Inhibition of Glyceraldehyde Phosphate
Dehydrogenase by Salts other than
Lithium Chloride
by RICHARD G. HAM1 and ROBERT E. EAKIN
From the Biochemical Institute and the Department of Chemistry, the University of Texas, Austin
(1954) has shown that 0-4 M lithium chloride strongly inactivates
glyceraldehyde phosphate dehydrogenase—a finding which might partially
explain some of the developmental changes found in lithium-treated embryos.
In an attempt to establish an enzymatic basis for the morphological effects of
lithium ion on Hydra which have been observed in this laboratory (Ham &
LALLIER
TABLE 1
Effects of various salts on glyceraldehyde phosphate dehydrogenase
DPNH+formed
(molesx \0~l2lcm.3lsec.)
Salt
None
LiCl .
KC1 .
NaCl
NaSCN
.
.
.
.
.
NaNO 3 .
NaAcetate .
LiAcetate .
.
.
(Moles/L.)
Before addition
After addition
—
0-2
155
174
—
88
0-4
0-8
155
161
35
5*
0-2
139
54
0-4
0-4
008
0-4
0-4
0-4
0-4
155
148
126
150
161
152
131
24
21
32
0
lit
75
38
Activity
remaining
(%)
100
51
23
3
39
15
14
25
0
7
49
29
* Initial rate after addition; 2 minutes later rate had fallen to 0.
t Initial rate after addition; 4 minutes later rate had fallen to 0.
Eakin, 1955), we have repeated the enzyme study with lithium chloride and
extended it to include a number of other salts as controls.
From typical data (Table 1), it is obvious that the inhibition of glyceraldehyde
phosphate dehydrogenase activity is in no way a specific effect due to lithium
ions. Both sodium chloride and potassium chloride produced a greater inhibition
1
National Science Foundation Predoctoral Fellow.
Authors' address: The University of Texas, Austin, Texas, U.S.A.
[J. Embryol. exp. Morph. Vol. 4, Part 1, pp. 93-95, March 1956]
94
R. G. HAM AND R. E. E A K I N — I N H I B I T I O N OF
than did lithium chloride. From the various sodium salts tested, it was found that
the anion may be of more importance than the cation in determining the degree
of inhibition, although the cation also has some effect. It is of particular interest
to note that sodium thiocyanate, which reverses the effect of lithium ions in many
biological systems, is five times as effective as lithium chloride in inhibiting the
enzyme system.
It would appear that the glyceraldehyde phosphate dehydrogenase enzyme
system is of no value for studying the biochemical mechanisms leading to the
morphological effects of lithium salts.
EXPERIMENTAL METHOD
Glyceraldehyde phosphate dehydrogenase activity was measured by spectrophotometric determination of reduced diphosphopyridine nucleotide (DPN) at
340 m/x (Cori, Slein, & Cori, 1948; Verlick, 1955). A zero order (enzyme limiting)
system was used which gave a constant rate of reaction for the first 6 minutes
and which deviated only very slightly by 10 minutes. The reactions were carried
out in a 1 cm. silica spectrophotometer cuvette containing a final volume of
3 6 c.c. The final composition of the reaction mixture was: disodium arsenate,
0011 M; sodium pyrophosphate, 00028 M; DPN, O0004 M; glyceraldehyde
3-phosphate (prepared enzymatically from fructose 1,6-diphosphate and used
without purification), 0 0014 M; and a crystalline preparation of glyceraldehyde
phosphate dehydrogenase (rabbit muscle: Worthington Biochemical Company),
approximately 6 mg. per cuvette. The reaction mixture was buffered at pH 8-58-7. Reaction was initiated by addition of substrate after the other components
had been allowed to stand at least 15 minutes at room temperature to assure full
activation of the enzyme. The substrate was prepared by incubating 0010 M
fructose 1,6-diphosphate with crystalline aldolase (Nutritional Biochemical
Company) for 3 hours at 37° C.
Since the glyceraldehyde phosphate dehydrogenase was added in a very small
volume (005 c.c), considerable variations occurred from test to test. Errors due
to these variations were eliminated by allowing each system to serve as its own
control. The reaction was allowed to proceed exactly 2 minutes before appropriate amounts of 6 M solutions of the test salts were added. The reaction rate
was then followed for another 8 minutes. The degree of inhibition was then
determined from the rates before and after addition. Optical densities were read
every 20 seconds, and converted to moles of reduced DPN using a = 622 x
106 cm.2 moles" 1 (Kornberg & Horecker, 1953). Except as noted the rates of
reaction after addition of salt solutions were also constant.
SUMMARY
1. The previously reported inhibition of glyceraldehyde phosphate dehydrogenase by 0 4 M lithium chloride was confirmed.
GLYCERALDEHYDE PHOSPHATE DEHYDROGENASE
95
2. A number of salts of other cations were found to give comparable or greater
inhibition than lithium chloride.
3. Sodium thiocyanate, a biological antagonist of lithium ion, was found to be
five times as active as lithium chloride in inhibiting the enzyme.
4. It was concluded that the inhibition of glyceraldehyde phosphate dehydrogenase by lithium chloride does not constitute a suitable system for the study of
biochemical mechanisms leading to lithium-induced morphological changes.
REFERENCES
Cow, G. T., SLEIN, M. W., & CORI, C. F. (1948). Crystalline d-glyceraldehyde-3-phosphate
dehydrogenase from rabbit muscle. /. biol. Chem. 173, 605-18.
HAM, R. G., & EAKIN, R. E. (1955). Unpublished observations.
KORNBERG, A., & HORECKER, B. L. (1953). Diphosphopyridine nucleotide. Biochemical Preparations (E. E. Snell, Editor), 3, 20-24. New York: John Wiley & Sons.
LALLIER, R. (1954). Chlorure de lithium et biochimie du developpement de l'ceuf d'Amphibien.
J. Embryol. exp. Morphol. 2, 323-39.
VELICK, S. F. (1955). Glyceraldehyde-3-phosphate dehydrogenase from muscle. Methods in
Enzymology (Colowick, S. P., & Kaplan, N. O., Editors), 1, 401-6. New York: Academic
Press.
(Manuscript received 18:viii:55)