We Have Previously Reported' the Isolation of Guanosine

VOL. 48, 1962 BIOCHEMISTRY: HEATH AND ELBEIN 1209

9 Ramel, A., E. Stellwagen, and H. K. Schachman, Federation Proc., 20, 387 (1961). 10 Markus, G., A. L. Grossberg, and D. Pressman, Arch. Biochem. Biophys., 96, 63 (1962). "1 For preparation of anti-Xp antisera, see Nisonoff, A., and D. Pressman, J. Immunol., 80, 417 (1958) and idem., 83, 138 (1959). 12 For preparation of anti-Ap antisera, see Grossberg, A. L., and D. Pressman, J. Am. Chem. Soc., 82, 5478 (1960). 13 For preparation of anti-Rp antisera, see Pressman, D. and L. A. Sternberger, J. Immunol., 66, 609 (1951), and Grossberg, A. L., G. Radzimski, and D. Pressman, Biochemistry, 1, 391 (1962). 14 Smithies, O., Biochem. J., 71, 585 (1959). 15 Poulik, M. D., Biochim. et Biophysica Acta., 44, 390 (1960). 16 Edelman, G. M., and M. D. Poulik, J. Exp. Med., 113, 861 (1961). 17 Breinl, F., and F. Haurowitz, Z. Physiol. Chem., 192, 45 (1930). 18 Pauling, L., J. Am. Chem. Soc., 62, 2643 (1940). 19 Pressman, D., and 0. Roholt, these PROCEEDINGS, 47, 1606 (1961).

THE ENZYMATIC SYNTHESIS OF GUANOSINE DIPHOSPHATE COLITOSE BY A MUTANT STRAIN OF ESCHERICHIA COLI* BY EDWARD C. HEATHt AND ALAN D. ELBEINT RACKHAM ARTHRITIS RESEARCH UNIT AND DEPARTMENT OF BACTERIOLOGY, THE UNIVERSITY OF MICHIGAN Communicated by J. L. Oncley, May 10, 1962 We have previously reported' the isolation of guanosine diphosphate colitose (GDP-colitose* GDP-3,6-dideoxy-L-galactose) from Escherichia coli 0111-B4; only 2.5 umoles of this sugar nucleotide were isolated from 1 kilogram of cells. Studies on the biosynthesis of colitose with extracts of this organism indicated that GDP-mannose was a precursor;2 however, the enzymatically formed colitose was isolated from a high-molecular weight substance and attempts to isolate the sus- pected intermediate, GDP-colitose, were unsuccessful. We now wish to report the enzymatic synthesis of GDP-colitose from GDP-mannose (Fig. 1) using extracts of a mutant strain derived from E. coli 0111-B4. This mutant (designated E. col; J-5) was isolated from aged cultures of E. coli 0111-B4, and appears to have properties similar to those of the mutant strains of Salmonella typhi-murium and Salmonella enteritidis previously reported.3' 20 Thus. E. coli J-5 exhibits the following characteristics: (1) inability to ferment galactose, (2) galactose sensitivity, (3) accumulation of uridine diphosphate galactose (when growth media contain galactose), (4) accumulation of GDP-colitose. In addition, analysis of the cell-wall lipopolysaccharide isolated from the parent and mutant organisms agreed with these findings. Thus, the parent organism produces lipopolysaccharide containing glucose, galactose, and co'itose.4 When the mutant is grown in the absence of galactose, the lipopolysaccharide contains no galactose and little or no colitose; supplementation of the growth medium with galactose yields lipopolysaccharide that appears similar to the normal product. Materials and Methods.-Tyvelose was prepared by mild acid hydrolysis of the cell-wall lipopolysaccharide of Salmonella typhi4' followed by neutralization, deionization, and chromatography. 3-Deoxy-D-ribohexose was a generous gift of N. K. Richtmyer, National Institutes of Health, Bethesda, Md. Uniformly labeled L-fucose was kindly provided by H. S. Isbell of the Downloaded by guest on October 1, 2021 1210 BIOCHEMISTRY: HEATH AND ELBEIN PROC. N. A. S.

CH2OH National Bureau of Standards. All o o other chemicals were obtained from CH3 commercial sources. --PPRG -- H o- PPRG Guanosine diphosphate D-glucose H was a generous gift from D. M. Carlson of this laboratory. GDP- GDP-MANNOSE GDP- COLI/OSE mannose and guanosine diphos- GUANOSINE DIPHOSPHATE GUANOSINE DIPHOSPHATE phate L-fucose (GDP-fucose) were a- D- MANNOPYRANOSIDE P(?)- 3,6-DIDEOXY-L -GALACTOPYRANOSIDE chemically prepared by condensing FIG. 1.-The conversion of GDP-mannose to GDP- the corresponding hexose 1-phos- colitose. phate with GMP morpholidate.6 Mannose 1-phosphate was prepared by the method of Posternak and Rosselet.7 The synthesis of L-fucose 1-phosphate has not been previously described. In the present studies, L-fucose was converted to L-fucose 1-phosphate by a series of procedures analogous to those used for the preparation of mannose 1-phosphate; thus, fucose - (crystalline) tetra- acetyl-fucose (crystalline) 1-chloro-triacetyl L-fucose L 1-diphenyl-phosphoryl-triacetyl-L- fucoside- L-fucose 1-phosphate. Although the anomeric configuration of L-fucose 1-phosphate and therefore of GDP-fucose is not known with certainty, it is assumed to be the ,B-L-pyrano- side. Thus, when 1-chlorotriacetyl-L-fucoside was converted to methyl-2,3,4-triacetyl-L-fuCopy - ranoside, only the ,8-glycoside (mp 96° could be isolated; this derivative is readily distinguish- able8 from the a-anomer (mp 670). Chromatographic solvent systems used in these studies were as follows: I. Ethanol: 1 A ammonium acetate, pH 7.4 (7:3); II. Isobutyric acid: ammonium hydroxide: water (57:4:39); III. 0.1 M phosphate, pH 6.8: ammonium sulfate: n-propanol (100: 60:2); IV. Ethyl acetate: acetic acid:water (3:1:3); V. n-Butanol:pyridine:water (6:4:3); VI. n-Butanol:ethanol: water (10:4:3); VII. n-Butanol:pyridine:0.1 N hydrochloric acid (5:3:2); VIII. n-Butanol: acetic acid:water (4:1:5). For the determination of radioactivity on paper chromatograms, guide strips were scanned for radioactivity in a windowless 47r scanner. For quantitation, appropriate areas of the paper were cut out, suspended in a toluene solvent,9 and counted in a Packard liquid scintillation spec- trometer. Phosphate was determined by the method of Fiske and Subbarow;"° anthrone reagent' was used for the estimation of hexose; diphenylamine reagent" was used for the estimation of deoxyribose; dideoxyhexose was determined by the thiobarbituric acid procedure." Due to the extreme acid-lability of GDP-colitose (see Fig. 3), the acid conditions employed in the thiobarbituric acid test caused considerable hydrolysis of the nucleotide even at 37°. It was therefore necessary to perform the periodate oxidation at pH 7. Under these conditions, free colitose exhibited approximately one half the molar absorbancy index as that observed under standard conditions. Conversion of GDP-Mannose to GDP-Colitose.-E. coli J-5 was grown in Trypti- case Soy broth at 370 for 12 hr with shaking. Cells were harvested by centrifugation and washed with cold 0.15 M KC1. Extracts were prepared by suspending cells in 3 volumes of water, sonicating for 5 to 10 min, and centrifuging at 25,000 X g for 30 min. The incubation mixture contained the following (/umoles in a final volume of 36.5 ml): GDP-mannose-C"4 (34,100 cpm//Amole), 12; TPN, 50; glucose 6-phosphate, 50; potassium fluoride, 500; Tris buffer, pH 7.2, 5,000; and 20 ml of crude extract. Disappearance of GDP-mannose and appearance of a product were followed by removing 0.4 ml aliquots at the indicated times (Fig. 2) and transferring to 1 ml of warm ethanol to stop the reactions. After cooling and centrifuging, the supernatant fluids were concentrated in vacuo to 0.2 ml, applied to What- man 3MM paper in one inch bands, and chromatographed in solvent I. After developing the chromatograms for 17 hr, the areas of each strip corresponding to Downloaded by guest on October 1, 2021 VOL. 48, 1962 BIOCHEMISTRY: HEATH AND ELBEIN 1211 GDP-mannose (Rf = 0.16) and 40 ' ' GDP-colitose (Rf = 0.27) were cut A out and their radioactive content 32 determined as described. These re- N

suits (Fig. 2) indicated that GDP- x° 242 mannose was rapidly converted to E A GDP-Colifose a compound with chromatographic 6 GDP-Mannose properties similar to GDP-colitose. In addition, 85 to 100 per cent of / \ the total radioactivitywasaccounted 8 1

for in these two areas of the chro- u matograms for each aliquot tested, 20 40 60 80 100 120 indicating that GDP-colitose formed MINUTES by these extracts is not further FIG. 2.-The rate of enzymatic conversion of GDP-mannose to GDP-colitose. The details of the metabolized under these conditions.conditions experiment are described in the text. After 120-min incubation, the re- mainder of the mixture was added to 100 ml of warm ethanol and centrifuged, and the supernatant fluids were chromatographed in the same manner as described for the aliquots. The radioactive band was eluted with water and rechromato- graphed on Schleicher and Schuell 589-Blue Ribbon paper first in solvent II and then in solvent I. A single radioactive band was observed in each case which corresponded in mobility to GDP-colitose (isolated from E. coli J-5). Using these purification procedures, 6.6 umoles of GDP-colitose were isolated. Homogeneity of the Product.-The nucleotide appeared homogeneous (on What- man 1 paper) in solvents I (Rf = 0.35), II (Rf = 0.37), and III (Rf = 0.36) and by paper electrophoresis in 0.05 M potassium phosphate buffer, pH 7.5, and in 0.05 M citrate buffer, pH 4.6; in each case, the radioactive and ultraviolet-light- absorbing spots coincided. The chromatographic and electrophoretic mobilities of the enzymatically synthesized material were identical in all instances with those of GDP-colitose which had been isolated from cells. Characterization of GDP-Colitose.-The nucleotide exhibited ultraviolet-light- absorption spectra at pH 1, 7, and 11 which were indistinguishable from authentic GDP. Analysis of the nucleotide gave the following results (molar ratios): guanosine, 1.00; phosphorus, 2.04; 3,6-dideoxyhexose, 1.04. Inorganic phosphorus was not detected in the preparation. Guanosine was estimated from the absorbancy of the sample at 252 mAt at pH 7, assuming a molar absorbancy index of 13.7 X 103. The nucleotide gave negative reactions with diphenylamine and with anthrone indicating the absence of deoxyribose and of hexoses. The specific radioactivity of the nucleotide was found to be 30,200 cpm/,4mole agreeing with that of GDP- mannose (34,100 cpm/,jmole). The nucleotide is considerably more labile to acid hydrolysis than other nucleotide diphosphate hexoses. As shown in Figure 3, both GDP-colitose (isolated from the cells) and the enzymatically prepared GDP-dideoxyhexose were completely hydrolyzed at pH 2 in 1 min at 1000, as compared to about 6 minutes for GDP- mannose. The dideoxyhexose was shown to be glycosidically bound to the nucleotide since (1) it gave a negative reducing sugar test, (2) the carbonyl group was resistant to Downloaded by guest on October 1, 2021 1212 BIOCHEMISTRY: HEATH AND ELBEIN PROC. N. A. S.

,,, reduction by sodium borohydride, and (3) it gave a negative thio- 100 - barbituric acid test. After mild acid hydrolysis, the N 80 / nucleotide diphosphate moiety 0 E / / was characterized as GDP by 60 chromatography in solvents II and III. In both instances, the U 0: 40 g / / major ultraviolet-light-absorbing CL ^ / ;/ o GOP-Colilose(Enzymolic) components were indistinguish- 20 if / /f ^ GDP-Co/ltose (From cel/s) able from GDP (1, R. = 0.3; II, 20 /o GDP-Munnose Rf = 0.46), while traces of ma- * E. Co/i 0-/// sol- 0Lpopo/ysocchoride5 10 15 ventsterial werewhichobservedcorrespondedin both to MINUTES GMP. GDP is readily distin- FIG. 3-Acid-lability of GDP-colitose. The rate of guishable from ADP, UDP, and hydrolysis of GI)P-colitose was compared to that of ' (IDP-mannose and lipopolysaccharide (from E. coli CDP in these solvent systems. 0111-B4) at pH 2 at 1000. In all cases, samples were In addition the nucleotide di- adjusted to pH 2 in an ice bath and placed in a boiling water bath, and, at appropriate intervals, aliquots were phosphate was further shown to removed and pipetted into 0.1 ml of ice cold 0.5 M be a pentose nucleotide as it con- phosphate buffer. In the case of GDP-colitose and lipopolysaccharide, the rate of hydrolysis was meas- sumed periodate when the chro- ured by the thiobarbituric acid test' (using neutral matograms were treated with periodate as described). For GDP-mannose, the rate of hydrolysis was determined by the Park-Johnson periodic acid and benzidine.15 reducing sugar method.14 The values presented for After mild acid hydrolysis of the rate of hydrolysis of lipopolysaccharide represent only colitose release; thus, these values do not reflect the sugar nucleotide, the radioac- hydrolysis of the other sugar components. tive dideoxyhexose was purified by deionization with mixed-bed ion-exchange resin (Dowex-1-CO3= and Dowex-50-H+) and was tentatively identi- fied as colitose by cochromatography with the authentic compound as indicated in Table L. Of the four possible pairs of enantiomorphs of the dideoxyhexoses, only TABLE 1 CHROMATOGRAPHY OF SUGAR FROM ENZYMATICALLY SYNTHESIZED NUCLEOTIDE Solvent systems* Compound V VI VII VIII Unknown 0.66 0.56 0.64 0.55 Colitose 0.65 0.57 0.64 0.56 Tyvelose 0.73 0.64 0.74 0.63 3-deoxy-D-ribo-hexose 0.51 0.39 0.47. 0.35 i-fucose 0.44 0.33 0.39 0.31 2-deoxy-D-ribose 0.57 0.47 Unknown (reduced) 0.58 - 0.54 Colititol 0.58 0.55 * See text for description. tyvelose (3,6-dideoxy-D-arabinohexose) and colitose (3,6-dideoxy-L-xylohexose) were available as standards. In all instances, the unknown sugar cochromato- graphed with authentic colitose, and the radioactivity corresponded precisely with the colitose area of the chromatograms as determined with the ammoniacal silver reagent'6; no other reducing substances were detectable on the chromatograms. Further, after reduction of the sugar with sodium borohydride, a product was ob- Downloaded by guest on October 1, 2021 VOL. 48, 1962 BIOCHEMISTRY: HEATH AND ELBEIN 1213

tained that was indistinguishable from authentic colititol by paper chromatography in solvents V and VII. In addition, the sugar alcohol gave a positive thiobarbituric acid test with an absorption maximum at 532 mju; these results would not be obtained if the parent sugar were a 2-deoxyhexose since the corresponding alcohol could not yield malondialdehyde upon periodate oxidation. The identity of the dideoxyhexose was definitely established as colitose by preparation of a large quantity of the nucleotide and conversion of the sugar moiety to colititol. In this case, the incubation mixture contained the following (Amoles in a final volume of 32 ml): GDP-mannose-C 4 (2,900 cpm/,Lmole), 61; TPN, 50; glucose 6-phosphate, 50; potassium fluoride, 500; Tris buffer, pH 7.2, 5,000; and 20 ml of crude extract from E. coli J-5. The mixture was incubated for 4 hr at 370, treated with warm ethanol and streaked on Whatman 3MM filter paper as described. The GDP-colitose area of the chromatogram was eluted with water, heated in 0.01 N H2SO4 for 10 min at 1000, neutralized with solid BaCO3, and centrifuged. The supernatant fluid was treated with mixed-bed ion-exchange resin, concentrated in vacuo to a small volume, and streaked on Schleicher and Schuell 589-Green Ribbon paper which was then developed with solvent IV for 3 hr. Guide strips were cut from the edge of the chromatograms and stained with the ammoniacal silver reagent, revealing a band of material which corresponded to colitose (Rf = 0.57; representing all of the radioactivity on the chromatogram) and only a trace of some other reducing substance near the origin (Rf = 0.10). The band corresponding to colitose was eluted with water and concentrated in vacuo to about 5 ml. Analysis of the solution indicated the presence of approximately 60 ,umoles of dideoxyhexose. The solution was treated with 75 mg of sodium borohydride at room temperature for 30 min, adjusted to pH 1 with 2 N HCl and concentrated in vacuo to dryness. The residue was dissolved in methanol and concentrated in vacuo to dryness, and this process was repeated four times. Finally, the aqueous solution was deionized with mixed-bed ion-exchange resin and concentrated to a clear, colorless syrup from which water was removed by dissolving the syrup in ethanol and concentrating to dryness several times. The syrup was seeded with authentic colititol and stirred for several min. A few drops of acetone were then added, and the suspension was placed in an ice bath and allowed to crystallize for about 1 hr. The crystalline material was harvested by centrifugation, washed with cold acetone and ether, and dried in vacuo. The mother liquor was again carried through the procedure and a second crop of crystals was obtained. The total yield of crystalline material was approximately 6 mg. Thus, a total of 40 Amoles (2,240 cpm/,4mole) of crystalline colititol was obtained from approximately 61 Mmoles of GDP-mannose (2,900 cpm/,4mole) which had been used as substrate, or a yield of about 66 per cent. The physical constants which have been reported for synthetic colititol'7 are: mp 92-940, [a]D -51 (c = 2.5 in methanol); and the constants for authentic colititol prepared in our laboratory were, mp 89-910, [a]D23 -50.60 (c = 0.25 in methanol). The dideoxyhexitol derived from the enzymatically synthesized GDP-colitose exhibited the following values: mp 89-910, [aCD23 -50.30 i 0.60 (c = 0.32 in methanol).'8 These results clearly establish the identity of the dideoxy-hexitol as colititol, 3,6-dideoxy-L-xylo-hexitol and clearly distinguish it from any of the other three possible pairs of enantiomorphs, 3,6- dideoxy-arabino-hexitol, 3,6-dideoxy-ribo-hexito, and 3,6-dideoxy-lyxo-hexitol.4 Downloaded by guest on October 1, 2021 1214 BIOCHEMISTRY: HEATH AND ELBEIN PROC. N. A. S.

From these data, it was concluded that the sugar moiety of the isolated guanosine sugar nucleotide is colitose, or 3,6-dideoxy-L-galactose. In assigning the structure of the nucleotide as guanosine diphosphate colitose (Fig. 1), the following assump- tions have been made: (1) the colitose moiety is attached to the terminal phosphate group of the nucleotide diphosphate; (2) the colitose moiety of the nucleotide is in the pyranose ring form; and (3) the anomeric configuration of the glycosidic bond is in the fl-form by analogy to the position of this bond in the substrate, GDP- mannose. Specificity of the Reaction-To determine the specificity of the enzyme system for GDP-mannose, the extract was incubated with the other known guanosine hexose nucleotides, GDP-glucose and GDP-fucose. The results of these studies, shown in Table 2, clearly indicate that only GDP-mannose is capable of serving as a pre-

TABLE 2 SPECIFICITY STUDIES Total Radioactivity (Cpm) in Substrate* Colitose Mannose Glucose Fucose GDP-mannose, 3,600 2920 14 GDP-mannose, t 3,600 4 3220 GDP-glucose, 5,000 12 3153 GDP-fucose, 2,500 5 - 2250 * Incubation mixtures contained the following (pmoles in final volumes of 0.35 ml): either GDP- mannose, 0.1, GDP-glucose, 0.03, or GDP-fucose, 0.25; TPN, 0.5; glucose 6-phosphate, 0.5; potassium fluoride, 5; Tris buffer, pH 7.2, 50; and 0.2 ml of crude extract of B. coli J-5. Incubations were conducted at 370 for 1 hr. The mixtures were then treated with ethanol, hydrolyzed, deionized, and chromatographed on Whatman 1 paper with solvent VII. The strips were first scanned for radioactivity, and then the radioactive areas and the colitose areas of each strip were counted as described. t The extract used in this experiment was heated at 1000 for 2 min before it was added to the incubation mixture.

cursor of GDP-colitose. In addition, the data indicate that these extracts are in- capable of converting GDP-glucose to GDP-mannose and, further, that GDP- fucose cannot be an intermediate in the conversion of GDP-mannose to GDP- colitose. No definitive information is presently available concerning the cofactor require- ments of this enzyme system. The ability of the extracts to convert GDP-mannose to GDP-colitose was stimulated only 10 to 20 per cent by the addition of TPNH or a TPNH generating system. Exhaustive dialysis of the extracts against water, buffers, or EDTA resulted in substantial losses (50 per cent or more) of activity, although they could be reactivated only slightly by the addition of TPNH (or a TPNH-generating system) or by divalent cations. Discussion.-Nikaido3 suggested from studies with mutants of S. typhi-murium and S. enteritidis, which are similar in their properties to E. coli J-5, that in the absence of a galactosyl donor (UDP-galactose), the organisms are able to synthesize only the glucan portions of their cell-wall lipopolysaccharides; when a galactosyl donor is available (cells grown in the presence of galactose), cell-wall lipopolysaccharides are present which contain a normal complement of sugars (glucose, galactose, mannose, rhamnose, and either abequose or tyvelose in S. typhi-murium and S. enteritidis, respectively). The present studies with E. coli J-5 support this con- tention in so far as glucose-grown cells possess essentially only glucose in their lipopolysaccharides, while cells grown on galactose-supplemented media possess glucose, galactose, and colitose in amounts similar to the parent organism, E. coli Downloaded by guest on October 1, 2021 VOL. 48, 1962 BIOCHEMISTRY: HEATH AND ELBEIN 1215

0111-B4. In addition, the studies with E. coli J-5 indicate that colitose is probably attached terminally to the polysaccharide via galactose. The direct formation of GDP-colitose from GDP-mannose substantiates the in vivo isotope experiments reported by Cynkin and Ashwell,'9 which indicated that colitose was formed from glucose without inversion or cleavage of the carbon chain. The conversion of GDP-mannose to GDP-colitose would appear to involve a complex series of enzymatic transformations; thus, this conversion requires reduction at carbon atoms 3 and 6 in addition to epimerization at carbon atom 5. Although the isolation of CDP-abequose and CDP-tyvelose has been reported,20 the present studies are the first report of the enzymatic synthesis of any of the dideoxy-hexose nucleotides, and therefore no precedence has been established for a possible mechanism for the biosynthesis of these unique sugar nucleotides. Gins- burg,2' in his study of the conversion of GDP-mannose to GDP-fucose and Glaser and Kornfeld22 and Okazaki et at.23 in their studies of the conversion of TDP- glucose to TDP-L-rhamnose, in each instance, concluded that one of the intermediates in the biosynthesis of both of these 6-deoxyhexose nucleotides was a 4-keto-6- deoxyhexose nucleotide. It appears from the previous studies, therefore, that the 6-deoxy group is formed without any net reduction of the molecule and that TPNH functions to reduce the compounds ultimately to the level of fucose or rhamnose after the formation of the 4-keto-6-deoxy intermediate. Whether or not analogous intermediates are involved in the biosynthesis of GDP-colitose remains to be established. In this regard, it is of interest to note in the present studies that GDP->- fucose does not serve as a precursor of GDP-colitose. Further work is in progress in an attempt to elucidate the details of the mechanism of the biosynthesis of GDP-colitose from GDP-mannose.

* The Rackham Arthritis Research Unit is supported by a grant from the Horace H. Rackham School of Graduate Studies of The University of Michigan. This investigation was supported by a grant from the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health. t Research Career Development Awardee, U.S. Public Health Service. t Post-doctoral Fellow, National Institute of Allergy and Infectious Diseases, National Insti- tutes of Health. I Heath, E. C., Biochim. Biophys. Acta, 39, 377-378 (1960). 2Heath, E. C., Federation Proc., 19, 85 (1960). 3Nikaido, H., Biochim. Biophys. Acta, 48, 460-469 (1961). 4Westphal, O., and 0. Luderitz, Angew. Chem., 72, 881-891 (1960). 5 This preparation was a generous gift from A. G. Johnson of this University. 6Roseman, S., J. J. Distler, J. G. Moffatt, and H. G. Khorana, J. Am. Chem. Soc., 83, 659- 663 (1961). 7Posternak, T., and J. P. Rosselet, Helv. Chim. Acta, 36, 1614-1623 (1953). 8 Hockett, R. C., F. P. Phelps, and C. S. Hudson, J. Am. Chem. Soc., 61, 1658-1660 (1939). 9 Hayes, F. N., D. G. Ott, V. N. Kerr, and B. S. Rogers, Nucleonics, 13, No. 12, 38-41 (1955). 10 Fiske, C. H., and Y. Subbarow, J. Biol. Chem., 66, 375-400 (1925). 11 Loewus, F. A., Anal. Chem., 24, 219 (1952). 2 Seibert, F. B., J. Biol. Chem., 133, 593-604 (1940). 13 Cynkin, M. A., and G. Ashwell, Nature, 186, 155-156 (1960). 14 Park, J. T., and M. J. Johnson, J. Biol. Chem., 181, 149-151 (1949). 6 Gordon, H. T., W. Thornburg, and L. N. Werum, Anal. Chem., 28, 849-855 (1956). 16 Trevelyan, W. E., D. P. Procter, and J. S. Harrison, Nature, 166, 444 (1950). 7 Luderitz, O., A. M. Staub, S. Stirm, and 0. Westphal, Biochem. Zeit., 330, 193-197 (1958). Downloaded by guest on October 1, 2021 1216 BIOCHEMISTRY: HUANG AND BONNER PROC. N. A. S.

18 The authors are indebted to J. J. Distler of this laboratory for performing the polarimetry studies. 19 Cynkin, M. A., and G. Ashwell, Bact. Proc., 161 (1960). 20 Nikaido, H., and K. Jokura, Biochem. and Biophys. Res. Comm., 6, 304-309 (1961). 21 Ginsburg, V., J. Biol. Chem., 236, 2389-2393 (1961). 22 Glaser, L., and S. Kornfeld, J. Biol. Chem., 236, 1795-1799 (1961). 23 Okazaki. R., T. Okazaki, and J. L. Strominger, Federation Proc., 20, 85 (1961).

HISTONE, A SUPPRESSOR OF CHROMOSOMAL RNA SYNTHESIS* BY RU-CHIH C. HUANG AND JAMES BONNER

DIVISION OF BIOLOGY, CALIFORNIA INSTITUTE OF TECHNOLOGY Communicated May 22, 1962 We have previously reportedl1 2 that chromatin isolated from pea embryos pos- sesses the ability to carry out the DNA-dependent synthesis of RNA from the four riboside triphosphates.3 The present paper concerns the roles in such synthesis of the several components of chromatin. It will be shown that the DNA of pea embryo chromatin is present in at least two forms, namely, as DNA itself and as DNA bound in nucleohistone complex. It will be further shown that DNA fully com- plexed with histone is inactive in the support of DNA-dependent RNA synthesis. Materials and Methods.-Pea embryos: Pea seeds (var. Alaska) were germinated in 35-gallon barrels in lots of 25 lb. The seeds were soaked for 5 hr in running water at 200C and then gently sprayed with water for an additional 35 hr. The embryonic axes, approximately 1 cm in length, were next separated from the cotyledons in a semiautomatic 3-stage disassembly line. Fifty pound dry weight of seeds yield approximately 1 kg fresh weight of embryos. Preparation of chromatin: The chilled, sterilized (with 10OX diluted Clorox) embryos were ground for approximately 1 min in a Blendor with an equal weight of grinding medium (sucrose 0.25 M, tris pH 8.0,0.05 M, ,3-mercaptoethanol, 0.01 M, MgCl2, 0.001 M) and filtered successively through cheesecloth and miracloth to remove cell wall debris. The filtrate was then centrifuged for 30 min at 4,000 X g. Under these circumstances, mitochondria and smaller particles remain in suspension while starch and chromatin sediment. The gelatinous chromatin layer was scraped from the underlying, firm starch layer and washed by successive recentrifugation (10,000 X g) in grinding medium (1X), sucrose, 0.25 M (2X), and tris, 0.05 M, pH8.0(2X). ,3-Mercapto- ethanol, 0.01 M, was included in all of the above media. The final pellet was resuspended in the tris buffer, 30 ml per kg initial embryos. The yield of such crude chromatin is approximately 0.5 gm per kg embryos; the yield of DNA, 50 mg per kg embryos. Purification of crude chromatin: The crude chromatin prepared as described above contains ca. 95% of the DNA of the embryo but is contaminated by nonchromosomal protein, removable by sucrose gradient centrifugation. This was accomplished by layering 5 ml of crude chromatin suspension on 25 ml of 2 M sucrose (0.01 M in j-mercaptoethanol). The upper third of the tube was then gently stirred to form a rough gradient and the tubes centrifuged in the SW-25 swinging bucket head at 20 krpm for 3 hr. The resulting pellet of which the major constituents are DNA and histone will be referred to as purified chromatin. Approximately 70% of the DNA of crude chromatin is recovered in the purified chromatin. Analyses: DNA and RNA were determined principally by the Schmidt-Tannhauser procedure according to Ts'o and Sato.4 The diphenylamine method of Burton6 was used for determination of DNA in the presence of much protein. Total protein was determined by the Folin-phenol method as described by Lowry.8 Histone protein was separated from total protein on the basis of the solubility of the former in 0.2 N HC1 and determined on the acid extract by the Lowry method. Melting point determinations of DNA and nucleohistone were carried out Downloaded by guest on October 1, 2021