558th MEETING. EDINBURGH
985
fermentations (Williams, 1974). Thus, not only was the rate of polysaccharide formation
influenced by environmental factors (pH, temperature) and the input concentrations of
the essential nutrients nitrogen, carbon and phosphorus, but the incorporation rate was
also related to the growth rate and stage of growth of the organism.
Induction studies
Induction experiments were performed with washed suspensions of carbon- or nitrogen-limited cultures, in which the rate of exopolymer formation was determined in
incubation buffers supplemented with excess of carbon substrate, a nitrogen source
and/or a protein-synthesis inhibitor, either singly or in combination. The inclusion of
actinomycin D (5-25pg/ml) prevented neither the formation of the exopolysaccharide
nor the increase in activity of the synthesizing system in carbon-limited cells, suggesting
a constitutive system, since protein synthesis was not an essential pre-requisite for the
formation of polysaccharide by carbon-limited non-producing cells on transfer to
conditions favouring exopolymer formation. Pullulan elaboration by the fungus
Pullulariapullulans, however, is inducible and can be inhibited by cycloheximide (Catley,
1972). Polysaccharide formation by suspensions of the pseudomonad was inhibited by
chlorampheiiicol(20-500pg/ml)which is known to interfere specifically with the transfer
of monosaccharide moieties from the precursor nucleotides to intermediate lipid carrier
(Stow et al., 1971; Sutherland et al., 1971).
Enzyme specific activities
The specific activities of the four enzymes hexokinase, UDP-galactose 4-epimerase,
GDP-mannose pyrophosphorylase and dTDP-L-rhamnose synthetase, involved in the
synthesis of the precursor sugar nucleotides, as measured in the 115OOOgcell supernatant
fraction, remained relatively constant irrespective of growth conditions and were only
markedly affected at extremes of temperature and pH. The enzyme specific activities
did not reflect either the amount of polysaccharide produced or the rate at which glucose
was incorporated into exopolymeric material, and were not indicative of an inducible
system.
A. G . W. acknowledges the financial support of the S.R.C. and 'rate and Lyle Research Ltd.
Catley, R. J. (1972) FEBSLeft. 20,174-176
Dubois, M . , Gilles, K. A., Hamilton, J. K., Rebers, P. A. & Smith, F. (1956) Anal. Chem. 28,
350-356
Norval, M. (1969) Ph.D. Thesis, University of Edinburgh
Stow, M., Starkey, B. J., Hancock, I. C. & Baddiley, J. (1971) Nature (London) New Biol. 229,
56-57
Sutherland, I. W. & Norval, M. (1970) Biochem. J. 120,567-576
Sutherland, I. W., Norval, M. &Poxton, I. (1971)J. Gen. Microbiol. 68, v
Wilkinson, J. F. & Stark, G . H. (1956) Proc. R . Phys. SOC.Edinburgh 25, 35-38
Williams, A. G. (1974) Ph.D. Thesis, University of Wales
Williams, A. G . ,Wirnpenny, J. W. T. & Lawson, C. J. (1973)J. Gen. Microbiol. 77, xiii
a-Glucose 1-Phosphate, a Precursor in the Biosynthesis of Maltose in
Higher Plants
NORBERT SCHILLING and OTTO KANDLER
Bofanisches Institut der Universitat Munchen, 8OOO Miinchen 19,
Menringer Strusse 67, West Germany
Maltose is a common oligosaccharide in plants, although it is rarely detected on chromatograms by sugar reagents, owing to its usually very low concentration. It is, however,
detectable in many plants after photosynthesis in I4CO2 (Norris et al., 1955; Nishida,
VOl. 3
986
BIOCHEMICAL SOCIETY TRANSACTIONS
Table I . Distribution of 14Cin maltose after photosynthesis of leaves in l4CUZ
( a ) % of I4C in maltose after I4COz fixation; (b)
% of I4C in the non-reducing end of
free maltose; (c) % of "C in the non-reducing end of maltose derived from starch by
the action of 8-amylase; n.d., not determined.
-Spinacia oferacea
r
1
1
3
4
5
7
I O(20)
?
Leaves
Photosynthesis in
I4CO2
(min)
0.3
0.50
0.85
1.2
1.8
1.0
0.4
n.d.
91
89
77
70
52
50
Chloroplasts
(a)
(6)
(4
(b)
(4
n.d.
58
58
47
n.d.
50
0.2
0.4
1.0
62
63
n.d.
n.d.
54
57
54
n.d.
n.d.
52
48
0.5
54
52
74
72
66
n.d.
n.d.
56
n.d.
n.d.
n.d.
n.d.
n.d.
45
49
46
1962; Kandler, 1964, 1967). Whereas it is generally assumed (Gibbs, 1966; Fekete &
Vieweg, 1974) that maltose and its related homologues are degradation products of
starch, the kinetics of maltose labelling in young leaves of Gentianalutea and other plants
during photosynthesis in 14C02+air(0.1 :99.9) illustrate that maltose is an intermediate
in starch synthesis (Linden et al., 1975). As shown in Table 1, the percentage of I4C in
maltose reaches a maximum within 5min as is the case with other intermediates, i.e.
sugar phosphates and sugar nucleotides. It also shows that the non-reducing end of free
maltose is much more rapidly labelled than the reducing end, whereas in maltose derived
from starch both glucose moieties are practically equally labelled even after the shortest
period investigated. Similar results were obtained in experiments with isolated spinach
chloroplasts (for details of methods see Linden et al., 1975).
Thecomparison of the distribution of label in free maltose and in maltosederived from
starch 8-amylase action shows that the free maltose in leaves and in chloroplast preparations cannot have been formed as a result of j3-amylase action. Our data firmly indicate
that a maltose-synthesizing system, until now only described for bacteria (Fitting &
Doudoroff, 1952), also exists in green plants.
To demonstrate the enzymic synthesis of maltose in leaf extracts, young leaves were
ground in liquid Nz after addition of Polyclar, and extracted in the cold with 0.1 MHepes [2-(N-2-hydroxyethylpiperazin-N-'yl)ethanesulphonic acid] buffer (pH7.2)
containing 0.001M-dithioerythritol, 0.01 M-Mg*+ and 0.005M-Mn2+. The mixture was
filtered through four layers of cheesecloth and the filtrate centrifuged at 20000g for
15min. The protein was precipitated with 80%-satd. (NH4)2S04,dissolved in 1.51111 of
the Hepes buffer used for the extraction and applied to a Sephadex G-25 column. The
whole protein fraction was used as an enzyme preparation.
The incubation mixture contained 0.3 ml of the enzyme solution and substrates at final
concentrations of 3 m ~ Labelled
.
substrates were added with activities of 1-2pCi.
Incubation took place at 30°C and was terminated by adding 1 .5 ml of hot 96% ethanol.
The products formed were separated or isolated by paper chromatography [for details
see Linden et al. (1974)] or paper electrophoresis in 0.05~-sodiumtetraborate (pH9.8)
by the method of Weigel(l962). To investigate the label distribution within the molecule,
maltose was reduced with borohydride and the products resulting from the following
acid hydrolysis were again identified by paper chromatography and counted for radioactivity.
As shown in Table 2, labelled maltose is only formed when glucose 1-phosphate is
1975
558th MEETING, EDINBURGH
987
Table 2. Formation of maltose from glucose and various glucosyl donors by an enzyme
preparation ,from Spinacia oleracea var. Vital R
Type of
expected reaction
Control
Synthesis
Synthesis
Synthesis
Synthesis
Synthesis
Exchangc
Acceptor
[14C]Glc
[14C]Glc
[14C]Gl~
[14C]Gl~
['4C]Glc
['4C]Glc
[14C]Glc
Donor
None
UDP-Glc
ADP-Glc
GDP-Glc
Glc-1-P
['4C]Glc-I-P
Maltose
['4C]Maltose
[14C]Maltotriose
(c.p.m. on paper) (c.p.m. on paper)
-
-
-
-
51 250
68 860
93 350
15400
15105
16600
used as the glucosyl donor, whereas nucleotide-bound glucose cannot function as the
donor. There is also an exchange between the reducing end of maltose and [14C]glucose
as described by Linden ef a/. (1974). Besides maltose, maltotriose is also labelled. No
labelled starch was formed, even though soluble starch was added together with [14C]glucose 1-phosphate in some experiments. Also, labelled glucose is found which is due
to phosphate still present in the crude enzyme preparation.
When [14C]glucosewas supplied only the reducing end of maltose was labelled, whereas most of the radioactivity was found at the non-reducing end when glucose 1-phosphate
was the labelled substrate. In the latter case a significant portion of I4C was also found
at the reducing end, owing to the formation of labelled glucose from ['4C]glucose
1-phosphate by the action of phosphatase.
To demonstrate the reverse reaction, ['4C]maltose and 32Pwere added to the reaction
mixture and the products were identified by electrophoresis followed by paper chromatography. [14C]glucosel-[32P]phosphateand ['4C]glucose were the only products detected.
These findings indicate the presence of a maltose phosphorylase in young spinach leaves
catalysing the reaction :
Glucose+ Glc-l-P+ maltose
+ P,
Similar results were obtained with young leaves of Gentiana lutea and Acer pseudoplatanus. No activity was found in mature leaves, however. Some experiments indicate
that only part of the synthesized maltose is formed by the reaction mentioned above.
Another part may be formed by the transfer of a glucosyl residue from glucose I-phosphate to another molecule of glucose 1-phosphate resulting in maltose 1-phosphate,
although the latter compound has not been unequivocally found in the reaction mixture
so far. Further purification of the enzyme is necessary to clarify the exact mechanisms
of maltose synthesis from glucose I-phosphate in higher plants.
This work was supported by a grant from the Deutsche Forschungsgemeinschaft.
Fekete, M. A. R. & Vieweg, H. (1974) in Plant Carbohydrate Biochemistry (Pridham, J. B., ed.),
pp. 127-144, Academic Press, London
Fitting, C. & Doudoroff, M. (1952)J. Biol.Chem. 199, 153-163
Gibbs, M. (1966) in Plant Physiology: A Treatise (Steward, F. C., ed.), vol. IV B, pp. 3-115,
Academic Press, New York and London
Kandler, 0. (1964) Ber. Dfsch. Bof. Ges. 77,62-73
Kandler, 0.(1967) in Haruesting fhesun;photosynthesis inplant life (San Pietro, A., Greer, F. A.
&Army, T. J., eds.), pp. 131-152, Academic Press, New York
Linden, J. C., Tanner, W. & Kandler, 0. (1974) Plarzt Physiol. 54,752-757
Linden, J . C., Schilling, N., Brackenhofer, H. & Kandler, 0. (1975) Z.Pflanzenphysiol. 76,
176-181
Nishida, K. (1962) Physiol. Plant. 15,47-58
Norris, L., Norris, R. E. & Calvin, M. (1955) J . Exp. Bof. 6, 64-74
Weigel, H. (1962) Adv. Carbohydr. G e m . 18, 61-78
Vol. 3