Molecular and Cellular Biochemistry 62, 121-132 (1984). © 1984, Martinus Nijhoff Publishers, Boston. Printed in the Netherlands.

Inhibitors of cytoplasmic protein synthesis purified from rat liver mitochondria

Jos6 Luis P6rez, Blas Dorta and Nestor Gonzfilez-Cadavid* Departamento de Biologia Cellular, Facultad de Ciencias, Universidad Central de Venezuela, Apartado 21201, Caracas 1020, Venezuela

Summary

Purified mitochondria from rat liver were found to contain protein synthesis inhibitors, that could be extracted by disruption of mitochondrial membranes and fractionated by gel filtration into two fractions of low and high molecular weight. Small size inhibitors were also released from the latter peak by high ionic strength followed by gel filtration. Both types of factors inhibit incorporation of radioactive amino acids into protein by liver cytoplasmic polysomes programmed with endogenous mRNA or poly U, and by rabbit reticulocyte lysates programmed with added globin mRNA and by incubations of Walker carcinoma cells. They decrease to the same level the cytoplasmic synthesis of proteins for the mitochondrial and extra-mito- chondrial compartments in intact cells, but do not appear to inhibit substantially endogenous mitochondrial protein synthesis. Inhibitors were purified by paper chromatography and reverse phase high performance liquid chromatography into fractions which block with the same kinetics the incorporation of [14]leucine and [35]methionine into protein in systems able to initiate protein synthesis, such as reticulocyte lysates or intact cells, but differ in this respect in incubations of liver ribosomes where re-binding of mRNA is a limiting step. Some of these factors behave as oligopeptides that are assumed to inhibit in vitro primarily the initiation stage but whose function in vivo is still undetermined.

Introduction

It is well known that most mitochondrial pro- teins are made on cytoplasmic ribosomes and then transferred to the organelle in a process involving post-translational modifications (1, 2). However, the reverse situation, that is the transport to the cytoplasm of mitochondrially synthesized peptides has not practically been shown, except for two early reports proposing that they might play a role in the regulation of nuclear gene transcription (3, 4), and another one (5) that did not relate them to regulato- ry events. Thus, mitochondrial/cytoplasmic inter- action in protein synthesis, aside from the ATP flow required for some energy-depending steps, ap-

* To whom all correspondence should be addressed.

pears to be a mainly one-sided mechanism directed towards the organelle.

The coordination between protein synthesis in both cell compartments is likely to be mediated by the transfer of regulatory substances able to control transcriptional or translational events outside the organelle. No such factors have been isolated so far, but is is not too speculative to assume that regulato- ry molecules located in the mitochondrial com- partment could under certain conditions modulate the extra-mitochondrial formation of mitochon- drial proteins, or alternatively participate in the overall process of cytoplasmic protein synthesis without selection of any particular class ofsubcellu- lar proteins.

As a preliminary approach to the latter possibili- ty, we show here that purified mitochondria from

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liver and the Walker carcinosarcoma of the rat contain low molecular weight inhibitors of in vitro cytoplasmic protein synthesis, that are partially bound to macromolecules inside the organelle and that when added to intact cell incubations inhibit to the same extent the synthesis of mitochondrial and non-mitochondrial proteins in the cytoplasm.

Materials and methods

Materials

L-U-[14C]leucine (sp. radioactivity 300 mCi/ mmol), L-G-[3 H]leucine (sp. radioactivity250 mCi/ mmol), and DL-3-[14C]phenylalanine (sp. radio- activity 50 mCi/mmol) , were purchased from the Commissariat a l'Energie Atomique (CEA), Gif- sur-Yvette, France. L-[3SS]methionine (sp. radio- activity 1 255 Ci /mmol) was purchased from Amersham Intern., Buckinghamshire, England. The rabbit reticulocyte lysate treated with micro- coccus nuclease was initially prepared according to Pelham and Jackson (6) and later obtained from BRL, Taithersburg, Maryland, U.S.A. Insoluble trypsin, RNAse-free pancreatic DNAse, RNAse T1, dithiothreitol, cycloheximide, D-threo chloram- phenicol, L-leucine, polyuridylic acid (type II), Sephadex G-50 and 15 (fine), L-amino acids and cytochrome c (type III), were from Sigma Chemical Co., St. Louis, Missouri, U.S.A.

Cell culture media were f rom GIBCO, New York. Trypsin 1:250 (frog pancreas) was from 1CN Pharmaceuticals Inc., Cleveland, Ohio, U.S.A. Twice distilled water was used throughout. All oth- er reagents were as indicated by Carmona and Gon- zfilez-Cadavid (7).

Animals

Female Sprague-Dawley rats (150 + 10 g body wt) were used for liver excision and Walker carci- nosarcoma propagation. The solid subcutaneous carcinoma was maintained as described (8) for 8 days. When ascites cells were required, a 1-2 g fragment was washed with Earle's solution, minced, and 0.3 ml of the thick suspension was implanted in the intraperitoneal region. Rats were killed at the 3rd or 4th day, the peritoneal fluid (approx. 25 35 X 10 6 cells/ml) was aseptically removed, 0.3 ml

were re-injected intraperitoneally, and the process repeated to obtain a two-passage ascites fluid.

Isolation of mitochondrial inhibitory factors

All operations were carried out at 0 2 ° C. Tho- roughly purified mitochondria were obtained from rat liver by applying essentially the procedure of O'Brien and Kalf(9) on a homogenate prepared in 6 ml of 0.34 M-sucrose, 2 mM Tris/HC1 buffer, pH 7.4 at 20 ° C, per g of fresh tissue, using 6-8 strokes with the Potter-Elvehem homogenizer (clearance : 0.13 0.18 mm). A mitochondrial fraction with mi- nor contamination by endoplasmic reticulum fragments was obtained from the Walker carcino- sarcoma following the method of Gonzfilez-Cadav- id and P6rez (I0, 11) on a homogenate prepared in2 ml of 0.25 M sucrose, 2 mM Tris/HC1 buffer, pH 7.4 at2 ° C, 1 mM EDTA, perg of fresh tissue, using 20 strokes with the same homogenizer, and finally collecting the '4-6 ' mitochondrial fraction from the sucrose gradient.

The crude mitochondrial inhibitors were isolated from the purified mitochondria suspended in 10 mM Tris/HC1 buffer, pH 7.4 at 2 ° C, at 0.1 ml /g of liver or 0.05 ml /g of carcinoma, and ultrasoni- cated twice for 30 sec at approx. 5/~ amplitude. The suspensions were left standing for 15 min and then centrifuged for 30 min at 40 000 g. The clear super- natant was named MF and samples of up to 3.5 ml (50 mg protein) were submitted to gel filtration through a refrigerated Sephadex G-50 column (74 ml bed volume ; 30 cm height) equilibrated with 10 mM Tris/HC1 buffer, pH 7.4 at 2 ° C. Elution was carried out with the same buffer, collecting frac- tions from the moment the sample was put on to the column. The inhibitory activity was assayed as des- cribed below and pools of the active fractions were named MFI for the first, high molecular weight, peak, and MFII for the second, low molecular weight, peak.

Purification of mitochondrial inhibitors

Fraction MFI was lyophilized, resuspended in 2.5 ml water, clarified by centrifugation in the Ep- pendorf centrifuge for 3 min, brought to 0.1 M NaC1 with 5 M NaC1, left at 0 °C for 15 min and then submitted to gel filtration under the same con- ditions as above or with 0.1 M NaC1, 10 M

Tris/HC1, pH 7.4 at 2 ° C as the elution buffer. The inhibitory activity was assayed, and the peak at the exclusion zone was pooled and designated M F I - H - SII.

Fraction M F I - H S I I (gel filtrated without 0.1 M NaC1 in the elution buffer) was lyophilized, retaken in 200 /~1 of water and submitted to descendent chromatography on 40 × 46 cm Whatman paper No. 1 with n-butanol/acetic acid/water (12:3:5) as mobile phase (12), using 10 #1 spots per lane. Ami- no acid mixtures were run as standards. One lane of each kind of sample and of the amino acid stand- ards was routinely stained with ninhydrin (13), and, when indicated, with ammoniacal silver nitrate (14) and aniline hydrogen phthalate (15) for sugars, I 2 vapours for polysaccharides and insaturated lipids (16), molybden oxide for phosphatides (17), Sudan I lI for lipids (18) and fluorescein for nucleotides and derivatives (19). Direct inspection of unstained lanes with UV light was also applied for the latter.

The other lanes were cut along the length of the paper into 0.5 cm pieces and submitted to two rounds of elution with 1 ml of water, first overnight at 4 ° C and then for 4 hr at room temperature. Each pooled extract was separately tested for the inhibi- tion of protein synthesis, either as such or after a 10-fold concentration by lyophilization. Pools of active fractions were made as indicated in the 'Re- sults' section and designated MFI-HSII (E) or MFI- HSII(F) , respectively. Each pool was lyophilized again, retaken in 100-200 Fzl water and 50 100 #1 used for reverse phase high-performance liquid chromatography ( R P - H P L C ) in a Varian Instru- ments unit, model 5060, on a Micropack MCH-10 ODS, 25 cm column (Varian Instruments), with 0.1% trifluoroacetie acid in a linear 0-66% acetoni- trile gradient in water (20). Flow rate was 1 ml/min, with a total run time of 60 rain, and elution was checked by recording absorption at 210 nm. Frac- tions were collected every 30 sec and brought to dryness in a Buchler Vortex Evaporater at 40 ° C. In instances when only the first 50 fractions were ana- lyzed, a lyophilizer was used for concentration.

Assay of inhibitory activity on protein synthesis by cytoplasmic ribosomes

Polysome-enriched detergent-treated ribosomes (C-ribosomes) were isolated from rat liver as pre-

123

viously described (7) and a similar procedure was applied for the preparation of ribosomes from the Walker carcinosarcoma, but in the absence of de- tergent. Results obtained in this laboratory had shown that the majority of the polysomes in this tumor are free in the cytoso], and that treatment with deoxycholate to isolate them jointly with the population originally bound to endoplasmic reticu- lum membranes, decreased considerably their pro- tein synthesizing activity. The pH 5 fraction was obtained by the method of Falvey and Staehelin (21) at a final concentration of approx. 25 mg pro- tein/ ml in a buffer containing 60 mM Tris/HC1, pH 7.6, 100 mM KC1, 10 mM MgC12, 0.5 mM EDTA, 2 mM dithiothreitol. Assays of radioactive amino acid incorporation into protein were carried out by the method of Schreier and Staehelin (22) with minor modifications, by incubating ribosomes at 250 #g r R N A / m l in 25 #1 of a medium containing : 1.4 mg/ml liver, pH 5 fraction protein (unless indi- cated), 67 mM K +, 6 mM Mg 2+, 0.2 mM EDTA; 1 mM dithiotreitol, 30 mM Tris/HC1, pH 7.6, 10 ~ l /ml glycerol; 20 mM NH4 +, 2 mM ATP, 10 mM phosphoenolpyruvate; 0.25 mM GTP, 25 U /ml pyruvate kinase, L-amino acids (18 amino acids, 0.03 mM each, no leucine) and 0.4 0.9 #Ci /ml of [14C]leucine (35-75 pmoles). Contribution of endo- genous leucine by the pH 5 fraction was 92 pmoles in the 25 ~1 assay.

For the different assays, 2.5 #1 samples of the fractions or suitable dilutions were used. In the liver ribosomal system we added 7.5 #1 of a master mix 'A', containing the energy source and amino acids, and then 15 #1 of a master mix 'B ' with the subcellu- lar constituents. Incubations were carried out at 30 ° C for 40 min.

The assay of exogenous natural mRNA transla- tion was carried out with the nuclease-treated rab- bit reticulocyte lysate programmed with 50 ng of globin m R N A in 15 ~1 incubations containing 7 ~Ci /ml [14C] leucine (350 pmoles) or 5 ~zCi/ml [35S]methionine for 60 min at 30 ° C, as described by the suppliers. Endogenous leucine was calculat- ed to be 160 pmoles in the 15/~1 assay.

The assay of synthetic polynucleotide translation was carried out using ' run off' ribosomes (22) and the same system as for endogenous m R N A transla- tion, except for the following: a) poly U was added at 0.25 mg/ml; b) Mg 2+ concentrations was raised

124

to 12 raM; c) [14C]phenylalanine (0.5 #Ci/ml) was substituted for [14C]leucine; d) rat liver initiation factors were added at 20 mg protein/ml.

The incorporation of radioactive amino acids was stopped by cooling down to 0 ° C and adding 2 #1 of the non-radioactive precursors in 10 mg/ml solutions.

Assay of inhibitory activity on protein synthesis by whole cells

For the isolation of the ascites form of the Walker carcinoma, the peritoneal fluid was re- moved aseptically in the presence of 5 U/ml of Na heparine, cooled down to 0 ° C and submitted to a slight hypotonic shock to lyse the erythrocytes by diluting 2.5-fold with distilled water during 1 min. All operations thereafter were carried out at 2 4 ° C. Isotonicity was restored with 0.1 vol ofN NaCI in 1 vol of Earle's medium, and the cells sedimented at 80 gav for 5 min. After One washing by gentle resus- pension in Earle's solution and centrifugation, the pellet was suspended in fresh Earle's medium and viable cell concentration (trypan blue-excluding cells) adjusted to the appropiate concentration of l X 10 6 cells/ml. Each ml of ascites fluid gave 2.5-3.0 X 107 cells. These cells were used for the incubations where the inhibition of radioactive amino acid and incorporation into protein was studied, either as such or maintained in suspension culture as an established cell line in Dulbecco's modified Eagle medium with 5% calf serum.

Cell incubations for total protein synthesis were carried out in 1.5 ml Eppendorf microcentrifuge tubes in a rotatory shaker at 37 ° C for 90 min in a 5% CO 2 atmosphere in a leucine- or methionine- free medium with dialyzed serum, using cells washed three times with the same medium. The 0.1 ml samples contained 105 cells, and either 0.2 #Ci of [14C]leucine or 0.1 #Ci of [3SS]methionine and the incorporation of radioactivity was stopped after 60 min by adding 0.5 mg of L-leucine or L-methionine and cooling to 0 ° C. Endogenous leucine in the 105 cells during incubation was 2 500 pmoles.

In a few instances where the mitochondrial and post-mitochondrial fractions were separated, incu- bations were carried out at 0.4 × 10 6 cells/ml in Spinner flasks with a total volume of 20 ml. Subcel- lular fractionation of the washed cells after incuba- tion was carried out as described (11).

RadioactiviO, measurements

Samples were plated onto 2.4 cm glass fibre discs, air-dried and treated for 2 3 hr with 10% w/v trich- loroacetic acid containing 0.25 mg/ml leucine or methionine. The discs were then heated for 15 min at 90 ° C in 5% w/v trichloroacetic acid, washed with the same solution at room temperature, then with ethanol/ether (3/1), and ether, and finally counted for radioactivity in vials with 2 ml of a scintillation liquid containing 0.4% of PPO (2,5-di- phenyloxazole) and 0.005% of POPOP (1,4-bis-(5- phenyloxazol-2-yl) benzene) in toluene. Efficien- cies for [J4C] and for [35S] were 80%.

The precipitates from the trichloroacetic acid- treated suspensions of subcellular fractions from incubated cells were washed twice with 5% trichlo- roacetic acid and suspended in 0.1 ml of formic acid, transferred to vials and counted for radioac- tivity with 5 ml of a similar scintillation mixture in 60% toluene-40% Triton X-100. Efficiencies for double labeling counting were 60% [lac] and 27% [3H].

Miscellaneous determinations

Amino acids analysis on ion-exchange columns was performed on lyophilized samples, taken up in pH 2.2 buffer and chromatographed on a Beckman Multichrom liquid column chromatograph 4 255. Nucleotide analysis was carried out in lyophilized samples dissolved 0.1 M-KH2PO4, pH 3.35 and chromatographed on a Varian Aerograph Liquid Chromatograph LCS-1 000 with pellicular anion exchange resin. Specific conductance determina- tions were done in a Varian Techtron. Model 1 000, Atomic Absorption Spectrophotometer, using 10 mM KC1 as standard and the mEq/1 of total ions was calculated by multiplying this value in #mhos/ cm by a factor of 10 2 (23). The concentration of Mg 2+ and Ca 2+ was determined in supernatants from 5% w/v trichloroacetic acid treatments, in the presence of 65 mM KC1 and 19 mM LaC13. Blank values for 10 mM Tris were subtracted in these estimations, as well as in those of specific conduc- tance. All protein assays were done by the method of Lowry et al. (24).

Results

Preparation of extracts from rat liver mitochondria with inhibitoo' activity on protein synthesis by cy- toplasmic ribosomes

The main aim of this work was to detect and isolate mitochondrial factors that could be easily diffusable through the inner and outer membranes and affect protein synthesis in the cytoplasm. Therefore, we decided to concentrate our search on extracts obtained from purified organelles by a mild release based on hypotonic shock and ultra- sonication. The purity of the mitochondrial frac- tion from rat liver was assured by using an isolation procedure already employed for the preparation of mitochondrial ribosomes (9) and matrix proteins (Casanova and Gonz~lez-Cadavid, unpublished re- sults), and it was substantially free of cytoplasmic contaminat ion as tested by enzymic markers and electron microscopy (ibid.). The soluble extract from the high speed centrifugation of the disrupted mitochondria should contain any putative diffusa- ble component together with other matrix constitu- ents but no membrane fragments. In a series of 7 separate preparations the average mitochondrial yield was 2.3 mg prote in/g of liver and the soluble protein amounted to 64% of the total mitochon- drial protein submitted to extraction, giving an ex- tract with a protein concentration of 6 to 15 rag/ml.

Preliminary experiments showed that these ex- tracts inhibited protein synthesis by rat liver cyto- plasmic ribosomes by more than 90%, both with endogenous mRNA or programmed with poly U, and that this activity was heat-stable at 90 ° C for 20 rain; did not precipitate with 10% trichloroacetic acid or 90% saturation with (NH4):SO 4 and was partially retained by dialysis against the extraction buffer. These extracts were routinely fractionated by gel filtration on Sephadex G-50 columns equili- brated with the same buffer into two peaks of inhib- itory activity. Figure 1A shows the profile of a typical experiment. A first fraction comes out with the void volume and was named MFI (for 'mito- chondrial factors'), and a second broader peak emerging with a Kay of-----0.9 was designated MFII .

Each individual fraction having higher than 50% inhibitory activity was assayed at several dilutions and then pooled to constitute MFI and MFII . These pools were also tested in the same assay (Fig.

125

1B, insert). The plot of % inhibition against the volume of MF added showed that the IAs0 (amount inhibiting protein synthesis by 50% in the 25 #1 incubations) for MFI was equivalent to 4.4 #g of total mitochondrial protein submitted to extraction (2.7 #g soluble protein) in a test system having 120 #g of pH 5 fraction protein and 25 #g of ribo- somal protein. For MFII (protein-free fraction), the IAs0 was 1.7 #g of mitochondrial protein sub- mitted to extraction.

Using this information, the profiles shown in Fig. 1A could be recalculated as units of inhibitory ac- tivity present in each fraction. The resulting curves (Fig. IA, broken line) show narrower peaks and it can be readily noticed than MFI appears to have half the activity of MFII . In fact, it could be calcu- lated that 70% of the inhibitory activity was found in MFII . However in other fractionations the dis- tribution obtained was about the same for MFI and II. Storage of MFI and MFII at -20 ° C for several months did not decrease activity of MFII , but the irreversible precipitation of some proteins in the case of MFI diminished activity after clarification by centrifugation.

The fact that the inhibitory substances present in MFI do not behave like macromolecules when heated or deproteinized implies that the activity can be released from a loose association with the large constituents initially separated by gel filtration. To test this hypothesis, samples of clarified MFI were brought to 0.1 M NaC1 and submitted to gel filtra- tion through Sephadex G-50 maintaning the high salt concentration. The inhibitory activity shifted to a zone of K d ~0.9, with less than 5% of the original units remaining in the exclusion peak. Figure I B shows the results of a typical experiment, where the whole MFI fraction was used for the preparation of the 'high salt' factors behaving like MFII and de- signated MFI-HSII . About 60% of the initial activi- ty of MFI was recovered. Identical results were obtained by heating MFI at 90 °C for 30 min and submitting it to gel filtration through Sephadex G-50 under the usual low ionic strength conditions. The molecular weight of the factors present in MFII or MF1-HSII , could not be established by gel filtration on Sephadex G-15 and G-10 because of absorption effects that retarded elution. The use of solvents know to counteract these interactions was ruled out, due to interference with the protein syn- thesis assays applied for detection.

126

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Fig. 1. Fractionation by gel filtration of mitochondrial factors with inhibitory activity on protein synthesis. A. An extract (4.8 ml) containing 6.1 mg/ml of mitochondrial soluble protein was submitted to gel filtration as described in the text and each 1.8 ml fraction was tested on protein synthesis by liver cytoplasmic ribosomes with [14C]leucine. The inhibitory activity was calculated from a plot where suitable dilutions of MF were assayed to determine the IAs0, one unit defined as the amount of MF necessary to inhibit by 50% amino acid incorporation into protein by liver cytoplasmic ribosomes (see insert in B). B. Peak M FI was collected and a 1 / 2 aliquot was treated and resubmitted to gel filtration with 0.1 M NaCI, 10 mM Tris, pH 7,4 as indicated under 'Methods'. O - ~ O : % inhibition of radioactivity incorporation; O -O: inhibition expressed in IAs0 units. 1 nsert: 1 #1 of solution is equivalent to the following amounts of mitochondrial protein submitted to extraction: 4.6 #g (MFI: O - - O ) and 2.6/~g (MFIh z~--z~), respectively.

The dissociation of M F I - H S I I appears to be at least partially reversible because when gel filtration of MFI after treatment with 0.1 M NaC1 was car- ried out under low ionic strength conditions, nearly 20% of the original activity remained in the exclu- sion peak. However, no reassociation took place between MFII and 'stripped' MFI by pooling them and lowering the ionic strength to 10 mM.

The inhibitory activity of MFII on the incorpora- tion of [laC]leucine can be attributed only to a partial extent to unspecific factors such as dilution by the non-radioactive amino acid present in MFII (40 pmol/unit) , as shown by the facts that a) the 25 #lliver polysomal incubation contains 75 pmoles of added leucine and 92 pmoles of endogenous

leucine, as determined by isotopic dilution, which would give only 20% and 32% inhibition for 1 and 2 units added, respectively, against the 50 and 69% actually observed; and b) the constant inhibition achieved by increasing amount of pH 5 fraction (150-500 pmoles of L-leucine/25 ~1 assay, as de- termined also by isotopic dilution) or unlabeled leucine added up to 400 pmoles. Neither deleterious amounts of other molecules were present, such as ADP, ATP or other nucleotides (excluded by liquid chromatography), Mg 2÷ and Ca 2+ (determined by absorption spectrophotometry), heavy metals (EDTA chelation), or total ions (measured by spe- cific conductance). However M F I - H S I I was chos- en for most further studies because it should be

practically free of contaminating small size com- pounds and the parent fraction (MFI) does not exhibit ribonuclease activity, as shown by the lack of effect on the polysome profile. Its inhibitory activity was not substantially affected by digestion with DNAse (100 #g/ml) , insoluble trypsin (6 rag/ml), or HCI(1 N, 1 hr, 100 °C). Treatment with NaOH (1 N, 17 hr, 37 °C) decreased activity by 4O%.

Resolution of two different types on inhibitoo' ac- tivity by paper chromatography

Both MFII and MF1-HSII were concentrated and submitted to paper chromatography, using some of the lanes for detection tests and the re- mainder for fractionated elution all along the direc- tion of the run. MFII behave similarly to MFI-H- SI1 during fractionation, but to simplify the analysis results considered here are restricted to the more pure fraction (MFI-HSII ) . Figure 2 shows that at least six major spots are visible with ninhy- drin. When each eluted fraction from a pool of the lanes not submitted to detection tests, was assayed on the incorporation of [14C]leucine or [35S]methi- onine into protein by the liver cytoplasmic poly- somes, the inhibitory activity recovered (240% in the sum of both fractions) was restricted to two zones. One was slightly advanced to the fastest ninhydrin-positive spot, always running faster than leucine and often ahead of isoleucine. It was desig- nated fraction 'F' . The second zone was in a region coinciding with a ninhydrin-positive spot, and it generally ran slightly behind methionine. It was designated fraction °E'. Concentrated eluates form- ing each fraction were shown to inhibit the incorpo- ration of both radioactive amino acids. Upon l0 100-fold dilution, fraction 'F ' tubes were still inhibitory for [J4C]leucine, but not for [35S]methio- nine, whereas the reverse occurred with the eluates in zone 'E'.

Active eluates were pooled according to the re- gion they belonged into fractions MFI-HSII(E) , or MFI-HSII (F) , respectively. Assay of their inhibito- ry activity with increasing dilution of the pools confirmed that at high concentrations fractions 'F ' and 'E ' inhibited the incorporation of both radioac- tive amino acids into protein by liver cytoplasmic ribosomes (Fig. 3A and B), whereas upon dilution the inhibition of [14C]leucine incorporation was

127

restricted to fraction 'F ' (Fig. 3A), and that of [35S]methionine was found only in f ract ion 'E ' (Fig. 3B). The liver cytoplasmic ribosomes used in the in vitro assays are not however an adequate system for testing unknown inhibitors of protein synthesis, because those acting on the initiation stages will not be readily characterized in a system very inefficient in re-binding endogenous mRNA or forming the initiation complex. It is most likely that this test is not sensitive enough for accurately following a primary effect on initiation at low concentration of the factors, but is useful for detecting a secondary effect discriminating between amino acids, such as for instance the charge of specific tRNAs.

To test this hypothesis we repeated the determi- nations using the rabbit reticulocyte system pre- treated with micrococcus nuclease to eliminate en- dogenous mRNA, and re-programmed with added globin mRNA, which is a highly efficient initiation and elongation system of protein synthesis. Now, f rac t ion 'F ' inhibited with identical alope the incor- poration of [14C]leucine (Fig. 3A) and [35S]methio- nine (Fig. 3B). The IAs0 was absolutely identical for both amino acids in each case, when expressed as the amount of mitochondrial protein submitted to extraction to yield the factors able to reduce by 50% the incorporation of either radioactive amino acid in each 15 ~1 assay (116 #g). Fraction 'E' behaves in a similar fashion regarding the slopes of inhibitory activity on the incorporation of either amino acid, with IAs0 of 45 #g in the case of [35S]methionine (Fig. 3B). The higher sensitivity of the latter may be due to the secondary effect mentioned above.

Further purification by reversed-phase high-per- formance liquid chromatography

Fractions 'E' and 'F ' contain ninhydrin-positive substances which were tentatively assumed to be small peptides or amino acids. The presence of other substances, at the level of detection of the different procedures applied to the paper chroma- tography separation, was excluded by the proce- dures described under 'Materials and methods'. The UV spectrum was identical for the two frac- tions, with no prominent .peak at 205 nm and no significant shoulders visible at either 280 or 260 nm. On re-chromatography in the same system frac- tions 'E ' and 'F ' ran essentially as before but some minor spots were also detected with ninhydrin sug-

128

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' Fig. 3. Inhibitory effect of mitchondrial factors on [J4C]leucine and [35S]methionine incorporation into protein by liver polysomes and rabbit reticulocyte lysates. Assays were performed with MFI-HSII as described in the text. A: [lnC]leucine; B: [35S]methionine; A - - A : fraction 'F', reticulocyte lysate; A - - - ~ : fraction 'F', liver polysomes; • --: fraction 'E', reticulocyte lysate; O O: fraction 'E', liver polysomes. 1 ~1 of solution is equivalent to 640 #g of extracted mitochondrial protein.

129

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Fig. 4. Fractionation of mitochondrial inhibitory factors by RP-HPLC. Inhibitors extracted from 30 mg mitochondrial protein were fractionated by paper chromatography and then analyzed by RP-HPLC. A: MFI-HSll(F); B: MFI-HSII(E). Arrows indicate the position where the peak of inhibitory activity was found.

gesting partial degradation or dissociation occur- ring during this process.

For further purification we chose RP-HPLC on an octadecenyl column as a procedure particularly suitable for fractionation and recovery of peptides smaller than 18-20 residues (25). Elution times are the result of the algebraic contribution of the reten- tion coefficients of each constitutive amino acid. Both the free amino acids and the small polar pep- tides come out in the first 1/3 of the gradient. Figure 4 shows the actual elution profiles at 210 nm restricted to the initial 30 min, since no active peak was detected in the remainder fractions. Zones 'E' and 'F ' differ in their patterns, with a very distinc- tive peak at 17 rain in 'F ' , which is absent in 'E' , and conversely, one in 'E' at 13 rain not present in 'F', plus some other differences.

When each eluted fraction was concentrated and tested on the liver ribosomal system, there was a main peak of inibitory activity at 12-13 min in fraction MFI-HSII(F) not strictly corresponding to any peak of optical absorption, and another one at 8 min in fraction MFI-HSII(E) in-between two peaks in the 210 nm profile. The inhibitory activity was detected with both [14C]leucine or [35S]methionine, but upon dilution peaks of fractions 'F ' and 'E' discriminated between both amino acids. However, as in the case of the fractions prior to RP-HPLC, the purified peaks were equally inhibitory on the incorporation of both amino acids in the reticulo- cyte lysate.

Paper chromatography of the most active RP- HPLC eluate of fractions 'F ' showed a single nin- hydrin-positive spot running coincidently or slight- ly ahead of isoleucine or nor-leucine (Fig. 5). Upon 24 hr hydrolysis in 6 N HC1, there was a spot slightly retarded to that of the non-hydrolyzed sample, and three to four minor spots moving in the regions of alanine, threonine, arginine and lysine. In the case of the RP-HPLC peak of fractions 'E' no spots could be detected with ninhydrin, despite the fact that 12 units of IAs0 were applied to the chromato- gram. The inhibitory activity present in fraction 'F ' or in its RP-HPLC peak was reduced bye60 to 75% after hydrolysis.

Inhibition o f o, toplasmicprotein sI'nthesis in whole cells

All fractions prior to RP-HPLC purification were tested for their ability to inhibit the incorpora- tion of radioactive amino acids into protein by intact cells from the Walker carcinoma with two main purposes: to demonstrate that the true effect on protein synthesis observed in vitro in the reticul- ocyte lysate occurred also in vivo, and to determine whether the factors were active on mitochondrial protein synthesis. Preliminary assays had shown that in spinner flaks MFII inhibited the incorpora- tion of[14C]leucine into protein with essentially the same pattern as in the case of cell-free systems.

130

A) Before Hydrolysis

Nor-Leu t Ile

It-- lw Lou t

Vo~l Mot

o,,, tl, Tflr la

Glu i OH-Pro .,, Gly ~ f • ~A$p ~ , ~ _Ser v ~pr=

His Lys

~ p Cys Cys- Cys

• • • • •

Standards

B) After Hydrolysis

Nor - Lou

• •

25 26

MFI-HSTT

to t6tl,."l

e |

# o'° I O ~ Cys

Cys-Cys

25 26

MFT.-HS~. Standards

Fig. 5. Schematic representation of ninhydrin-positive substances detected after paper chromatography of the mitochondrial inhibitory factors of MFI-HSII(F) separated by RP-HPLC. Samples from individual fractions collected after RP-HPLC were either left as such or submitted to hydrolytic treatment in 6 N H CI, and then analyzed by paper chromatography as described in the text. Spots obtained after ninhydrin staining are represented according to their actual positions and shapes but no indication of their relative color intensity is shown here. A dotted line joins the centers ofleucine spots. Zone E was submitted to the same analysis but no ninhydrin-positive spots were visible.

When the tests were repeated in 0.1 ml samples no discrimination was observed for fractions 'E' and 'F ' from MFI-HSII . This confirmed the results ob- tained with the reticulocyte lysate, thus showing that in systems efficient in initiating protein synthe- sis there is a clear inhibition of the process, with little, if any, secondary effect on aminoacylation of tRNAs or other pre-translational steps. However, intact cells are much less sensitive to the inhibitors, with an IAso 20-fold lower than in the reticulocyte assay. This decreased activity is partly due to a mere dilution effect by the higher volume ratio (100/15)

of the respective assays, but most likely reflects the membrane barrier absent in the reticulocyte lysate.

In intact cells there was no preferential effect of the mitochondrial inhibitors on the cytoplasmic synthesis of mitochondrial proteins in comparison to the remainder cell proteins, as shown with MFII rather than MFI-HSII , due to the cell numbers incubated which demanded too large amounts of inhibitors. 107 Walker carcinoma cells were incu- bated in spinner flasks (20 ml volume) with increas- ing concentrations of MFII for 60 min, with 5 #C of [3H]leucine, and the post-mitochondrial and mito-

chondrial fractions were obtained. From 90 to 95% of the proteins present in the purified mitochondria are synthesized in the cytoplasm, so that the ra- dioactivity found in this fraction can be taken as representative of the mitochondrial protein popula- tion made on cytoribosomes. No significant differ- ence was found between the inhibition of the label- ing of both this fraction and the total cytoplasmic proteins. On the other hand, preliminary experi- ments where the contr ibution of cytoplasmic pro- tein synthesis was blocked with cycloheximide, in- dicated that MFII did not appear to inhibit mito- chondrial protein synthesis at concentrations that reduce cytoplasmic protein synthesis by 70 80%.

Mitochondria isolated from the Walker carci- noma cells (55% of soluble protein) contain what appears to be the same inhibitors extracted from liver mitochondria, although in lower amounts, particularly when MFII is considered. Results from three differente experiments gave M F I I / M F I values of 0.6 against 2.7 for liver. The effect of M F I - H S I I on the incorporation of [t4C]leucine in

vitro by cytoplasmic polysomes from these cells is similar to the one observed for liver ribosomes.

Discuss ion

Our results show that purified mitochondria from at least two types of tissues, liver and Walker carcinosarcoma, contain substantial amounts of soluble compounds that can inhibit protein synthe- sis by cytoplasmic ribosomes from both origins, intact cell incubations, and rabbit reticulocyte ly- sates. These substances are presumably located in the matrix, since they can be extracted by hypoton- ic shock in a low ionic strength buffer followed by ultrasonication, working at 0-2 °C to minimize proteolytic attack. Contribution by other subcellu- lar structures can be ruled out from the fact that liver mitochondria were obtained by an exhaustive purification procedure devised for the isolation of mitoribosomes free from endoplasmic reticulum contaminants. If the case of the Walker carcinoma mitochondria, the procedure used (11) gave a rea- sonably purified fraction. Experiments are in pro- gress to determine with more sensitive procedures whether small pools of similar inhibitors can be found in other cell locations, but their considerable concentration in mitochondria allows us to con-

131

clude that they are mainly, if not exclusively, origi- nated in the organelle.

The behaviour in gel filtration after heating, or raising the ionic strength, indicates that all of these inhibitors are of low molecular weight, but that when freshly extracted they are partially associated with macromolecules present in the so-called MFI peak. We have not yet been able to demonstrate that reassociation can take place, but it is not un- likely that a reversible binding to large molecules could provide a physiological mechanism for regu- lating the intra-mitochondrial amount of free inhib- itor and any subsequent passage through the orga- nelle membranes. Some features, such as size, spectrum and partial absorption to Sephadex and behaviour in paper and high performance liquid chromatography, suggest that at least part of the bound inhibitory factors in MFI are essentially the same as some of those extracted as MFII . When released as M F I - H S I ! they constitute a much purer fraction than MFII , due to the isolation procedure where most of the small molecular weight contami- nants are separated during the first gel chromatog- raphy. Particular care was exerted to rule out the presence of a pool of unlabeled amino acids which could introduce isotopic dilution artifacts. The fact that upon hydrolysis about 2/3 of the inhibitory activity is eliminated, and the comparison of RF values of the active zones both before and after hydrolysis, indicate that we are dealing with small molecules that are different from the amino acids normally present in proteins.

At the high level of purification we achieved, the usual staining procedures for paper chromatog- raphy, and the very high sensitivity recording at 210 nm in liquid chromatography are insufficient for following up factors that can only be detected by their effect on the incorporation of radioactive amino acids into protein. Therefore, at this stage we can only assume that one of the two paper chroma- tography fractions (F) appears to contain a small peptide, but this remains to be proved more conclu- sively with analytical procedures of higher sensitivi- ty. The active factors, irrespective of whether they are in fraction 'F ' or 'E ' , appear to inhibit primarily the initiation of translation as shown by the very similar effects on the incorporation of either leucine or methionine into protein in systems able to re-ini- tiate protein synthesis, as opposed to the poor level of this kind of activity found with a system (liver

132

r ibosomes) practically restricted to elongat ion. Oli-

gopept ides have been isolated f rom calf thymus

nuclei that, similarly to what we observed with our

purified fractions, can inhibit ini t iat ion of transla-

t ion in the rabbit reticulocyte lysate (26).

The discr iminat ion by fractions 'E ' and 'F ' at

med ium and low concent ra t ions on the inhibi t ion

in vitro of the incorpora t ion of methionine and

leucine into protein by liver r ibosomes, not iceable

in the absence of ini t iat ion seems to be a secondary

effect perhaps at the level of cytoplasmic aminoacy-

lat ion of t R N A . It is possible that, at least in the

case o f ' F ' , the putat ive small peptide would inhibit

compet i t ively this react ion due to a const i tuent

amino acid analog which in itself could carry some

activity, thus expla ining the small residual effect

surviving t rea tment with 6 N HC1 at 100 ° C. If this

type of inhibi t ion occurs, it should be impor tan t to

determine whether it is related in vivo to pre-trans-

lat ional regulatory events (27).

The fact that in incubat ions of intact cells, at least

one class of mi tochondr i a l factors at the initial

stage of pur i f ica t ion ( M F I I ) does not affect prefer-

ential ly the fo rma t ion in the cytoplasm of a specific

protein popula t ion to be imported in the organelle,

or does not appear to decrease endogenous mito-

chondr ia l prote in synthesis, suggests that if those

inhibi tors play indeed a role in the in vivo regula-

t ion of pro te in synthesis it has to be on the overall

rate of cytoplasmic protein synthesis rather than on

the modula t ion of protein product ion for the mito-

chondria . The demons t ra t ion that any of the more

highly purified inhibitors part icipate in this type of

control requires their quant i ta t ive de te rmina t ion in

the cy top lasm under exper imenta l condi t ions that

would indicate a physiological t ransport through

the mi tochondr ia l membrane. Exper iments are in

progress to find them in the cytosol among the large

amoun t of small molecules potential ly able to affect

pro te in synthesis in the tests with ret iculocyte ly-

sates, by specific isolat ion through affinity binding

to immobi l ized macromolecules such as the ones

present in the M F I fraction.

Acknowledgements

The coopera t ion of Dr I tala Becemberg in the

nueleotide analysis, and of Miss Eneida R o m e r o in

the Mg 2+ and Ca 2+ est imations, are gratefully ac-

knowledged. This work was supported by grants

f rom the Consejo de Desar ro l lo Cientif ico y Hu-

manlst ico of the Univers idad Central de Venezuela and f rom C O N I C I T .

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Received in revised form 14 November 1983.