VOL.

48, 1962 BIOCHEMISTRY: LOEWUS, KELLY, AND NEUFELD 421

20 Chance, B., and R. Sager, Plant Physiol., 32, 548 (1957).

21 Lundergardh, H., Nature, 192, 243 (1961).
22 Bishop, N. I., these PROCEEDINGS, 45, 1696 (1959).
28 Krogman, D. W., Biochem. Biophys. Res. Commun., 4, 276 (1961).
24 Hill, R., and F. Bendall, Nature, 186, 136 (1960).
25 Levine, R. P., and E. E. Levine, unpublished.

METABOLISM OF MYO-INOSITOL IN PLANTS: CONVERSION TO

PECTIN, HEMICELLULOSE, D-XYLOSE, AND SUGAR ACIDS
BY FRANK A. LOEWUS, STANLEY KELLY,* AND ELIZABETH F. NEUFELDt
WESTERN REGIONAL RESEARCH LABORATORY, ALBANY, CALIFORNIA, AND THE
UNIVERSITY OF CALIFORNIA AT BERKELEY

Communicated by W. Z. Hassid, January 22, 1962

myo-Inositol is a ubiquitous constituent of higher plants, both in the free state
and as the hexaphosphate, phytic acid;I-3 yet its metabolism in plant tissue has thus
far remained obscure. myo-Inositol is known to be converted to D-glucuronic acid
by an oxidase in rat kidney4 and in a yeast,5 and preliminary evidence suggests that
a similar reaction occurs in barley roots.6 D-Glucuronic acid and its lactone have
been shown to be directly converted by detached plant tissues to i-gulonic acid and
to the D-galacturonosyl residues of pectin, and, by decarboxylation, to free D-xylOse
and to the D-xylosyl and -arabinosyl residues of hemicellulose.7-10 If myo-inositol
is metabolized via D-glucuronic acid, one would expect it to be converted to the same
end products. Evidence for such a conversion of myo-inositol to D-glucuronic
acid, D-Xylose, L-gulonic acid, pectin, hemicellulose, and related metabolites is
presented in this report.
Materials and Methods.-myo-Inositol-2-H3 was prepared from myo-inososell
by reduction with NaBH312. The resulting mixture of myo- and scyllo-inositol
was converted to hexa-O-acetyl derivatives which could be separated by means of
their differential solubilities in ethanol. Hydrolysis of hexa-O-acetyl myo-inositol
with Ba(OH)2 in methanolic solution'3 yielded free myo-inositol, which was re-
crystallized from water and ethanol. myo-Inositol-2-C'4 was generously provided
by Laurens Anderson.'4
The labeled compounds were administered in aqueous solution through the cut
stems of parsley (Petroselinum) leaves or strawberry (Fragaria) fruits. Pertinent
details are listed for each experiment in Table 1. After the indicated period of me-
tabolism, cellular constituents were separated as previously described.7
Radioactive determinations were made in a napthalene-dioxane-diglyme system'5
using a liquid scintillation counter with an efficiency of 14% for H3 and 49% for
C14.
Results.-The distribution of label in various fractions at the end of each experi-
ment is listed in Table 1. From 35 to 56 per cent of the administered label remained
in the insoluble residue after repeated washes with 70 per cent ethanol. Hydrolysis
of these residues revealed that a considerable portion of the activity (as much as
24% of that administered in experiment 2) was in the D-galacturonosyl residues of
422 BIOCHEMISTRY: LOEWUS, KELLY, AND NEUFELD PROC. N. A. S.

pectin (Table 2). D-Galacturonic acid was isolated as the sodium-calcium salt16
and further identified by reduction to L-galactonic acid. The latter was lactonized
and identified by isotopic dilution with L-galactono-'y-lactone and crystallization,
followed by conversion of the lactone to the amide and recrystallization. The
specific activities of these compounds are listed in Table 2.
In addition to liberating D-galacturonic acid, treatment with pectinase released
several radioactive neutral compounds (Table 1). About one third of the activity
TABLE 1
EXPERIMENTAL DATA AND RADIOISOTOPE DISTRIBUTION AMONG FRACTIONS RECOVERED FROM
PARSLEY LEAVES AND STRAWBERRY FRUITS
Experiment I Experiment 2 Experiment 3
Isotope and position -2-H3 -2-H3 -2-C'4
Specific activity of myo-inositol (mc/mmole) 0.4 6.7 0.79
Activity administered (jc) 111. 181. 0.7
Tissue used (gm) Parsley, 5.9 Strawberry, 14.4 Strawberry, 20.2
Time of label uptake (hr) 12 2 2
Total period of metabolism (hr) 44.5 67 70
Isotope distribution Per cent of administered radioactivity
70 per cent Ethanol-insoluble fraction* 39 56 35
Activity solubilized by pectinase 9 34 20
Acidic constituents 7.3 27 14
Neutral constituents 1.4 5.8 5.4
70 per cent Ethanol-soluble fraction 60 41 53
Activity in extract and 1st wash 53 37 48
Acidic constituents 12 3 7
Neutral constituents 41 30 33
Activity in respired CO2 (C'4) - 12
Activity exchanged into water (H3) 1 3
* Calculated as the difference between radioactivity administered and the sum of activities in the 70% ethanol
soluble fraction, subsequent 70% ethanol washes and water or respired C02.

in this neutral fraction was identified as L-arabinose by chromatographic separation,

crystallization with authentic L-arabinose, oxidation to L-arabonic acid,'7 and re-
crystallization (Table 2). A second radioactive compound was shown to be D-xylOse
by crystallization with authentic material (Table 2). The remaining activity
was shown by chromatography in pyridine: ethyl acetate: water (2:8:1) to be
associated with glucose and several unidentified slow-moving compounds. It
is assumed that these sugars had been constituents of the hemicellulose fraction of the
cell wall.
The acidic constituents of the ethanol-soluble fraction were separated by gradient
elution from an anion-exchange resin, as previously described, into several charac-
teristic acid "regions."7 The predominant constituent of the uronic acid region
was identified as D-glucuronic acid by the following procedure. It was reduced to
L-gulonic acid, which was recovered by carrier dilution and further characterized
by crystallization as the Py-lactone and the amide (Table 2).
The radioactive peak in the dicarboxylic acid region contained a substance with
the electrophoretic mobility of a hexaric acid at pH 3.7. Further identification of
this compound is in progress.
L-Gulonic acid accounted for two thirds of the activity in the aldonic acid region
in the three experiments. It was recovered by carrier dilution and characterized
by crystallization as the -y-lactone and the amide (Table 2).
Only an insignificant amount of radioactivity was found in ascorbic acid (Table
2). Such an observation would be expected in Experiments 1 and 2 if H' from inos-
VOL. 48, 1962 BIOCHEMISTRY: LOEWUS, KELLY, AND NEUFELD 423

TABLE 2
RADIOACTIVITY OF CONSTITUENTS RECOVERED FROM PARSLEY LEAVES AND STRAWBERRY FRUITS
ADMINISTERED mYO-INOSITOL-2-H3 (EXPERIMENTS 1 AND 2) OR myo-INOSITOL-
2-C14 (EXPERIMENT 3)
Specific activity of Per cent of
Experi- Amount Diluent diluted constituent administered
ment Frac- recovered added or derivative labelt
No. tions* Constituent (mg) (mg) (sc/mmole) (%)
1 i D-Galacturonic acid 80 None 18.6 5.3
L-Arabinose 4.8 145 0.55 0.5
0.56 (K arabonate)
D-Xylose 0.4 68 0.15 0.07
s L-Gulonic acid Trace 100 1.5 (-y-lactone) 2.7
1.5 (amide)
L-Ascorbic acid 3.1 180 <0.01 (3rd recryst.) <0.01
D-Xylose 0.22 158 0.08 0.07
2 i D-Galacturonic acid 109 None 89.1 24
86 (L-galactonamide)
L-Arabinose 5.6 196 2.34 1.7
2.26 (K arabonate)
D-Xylose 0.5 150 0.27 0.15
s L-Gulonic acid Trace 90 0.34 (,y-lactone) 0.2
0.36 (amide)
L-Ascorbic acid 5.2 150 <0.01 (3rd recryst.) <0.01
D-Glucuronic acid Trace 100 0.46 (L-gulono--y- 0.1
lactone)
0.46 (L-gulonamide)
D-Xylose 5.0 980 1.99 7.2
3 i D-Galacturonic acid 143 None 0.10 11
0.10 (L-galactonate)
L-Arabinose 8.9 128 0.13 2.0
0.13 (K arabonate)
D-Xylose 0.9 66 0.02 1.0
s L-Gulonic acid Trace 76 0.002 (-y-lactone) 0.1
0.002 (amide)
i-Ascorbic acid 6.3 100 0.000 0.00
D-Xylose 11 435 0.04 17.5
* i = obtained from fractions insoluble in 70 per cent ethanol, treated with pectinase. s = obtained from frac -
tions soluble in 70 per cent ethanol.
t These are minimal values, as they represent the yield of recrystallized material.

itol-2-H3 were retained on carbon atom 5 of D-glucuronic acid4 and then lost during
the subsequent oxidation to L-ascorbic acid.18 However, no label was incorporated
into iascorbic acid in Experiment 3, even though in this experiment the tracer was
C14 and hence not subject to removal during oxidation.
Distribution of radioactivity among the ethanol-soluble neutral constituents ap-
peared to depend upon the nature of the plant tissue. In parsley leaf, label was
found in sucrose, glucose, fructose, xylose, and ribose along with an appreciable
amount of unmetabolized myo-inositol. By contrast, in the strawberry fruit (Ex-
periments 2 and 3), almost all radioactivity not associated with myo-inositol was
inseparable from D-xylose upon co-chromatography or by carrier dilution with
authentic D-xylose followed by recrystallization. This compound accounted for
about 7 to 17 per cent of the label administered to the berries.
Discussion.-The similarity between the products of myo-inositol metabolism
and those of D-glucuronic acid metabolism7 supports the hypothesis that the first
step of rnyo-inositol breakdown in plant tissues is the oxidative cleavage to D-glu-
curonic acid. A portion of the D-glucuronic acid may be further metabolized to
424 BIOCHEMISTRY: LOEWUS, KELLY, AND NEUFELD PROC. N. A. S.

hexonic and hexaric acids7 19 and to D-Xylose.7' 20 The latter then enters the pen-
tose cycle and undergoes further metabolic conversions.2'
The remainder of the D-glucuronic acid may be phosphorylated, incorporated
into UDP-D-glucuronic acid, and utilized for the synthesis of pectin and hemicellu-
lose.22-24 The steps involved in these processes are outlined in Figure 1.
D-glucose .-- myo-inositol - - -hexaric acid
D-glucose 6-P D-glucuronic acid --- -- L-gulonic acid
Jr pentose
D-xylose - hexose phosphates
D-glucose 1-P D-glucuronic acid 1-P + cycle and related
Jr J metabolites
UDP-D-glucose - UDP-D-glucuronic acid - UDP-D-xylose=UDP-L-arabinose
UDP-D-galacturonic acid hemicellulose
pectin
FIG. 1.-Proposed pathways of myo-inositol metabolism and pectin and hemicellulose bio-
synthesis in higher plants. The solid arrows denote enzymatic reactions which have been demon-
strated in plant tissues, while broken lines denote steps which have not yet been elucidated.
The absence of label in -ascorbic acid in Experiment 3 is reminiscent of the nega-
tive results obtained when D-glucuronic acid was supplied to strawberries.25 By
contrast, D-glucuronolactone is an effective precursor of ascorbic acid.7 These data
may be interpreted as indicating the absence of a mechanism for interconverting
D-glucuronic acid and its lactone in plant tissue.
The alternate pathway shown in Figure 1, in which D-glucose is converted to
UDP-D-glucuronic acid via UDP-D-glucose,26 has until now been considered the only
significant route for pectin and hemicellulose biosynthesis. However, the consider-
able incorporation of label into pectin and hemicellulose obtained in the present
experiments, especially Experiment 2, indicates that myo-inositol supplied exo-
genously is an excellent carbon source for these cell wall constituents. Presum-
ably, endogenous myo-inositol is utilized in the same way.
Present data do not allow any judgment as to the relative importance of the two
pathways at various stages of plant growth. Yet it should be noted that in the
ripening strawberry, myo-inositol is a much better precusor of pectin and hemicellu-
lose than is D-glucose.8' 27 It may also be speculated that during seed germination,
when large reserves of phytic acid are hydrolyzed,2 the appreciable quantity of ino-
sitol released could be utilized to form cell wall polysaccharides.
Summary.-Radioactive myo-inositol (-2-H3 or -2-C14) supplied to strawberry
fruit or parsley leaves was converted primarily to the D-galacturonosyl residues of
pectin. Other radioactive products included, in order of quantitative importance,
D-xylose, L-arabinosyl, and D-xylosyl residues of hemicellulose, i-gulonic acid, and
D-glucuronic acid. No label was incorporated into L-ascorbic acid. The similarity
of the end products of myo-inositol metabolism to those previously shown to arise
from D-glucuronic acid indicates that myo-inositol is converted to D-gluCuronic acid
in plant tissues.
*
Western Regional Research Laboratory, Albany, California (Western Utilization Research
and Development Division, Agricultural Research Service, U.S. Department of Agriculture).
t Department of Biochemistry, University of California, Berkeley. The work of this investi-
gator was supported in part by a grant (No. A-1418) from the U.S. Public Health Service.
VOL. 48, 1962 BIOCHEMISTRY: OTAKA, MITSUI, AND OSAWA 425

1 Ballou, C. E., in Encyclopedia of Plant Physiology, vol. X (Berlin: Springer-Verlag, 1958),

p. 442.
2 Malangeau, P., Qualitas Plantarum et Materiae Vegetabilis, 3-4, 393 (1958).
3 Angyal, S. J., and L. Anderson, Adv. Carbohydrate Chem., 14, 135 (1959).
4 Charalampous, F. C., J. Biol. Chem., 234, 220 (1959); J. Biol. Chem., 235, 1286 (1960).
5 Jungwirth, C., A. Sivak, 0. Hoffmann-Ostenhof, and R. G. Janke, Monatsh. Chem., 92, 72
(1961).
6 Gillett, J. W., and C. E. Ballou, personal communication.
7 Loewus, F. A., Ann. N. Y. Acad. Sci., 92, 57 (1961).
8 Loewus, F. A., R. Jang, and C. G. Seegmiller, J. Biol. Chem., 232, 533 (1958).
9 Neish, A. C., Can. J. Biochem. and Physiol., 36, 187 (1958).
10 Slater, W. G., and H. Beevers, Plant Physiol., 33, 146 (1958).
11 We are indebted to Dr. C. E. Ballou for the myo-inosose used in this synthesis.
12 Reymond, D., Helv. Chim. Acta, 40, 492 (1957).
13 Posternak, T., Helv. Chim. Acta, 24, 1045 (1941).
14 Drummond, G. I., J. N. Aronson, and L. Anderson, J. Org. Chem., 26, 1601 (1961).
15 Loewus, F. A., Intern. J. Appl. Radiation Isotopes, 12, 6 (1961).
16 Isbell, H. S., and H. L. Frush, J. Res. Nat. Bur. Standards, 32, 77 (1944).
17 Dimler, R. J., and K. P. Link, J. Biol. Chem., 150, 345 (1943).
18 Preliminary experiments have shown that C-5 of the n-galacturonosyl residue corresponds to
C-2 of myo-inositol (Loewus, F. A., and S. Kelly, Fed. Proc., in press). This labeling pattern is
expected if myo-inositol is cleaved to D-glucuronic acid by a mechanism similar to that operating
in rat kidney,4 and the D-glucuronic acid then directly incorporated into pectin.7
19 Kessler, G., E. F. Neufeld, D. S. Feingold, and W. Z. Hassid, J. Biol. Chem., 236, 308 (1961).
20 Loewus, F. A., and S. Kelly, Arch. Biochem. Biophys., 95, 483 (1961).
21 Loewus, F. A., and R. Jang, J. Biol. Chem., 232, 521 (1958).
22 Feingold, D. S., E. F. Neufeld, and W. Z. Hassid, Arch. Biochem. Biophys., 78, 401 (1958).
23 Neufeld, E. F., D. S. Feingold, and W. Z. Hassid, Arch. Biochem. Biophys. 83, 96 (1959).
24 Feingold, D. S., E. F. Neufeld, and W. Z. Hassid, J. Biol. Chem., 235, 910 (1960).
25 Loewus, F. A., R. Jang, and C. G. Seegmiller, J. Biol. Chem., 222, 649 (1956).
26 Strominger, J. L., and L. W. Mapson, Biochem. J., 66, 567 (1957).
27 Seegmiller, C. G., R. Jang, and W. Mann, Jr., Arch. Biochem. Bioph~ys., 61, 422 (1956).

ON THE RIBONUCLEIC ACID SYNTHESIZED IN A CELL-FREE SYSTEM

OF ESCHERICHIA COLI*
BY E. OTAKA, H. MITSUI, AND S. OSAWA
INSTITUTE FOR MOLECULAR BIOLOGY, NAGOYA UNIVERSITY, NAGOYA

Communicated by A. E. Mirsky, January 8, 1962

In a previous communicationI we described a method for the preparation of
"undegraded" messenger-RNAs from bacterial cells (E. coli). Messenger-RNAs
have been described before, and the selective synthesis of this type of RNA under
special culture conditions has been reported by Hayashi and Spiegelman.3 These
RNA fractions are rapidly labeled in isotope experiments and have base composi-
tions resembling that of the bacterial DNA.2 In our experiments the "undegraded"
messenger-RNA fraction was prepared by the phenol method after first breaking
the cells in the presence of Duponol in order to assure the immediate inhibition of
ribonuclease activity. Messenger-RNA could be distinguished from ribosomal
RNA by chromatography on columns of methylated serum albumin or by centrifu-