Studies of the nuclear residual proteins

ARCHIVES

OF

BIOCHEMISTRY

AND

Studies G. PATEL, Department

BIOPHYSICS

128,

654-662 (1968)

of the Nuclear V. PATEL,

Residual

T. Y. WANG,

AND

Proteins C. R. ZOBEL

of Zoology,

University of Georgia, Athens, Georgia 30601; and Departments Biophysics, Stale University of New York, Buffalo, New York LQ’1.J Received

July

of Biology

and

1, 1968

The nuclear residual proteins of rat liver have been prepared by solubilization with sodium deoxycholate and Sephadex gel filtration. These proteins were examined by analytical ultracentrifugation, DEAE-cellulose chromatography, starch gel electrophoresis, amino acid composition, and alkali-labile phosphorus analysis. The results show that these proteins are heterogeneous, having a high glutamic acid content, and contain phosphoprotein. Partial fractionation was achieved by DEAE-cellulose chromatography and starch gel electrophoresis. Some of these proteins appeared as spheroidal particles with diameters of 100-400 8. In the presence of sodium deoxycholate, the particles dissociated into smaller units that reassociated upon removal of the detergent.

The nuclear fraction that remains as insoluble residue after isolated mammalian cell nuclei are successively extracted with buffered 0.14 M NaCl and l-2 M NaCl has been considered to contain components of nucleoli, the nuclear envelope and structures resembling the residual chromosomes (l-6). Using dilute alkali various investigators have solubilized from this residue a group of acidic proteins that contain RNA, DNA, and lipid (7-10). Busch (11) and co-workers have studied extensively the chemical characteristics of these alkali-soluble acidic proteins. However, the use of alkali leads to possible denaturation of proteins, and consequently to difficulty in their fractionation and biochemical studies. Recently, Wang (12) has reported solubilization of these nuclear residue acidic proteins in dilute Tris buffer by treatment with sodium deoxycholate, and their partial fractionation. In the studies reported here we have removed the detergent by gel filtration on Sephadex columns prior to fractionation, and reexamined and extended our studies of these proteins. Complete removal of the detergent affects the fractionation and properties of these proteins. The results aIso show that upon removal of

the detergent some of the residual proteins aggregate into spherical particles of higher sedimentation coefficients than reported previously (12). These particles dissociate reversibly in the presence of deoxycholate. Fractionation of the detergent-solubilized proteins by DEAE-cellulose chromatography and starch-gel electrophoresis, as well as other properties are also described. MATERIALS

AND

METHODS

Preparation of the nuclear residue proteins. Male Sprague-Dawley rats weighing 200-250 g were used throughout this work. The isolation of liver nuclei, the extraction of the nuclear sap and the nucleohistones, and the solubilization of the nuclear residual proteins by sodium deoxycholate were carried out as reported previously (12). Briefly described, the rat-liver nuclei were isolated essentially according to the procedure of Chauveau et al. (13). The isolated nuclei were successively extracted three times with 0.05 M Tris-HCI containing 5 mM MgCI,, and twice with 1 M NaCI. The nuclear residue thus obtained was suspended in 0.05 M Tris-HCI, pH 8.5. Enough deoxycholate was then added to make the suspension 1% with respect to the detergent. The mixture was allowed to stand for 20 min in the cold and was then centrifuged at 27,000g for 30 min. The supernatant solution contained the t.otal soIubilized residual proteins. 654

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Fractionation of the residual proteins. The deoxycholate-solubilized residual proteins were centrifuged at 105,OOOg for 60 min to pellet a residual ribonucleoprotein fraction that is referred to a RRNP. The RRNP pellet was gently suspended in 0.05 M Tris buffer, pH 8.5, with the aid of a glass rod. Most of the pellet was dissolved in the Tris buffer by standing overnight at 4’. The supernate from 105,OOOg centrifugation was passed through a Sephadex G-25 column with bed volume five times that of the sample size, and the effluent protein solution was successively adjusted to pH 6 and to pH 5 by the addition of 1 N acetic acid. The proteins that precipitated at pH 6 and at pH 5 were collected by centrifugation and are referred to as the pH 6 and the pH 5 fractions respectively. The proteins in the pH 5 supernatant solution were precipitated at 80% saturation with ammonium sulfate (A.S. fraction). The four fractions thus obtained were dialyzed against 0.05 M Tris buffer, pH 8.5, and used for the studies reported here. Analytical ultraeentrifugation. Sedimentation analyses of the protein fractions were carried out in a Spinco model E analytical ultracentrifuge using Kel-F centerpieces, and the observed sedimentation coefficients were corrected to 20” in water. For sucrose gradient centrifugation, 0.8 ml of the protein sample was layered on top of a linear gradient of 520% sucrose in 0.05 M TrisHCI, pH 8.5, and centrifuged in a Spinco SW 25.1 rotor at 25,000 rpm for 5 hr. Fractions of 50 drops each were collected through a 20-gauge needle inserted into the bottoms of the tubes and their absorbancies at 280 rnp were read on a Zeiss spect,rophotometer. Amino acid unaZy.sis. Amino acid analyses were carried out on protein samples that were extract,ed twice with 5y0 trichloracet.ic acid at 90” for 30 min. The precipitate was extracted with alcohol: ether (1:l) and ether, and then dried under vacuum. The protein powder was hydrolyzed in constant boiling HCl for 22 hr at 110’ and the hydrolyzate analyzed in a Spinco model 120 c automatic amino acicl analyzer (14). TABLE

I

QUANTITATIVE DISTRIBUTION OF THE NUCLEAR RESIDUAL PROTEIN FRACTIONS Fraction RRNP PH 6 pH5 A.S. fraction * Average

% of total solubilized proteina 25.3 55.4 12.8 6.51

f f f f

values from 12 preparations.

4.07 4.14 2.24 1.07

RESIDUAL

655

PROTEINS TABLE

II

AMINO ACID COMPOSITION OF NUCLEAR RESIDUAL PROTEINS OF RAT LIVER” Amino acid

RRNP

Lysine Histine Arginine Aspart.ic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Ratio, aspartic $ glutamic acid, to lysine + his t,idine + argi nine a Expressed

7.12 2.31 6.50 9.00 5.08 5.85 12.85 5.61 7.02 7.73 1.60 6.19 2.25 4.62 9.71 2.68 3.82 1.37

--

PH 6

pH 5

PL.S.ppt

6.23 1.94 5.70 9.39 5.45 6.28 12.60 5.08 6.70 7.64 0.99 6.65 1.78 5.29 11.29 2.62 4.45 1.59

6.48 1.92 5.68 10.x 5.20 6.16 13.20 4.48 6.64 7.52 1.28 6.48 2.16 5.12 10.64 2.27 4.16 1.66

7.22 1.56 4.36 10.86 5.40 6.38 16.19 5.54 7 .73 7.39 0.44 6.05 1.34 4.42 8.29 2.12 4.53 2.06

as moles/LOO moles recovered.

Electronmicroscopy. Samples were prepared for elect,ronmicroscopy by the shadow replica technique (15) and examined with a Siemens Elmiskop I microscope. DEAE-cellulose chromatography. Chromatography of the residual proteins was performed as described previously (16) for the nuclear sapsoluble proteins. DEAE-cellulose (Type 40), purchased from Carl Schleicher and Schuell (Keene, New Hampshire), was treated according to Peterson and Sober (17) and equilibrated with the starting buffer (5 mM Tris-phosphate, pH 8.0). The coIumn (30 X 1 cm) was packed at, 30 psi and washed with the starting buffer before use. The protein, dialyzed thoroughly versus the starting buffer, was applied to the column under gravity. After adsorpt.ion of the protein, the column was washed with 150 ml of the starting buffer to elute the unabsorbed material. The proteins were t,hen elut,ed from the column with a pa.rabolic gradient of N&l. The salt gradient was obtained with three mixing flasks connected in series; each of the first two flasks contained 200 ml of the start,ing buffer while the third flask contained 200 ml of 1 M NaCl in the starting buffer. The flow rate of the eluant (25-30 ml/hr) was cont,rolled by a peristaltic pump between the column and the gradient mixer.

656

PATEL,

PATEL,

WANG,

The protein fractions eluted from the column were pooled as indicated in Fig. 6 and concentrated by ultrafiltration. The proteins not eluted by NaCl were finally eluted with 0.1 N NaOH at room temperature. S/arch gel eZectrophoresis. Vertical starch gel electrophoresis was performed by the method of Smithies (18). Hydrolyzed starch (Connaught Labs) was suspended in 5 rnM Tris-citrate (pH 8.6) containing 10 mM 2mercaptoethanol for the preparation of the gel, and 0.3 M sodium borate (pH 8.3) was used as the electrode buffer (19, 20). The electrophoresis was run in a cold room for 3 hr at 300 V. At the end of the run, the gel was sliced and the bottom half was stained in amido black B (17, dye solut,ion in 5y0 acetic acid) and destained in an electrophoretic destainer. Other methoda. The solubility of t,he residual proteins in HCl and the extent of their precipitation byMgC1, were measured by mixing theprotein samples with equal volumes of 0.50 N HCl or MgC12 of varying concentrations. The mixture was allowed to stand in the cold for 1 hr. The precipitate formed was separated by centrifugation at 7000g for 10 min. The protein concentration in the supernatant fluid was measured by the method of Lowry et al. (21) using human serum albumin as a standard. Protein phosphorus was determined by the method of Berenbaum and Chain (22) as described by Kleinsmith et al. (23). RESULTS

Table I shows the relative proportions of the four fractions of the deoxycholatesolubilized nuclear residual proteins. These values vary from those reported previously (12). The proportion of the RRNP fraction has increased as a result of the overnight dissolution of the ultracentrifuge pellets in

AND

ZOBEL

the buffer. The change in the relative proportions of the other three fractions is apparently due to removal of the detergent, which may affect their precipitation, prior to fractionation. These changes necessitated a reexamination of their amino-acid compositions. The results of these analyses are shown in Table II. The ratios of the acidic to basic amino acid residues are higher than those reported previously for all the four fractions of the nuclear residual proteins. Glutamic acid predominates in all four fractions, and along with leucine shows greatest variability among all the fractions. With the exception of the AS. fraction, the other t’hree fractions seem to have similar amino acid compositions. Excluding the consideration of amide contents, which were not determined in the present study, the nuclear residual proteins solubilized by deoxycholate may be considered as acidic. Their over-all amino acid composition is similar t,o that of the nuclear alkali-soluble proteins reported by Busch and Steele (24) and by Dounce and Hilgartner (25). The presence of deoxycholate in the residual proteins not only influences, quantitatively, their fractionation but also affects their sedimentation behavior, especially of the pH 6 fraction. This can be seen by their sedimentat,ion pat#terns shown in Pig. 1. The RRNP fraction is not included here since the procedure of its preparation has not changed significantly from the previous report (12). All three fractions exhibit considerable molecular heterogeneity. In fact,

FIG. 1. Centrifugation of rat-liver nuclear residual protein fractions at 24,630 rpm. Concentration was 7.0 mg/ml in 0.05 M Tris-HCl, pH 8.5, in all cases. Pictures were taken at 6960 (pH 6), 6280 (pH 5), and 11,580 (A. S. ppt fraction) seconds after reaching speed.

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the low speed used to obtain the patterns in this figure was necessit’ated by the extreme polydispersity of the pH 6 fraction. At higher speeds we were unable t’o obtain satisfactory peaks t,hat, would migrate in the centrifugal field as a function of t’ime. Instead, t’he peaks flattened rapidly making any calculation of t,he S values impossible. The sedimentation patterns of the pH 5 and AS. fractions are also shown at this speed for the sake of consistency. We have examined these fractions at higher speed and calculated t’he same S values as reported here. The majority of the pH .5 fraction sediments as a 6 S peak with a shoulder of average 21 S, while the A.S. fraction sediments as a 2.5 S peak as reported previously. The pH 6 fraction appears as t’wo peaks of average 32 S and 6 S. The fast-sedimenting 32 S component’ was not observed previously when the fract,ionation was conducted in t’he presence of deoxycholate. Furthermore, contrary to other components, its sedimentation is concentration dependent (Fig. 2). It appears that, the det)ergent may cause dissociation of the 32 S proteins into smaller molecular species with an average sedimentation coefficient of 6 S. If such is t’he case, addition of the detergent t’o the pH 6 fraction should change it’ to mainlv a 6 S component. Evidence of this d&o&a&on can be seen in Fig. 3, which shows the sedimentation

.-.-

PROTEIN CONC. b-w/ml)

FIG. 2. Dependence of sedimentation cients of the rat-liver nuclear residual on protein concentration.

coe& proteins

RESIDUAL

PROTEINS

657

FIG. 3. Centrifugation of rat-liver nuclear residual pH 6 fraction in the presence of sodium deoxycholate at 42,040 rpm. Concentration was 7.0 mg/ml in 0.05 M Tris-HCI, pH 8.5, containing 0.5~~ sodium deoxycholate. Picture was taken at 4140 seconds after reaching speed.

patt,ern of the pH 6 fraction in the presence of 0.5 % sodium deoxycholate. Thus, it seems clear that deoxycholat’e affects dissociation of the pH 6 proteins, and this effect is reversible upon removal of the detergent. Partial separation of the 32 S and 6 S component,s of the pH G fraction was achieved by centrifugation in a Tj-20 9%linear sucrose gradient. Analyses of the fract8ionat,ed component,s showed that, they have similar amino acid compositions, wit,h observable difference in t,heir glutamic acid content. The elect,ron micrograph of t,he pH 6 fraction (Fig. 4) shows0 spheroidal particles ranging from 100-400 A in diameter. Some of the aggregated particles suggest a subunit, structure. Addition of deoxycholate to t,his fraction resulted in a complete disappearance of the particles. These electron-dense particles probably represent t’he 32 S peak. I‘rom the size range of these particles the 32 S peak is not a homogeneous component. Elect’ron microscopic observations of the pH 5 and AS. fractions did not, reveal an:, particular structures. The residual prot,ein fractions also contain phosphoproteins. The percentage distribution of alkali-labile phosphorus among t,he four protein fractions is listed in Table III.

658

PATEL,

FIG. 4. Electron

photomicrograph

PATEL,

WANG,

of rat-liver

Langan and Smith (27) have shown that phosphoproteins can be precipitated by Mg2+. Such a characteristic can also be demonstrated with the solubilized residual proteins, as shown in Fig. 5. However, the precipitation of these proteins by Mg”+

AND

nuclear

ZOBEL

residual

pH 6 fraction.

X 114,000.

apparently is not proportional to their phosphoprotein content. With the exception of the A.S. fraction, which manifests only feeble precipitability by Mg2+ and only at higher Mg2+ concentrations, all the other residual protein fractions may be precipi-

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tated by this divalent cat’ion. The effective concentration of Mg2+ for t’he maximal precipitation of the pH 6 and t’he pH 5 fractions is the same, but different from t.hat for the RRNP and the AS. fraction. The deoxycholate-solubilized residual proteins are quite soluble in dilute HCl. The data in Table IV show that all four residual fractions are soluble in 0.25 N HCl to various degrees. The acid-solubility of these residual proteins is in agreement with the observation of Grogan, Desjardins, and Busch (26) who reported that some of the acidic proteins of isolated nucleoli are acid-extractable. Chromatography of the residual proteins on DEAE-cellulose revealed variable elutibility of these proteins. The RRNP fraction yielded poor recovery and therefore was not included in the present study. The DEAEcellulose chromatographic profiles of the pH 6, pH 5, and A.S. fract,ions are shown in TABLE PHOSPHOPROTEIN NUCLEAR

Protein fraction

RRNP PH

6

pH 5 A.S. fract,ion

RESIDUAL

TABLE SOLUBILITY

659

PROTEINS

OF RAT PROTEINS Protein fraction

RRNP PH 6

pH 5 A.S. fraction

IV

LIVER NUCLEAR IN 0.25 N HCL

RESIDUAL

% soluble in HCI

60 80 91 77

/-1---T

III

CONTEXT RESIDUAL

OF RAT PROTEINS

LIVER

Protein (w)

Alkalilabilephosphorus dug)

‘% Alkalilabile phosphorus in protein

6.50 14.4 13.9 5.42

15.5 39.0 17.8 5.2

0.238 0.272 0.128 0.096 0 lo 20 30 40 50 60 70 80 ?k FRACTION NUMBER FIG. 6. Fractionation of rat-liver nuclear residual proteins by DEAE-cellulose chromatography. The experimental details are described in the text. Arrows indicate the start of the NaCl gradient.

* 0

0.1

1.0

2.0

3.0

mM MgCl2 5. Precipitation of rat-liver nuclear residual proteins by MgCl? under conditions as described in the text. 0, RRNP fraction; q , pH 6 fraction; l , pH 5 fraction; and A, A.S. ppt fraction. FIG.

Fig. 6. In general, t’he three fractions have distinct patterns. The pH 6 fraction is the least resolved chromatographically, while the AS. fraction shows a satisfactory resolution. As show-n in Table V, the recovery from the DEAE-cellulose column of the pH 6 protein eluted by NaCl is also the poorest among these three fractions. Only 32% of the total pH 6 protein applied to the column could be recovered. Elution by additional NaOH gave the total recovery of 83 % of the applied pH 6 protein. In the case

660

PATEL, TABLE

RECOVERY TEINS

PH6 PH 5 AS. ppt.

WANG, AND ZOBEL

V

7b

OF DOC-SOLUBILIZED FROM DEAE-CELLULOSE

Residual protein subfraction

PATEL,

RESIDUAL PROCOLIJXNS To Recovery

By NaCl

32.0 43.5 85.0

By additional 1 0.1 s lUaOH

51.2 50.8 15.0

7a

FIG. 7. Starch gel electrophoreses of rat-liver nuclear residual proteins. (a) pH 6 fraction; (b) pH 5 fraction; and (c) A.S. ppt fraction. Each electrophoretic pattern corresponds, from left to right, to the respective DEAE-chromatographic peaks as indicated in Fig. 6, beginning with the breakthrough peak.

of the pH 5 fraction, about equal amounts are eluted by NaCl and NaOH, giving a total recovery of 94%. The AS. fraction protein is quantitatively eluted by NaCl and NaOH, 85 % of the proteins being eluted by the NaCl gradient. Starch gel electrophoreses (Fig. 7) of the protein subfractions obtained by DEAE-cellulose chromatography show distinctly different patterns among the three residual fract’ions. Except for the protein bands that move toward the cathode (breakthrough and No. 2 peak of the pH 6 fraction), the pH 6 proteins (Fig. 7a) and the major components of the pH 5 fraction (Fig. 7b) do not resolve into distinct

FIG. 7 (b-c)

bands. Other components of t’he pH 5 proteins, and especially proteins of the A.S. fraction, reveal satisfactory separation by this technique. Most proteins in the break-

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through peak of the A.S. fraction move toward the cathode. These proteins and those of the pH 6 fraction with similar mobility may be basic, probably representing some cont,amination of histones. I)ISCUSSION

The term “residual proteins” has appeared in literature to describe different nuclear fractions. It has been applied to the protein fraction of isolated nuclei that remain insoluble aft.er successive ext,ract,ions with with 0.14 M NaCl and/or 0.1 N HCI and alcohol:ether mixture (25); with dilute Tris buffer and 1 XI KaCl (2S, 29); and with 0.14 l< NaCl, dilute Tris buffer, 2 1\1NaCl, and 0.05 N NaOH (30). The alkali-insoluble residual proteins in the last, reference resemble collagen in amino acid composition and in electron microscopic appearance, and were considered to cont,ain nuclear envelope proteins. Since t#he deoxycholate-solubilized proteins in our study do not give any indication of collagen composition by eit.her of these criteria, they are simiIar to the alkalisoluble proteins studied by Steele and Busch (10). In addit,ion to the nuclear envelope material, the Tris buffer and l-2 M NaCIinsoluble fraction of nuclei has been shown to also contain nurleolar and residual chromatin components (1). Therefore the deoxycholate-solubilized nuclear residual proteins cont#ain at least some of t,he nucleolar proteins. Fractionation of proteins from purified rat liver nucleoli by DEAE-cellulose chromatography, by ext’raction with various solvents, and by starch-gel electrophoresis has recent,ly been reported by Busch and associates (26, 31). Most of the nucleolar proteins have been shown to be acidic. Some of these, however, are extractable by NaCl and therefore would not be expected to remain in the nuclear residue described in our study. The result,s described in this report on the chromat,ography and DEAE-cellulose st,arch-gel electrophoresis of the solubilized residual proteins show only partial fractionation. The majority of the proteins, of which the pH 6 fract’ion represents a predominant portion, is not, well resolved. This difficulty may be due to its lipoprotein nature (7, 32) or, as suggested by Grogan and Busch (31),

RESIDUAL

PROTEINS

661

due to their high molecular weight and/or their RNA content. Nevertheless, our studies demonstrate considerable heterogeneity in these proteins wit’h respect to t’heir molecular size and net charge. The existence of phosphoproteins in the cell nucleus has been reported by several investigators (23, 27). It. has further been shown that phosphoproteins (33) and nonhist’one chromosomal proteins (34) purified from rat liver nuclei can bind histones to reverse the inhibition by histones of DKAdependent RKA synt,hesis in v&0, implicating a de-repressor role. Bot>h the nonhistone chromosomal proteins (35) and the residual proteins described in t’his report contain phosphoprot,ein. They have also been shown to form complexes with histones (12, 35, 36). Although their content of alkali-labile phosphorus is comparatively low, the degree of their phosphorylation in vivo could be varied by protein phosphokinases (37, 38). Thus these acidic proteins may function as gene regulators by varying then quantities as well as the degree of their phosphorylation. Although t,he amino acid composition of the A.S. fraction shows it to be more acidic than the other fractions, its binding with histories is weaker than the other fract.ions (12). This suggest,s that t.he phosphorous content rather than acidic amino acid residues may be important in their interaction with histones. The biochemical function of the nuclear residual proteins described here is as yet unknown. The present studies show that they are heterogeneous, and one can a.ssume them to have a variety of biochemical functions. Some of t’hem may be involved in gene regulation by competing with DNA for interaction with histones. Others, like the pH 6 fraction, may contain the aggregate nucleic acid polymerases suggested by Busch et al. (11). Still others, like the RRNP fraction, may be important for nuclear protein synthesis (39). ACKNOWLEDGMENTS This investigation was supported by grants from the American Cancer Society (E-44) and the U.S. Public Health Service (GM 11698-05).

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WANG,

REFERENCES 1. ZBARSKY, I. B., AND GEORGIEV, G. P., Biochim. Biophys. Acta 32, 301 (1959). 2. GEORGIEV, G. P., AND CHENTSOV, J. S., Exptl. Cell Res, 27, 570 (1962). 3. ZBARSKY, I. B., AND GEORGIEV, G. P., Expll. Cell Res. 27, 573 (1962). 4. SII~ATANI, A., DE KLOEF, S. R., ALLFREY, V. G., AND MIRSKY, A. E., Proc. Natl. Acad. Sci. U.S. 48, 471 (1962). 5. SMETANA, K., STEELE, W. J., AND BUSCH, H., ExpL Cell Res. 31, 198 (1963). 6. MIRSKY, A. E., AND RIS, H., J. Gen. Physiol. 31, 1 (1946). 7. WANG, T. Y., MAYERS, D. T., AND THOMAS, L. E., Exptl. Cell Res. 4, 102 (1953). 8. DALY, M. M., ALLFREY, V. G., AND MIRSKY, A. E., J. Gen. Physiol. 36, 173 (1952). 9. FLAMM, W. J., AND BIRNSTIEL, M. L., in “The Nucleohistones” (J. Bonner and P. 0. P. T’so, eds.), p, 230. Holden-Day, San Francisco (1964). 10. STEELE, W. J., AND BUSCH, H., Cancer Res. 23, 1153 (1963). 11. BUSCH, H., “Histones and Other Nuclear Proteins,” Academic Press, New York (1965). 12. WANG, T. Y., J. Biol. Chem. 241, 2913 (1966). 13. CHAUVEAU, J., MOULE, Y., AND ROUELLER, C., Exptl. Cell Res. 11, 317 (1956). 14. MOORE, S., SPACKMAN, D. H., AND STEIN, W. II., Annul. Chem. 30, 1185 (1958). 15. HALL, B. D., J. Biophys. Biochem. Cylol. 2, 625 (1956). 16. PATEL, G., AND WANG, T. Y., Exptl. Cell Res. 34, 120 (1964). 17. PETERSON, E. A., AND SOBER, H. A., J. Am. Chem. Sot. 78, 751 (1956). 18. SMITHIES, O., Biochem. J. 71, 585 (1959). 19. POULIK, &I. D., Nature 180, 1477 (1957).

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20. MOORE, B. W., AND MCGREGOR, D., J. Biol. Chem. 240, 1647 (1965). 21. Lo\~RY, O., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 22. BERENBAUM, I., AND CHAIN, E., Biochem. J. 32, 295 (1938). 23. KLEINSMITH,L. J.,ALLFREY,V. G., AND MIRSKY, A. E., Proc. Natl. Acad. Sci. U.S. 66, 1182 (1966). 24. BUSCH, H., AND STEELE, W. J., Adv. Cancer Res. 8, 41 (1964). 25. DOUNCE, A. L., AND HILGARTNER, C. A., Expll. Cell Res. 36, 228 (1964). 26. GROGAN, D. E., DESJAHDINS, R., AND BUSCH, H., Cancer Res. 26, 775 (1966). 27. LANGAN, T., AND SMITH, L., Information Exchange Group, No. 7 (1965). 28. ALLFREY, V. G., DALY, M. M., AND MIRSKY, A. E., J. Gen. Physiol. 38, 415 (1955). 29. ALLFREY, V. G., AND MIRSKY, A. E., Proc. Nail. Acad. Sci. U.S. 43, 821 (1957). Cell 30. STEELE, W. J., AND BUSCH, H., Exptl. Res. 33, 68 (1964). 31. GROGAN, D. E., AND BUSCH, H., Biochemistry 6, 573 (1967). 3.2. ROSE, H. G., AND FRENSTER, .I. H., Bioehim. Biophys. Acta 106, 577 (1965). 33. LANGAN, T., in “Regulation of Nucleic Acid and Protein Synthesis” (N. J. Koningsberger and L. Bosch, eds.), p. 241. Elsevier, Amst,erdam (1967). 34. WANG, T. Y., Exptl. Cell Res., in press. 35. WANG, T. Y., J. Biol. Chem. 242, 1220 (1967). 36. WANG, T. Y., AND JOHNS, E. W., Arch. Biothem. Biophys. 124, 176 (1968). 37. BURNETT, G., AND KENNEDY, E. P., J. Biol. Chem. 211, 969 (1964). 38. RABINOWITZ, M., AND LIPMANN, F., J. Biol. Chem. 236, 1043 (1960). 39. WANG, T. Y., AND PATEL, G., Life Sci. 6, 413 (1967).