Protein synthesis by isolated nucleoprotein particles

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 86, 159-170 (1959)

Protein

Synthesis by Isolated

Nucleoprotein

Particles’

George C. Webster From the Department of Agricultural Biochemistry, Columbus, Ohio Received

April

The Ohio State University,

14, 1959

INTRODUCTION

Ket protein synthesis from amino acids has been reported wit’h cell-free preparations of Staphylococcus aureus (a), peas (3, 4), Bacillus megatetium (5), Alicaligenes faecalis (6), and Tetrahymena myriformis (7). In each instance the experimental system has been relatively crude, and the amount of protein formed was usually small. However, the systems do offer the opportunity for the study of actual protein synthesis, in contrast to the study of amino acid incorporation into protein, where it is often difficult to judge whether the incorporation measured represents protein synthesis. In order to understand the mechanism of protein synthesis, therefore, it is of some interest to purify an enzyme system involved in net protein synt’hesis and to learn more concerning t,he characteristics of the protein synthesis process. An experimental system that is capable of net protein synthesis (4) has now been purified somewhat from cell-free extracts of peas. The system consists of ribonucleoprotein particles which exhibit one major component in the ultracentrifuge (S), and which closely resemble, in physical and chemical characteristics, the microsomal nucleoprotein particles which have been found in extracts of a variety of plants, animals, and bacteria (8, 9). The particles are able to incorporate amino acids into their protein (lo), but, more importantly, as is reported in the present communication, they are able to form a group of ‘koluble” proteins which appear to have at least some biological activity. EXPERIMENTAL Ribonucleoprotein

particles

were isolated

from

S-day-old

peas as described

pre-

viously (10). Insofar as possible, the temperature was maintained at 1°C. during 1 Supported by grants from the Herman Frasch Foundation and the National Science Foundation. Presented before the annual meeting of the American Society of Biological Chemists, Atlantic City, Ic’. J., April, 1959. A preliminary report concerning this investigation has appeared elsewhere (1). 159

160

WEBSTER

isolation. The particles were shaken in a Dubnoff metabolic incubator at 38°C. with the reaction mixtures described with the accompanying tables and figures. At the end of the incubation period, the mixtures were cooled rapidly to about 3”C., in an ice-water bath. The particles were then sedimented by centrifugation at 105,000 X g (40,000 r.p.m. in the No. 40 rotor) for 90 min. in a Spinco preparative centrifuge. The supernatant solution was carefully separated from the sedimented particles, and both particles and supernatant solution were assayed for protein by the biuret method (11). In early experiments, protein was also determined by both the modified phenol method of Lowry et al. (12)) and by the micro-Kjeldahl procedure (13). The apparent polynucleotide factor required for protein synthesis by the nucleoprotein particles was prepared in the following manner. The supernatant solution remaining after a cell-free extract of peas (10) had been subjected to centrifugation at 105,000 X g for 60 min. was mixed with an equal volume of Mallinkrodt “Giltlabel” liquid phenol. Polynucleotide was isolated from this mixture and purified by methoxyethanol as described by Kirby (14). Amino acid polynucleotide was prepared in the same manner as reported previously (15). Hydrolysis of ATP2 was measured by incubation of 0.1 ml. of protein solution with 0.1 M Tris-HCl (pH 7.5), 0.01 M sodium ATP, and 0.005 2M MgSOh (in a total volume of 1 ml.) for 15 min. at 38°C. Inorganic phosphate released was determined by the method of Fiske and SubbaRow (16). Electrophoresis was performed in a Perkin-Elmer model 38 electrophoresis apparatus. Amino acid incorporation into protein was measured as described previously (10). The mixture of C%labeled amino acids consisted of a hydrolyzate of W-labeled yeast protein (Schwarz Laboratories) supplemented with tryptophanCl4 (New England Nuclear Corp.), nonlabeled asparagine, and glutamine-CY4, prepared from glutamate-C4 by the action of highly purified glutamine synthetase (17). Determination of the possible N-terminal nature of incorporated alanine-Cl4 was performed by reaction of the isolated ribonucleoprotein particles, following incubation with the reaction system, with fluorodinitrobenzene as described by Sanger (18). The particulate protein was hydrolyzed for 24 hr. at 100°C. in 5.7 N HCl. Carrier N-dinitrophenylalanine (5 mg.) was added to the hydrolyzate, and any labeled Ndinitrophenylalanine resulting from hydrolysis was isolated by chromatography on silica gel as described by Porter (19). N-Dinitrophenylalanine was prepared as described by Sanger (18).

RESULTS

When a suspension of ribonucleoprotein particles is incubated with an amino acid mixture, ATP, GTP, Mn++, phosphoglycerate, and a polynucleotide fraction (see Experimental section), the protein level of the particles does not change appreciably (Fig. 1). However, the protein level of the medium surrounding the particles steadily increases for about 30 min. Table I shows that this increase in nonparticulate protein is essentially the same whether measured by the biuret reagent (ll), the modified phenol method of Lowry et al. (12), or the micro-Kjeldahl procedure (13). The nonparticulate protein formed during this reaction is almost completely precipitated by 5% trichloroacetic acid, by exposure to 100°C. for 3 min., 2 Abbreviations used : ATP, adenosine triphosphate ; Tris, tris (hydroxymethyl) aminomethane; GTP, guanosine triphosphate; DNP, dinitrophenyl.

-

PROTEIN

161

SYNTHESIS

TIME

(MIN

1

FIG. 1. Particulate and nonparticulate protein levels during the incubation of ribonucleoprotein particles with an amino acid mixture. Complete system contained: 0.05 M Tris-HCl (pH 7.5); 6.0 mg. of an amino acid mixture containing 0.3 mg. of each of the following L-amino acids and amides : alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, histidine, isoleucine, leucine, lysine, tryptophan, tyrosine, and methionine, phenylalanine, proline, serine, threonine, valine; 0.0001 M ATP; 0.0003 M GTP; 0.0003 M MnClz ; 0.0001 M KCl; 0.01 M phosphoglycerate; 3.0 mg. of purified polynucleotide (prepared as described in the Experimental section) ; and 8.0 mg. of ribonucleoprotein particles (containing 4.8-4.9 mg. protein) in a total volume of 10 ml. Particulate protein refers to the protein of “soluble” protein refers to that protein not sedithe ribonucleoprotein particles; mentable with the ribonucleoprotein particles. TABLE Formation Reaction

of “Soluble” system Time

min. 0 30

Protein

and procedure

I

as Determined

by Various

was the same as described

Assay

with

Methods

Fig. 1.

Protein formed, mg. Biuret

Phenol reagent

0.0 1.9

0.0 1.8

micro-Kjeldahl

0.0 2.3

by 70% ethanol, and by saturated ammonium sulfate. The precipitates from trichloroacetic acid and from boiling are no longer soluble in 0.1 M Tris buffer of pH 7.5. The precipitate from 70% ethanol, however, is partially soluble, and the precipitate from ammonium sulfate is completely soluble. The nonparticulate protein disappears upon hydrolysis in 5.7 N

162

WEBSTER

TIME

(h4IN.I

2. Amino acid’incorporation into particulate and nonparticulate protein during the incubation of ribonucleoprotein particles with a mixture of C14-amino acids. Reaction system and conditions were the same as those described with Fig. 1, except the amino acid mixture was replaced with an equal concentration of Cl*amino acids having a specific activity of 50,000 counts/min./mg. of amino acid mixture. Total “soluble” protein formed after 60 min. was 1.9 mg. FIG.

HCl at 100°C. for 24 hr. Two-dimensional paper chromatography of the hydrolyzate (phenol-water in one direction, butanol-acetic acid-water in the other) produces a pattern of ninhydrin-reactive spots essentially the same as that obtained from a hydrolyzate of the nonparticulate protein of peas. The material formed by incubation of ribonucleoprotein particles with an amino acid mixture and the cofactors, therefore, reacts in every case as protein. Figure 2 shows the results of protein formation in the presence of a mixture of C4-amino acids. It can be seen that the particles incorporate only a relatively small amount of amino acid. In contrast, the ‘koluble” protein, which appears in the medium surrounding the particles, incorporates relatively great amounts of amino acid, and has a specific activity similar to that of the W-amino acid mixture. This demonstrates that the protein which appears in the medium surrounding the particles is formed from the mixture of free amino acids and not from the particulate protein. The protein formed from free amino acids in the reaction described above exhibits two major and three minor components upon electrophoresis (Perkin-Elmer model 38 apparatus) in pH 8.6 Verona1 buffer of 0.1 ~1ionic strength. Although further physical characterization of the synthesized protein has not yet been performed, the electrophoretic pattern indicates that more than a single species of protein is formed by the reaction.

PROTEIN

163

SYNTHESIS

TABLE

II

Requirements for Protein Formation “Soluble” protein formed

System”

Complete Minus nucleoprotein particles Minus ATP or GTP Minus polynucleotide Minus Mn++ Minus glutamine and asparagine Minus leucine Minus lysine Minus tryptophan Plus ribonuclease Plus chloramphenicol

m&Y. 1.8 0 0

0.3-0.5 0 0 0 0 0 0.2-0.6 0.3

a Complete system was the same as described with Fig. 1. The reaction mixtures were shaken for 30 min. at 38°C. Ribonuclease concentration was 0.1 mg./ml., and chloramphenicol was 0.001 M. Complete inhibition of protein synthesis is obtained if the particles are preincubated for 15 min. with ribonuclease.

Characterization

of the Synthesis

Reaction

Table II presents the effects of omission of various components of the reaction system on protein formation. It is evident that maximal protein formation requires the presence of ATP, GTP, Mn++, and polynucleotide. ATP and GTP cannot be replaced by other nucleoside triphosphates. Mn++ can be partially replaced by Co++ or Mg*, but other bivalent cations are inactive. Maximal synthesis is obtained if a low concentration (1O-4 LV) of magnesium ions is present in addition to manganese ions. Protein formation also requires phosphoglycerate or phosphoenolpyruvate, presumably for the regeneration of ATP. As might be expected, omission of the amino acid mixture completely eliminat,es protein formation. However, it is of more interest that omission of any of several individual amino acids or the two amides, glutamine and asparagine, also completely abolishes synthesis (Table II). Not only are amino acids and the various cofactors required for protein synthesis, but the synthesis process is greatly dependent upon the concentration of each of the required components of the reaction system. This is shown by the data of Fig. 3, which illustrates the dependence of protein formation on pH, and on the concentration of amino acids, ATP, GTP, and bivalent cations. Although Mn++ can be replaced partially by either Mg++ or Co++, each cation differs somewhat in its optimal concentration. The experimental system is relatively unstable. During preparation, the nucleoprotein particles are kept at a temperature of 1°C. insofar as possible. Table III shows the effect on subsequent protein formation of higher tem-

164

WEBSTER

PH 20-

2.0 -

I2

12

: I H

x

:,!/Jf!

AMINO MIXTURE ACID

04

04 60

70

80

02

0.6

1.0

a5

a3 MICROMOLES

PER ML

FIG. 3. Effect of reaction conditions on protein formation. Ordinate: total protein formed, milligrams. Abscissas: pH units for pH, mg./ml. for amino acid mixture, and pmoles/ml. for ATP, GTP, Mn++, Mg++, and Co ++. Reaction conditions were those given in Fig. 1, with the exceptions noted in the above figure. The reaction mixtures were shaken for 60 min. at 38°C. TABLE Effect

III

of Various Temperatures

during Particle Isolation of the Particles to Form Protein

on the Subsequent

Ability

Nucleoprotein particles were isolated, insofar as possible, at? the temperatures given above. The particles were then incubated under the conditions described with Fig. 1. Approximate. temperature during particle isolation “C.

1 5 10 20 30

Protein formed by isolated particles w.

1.9 0.8 0 0 0

peratures during the preparation of the particles. It can be seen that increased temperatures apparently result in the inactivation of the proteinforming ability of the particles. In addition, isolation of particles at pH values on either side of pH 7.5 (especially on the alkaline side) or in the presence of 0.001 M or greater concentrations of orthophosphate or of either univalent or bivalent cations results in variable amounts of inactivation. Occasional preparations which have been isolated under apparently

PROTEIN

165

SYNTHESIS

optimal conditions have also failed to produce protein, suggesting that some as yet unrecognized factor may be involved in the stability of the protein-forming ability of the particles. Apparent Enzyme Synthesis Although the above results all support the thesis that the reaction system is capable of net protein synthesis, it is of interest to learn whether t.he reaction results in the formation of polypeptide, consisting of a random aggregation of amino acids, or whether specific proteins are formed. For example, if the reaction results in the formation of protein with enzymic activity, then one might wonder if the amino acids are not being arranged in a specific sequence. Young and Varner (20) have reported that peas, upon germination, rapidly synthesize a “soluble” enzyme which catalyzes the hydrolysis of ATP. Their experiments suggested that the initial site of synthesis of this enzyme might be the microsomal particles of peas. Therefore, the nucleoprotein particles employed in t,he present experiments were examined for their ability to form this enzyme. Figure 4 shows that incubation of the particles with the amino acid mixture and cofactors causes only a slight increase in the ability of the particles to hydrolyze ATP. In contrast, the protein formed in the medium exhibits a marked ability to hydrolyze ATP, and the ability increases in a manner parallel to t,he increase in protein. The

7

I IO

I 20 TIME

I 30 (MIN)

I 40

I 50

FIG. 4. ATP hydrolysis by particulate and nonparticulate fractions. Reaction system was the same as described with Fig. 1. At the times indicated the particulate and nonparticulate fractions were separated and assayed for their ability to hydrolyze ATP as described in the Experimental section.

166

WEBSTER

ability to hydrolyze ATP is completely destroyed by exposure of the “soluble” protein to 100°C. for 3 min. Increase in enzyme activity requires the presence of ATP, GTP, Mn++, polynucleotide, and the amino acid mixture in the same manner as does the increase in protein. Preliminary

Observations on the Mechanism of Protein Synthesis

The results presented above indicate that the nucleoprotein particles are able to form protein from amino acids in the presence of appropriate cofactors. It is of considerable interest to learn about the nature of this reaction. In particular, two questions arise immediately. First, what is the relationship between amino acid incorporation by the nucleoprotein particles and the formation of protein? Second, is the process of amino acid activation described by Hoagland (21-23) an intermediate state in net protein synthesis by the particles? It has already been shown (10) that the nucleoprotein particles incor‘porate individual amino acids into their protein. If the particles, following incubation with a single CY4-amino acid (10) are treated with fluorodinitrobenzene and hydrolyzed, most of the incorporated amino acid can be recovered as dinitrophenylamino acid (Table IV), even after the particles are “saturated” with amino acid. In contrast, when the same U4-amino acid is incubated in the presence of a mixture of unlabeled amino acids, only a small fraction of the labeled amino acid can be recovered as dinitrophenylamino acid. This indicates that a single incorporated amino acid is bound to the ends of peptide chains associated with the particles. In the presence of a mixture of amino acids, however, by far the greatest amount, TABLE Amino

Acid

Reactive

with

IV

Fluorodinitrobenzene after Amino into Particulate Protein

Acid

Incorporation

Particles were incubated with either alanine-Cl4 alone (total activity, 10 PC.) or with an amino acid mixture containing alanine-Cl4 (10 PC.). In each case, the cofactors listed with Fig. 1 were included. The particles were isolated by centrifugation (105,000 X 9 for 90 min.) and washed as described previously (10). Reaction with fluorodinitrobenzene and subsequent processing was as described in the Experimental section. Total amounts of radioactivity incorporated were: for alaninealone, 1125 counts/min. after 10 min.; for alanine-Cl4 plus an amino acid mixture, 8350 counts/min. after 10 min. Per cent Alanine-Cl4

min. 1 5 10 -

DNP

93 95 93

of total alone Non-DNP

7 5 7

radioactivity

incorporated Alanine-Cl* plus amino acid mixture DNP

Non-DNP

37 7 5

63 93 95

PROTEIN

SYXTHESIS

167

of amino acid is apparently incorporated into the interior of peptide chains. The only exception to this comes from a I-min. incubation. This suggests that N-terminal binding of amino acids may also occur at very short time periods during net protein synthesis. The observation of addition of amino acids to the N-terminal ends of peptide chains suggests that carboxyl activation of amino acids may indeed be an intermediate state in protein formation by the particles. It has not been possible to obtain unequivocal evidence for this, however. Previous experiments (10) showed that extraction from the particles of a protein fraction (capable of activating amino acids) completely stopped amino acid incorporation by the particles. Incorporation activity could be restored by addition of the extract. Similar results have now been obtained with regard to net protein formation. It is not known, however, that the activating enzymes constitute the essential portion of the extract. Likewise, it has been shown that amino acid-activating enzyme preparations form amino acid-polynucleotide compounds from amino acids, ATP, and polynucleotide (15, 23), and that the amino acid of such compounds is transferred to the protein of microsomes (23) and ribonucleoprotein particles (15). The polynucleotide binds only small amounts of amino acids, however, and it has not been possible to obtain sufficient amino acid bound to polynucleotide to test adequately the possibility that amino acid-polynucleotide is an intermediate in net protein synthesis. Such a test apparently must await the isolation of the active polynucleotide (or polynucleotides) in a more pure form. DISCUSSION

The evidence presented in this paper indicates that cellular nucleoprotein particles are able, under certain conditions, to catalyze the synthesis of protein. Unlike amino acid incorporation by these particles (lo), protein synthesis requires the presence of cytoplasmic polynucleotide. Synthesis is also strongly dependent upon t,he employment of the optimum concentration of each component of the reaction system. The system exhibits a rather striking instability, and the observation that occasional preparations fail to form any protein, despite their isolation and incubation under apparently optimal conditions, suggests that there are further, as yet unrecognized, factors required for protein synthesis by this system, This possibility is also suggested by the failure of protein synthesis to continue for extended periods of time. n’evertheless, despite the many difficulties associated with its successful operation, the system does provide a means for the study of the mechanism of actual protein synthesis, instead of amino acid incorporation into protein. It has been proposed (23, 24) that protein synthesis proceeds through the

168

WEBSTER

formation (catalyzed by amino acid-activating enzymes) of amino acidpolynucleotide intermediates. Unequivocal evidence for the participation of such intermediates, or of amino acid-activating enzymes, in the present system has not been obtained. However, the dependence of protein formation on the presence of a protein fraction which contains amino acid-activating enzymes, and on the presence of “soluble” polynucleotide suggests that amino acid-polynucleotide could be an intermediate in net protein synthesis. Likewise, the findings with fluorodinitrobenzene are open to more than one interpretation. The findings are that incorporation of a single labeled amino acid results in essentially all of the incorporated amino acid remaining in the X-terminal position, while incorporation in the presence of a mixture of unlabeled amino acids results in most of t#he label being in a non-N-terminal position. One explanation of such findings is that the incorporation of a single amino acid is not related t,o the protein synthesis found in the presence of a mixture of amino acids. Thus, single amino acids, in the absence of protein synthesis, might simply be bound to the Nterminal ends of peptide chains on the particles by a mechanism not directly related to protein synthesis. In the presence of all amino acids, protein formation could occur by the activation of the amino groups of amino acids, giving rise to the lack of N-terminal amino acids in the presence of an amino acid mixture. However, a more reasonable explanation of the dinitrophenyl results is that protein synthesis proceeds by the sequential addition of carboxyl-activated amino acids. Thus, incorporation of a single amino acid would result in its attachment to the N-terminal ends of partly formed peptide chains. In the absence of other amino acids, the incorporation would be expected to stop when all reactive sites had added an amino acid, and all of the amino acid would be reactive with fluorodinitrobenzene. In the presence of a mixture of unlabeled amino acids, the peptide chain would be expected to lengthen rapidly by addition of various unlabeled amino acids in addition to the labeled amino acid, and little of the labeled amino acid would be N-terminal. Thus, both direct experiments and the results with fluorodinitrobenzene are compatible with, but do not provide direct evidence for, the participation of amino acid-activating enzymes and carboxyl-activated amino acids in net protein synthesis. It would seem that the most important problem in the study of the mechanism of protein synthesis is a careful evaluation of the manner of activation of amino acids prior to their condensation to protein, and of whether the amino acid-activating enzymes discovered by Hoagland (21) are responsible for this activation. Some excellent beginnings (25-27) have been made, but the activation of many amino acids has hardly been investigated, although evidence exists for the occurrence of enzymes activating all of the amino acids normally found in protein (15, 28-29).

169

PROTEIN SYNTHESIS

Until these enzymes and their apparent polynucleotide cofactors are wellcharacterized and their role in protein synthesis is clearly evaluated, the mechanism of protein synthesis will probably remain in doubt.

The excellent

technical

assistance

of Sandra Whitman

is gratefully

acknowledged.

SUMMARY

Isolated ribonucleoprotein particles from peas catalyze a net synthesis of “soluble” protein in the presence of adenosine triphosphate, guanosine triphosphate, manganese ions, phosphoglycerate, soluble polynucleotide, and a mixture of the 18 amino acids and two amides usually found in protein. The protein thus formed exhibits more than one electrophoretic component, and has enzymic activity. If protein synthesis proceeds in the presence of C14-labeled amino acids, the protein formed has a specific activity close to that of the free amino acids. The enzyme system is rather unstable. REFERENCES 1. WEBSTER, G. C., 2. GALE, E. F., AND 3. WEBSTER, G. C., 4. WEBSTER, G. C., 5. 6. 7. 8.

9. 10. 11. 12.

13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

Federatior~ Proc. 18, 348 (1959). FOLKES, J. P., Biochem. J. 69,661

(1955).

Plant Physiol. Suppl. 30, 28 (1955). in “Chemical Basis of Heredity” (McElroy, W. D. and Glass, B., eds.). Johns Hopkins Press, Baltimore, 1957. SPIEGELMAN, S., in “Chemical Basis of Heredity” (McElroy, W. D., and Glass, B., eds.). Johns Hopkins Press, Baltimore, 1957. OCHOA, S., AND BELJANSKI, M., Science 127, 1062 (1958). SEAMAN, G., Bacterial Proc. 68, 101 (1958). Tso, P., BONXER, J., AND VINOGRAD, J., J. Biophys. Biochem. Cytol. 2,451 (1956). H., Arch. Biochem. Biophys. 76, 225 (1958). Tso, P., BONNER, J., AND DINTZIS, WEBSTER, G. C., J. Biol. Chem. 229, 535 (1957). GORNALL, A. G., BARDAWILL, C. J., AND DAVID, M. M., J. Biol. Chem. 177, 751 (1949). LOWRY, 0. H., ROSEBROUGH, N. J., AND FARR, A. L., J. Biol. Chem. 193, 265 (1951). HILLER, A., PLAZIN, J., AND VAN SLYKE, D. D., J. Biol. Chem. 176, 1401 (1948). KIRBY, K. S., Biochem. J. 64,405 (1956). WEBSTER, G. C., Arch. Biochem. Biophys. 82, 125 (1959). FISKE, C. H., AND SUBBAROW, Y., J. Biol. Chem. 66,375 (1925). VARNER, J. E., AND WEBSTER, G. C., Plant Physiol. 30,393 (1955). SANGER, F., Biochem. J. 39, 507 (1945). PORTER, R. R., in “Methods in Enzymology” (Colowick, S. P., and Kaplan, N. O., eds.), Vol. IV. Academic Press, New York, 1957. YOUNG, J. L., AND VARNER, J. E., Arch. Biochem. Biophys., 84, 71 (1959). HOAGLAND, M. B., Biochim. et Biophys. Acta 16, 288 (1955). HOAGLAND, M. B., KELLER, E. B., AND ZAMECNIK, P. C., J. Biol. Chem. 218, 345 (1956).

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23. HOAQLAND, M. B., STEPHENSON, M. L., SCOTT, J. F., HECHT, L. I., AND ZAMECNIK, P. C., J. Biol. Chem. 231, 241 (1958). 24. ZAMECNIK, P. C., STEPHENSON, M. L., AND HECHT, L. I. Proc. Natl. Acad. Sci. u. s. 44,73 (1958). 25. DAVIE, E. W., KONINGSBERGER, V. V., AND LIPMANN, F., Arch. Biochem. Biophys. 66, 21 (1956). 26. VAN DEN VEN, A. M., KONINGSBERGER, V. V., AND OVERBEEK, J. T. G., Biochim. et Biophys. Acta 26, 134 (1958). 27. SCHWEET, R. S., AND ALLEN, E. S., J. Biol. Chem. !W, 1104 (1958). 28. NISMAN, B., BERGMANN, F. H., AND BERG, P., Biochim. et Biophys. Acta 26, 639 (1957). 29. LIPMANN, F., Proc. Natl. Acad. Sci. U. S. 44, 67 (1958).