THE BREAKDOWN OF COMMERCIAL STARCHES BY
MUSCLE EXTRACT
by P. PANTL AND M. N O E L ROMEi
(Prom the Baker Institute for Medical Research, Alfred Hospital, Melbourne).
(Accepted for p^d)lication 20th July, 1942.)
Fresh muscle extracts, poisoned by iodoacetic acid, can be used for the preparation of either glucose-1-phosphate or the "equilibrium ester" (Kiessling,
1938). As shown previously, it is convenient to use starch as substrate (Pantl and
Anderson, 1941). With most commercial starches no difficulties have been encountered in obtaining good yields of glucose-1-phosphate, but, with certain samples
of wheat starch, we failed to isolate hexose-6-phosphate in the expected amounts.
An analytical study gave results recorded in Table 1.
TABLE 1.
Breakdown of starches by rahhit muscle extract.
Reaction Mixture: 100 ml. extract (Kiessling) containing 0-14 p.c. iodoacetio acid, 0-66 p.c.
starch, M/5 PO4, pH = 6-50 (glass electrode). Incubated in an atmosphere of Nj — 5 p.c. COg
at 23° C.
After 15 mins. incubation.
After 45 mins. ineubation.
Starch used. Glucose-l-PO4.
Hexose-6-PO4.
Glucose-l-PO4.
Hexose-6-PO4.
Maize
41-6
19-9
30-7
42-7
Potato
37-8
20-6
29-0
48-2
Bice
34-8
24-4
29-0
44-0
Wheat
44-8
10-2
47-0
17-7
The figures are given in mg. P. per 100 ml. reaction mixture. The estimations were carried
out in trichloracetic acid filtrate after removal of inorganic phosphate by magnesia mixture.
Glucose-1-phosphate was estimated by 10 mine, hydrolysis in N.H2SO4 at 100° C. and hexose-6phosphate by 10 mins. hydrolysis in N.KOH. In addition to the phosphorus estimations, the
barium salts of the reaction products were prepared. The yields were approximately the same in
all cases. Here the hexose component was also estimated and the results were similar to those
shown in Table 1.
Since all starches used showed a total phosphorus uptake not differing by more than 10 p.c.
it is evident that the first step of the degradation yielding glucose-1-phosphate was not inhibited.
However, the ratio of 1 to 6 ester was significantly changed in the case of wheat starch which
suggested that this starch either contained an inhibitor of phosphoglucomutase or lacked an
accelerator which was present in the other starches.
From the results of Cori et al. (1938), Lehmann (1939), and Gill and Lehmann (1939)
it is known that metal ions have either an accelerating (Mg, Mn, Ni, Co) or inhibiting (Cu, Fe, Zn)
influence on phosphoglucomutase. Therefore, ashes of the starches were prepared and their
solutions added to the reaction mixtures.
TABLE 2.
Influence of ashes from commercial starches on hexose—phosphate formation.
Conditions of experiments similar to those given in Table 1. Incubation time 45 mins., pH = 6-45,
temp. 25° C. Ash prepared from an amount of starch equal to that taken for each experiment.
Starch used.
Addition.
Glucose-1-phosphate.
Hexose-6-phosphate.
Potato
nil
30-7
29-5
Potato
Ash from wheat
52-1
16-2
Wheat
nil
52-3
11-7
Wheat
Ash from potato
52-9
14'6
The values are given in mg. P. per 100 ml. reaction mixture. The figures recorded in Table 2
indicate that 50 p.c. of the hexose-phosphates formed from the potato starch was converted into
hexose-6-phosphate and that the ash solution of the wheat starch exerted an inhibitory influence
on this conversion. The potato ash, added to the wheat starch, had no accelerating influence on the
formation of hexose-6-phosphate.
1 Working under a full time grant from the National Health and Medical Besearch Council.
188
P. FANTL AND M. NOEL ROME
The same ratios were obtained by estimating the sugar components in the isolated barium
salts using the reducing power before and after 10 mins. hydrolysis in N.H2SO4.
Analyses of the starch ashes are shown in Table 3.
Ash content
Starch. in p.c.
TABLE 3.
Zinc estimated
Spectrographic analysis*
by dithizone t
Ca Zn
Cu
Mg Mn P
Si
B Fe Pb
mg. p.c.
2
Wheat
0-16
10
tr.
4
tr.
tr.
A
A
A A
3-95
4
A
3
tr.
A tr.
Potato
0-27
tr.
A tr.
A
0-8
0-7
10
A
10
Bice
10 10
10
10 tr. V. f.tr.
tr.
5
A
5
A
tr.
A
tr. A A
Maize
0-12
V. f. tr
A signifies absent in spectrum; tr. signifies trace only; v.f.tr. signifies very faint trace. A
value of 10 has been assigned to the sample in which the element occurred in the greatest amount,
and the other samples graded accordingly.
• We are indebted to Mr. Keith Winsor of the E.M.F. Electric Co. Pty. Ltd. for these analyses
and evaluation of the data.
t Method used for zinc estimation described by Sylvester and Hughes (1936), Walkley (1942)
and O'Brien (1942).
6
v
--.
y K J
These analyses suggest that the zinc content of the wheat starch was responsible for the
inhibition of the phosphoglucomutase. Commercially-prepared starches vary considerably in their
ash content and composition. Therefore, an adverse infiuence on the formation of either glucose-1or hexose-6-phosphate may be expected. However, it is still possible to use such starches without
further purification by making use of the specific infiuence of different metal salts as illustrated
in Table 4.
TABLE 4.
Reaction mixture of similar composition to Table 1, Temp. £7° C.
After 15 mins. incubation.
Starch.
Glucose-l-PO4.
Hexose-6-P04.
Potato
53-2
9-2
Potato + 2 X 10~'
M. ZnS04
62-2
6-5
Wheat
61
6-8
Wheat + 2 X lO"'
M. MnS04
28-8
22-7
After 45 mins. incubation.
Gluco8e-l-PO4.
Hexose-6-PO4.
52-1
22
65 4
69
111
11-5
20-8
38
These results indicate that an addition of 2 X 10"° M. Zn is of advantage in the
preparation of glucose-1-phosphate, whereas, in the case of the preparation of
hexose-6-phosphate, an addition of manganese is advisable to abolish any inhibition
by zinc.
SUMMAEY.
Certain commercial samples of starches were fonnd to contain small amounts
of zinc which were sufficient to inhibit, to a large extent, the formation of hexose-6phosphate by rabbit muscle extract. Addition of manganese abolishes this inhibition.
Acknowledgment.
estimations.
We are indebted to Mr. J. F. Nelson for carrying out the phosphorus
EEFERENCES.
Cori, G. T., Colowick, S. P. and Cori, C. F. (1938): J. biol. Chem., 124, p. 543.
Fantl, P. and Anderson, C. M. (1941): Austral. J. exp. Biol., 29, p. 117.
Gill, P. M. and Lehmann, H. (1939): Bioehem. J., 33, p. 1151.
Kiessling, W. (1938): Bioehem Z., 298, p. 421.
Lehmann, H. (1939): Bioehem. J., 33, p. 1241.
O'Brien, E. J. (1942): J. and Proc, Austral, chem. Inst., 9 (2), p. 35.
Sylvester, N. D. and Hughes, E. B. (1936): Analyst, 61, p. 734.
Walkley, A. (1942): J. and Proc, Austral, chem. Inst., 9 (2), p. 29.