PREPARATION AND PROPERTIES OF THYMOSTA ... - Europe PMC

PREPARATION AND PROPERTIES OF THYMOSTA TIN, A NEW THYMIC INHIBITOR OF DNA AND RNA SYNTHESIS* BY ALLAN L. GOLDSTEIN, SIPRA BANERJEE, AND ABRAHAM WHITE DEPARTMENT OF BIOCHEMISTRY, ALBERT EINSTEIN COLLEGE OF MEDICINE, YESHIVA UNIVERSITY, NEW YORK

Communicated by Alfred Gilman, January 23, 1967

In a recent publication from this laboratory,1 we have described the purification of thymosin, a lymphocytopoietic factor from calf thymus. Thymosin stimulated incorporation of isotopically labeled precursors into DNA, RNA, and protein of mouse peripheral lymph nodes when injected in vivo as well as when added directly in vitro to rat or rabbit mesenteric lymph node cell suspensions. In the course of purification of thymosin, a fraction was isolated that, in contrast to the stimulatory effects of thymosin, markedly inhibited in vitro and in vivo the incorporation of labeled nucleosides into the DNA and RNA of lymphocytes. Subsequently, it was found that this factor also inhibited incorporation of labeled nucleosides in vitro into other cell types, both lymphoid and nonlymphoid. In this communication we wish to report the preparation and partial purification from calf thymus of this inhibitory factor, which we designate as thymostatin, and to describe some of its biological activities. Materials and Methods.-Animals and tissues used: Used as a source of mesenteric lymph node cells and thymocytes for the in vitro assays were 8- to 10-week-old male New Zealand white rabbits. CBA male mice, 60 days of age, were used for the assays in vivo. The Murphy-Strum transplantable lymphosarcoma was grown in 3-week-old Wistar strain rats.2 HeLa cells were cultured in Eagle's complete spinner culture medium.3 Radioactive precursors: H3-thymidine (3.0 c/mmole), H3-deoxycytidine (2.4 c/mmole), and H3-valine (5.0 mc/mmole) were purchased from Schwarz BioResearch, Inc.; H3-uridine (1.8 c/mmole) and H3-leucine (5.0 mc/mmole) were purchased from New England Nuclear. Chemicals and reagents: Eagle's spinner culture medium (MEM), without phosphate and calcium, was obtained in powdered form from General Biochemical Corp. Sodium phosphate was added during preparation of the medium. Certified grade acetone, anhydrous chloroform and anhydrous ethyl ether were purchased from Fisher Scientific Co.; spectrophotometric grade absolute methanol from Baker Chemical Co.; Bio-Gel P-2 (100-200 mesh) from Bio-Rad Laboratories; 0-(diethylaminoethyl) cellulose (DEAE-cellulose), 0.85 mEq/gm, from Schleicher and Schuell; carboxymethyl Sephadex, C-50, 4.5 mEq/Gm, particle size 40-120 ,g, from Pharmacia Fine Chemicals, Inc. All other chemicals were of analytical or reagent grade and used without further purification. Bioassay: Assay of thymic inhibitory fractions in vitro was based on a procedure described previously.' Rabbit mesenteric lymph nodes were removed and placed in ice-chilled Eagle's spinner culture medium, previously equilibrated with 10% CO2. The cells were teased out gently and passed through a 250-mesh stainless-steel wire screen and then collected by centrifugation at 150 X g for 2 min in a Clay-Adams clinical centrifuge. The supernatant fluid was removed with a pipette, and the cell pellet dispersed by gentle tapping on the side of the test tube. The cells were then washed twice with the same culture medium, counted, and diluted to 1.5 X 107 cells/ml. The cell suspension was incubated at 370C for 30 min with 10% CO2 in air as the gas phase. Aliquots (0.5 ml) of this cell suspension were then used in each test tube which received the preparations to be assayed dissolved in 50 X of either 0.001 M phosphate buffer, pH 5.7, or 0.15 M NaCl. Incubation was continued for 3 hr with 10% CO2 in air as the gas phase. The cells were then pulse-labeled for an additional hour with a labeled precursor. The tubes were then chilled in an ice bath and centrifuged. After removal of the supernatant fluid, the cell pellets were

821

CALF THYMIC TISSUE HOMOGENIZE WITH 0.15 M NoCI; CENTRIFUGE, 1200 x 9, 15'

PRECIPITATE (DISCARD)

SUPERNATANT SOLUTION CENTRIFUGE, 105,000 x g, 60'

l PRECIPITATE

SUPERNATANT SOLUTION

(DISCARD) HEAT AT 1000C, 15'

PRECIPITATE (DISCARD)

SUPERNATANT SOLUTION ACETONE PRECIPITATION


PRECIPITATE (CRUDE THYMOSIN)

FLASH EVAPORATE ACETONE; LYOPHILIZE REMAINING SOLUTION (FRACTION A)

CHLOROFORM: METHANOL (2:1) EXTRACTION 90'

PRECIPITATE


(DISCARD) FLASH EVAPORATE

I ANHYDROUS ETHER EXTRACTION, 150' I


PRECIPITATE H20 EXTRACTION, LYOPHILIZE (FRACTION B)

(DISCARD)

I PHOSPHATE BUFFER EXTRACTION

~~~~~~~I

I SOLUTION P-2 POLYACRYLAMIDE GEL

RESIDUE (DISCARD)

Il FRACTIONS (FRACTION C)

INACTIVE FRACTIONS

ACTIVE

(DISCARD)

DEAE

-

CELLULOSE

ACTIVE FRACTIONS (FRACTION D)

INACTIVE FRACTIONS (DISCARD)

CM - SEPHADEX

I

ACTIVE FRACTIONS (LYOPHILIZE) (FRACTION E)

I

INACTIVE FRACTIONS

(DISCARD)

FJe. 1.-Diagram of fractionation procedure for purification of thymostatin.

BIOCHEMISTRY: GOLDSTEIN ET AL.

Voi,..57, 1!)67

823

broken up with the aid of a Vortex mixer, and 1 ml of cold 5% trichloroacetic acid added. The contents of the tube were transferred to a Millipore filter (0.45-1i pore size) with generous washilig out of the tuhe with (old 5% trichloroacetic acid. The filters holding the acid insoluble fractions were placed in polyethylene vials containing 15 ml of Bray's solution. The vials wert shaken thoroughly and then counted in a Packard scintillationi couinter. Stispensions of thymocytes, lymphosarcoma cells, and I eLa cells were plepared, washed, and used in assays as described above. Assays in vivo were conducted as described previously' except that animals were sacrificed at an earlier time, namely at 1 and 3 hr after receiving thymostatin rather than at 24, 48, and 72 hr, as has been the practice in the thymosin assays. The shorter period used for assay of thymostatin activity in vivo is due to results of studies indicating that this inhibitory factor, when injected, has an acute effect that is of relatively short duration.4 Total protein was determined by the Lowry method5 on precipitates obtained by treating aliquots of cell suspensions with 4 vol of cold 5% trichloroacetic acid, centrifuging the precipitate, and washing it twice with 95% ethanol. A standard protein curve was prepared utilizing bovine sertum albumin. Fractionation procedure: Figure 1 is a diagram of the fractionation procedure; all steps were carried out in the cold (0-50C). Fresh or frozen calf thymus was obtained from a local abattoir6; 500 gm were cleaned, defatted, and homogenized in 0.15 M NaCl (tissue: saline = 1: 3) in a Waring Blendor. The homogenate was centrifuged at 1,200 X g in an International refrigerated centrifuge for 15 min. The supernatant fluid was then centrifuged at 105,000 X g in a Spinco model L ultracentrifuge for 1 hr. The supernatant fluid was passed through glass wool to remove floating particulate material, the clear extract heated in a water bath at 1000C for 15 min, and then cooled in an ice bath. A large white voluminous precipitate that formed was removed by centrifugation at 50,000 X g for 15 min. The resulting clear supernatant fluid was added slowly to 10 vol of cold acetone (< - 10'C) with constant stirring. The precipitate that forms contains the crude thymosin, while the supernatant solution contains the crude inhibitor (thymostatin). The former is removed by filtration. The aqueous acetone solution remaining after removal of the crude thymosin was concentrated in vacuo in a rotary evaporator. The acetone-free solution was then lyophilized; the dried powder was designated fraction A. Fraction A was extracted with chloroform: methanol (2: 1) for 11/2 hr using 100 ml of the solvent mixture for each gram of fraction A. Thymostatin activity was completely soluble in chloroform: methanol. The insoluble residue was removed by centrifugation and the clear supernatant fluid was evaporated in vacuo. The dry residue was then extracted with anhydrous ether (100 ml ether for each gram of fraction A) for 21/2 hr, and the ether-soluble fraction removed by centrifugation and decanting. The residue was dried in vacuo and then extracted with H20 (35 mg of precipitate/ml H20). A small amount of insoluble material was removed by centrifugation and the orange-colored supernatant fluid lyophilized. The residue was designated fraction B. Fraction B was subjected to gel filtration on a column of Bio Gel P-2 polyacrylamide (2.5 X 25 cm) which had previously been washed extensively with 0.001 M phosphate buffer (pH 5.7) and decanted several times to remove fines. Each 300 mg of fraction B dissolved in 6 ml of the same buffer was placed on the column; elution was accomplished using an excess of the buffer. As shown in Figure 2, three major fractions were obtained. Inhibitory activity (tubes 24-30) was present in the ascending limb of the second peak. Tubes 24-30 were combined and designated fraction C. The latter was further purified by chromatography on a DEAE-cellulose column 35-

\260 mp

3.0-

FIA. 2.-Gel filtration of a soluble extract of a thymic fraction (fraction B) on a Bio-Gel polyacrylamide P-2 column (2.5 X 25 cm). ,! 280nw \ The eluant was 0.001 M phosphate buffer 2 1.5o lo.0 \ (pH 5.7). Fractions were collected in 5-ml 24-30 contained the major it 0-5 -NUS,/ ~vols;fractions of the biological activity. LI -1 portion ! In 14 18 22 26 30 34 38 42 46 50

rn W

2.5-

,.

FRACTION NUMBER

\


824

PRoc. N. A. S.

.8 _ .7 .6-

260 mjU

.5~ ~ ~ ~ ~ ~~~~~5 M ~

C, z

FRAC4ION

2N80U7

05? 2 00W H 5.7 pH?74 280mp ~

CL

0

.2-

OU

A\

OM pH O574 pH?4 M

z0

3.0

I

II

7

pH 7

1.OM 74

OS M pH 74

M0. ~~~~~~~~~~0.01M r64, H74 pH

OOO01 M

pH

6

2.0

1.1 20

30

4050 8090

901)0

140

170

180

FRACTION NUMBER

LL

18

2

26

30

34

64

68

12

76

80

88

106 106

FRACTION NUMBER

FIG. 3.-Ion-exchange chromatography of a soluble thymic fraction (fraction C) on a DEAE-cellulose column (2.5 X 25 cm). Elution was carried out by means of a discontinuous gradient beginning with 0.001 M phosphate buffer (pH 5.7) and increasing to 1.0 M phosphate (pH 7.4). Fractions were collected in 5-iml vol; fractions 17-34 contained the major portion of the biological

activity.

FIG. 4.-Ion-exchange chromatography of fraction D on a CM-Sephadex C-50 column (2.5 X 25 cm). Elution was carried out by means of a discontinuous gradient beginning with 0.001 M phosphate buffer (pH 5.7) and increasing to 1.0 M phosphate (pH 7.4). Fractions were collected in 5-ml vol; fractions 18-36 contained the major portion of the biological activity.

(2.5 X 25 cm). Before use, each 100 gm of DEAE-cellulose was washed successively with 2 liters of each of the following: 1 M NaCl; H20; 0.5 N KOH; H20; 0.5 N HCl in 95% ethanol; H20; 0.5 N KOH; H20. The washed cellulose was then equilibrated with 0.001 M phosphate buffer (pH 5.7) after decanting several times to remove fines. After placing fraction C on the column, the latter was initially eluted with 0.001 M phosphate, p'1 5.7; then with 0.01 M phosphate, pH 7.4, followed by a discontinuous gradient up to 1 M phosphate, pH 7.4. Figure 3 shows that this fractionation resulted in five peaks; inhibitory activity was associated with the first. The tubes containing the inhibitory fractions (tubes 17-34) were combined and designatedfraction D. The latter was then fractionated on a CM-Sephadex column (2.5 X 25 cm). Before use, each 20 gm of CM-Sephadex was washed successively with 2 liters of each of the following: 0.5 N NaOH; H20; 0.5 N HCl; H20. The washed CM-Sephadex was then equilibrated with 0.001 M phosphate buffer (pH 5.7) after decanting several times to remove fines. The sample was placed on the column and a gradient elution of phosphate buffer, beginning with 0.001 M phosphate (pH 5.7), was used to elute fractions from the column; two major fractions were obtained (Fig. 4). Most of the inhibitory activity was associated with the first. The active fractions (tubes 18-36) were then combined and lyophilized. The resulting powder was designated fraction E (5 mg, expressed as protein, from 500 gm of thymus). Fraction E, the purified inhibitor, has been stored at -5SC in a bottle containing anhydrous CaSO4 for periods of at least 1 month without loss of activity.

Results.-The supernatant fluid obtained by centrifugation of the thymic extract at 105,000 X g for 60 minutes was inactive when assayed in vitro either for stimulatory or inhibitory activity. This was also true of the supernatant fraction after it was subjected to heat. However, following the acetone precipitation step (see Fig. 1), the acetone-soluble fraction (fraction A) exhibited inhibitory activity. The solubility of the thymic inhibitory material in chloroform: methanol (2:1) permitted its separation from the large quantity of salt present in the acetonesoluble fraction. Indeed, in vitro bioassays were not possible in the presence of these high salt concentrations. Table 1 contains data for the effects of various concentrations of thymic inhibitory fractions on the incorporation in vitro of H3-thymidine into the trichloroacetic acid-insoluble precipitate of rabbit mesenteric lymphoid cells. The most


VOL. .57, 1967

825

TABLE 1 EFFECT OF CRUDE AND PARTIALLY PURIFIED THYMIC FRACTIONS ON THE INCORPORATION OF

H3-THYMIDINE

INTO AN

ACID-INSOLUBLE FORM BY A SUSPENSION OF RABBIT MESENTERIC LYMPH NODE CELLS in vitro

Fractions assayed*

Protein conc. (jug/vessel)

Cpm/mg cell proteint

Change (%)

Phosphate buffer, Control B B B C C C D D D E E E

-

22,857 284 6,450 22,000 3,246 7,986 9,303 702 3,403 10,694 405 2,405 13,005

-99 -72 -4 -86 -65 -59 -97 -85 -53 -98 -89 -43

*

122 12.2 1.22 77.5 15.5 7.75 10 1.0 0.1 6.25 0.625 0.0625

See Figure 1 for method of preparation of fractions. mean of three individual experiments.

t Each value represents the

purified fraction (fraction E) produced a greater than 85 per cent inhibition when tested in concentrations of less than 1 ,ug per incubation vessel. The data in Table 2 illustrate the influence of fraction D over a four-hour period on incorporation in vitro of labeled precursors into DNA, RNA, and protein of mesenteric lymph node cells. The incorporation of H3-thymidine, HI-deoxycytidine, and H3-uridine is greatly decreased, while there is little or no effect on the incorporation of H3-valine or HI-leucine. A time study of the effect of fraction D on precursor incorporation into lymph node DNA, RNA, and protein is shown in Figure 5. Maximal inhibition of thymidine and uridine incorporation is seen, respectively, by 30 minutes and by 60 minutes after initiation of incubation. During a two-hour incubation, there is no apparent effect of thymostatin on the incorporation of IP-leucine. In contrast to the specificity seen with thymosin for mesenteric and peripheral lymph node cells,' thymostatin inhibited the incorporation of nucleosides into several other cell types, as seen from the data in Table 3. On the basis of approximately equal cell protein concentrations per incubation vessel, fraction D markedly TABLE 2 EFFECT OF PURIFIED THYMIC FRACTION (FRACTION D) ON THE INCORPORATION OF LABELED PRECURSORS OF DNA, RNA, AND PROTEIN INTO AN ACID-INSOLUBLE FORM BY SUSPENSIONS OF RABBIT MESENTERIC LYMPH NODE CELLS in vitro Fractions assayed

Saline Control Fraction D Saline control Fraction D Saline Control Fraction D Saline control Fraction D Saline control Fraction D

Precursors*

113-thymidine it H3-deoxycytidine " H3-uridine "

H3-valine " H3-4eucine "

Cpm/mg cell proteint

Change (%)

14,360 4,098 2,213 1,085

-71

17,226 5,415 2,311 2 085 6,866 6,518

-51

-69 -10

-5

The labeled precursors were added in aliquots of 25 X to the control and experimental vessels in the following concentrations: Hs-thymidine, 2.5 yc; Hs-deoxycytidine, 0.25 pAc; Hs-uridine, 1.25 yc; H-valine, 1.25 ye; Hsleucine, 2.5 ,uc. Labeled precursors were added in 0.9% NaCl. t Each value represents the mean of three individual experiments. t Fraction D when added was in an amount equivalent to 1.38 pg protein. *

BIOCHEMISTRY: 826826 100

3

PROC.

N.

ET AL.

inhibitory fracthymic FIG.(fraction 5.-Effect of a purified of the incorporation vitro on tion D) added in labeled precursors of D)NA, RNA, and

URIDINE

protein

0

-----

GOLDSTSEIN

80 zH3TY 60

acid-insoluible form by a suspension of rabbit

into

an

miesenteric

was prepared suispenisiona 30-min node cells.tbe The lymph equiffibas described text.cellFollowing ill

/i60 40 l 4020

period at 37'C, the cells were divided into I~ra ebratioi two test vessels; a volume of phosphate buffer

Z

con-

'

2E

taining

UCINE

fraction

(1.4

7.5

/g

106

cells)

+

..

120

30

60

TIME

(MINUTES)

was

phosadded to one vessel and a comparable phate buffer was added to the second vessel. At varitime periods, aliquots of cells from vessel were removed and placed in tubes containing volume

each

0.5-mi

ous

25X

of

,

of either H3-thymidine (2.5 c), H3-uridine (1.25

The cells were (2.5 inc)0.9% NaCI. ,u c),andorH3-leicine 30 the reaction terminated, the cells treated as described in the text foresi ipulsed formin,

radioactivity. inhibited the inceorporation of H3-thymidine into rabbit thymocytes, rat lymphosarcoma cells, and HeLa cells, in addition to mesenteric lymph node cells. The depressed incorporation of nucleosides into lymphocytes in the presence of thymostatin is not a result of an irreversible cytotoxic action, since cells inhibited by thymostatin show normal precursor incorporating activity when they are washed, incubated in fresh spinner culture medium, and pulse-labeled. Thymostatin appears to act in vivo in a manner similar to that observed in vitro. Preliminary results indicate that within one hour following a single subcutaneous injection of 1 mg of thymostatin into CBA mice, there is a pronounced absolute lymphopenia and a marked inhibition of incorporation of H3-thymidine into the DNA of lymphoid tissue.4 The physical and chemical properties of the partially purified inhibitory fraction suggest that biological activity is associated with a low molecular weight component. Preliminary amino acid analysis of fractionE indicates a low content of basic amino acids. High-voltage paper electrophoresis of fraction E in a pyridine: mation of incorporation of

acetic acid buffer (pH 6.0) revealed the presence of a single ninhydrin staining component, while at pH 3.5, two major and two minor components were observed; all moved as cations. The biological of fraction is heat-stable (15 at 1000C in 0.001 M phosphate buffer, pH 5.7); fraction E gives positive Lowry, biuret, anthrone, and orcinol reactions but no color with diphenylamine. Discussion.-Partial purification from calf thymus of a potent inhibitor, designated as thymostatin, of DNA and RNA synthesis in vitro has been achieved by

min

E

activity

TABLE 3

H3-THYMIDINE

EFFECT OF PURIFIED THYMIC FRACTION (FRACTION D) ON THE INCORPORATION OF LYMPH NODE INTO AN ACID-INSOLUBLE FORM BY SUSPENSIONS OF CELLS, AND HELA CELLS in vitro*

THYMOCYTES, LYMPHOSARCOMA

Cell type

Lymphocyte Thymocyte Lymphosarcoma It

it

HeLa it

Fractions assayed

Phosphate buffer Fraction D Phosphate buffer Fraction D Phosphate buffer Fraction D Phosphate buffer Fraction 1)

* Incubation procedure as described

MESENTERIC

Protein conc. (jg/vessel)

CELLS,

Cpm/mg cell

proteint

Change

(%)

13,712

1.4 1.0

1.4 1.4

in text.

t Each value represents the mean of three individual experiments.

2,256

-84

33,995

-65

109,152 15,576 33,170

-86

97,656

8,670

-74

Voi.. 57, 196i7


827

means of saline extraction and ultracentrifugation, followed by a heat step, acetone precipitation, organic solvent extraction, gel filtration, and ion-exchange chromatography. A number of investigators7 --"' have reported the isolation from thymic tissue of preparations that inhibited cellular metabolism in vivo and in vitro. Several of these products have been characterized chemically as histones or basic peptides. The effects of these substances on incorporation of labeled precursors into DNA, RNA, and proteins are well documented. Their mechanism of action, however, appears to be complex and not completely understood. The cellular inhibitions reported for these preparations include several metabolic functions ranging from transport and oxidative phosphorylation to a possible role in the transcription and control of genetic information. In addition, certain products have exhibited an antibiotic-like activity. Dubos and Hirsch8-'0 reported the isolation of a basic, water-soluble peptide from calf thymus that under specific conditions in vitro possessed potent antimycobacterial activity. Szent-Gy6rgyi and his co-workers'2-'6 obtained from thymus, as well as other organs, a methylglyoxal derivative termed "retine" that was cytotoxic to various types of cancer cells in vitro and inhibited tumor growth in vivo. Recently, in a report of a conference, it was stated"8 that Hinshaw and Jolly have isolated lysine and arginine-rich peptides which when injected into mice inhibited the growth of transplanted sarcoma-180 or C-6 myeloma tumors. Also, Holubek'9 has prepared from the nuclei of Ehrlich ascites cells arginine- and lysine-rich histones that, respectively, suppressed or stimulated in vitro the incorporation of labeled uridine or thymidine into Ehrlich ascites cells. On the basis of chemical composition and biochemical actions in vitro, thymostatin does not appear to be identical with any of the previously described inhibitory substances from thymic tissue. The electrophoretic properties and amino acid composition of thymostatin do not resemble those of a histone and its biochemical effects are more restricted. Whereas protamines or histones, and polycations such as polylysine, in concentrations greater than 75 lug per vessel inhibited incorporation of labeled precursors into DNA, RNA, and proteins,4 thymostatin inhibits only the incorporation of nucleosides under the conditions of the assay used. In addition, the inhibitory effects seen with thymostatin are readily reversible. The thymic peptide of Dubos and Hirschg-l' was, in contrast to thymostatin, acetoneinsoluble and did not contain carbohydrate. The retine preparation of SzentGyorgyi et al.'2' 16 was reported to be very unstable, volatile, and was extractable into chloroform. Thymostatin is stable, is not volatile, and is not soluble in chloroform. In some of its properties, thymostatin resembles thymosin, the lymphocytopoietic hormone isolated from the same tissue.' Both are relatively small, heat-stable substances that give protein color reactions, contain carbohydrate, and do not adsorb to DEAE-cellulose that has been equilibrated with 0.001 M phosphate buffer, pH 5.7. However, the biological activities of thymosin and thymostatin differ. Thymosin stimulates the incorporation of nucleosides into lymph node DNA and RNA as well as the incorporation of amino acids into protein. Thymostatin, on the other hand, inhibits the incorporation of precursors into DNA and RNA, but, over a four-hour incubation period, has little effect on protein synthesis.

828


PROC. N. A. S.

The two active principles are further distinguishable by the limitation of thymosin activity to peripheral lymphoid cells while the in vitro inhibitory effects of thymostatin are also seen with other cell populations. The biological activity of the tN o thymic factors can be titrated in vitro against one another. Based on protein concentration, thymostatin is significantly more potent than thymosin at the present state of purity of each. In order to prevent the decrease in H3-thymidine incorporation into mesenteric lymphocyte DNA seen with thymostatin, approximately 10 times as much thymosin is needed in the incubation medium. The fact that thymostatin appears, under the experimental conditions described, to inhibit precursor incorporation into DNA and RNA, but not into protein of the cell types studied, suggests that thymostatin may prove a useful agent for elucidation of processes regulating synthesis of macromolecules. It is postulated that thymosin and thymostatin are humoral agents that have a role in the homeostatic regulation of lymphoid tissue structure and function. Summary.-A procedure is described for the preparation and partial purification from calf thymus of a product that inhibits in vitro and in vivo the incorporation of labeled nucleosides into lymph node DNA and RNA. This factor, designated as thymostatin, also inhibits nucleoside incorporation in vitro into other cell types. Thymostatin does not affect incorporation in vitro of labeled amino acids into cellular protein under the assay conditions used. The properties of the most purified fraction suggest that the inhibitory activity is associated with a relatively heatstable, carbohydrate-containing peptide v ith a particle size of less than 2000. Other possible effects of this preparation in vivo and its relationship to thymosin and other cellular control mechanisms are presently under investigation. Grateful appreciation is expressed to Dr. A. K. Banerjee for aid in electrophoretic studies and to Mrs. Florence D. Slater and Miss Norma Robert for valuable technical contributions. * This investigation was aided by grants from the National Cancer Institute, National Institutes of Health, USPHS (CA-07470-03), the National Science Foundation (GB-2711), and the American Cancer Society (P-68-H). 1 Goldstein, A. L., F. D. Slater, and A. White, these PROCEEDINGS, 56, 1010 (1966). 2 Kindly provided by I)r. L. Graf. I Kindly provided by Dr. J. E. 1)arnell. Unpublished observations. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem., 193, 265 (1"51 ). 6 Insel and Cohen Abattoirs, Newark, N. J. I Busch, H., Histones and Other Nuclear Proteins (New York: Academic Press, 1965). 8 Dubos, R. J., and J. G. Hirsch, J. Exptl. Med., 99, 55 (1954). 9 hirsch, J. G., and R. J. D)ubos, J. Exptl. Med., 99, 65 (1954). 10 Hirsch, J. G., J. Exptl. Med., 99, 79 (1954). Becker, P. F., and H. Green, Federation Proc., 18, 468 (1959). 12 Szent-Gy6rgyi, A., A. Hegyeli, and J. A. McLaughlin, these PROCEEDINGS, 48, 1439 (1962). 13 Allfrey, V. G., in The Molecular Basis of Neoplasia (Austin: University of Texas Press, W162), p. 581. 14 McEwen, B. S., V. G. Allfrey, and A. E. Mirsky, J. Biol. Chem., 238, 738 (1963). 15 Allfrey, Ar. G., V. C. Littau, and A. E. Mirsky, these PROCEEDINGS, 49, 414 (1963). 16 Szent-Gyorgyi, A., Science, 149, 34 (1965). 17 Sherbet, G. V., J. Embryol. Exptl. Morphol., 16, 159 (1966). 18 Hinshaw, D. B., and W. B. Jolly, cited in J. Am. Med. Assoc., 197, 36 (1966). 19 Holubek, V., J. Cell. Biol., 31, 49A (1966). I

5