‘TII~: Jou~rn~ OF I~IOLOGIC~L CHEMISTRY Vol. 2.13, No. 9, Isvue of May 10, pp. 3220-3227, 1973

Printed in U.S.A.

Translation of Liver Messenger Ribonucleic Acid in a

Messenger-dependent Reticulocyte Cell-free System

PROPERTIES OF THE SYSTEM AND IDENTIFICATION OF FERRITIN IN THE PRODUCT*

(Received for publication, October 30, 1072)

DAVID A. SHAI~~RITZ, JAZZES W. DRYSDALE, AND Nuns J. ISSELBACHER

From the Departments of Medicine, Harvard Medical School and Massachusetts General Hospital (Gastroin- testinal Unit), and the Department of Biochemistry and Pharmacology, Tufts Ukversity School of Medicine, Boston, Massachusetts O,S’II4

SUMMARY

An RNA fraction with messenger activity has been isolated from free polysomes of rabbit liver. This liver RNA was translated in a reticulocyte cell-free system, which had been rendered dependent on exogenous messenger RNA by mild ribonuclease treatment. After ribonuclease was removed from the ribosomes, protein synthesis was almost totally dependent on 0.5 M KC1 ribosomal wash protein and exoge- nous RNA isolated from either reticulocyte or liver polysomes. In contrast to reticulocyte RNA, which directed the synthesis of LY and @ globin chains almost exclusively, liver RNA directed synthesis of polypeptides over a broad range of molecular weights. Little globin chain synthesis was de- tected with liver RNA. Evidence for the specific synthesis of ferritin was obtained by chemical purification and crystal- lization of this protein from the cell-free reaction product, by immunoprecipitation of the purified material with a mono- specific antibody to rabbit liver ferritin, and by coincident migration of radioactivity with purified ferritin in both polyacrylamide and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Since this highly active, fractionated, reticulocyte cell-free system is capable of translating mRNA fractions from both the liver and the reticulocyte into specific natural proteins, it may be useful in future studies dealing with the regulation of protein synthesis with messenger RNA from various animal sources.

Iluring the past several years, methods for isolating and char- acterizing mammalian mRNAs have been greatly improved and a number of investigators have reported translation of specific proteins from these messengers. These include rabbit, mouse, duck, and human globin (l-8), chicken ovalbumin, myosin, and lens crystallins @la), mouse myeloma cell immunoglobulins (13, 14), bovine lens crystallins (15), rat liver catalase and al- bumin (16), and many specific animal virus proteins (17-21).

* This work was supported by Grants AM-01392, AM-03014, and AM-14359 from the National Irlstitutes of Health.

Most of these studies have employed a reticulocyte or ascites tumor cell-free system to translate homologous or heterologous mRNA and have relied upon preincubation of the ribosomal ma- terial to reduce endogenous messenger activity.

Recently Crystal et al. (22) reported a highly active, reticulo- cyte cell-free system, in which exogenous messenger RNA de- pendence was obtained by preincubation of the ribosomes with pancreatic ribonuclease A. After the ribonuclease was removed by sedimentation of the ribosomes through 1 M sucrose containing 0.5 M KCl, the synthesis of globin cy and 0 chains was dependent on both 0.5 M KC1 ribosomal wash protein and exogenous reticu- locyte messenger RNA. In the present study this fractionated, reticulocyte cell-free system has been utilized to examine an RNA extract from liver polysomes for messenger activity. The present communication describes the properties of this heterologous sys- tem for translation of liver RNA into non-globin polypeptides of high and varied molecular weights. Identification of labeled ferritin in this cell-free product served as an example for the synthesis of a specific natural liver protein under the direction of intact liver mRNA. These results indicated that the fraction- ated messenger-dependent reticulocytc cell-free system was capa- ble of translating liver and reticulocyte mRNA fractions into specific proteins characteristic of the source from which the messenger RNA was derived.

EXPERIMENTAL PROCEDURE

Materials-All chemicals used were freshly prepared and of analytical or reagent grade. Enzyme grade ammonium sulfate, ribonuclease-free sucrose, and [aH]poly(U), K+ salt (specific ac- tivity 7.76 mCi per mmole of phosphorus) were purchased from Schwarz-Mann, Orangeburg, N. Y. L-[ U-14C]Valine (specific ac- tivity 260 mCi per mmole), L-[ U-Wlleucine (specific activity 331 mCi per mmole), L-[4, 5-aH]leucine (specific activity 36 Ci per mmole), and L-[ U-14C]phenylalanine [specific activity 513 mCi per mmole) were purchased from Amersham-Searle Corp., Ar- lington Heights, Ill., and were diluted with unlabeled L-amino acid to the specific activities indicated in the appropriate figure legends. Rabbit liver tRNA (stripped) was obtained from Gen- eral Biochemicals, Inc., Chagrin Falls, Oh., poly(U) (NH4+ salt) from Miles Laboratories, Elkhart, Ind., GTP, ATP, P-enol- pyruvate, pyruvate kinase (rabbit muscle), and dithiothreitol

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from Calbiochem, San 1: iego, Calif., and preswollen microgranu- Preparation of mRNA Fractions-Total crude RNA was ex- lar carbosymethylcellulose (1.0 meq per g), Whatman CM-52, tracted from liver polysomes or reticulocyte lysate by the method from Reeve Angel Co., New York, N. Y. of Crystal et al. for the rabbit reticulocyte system (22), escept

Preparation of Ribosomes and Enzyme Fractions-Unfasted, 3.5-pound New Zealand white rabbits were used for all studies. The rabbits were killed by intracardiac injection of air. The livers were excised rapidly and rinsed in cold 0.14 51 KaCl-10 mM Tris-HCl, pH 7.4. All subsequent procedures were performed at O-4”. The livers were transferred to homogenizing solution (0.25 M sucrose, 5 mM MgC12, 2 mM dithiothreitol, 0.1 mM EDTA, pH 7.4, and 150 pg per ml of heparin), minced with a scissors, rinsed three times in homogenizing solution, and homogenized in 1.5 to 2 volumes of homogenizing solution using 12 to 15 strokes in a conical Potter-Elvehjem homogenizer. A post-mitochon- drial supernatant fraction was prepared from the homogenate by centrifugation at 10,000 X g for 5 min and 17,300 X g for 10 min, discarding the pellet. Free polysomes were isolated from this fraction by a series of two discontinuous sucrose gradients, using a modification of the procedure of Bloemendal et al. (23). Aliquots of the post-mitochondrial supernatant (16 ml) were placed over bilayer sucrose gradients. The lower layer (6.5 ml) contained 1.8 M sucrose in homogenizing solution minus heparin to collect free polysomes and the upper layer (7.2 ml) contained 1.45 M sucrose in homogenizing solution minus heparin to trap membrane-bound polysomes. Centrifugation was performed at 78,000 X g for 7 hours in a Beckman No. 30 rotor. The free polysomes, loosely sedimented to the bottom of the centrifuge tube on the surface of the clear glycogen pellet, were suspended in sucrose solution (0.25 M sucrose, 5 mM MgCls, 1 mM dithio- threitol, and 0.1 mM EDTA, pH 7.0). To increase purity of the

that both the phenol-m-cresol-S-hydroxyquinoline estraction and the ethanol precipitation step were performed twice. The final material was dialyzed for 24 hours against several changes of 4 liters of 5 mM Tris-HCl, pH 7.4, and 2 1llM KCl, and centrifuged at 178,000 x g for 1 hour to remove aggregated material. A fraction enriched in messenger activity was then obtained by sucrose gradient centrifugation, using the procedure of Evans and Lingrel (28). Crude RNAs, 75 A260 units in 1.0 ml, were layered over identical 12-ml, 5 to 20y0 exponential sucrose gradients prepared with a Technicon AutoAnalyzer pump as described by No11 (29). After 12 to 14 hours of centrifugation at 201,000 X g in an SW 41 Beckman rotor, 0.5.ml fractions were collected from the bottom of each tube. Absorbance at 260 run was measured on 50.~1 aliquots from each fraction with a ‘Zeiss PMQ II spectro- photometer.

Polymerization Assays-Incorporation of r$4C]valine, under the direction of natural mRNA or L-[14C]pl~enylalanine, under the direction of polyuridylic acid (poly(U)), was performed as previ- ously reported (30). A detailed list of reaction components and incubation conditions is given in Table I. Reactions were stopped by the addition of ice-cold 10yo trichloroacetic acid and protein synthesis determined by incorporation of radioactivity into hot trichloroacetic acid-insoluble material (27).

Isolation of Rabbit Liver Fe&fin-Adult New Zealand white rabbits were injected intramuscularly with 2 ml of Imferon (an iron-dextran solution) to increase the ferritin content of their tissues. After 2.5 to 3 weeks, the livers were excised and ferritin

free polysomes, discontinuous sucrose gradient centrifugation was isolated and purified by the method of Drysdale and Munro was repeated with the crude fraction in a Beckman SW 27 rotor (31). This procedure included heat coagulation (75” for 2 min), at 135,000 x g for 8 hours. .4 one-fifth volume of crude rabbit pH 5.0 precipitation (saving the aqueous fraction), carboxymeth- liver 105,000 X g supernatant, isolated by previous centrifuga- ylcellulose chromatography, fractional ammonium sulfate pre- tion, was included in each sucrose layer as a ribonuclease inhibitor cipitation, and Sephadex G-200 chromatography. The final ma- (24), so that this method encompassed the essential features of terial contained 23 to 24 y0 iron by weight, as previously observed the procedures of Blobel and Potter (25) and Scornik el al. (26). (31, 33), and showed only one minor noll-iron-staining protein

Reticulocyte lysate, ribosomes, supernatant protein, and a band on polyacrylamide gel electrophoresis (see methods below). 0.5 M KC1 ribosomal wash fraction were prepared as described by This minor band probably represents a trace of the apoferritin Shafritz and Anderson (27). The ribosomal wash fraction was subunit (32). Ferritin was crystallized from this final material precipitated with ammonium sulfate (33 to 65y0 fraction) and as the cadmium salt (33). The protein pattern of the crystalline dialyzed for 12 hours against 4 liters of Buffer A (10 mM Tris-HCl, ferritin by either polyacrylamide or SDS”-polyacrylamide gel pH 7.4, 100 mM KCl, 1 mM MgC$, 1 mM dithiothreitol, and 0.1 electrophoresis was indistinguishable from the Sephadex G-200- mM EDTA) . Reticulocyte ribosomes were rendered exogenous purified material. mRNA-dependent for protein synthesis by mild digestion with Preparaiion of Antiferritin Antibody and Immunoprecipiiaticn pancreatic ribonuclease A (Worthington Biochemicals, Freehold, Reaction-An antiserum to purified rabbit liver ferritin was pro- N. J.) as described by Crystal et al. (22). Crude reticulocyte duced in the goat by a series of three alternate weekly intramuscu- polysomes were suspended to a final concentration of 75 ASO lar injections of 5.0 mg of purified antigen (ferritin) in complete units per ml in 0.25 M sucrose, 20 mnr Tris-HCl, pH 7.4, 2 mM Freund’s adjuvant. The antiserum contained a monospecific MgCl*, 1 mM dithiothreitol, 0.1 my EDTB, to which pancreatic antibody to rabbit ferritin, as judged by immunodiffusion and ribonuclease rl was added at a final concentration of 0.05 pg per immunoelectrophoresis. Antibody in the crude goat serum was ml. This solution was incubated at 3;’ for 10 min, layered over present at a titer of 1: 128 and was used for all studies. Immuno- 4.5 ml of buffered sucrose solution (1 M sucrose, 0.5 M KCl, 50 mu precipitation of ferritin (as described below) was not observed Tris-HCl, pH 7.4, 2 mM MgCl,, and 1 mM dithiothreitol), and with control rabbit serum or with goat serum from a nonim- centrifuged at 178,000 X g for 4 hours. The aqueous layers were munized animal. removed, the side walls of the centrifuge tubes and the pellet were Precipitation of ferritin by the above antibody was performed drained and rinsed carefully three times with buffer, the ribo- by the method of Means et al. (9). This method uses the deter- somes were suspended in 0.25 M sucrose, 10 mM KCl, 2 mM gents sodium deoxycholate and Triton X-100 to reduce non- MgCl?, and 1 mM dithiothreitol, and the final suspension was specific immunoprecipitation and adsorption of isotope to precip- stored in small aliquots in liquid N2. These ribosomes showed itated material. For these studies, a reaction mixture of 100 ~1, no residual ribonuclease activity, as judged by their lack of hy- with proportionate increases in all components, was used together drolysis of [3H]poly(U), even at concentrations 10 times the amount used routinely for cell-free protein synthesis. 1 The abbreviation used is : SDS, sodium dodecyl sulfate.

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with [3H]leucine, specific activity 6 Ci per mmole. After 30 min of incubation, a loo-fold excess of [Wlleucine was added and incorporation of radioactivity into hot trichloroacetic acid-in- soluble polypeptide was determined on a lo-$ aliquot. Carrier ferritin, 10 pg of protein, was added to the remaining 90 ~1 and incubation was continued for 10 min at 30”. Sodium deoxy- cholate and Triton X-100 together with the goat antiserum and 10 m&f KPOl buffer, pH 7.4-150 mM NaCI-1 mM [i2C]leucine were then added as described by Means et al. (9). The mixture was again incubated at 30” for 30 min with occasional shaking and then left at 0” for 2 hours. Immunoprecipitates were sedimented in an Eppendorf microcentrifuge (Brinkmann Instruments), re- suspended, and centrifuged again in 3% Triton X-100-10 mM KPOI, pH 7.4-150 mM NaCI-1 mM [‘Vlleucine. The precipitates were collected on nitrocellulose filters, washed with 20 ml of the same solution, and counted by liquid scintillation spectrometry. The same procedure was used for immunoprecipitation of the cell-free reaction product purified for ferritin. From the meas- ured amount of iron in the precipitate (31), the efficiency of im- munoprecipitation in the presence of the detergents was esti- mated at 66’$& Blanks were usually in the range of 200 cpm for a loo-p1 reaction mixture.

Polyacrylarnide Gel Electrophoresis-Polyacrylamide gel elec- trophoresis was performed by the method of Davis (34), using 6 y0 acrylamide-methylenebisacrylamide gels. Gels were cast in glass tubing with dimensions of 5 x 75 mm, and electrophoresis in a Canalco apparatus was performed in 0.065 M Tris-borate buffer, pH 9.0, at room temperature for 1 hour, using a current of 3 ma per gel. Gels were extruded, fixed in 25% isopropyl al- cohol-IO% acetic acid and stained for protein with Coomassie blue or for iron with potassium ferrocyanide (Prussian blue stain). SDS-polyacrylamide gel electrophoresis was performed by the method of Fairbanks ef al. (35), except that 6.5% acrylamide- methylenebisacrylamide and 0.5% SDS (Schwarz-Mami) were used in formation of the gel. Molecular weight standards of albumin (68,000)) ovalbumin (45,000)) chymotrypsinogen A (25,000), and ribonuclease A (13,700) were obtained from Phar- macia Fine Chemicals, Piscataway, N. J. For determination of radioactivity, gels were sliced into 1.25-mm sections, digested for 6 hours in 0.5 ml of NCS tissue solubilizcr (Amcrsham-Searle Corp.), and counted by liquid scintillation in NCS-ammonia- toluenc-1,4-bis[2-(5.phenyloxazolyl)]benzene (POPOP)-2,5-di- phenyloxazole (PPO) (36).

RESULTS

It was previously shown (22) that ribosomes prepared from reticulocyte polysomes by brief exposure to pancreatic ribo- nuclease retain their ability to function in cell-free protein syn- thesis but show a marked reduction in endogenous messenger ac- tivity. As indicated in Table I, natural protein synthesis with these ribosomes was found to be virtually completely dependent on the addition of both RNA and the 0.5 M KC1 reticulocyte ribosomal wash fraction. A crude RNA extract from liver poly- somes produced a 40. to 50-fold stimulation of cell-free protein synthesis. Under similar conditions, with low Mgzf concentra- tion in the presence of the ribosomal wash fraction, polyuridylic acid served as an excellent messenger template for polyphenylala- nine synthesis. The use of such an artificial messenger, not contaminated by ribosomal or other types of RNA, confirmed both the general viability of the RNase-treated reticulocyte ribosomes and the specific ability of these ribosomes to be stimu- lated by exogenous messenger RNA.

polysomes enriched in mcsscnger activity and partially separated from other types of RNA was obtained by sucrose gradient cen-

trifugation (Fig. 1). Fractions of 0.5 ml were collected from each gradient, and stimulation of protein synthesis in the reticulocyte messenger-dependent system was determined on 25.~1 aliquots from each fraction. Fractions producing the greatest stimula- tion of protein synthesis were located between 4 S (tRNA) and 18 S (ribosomal RNA). As shown in Fig. 1, when the data were expressed as picomoles of [14C]valine incorporated per A260 unit of RNA added, Fractions 16 and 17 had the highest apparent specific activity. Fractions 12 to 19 were pooled, concentrated by vacuum dialysis, and used for subsequent studies. A similar reticulocyte RNA fraction (7 to 14 S) isolated from reticulocyte

lysate by this sucrose gradient method, produced a 75- to 80.fold stimulation of cell-free protein synthesis.

Characterization of Cell-free System-As shown in Fig. 2A, pro- tein synthesis was linear with increasing amounts of ribosome to

0.15 A260 unit. Stimulation of protein synthesis by 0.5 M KC1 ribosomal wash protein produced a xigmoidal curve (Fig. 2B), with apparent saturation at approximately 150 pg of protein. For subsequent studies, 0.1 A260 unit of ribosome and 150 pg of

ribosomal wash protein were used. Under these conditions, pro-

Isolation of Liver RNA Fraction-An RNA fraction from liver

tein synthesis increased in a near-linear fashion with added liver RNA up to 0.36 AzGO unit (Fig. 3). With this partially purified RNA fraction (from the sucrose gradient), stimulation of protein

Requirements for cell-free protein synthesis with RNase and 0.5 M KCI-treated reliculocyle ribosomes

For the complete system, incubations in a total volume of 50 ~1 were performed at 30” for 30 min for natural protein synthesis and 37” for 10 min for polyphenylalanine synthesis. Reactions contained 15 mM Tris-HCI, pH 7.5,l rnrvr ATP, 0.2 mM GTP, 3 mM phosphoenolpyruvate, 0.2 i.u. of pyruvate kinase, 1 mrvr dithio- threitol, 0.08 X 1W3 M 19 minus 12C-amino acids, 0.1 A260 unit of RNase and 0.5 AX KC1 treated reticulocyte ribosomes, 0.06 A260 unit of deacylated rabbit liver tRNA, 300 pg of 105,000 X g super- natant protein, and 150 fig of 0.5 M KC1 ribosomal wash protein. For natural protein synthesis 4 mM MgCl,, L-[14C]valine (specific activity 260 mCi per mmole), 80 mM KCl, and 1.45 A260 units of crude liver free polysomal RNA were utilized. This amount of liver RNA gave maximal stimulation. For polyphenylalanine synthesis 5 mM MgC12, n-[‘%]phenylalanine (specific activity 85 mCi per mmole) and 0.56 A260 unit of polyuridylic acid (poly(U)) were used. Blanks of 0.36 pmole for natural protein synthesis and 0.83 pmole for polyphenylalanine synthesis, representing isotope retention by the nitrocellulose filter in the absence of ribosomes, protein factors, and mRNA, wcrc subtracted from each value.

I Amount incorporation

L-[WJPhenyl- L-[*NZ]Valine, alanine,

4 nud Mgl+ 5 mu Mg?+ (natural pro-

tein synthesis) (P;y$;fYl-

synthesis)

Complete system Liver free polysomal RNA. Poly(U)

Deletions Ribosomes 0.5 M KC1 ribosomal wash Liver free polysomal RNA Poly(U)

I pmo1es

14.6

0.02 0.18 0.33

89.0

1.1 1.4

0.2

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0 5 10 15 20 25

FRACTION

FIG. 1. Sucrose gradient analysis of liver RNA extract. Crude RNA (75 Ate0 units), isolated from free polysomes of rabbit liver as described under “Experimental Procedure,” was placed over a 12-ml, 5 to 20% exponential sucrose gradient in 10 mM Tris-HCI, pH 7.5-10 mM KCl. Centrifugation at 201,000 X gay was per- formed at O-2” in a Beckman SW 41 rotor for 12 to 14 hours. Frac- tions of 0.5 ml were taken from the bottom of each tube and ab- sorbance at 260 nm was measured on 50.~~1 aliquots. Stimulation of protein synthesis in the reticulocyte cell-free system was de- termined on 25-J aliquots from each fraction, using the conditions described in Table I, except that loo-p1 reaction mixtures were used. A blank of 0.32 pmole, representing activity in the ab- sence of added liver RNA, was subtracted from each value.

RBOSOME CONC /AzSo units /SOyL / RIBOSOMAL WASH +Q ~mieinl

FIG. 2. Incorporation of [“V?]valine into protein as a function of ribosome and 0.5 M KC1 ribosomal wash protein concentration. For these studies 0.36 A260 unit partially purified liver RNA was used. In A, 150 pg of ribosomal wash protein were used, and in B, 0.1 Ax60 unit of ribosome was used. Other experimental con- ditions were as noted in Table I.

synthesis was equivalent to that obtained with crude liver RNA extract (cf. Fig. 3 and Table I). This represented a 4-fold in- crease in messenger specific activity. In this system protein syn- thesis was linear for 40 min and was still increasing after 1 hour (Fig. 4). Under these conditions approximately 90% of the radioactivity incorporated into polypeptides was released from the ribosome into the supernatant fraction after 20 to 30 min of incubation (Fig. 5). In addition, the Mg”+ concentration op- timum for liver mRNA translation in this system was approxi- mately 4 mM (Fig. 6).

Characterization of Cell-free Reaction Products-Evidence for synthesis of non-globin polypeptides under the direction of liver

RNA was obtained by SDS-polyacrylamide gel electrophoresis and carboxymethylcellulose chromatography of the cell-free reac- tion product. This material was compared to the reaction product produced with a comparable reticulocyte RNA fraction.

In separate incubations, the translation of reticulocyte RNA was performed with @C]leucine and liver RNA with LJ3H]leucine.

1 1.0

Azso UA’/TS L lVER WA ADDED

FIG. 3. Stimulation of protein synthesis in response to exoge- nous liver RNA. For these studies, the partially purified sucrose gradient liver RNA fraction was used in increasing amounts in conjunction with 0.1 A260 unit of RNase-treated reticulocyte ribosomes and 150 Pg of 0.5 M KC1 ribosomal wash protein. Other experimental conditions are given in Table I.

U/NU E5.S

FIG. 4. Time course of protein synthesis under the direction of exogenous liver RNA. For these studies 0.36 Asso unit of partial purified liver RNA was used. See Table I for further experimental details.

After protein synthesis was completed, the two reaction mixtures were combined as a single sample, so that the subsequent results would be obtained in a simultaneous double label fashion. On SDS-gel electrophoresis (Fig. 7), reticulocyte RNA produced one sharp peak of labeled protein ([14C]leucine label). When com- pared to standard molecular weight markers, this material had an approximate molecular weight of 18,000, the expected value for both the Q( and the p polypeptide chain of hemoglobin. In contrast, liver RNA directed the synthesis of material over a much broader molecular weight range ([3H]leucine label), with considerable material between 25,000 and 68,000 molecular weight (Fig. 7).

Since material in the 18,000 molecular weight region was pro- duced with both mRNA fractions (Fig. 7), carboxymethyl- cellulose chromatography was performed by the method of Dintzis (38) to measure a! and /3 globin chains directly. For these experiments, the reticulocyte mRNA cell-free product was

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a I “Released”Polypeptides I

MINUTES

FIG. 5. Released (soluble) polypeptide versus nascent (riboso- m&bound) polypeptides as a function of time. Protein synthesis in the reticulocyte cell-free system under the direction of liver RNA was performed as noted in Fig. 4. After the appropriate time intervals protein synthesis was stopped by the addition of ice-cold buffer (20 mM Tris-HCl, pH 7.4-100 rnM KCl-5 mM MgC12) to a final volume of 1.0 ml. Aliquots of 100 ~1 were removed and hot trichloroacetic acid-insoluble polypeptides determined. The remaining portion of the reaction mixture was centrifuged at 105,000 x g for 2 hours at O-4”. The aqueous portion was removed, the ribosomal pellet suspended in the above buffer solution, and hot trichloroacetic acid-insoluble polypeptides determined. Re- leased polypeptides represent total protein synthesis minus poly- peptides which had remained in the ribosomal-bound fraction (nascent chains).

I I I I 0 2 4 6 8 IO

Mg++COnlC. , mM

FIG. 6. Stimulation of [%]valine incorporation by exogenous liver RNA as a function of Mgz+ concentration. Partially puri- fied liver R.NA, 0.36 Aas0 unit, was used. Ot,her experimental con- ditions are given in Table I.

again labeled with [14C]leucine and the liver mRNA cell-free prod- uct with [3H]leucine. Approximately equal counts per min of [14C]leucine- and [3H]leucine-labeled protein were mixed together with unlabeled carrier hemoglobin and the mixture dialyzed against Hz0 for 24 hours at 0”. From the dialyzed mixture, an acid-acetone powder was prepared. This material was dissolved in 0.02 1u pyridine-0.2 M formic acid and chromatographed over a column (1 X 22 cm) of CXcellulose as described in Fig. 8. ‘I’he positions of the carrier (Y and fi globin chains in the eluted frac- tions were determined by ultraviolet absorbance at 280 nm. Most of the product programmed by reticulocyte RNA chromato- graphed as either the a or the /3 globin chain (Fig. 8). The amount of radioactivity in these two peaks represented greater than SOY0 of the total 14C-labeled polypeptide applied to the col-

/ / 0 IO 20 30 -40

ORIGIN FRONT GEL FRACJlOh’

FIG. 7. SDS-polyacrylamide gel electrophoresis of the cell-free reaction product under the direction of liver and reticulocyte RNA. The cell-free reaction products labeled with [Wlleucine for reticulocyte RNA and [3H]leucine for liver RNA, were mixed together and dialyzed for 24 hours against two to three changes of 10 mM NaHCOa-5mM p-mercaptoethanol. Subsequent steps fol- lowed the procedure of Fairbanks et al. (35), except that 0.5% SDS and 6.5% polyacrylamide were used in the preparation of the gel. Seventy-five microliters of the reaction mixture, including 5 to 10% sucrose and pyronin Y (tracking dye), were layered over the gels followed by electrophoresis at room temperature with 5 ma per tube. After electrophoresis, gels were immediately frozen and thawed for slicing into 1.25-mm sections. Material was di- gested from the gel slices with 0.5 ml of NCS tissue solubilizer to which 0.1 ml of 4 M NHGOH and 5.0 ml of toluene-1,4-bis[2(5- phenyloxazolyl)]benzene-2,5-diphenyloxazole were added. Gel slices were then counted by liquid scintillation with 22% efficiency for 3H (less than 0.1% spillover into the W-channel), and 49% efficiency for ‘“C (2Cy0 spillover into the 3H-channel). Standard proteins for molecular weight calibration of the gels included a, albumin (68,000 mol wt) ; b, ovalbumin (45,000 mol wt) ; c, chymo- trypsinogen A (25,000 mol wt) ; and cl, ribonuclease A (13,700 mol wt). As found by previous investigators (37), the apparent mo- lecular weight for ribonuclease A was slightly higher than the theoretical value.

umn. In contrast, with liver messenger RNA little radioactivity eluted with carrier (Y and /3 globin chains. This represented less than 10yO of the total Wlabeled material applied to the column. Most of the [3H]leucine-labeled liver proteins remained attached to CM-cellulose after elution of the globin chains, but an addi- tional 50% could be eluted with 0.1 RI Tris-HCl, pH 7.4.

ZdeniiJication of Ferriiin in Cell-jree Reaction Product-lTndel the directiou of liver RNA, the synthesis of material with the properties of ferritin was shown by chemical purification, im- munoprecipitation, and electrophoresis in two separate systems. For these studies [3H]leucine (specific activity 6 Ci per mmole) was used in a total reaction mixture of 1.0 ml with proportionate increases in all components. In this case, incorporation of radio- activity into hot trichloroacetic acid-insoluble polypeptides was routinely 500,000 cpm. After 30 min of incubation, rabbit liver ferritin was added along with a loo-fold excess of [‘T]leucine and incubation \yas continued for 10 min. Ferritin was then reiso- lated and purified by chromatographic procedures (31) with 51 y’, total recovery as estimated by iron determination. The final step in the purification was Sephades G-200 chromatography with the large ferritin complex (mol wt > 450,000) eluting slightly behind the peak position of blue destran, which was first used to determine the void volume of the column. A large portion of the applied radioactivity co-eluted Iv-it11 the ferritin fraction. This material was concentrated by vacuum dialysis and used in subsequent studies. Ferritin was crystallized from a portion of this chemically purified material (Fig. 9). Approximately 60 to

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FIG. 8. Carboxymethylcellulose chromatography of the cell- free reaction product under the direction of the reticulocyte and the liver RNA fraction. For each exneriment. a column. 1 X 22 cm, of microgranular carboxymethylc~llulose (Whatman bM-52), equilibrated with 0.2 M formic acid-O.02 M pyridine, was used. Carrier rabbit hemoglobin was added to a mixture of the cell-free reaction products, which were labeled with [%]leucine for reticu- locyte RNA and [aH]leucine for liver RNA. The solutions were dialyzed at 0” for 24 hours against H,O, saturated with toluene. An acid-acetone precipitate was prepared from each mixture, con- taining equal counts per min for [‘“Cl- and [3H]leucine-labeled pro- tein, and the dried acetone powder dissolved in the column buffer. A linear gradient from 0.2 M formic acid-O.02 M pyridine (125 ml) to 2.0 M formic acid-O.2 M pyridine (125 ml) w&&ed to elute the rabbit (Y and B rrlobin chains. Fractions (2.5 ml) were collected. and the position of unlabeled carrier (Y and‘p glob& chains was de: termined by ultraviolet absorbance at 280 nm.

FIG. 9. Crystals of ferritin isolated from the purified cell-fre reaction product in the presence of carrier. The Sephadex G-200- purified ferritin fraction was concentrated by vacuum dialysis and an aliquot removed for crystallization by the method of Granick (33). Additional carrier ferritin was added to a final concentra- tion of 5 mg of protein per ml. Twenty per cent CdSOd in Hz0 was added slowly over a half-hour to a final concentration of 5%. Crystallization,which started immediately, was allowed to c&- tinue at 0” for 4 hours. The crystals were packed by centrifuga- tion, and prepared for photography on a microscopic slide at a magnification of X 250.

7070 of the carrier ferritin was recovered in these crystals with a proportionate amount of the expected radioactivity.

Additional evidence for the presence of radioactivity in ferritin was obtained by polyacrylamidc and SDS-polyacrylamide gel clectrophoresis. These experiments utilized the purified ma- terial, which represents repurified carrier ferritin plus labeled protein from the cell-free system co-purifying with this ferritin. With gel electrophoresis (Fig. 10) this material showed two pre- dominant bands and several minor bands of protein. A duplicate gel stained for iron with potassium ferrocyanide (Prussian blue stain), indicated that the two major bands and the top of the gel

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0 IO 20 30 40 50 CATHODE ANODE

GEL FRACTlOh

FIG. 10. Polyacrylamide gel electrophoresis of the cell-free re- action product purified for ferritin. Six per cent acrylamide- methylenebisacrylamide gels were cast in glass tubes, 5 X 75 mm, and used for electrophoresis as described under “Experimental Procedure.” Samples of 100 ~1 were applied to each gel and elec- trophoresis was performed at 3 ma per gel, using bromphenol blue as the tracking dye. Gels containing [3H]leucine-labeled material were stained for protein with Coomassie blue, scanned with a Gilford linear gel scanner, sliced into 1.25-mm sections, and counted by liquid scintillation (36).

represented ferritin and ferritin aggregates, respectively. When the gel was sectioned and counted, peaks of radioactivity cor- responded to each band which had stained for both iron and protein. One of the smaller non-iron-staining protein bands also contained radioactivity. This may represent the apoferritin subunit (32). With SDS-polyacrylamide gel electrophoresis (Fig. 11, upper frame), radioactivity again corresponded to the major bandof ferritin (mol wt > 450,000), as well as other ferritin aggregates. In order to dissociate the ferritin complex into the subunit (mol wt - 20,000), a second aliquot was heated to 60” for 30 min (32). After heating (Fig. 11, lower frame) SDS-poly- acrylamide gel electrophoresis revealed two partially resolved bands over a total molecular weight range of 18,000 to 23,000. In the gel scan (Fig. 11, lower frame) the higher molecular weight portion appeared as a shoulder on the major protein band. When the gel containing the heat-treated material was sectioned and counted, radioactivity had moved from the position of ferritin to the position of the apoferritin subunit (primarily into the 21,000 to 23,000 mol wt region). Additional radioactivity was also found in smaller molecular weight polypeptides, which were thought to be further heat degradation products of ferritin.

Immunoprecipitation of the cell-free reaction product was per- formed both before and after purification of ferritin (Table II). Approximately 2.0 to 2.5% of the crude material with liver mRNA was precipitated by goat antibody to rabbit liver ferritin. With reticulocyte mRNA, only 0.12% of the total product was precipitated by the antibody. Nonimmune goat serum precipi- tated only 0.22 To of the total protein produced under the direction of liver mRNA. When ferritin was purified from the cell-free product programmed by liver mRNA, the amount of labeled protein precipitated by the antibody increased to 44%. If the efficiency of the immunoprecipitation procedure is taken into consideration (see “Experimental Procedure”), then 67 y. of the radioactivity in the purified material represented immuno- logically competent ferritin. When the chemical purification procedure was used to compare ferritin synthesis under the direc- tion of liver and reticulocyte mRNA, the amount of radioactivity

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/ 1

t

BEFORE UEA TING 100

2 AFTER HEATING

150

100

50

/

1 0 .-. I I I I I I 0 10 20 30 40 50

ORIGIN FRONT

GEL FRACT/O/V

FIG. 11. SDS-polyacrylamide gel electrophoresis of cell-free reaction product purified for ferritin. SDS was added to an aliquot of the cell-free reaction product purified for ferritin (in 20 mM Tris-HCl, pH 7.4) to a final concentration of 0.5a/ The aliquot was divided into two portions. One portion was heated to 60” for 30 min, while the other portion was kept at room temper- ature. Sucrose and pyronin Y were then added and electrophore- sis performed as noted under “Experimental Procedure.” The gels were stained for protein with Coomassie blue, scanned with a Gilford linear gel scanner, sliced into 1.25.mm sections, and counted by liquid scintillation. The position of molecular weight standards for calibration of the gel was determined as noted in Fig. 6.

obtained with liver mRNA was 30 times greater than that ob- tained with reticulocyte mRNA.

DISCUSSION

In the present studies an RNA fraction from liver-free poly- somes stimulated protein synthesis 40- to 50-fold in a fraction- ated mRNA-dependent reticulocyte cell-free system. This stimulation was essentially linear for 40 min and required the separate addition of 0.5 M KC1 ribosomal wash protein. Since approximately 90 70 of the total labeled polypeptide was released from the ribosomes into the supernatant fraction after 20 to 30 min, this system showed the complete mechanism for protein synthesis. It also provided a sensitive assay for the detection of messenger RNA and could be utilized to examine RNA fractions directly from sucrose gradients. With crude liver RNA, messen- ger activity was distributed throughout the gradient suggesting a rather heterogeneous nature for liver mRNA as indicated by others in various labeling studies (39-41). However, the greatest activity and highest specific activity was found between 7 and 14 S. Unlike the ‘(9 S” mRNA fraction from reticulocytes, liver RNA stimulated the synthesis of polypeptides over a broad molecular weight range, with considerable material between 25,000 to 68,000. In addition, less than 10% of the liver RNA-

TABLE II

Identification of 'tferritin-like" material by inlmunoprecipitation of reticulocyte cell-free reaction products

Liver . . . . . . . . . . . . . lteticulocyte Liver (nonimmune serum)

Liver (cell-free product pu- rified for ferritin)

cpnt cm % 72,000 1,675 2.3

315,000 375 0.12 87,600 200 0.22

132 44a

a If the data were corrected for the efficiency of immunopre- . cipitatlon (667,, see “Experimental Procedure”), this value

would be increased to 67oj,.

directed polypeptides eluted with carrier rabbit 01 and @ rabbit globin chains by carboxymethylcellulose chromatography. This small amount of material probably represented residual endog- enous messenger activity on the ribosomes, or traces of mRNA in the ribosomal wash (6) or crude supernatant fraction (42).

Direct evidence for translation of liver mRNA into specific liver protein in the reticulocyte system was obtained by identifi- cation of ferritin in the cell-free reaction product. Ferritin was chosen because it is a stable, well characterized protein produced by the liver cell. At first glance, the selection of this substance (mol wt N 450,000) would seem a poor choice for demonstrating natural protein synthesis in a cell-free system. However, each apoferritin molecule is composed of an aggregate of 20 or 24 similar subunits (mol wt h 20,000) (43, 44) which are noncova- lently linked and freely exchangeable (45). Therefore, in order to incorporate radioactivity into ferritin in a cell-free system, one need only synthesize the apoferritin subunit and incubate this material with carrier as performed in the present studies.

In a conventional purification process, a portion of the cell-free product eshibited various properties characteristic of authentic ferritin. These included heat extraction and stability at 80°, pH 5, and ammonium sulfate fractionation, and chromatography on both carboxymethylcellulose and Sephadex G-200. Other methods for ferritin identification included crystallization with purified carrier, immunoprecipitation with a monospecific anti- body, and polyacrylamide gel electrophoresis in the presence and absence of SDS. In the final identification step a portion of the purified labeled material was dissociated in SDS at 60” to produce free iron and the apoferritin subunit (32). When this material was analyzed by SDS-polyacrylamide gel electrophoresis, the bulk of the protein and a corresponding proportion of radioactvity shifted from the position of ferritin to the position of the apo- ferritin subunit. This clearly showed that the apoferritin poly- peptide subunit was synthesized in the reticulocyte cell-free sys- tem and was subsequently incorporated into the apoferritin shell. For reasons which remain unclear, the subunit fraction showed two incompletely resolved components, with radioactivity mi- grating predominantly into the larger component. Although various explanations are possible, this finding may be related to the heterogeneity of ferritin (46 to 49).

The kinetic data and rharacterist,ics of the cell-free system would further suggest that liver mRNA has directed de lzooo syn- thesis of the apoferritin subunit in this heterologous system. However, a comparison of tryptic peptides from the purified cell-

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free reaction product with the tryptic peptides from uniformly labeled ferritin synthesized in vivo will be needed to establish complete fidelity for this translation process. In studies to be reported elsewhere, we have observed protein synthesis with mRNA extracts from membrane-bound polysomes, and have identified albumin as another specific protein product. There- fore, the methods used in the present studies for mRNA isolation and translation may have a more general applicability for both membrane-bound and free polysomes.

Since translation of liver mRNA in the reticulocyte cell-free system was obtained without the addition of liver protein factors, there does not appear to be an absolute requirement for liver- specific factors in this process. These results are in accord with observations by various investigators in other heterologous sys- tems (4, 5,8-10, 12-14, 50, 51). However, they do not eliminate the possibility that the rate or efficiency of translation of a specific messenger might be influenced by messenger-specific factors. Comparative experiments with purified messengers, factors, and ribosomes from both the liver and reticulocyte will therefore be needed to understand the complex interaction of these compo- nents in the regulation of eucaryotic protein synthesis.

Acknowledgments-We thank Dr. W. F. Anderson for making available the methods for RNase treatment of reticulocyte ribo- somes and RNA extraction prior to publication, Dr. R. G. Crys- tal for supplying initial samples of these materials and for helpful discussions, and Misses J. Finn and M. Tse for excellent technical assistance.

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3. 4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

REFERENCES

LINGHI~:L, J. B., LOCICARD, R. E., JONES, R. F., BURR. H. E., .~ND HOLDER. J. W. (1971) Ser. Haematol. 4, 37-69

SCH.~PIR.~, G.,. DR~YFUS, J. C., ‘\ND MALEKNI~, N. (1968) Biochem. Biowhus. Res. Commun. 32, 558-561

LAYCOCK, D. G:, .~ND HUNT, J. A. (1969) Nature 221, 1118-1122 PEMBERTON, R.‘E., Hou&~N, I>., LODISH, H. F., AND BIG-

LIONI. C. (1972) Nature New Biol. 236, 99-102 HOUSM.;N, c., P&I~I~I<TON, I~., AND T&ER, R. (1971) Proc.

Nat. Acud. Sci. U. S. A. 68, 2716-2719 NIENHUIS, A. W., AND ANDERSON, W. F. (1971) J. Clin. Invest.

60, 245882460 BKNZ, E. J. JR., AND FORGET, B. G. (1971) J. Clin. Znvest.

60, 2755-2760

39.

40. 41.

42.

MI<TAFOR.\, S., TI~R.~D.\, M., Dow, L. W., MARKS, P. A., AND BANK, A. (1972) PTOC. i\iat. Acad. Sci. U. S. A. 69, 1299-1303

MIUNS, A. R., COMSTOCK, J. P., ROSENFELD, G. C., AND O’MALLEY B. W. (1972) PTOC. Nat. Acad. Sci. U. S. A. 69, 1146-1150

43.

44. 45.

RHO.YDS, R. E., MCKNIGHT, G. S., AND SCHIMK~;, R. T. (1971) J. Biol. Chem. 246, 7407-7410

ROUI~I~E, A. W., AND HEYWOOD, S. M. (1972) Biochemistry 11, 2061-2066

46.

47.

WILLIAMSON, R., CLAYTON, R., AND TRUMAN, D. E. S. (1972) Biochem. Biophys. Res. Commun. 46, 1936-1943

STAVNEZ~R, J., AND HUANG, R. C. C. (1971) Nature New Biol. 230, 172-176

SW.~N, D., AVIV, H., AND LF,DER, P. (1971) Proc. Nut. Acad. 81%. U. 8. A. 69, 1967-1971

48.

49.

50.

MATHEWS, M. B., OSBORN, M., BURNS, A. J. M., AND BLOEMENDAL, H. (1972) Nature New Biol. 236, 5-7

UENOYAMA, K., AND ONO, T. (1972) J. Mol. Biol. 66, 75-89

51.

17.

18.

19.

20.

21.

22.

23.

24. 25.

26.

27.

28.

29.

30.

31.

32. SMITH-J• HANNSI~N, H., AND DRYSDALI~~, J. W. (1969) Biochim. Hiophys. Acta 194, 43-49

33. Gna~rcrc, S. (1946) Chem. Rev. 38, 379 34. D.~vIs, B.. J. (1964) Ann. N. Y. Acad. Sci. 121, 404-427 35. FAIRBANI~S. G.. STECK. T. L.. AND WALL~CH, D. F. H. (1968)

36.

37.

38.

MATHI~:~S, M. B., .~ND KORNER, A. (1970) Eur. J. Biochem. 17, 328-338

LEVIN, D. K., KYNER, D., AND Acs, G. (1971) Biochem. Bio- phys. Res. Commun. 42, 454461

BOIME. I., AVIV, H., AND LEDER, P. (1971) Biochem. Bioph,ys. Res.‘Commun. 46; 788-795

.

VIDRINIG. J. G.. WANG. C. S.. AND ARLINGHAUS. R. B. (1972) Bioche’m. Bidphys. Res. Co’mmun. 46, 538-544’ ~

GRAZIACLEI, W. D., III, ,\ND LENGYJCL, P. (1972) Biochem. Biophys. Res. Commun. 46, 1816-1823

CRYSTAL, R. G., NIENHKJIS, A. W., ELSON, N. A., .~ND ANDISI<- SON, Mi. F. (1972) J. Biot. Chem. 247, 5357-5368

BLOEMICNDAL, H., BONT, W. S., DEVRIES, M., AND BEN’EDETTI,

E. L. (1967) Biochem. J. 103, 177-182 SHORTMAN, K. (1961) Biochim. Biophys. Acta 61, 37-49 BLOBEL. G.. I\ND POTTER. V. R. (1966) PTOC. Nat. Acad. Sci.

U. K’A. 66, 1283-1287 ’ ’ SCORNIK, 0. A., HO~GLAND, M. B., PFJCFFL.:KI<ORN, L. C., AND

BISHOP, E. A. (1967) J. Biol. Chem. 242, 131-139 SHAFRITZ, D. A., AND ANDERSON, W. F. (1970) J. Biol. Chem.

246, 5553-5559 EVANS, M. J., AND LINGHEL, J. B. (1969) Biochemistry 8, 829

831 NOLL, H. (1969) in Techniques in Protein Biosynthesis (C-IMP-

RI~LL. P. N.. AND SARGENT. J. R... eds) Vol. II. Ch. 3. n. loll 179, kc,zd&ic Press, New’York’ ’ ’ ’ ’

SHAFRITZ, D. A., AND ISSGLRACHER, K. J. (1972) Biochem. Biophys. Res. Commun. 46, 1721-1727

DRYSDALI.:, J. W., AND MUNRO, H. N. (1965) Biochem. J. 96, 851-858

Biochemistry ‘10, 26062617 ’ W.~RD, S., WILSON, D. L., AND GILLIAM, J. J. (1970) Anal.

Biochem. 38, 90-97 SH~PII~O, A. L., AND VIRUIXLA, E. (1967) Biochem. Biophys.

Res. Commun. 28, 815-820 DINTZIS, H. M. (1961) PTOC. Nut. Acad. Sci. U. S. A. 47, 247-

261 HENSH~~, E. C., REVEL, M., AND HIATT, H. H. (1965) J. Mol.

Biol. 14, 241-250 OLSNTSS, S. (1971) Eur. J. Biochem. 18, 242-251 Lxx, S.‘Y., MICN~ECKI, J., AND BRAWERMAN, G. (1971) PTOC.

Nat. Acad. Sci. U. 8. A. 68. 1331-1335 JACOBS-LORENA, M., AND BAG~IONI, C. (1972) PTOC. Nut. Acad.

Sci. U. S. A. 69, 14251428 BRYCI.:, C. F. A., AND CRICHTON, R. R. (1971) J. Biol. Chem.

246, 4198-4205 BJORK, I., AND FISH, W. W. (1971) Biochemistry 10, 2844-2848 DRYSDALE, J. W., HAGGIS, G. H., AND HARRISON, P.M. (1968)

Nature 219, 1045-1046 ALFRIBY, C. P., JR., LYNCH, E. C., AND WHITLEY, C. E. (1967)

J. Lab. Clin. Med. 70. 419-428 GABUZDA, T. G., AND @F,ARSON, J. (1968) Nature 220, 1234-

1235 DRYSDALIS, J. W. (1970) Biochim. Biophys. Acta 207, 256-

258 LINDI~R-HOROWITZ, M., RUETTINGER, R. T., AND MUNRO,

H. N. (1970) Biochim. Biophys. Acta 200, 442-448 L.~NE, C. D., MARBAIX, G., AND GURDON, J. B. (1971) J. Mol.

Biol. 61, 73-91 PICCIANO, D. J., PRICHARD, P. M., MERRICK, W. C., SHAFRITZ,

D. A., GRSF, H., CRYSTAL, R. G., AND ANDERSON, W. F. (1973) J. Biol. Chem. 248, 204-421

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David A. Shafritz, James W. Drysdale and Kurt J. IsselbacherIDENTIFICATION OF FERRITIN IN THE PRODUCT

Reticulocyte Cell-free System: PROPERTIES OF THE SYSTEM AND Translation of Liver Messenger Ribonucleic Acid in a Messenger-dependent

1973, 248:3220-3227.J. Biol. Chem. 

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