Sept. 20, 1957
INTERACTION OF BACTERIAL POLYGLUCOSANS WITH CONCANAVALIN-A
Cl,, c 0.8), [aIsaD +4' (glacial HOAC, C 0.8), [a]*'D -32" (SOCls, c 0.6), [ a ] * l -22" ~ (dioxane, C 1). Anal. C a l ~ d .for ~ ~ClgHl~O9&!1: C, 49.44; H, 5.46; C1, 1.03; OAc, 43.63. Found: C, 48.53; H, 5.22; C1, 1.03-1.60; OAc," 43.07. Decarboxylation of Reacetylated Reduced Oxidized Cellulose Acetate.-The product formed by the reaction of reacetylated reduced oxidized cellulose acetate with thionyl chloride was treated with silver oxide and bromine following the procedure used for the product obtained from the reaction of oxidized cellulose acetate with thionyl chloride; [cY]"D -12.3' (C2H2Clir C 0.4)) [a]"D k0.0" (ghCkZl HOAC, c 0.6), [a]laD -14' (dioxane, c I), [aIz4D -13.5' (SOCln, c 1.7). Anal. Calcd.26 for ClclH1870saBr:C, 48.86; H, 5.44; Br, 2.31; OAc, 43.42; COZ,6.8 ml. of 0.1 N NaOH per g. Found: C, 48.47; H, 5.22; Br, 2.50; OAC," 43.07; c o z , 6.1 ml. of 0.1 N NaOH per g. Reduction of Decarboxylated Reacetylated Reduced Oxidized Cellulose Acetate with Sodium Borohydride..4n amount of 5 . g. of the decarboxylated reacetylated reduced oxidized cellulose acetate was dissolved in 500 ml. of dioxane (distilled from sodium). Sodium borohydride (2 g.) was added and the mixture stirred for 4 hours a t 40-50'.
[CONTRIBUTIONFROM
THE
5055
Stirring was continued overnight a t room temperature, at the end of which time a thick thixotropic gel had formed. The gel was broken up, filtered and the solid triturated with water (2 X 500 ml.), filtered, and washed with aqueous half-saturated sodium bicarbonate (2 X 100 ml.), water ( 2 X 500 ml.) and absolute ethanol (2 X 500 ml,). The polymer was dried overnight under a high vacuum at room temperature and then dissolved in 500 ml. of dioxane and precipitated by the addition of approximately two volumes -20" (CoH2C14: of ether; yield approximately 5 g.; [ a ] l * ~ C 0.4), [aI2'D +5' ( g h C i a l HOAC, C 0.8), [ a I t 1 D -21 (soc12, cO.4); [a]18D-19" (dioxane, c 1). Anal. Calcd.*6for ClllH188091:C, 49.98; H , 5.60; OAc, 44.44. Found: C, 49.85; H , 5.55; O A ~ , ~ ~ 4 2 . 5 6 .
Acknowledgment.-We wish to thank Professor J. H. Shereshefsky for the sample of cellulose acetate of molecular weight 29000 and Dr. c. H. Penning of Eastman Products, Inc., Kingsport, Term., for the oxidized cellulose acetate. We also wish to thank Mrs. P. P. Wheeler for the microanalyses. INDIAN HEAD,MD.
DEPARTMENT O F AGRICULTURAL BIOCHEMISTRY, UNIVERSITY OF MINNESOTA]
The Interaction of Bacterial Polyglucosans with Concanavalin-A BY J. A. CIFONELLIAND F. SMITH^ RECEIVED MARCH29, 1957 The globulin, concanavalin-A, obtained from Jack Bean meal, which has been shown previously to give a precipitate with glycogen and not with the apparently closely related amylopectin, has been used t o ascertain whether certain bacterial polyglucosans are related t o glycogen or to amylopectin.
Glycogen and amylopectin have been reported to be similar in respect to properties and structures.2-6 Some of the properties displayed by the polyglucosans obtained from Neisseria perJEava appear to be intermediate between glycogen and a m y l ~ p e c t i n . ~One ~ ~ preparation was found3.'3 by periodate oxidation and by methylation to have, like glycogen, an average repeating unit of about 12 residues whereas it behaved like amylopectin in giving a reddish-purple color with iodine and undergoing a 55-597, degradation upon treatment with 8-amylase. In view of the apparent relationship of the Neisseria polyglucosans to both glycogen and amylopectin i t was decided to investigate their behavior when treated with concanavalin-A, a globulin from jackbean mealI8-l2 since i t had been observed ( I ) Paper No. 3300, Scientific Journal Series, Minnesota Agricultural Experiment Station, University of Minnesota, St. Paul, Minnesota. (2) J. Lamer, Barbara Illingworth, Gerty T. Cori and C. . '5 Cori, J. Biol. Chem., 199,641 (1952). (3) S. A. Barker, E. J. Bourne and M. Stacey, J. Chcm. Soc., 2884 ( 1950). (4) D.J. Bell and D. J. Manners, ibid., 3641 (1952). (5) M.Stacey, Adu. Enaymol., 16, 301 (1951). (6) E. J. Hehre and D. M. Hamilton, J. B i d . Chcm., 166, 777 (1946). (7) E. J. Hehre, D. M. Hamilton and A. S. Carlson, ibid., 171, 267 (1949). (8) J. B. Sumner and D. J. O'Kane, Enrymol., 13, 251 (1948). (9) J. B. Sumner and S. F. Howell, J . Biol. Chem., 116, 583 (1936). (IO) S. F. Howell, Federnlion Proc., 12, 220 (1953). (11) J. A. Cifonelli and F. Smith, A n d . Chcm., 37, 1639 (1955). (12) J. A. Cifonelli, R. Montgomery and F. Smith, THIS JOURNAL, 78, 2485 (1956).
that this protein gives a precipitate with glycogen9s11*12 but not with amylopectin.12 Certain of the enteric bacterial polyglucosans were likewise investigated as were various dextrans t o ascertain whether they reacted with concanavalin-A. Methods.-The preparation of concanavalin-A reagent from jackbean meal was described earlier.11d2 The procedure for estimating "glycogen-values'' is based on the absorbancy obtained upon treatment of one mg. of the material with the concanavalin-A reagent, in relation to the absorbancy obtained upon similar treatment of one mg. of a standard glycogen.11J2 a-Amylolysis was carried out by treating 0.2% solutions of the polysaccharides a t 25' with 0.1 volume of diluted saliva (1 :IO) buffered to pH 7.0 with phosphate. 6-Amylolysis was performed a t pH 4.5 and 25", using 1 mg. of Wallerstein 6-amylase of analytical purity with 100 mg. of polysaccharide in 50 ml. of 0.01 M acetate buffer solution.
Results and Discussion
It had been noted earlier12 that neither amylopectin nor its B-limit dextrin were precipitated on treatment with concanavalin-A reagent, whereas all of the glycogens examined did so. Since Neisseria perfiva cultures which contained sucrose were stated t o produce amylopectin-like polysaccharides having in some respects properties intermediate between those of glycogen and amylopectin, 3,6 i t appeared of interest to examine the Neisseria polysaccharides with concanavalin reagent. The results are shown in Table I. Contrary to the results obtained with plant amylopectins, the Neisseria amylopectin-like sub-
THEIXTERACTION OF POLYGLUCOS ASS WITH CONCANAVAI >IX-A Preparation
19-34 11-1 11-2 19-34 4943" F90A" NRKL B-512" P(CL) 3-1 A12, KOH prep. 9 1 2 , sonic prep.
Organism used
Apparent glucosan type
Absorbancy
Glycogen value
N. perjlava (live culture) N. perflava (live culture)
Amylopectin 0.137 0.80 .125 .75 Amylopectin perjlava (live culture) .125 .75 Amylopectin N . perflava (cell-free enzyme) ,170 1.00 G1y cogen Acetobacter capsulatum Dextran 0.60 .099 Streptococcus (group H ) Dextran .000 .oo Leuconostoc mesenteroides .05 ,006 Dextran Salmonella montevide2 ,232 1.35 Glycogen il erobacter aerozenesc 1.10 ,188 Glycogeii -1, aerogenesd ,177 1.05 Glycogen Dissolved with the aid of alkali (.V NaOH) and neutralized with A' HC1. Grown on 1% mannose-nutrient agar and isolated by extraction with KOII atid cthauol precipitatioii. Grown on 1% glucose-nutrient agar and isolated a s in b. A s in 0, but a sonic lysate.
stances all show significant reactions with the concanavalin reagent. Though the glycogen values of the substance were somewhat lower than are generally obtained with animal or plant glycogens, it will be noted from Table I1 that fractionation of one of the Neisseria polysaccharides (11-1) gave three fractions (I, 20yo; 11, 605; and 111, 2070, respectively, of the material fractionated) with increasing glycogen values. I t thus appears that these products may indeed be largely glycogen-like and that any amylopectin-like material is probably present to L: smaller extent. TABLE I1 EFFECTOD FRACTIONATION^ ON THE GLYCOGEN VALUESOF C E R T A I N BACTERIAL Gl.UCOS.4NS Preparation
Fraction 1 G. value
Fraction 2 G. value
Fraction 3 G. value
N. per$aaa 11-1 A . capsulatum 4943
0.70 0.95 1.05 .75 .60 a Fractionation was effected in the usual way by adding alcohol to a n aqueous solution of the polysaccharide.
On the other hand, the cell-free system from Neisseria perJ?aua cultures produces a glycogen-like substance when incubated with sucrose. The material obtained in this way from strain 19-34 had a glycogen value corresponding to those shown by normal human and rabbit liver glycogens.'* Periodate oxidation indicated an average chain length of 11 ini its.'^ From these results it appears that both the cell-free and the cell-containing systems produce glycogen-like substances while any amylopectin-like material that is produced appears in cultures of the growing cells. The fact that the amylopectin-like preparation, 11-1, was found3 by methylation studies to have a chain length of 11-12 residues, further adds to the probability that glycogen is the main constituent. The possibility that the substance is homogeneous and represents a polysaccharide with properties intermediate between glycogen and amylopectin appears to be ruled out by the fractionation results (see Table 11) which show that increasing glycogen values are obtained for successive fractions. Reactions such as iodine coloration and extent of degradation by a-amylase are inadequate criteria for distinguishing between the two types of polysaccharides. Thus, while preparation 11-1showed (13) SI. Ahdel-Akher and F. Smith, THISJ O U R N A L , 1 3 , 994 (1951),
P-amylolysis of .53-.30%, values of 25-569;'0 have been recorded4*'*for various animal glycogens and a rabbit liver glycogen sample, reported to have an IS unit branch length15v16was found to be degraded 5370 by P-amylase. Similarly, the redpurple color which the amylopectin-like substances produce with iodine is much like that obtained with rabbit leg muscle glycogen.'* The similarity in properties of glycogen and amylopectin makes i t difficult to distinguish glycogen when admixed with amylopectin and it would not be surprising that a mixture of the two should be looked upon as being amylopectin-like. It would be interesting to investigate with concanavalin-A other glucosans with apparently overlapping properties such as the Floridean and Ulva starches described by Meeuse and KregerI7 which give X-ray powder diagrams much like potato starch but have average chain lengths, as determined by periodate oxidation, similar to that of glycogen. An interesting behavior of the Neisseria polyglucosans was encountered when these were treated with salivary a-amylase. Each of the polysaccharides failed to be degraded sufficiently during 5 hours incubation with diluted saliva buffered a t pH 7.0 to diminish completely the reaction with the concanavalin reagent. The extent of degradation as reflected by reaction with concanavalin varies considerably for the different preparations. HOWever, one of the preparations (11-1) was fractionated with ethanol and it was then found that the fmctions obtained were degraded in 10 minutes by the salivary a-amylase to the extent that no concanavalin reaction was obtained. Similar results were noted with certain muscle glycogens.'? Another unusual reaction concerns the synthesis's of a dextran by an Acetobacter capsulnfunz enzyme system acting upon dextrins instead of sucrose which is the substrate for the formation of dextran by Leuconostoc mesenteroides. Equally unusual is the result obtained in the treatment of (14) M. Liddle and D. J. Manners, Biochem. J . , 61, XI1 (1955). (15) T. G. Halsall, E. L. Iiirst, L. Hougb and J. K. ?I. Jones, J . Ciirm. SOC.,3200 (1949). (16) W. N. Haworth, E. I,. H i r s t and F. A. Isherwood, ibid., 784 (1937). (17) J. D. hfeeuse and D. R. Kreger, Biochim. Biophys. Acta, 13, 593 (1954). (18) E. 5. I-lehre and D. 11. R n r n i l t ~ , ~.I,~ .B i d . C h ~ v z . ,192, lC,l ( 1 'I5 1).
Sept. 20, 1937
NEW PIIOSPIIORYI,,\TED PENTOSE DERIV?ITIVES
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Reports on the isolation of glycogen-like subthe Acetobacter dextran with concanavalin reagent. Thus, while the dextrans formed by Leuconostoc stances from bacteria other than Neisseria have mesenteroides and Streptococcus group H, show no been few. Glycogens have been isolated from significant reaction with concanavalin, the poly- avian tubercle bacilli,2o from Bacillus megathesaccharide formed by Acetobacter capsulatum shows riumZ1and most recently from enteric bacteria.22 Glycogens from Aerobacter aerogenes and Sala glycogen-value equivalent to 60% of that shown monella montevideo (enteric bacteria) grown on nuby normal human liver glycogen (see Table I). Fractionation of the polyglucosan synthesized trient agar containing 1% mannose for the Salby Acetobacter capsulatum on a limited scale gave monella and 1% glucose for the Aerobacter cultures two fractions, one of which showed a small in- were examined by means of the concanavalincrease in the glycogen value (Table 11). Unlike polysaccharide reaction. The results which are the Neisseria polysaccharide which gave rise to shown in Table I indicate that the bacteria profractions that became less opalescent in aqueous duced glycogens of the normal type, although the solution as the glycogen value increased, the Salmonella glycogen gave a somewhat higher than Acetobacter polysaccharide fraction which formed average glycogen-value. Treatment of the three preparations from the enthe more opalescent solution showed the larger teric bacteria with diluted saliva resulted in their glycogen value. The above results indicate that the Acetobacter rapid degradation to the point where they no longer polysaccharide must differ in some respect from the reacted visibly with concanavalin reagent. It other dextrans tested. The nature of this differ- was interesting, however, that the Salmonella ence has yet to be ascertained. T h a t the results glycogen which was slightly impure as obtained may be due to the presence of glycogen-like mate- from Dr. Levine was not degraded by p-amylase rial is possible but unlikely since periodate oxidation, until it was further purified. All three of the puriin which the periodate consumption was 1.89 moles fied glycogens underwent a 30-40% hydrolysis per anhydroglucose unit, resulted in the formation when treated with P-amylase, a result in agreeof 0.82 mole of formic acid per anhydroglucose ment with the values shown by most animal glycounit. Such results favor a dextran type of struc- gens. Acknowledgment.-The authors thank Dr. E. J. ture for the Acetobacter polyglucosan in which the ratio of 1,6-linkages t o other types is 5:l. It is Hehre for the samples of Neisseria, Acetobacter and conceivable that the difference between the Leu- Streptococcal dextrans, Dr. Allene Jeanes for the conostoc dextran and the Acetobacter polyglucosan is Leuconostoc dextran and Dr. Seymour Levine for due to the presence of linkages in the latter which the enteric bacterial glycogens. are not of the 1,6-type. The formation of dextrans (20) E. Chargaff and D. H. Moore, J . B i d . Chem., 155,493 (1944). (21) C. Barry, R. Gavard, C. Milhaud and J. P. Aubert, Comfit. with varying types of linkageslg may make i t pos936, 1062 (1952). sible to test this hypothesis and information bear- rend., (22) S. Levine, H. J. R. Stevenson, E. C. Tabor, R. H. Bordner and ing on this point is now being sought. L. A. Chambers, J . Bad., 66, 664 (1953). (19) J. C. Rankin and A. Jeanes, THISJOURNAL, 76, 4435 (1954).
[CONTRIBUTION
FROM THE
ST.PAUL1, MINNESOTA
DEPARTMEST O F -4GRICULTURAL BIOCHEMISTRY, COLLEGE CALIFORNIA ]
OF
AGRICULTURE, UNIVERSITY
OF
Anomeric 1-Dicyclohexylarnmonium Phosphate Esters of D-Glucopyranose, D-Galactopyranose, D-Xylopyranose and L-Arabinopyranosel BY E. W. PUTMAN AND W. 2. HASSID RECEIVED APRIL 30, 1957 Three new phosphorylated pentose derivatives, p-D-xylose 1-phosphate and the a- and p-forms of L-arabinose 1-phosphate have been obtained as crystalline cyclohexylammonium salts. In addition the cyclohexylammonium salts of the l-phosphoric acid esters of a-D-xylose, a-and P-D-glUCOSeand a-and 8-D-galactosehave been prepared. The cyclohexylammonium salts of these phosphorylated sugars may be purified readily by recrystallization, in contrast to the barium salts which are amorphous and usually contain anomeric impurities. Application of Hudson’s isorotation rules t o the a- and p-anomers of the four phosphorylated sugars shows the 2A values to be consistent with the first isorotation rule, the average value being 25,400. However, the 2B values are greater than those of the corresponding methyl glycosides by about 9,000 molecular rotation units.
Previous investigations revealed that mung bean seedlings contain uridyl pyrophosphorylases capable of catalyzing the reversible formation of uridine diphosphate D-glucose from uridine triphosphate and a-D-glucose 1-phosphate, as well as the
formation of uridine diphosphate D-xylose from uridine triphosphate and a-D-xylose l-phosphate.2 For continuation of these studies pure a- and Pforms of 1-phosphate esters of D-galactose, Dglucose, D-XylOSe and L-arabinose were required.
(1) This work was supported in part by a research contract with the United States Atomic Energy Commission.
(2) V. Ginsburg, E. F. Neufeld and W. 2. Hassid, Proc. Null. Acad. sci. u. s.,la, 333 (1956).
