THE JOURNAL OF BIOLOQICAL CHEMISTRY Vol. 237, No. 3, March 1962

Printed in U.S.A.

Synthesis of Hemoglobin in a Cell-free System

I. PROPERTIES OF THE COMPLETE SYSTEM

ESTHER H. ALLEN* AND RICHARD S. ScHwEmt

From the Biochemistry Department, City of Hope Medical Center, Duarte, California and the Department of

Biochemistry, College of Medicine, University of Kentucky, Lexington, Kentucky

(Received for publication, June 14, 1961)

The incorporation of Ci4-amino acids into protein, with cell- free systems containing ribosomes and soluble enzymes, has been studied extensively since the first reports by Zamecnik and Keller (1). This topic has been reviewed in detail by Hoagland (2). One limitation with many of the cell-free systems has been the failure to find incorporation into known proteins. Incorpo- ration of CY-amino acids into ribosome-bound serum albumin (3) and soluble hemoglobin (4) was reported in 1958. More recently, incorporation into albumin (5-7), silk fibroin (8), and /3-galactosidase (9) has been observed with similar cell-free sys- tems. Incorporation of CF-amino acids into protein in the cell- free system from rabbit reticulocytes appears to correspond to a limited synthesis of hemoglobin. Evidence for this conclusion was the stimulation of incorporation by a mixture of unlabeled amino acids (4), the agreement between the ratios of incorpora- tion of labeled valine, leucine, and isoleucine and the amounts of these amino acids in crude globin (4), the species specificity of hemoglobin labeling when ribosomes from either mouse or rabbit reticulocytes were used (lo-12), and the comparison of NHz-terminal and internal Cr4-valine labeling (13). Detailed studies of the incorporation of C14-amino acids into protein with ribosomes and partially purified enzymes from rabbit reticulo- cytes will be presented here. The incorporation of Cr4-amino acids into protein, starting with amino acyl-ribonucleic acid and highly purified transfer enzyme fractions’ (14), and studies of the final stages of completion and release of soluble protein from the ribosomez (15) will be reported in future papers.

EXPERIMENTAL PROCEDURE

Materials and Methods

Nucleosides, nucleotides, and nucleoside di- and triphosphates were obtained from Pabst Laboratories; creatine phosphate and Tris, from Sigma Chemical Company. Uniformly labeled Cr4- amino acids were obtained from Nuclear-Chicago Corporation, and nn-leucine-l-Cl’, from the California Corporation for Bio-

*Public Health Service Predoctoral Research Fellow of the National Cancer Institute.

t Part of these studies were performed during the tenure of an Established Investigatorshin of the American Heart Association. Supported in part by research grants from the National Science Foundation and the United States Public Health Service.

1 J. Bishop, J. Leahy, and R. Schweet, unpublished observa- tions.

2 A. Morris, and R. Schweet, unpublished observations.

chemical Research. ATP-S-Cl4 was obtained from Schwarr Laboratories, Inc.

Creatine kinase was prepared from rabbit muscle and purified through the alcohol fractionation stage as described by Kuby, Noda, and Lardy (16). Protein was determined by the method of Lowry et al. (17), and phosphate, by the method of Dryer, Tammes, and Routh (18). The dry weight of ribosomes was determined directly on samples put through the usual washing procedure described below. After washing and plating, these samples contained 30 to 40% RNA based on the ribose content (19). Other methods and preparations have been described (20, 21).

Ribosome Preparation-Reticulocytosis was produced in rab- bits by a modification of the method of Borsook et aZ. (22). New Zealand rabbits weighing 5 to 6 pounds were treated daily with subcutaneous injections of 1.0 ml of 2.5% phenylhydrazine solution for 5 days. The phenylhydrazine solution was prepared and neutralized to pH 7.0 with NaOH, filtered, and used at once; or it was stored at - 15” with 0.001 M GSH added. Each day’s supply was frozen in an individual vial. The rabbits received no injections on the 6th day and were bled by heart puncture on the 7th day.3 The heparinized blood was cooled to 4” and centrifuged for 15 minutes at 2500 x g, and the plasma was dis- carded. All further procedures were done at 4” unless other- wise indicated. The cells were resuspended in a solution con- taining 0.13 M NaCl, 0.005 M KCI, and 0.0075 M MgC12, with a volume equal to the plasma. After filtering through gauze, the cells were centrifuged as before. The packed cells (usually 50 ml from three rabbits) were lysed by adding 4 volumes (200 ml) of 0.002 M MgCh and stirring gently for 10 minutes. One vol- ume (50 ml) of 1.5 M,sucrose containing 0.15 M KC1 was then added. The mixture was stirred and centrifuged at 15,000 X g for 10 minutes. The clear supernatant fluid was carefully drawn off with a large pipette and centrifuged for 90 minutes at 78,000 x g, or for 60 minutes at 105,000 x g. The whole supernatant solution was saved for enzyme preparation. The pellets in each tube were rinsed with Medium B (0.25 M sucrose, 0.0175 M

KHC03, and 0.002 M MgCh), and then combined and gently homogenized by hand in Medium B with a glass homogenizer with a Lucite or Teflon pestle. The faintly opalescent solution was diluted to 200 ml with Medium B and centrifuged at high speed as before. The final ribosome pellet was homogenized in

3 Recently, daily injections for 4 days, followed by 2 days with- out injections before bleeding, have given good reticulocytosis and practically no mortality.

760

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0.25 M sucrose and centrifuged at 15,000 x g for 5 minutes. The final solution (12 ml) was slightly yellow and faintly opales- cent and usually contained 10 to 12 mg of ribosomes per ml.4

Preparation of Soluble Enzyme Fraction-Usually, freshly pre- pared whole supernatant solution was used for enzyme fractiona- tion. The solution (250 ml) was made 0.1 M in Tris buffer by adding 13.0 ml of 2 M Tris chloride buffer, pH 7.5.5 Neutralized protamine sulfate solution (10 mg per ml) was added to a final concentration of 0.17 mg of protamine sulfate per ml. After 30 minutes, the mixture was centrifuged, and the protamine- RNA precipitate was saved for the preparation of soluble RNA as previously described (21). Powdered ammonium sulfate (58.8 g) was added slowly to the supernatant solution to 40% saturation. After 1 hour, the mixture was centrifuged, the precipitate was discarded, and powdered ammonium sulfate (54.6 g) was added to the supernatant solution to 70% satura- tion. After standing for 2 hours with occasional stirring, the precipitate was collected by centrifugation at 15,000 x g for 30 minutes. The supernatant solution, largely hemoglobin, was discarded. The precipitate was suspended with gentle homoge- nization in approximately 100 ml of 0.1 M Tris buffer, pH 7.5, which was 70% saturated with ammonium sulfate (43.6 g dis- solved in 100 ml of the buffer). The suspended protein was centrifuged as before, and the precipitate was dissolved in 50 ml of 0.1 M Tris buffer, pH 7.5, and dialyzed for 18 hours against two changes of 0.02 M Tris buffer, pH 7.5, containing 0.001 M

GSH and 0.0001 M EDTA. The dialyzed enzyme fraction (ASTo) was stored at -20” in the presence of 0.025 M GSH and 0.0025 M EDTA.‘j This enzyme preparation was more stable and much more active than the “pH 5 enzyme” prepared as previously described (20). Amino acid-activating enzymes were found largely in the supernatant solution after precipitation of reticulocyte whole super at pH 5.

Assay Procedure for Amino Acid Incorporalion-The complete reaction mixture contained 0.5 ml of ribosomes (4 to 8 mg); 1 to 2 mg of enzyme fraction AST~; 50 to 100 pg of transfer RNA7 (21), or 100 to 200 pg of soluble RNA; 120 pg of creatine kinase; 1.0 pmole of dipotassium ATP adjusted to pH 7.5 with KOH; 50 pmoles of Tris chloride buffer, pH 7.5; 10 pmoles of creatine phosphate adjusted to pH 7.0 with HCl; 20 pmoles of GSH; 50 pmoles of KCI; 5 pmoles of MgC12; 0.25 pmoie of GTP; 0.1~ pmole of C14-amino acid; and 0.05 ml of amino acid mixture, in a final volume of 1.4 ml. The reactants were pipetted into tubes kept in an ice bath, and MgCL, enzymes, ribosomes, and CWamino acids were the last four constituents, added in that order just before incubation. The amino acid mixture used was that described by Borsook, Fischer, and Keighley (23), minus the W-amino acid under study.

The mixture was incubated for 40 minutes at 37”, 6 mg of

4 Ribosomes prepared in this way were about twice as active as those made with Medium A (4).

6 The Tris buffer is prepared at room temperature. It is used here at 4”, at which temperature the pH is 8.1.

6 Ammonium sulfate fractionation at pH 6.5 has yielded an enzyme preparation which was highly active and almost free of hemoglobin. Recently, with the phenylhydraxine injection pro- cedure given in footnote 4, twice as much protamine was reauired to yield an RNA-free enzyme.

7 Transfer RNA is defined (20) as that fraction of the soluble RNA which can form amino a&-RNA compounds. A purified fraction of the soluble RNA which appeared to contain largely this type of RNA was used in this case.

TABLE I Requirements for optimal incorporation of Q4-leucine into protein

The complete system contained the standard assay constitu- ents, including 5 mg of ribosomes, 2 mg of ASTo, and 125 rg of reticulocyte-soluble RNA. The results are given as c.p.m. per mg of ribosomes used.

Reaction mixture Activity

c.).m./ng

Complete system.................................. 1722 Complete, minus ATP and generating system.. 9 Complete, minus ribosomes. . 0 Complete, minus ASTo enzymes. 165 Complete, minus amino acid mixture. 743 Complete, minus soluble RNA. _. 739 Complete, minus GSH. 792 Complete, minus MgC12. . 82

casein were added, and the proteins were precipitated with 5% trichloroacetic acid. After standing for an hour at room tem- perature with occasional stirring, the mixture was centrifuged, and the protein precipitate was washed (resuspended and re- centrifuged) again with trichloroacetic acid. The protein pre- cipitate was then dissolved in 0.5 ml of 1 N NaOH and, after exactly 2 minutes, reprecipitated with trichloroacetic acid. The precipitate was washed again with trichloroacetic acid and then stirred for 5 to 10 minutes in 3 ml of 95% ethanol.8 Two vol- umes of ether were added, and the mixture was stirred thor- oughly and allowed to stand for 20 minutes to assure precipita- tion of protein. The precipitate was washed twice with ether, dried at 40”, and plated, and the dried precipitates were weighed and counted with a Nuclear gas flow counter with a Micromil window. All results were corrected for self-absorption. (-74-

amino acids were used at a specific activity of 7 PC per pmole, or results were corrected to that activity, and these gave 3 x lo6 c.p.m. per pmole.

RESULTS

General Requirements for Incorporation-Incorporation of C’4- leucine into total protein was dependent on the components shown in Tabie I. The total incorporation was equivalent to approximately 1.0 mpmole of C14-leucine per mg of ribosomal protein. Ribosome activity was highly reproducible, and low incorporation was usually due to the particular enzyme prepara- tion used. In order to show requirements for added enzymes, the washing procedure and medium described must be followed. Ribosomes washed once instead of twice, or washed in the ab- sence of MgCL, with higher levels of MgCL, or at a lower pH, did not give the low basal activity shown. It was particularly important to include GSH to study the basal activity, since the enzymes present as ribosomal contaminants in such cases were almost inactive unless GSH was present. Addition of soluble enzymes to ribosomes in the absence of GSH may stimulate incorporation because of the reducing properties of the prepara- tion, or for other reasons, rather than because of a direct involve- ment of the enzyme fraction in protein synthesis. When micro- somes, rather than ribosomes, are used, the effects of added enzyme components are particularly difficult to interpret, since

* Heating at 60” gave similar results and appeared to offer no advantage over the procedure described.

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762 Hemoglobin Synthesis. I Vol. 237, No. 3

3 4 5 6 7 6 RIBOSOME CONCENTRATION (MG)

FIG. 1. Incorporation of CWleucine into protein with different amounts of ribosomes. The values are given per assay tube with standard assay conditions with 2 mg of ASTo.

Oo- ---xi ENZYME CONCENTRATION (MQ

FIG. 2. Incorporation of C”-leucine into protein at various enzyme concentrations. The specific activity is given in counts per minute per mg, and the enzyme concentration, in milligrams per assay tube, with standard assay conditions with 4.3 mg of ribosomes .

I Oo

4 I 5 IO 20 “40 60

1

GSH CONCENTRATION (uMOLE9

FIG. 3. Effect of GSH concentration on C”-leucine incorpora- tion into protein. The specific activity is given in counts per minute per mg, and the GSH concentration, in micromoles per assay tube. Standard assay conditions were used, with 2.1 mg of freshly prepared enzyme. The tube without added GSH con- tained 0.01 pmole of GSH from the dialysis (see “Methods”).

these particles cannot be freed of soluble proteins by washing. The characterization of the reticulocyte particles as ribosomes has been reported by Dintzis, Borsook and Vinograd (24).

Effects of Enzyme and Ribosome Concentration-In the stand- ard assay system the ribosomes are the limiting component. These are saturated and will no longer incorporate after the standard incubation. Incorporation was therefore dependent

on ribosome concentration (Fig. 1). The specific activity of different ribosome preparations was quite similar, and this ac- tivity was maintained for 4 to 6 weeks when the preparations were stored at -20” in 0.25 M sucrose. For example, ribosomes which incorporated 1450 c.p.m. per mg initially incorporated 1100 c.p.m. per mg after 3 weeks, and 1015 c.p.m. per mg after 6 weeks of storage. Ribosomes stored at -20” in media con- taining MgClz aggregated and lost activity rapidly.

With lower amounts of soluble enzyme fractions, incorpora- tion was roughly proportional to enzyme concentration (Fig. 2). For accurate assays of enzyme activity, only the lower amounts of enzyme can be used. The AS0 fraction has been largely freed of hemoglobin and was approximately twice as active per unit of protein as the whole supernatant solution. Also, inhibitory factors were apparently removed in the purification, since the highest level of A& gave considerably more total incorporation than the highest levels of whole supernatant solution. The ASTo fraction could not be stored or dialyzed in the absence of GSH without large losses in activity, but even under the condi- tions described, approximately 50% of the activity was lost after 10 days’ storage at -20’. Similar protection by GSH has been reported for a protein fraction which stimulated amino acid incorporation by liver microsomes (25). Further purifica- tion of the soluble enzyme fraction has met with little success, and has not been pursued because of the presumably large number of enzymes necessary for protein synthesis. Various other fractions, such as the AS-l and AS-2 fractions from guinea pig liver (20) had low activity, whereas the pH 5 enzyme frac- tion from guinea pig liver was usually only 50% as active, even at the highest level, as the A& fractions.

Effect of -SH Compounds-A requirement for GSH during the preparation and storage of enzyme fractions was discussed above. In addition, GSH stimulated incorporation when added to the incubation mixture (Fig. 3). Cysteine and mercapto- ethylamine were not quite as effective as GSH. The function of GSH during the incubation is probably also an effect on preservation of enzyme activity, rather than any direct role in protein synthesis. This conclusion is based on the lack of speci- ficity for the -SH compound, the high level of GSH required for optimal activity (Fig. 3), and the demonstration of such a role for GSH in amino acid activation (20) and amino acid transfer from amino acyl-RNA (14).

Effect of ATP and Related Compounds-When the standard assay conditions were used, there was no incorporation in the absence of ATP (Fig. 4). The optimal ATP level varied in different experiments between 0.5 and 2.0 pmoles of ATP, and incorporation levels in this range were similar. High ATP levels were completely inhibitory, but when the MgC12 level was kept at twice the ATP level, the inhibition was much less (Fig. 4). The effect of high ATP levels was probably due to ribosome breakdown (15). I f the ATP-generating system was omitted, incorporation with U-and ‘i:U”pmoie of ‘AT?! ‘was i5and %.SyG respectively, of the complete system. Creatine phosphate plus creatine kinase, or phosphoenolpyruvate plus phosphoenolpyru- vie kinase, gave similar results. The optimal creatine phosphate concentration was usually between 5 and 10 pmoles per assay tube, and higher levels were somewhat inhibitory.

No incorporation was observed in the absence of MgC12, and, as with other components, an optimal level with inhibition at high levels was found (Fig. 5). The requirement for MgC& was

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rather specific, and MnCls at 5 pmoles per assay tube was only 63% as active.

The effects of nucleoside triphosphates were irregular. In earlier studies with soluble enzymes from guinea pig liver (4), a consistent stimulation by GTP was observed. With ASo, this effect was not usually found. However, GTP was included in the reaction mixture, since its role in protein synthesis has been well documented (2), and added GTP was required for CY4-amino acid transfer from amino acyl-RNA to reticulocyte ribosomes (ll), as in other systems (2). Enzymes which form GTP in the presence of the ATP-generating system have been found in AS7o.l No consistent stimulation with UTP or CTP could be shown.

E$ect of Amino Acid Concentrations-A number of concentra- tions of the amino acid mixture were tested with each concen- tration of CY-leucine. The level of Cl4-leucine did not appear to be too critical, and similar results were obtained with 0.05, 0.1, and 0.2 pmole. The concentration of the amino acid mix- ture, however, showed an optimal stimulation at 0.05 ml and some inhibition at higher levels (Fig. 6). At the optimal level, the amounts of individual amino acids in the mixture varied from 5 pg for glycine to 0.5 pg for isoleucine. The effect of varying individual amino acid levels has not been studied. With freshly dialyzed enzyme, a 2- to 3-fold stimulation of the

-- (u ‘6

‘0 x 15 -N

c -N

----o

5 g 12 a

g9 G aw ~06

lc 4

0 ’ 0.1 0.5 to 2.0 5.0 10.0 ATP CONCENTRATION @MOLES)

FIG. 4. Effect of ATP concentration on C”-leucine incorpora- tion into protein. The units and assay conditions are the same as Fig. 3. The solid circles show results with 5 pmoles of MgC12; the open circles, with twice the concentration of MgCL as ATP (10 and 20 rmoles, respectively).

Mg Cl2 CONCENTRATION (pMOLES)

FIQ. 5. Effect of MgClz concentration on CY-leucine incorpora- tion into protein. The units and assay conditions are those previously noted.

24t -

I OO

I 0.025 0.05 - I 0.1 0.2

AMINO ACID MIXTURE (MU

FIG. 6. Effect of concentration of amino acid mixture on C14- leucine incorporation. The units and assay conditions are those previously noted.

incorporation of C14-leucine and other C14-amino acids was ob- tained regularly.9

E$ect of Salt Concentrations-A consistent stimulation of amino acid incorporation was obtained by the addition of KC1 or NaCl to the reaction mixture. The optimal level of KC1 was 50 pmoles per assay tube. In some experiments, 100 pmoles of KC1 gave results similar to those obtained with 50 pmoles, but 100 pmoles were occasionally, and 200 pmoles, usually, quite in- hibitory. When the KHC03 of Medium R was replaced with Tris chloride, the incubation medium still contained a total of 24 pmoles of Na and K ion. Under these conditions, a 3-fold stimulation of incorporation with 60 pmoles of KC1 was ob- tained. The incorporation observed without added KC1 would have been decreased, presumably, if the cations of the ATP and creatine phosphate had been absent. NaCl was 75% as effective in stimulating incorporation as KCl. These stimulations re- semble, at least qualitatively, those reported for liver micro- somes by Sachs (25), but NaCl was ineffective in that case.

Time Course of Incorporation-Incorporation into total protein was linear for the first 10 minutes and completed usually by 30 minutes. Occasionally incorporation continued for longer peri- ods up to 50 minutes, but in these cases the total incorporation was somewhat less. When the ribosomes and supernatant pro- tein were separated after incubation and counted separately (Fig. 7), the ribosomes were labeled first and reached a plateau, whereas the radioactivity in soluble protein continued to rise. The radioactivity in soluble protein often reached twice that in the ribosomes. Similar kinetic studies with intact cells have provided evidence that ribosomes are the site of protein synthe- sis (2).

The reasons for cessation of incorporation are not known. However, the failure is probably due to some ribosomal compo- nent. This was shown by studies in which various components of the system were added after incorporation had ceased. Ad- dition of fresh soluble components either singly or together had little effect, but ribosomes added to the original mixture incorpo- rated almost as well as the first ribosomes. These findings were investigated in more detail by preincubation experiments (Table II). Soluble components were stable, as were ribosomes alone,

0 Recently, similar results have been obtained with an amino acid mixture which furnished 0.05 pmole of each amino acid per assay tube (G. Favelukes and S. Favelukes, unpublished observa- tions).

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764 Hemoglobin Synthesis. I Vol. 237, so. 3

0 IO 20 30 40 50 60 TIME IN MINUTES

FIG. 7. Time course of CY-leucine incorporation. The units and assay conditions are those previously noted. After incuba- tion, the contents of each assay tube were cooled and transferred to centrifuge tubes, diluted to 3.5 ml with Medium B, and cen- trifuged for 1 hour at 105,000 X g. The supernatant protein (solid. circles) and precipitated ribosomes (open circles) were counted separately.

TABLE II

E$ect of preincubation on CWeucine incorporation The preincubation mixture was shaken at 37” for 20 minutes;

then the omitted constituents were added to make the complete system. The mixture was then incubated as usual. The “com- plete” system preincubation contained everything but C”-leu- tine; “ribosomes alone” were suspended in Medium B; “complete, no ribosomes” is self-explanatory; “complete, no energy” con- tained everything but ATP, GTP, and creatine phosphate. The standard assay, not preincubated, was assigned the 100% value.

Preincubation mixture Total incorporation after regular assay

Complete, no Ci4-leucine ...................... Ribosomes alone. .............................. Complete, no ribosomes. ...................... Complete, no energy. .........................

% 31.5 77.0 85.2 65.0

since these incorporated nearly as well after the preincubation as did unincubated controls. The largest difference was seen when all the constituents with and without the energy-generat- ing components were compared (Table II). The addition of the energy-generating components during the preincubation resulted in inactivation. It should be noted that under these conditions (complete system minus Q4-leucine), endogenous CWleucine would permit CWamino acid incorporation to take place. Since the addition of energy-generating components had no effect in the cold, it seems likely that inactivation was asso- ciated with the incorporation process itself.

Soluble Intermediates in Amino Acid Incorporation-The stimulation of incorporation by added transfer RNA or soluble reticulocyte RNA (Table I) suggested that amino acyl-RNA compounds were intermediates in hemoglobin synthesis. Such effects on amino acid incorporation had been observed in other systems (26, 27). Further evidence that the stimulation by RNA was related to its ability to form amino acyl-RNA com- pounds has been reported (28). The role of amino acyl-RNA compounds, and other possible soluble intermediates, was stud- ied further by “chase” experiments. All of the soluble compo- nents were preincubated with C14-leucine; then a loo-fold excess

of C12-leucine was added along with ribosomes. Incorporation into protein under these conditions would come from all soluble intermediates formed in the absence of ribosomes and only from such intermediates. The total radioactivity in protein in these experiments (Table III) agreed closely with the amount of amino acyl-RNA formed. Thus, all the C4-leucine in protein passed through C14-leucyl-RNA, but the results do not exclude the possibility of a second soluble intermediate present in small amount in comparison with amino acyl-RNA compounds. In another part of these experiments, only ribosomes were added (no CY-leucine), and the total incorporation was measured (Table III). A number of such experiments showed that the amount of extra CWleucine incorporated into protein because of the addition of soluble RNA was 15 to 40 times the CY4-leucyl- RNA formed by that amount of RNA (Table III). These re- sults demonstrated a “catalytic” role for transfer RNA in pro- tein synthesis (28).

In line with this catalytic role, it was suggested that transfer RNA did not break down during the process of forming amino acyl-RNA compounds and tranferring them to protein (28). This was tested here by the use of soluble RNA labeled with ATP-8-C14, essentially as described by Hecht, Stephenson, and Zamecnik (29). The V-RNA, labeled predominantly in the terminal adenosine, was incubated in the regular assay system for amino acid incorporation, except that CY-amino acids were used. After incubation, 18% of the original radioactivity was associated with ribosomal RNA, and 55% was recovered as soluble RNA. Since an excess of Ci2-ATP was present in the reaction mixture, and, as noted above, the RNA “turned over” 15 to 40 times as far as amino acid transfer was concerned, the amount of transfer RNA breakdown or integration with ribo- somal RNA was very small. In particular, these results seem to exclude the removal and replacement of terminal nucleotides as a step in the transfer of amino acids from transfer RNA to ribosome protein.

Effects of Inhibitors on Amino Acid Incorporation-As an ap-

TABLE III

Transfer of C”-leucine from soluble intermediates to protein

The “preincubation” contained all of the soluble components, but no ribosomes, GTP, or KCl. Either 100 pg of transfer RNA or 300 pg of reticulocyte-soluble RNA were used, as indicated. These mixtures were incubated in triplicate for 5 minutes. One tube was used for the determination of CWleucyl-RNA. Ribo- somes, GTP, and KC1 were then added, and the usual “incuba- tion” was done with the-other two_tubes Oneof _these contained 10 pmoles of CWleucine. The total radioactivity is given.

Preincubation I

Incubation

Transfer RNA. None Transfer RNA. Plus Cn-leucine Transfer RNA. No Cn-leucine*

Reticulocyte RNA. None Reticulocyte RNA. Plus CWleucine Reticulocyte RNA. No Ciz-leucine*

wleyl-

c.p.m. 430

678

W-leucine in protein

c.p.m.

375 9,848

668 11,200

* These counts are those incorporated because of the added RNA. A control without added RNA was always included, and the Ci4-leucine incorporated into protein under these conditions has been subtracted (28).

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preach to the question whether the cell-free system resembled hemoglobin synthesis in intact cells, the effects of several inhibi- tors were compared (Table IV). Results in both systems were similar and support the conclusion that a limited amount of hemoglobin was synthesized in the cell-free system. Chloram- phenicol, which is an inhibitor of protein synthesis in bacterial systems (30), did not inhibit either system,‘0 whereas puromy- cinli inhibited both systems. Puromycin has been reported to inhibit amino acid incorporation into liver microsomes (31). It was suggested that the inhibition was related to the structural similarity between the antibiotic and the terminal amino acyl- adenosine of soluble amino acyl-RNA. Inhibition of amino acid incorporation by puromycin has been studied in detail in the present system. The incorporation of CWvaline, Wleucine, and CWlysine was inhibited to an equal extent by various levels of puromycin. Thus, it was not acting as an amino acidanalogue. Very low levels of puromycin were inhibitory in the complete sys- tem. At puromycin concentrations of 7 X lo-+ M, 1.5 X lO+ M,

and 4 X 10-fi~, C”-leucine incorporation was inhibited 88.1’+& 65.0 %, and 44.0 %, respectively. Puromycin inhibition was not decreased by increasing the enzyme, the GTP, or the soluble RNA concentrations Infact, the same inhibitionof incorporation ~a+s observed in the presence and absence of transfer RNA. In a repre- sentative experiment, incorporation in the complete system was 1413 c.p.m. per mg and, in the absence of added transfer RNA, was 890 c.p.m. per mg. When 1.5 X 1W5 M puromycin was nresent,.incorp-oration was 586 and 315 c.p>m. per mg,respec- ihfy.- Lkdn-b~e~~+A; th-erefwe; d+&- ii&-i emrse- ihe-~&CL-VI*- puromycin but gave the same relative stimulation of incorpora- tion. These results indicate that puromycin inhibition is due to a direct effect of the antibiotic on the ribosome. More detailed studies which confirm the direct effect of puromycin on ribo- somes have been reported (11, 15) (see “Discussion”).

Incorporation in this system was extremely sensitive to RNase addition. As little as 0.1 pg of pancreatic RNase added at the start of the incubation gave more than 90% inhibition. At these levels, it is possible that transfer RNA and possibly ribo- somal RNA were affected. However, by preincubation with low levels of RNase, a direct effect on the ribosome was found. Ribosomes were preincubated for 5 minutes at 37” in the pres- ence of 1 pmole of MgClz plus 0.005 gg of RNase. The usual assay constituents were then added. Incorporation was in- hibited 43%, compared with the control in the absence of added transfer RNA. Furthermore, added transfer RNA had little effect, so that, compared with the control tith added transfer RNA, the inhibition was 66%. The control with 0.005 pg of RNase added initially showed only 13% inhibition. Similar results were obtained when ribosomes were preincubated with RNase, then diluted and centrifuged at 105,000 X g in order to reduce the RNase level in the assay. The most likely interpre- tation of these results is that some ribosomal nucleic acid com- ponent is quite sensitive to destruction by RNase.

Many other types of inhibitors have been tested. High salt concentrations, pyrophosphate, and high ATP all inhibited in- corporation. These effects were primarily due to ribosome breakdown, in contrast to the results with RNase and puromy-

10 At higher levels of chloramphenicol, considerable inhibition of incorporation in the whole cell was noted. Studies are in progress to determine whether this is a direct effect on protein synthesis.

11 Kindly provided by Dr. E. Stokstad, Lederle Laboratories.

TABLE IV Effect of inhibitors on CWeucine incorporation in

intact cell and cell-free system

Intact cells were incubated for 10 minutes under the conditions described by Borsook, Fischer, and Keighley (23). Standard cell-free incubations were used. Chloramphenicol, spermine, and puromycin were present at concentrations of 10-3, 10-8, and 10-’ M, respectively.

C4eucine incorporated Additions

Intact cells Cell-free system

% % No inhibitor. 100.0 100.0 Plus chloramphenicol. 79.0 97.0 Plus puromycin.. 2.0 5.2 Plus spermine. 1.0 24.0*

* In other experiments, this level of spermine resulted in less than 5% incorporation.

tin (see “Discussion”). Incorporation was inhibited greatly by low levels of sulfhydryl inhibitors in the absence of GSH, and p-chloromercuribenzoate, for example, was an excellent inhibitor. This was expected on the basis of its inhibition of amino acid- activating enzymes (20) and amino acid transfer enzymes (14).

A number of amino acid analogues, such as 5-methyltrypto- phan, norleucine, norvaline, methionine sulfoximine, pfluoro- phCBy4&l~Ti~C~ LiTid ~t~T&niili~; ~-ii~t-;l~-at-O.~~ ’ M-&en-~ tested in the absence of added amino acids. Although some of these analogues may have been incorporated in place of the cor- responding amino acid and so shown no effect, inhibition by at

least one of these analogues might have been expected. The failure of amino acid analogues to inhibit incorporation is being studied further.

DISCUSSION

The system used in these studies differs from many of the earlier amino acid-incorporating systems in several respects. The use of washed ribosomes plus partially purified, RNA-free, soluble enzymes has made it possible to delineate more clearly the optimal requirements for amino acid incorporation. Sec- ondly, the formation of soluble C14-labelled protein, consisting largely of hemoglobin, has led to direct evidence for protein syn- thesis in a cell-free system and to detailed studies of the intra- ribosomal events in this process.

The conclusion that amino acyl-RNA compounds were inter- mediates was indicated by the stimulation of incorporation with added RNA as in other systems (26, 27), and, as reported previ- ously, only by soluble RNA capable of forming amino acyl-RNA (28). Amino acyl-RNA compounds were the only soluble in- termediates found. Added transfer RNA acted catalytically, carrying 15 to 40 residues of amino acid into protein per RNA molecule. Little or no breakdown of the RNA was observed during amino acid incorporation. Recent studies with amino acyl-RNA compounds have also provided evidence for the cata- lytic role of transfer RNA (32, 33). The failure to obtain more than 3-fold stimulation of incorporation with added transfer RNA has not been explained, but may be due to the presence of transfer RNA in the ribosome (28). The problem of various types of RNA in the ribosome has been discussed previously (11). Further studies, starting with Cr4-amino acyl-RNA com-

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766 Hemoglobin Synthesis. I Vol. 237, No. 3

pounds, have provided direct evidence that these compounds were hemoglobin precursors, as CY-hemoglobin was isolated starting with CY4-leucyl-RNA (10, 11).

One of the most interesting points which has come from the detailed study of the requirements for incorporation has been the narrow optimal ranges for most of the components. Most of the components added were inhibitory at higher than optimal concentrations, and the optimal concentration also depended on the concentration of other components of the system. These interactions result in a very delicately balanced system. Wide variations in amino acid incorporation from day to day may occur when these numerous variables are not controlled. In earlier studies with other systems (2), KC1 and GSH were often omitted. The function of KC1 is not known, but it may be related to the effect of KC1 on ribosome structure (34). The function of GSH was to preserve the activity of amino acid- activating (20) and transfer (14) enzymes.

Another new aspect of this system was the extensive forma- tion of labeled soluble protein. The reasons for this are not known, but may be related to the high level of incorporation found in this system. That is, since only completed protein was released (15), low incorporation might not give any finished protein, and therefore amino acid incorporation into ribosomes or microsomes, but not into soluble protein, might result. This has been the experience with the present system. When poor incorporation was observed, because of a poor enzyme prepara- tion or other reasons, the labeling of soluble protein was usually reduced much more than ribosome labeling. There may also be enzymatic or other factors involved in the release of protein from ribosomes (see below), but there does not appear to be any requirement for the membrane or phospholipid components of microsomes in order to produce finished protein in the reticulo- cyte system, as suggested earlier (2). The early labeling of ribo- somal protein, followed by that of soluble protein (Fig. 7), pro- vided indirect evidence that ribosomes were the site of protein synthesis. However, this and the more elegant “chase” experi- ment either with intact cells (2) or in a cell-free system (35) does not provide definitive evidence on this point. This type of data indicates that an “intermediate” for proteir synthesis is formed in the ribosome. This could be a store of “activated” amino acids, or other compounds. For the ribosome to be the site of synthesis, the “chase” experiment must show that the radioac- tivity lost from the ribosome is quantitatively found in protein in the presence of unlabeled soluble intermediates, and that the radioactive intermediates in the ribosome have the sequence of the soluble protein formed, Tn the present system, the first criterion has been satisfied (15)) and evidence indicating that the second is true has been reported (13).

One of the many unsolved problems in this system revolves around the question of why incorporation ceases after some time (Fig. 7). This was probably due to failure of a ribosomal com- ponent, rather than any of the soluble components, and ap- peared to be associated with the incorporation process itself. That is, preincubation with all the components of the system gave the greatest loss of incorporating ability (Table II). Stud- ies in the analytical ultracentrifuge have shown that ribosomes did not dissociate during the incubation and were largely 80-S particles after cessation of incorporation2 Thus ribosome breakdown is not the cause of the failure to incorporate. Tis- s&es, Schlessinger, and Gros (35) have suggested that in an Escherichia coli ribosome system, incorporation failure is due to

the failure to relase finished protein from the ribosome. In their model, the ribosome dissociates in order to release finished pro- tein. No evidence for ribosome breakdown was found in this system, even though soluble protein radioactivity often reached twice that in the ribosomes. Our results also suggest that failure to release completed chains is not the reason why in- corporation ceases. Some evidence for this is seen in Fig. 7, where the radioactivity in the ribosomes decreased somewhat toward the longer time periods. This was not always the case but suggests that, when it occurred, ribosomes with a decreasing ability to incorporate could still release chains previously com- pleted. Depletion or loss of a ribosomal component in the proc- ess of amino acid incorporation is therefore indicated as the cause of the cessation of incorporation. In order to study this problem in detail, it will be necessary to dissociate the incorpo- ration process, i.e. synthesis of protein chains on the ribosome (13), from release of such chains. Two methods which permit, release of labeled protein in the absence of incorporation are being studied. The treatment with small amounts of RNase, which completely inhibited incorporation, did not inhibit release of labeled protein2 (11). The inhibition of incorporation by small amounts of RNase, as noted, was probably due to the cleavage of a few bonds in some RNA moiety of the ribosome which is critical for amino acid incorporation. No detectable RNA hydrolysis was observed under these conditions.2 Since en- zymatic components were required for release of labeled protein from RNase-treated ribosomes, these results suggest that a specific mechanism is required for release.

Inhibition of amino acid incorporation by puromycin resulted from a direct effect of this antibiotic on the ribosome, as in- hibition was not reversed by the addition of soluble components. This conclusion is supported by the direct effect of puromycin on ribosomes reported previously. Ribosomes alone, incubated with the same levels of puromycin used for inhibition, released labeled hemoglobin precursors without detectable ribosome breakdown (11, 15). Puromycin inhibition of protein synthesis, therefore, appears to result from the attachment of puromycin to one or more sites on the ribosome, displacing the hemoglobin precursors which are present and also blocking further peptide bond formation. It should be noted that the foregoing discus- sion has implied that each ribosome in the cell-free system can, at most, synthesize and release once. In contrast, in the intact cell, each ribosome synthesizes many molecules of hemoglobin (24). Evidence for this viewpoint and for the concept that globin chains are assembled in the ribosome in sequence starting with the NH&erminal amino acid has been reported (13, 36).

SUMMARY

1. The requirements for optimal incorporation of CY4-amino acids into protein with the reticulocyte ribosome system have been described.

2. Incorporation was stimulated by the addition of transfer ribonucleic acid, and amino acyl-ribonucleic acid compounds were the only free soluble intermediates found. Transfer ribonucleic acid acted catalytically and was not destroyed in the course of the incubation.

3. Several inhibitors showed similar effects in the intact cell and the cell-free system, thus providing further evidence that incorporation in this system represented hemoglobin synthesis. Puromycin at low levels inhibited incorporation, and its action was localized to an effect on the ribosome. Tiny amounts of

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March 1962 E. H. Allen and R. X. Schweet 767

ribonuclease also inhibited incorporation, probably because of its 15. action on a ribosomal component.

4. Evidence was presented that the assembly of peptide 16.

chains occurred in the ribosome. The cessation of incorporation 17. in this cell-free system appeared to result from the loss or in- activation of a ribosomal component during synthesis, but not 1% from a failure to release completed chains or from ribosome break- down. The problem of the release of completed chains and the

I9 .

use of puromycin and ribonuclease in the study of this problem 20. were discussed.

21.

1.

2.

3.

4.

5.

6.

7. 8. 9.

10.

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Esther H. Allen and Richard S. SchweetCOMPLETE SYSTEM

Synthesis of Hemoglobin in a Cell-free System: I. PROPERTIES OF THE

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