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J. Biol. Chem.-1957-Busch-377-87
J. Biol. Chem.-1957-Busch-377-87
BY FLUOROPYRUVIC ACID*
Results
Inhibition of Lactic Dehydrogenase-The marked inhibition of activity
of lactic dehydrogenase by fluoropyruvic acid is illustrated in Fig. 1. Addi-
tion of 2 and 5 pmoles of fluoropyruvic acid to the system resulted in a 75
and a 95 per cent suppression of activity, respectively. Increasing con-
centrations of pyruvic acid did not increase the rate of the reaction. The
rate of the enzymatic reaction was approximately the same in the presence
and absence of pyruvate when 2 pmoles of fluoropyruvic acid were present
(Fig: 1). The concentrations of pyruvate and fluoropyruvic acid utilized
ranged from 1 X 1O-4 M to 2 X 1O-3 M, since these and other experiments
0
60
SEZNDS
FIG. 1. Rates of oxidation of DPNH in systems containing 4 y of lactic dehy-
drogenase, 5 pmoles of Na pyruvate, and 1 mg. of DPNH in 2.8 ml. of 0.1 M phosphate
buffer at pH 7.4. In the control system the rate was linear for 60 seconds and was
suppressed 75 per cent by 2 @moles of fluoropyruvic acid and 95 per cent by 5 pmoles
of fluoropyruvic acid. In the system containing 2pmoles of fluoropyruvic acid with-
out pyruvate, the rate was equal to that of systems containing both fluoropyruvic acid
and pyruvate.
linic, oxamic, and oxalic acids inhibited the reaction to 50 per cent of the
control values, and for these 50 per cent inhibition was obtained at con-
centrations of 0.036 M, 0.0008 M, and 0.0008 M, respectively. The com-
parative inhibitory effects of oxalic and fluoropyruvic acids are presented
in Fig. 3. The inhibitory effects of fluoropyruvic acid were more marked
than those of either oxalic or oxamic acid at all concentrations studied;
the concentration producing 50 per cent inhibition of activity of the enzyme
ranged from 0.0002 to 0.0004 M. The inhibitory activity of both oxamic
and oxalic acids was found to be competitive, with respect to pyruvate,
and was reversible (14). The degree of inhibition was unaffected by the
order of addition of the reactants, as contrasted to that of fluoropyruvic
Y
2
z
%,-
a
d PYRUVATE CONCENTRATION
a AND L&H ACTIVITY
7
036 .oi2
MOLARITY OF PYRUVATE
FIG. 2. Effect of pyruvate concentration on DPNH oxidation in systems contain-
ing DPNH and purified lactic dehydrogenase in concentrations equivalent to those
of Fig. 1.
TABLE I
Inhibition of Lactic Dehydrogenase by Various Compounds
aximal inhibi-
Compound Concentration m in per cent ICSO
range studied ,f control (60
seconds)
M Al
K oxamate...................................O.0008-0.00 4 80 0.0008
“ oxalate or oxalic acid. ..................... 0.0002-0.004 a7 0.0008
a-Chloropropionic acid. ...................... 0.008-0.02 38
@-Chlkwopropionic “ ..................... ..0.004-0.02 0 22
a-Bromopropionic “ ....................... 0.012-0.02 35
““1 f /
ov I I
15 30
pg. L.D.H.
FIG. 4. Increased rate of oxidation of DPNH with increased enzyme concentration
in fluoropyruvic acid-inhibited systems compared with control system
TABLE II
Efects of Order of Additions on Inhibition of Enzymatic
Activity by Fluoropyruvic Acid
was noted; a similar result was obtained when fluoropyruvic acid and
DPNH were mixed together before addition of LDH. By contrast,
oxamic and oxalic acids inhibited the enzyme equally well, whether added
before or after DPNH. These data suggest that a stable ternary complex
is formed between enzyme, DPNH, and fluoropyruvic acid. The site of
this binding does not seem to be a sulfhydryl group, inasmuch as cysteine
exerted no protective effect against the inhibition (Table III).
384 INHIBITION OF LACTIC DEHYDROGENASE
TABLE III
Effect of Cysteine on Inhibition of LDH by Fluoropyruvic Acid
Control................................................... 0.300
F-P added after DPNH, LDH, and cysteine, 20 pmoles. 0.020
Add 26 pmoles of cysteine after above additions. 0.020
Add fresh LDH to above system. 0.295
125-
o-
0 IO 20
MICROMOLES FLUOROPYRUVIC ACID
FIG. 6. Inhibition of oxidation of malate and pyruvate in homogenate systems.
To each flask was added 0.8 ml. of 10 per cent rat liver homogenate or 0.5.ml. of rat
kidney homogenate, 20 pmoles of Na pyruvate, 5 pmoles of K malate, and 30 pmoles
of KPOa buffer at pH 7.4. The final volume was made to 3.0 ml. with isotonic KCl.
When oxidation was observed to be proceeding linearly, fluoropyruvic acid was
added to the main chamber from the side arm.
the reaction. Moreover, fluoropyruvic acid did not significantly alter the
rate of reduction of oxalacetic acid by malic dehydrogenase.
Effects on Homogenate Systems-In both liver and kidney homogenate
systems, oxidation of a mixture of pyruvate and malate or acetate and
malate was progressively inhibited with increasing concentrations of
fluoropyruvic acid (Fig. 6). However, in concentrations up to 0.003 M
there was no significant inhibition of oxidation of citrate, glutamate, and
H. BUSCH AND I’. V. NAIK 385
DISCUSSIOS
pounds of the citric acid cycle was not suppressed by fluoropyruvic acid.
However, oxidation of malic and pyruvic and malic and acetic acids was
markedly inhibited by fluoropyruvic acid. These data suggest that fluoro-
pyruvic acid directly inhibits pyruvic oxidase and also can form fluoroacetyl
coenzyme A, which prevents the utilization of acetate by these systems.
Not only is there no citrate accumulation in these in vitro systems as has
been found with fluoroacetate, but also there is inhibition of formation
of citrate (17). It would appear that enough fluoroacetyl coenzyme A
forms to inhibit acetate utilization, but only a relatively small amount
forms fluorocitrate, and hence sufficient aconitase and other enzymes of
the citric acid cycle remain available for oxidation of the citrate which
SUMMARY
BIBLIOQRAPHY
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