.

branes were lipoperoxidized. Comparing data from a Scheme 1 incubation (Fig. 1A) with data from a Scheme 2 schedule (Fig. lB), it can be seen that the incorporative activity with the latter was nearly as great as with membranes incubated according to Scheme 1 but in the absence of NADPH (Fig. 1A vs 1B). Moreover, the incorporation by Scheme 2 membranes was essentially the same with or without the presence of NADPH in the incubation scheme (Fig. 1B). The results with Scheme 2 suggested that the inhibition of incorporative activity observed with concurrent lipoperoxidation (Scheme 1) was partially reversible and probably transient. Essentially the same results were obtained with Scheme 2 experiments carried out with [3Hlphenylalanine in the incorporation mixture instead of [3H]leucine. Comparing the status of the membranes in Schemes 1 and 2, it will be seen that the rough membranes in Scheme 2 have al-

of system

OF

Malonyldialdeh de Produced (nmo F in 1 ml) With 1rnM NADPH

N A%H

hie alone pH 5 alone E alone aa alone D’lT alone

7.8 1.2 0.0 0.0 0.0

0.8 0.7 0.0 0.0 0.0

Bit + PI-I 5 pH5+E Rmic + E Rm,, + Dm

5.6 0.9 7.0 7.7

1.9 0.0 0.9 0.7

R,,,,+pH5+E+DT”f F&C + pH 5 + E + aa + D?T

4.9 5.0

1.6 1.5

a Rmic, rough membranes, 1 mg in each incubation; E, the energy system; aa, 20 unlabeled amino acids; DlT, dithiothreitol. Concentrations of all elements are the same as those described in Materials and Methods. TABLE

II

POSITION OF POLYSOMES ON MEMBRANES AND THEIR SUSCEPTIBILITY TO LIPOPEROXIDATIVE INHIBITION OF AMINO ACID INCORPORATION(’ Compound

added

Control NADPH (1 mM) Malonyldialdehyde (50 nmol/ml)

13H]leucine

incorporated (dpm)

Rough microsomes

Polysomes

2228 908 2233

19,585 20,901 19,311

Polysomes + smooth membranes 15,092 5,020 14,353

n Each incubation tube contained 1 mg of rough microsomes (based on protein) or 100 kg of polysomes (based on RNA), or the combination of 1 mg of smooth microsomes and 100 pg of polysomes. All tubes contained the complete amino acid incorporating system described in Materials and Methods. Control tubes contained no NADPH or malonyldialdehyde. The RNA/protein ratio of the rough microsoma1 preparations was 0.19.

AMINO Incubation Mixture

ACID

INCORPORATION

Scheme 1 of: rough membranes, pyridine nucleotide (NADPH, NADP etc.), pH 5 fraction, energy, amino acids, r3Hlleucine

BY

[ 1

Incubation Scheme step 1 Mixture of: rough pyridine (NADPH,

5-30

min

ROUGH

MICROSOMAL

~

701

MEMBRANES

Cl,AcOH

precipitation

25°C

2

membranes, nucleotide NADP etc.),

Step 2 Add: pH 5 fraction, amino energy, acids, 3H-leucine

1 [ +

30’

25°C

ready undergone 30-min incubation before their amino acid incorporative capacity was tested. Further, Scheme 2 membranes were incubated with NADPH in the absence of the pH 5 fraction and therefore suffered higher levels of peroxidation than Scheme 1 membranes whose lipoperoxidation occurred in the presence of a complete incorporation system (cf. Fig. 3 and Table I). Despite this inequality, the results of Scheme 2 incubations constitute a reasonable demonstration of reversibility of the inhibition of incorporation seen with Scheme 1 experiments. Measurable lipoperoxidation was greater in Scheme 2 than 1 and, therefore, the anticipated depression of incorporation greater, and the incorporative capacity in Scheme 2 membranes was restored from a level of greater inhibition than was reached in Scheme 1 experiments. Clarification of any involvement of the pH 5 fraction in the results with either Scheme 1 or 2 is essential. Conceivably during concurrent lipoperoxidation and incorporation, the observed inhibition might originate from a peroxidation-linked inactivation of the pH 5 proteins, and the restoration in the incorporative capacity of the rough membranes seen in Scheme 2 with delayed addition of the incorporation system could be due to fresh pH 5 fraction in the mixture at a time when the most active phase of lipoperoxidation has terminated. To test this possibility (Fig. 41, the pH 5 fraction was pre-incubated by itself, with NADPH, or with rough membranes and NADPH, and then at varying time

1J

5-30 min ------+ ^_^^

Cl,AcOH

precipitation

25°C:

intervals hydroxybutyl toluene was added and the ability of the fraction to support incorporation was tested immediately. As Fig. 4 shows, there was some decline in the capacity of the pH 5 fraction to support incorporation, and this decline was intensified by the combined incubation of NADPH and membranes. However, the observed peroxidation-dependent decline in the ability of the pH 5 fraction to support incorporation did not seem sufficiently pronounced to provide an explanation for the net observed inhibition of incorporation. Evidence for a more direct influence of lipoperoxidation on the rough membrane per se and its ability to incorporate amino acid was sought by incubating rough microsomal membranes with NADPH for various time intervals, stopping the peroxidation with hydroxybutyl toluene, and immediately assaying the capacity of the membranes to incorporate 13Hlleucine (Fig. 4). As Fig. 4 shows, the lipoperoxidizing membrane had a markedly reduced incorporating capacity after 5 min of incubation, but the level of this inhibition decreased for the next 15 min thereafter. This pattern of inhibition with its deep trough early in the course of lipoperoxidation added support to indications that inhibition was transient. If assay for incorporation was not carried out immediately after addition of hydroxybutyl toluene but was delayed lo-15 min, the trough disappeared (data not shown). This suggested that inhibition of incorporation involved the lipoperoxidative production of some

702

T. K.

SHIRES

erence to the time course of lipoperoxidation (Fig. 3) showed that its rate was greatest during first few minutes of incubation and began to decrease somewhat before 5 min was reached. The Condition of Polysomes on Lipoperoxidized Rough Membranes

FIG. 4. The time-dependent effects of lipoperoxidation on the ability of the rough microsomal membranes to incorporate amino acid and of the pH 5 fraction to support incorporation. The pH 5 fraction in 0.6 ml was incubated at 25°C with 1 mM NADPH alone (A,---) or with 1 mg of membranes and NADPH (A,-). At each time interval, 50 PM hydroxybutyl toluene was added and the mixture chilled. Tubes with membranes were spun in an SW 56 rotor for 15 min to pellet the membranes. The supernatant fluid was then removed and to it was added 1 mg of fresh membranes, energy system and amino acids. The mixture was then incubated at 25°C an additional 30 min. Tubes without membranes were kept chilled for 15 min, and membranes and the remainder of the incorporating system added and together incubated for 30 min. Timedependent lipoperoxidation effects on rough membranes were observed by incubating the membranes with (0) or without (0) 1 mM NADPH. Hydroxybutyl toluene was added at the indicated time intervals, followed immediately by the complete amino acid incorporating system. Incubation was then continued for an additional 30 min.

short-lived intermediate whose accumulation is perhaps already maximal by 5 min of incubation and therefore not effected significantly by hydroxybutyl toluene. Ref-

Several studies have found that polysomesnot attached to membranes are considerably more active in amino acid incorporation than those attached to membranes (15, 34-36). If lipoperoxidation of rough membranes caused disjunction of functional polysomes from the membranes, the incorporative capacity of the entire preparation might appear elevated, with the freed polysomes offsetting the depressed capacity of those still attached. Possibly, such a situation might occur during the “reversal” of lipoperoxidation inhibition. Initial assessmentssuggested possible disjunction (Fig. 5A). Rough membranes bearing tritium-labeled polysomes were incubated with NADPH and, while still in the presence of pro-oxidant, centrifuged to separate membrane-bound polysomes(which in this system migrate centripetally) from the free, detached polysomes (which pellet). It appeared that incubation with increasing NADPH concentrations caused an increasing proportion of polysomesto become pelleted, and this did not occur in the absence of lipoperoxidation (Fig. 5A). However, if rough membranes were isolated with their phospholipid constituents labeled with 13Hlcholine (211, as shown in Figure 5B, a considerable amount of lipid was found pelleted along with the polysomes. The extent of this pelleting was partially a function of the NADPH concentration used for incubation. Larger percentages of the labeled choline traveled neither with membrane nor pellet but appeared in a nonsedimentable or supernatant fraction (Fig. 5B). If hydroxybutyl toluene was added to tritium-labeled rough membranes after a 30-min incubation with. NADPH but before the centrifugal separation of attached and unattached polysomes was carried out, little detachment of polysomes could be demonstrated (Fig. 5A), nor did

AMINO

mN

01 05 NADPH

1 lo! 25’

ACID

2 for

INCORPORATION

BY

3 30

mini

mM

NADPH

(01 25”

for

30

mtnl

FIG. 5. (A) Dispersal of tritium-labeled polysomes from rough microsomal membranes by lipoperoxidation. Rough membranes bearing 3H-labeled polysomes were incubated at 25°C for 30 min in the presence of increasing concentrations of NADPH. Hydroxybutyl toluene (HBT) (50 PM) was added to the one set of tubes (lower line) but not to a second set (upper line). The mixtures were chilled and sucrose added to make a final concentration of 1.8 M. Following the flotation assay for the extent to which the labeled polysomes remain associated with the membranes, the tubes were centrifuged and fractionated as indicated in Materials and Methods. The ordinate shows the percentage of the total recovered radioactivity that pelleted to the bottom of the centrifuge tubes. (B) Dispersal of radioactivity from rough microsomal membranes labeled with 13H1choline after lipoperoxidation. Experiments were carried out as in (Al. Dashed lines show the percentage of radioactivity appearing in nonsedimentable fractions of the centrifuge tube, and solid lines the percentage in the pellet.

ROUGH

MICROSOMAL

MEMBRANES

703

pelleting as due to a disjunction from the membrane but rather as due to a kind of membrane disruption. It should be noted that this disruption is not demonstrable until after those incubation times used for the incorporation studies (up to 1 h). Thus, this membrane fragmentation plays no role in either the inhibition of incorporation or its reversals. Partial or complete reversibility of peroxidative inhibition of amino acid incorporation entails that the basic structural components of membrane-bound polysomes remain intact and potentially functional. RNA was isolated from rough membranes incubated at 25°C (Fig. 6A), with NADPH at 25”C, as in the first step of Scheme 2 (Fig. 60, and from membranes incubated with a complete amino acid incorporating system and NADPH (Scheme 1) (Fig. 6B). Lipoperoxidation of rough membranes produced few detectable changes in the RNA patterns (Fig. 6C vs 6A). Some degradation of RNA was shown on membranes concurrently incubated with an incorporating system and NADPH as indicated by the heightened peaks between 18 and 2% (Fig. 6B). Release ofInhibitory Substances from Lipoperoxidizing Membranes

Lipoperoxidation is presumably an intramembranous event, yet the incorporative 13Hlcholine appear in the pellets (Fig. 5B). machinery of the rough microsomal memThus, the pelleted material seen when no brane is located on its surface. While on antioxidant was present seems to have re- the one hand a hypothetical, transient alsulted from changes taking place after the lipoperoxidation incurred during the 30min, 25°C incubation and was caused by the continued peroxidation taking place during centrifugation. Apparently, this continued peroxidation so weakens the membranes that the attached polysomes tear free and pellet, carrying along phospholipid-containing membrane fragments. Rough membranes incubated longer than 1 h at 25°C with 1 mM NADPH and then treated with hydroxybutyl toluene also exhibited this destructive pelleting of membrane fragments with the polysomes. FIG. 6. Acrylamide-gel scans of RNA isolated Since in this destructive situation the poly- from rough membranes incubated 30 min at 25°C somes have probably not cleanly disen- (Al, incubated with 1 mM NADPH CC), or incubated gaged from their membrane-binding sites, 30 min at 25°C with an amino acid incorporating it seems preferable not to interpret their system and NADPH (B).

704

T. K.

SHIRES

teration in the membrane substratum for polysomes might somehow influence their function, on the other hand the partial inactivation of pH 5 fraction activity by lipoperoxidating membranes raised the question of whether products of the peroxidizing process might be released from the hydrophobic interior of the membrane to the outside where they might directly interact with elements of the incorporating system. To test for release of such extravesicular inhibitors, the incorporative activity of polysomes was assessed in the presence of lipoperoxidizing membranes when the polysomes were not attached to them. If smooth microsomal membranes, which under our conditions have a low propensity to bind exogenous polysomes (171, are incubated with isolated polysomes and NADPH and can influence the incorporative activity of the polysomes, it could be concluded that direct contact of polysome and membrane is not required for peroxidative inhibition of incorporation. It has previously been shown that, based on equal amounts of RNA, polysomes by themselves are more active in incorporating amino acids in vitro than are rough membranes (15). Attachment of free polysomes to membranes results in an immediate decline in their incorporative capacity (15, 35). As shown in Table II, mixture of polysomes with washed membranes from the smooth microsomal fraction causes a decline of about 21% in the incorporation activity of the polysomes. The time course and magnitude of lipoperoxidation in the free polysome-smooth membrane mixture incubated together with 1 mM NADPH and a complete incorporating system was closely similar to that for rough membranes shown in Fig. 3. NADPH-stimulated lipoperoxidation caused a marked decline in the incorporation by the smooth membrane-polysome mixture (Table II). This inhibition represented a decrease of 67% from the amino acid incorporation of the nonperoxidized mixture, thus showing that polysome attachment to lipoperoxidizing membranes was not mandatory for the membrane changes to influence the polysomes’ activity. Attempts to study reversibility of the

lipoperoxidative inhibition of incorporation by free polysomes were not successful when a Scheme 2 set of incubations was used. In experiments where free polysomes and smooth membranes were first incubated at 25°C for 30 min in the absence of NADPH and subsequently assayed for incorporative ability, the decline in leutine incorporation was greater than 70%. Addition of ribonuclease inhibitor (as postmicrosomal supernatant) during the first incubation reduced but did not abolish the loss in incorporative capacity. Other approaches to study lipoperoxidative effects on free polysomes are in progress. Considerable amounts of material can be shown to be released from rough membrane vesicles undergoing lipoperoxidation, including protein and malonyldialdehyde (Fig. 7). The extent of release of both protein and malonyldialdehyde appeared

7cb 5: ./‘.

Incubation

Period

(Minutes)

FIG. 7. Release of protein and malonyldialdehyde from lipoperoxidized rough microsomal vesicles. The percentage of the total protein as determined by the Folin reaction (lower line) or malonyldialdehyde (upper line) released from rough membranes incubated at 25°C. After incubation the membrane mixture was centrifuged in an SW 56 rotor at 50,000 rpm for 30 min. The supernatant fluid was decanted and the pellets resuspended and assays for protein and malonyldialdehyde were performed on both.

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to roughly parallel the extent of measurable lipoperoxidation, as seen by comparing Fig. 7 with Figs. 2 and 3. The released protein contained no detectable glucose-gphosphatase or N-ethylmorphine demethylase activity but did contain some NADPH-cytochrome c reductase activity. Recalling Fig. 5B, tritiated choline incorporated into membrane phospholipid also was freed from pelletable membrane, but its appearance was not detected unless incubation of membrane and NADPH continued longer than the 1 h that produced the changes shown in Fig. 7. By means of the labeling scheme of Kriebich et al. (22), rough microsomal vesicles were prepared with radioactive label either in intravesicular protein (using a 30-min labeling time, [3H]leucine), intravesicular glycoprotein ([3H]glucosamine), or nascent polypeptide (using a 20-s labeling time, 13Hlleucine). As shown in Table III, lipoperoxidation did not heighten the appearance of nascent polypeptide in the extravesicular medium. However, labels in intravesicular components of rough vesicular preparations appeared in greater amounts outside of the vesicles (i.e., in the supernatant fractions) after lipoperoxidation, reminiscent of the results obtained by Kriebich et al. with microsomes exposed to low concentrations of desoxycholate. Since the lipoperoxidative product malonyldialdehyde is released from peroxidizing membranes and since it has been reported to interact with protein and to inhibit certain test enzymes (37-39), it seems a good candidate as an inhibitor of incorporation. In three systems, rough membranes, free polysomes, and a combination of free polysomes and smooth membranes, up to 50 nmol/ml of freshly prepared malonyldialdehyde had no effect on amino acid incorporation (Table II). Fifty nanomoles of this bifunctional reagent is severalfold greater than that usually produced by 1 mg of membrane with 1 mM NADPH. Additional experiments in which membranes and NADPH were first incubated together at 37°C for 10 min, then centrifuged, and the supernatant buffer used to suspend fresh membranes plus a complete incorporation system resulted in about a

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TABLE PERCENTAGE

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MEMBRANES III

OF Loss OF MEMBRANE AFTER LIPOPEROXIDATION~

Conditions

Labels 13Hlleutine, 30 8

Buffer only NADPH (1 mM1 NADPH + incorporation system

15.7 16.7 20.3

CONSTITUENTS

and labeling 13Hlleutine 30 m$

4.7 21.4 13.8

time 13Hlglucosamine, 30 min 6.5 30.3 19.3

a Rough membranes (1 mg/ml) with label were incubated at 25°C for 30 min in 0.44 M sucrosefTsT,,K,,M,. The mixture was spun in an SW 56 rotor at 50,000 rpm for 45 min. Samples for scintillation counting were taken from the resulting supernatant fluid and from the resuspended pellet. The results are expressed as the percentage of radioactivity of the total recovered disintegrations per minute that occurred in the supernatant fraction.

20% inhibition of liminary evidence inhibitors being released from the lipoperoxidation.

incorporation. This preis consistent with the among the substances vesicles as a result of

DISCUSSION

Lipoperoxidative inhibition of amino acid incorporation by rough microsomal membrane has been found to arise from peroxidative effects on both the polysomes and the soluble incorporation factors. During concurrent incubation of the rough membranes with pro-oxidant and an incorporating system, the inhibitory effect on polysomes occurs early in the incubation, while that on the pH 5 fraction builds up over the course of the incubation. There was no evidence that lipoperoxidation inhibits the energy system used for the in vitro incorporation. Malonyldialdehyde appears to be not responsible for the decline in the pH 5 fraction’s activity. The net inhibition during the first 5 min of the incubation demonstrably did not involve soluble factors. Polysomes located on rough membranes appeared to withstand lipoperoxidation, judging from the acrylamide-gel analysis of rRNA, and from the partial reversibility of the lipoperoxidative inhibition. How-

706

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ever, the membranes subjacent to these polysomes underwent significant changes during peroxidation including changes in the phospholipid implied by attack on polyenoic fatty acids (8, 9, 12-14), the marked decrease in activity of several membrane enzymes, and the escape of intravesicular contents into the extravesicular medium. Lipoperoxidizing membranes inhibited the incorporative activity of those polysomes physically attached to the membrane as well as polysomes which were not. Inhibitory substances generated during peroxidation are apparently able to leave the membrane and interact both in the medium and at the membrane surface. Given the complexity of the chain of lipoperoxidative intermediates (reviewed in Ref. (4) and (1411,the chemical modality of inhibition affecting the free polysomes conceivably might differ from that affecting the bound, and it thus remains presumptive that the free polysome inhibition is due to the same inhibitory intermediates affecting membrane-bound polysomes. Other aspects such as the reversibility of free polysome inhibition also remain unknown because of that population’s lesser stability during incubation in vitro which restricts use of incubation schemes successful with bound polysomes. Lipoperoxidative inhibition of polysome function as it appears in vitro could be viewed as an exaggeration of an ongoing process in uiuo. The susceptibility of polyunsaturated lipids in membranes and the need for constant supply of antioxidant compounds in the cell has long been appreciated. Although peroxidative attack on points of unsaturation is generally observed under experimental conditions of probably global membrane involvement, some kind of a limited but continuously occurring lipoperoxidation might influence function of rough endoplasmic reticulum, without affecting the free polysomes, with the influence perhaps being restricted to individual bound polysomes. In this light, it is interesting to consider the great differences in the amino acid incorporative abilities of free and bound polysomes. The more sluggish activity of bound polysomes, while attributed by

some investigators to a loss or damage of native mRNA (401, has also been observed when intact exogenous polysomes have been artificially bound onto microsomal membranes (15). Previous reports of an incorporation “inhibitor” present on microsomal membrane may be related (41). The fundamental mechanism of protein synthesis by membrane-bound polysomes is presumably the same as that established for unattached polysomes (34). However, in all likelihood, synthesis on rough microsomal membranes in vitro is significantly more limited than that found on the rough endoplasmic reticulum in uiuo. For instance, fragmentation of the rough endoplasmic reticulum that takes place during microsomal isolation perhaps also results in the fragmentation of the attached polysomes, producing polysomes and mRNA or reduced sizes (34). Nevertheless, because of the general similarity of synthetic processit is yet reasonable to look for lipoperoxidative effects in live cells similar to those in vitro. In carbon tetrachloride poisoning the inhibition of protein synthesis occurring concomitantly with lipoperoxidation in hepatocytes is, with appropriate dosage, reversible (42). The results reported here indicate that lipoperoxidative inhibition of amino acid incorporation may involve an intermediate product in the peroxidative chain. Malonyldialdehyde, one of the final products in the chain, has been shown not to be responsible. The argument for an intermediate peroxidative product as responsible for the inhibition rests on, first, the occurrence of the greatest inhibition early in the incubation and its coincidence with the highest rates of lipoperoxidation and, second, on the fact that inhibition is transient. However, in addition to determining the nature of the inhibitory intermediate(s), further investigation should be concerned with the manner in which the inhibitory intermediates accumulate and disperse. ACKNOWLEDGMENTS The author thanks Dr. J. Baron and Dr. T. Tephly for their assistance and support and Dr. L. Poyer and Dr. P. B. McCay for their helpful discussions and suggestions. Technical assistance was

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ably performed by Rolly Glasgow. The work has been supported by a grant from the National Institute for General Medical Sciences (No. GM 12675 10). REFERENCES 1. SHIRES, T. K., KAUFFMAN, S., AND PITOT, H. C. (1974) in Biomembranes (Manson, L. A., ed.), Vol. 5, p. 81, Pergamon, New York. 2. SHIOKAWA, K., AND Poco, A. 0. (1974) Proc. Nat. Acad. Sci. USA 71, 2658. 3. MILCAREK, C., AND PENMAN, S. (1974) J. Mol. Biol. 89, 327. 4. RECKNAGEL, R. 0. (1967) Pharmacol. Reu. 19, 145. 5. SMUCKLER, E. A., AND BENDITT, E. (1965) Biochemistry 4, 671. 6. SMUCKLER, E. A., AND ARCASOY, M. (1969) Znt. Rev. Exp. Pathol. 7, 305. 7. SMUCKLER, E. A., ISERI, 0. A., AND BENDITT, E. (1962) J. Exp. Med. 116, 55. 8. MAY, H. E., AND MCCAY, P. B. (1968) J. Biol. Chem. 243, 2288. 9. FONG, K. L., MCCAY, P. B., POYER, J. L., KEELE, B. B., AND MISRA, H. (1973) J. Biol. Chem. 248, 7792. 10. PEDERSON, T. C., BUEGE, J. A., AND AUST, S. D. (1973) J. Biol. Chem. 248, 7134. 11. BIDLACK, W. R., OKITA, R. T., AND HOCHSTEIN, P. (1973) Biochem. Biophys. Res. Commun. 53,459. 12. MAY, H. E., AND MCCAY, P. B. (1968) J. Biol. Chem. 243, 2288. 13. TAM, B. K., AND MCCAY, P. B. (1970) J. Biol. Chem. 245, 2295. 14. POYER, J. L. (1969) Chain-Scission of Microsoma1 Phospholipids Occurring during the Enzymatic Oxidation of NADPH; Reaction Mechanism and Products, Ph.D. dissertation, University of Oklahoma, Norman, Okla. 15. SHIRES, T. K., EKREN, T., NARURKAR, L., AND PITOT, H. C. (1973) Nature New Biol. 242,198. 16. SHIRES, T. K., EKREN, T., HINDERAKER, P., AND PITOT, H. C. (1974) Biochim. Biophys. Acta 374,59. 17. SHIRES, T. K., MCLAUGHLIN, C. M., AND PITOT, H. C. (1975) Biochem. J. 146, 513. 18. MOYER, G. H., MURRAY, R. K., KHAIRALLAH, L. H., Suss, R., AND PITOT, H. C. (1970) Lab. Znuest. 23, 108.

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19. SHIRES, T. K., NARURKAR, L., AND PITOT, H. C. (1971) Biochem. J. 125,67. 20. RAGLAND, W., SHIRES, T. K., AND PITOT, H. C. (1971) Biochem. J. 121,271. 21. HALLINAN, T., NAGLEY, P., MURTY, C. N., BENNETT, J., AND GRANT, J. H. (1969) Biochim. Biophys. Acta 173, 554. 22. KREIBICH, G., DEBEY, P., AND SABATINI, D. D. (1973) J. Cell Biol. 58, 436. 23. NORDLIE, R. C., AND ARION, W. J. (1964) J. Biol. Chem. 239, 1680. 24. MASTERS, B. S. S., BARON, J., TAYLOR, W. E., ISAACSON, E. L., AND LOSPALLUTO, J. (1971) J. Biol. Chem. 246, 393. 25. NASH, T. (1953) Biochem. J. 55,416. 26. COCHIN, J., AND AXELROD, J. (1959) J. Pharmacol. Ezp. Ther. 125, 105. 27. O’ITOLENGHI, A. (1959) Arch. Biochem. Biophys. 79, 355. 28. MAY, H. E., AND REED, D. J. (1973) Anal. Biothem. 55, 331. 29. WILLS, E. D. (1969) Biochem. J. 113, 315. 30. KAMATKAI, T., AND KITAGAWA, H. (1973) Biothem. Pharmacol. 22.3199. 31. HOCHBERG, J., BERGSTRAND, A., AND JAKOB~ SON, S. (1973) Eur. J. Biochem. 37, 51. 32. WILLS, E. D. (1971) Biochem. J. 123, 983. 33. GLENDE, E. A. (1972) Biochem. Pharmacol. 21, 2131. 34. SHIRES, T. K., AND PITOT, H. C. (1973) Aduan. Enzyme Regul. 11, 255. 35. BONT, W. S., GEELS, J., HUIZINGA, A., MEKKEG HOLT, K., AND EMMELOT, P. (1972) Biochim. Biophys. Acta 262, 514. 36. SHAFRITZ, D. A., AND ISSELBACHER, K. J. (1972) Biochem. Biophys. Res. Commun. 46,172l. 37. CHIO, K. S., AND TAPPEL, A. L. (1969) Biochemistry 8, 2827. 38. ROUBAL, W., AND TAPPEL, A. L. (1966) Arch. Biochem. Biophys. 113, 150. 39. ROUBAL, W., AND TAPPEL, A. L. (1966) Arch. Biochem. Biophys. 113, 5. 40. LESLIE, R. A., AND MANSBRIDGE, J. N. (1970) Biochem. J. 117, 893. 41. SCORNIK, 0. A., HOAGLAND, M. B., PFEFFERKORN, L. C., AND BISHOP, E. A. (1967) J. Biol. Chem. 242, 131. 42. GERHARD, H., SCHULTZE, B., AND MAURER, W. (1970) Virchow’s Arch. Zellpathol. 6, 38.