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ATP utilization
associated
in anaerobic frog muscle
with
recovery
ROBERT
R. DEFURIA
AND MARTIN
J. KUSHMERICK
Department
of Physiology, Harvard Medical School, Boston,
anaerobic
metabolism;
muscle energetics;
recovery
energetics
LABORATORY
HAS
FOUND
an apparent
P/O ratio
Of 2
in frog sartorii, Rana pipiens, at 0°C during the metabolic restoration of ATP previously utilized during an
isometric tetanus (34). This measured value was significantly lower than predicted from observations of optimally coupled mitochondria. Similar results were obtained for single sartorii isolated from Rana temporaria
at 0°C and from Rana pipiens at 20°C (unpublished
data). These data have two simple biochemical interpretations: a) there was significant suprabasal ATP utilization after mechanical relaxation, during metabolic recovery, possibly due to metabolic substrate cycling (3,8,
9, 32, 40); b) the low P/O ratio could be interpreted as
partial uncoupling of oxidative phosphorylation in the
muscle mitochondria (7, 13, 36).
The experiments described in this paper were designed to find out if there was significant suprabasal
ATP breakdown in frog sartorii
during the metabolic
restoration of ATP utilized during prior contraction.
Our experiments rely on the fact that anaerobic glycolysis is the primary pathway for energy metabolism in our
preparation; the experiments to be described suggest
that this is true. Our strategy is based on the fact that
the ratio of the suprabasal ATP synthesized by simple
Embden-Meyerhof glycolysis and the suprabasal lactate
produced during this recovery period is 1.5. That is, 3
net mol of ADP are phosphorylated when 2 mol of
lactate are produced to give a P/lactate ratio of 3/2 (38,
39). We compared an experimentally determined P/lactate ratio with this known value of 1.5. ATP utilized
during an isometric tetanus and suprabasal lactate proOUR
c30
IWassachusetts
02115
duced by anaerobic sartorii during recovery from a tetanic stimulation were measured. Our experimental P/
lactate ratio was taken to be the quotient of these numbers. The total ATP turnover is thus equal to the sum of
the ATP utilized during the tetanus and any suprabasal
ATP utilization occurring during metabolic recovery.
Since the muscles are frozen just after relaxation of
isometric tension, the quick-freezing technique only
measures ATP turnover during the tetanus. Thus, an
experimental P/lactate ratio of less than 1.5 suggests
that suprabasal ATP utilization occurred during the
metabolic recovery period.
Suprabasal ATP turnover associated
with tetanic contractions after mechanical relaxation has not been
found (11, 20, 26, 31). However, these studies were concerned with the possibility of ATP splitting associated
with tetanic processes occurring during and shortly
after mechanical
relaxation
and were not concerned
with the broader issue of ATP breakdown above the
basal rate associated with metabolic recovery.
Estimates of P/lactate ratios have been previously
made (1, 6, 29, 46). Interpretation of such data depend
on the metabolic properties of the muscles used. In order
to interpret our experimentally measured P/lactate ratio, we tested the following
characteristics
of our preparations:
a) no significant
ATP was resynthesized by
glycolysis or oxidative phosphorylation during the tetanus; b) no significant ATP resynthesis by oxidative
phosphorylation
occurred
during
metabolic
recovery;
c)
recovery
did occur in anaerobic muscles, such that the
muscle content
of ATP, creatine phosphate, creatine,
inorganic phosphate, and lactate was not altered by a
stimulation-recovery
cycle; d) lactate was the primary
end product
of this anaerobic
recovery,
and glycogen
was the source of this lactate; e) total lactate collected in
the bathing medium was an accurate
measure
of the
amount
of lactate produced
by glycolysis
during
a recovery period.
PROCEDURES,
METHODS,
AND
EXPERIMENTAL
DESIGN
Rana pipiens were kept at 10°C in tap water for 2-10
wk. Pairs of sartorii were dissected
the day before an
experiment
and left overnight
at 0°C immersed
in airequilibrated Ringer-phosphate buffer: 115 mM NaCl, 2.5
mM KCl, 3 mM Na,HP0,/NaH,P04,
pH 7.1, 1.8 mM
CaCl,, 1 mM MgSOd, and chloramphenicol 50 pglml.
While at O”C, each muscle was subjected to two brief
isometric tetani to test its stability. The first (2 s) was
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.2 on June 17, 2017
DEFURIA,
ROBERT R., AND MARTIN J. KUSHMERICK. ATP
utilization
associated with recouery metabolism
in anaerobic
frog muscZe. Am. J. Physiol. 232(l): C30-C36, 1977 or Am. J.
Physiol: Cell Physiol. l(1): C30-C36, 1977. -The ATP turnover
during single isometric 0.5, 1-, and 3-s tetani in anaerobic frog
sartorii was measured at 20°C. In parallel
experiments,
we
measured the lactate production by sartorii during their metabolic recovery from such tetani. From these measurements,
we
obtained an experimental
P/lactate ratio which was less than
1.5, the expected value for simple Embden-Meyerhof
glycolysis
when glycogen is metabolized
to lactate. We interpreted
these
results as indicating
a substantial
amount of suprabasal ATP
breakdown
occurred during metabolic recovery. The operation
of the fructose phosphate
substrate cycle is suggested as a
cause of this additional
ATP breakdown.
metabolism
RECOVERY
ATP
c31
UTILIZATION
Recovery
of Added
above baseline
loo~5
Et
=,;;
2
8o
6o+
;
Fa -z
$
40-J
I3
” t
5”
;i
I2
l
l a.mmm*
Lactate
l .memm*
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IO
;
.**m
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****a-*
7
4
:
IO
added
20-A
OO
Observed
l
l
gE”
g=
Fraction
*mm*mam
1
l mm*l
mmm.0 l mm**
I
I
20
FRACTION
,
30
l *@
1
Lactate
Predicted
43.2
23.3
t2 5
6’
3.6
1::
?
.mm*m@
L
40
1
I
50
NUMBER
FIG. 1. A: base line
of lactate
production
by an unstimulated
muscle in a N,-bubbled
bath at 20°C, Number
of nanomoles
of lactate
in each serial lo-min
fraction
is plotted.
Precision
of lactate assay is
equivalent
to 1 nmol per fraction.
Volume
of muscle bath was 5 ml.
Affluent
and effluent
flow rate was 0.307 ml/min.
Dry weight
of
for these assays were calibrated with spectrophotometrically determined
concentrations
of NADH.
Glycogen
was assayed by the method of Handel (16). Our assay
standards
and lactate dehydrogenase
(L/2500), malic
dehydrogenase
(4lO-l3),
cu-glycerolphosphate
dehydrogenase (G6751), were obtained from the Sigma Chemical Company; the identifying
numbers in parentheses
are catalog numbers.
After most experiments,
the dry weight of the muscle
tissue was obtained by dessication for at least 24 h under
vacuum over phosphorus
pentoxide.
All the data for
tissue contents
reported
are normalized
to the dry
weight
of the tissue,
wet
Sometimes
only blotted
weights or a total protein by the method of Lowry et al.
(28) was obtained. The conversion
factors were independently measured and were: 4,2 blotted wet wt/g dry
wt and 764 mg protein/g dry wt.
Our experiments
were designed to measure the net
suprabasal
lactate produced by anaerobic muscles during metabolic recovery from a prior 0.5, 1-, or 3-s isometric tetanus, We also measured the ATP utilized by
muscles during 0.5-, I-, and 3-s isometric
tetani. The
ratio of these two members is an experimental
P/lactate
ratio which is to be compared with 1.5, the known value
of the P/lactate ratio of Embden-Meyerhof
glycolysis
when glycogen is metabolized to lactate. Recently, Scopes (38, 39) has assembled the glycolytic
enzymes in
vitro along with phosphorylase,
adenylate kinase, creatine kinase, and AMP deaminase and found that 3 mol
of ADP were phosphorylated
when 2 mol of lactate were
produced, verifying
the P/lactate ratio of 1.5. Although
there is the possibility
of uncoupling
carbon flux from
ADP phosphorylation
in glycolysis
(for example, the
hydrolysis
of the acyl intermediate
of glyceraldehyde
dehydrogenase),
which would lower the experimental
PI
lactate ratio, there is no evidence that these kinds of
processes occur (38, 39). Another metabolic simplif”lcation of our preparation
is that, although there are some
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.2 on June 17, 2017
soon after dissection; the second (1 s) on the morning of
the experiment
showed no deterioration.
The muscle
bath was brought to 20°C while the bathing solution was
bubbled continuously
with either O,-free N, or CO
(99.99%). When required, the muscles were stimulated
isometrically
at 10 V and 40 Hz to evoke a fused tetanus
(27). Muscle length on the apparatus
was set approximately to the in vivo rest length by loading with 0.005 N
tension.
The ATP turnover
during a tetanus was measured
according to the protocol of Kushmerick
and Paul (27)
with the quick-freezing
technique and assays previously
described (24, 25-26). In this paper, we report measured
APCrlg dry wt because no net breakdown
of ATP was
observed (see Table 4) and because there were no differences between APCr/g and A - P/g dry wt. The calculated values for A - P/g dry wt relevant to Table 4 are
for series A: 12.0 t 0.9, 15.4 t 1.1, and 36.2 t 2.2 (for
0.5, 1.0, and 3.0 s tetani, respectively)
and for series B:
14.1 t 0.9. These muscles were frozen 1 s after the
isometric tension fell to the base line. Some muscles
were allowed to recovery anaerobically.
The lactate producti .on of’ those muscles was measured
with a flowthrough
apparatus
where the effluent of the muscle
bath was pumped to a fraction collector. The lactate
content of each fraction was measured.
The volume of
the muscle bath was kept constant by maintaining
an
affluent flow rate from a Ringer reservoir
equal to the
effluent rate. Approximate
values for the muscle bath
volume were 4 ml and for the flow rates 0.3 mllmin.
Pump stability was checked periodically
by measuring
the weight of individual
fractions at constant bath volume; however,
bath volume and flow rate varied
slightly from one experiment
to another. The average
flow
r&e of a particular
experiment
was determined
from the weight of the reservoir
before and after the
experiment and the total duration of the experiment.
Figure lB shows data from a control experiment done
-periodically to test the flow stability and precision of the
apparatus used to measure the lactate production of our
isolated sartorius
preparation.
The suprabasal
lactate
content of each fraction after the injection can be predieted for a given muscle bath volume, amount of injetted lactate, flow rate, and fraction collection time.
These values were compared with experimental
values.
These control experiments
also test the validity of the
base-line subtraction
procedure which was used to obtain the suprabasal lactate content of each fraction. The
base line content of each fraction collected during the
injection recovery period was estimated by a linear extrapolation
of the base line of fractions before and after
the recovery
period. The suprabasal
content of each
fraction was taken to be its total lactate content minus
this base line estimate. Data in Table 3 and Fig. IA give
further justification
for this procedure.
In order to measure the tissue content of glycogen and
other relevant
metabolites
(Tables 1 and 2), muscles
were quickly denatured
and solubilized by immersion
into 5 N NaOH at 100°C for 5 min; this solution was then
chilled. Lactate,
pyruvate,
malate,
a-glycerol
phosphate, and alanine were assayed fluorometrically
by
standard enzymatic methods (18, 21, 37). The standards
c32
R. R. DEFURIA
conflicting views concerning the existence of the pentose
phosphate pathway
in muscle, this pathway
would not
contribute
to the metabolism
of any anaerobic tissue
(15, 35, 40).
TABLE
1
1
1
I
I
1
20
30
40
50
60
70
FRACTION
NUMBER
2. Basal lactate
production
of a muscle in a NP-bubbled
bath
at 20°C is interrupted
by a l-s isometric
tetanus
at indicated
points.
Data are plotted
as in Fig. 1. Total suprabasal
lactate
production
after each stimulation
is given
above
data points.
Muscle
bath
volume
was 3.8 ml. Flow rate was 0.295 mllmin.
Fractions
were
collected
every 8 min. Dry weight
of muscle was 25.0 mg.
FIG.
l*mm*555
l
nonomoles
k
l
l *”
2nd
L
20
30
CO
from N2 bubbled
change
10
siimulotion,
40
FRACTION NUMBER
I
50
1
60
No. of animals
11
240
0.5%
XL 1.2
5
2
0.7%
2 1.3
5
21
2.5%
4 5.0
5
Pyruvate
4
3.1%
+ 5.1
5
Alanine
6
0.6%
k 7.0
5
Malate
a-Glycerol
phosphate
units)
Muscle
content
of glycogen,
lactate,
pyruvate,
a-glycerol
phosphate, malate,
and alanine
in aerobic sartorius
muscles at 20°C after
overnight
storage
(see METHODS).
Mean
values for all muscles
are
given in the first column,
The second column is the average
percentage differences
between
members
of a pair. The last column gives the
number
of muscle pairs used.
cogen depletions are summarized in Table 2. To test
whether lactate accumulation accounted for the glycogen depletion when the muscles were stimulated, the
experiment summarized in Fig. 4 was made. The data in
Tables 1 and 2 and in Fig. 4 show that lactate production
accounts for most of the glycogen metabolized in our
preparations.
The data in Table 3 show that the muscle content of
ATP, creatine phosphate, creatine, inorganic phosphate, and lactate is not altered by a complete tetanusrecovery cycle,
The measured ATP turnover during a 5-s tetanus at
20°C for aerobic sartorii was 72.0 k 4.8 (SEM; n = 10)
pmol/g. In sartorii treated for 30 min with 0,5 mM
iodoacetate and made anaerobic with NZ, the ATP utilization for an identical tetanus was very similar, 68.4 5
5.3 (SEM; n = 10) pmol/g.
Table 4 shows the amount of ATP turnover during
tetanic stimulation durations of 0.5, 1, and 3 s and the
amount of suprabasal lactate produced during metabolic
recovery from such tetani. Our experimental P/lactate
ratios are determined from these two quantities and are
given for each tetanus duration. All experimental P/
lactate ratios were less than 1.5.
DISCUSSION
-b--
t
x 100
2 SE
to co
both
1
70
FIG. 3. Basal
lactate production
of a muscle in a N,-bubbled
bath
at 20°C was interrupted
by a l-s isometric
tetanus
at point indicated.
Experiment
is repeated
after changing
to a CO-bubbled
bath. Data
are plotted
as in Fig. 1. Suprabasal
lactate
production
after each
stimulation
is given above data points.
Muscle bath volume
was 5
ml, flow rate was 0.380 ml/min
with lo-min
collection
fractions.
Dry
weight
of muscle was 47,O mg.
If there was no suprabasal ATP utilization during the
metabolic recovery, then the ATP utilized at the end of a
tetanus by rapid-freezing techniques should have been
equal to the high-energy phosphate resynthesized by
glycolysis. The latter quantity is given by the suprabasal lacate production times 1.5, the known stoichiometric factor for the metabolism of glycogen coupled to
ATP synthesis in the simple Embden-Meyerhof glycolytic pathway. This prediction is made because the metabolite contents were found to be unaltered by a stimulation recovery cycle (Table 3), and lactate is the only
significant end product of the anaerobic recovery (Tables 1 and 2, Fig. 4). One way of comparing the tetanic
ATP turnover with the amount of suprabasal ATP re-
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.2 on June 17, 2017
1
IO
- Right/Left
+ 2.4
Glycogen
(glucosyl
-m&W-
Left
Mean
muscles
0.6%
Figure 2 shows data from a typical experiment
designed to measure the suprabasal
lactate produced by
our preparations
of isolated sartorii
during metabolic
recovery from a single isometric tetanus. Data for 0.5,
l-, and 3-s tetani were obtained similarly
in a series of
muscles, and these data are summarized
in Table 4.
The data in Fig. 3 show that for a l-s tetanus the
suprabasal
lactate production
is the same when the
muscle is bubbled with 100% N, or 100% CO.
The contents of relevant metabolites
were measured
in aerobic muscles stored overnight
at 0°C (Table 1).
Other paired muscles were dissected, treated similarly,
and divided into experimental
and control groups. The
control muscles were treated exactly as those muscles
summarized
in Table 1. Experimental
muscles were
made anaerobic at 20°C and were incubated
without
stimulation
in Ringer containing 1 mM NaCN for 25 h.
The metabolite content of those muscles was then measured and compared with the initial content in a pairwise fashion. The accumulated
metabolites and the gly-
noncmoles
J, KUSHMERICK
24
Lactate
269
M.
uf unstimulated
1. Metabolite content
Mean
Content,
prnol/g
dry wt
RESULTS
287nonunoles
AND
RECOVERY
ATP
c33
UTILIZAT1ON
TABLE
2.
OfglyC0gU-t
Co??ZJIarisOn
and metabolite
depletion
production
Production
Expt No.
Lactate
a-GIycerol
phosphate
Pyruvate
pmollg
1
2
3
4
5
169
218
124
171
166
3
7
7
8
1
-15
-13
-9
-12
-13
Depletion
Malate
Glycogen
glucosyl
units
Alanine
dry wt
-1
2
3
0
4
4
-1
1
0
-1
92.0
113.6
58.5
79.3
84.9
The total production
of lactate,
pyruvate,
ar-glycerol
phosphate,
malate,
and alanine
is compared
with the total glycogen
depletion
in
five experiments.
Paired
muscles
were used. The experimental
member
of each pair was incubated
without
stimulation
at 20°C for
25 h in Ringer
solution
containing
1 mM NaCN.
The control
members of the pairs were treated
as in Table 1. The data in this table are
the differences
in muscle content,
experimental
minus control.
Total
Lactate
Production
p moles
Clycogen
I Total
in Glucosyl
p moles
lO*
t
l Om
l
l
l
-h
l
T-4
Depletion
Units
I
2nd stimulation
1st stimulotion
V
t
IO
begin N2
20
30
FRACTION
40
50
60
70
NUMBER
FIG. 4. Total
lactate produced
by a muscle (right of a pair) at 20°C
in a Ne-bubbled
bath during two l-s tetanic stimulation
and recovery
periods is compared
with its glycogen
depletion
during
same total
period. Data for lactate content in each fraction
are plotted as in Fig.
1; sum of those quantities
during
whole anaerobic
period
is total
lactate
production.
Glycogen
content
of control
(Eeft) muscle at beginning
of experiment
was 233 pmol/g.
Glycogen
content
of experimental
(right)
muscle was determined
at downward
arrow.
Glycogen
depleted
during
anaerobic
incubation
was difference
between
control
and experimental
muscle contents.
Muscle bath volume
was 3.3 ml
and flow rate was 0.279 ml/min
with 8-min collection
fractions.
Dry
weight
of muscle was 24.3 mg.
at least 3 h prior to stimulation. Our results differ in
that no significant resynthesis of ATP occurred during
tetani .c contractions of up to 5 s durations at 20°C because ATP turnover in aerobic as well as i.odoacetatepoisoned, anaerobic preparations were the same.
The data presented in Figs. l-4 indicate that the
behavior of our anaerobic muscle preparation is predictable and reproducible. In Fig. 2, the suprabasal lactate
production after a l-s tetanus was repeatable to within
6%. We found an average basal lactate production of 109
t 10 (SEM: n = 16) nmol/g per min, similar to those
values reported by Karpatkin et al. (22). Since muscle
lactate content was unaltered by a stimulation recovery
cycle, Table 3, and the lactate content of fractions collected after recovery could be predicted from prestimulation collections, we conclude that a) there is an increase in the rate of lactate production by the muscle
during the course of an experiment; b) this increase in
the rate of lactate production did not influence our
measurement of the amount of lactate produced.
The data presented in Fig. 3 indicate that the suprabasal lactate production in N, and CO after a l-s tetanus
is the same. It is known that CO is a potent and effective
inhibitor of oxidative phosphorylation (41). Since we
have found that the ATP breakdown in first and second
tetani is the same (unpublished data) and because it is
not expected that CO altered the extent of metabolic
recovery (shown to be complete in our N,-bubbled preparation), these data suggest that there is no significant
resynthesis of ATP by oxidative phosphorylation during
metabolic recovery in our standard N,-bubbled preparation. Consistent evidence is that our average basal
lactate production in an N,-bubbled bath was similar to
that in 1 mM NaCN Ringer, 112.9 t 10 (SEM: n = 5)
nmol/g per min; NaCN is known to block oxidative
phosphorylation completely (41).
The data shown in Table 3 indicate that a stimulation
recovery cycle did not alter the cellular content of ATP,
creatine phosphate, creatine, inorganic phosphate, and
lactate. This suggests that metabolic recovery was complete and that bath lactate was a measure of the lactate
produced during the recovery period. It has been reported that anaerobic frog gastrocnemii may undergo
only partial metabolic recovery. This was presumably
because the muscles were in a N, gas phase and intracellular lactate was forced to accumulate (1).
The data presented in Table 1 indicate that at the
beginning of an experiment, the content of glycogen,
lactate, pyruvate, a-glycerol phosphate, malate, and
alanine in both members of a pair of sartorii are the
same. This fact allowed us to do the type of glycogen
depletion experiments whose data are given in Table 2
which show a carbon balance, The lactate content determined in the controls for these experiments (Table 1)
was substantially greater than those shown in Table 3,
because of different experimental manipulations and
analytical techniques. The result of this glycogen depletion experiment was that most of the glycogen metabolized can be accounted for by lactate production when
the unstimulated sartorii were incubated in I mM
NaCN Ringer. This conclusion is consistent with the
data shown in Fig. 4 where the lactate production by our
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.2 on June 17, 2017
synthesized
by glycolysis
is to compare the experimental P/lactate ratio with the known stoichiometric
factor
1.5. The experimental
P/lactate ratios obtained for different tetanic durations in two series of experiments
are
lower than 1.5 (Table 4). Thus, there was suprabasal
ATP utilized during the metabolic recovery after a tetanus. The amount of this additional ATP turnover can be
appreciated if the data are further
analyzed as in the
bottom of Table 4. Here it is seen that the calculated
additional ATP turnover during recovery is always significantly different from zero and is a significant
fraction of the ATP turnover
actually measured during the
isometric tetanus. Moreover,
the calculated additional
ATP turnover
during recovery is about one-quarter
of
the estimated base-line ATP utilization
during the entire recovery period.
Our measured ATP turnover at the end of an isometric tetanus in anaerobic frog sartorii is the same order of
magnitude as that reported by Canfield, Lebacq, and
Marechal (4). The experiments
are not exactly comparable, however, because our preparation
was anaerobic for
c34
TABLE
R. R. DEFURIA
3. Anaerobic recovery of initial
AND
Mean
dry wt
Fractional
Differences
2 &q/n*
57.0
(51.0)
45.6
(50.5)
42.0
(44.5)
42.3
(40.7)
38.0
(35.8)
-0.002
k 0.012
99.9
(96.4)
123.8
(122.1)
130.3
(129.6)
147,2
(147.2)
127.0
(118.0)
139.3
(138.4)
-0.001
* 0.003
Creatine
73.3
(69.7)
94.2
(89.9)
65.1
(72.6)
54.6
(59.7)
57,l
(59.7)
56.5
(58.2)
0,003
k 0.009
ATP
11.8
(11.1)
22.8
(19.6)
17.1
(21.5)
17.2
(18.7)
18.6
(18.6)
18.5
(18.3)
0.007
f 0.029
3.5
1.2
(1.4)
5.3
(5.3)
Creatine
phosphate
phosphate
Lactate
7.6
2.8
(6.9)
(2.4)
(3.2)
1.9
(2.1)
The ATP, creatine
phosphate,
creatine,
inorganic
phosphate,
and lactate contents
of left and right sartorius
from
was subjected
to a l-s isometric
tetanus
and allowed
to recover
for 4 h at 20°C in a N,-bubbled
bath. Its contralateral
but was otherwise
treated
identically.
Chemical
contents
were obtained
for each pair at the end of the recovery
*Mean
* SE.
stimulated
muscle of each pair are given in parentheses.
4. Experimental
P/lactate ratios
Series A
Tetanus
Duration,
0.5
I) Decrease
in creatine
phosphate
or
ATP
turnover
during
tetanus,
Fmollg dry wt*
(n)
in ATP content,
j.mol/g
dry
11.9 * 0.8
112>
0.9 -4 0.6
Series
-.
s
1.0
3.0
16.5 2 0.9
(13)
39.0 k 2.4
(11)
-0.3
0.9 + 0.6
t 0.6
B
1.0
14.2 2 0.8
(9)
0.3 2 0.1
wt*
3) Suprabasal
lactate
pmollg dry wt”
(n)
production,
4) Experimental
ratio
P/lactate
14.2 2 0.6
(31
1.2
5) Tension-time
integral
N =s/cm2*
ATP turnover
experiments
Lactate recovery
experiments
6) Suprabasal
ATP resynthesized
glycolysis
(data in row 2 x
pmollg dry wt*
11.8 + 0.5
(9)
.I7 % 4
109 * 3
by
1.5),
16.1 k 0.6
21.3 k 0.8
7) Difference
between
tetanic
ATP I
4.2 t- 1.7
4.8 k 2.5
turnover
and suprabasal
ATP re- ’ i D < 0.0137
P < 0.025t
synthesized
(row 6 - row
I), prnol/g
dry wt*
8) Estimated
basal ATP turnover
during recovery
(basal lactate produch
tion x 1.51, pmollg dry wt
16
19
1
1.2
1.2
287 5 30
308 t 12
113 k 4
90 2 4
47.7 + 0.6
17.7 + 0.8
8.7 L 4.7
P < 0.05t
3.5 2 1.1
P < 0.005-t
25
21
The ATP utilized
by anaerobic
sartorius
muscles after a complete
0.5, l-, and 3-s
isometric
tetanus at 20°C is given in row
I. Suprabasal
lactate produced
during anaerobic
recovery from similar
tetani
is given in row 3. These experiments3
were carried
out as
illustrated
by Fig. 2, and the data were collected for the first tetanus only. The experimental P/lactate
ratios obtained
for each tetanus duration
from the data given in rows I and 2
are given in row 4. The areas under the isometric
myograms
(tension-time
integral)
are
given in row 5. Rows 2,6, 7, and 8 contain further analyses of the data. Experimental
series
A was made with a single batch of frogs obtained
in September,
1975; series B was made
with several batches of frog obtained
during the winter months of 1974-75.
*Mean
*
SE.
t Student t test for independent
and uncorrelated
means.
N,-bubbled preparation was found to be nearly stoichiometric with glycogen depletion over two stimulation
and recovery cycles. We did not measure the muscle
content of glycolytic intermediates in that experiment,
but the return of the muscle to a predictable base line of
lactate production suggests that a stimulation-recovery
cycle did not significantly alter metabolite content. To
the extent that other end products of glycolysis accumu-
-0.017
2 0.081
six frogs. One of the pair
mate was not stimulated
period.
The data for the
lated [such as n-lactate (10) or succinate (l7)] or that
oxidative phosphorylation
contributed to the resynthesis of ATP in our N,-bubbled preparation, the value
of our experimental P/lactate ratio is overestimated,
and therefore our conclusion concerning the magnitude
of ATP turnover during recovery is underestimated.
There are several trivial explanations for the observed P/lactate ratio being lower than the known Embden-Meyerhof Pathway stoichiometry which can be excluded: a) systematic analytical errors which underestimate the ATP turnover or overestimate the lactate production were ruled out because the net amount of creatine phosphate breakdown was equal to the production
of inorganic phosphate, and the latter was measured by
an enzymatic assay calibrated in the same way as the
lactate assay with solutions of known concentration so
NADH (see METHODS).
b) Bacterial metabolism of lactate was excluded by the data shown in Fig. 1. c) The
total chemical energy utilized during the isometric tetani for the muscles used in ATP turnover measurements is not likely to have been different from those
muscles used for recovery lactate measurements, since
within each experimental series, A and& Table 4, frogs
came from one batch, the stimulus conditions and temperature were the same, the shapes of the isometric
myograms were not distinguishable, and because the
areas under the tension record [the parameter called
tension-time integral as used by Kushmerick and Paul
(27)] were very similar as shown in Table 4.
Our observed suprabasal ATP breakdown could be
due to an, as yet, unidentified net exothermic process
occurring during contraction but reversed after mechanical relaxation at the expense of ATP turnover. This
possibility originates from a current problem in muscle
energetics where more heat can be liberated during a
contraction than is explained by the measured chemical
changes (5, 12, 14, 19, 30, 44, 46).
Our observed suprabasal ATP breakdown could be
due to the restoration of electrochemical potentials to
basal values by the sarcoplasmic reticulum, mitochondria, and the surface membrane. Any discussion of
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.2 on June 17, 2017
42.1
(39.8)
Inorganic
21 Change
J. KUSHMERICK
metabolite composition
pmollg
TABLE
M.
RECOVERY
ATP
c35
UTILIZATION
since the 1-malate-NADP
oxidoreductase
pathway
is
thought to be near equilibrium
(40). Reaction A has not
been observed, presumably
because of the tight reciprocal covalent and allosteric control of glycogen phosphorylase and synthetase
(43). The enzymes in reaction B
are also thought to be under tight reciprocal control , but
it does occur in liver (9) and in bee muscle (8). This
fructose phosphate
substrate
cycle has not yet been
studied in frog sartorii,
but it can be measured by the
method of Bloxham et al. (3). The following
evidence
indicates that this cycle can operate in frog sartorii; the
enzymes are present (23) , can be active (2), and they
may not always be under tight reciprocal control in the
cell (42). The operation of these cycles are an energy cost
associated with the structure
of the metabolic pathways
which must perform the task of net ATP restoration.
If
fructose phosphate cycling occurs in sartorii,
it could
explain an apparent low anaerobi c P/lactate and low
aerobic P/O ratio, Cycling would not rule out the possibility of partially uncoupled mitochondria
also contributing to a low P/O ratio.
Received
for publication
19 August
1976,
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