ON THE RELATION BETWEEN TENSION AND ATP IN CROSS-
              STRIATED MUSCLE

M. BORBIRO AND A. SZENT-GY6RGYI 1

Institute for Muscle Research, Marine Biological Laboratory, Woods Hole, Massachusetts 2 and
   Experimental Biology and Medicine Institute, Laboratory of Physical Biology,
         National Institutes of Health, Bethesda, Maryland

According to the theory outlined in the preceding paper, the contractile matter of
muscle is built of functional units containing myosin, actin, and ATP. Since
muscle contains no free ATP, it can be expected that if the ATP concentration of
muscle decreases, the nurhber of contractile units decreases proportionately. The
ATP concentration of muscle decreases after the death of the animal (Th. Erd6s,
1943). The object of the present research was to see whether the ATP content
and the tension developed by muscle decrease proportionately.  Such a parallelism
would support the theory outlined, while a lack of parallelism would plead against it.
For this reason, we measured, simultaneously, the tension developed by the muscle
and the ATP concentration at various intervals after the death of the animal.

The material used was the musculus psoas of the rabbit.  At various intervals
after the death of the animal, strips of this muscle were cut out and frozen at once.
The tension developed on thawing was measured.

The methods hitherto used for the estimation of ATP were found to be un-
satisfactory for the following reason : we possess no direct method for the estimation
of ATP.  When this substance has to be estimated, extracts of the tissue are sub-
jected to limited acid-hydrolysis, and the quantity of ATP is calculated from. the
quantity of labile phosphate liberated.  Muscle contains ifz vivo a not inconsider-
able amount of free phosphate.  As the ATP is gradually. decomposed post mortewz,
the amount of hydrolyzable phosphate decreases while the amount of free phosphate
increases, and thus a slight error in the phosphate estimation makes the results of
the ATP estimation doubtful.  A new method of phosphate estimation had to be
constructed in which the free phosphate did not interfere with the estimation of
the ATP.

In the first part of this paper this method will be described. In the second
part, the results obtained by this method will be given.

METHOD OF PHOSPHATE ESTIMATION

The method is based on the ready solubility of phosphomolybdic acid in iso-butyl
alcohol, described by Berenblum and Chain (1938)) and on the yellow color with
which the acid dissolves in this reagent.  The muscle was extracted with tri-
chloracetic acid. Ammonium molybdate was added to the solution. The free
phosphate present combined with the molybdate and was shaken out with a mixture

1 Special Fellow, U. S. Public Health Service.
2 Sponsored by the American Heart Association.

                          162


ATP AND CONTRACTILITY               163

of iso-butyl alcohol and ethyl ether.  Then the fluid was hydrolyzed and the free
phosphate shaken out with iso-butyl alcohol and estimated calorimetrically.

After the trichloracetic acid extract of the muscle is shaken out with butyl alcohol
ether, it still contains a small quantity of phosphate.  This quantity can be estimated
and taken into account.  If the extract is shaken out a second time with alcohol
ether, no phosphate is left, and no correction has to be made on the final readings.
In the present paper the former method was used.s

Muscle extract contains substances which, after boiling with HCl, yield products
which interfere with the development of the yellow color.  These substances are
eliminated by the alcohol ether, since they are of lipoidic nature.

The detailed description of the procedure is as follows : the rabbit (2-3 kg.)
was decapitated, eviscerated, the side walls of the abdomen cut off and the psoas
exposed.  Two thin strips of the psoas were taken out, provided with ligatures, fixed
and frozen with dry ice at their resting length, as described in the preceding paper.
These strips were used for estimating the maximum tension developed by the muscle
on thawing at 15" C.  Simultaneously, a somewhat thicker strip of about one gram
weight was cut out from the same region, weighed and frozen.  This strip served as
material for the ATP estimation.  The remainder of the muscle, left z'n situ, was
covered with cotton wool wetted with Ringer.  The procedure was repeated once
every hour.  First the right and then the left psoas was used.  Such samples were
taken until the muscle showed no elasticity and no contractility after thawing.  One
hour later a last sample was taken.

Extraction : 25 ml. of 10 per cent trichloracetic acid was pipetted into a mortar
which was pre-cooled to - 20" C.  The fluid solidified to a brei.  The muscle, after
having been weighed, was placed into the brei in frozen condition and ground to
a fine suspension. On thawing, the suspension was transferred into a centrifuge
tube and spun.  The clear fluid was poured into a 50 ml. graduated measuring cyl-
inder provided with a ground glass stopper.  The volume was noted ; then for every 10
ml., 1 ml. of 10 per cent ammonium molybdate solution was added and the fluid mixed.
Then 1 ml. of iso-butyl alcohol was added for every 4 ml. of the fluid, and 4 ml. of
ether added for every ml. of butyl alcohol used.  The fluid was strongly shaken for
twenty-five seconds and allowed to separate.  If there was no ready separation of
the two phases, the fluid was centrifuged.  Then the ether butyl alcohol mixture was
sucked off through a capillary glass tube.  A few ml. of ether were added without
shaking in order to wash off the remaining alcohol ether.  The volume of the fluid
was noted.  If, after the shaking with alcohol ether, a heavy precipitate was.formed,
this was separated by centrifugation.  The fluid was divided into samples, each of
which corresponded to 100 mg. of muscle, and pipetted into test tubes.  Out of nine
samples four were put aside. To five samples, l/10 parts of concentrated HCl
(approximately 10 N) was added and the tubes placed into the boiling water-bath
for seven minutes and then rapidly cooled.  To the unboiled samples, the same
amount of HCl was added.  To all tubes one drop of 0.1 per cent potassium per-
manganate was added which stained the fluid a rose color.  This color persisted
for about half a minute.  This was done in order to oxidize any reducing agent
present which would reduce the phosphomolybdate.  Then 10 per cent ammonium

  3 If for any reason the quantity of free phosphate present in the muscle extract had to be

known, this could be estimated calorimetrically in the combined alcohol ether extracts.


164         M. BORBIRO AND A. SZENT-GYGRGYI

molybdate was added to the unboiled tubes, and 5 ml. iso-butyl alcohol to all
samples. The butyl alcohol used here was shaken out previously with water.
(This is necessary in order to prevent the butyl alcohol from taking up water
later.)  The fluid was shaken strongly for five seconds, the opening of the tube
being closed by the thumb covered by a rubber glove.  After the two phases sepa-
rated, the watery phase at the bottom was sucked off by means of a thin glass tube,
connected to the vacuum by a thin rubber tube which was pinched tight while the
tip of the tube was passing the alcohol.  Then the alcohol was poured over into the
calorimeter tubes which were marked at their 5 ml. volume.  Usually the volume of
butyl aIcoho1 is less than 5 ml.  It was filled up to 5 ml. with butyl alcohol which
was used to rinse the tubes that contained the extract previously.  Then to every
tube 1 ml. of ethyl alcohol was added and the color estimated in the Klett-Summer-
son coloritneter with the S 42 blue light filter (400-460 mp).

As a standard, a solution of KH,PO, was used, containing 0.01 mg. per ml.
Samples of 1, 2 and 3 ml. of this fluid were filled up with water to 4 ml., 0.5 ml. cc.
HCI and 0.5 ml. of 10 per cent molybdate were added; then the fluid was shaken
out with 5 ml. butyl alcohol which was treated as described above,

EXPERIMENTAL RESULTS

Before embarking on the problem proper, a few minor points had to be cleared
up.  First, is the method of P estimation reliable, and is the distribution of ATP in
the psoas homogeneous ?

A rabbit was killed and six samples of 1 g, were taken from different parts
of the. two psoas muscles.  In Table I the actual calorimeter readings are repro-
duced.  The six upper columns related to the unhydrolyzed extract are thus the zero
values.  The corresponding readings of the hydrolyzed samples are reproduced
in the lower columns.

As can be seen, the readings are very uniform.  The one value in the fourth
column, marked with an asterisk, is evidently due to some rough mistake and has
to be discounted.  The other single values do not differ from the average by more
than five per cent.  The average of the 0 value was sub&acted from the average of
the hydrolyzed product. From this the ATP was calculated. The standard with

TABLE I

---
Average
  ATP

40
39
39

202
200
204
190
196
195

198
3.55

38        30        29       24        29
3.5       30       69*       24        28
37        30        29       24        28

194       190       204       198       204
189       200       200       194        202
194       198       206      196       202
196       189       200       200       198
199       187      204       208        194
186       198       214      195       -195
    ___-I_-_                    -
194       194       205       198        199
3.50     3.47.     3.70      3.68    3.60 mg. per gm.


ATP AND CONTRACTILITY               165

0.02 mg. phosphate gave a reading of 80.  The quantity of P found was multiplied
by 8.4 to give the ATP, which is noted in the last horizontal line.  This shows
the ATP content of the psoas to be very uniform, 3,6 mg. ATP per gm.

According to the literature, muscle contains 2-2.5 mg. ATP per gm., thus con-
siderably less than psoas.  This difference is probably due to the shielded position
of the psoas and the consequent poverty of connective material.  In order to eluci-
date this point, samples of different muscles of a freshly killed rabbit were taken
and subjected to analysis.  Results are reproduced in Table II.

TABLE II

Psoas                     3.55 (mg. ATP per pm.)
Deep'muscles of the back               3.04
Big adductor muscle                  2.56
Musculus gracilis                     2.10
Superficial muscle of the back             2.10
Smaller muscles from the gluteal region       1.96

These values show that the more superficial the position and the richer the con-
nective tissue, the lower the ATP content.  The muscles of the whole animal would
give an average of about 2.5 mg. ATP per gm.

The third question which had to be cleared up was whether the ATP content
of the psoas decreases uniformly in all its parts after the death of the animal.
Preliminary experiments have shown that the rate of disappearance of ATP post
mortem depends on the temperature and the oxygen supply.  If the muscle is cut
into thin strips which are exposed to air, the disappearance becomes much slower.
While the ATP in the muscle left in situ may disappear within three to four hours,
muscle strips exposed to air may contain ATP and thus remain contractile at room
temperature even twenty-four hours after the death of the animal. Experiment
also showed that in the muscle left in situ, the ATP disappeared faster in the deeper-
lying dorsal than in the superficial ventral part.

Experiment: The rabbit was killed, the psoas exposed as usual and covered
with wet cotton wool.  Three hours later, five strips, weighing approximately 1 gm.,
were cut out and analyzed for ATP. One of the strips was taken fro.m the lateral edge
of the muscle, two from the ventral surface, two from the deeper-lying dorsal surface.
ATP (mg.) per gm:

edge:        2.53
ventral:      2.77      ,2.77
dorsal:      1.80      1.95

If the ATP content of muscle and its tension are to be measured simultaneously,
it is essential that strips from the same region be used for both measurements.
Even with this precaution, considerable scattering of results can be expected.
The relation between ATP content and tension was studied in eight experi-
ments.  The following example may be cited (Fig. 1) : Samples of muscle were
taken every hour after the death of the animal.  The ATP content in milligrams per
gram of muscle is marked in the curve by points.  They relate to the left ordinate.


166          M. BORBIRO AND A. SZENT-GYGRGYI
The tension developed is marked with circles and refers to the right-hand side
ordinate.  (The scale of this ordinate is arbitrary and is chosen in such a way that
the numbers, if multiplied by 100, give the total working capacity in calories calcu-
lated for 35,000 gm. myosin by. the formula: `/3 tension x length x 0.000023.)

As the curve shows, tension and ATP content run parallel.  At the end of the
third hour the muscle develops no more tension and does not contract on thawing,
and is found to be completely inelastic.  At this point, the ATP curve shows a
break and becomes roughly parallel to the abscissa.

3

HOURS   ,       2      3      4      5

As the curve shows, the muscle at this point still contains a not inconsiderable
amount'of labile phosphate.  Whether this hydrolyzable ATP is derived from ATP
or some other source ( ADP ?) cannot be stated at present.  If this hydrolyzable P
is derived from ATP, this ATP must be in some way different from the rest, be-
cause it is no longer split by the muscle (or is split only exceedingly slowly) and
has no influence on contractility and elasticity. This "residual" hydrolyzable
phosphate was found in approximately the same proportion in all experiments.

The second point, equally borne out by the other experiments, is that the de-
crease of ATP concentration is linear : the rate of its disappearance is independent of
its concentration.  The most likely interpretation of this rather unexpected fact is
that the splitting of ATP depends on some change in the contractile matter.  As
has been shown by A. Biro and A. E. Szent-Gyorgyi (unpublished), myosin is enzy-
matically active in its contracted condition only.

In two out of the eight experiments, the ATP concentration did not fall at all
during the first hour after death.  This can be explained by the presence of creatine-
phosphate which rephosphorylates the ADP formed.
All. experiments gave similar results.  In most of them the scattering was
stronger than in the quoted example.  Nevertheless, all experiments bore out the
close parallelism between tension developed and the quantity of ATP present.


ATP AND CONTRACTILITY              167

SUMMARY

A new calorimetric method of ATP estimation is described.  In the psoas of
the rabbit the post mortem decomposition of ATP and the loss of contractility are
parallel.

LITERATURE CITED

BERENBLUM, J., AND E. CHAIN, 1938. Biochena. Jour., 32: 295.
ERDBS, TH., 1943. Studies Inst. Med. Chewz. Saeged, 3: 51.