114R. E. H. NICHOLAS AND C. RIMINGTON
5. Specimens from Prof. C. J. Watson's labor-
atory have been examined. The uroporphyrin
methyl ester I had m.p. 292-293° and behaved as
uroporphyrin I containing a trace of uroporphyrin
III on paper chromatography. The 2080 ester
behaved chromatographically as a hexacarboxylic
Wewish tothankDrFalkand Miss Benson forperforming
the chromatographic examinations by their method, and
also Prof. C. J. Watson and Dr J. Canivet for kindly placing
materials at our disposal. A generous grant to one of us
(C. R.) from the Trustees of the Nuffield Foundation has
made possible the establishment ofa Unit for the Investiga-
tion of Pyrrole Pigment Metabolism and this grant is
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The Protein Components of the Isolated Myofibril
BY S. V. PERRY
Department of Biochemi8try, Univer8ity of Cambridge
(Received 10 February 1953)
The myofibrili s unique among the organized com-
ponents of cells in that it requires a common sub-
strate, adenosine triphosphate (ATP), for enzymic
and mechanical activity.
survive when the myofibril
from the cell, and although it is true that the main
protein component, actomyosin, has similar charac-
teristics, extraction of this complex completely
disorganizes the myofibrillar structure and results
in modification of both enzymic and, mechanical
Schick & Hass (1951) investigated the solubility
in salt solutions of varying ionic strength of myo-
fibrils obtained by a procedure which involved
tryptic digestion ofthe muscle tissue. These workers
used an arbitrary method of microscopic examnina-
tion to determine when solution was complete, but
in view of the pronounced effect oftrypsin onmyo-
sin (Gergely, 1950; Perry, 1951), and on proteins
Both these properties
is removed intact
generally, it is difficult to decide to what extent the
constituent proteins were modified during the pre-
paration of the myofibrils. Information about the
protein components of the myofibril other than
actomyosin is scanty; for example nothing is known
about the nature ofthe various banded features, all
of which must play a part in the function of the
contractile unit.Earlier electron-microscope in-
vestigations (Rosza, Szent-Gyorgyi & Wyckoff,
1950; Perry & Home, 1952) have provided evidence
for the washing out ofsome band components with
no apparent modification of the underlying basic
The present communication is concemed with
further investigations of the properties of myo-
fibrils prepared without the use of enzymes. In
particular, the nature of the protein components
extracted from the myofibril under varying ionic
conditions has been investigated.
PROTEINS OF THE MYOFIBRIL
Preparation of myofibrils. Myofibrils were prepared as
previously described (Perry, 1952), using as the medium for
suspension 0 078M-borate buffer, pH 7-1 (30 ml. 0*05m.
boraxand 970 ml. 0 2M-boricaciddilutedto2*5 1.; Palitzsch,
1922). In certain cases, to reduce the amount of protein
leaching out of the myofibrils, the preparation was carried
out in a solution of 0 025m-KC1 and 0-039m-borate buffer,
pH 7X1. All manipulations were carried out in the cold
Extractionofmyofibrils. Myofibrils weretreated at 0° with
the appropriate salt solutions so that extraction was carried
out atprotein concentrations ranging from 0 5 to 1.0mg./ml.
The original suspension was always in borate buffer and in
consequence a low concentration of the latter was usually
present during the first extraction. After a given extraction
time, the myofibrillar residues were sedimented by centri-
fugation at 00 for 10 min. at 100000g. To determine the
amount ofinsoluble fraction, the residue was resuspended in
salt solution and the extraction repeated 2-3 times. Finally
total N estimations were carried out on the appropriate
Viscosity measurements. The usual procedure was to mix
5 ml. of extract with 2 ml. of 0 16m-phosphate buffer,
pH 7 0, and take 3 ml. of this solution for viscosity deter-
mination at 0° in an Ostwald viscosimeter (time of flow for
water, 40-50 sec.). Total concentration of protein in the
viscosimeter was 3-4 mg./ml. ATP (0 03 ml.) was added in
the viscosimeter to give a final concentration of 0-0004m.
Adenosine triphosphata8e activity. The method was es-
sentially that described earlier (Perry, 1951); glycylglycine
buffer was used at pH 7-4 and glycine atpH 9 0.
Collagen estimation. Myofibrils were treated with 01
NaOH for 20 hr. at 150, centrifuged for 10 min. at 100000g
and N determinations carried out on the clear supernatant
and the original whole extract. Nitrogenous material not
rendered soluble by this procedure was considered to con-
sist of collagen and elastin (Lowry, Giligan & Katersky,
1941; Robinson, 1952).
Estimations. The acid-labileP contentofthemyofibril and
derived fractions was estimated as described in an earlier
communication (Perry, 1952). Total nucleic acid (including
phosphoprotein) P values were determined bythe method of
Schmidt & Thannhauser (1945), and N byamicro-Kjeldahl
technique. Protein concentration was arbitrarily taken as
6 x nitrogen concentration.
Materials. The ATP was prepared routinely in this
department by Mr E. J. Morgan. It was freed fromheavy-
metal impurities by treatment with 8-hydroxyquinoline,
stored as the barium salt and converted to the sodium salt
for use. Dr T.-C. Tsao kindly providedtheelectrophoretic-
ally pure, recrystallized sample oftropomyosin.
Electrophoresis. Experiments were carried out with the
usual Longsworth & McInnes Tiselius apparatus, and also
with the Perkin-Elmer model 38 instrument. Conductivities
were determined on the protein solutions as prepared for
Myofibril proteins soluble in 0-078M-borate, pH7-1
Freshly prepared myofibril suspensions in 0-078m-
borate buffer contained verylittle dissolvedprotein,
for the addition of 10% (w/v) trichloroacetic acid
(TCA) to an equal volume of the clear supernatant
obtainedwhenthesuspensions had beencentrifuged
for 10 min. at 10000 g, usually gave no detectable
turbidity. During storage, at 4° in the presence of
toluene, protein passed into solution with the result
that after 15 days 11-12% of the total nitrogen of
the suspension had become soluble.
storage under sterile conditions brought
additional nitrogen into solution, and dialysis ofthe
solution showed that it did not contain nitrogen
compounds of low molecular weight. This solution
ofmyofibrillar proteins is subsequently referred to
as the soluble fraction.
Age of preparation (days)
Fig. 1. Rate of solution of protein N on storing myofibriI
suspensions in 0-078m-borate buffer, pH 7-1, at 4°. The
results obtained with five preparations are shown, each
preparation being indicated by a different set of points.
Fig. 1 illustrates the results obtained with five
different myofibril preparations and shows that
afteracomparatively rapidsolution ofproteininthe
first day or two, the rate of extraction decreases
until after 15 days it is veryslow. Whenasuspension
ofmyofibrils, which had been stored until 11-2% of
the total protein had gone into solution, was centri-
fuged down and resuspended in fresh borate buffer,
the rate ofsolutionwas not increased. This indicates
that the process was not a distribution of myo-
fibrillar proteins between the gel and sol phases but
more soluble in the buffer than the bulk ofthe myo-
The soluble fraction was obtained as a clear,
slightly viscous solution of protein with minimum
in the range pH 4-85-4.
saturation with ammonium sulphate little protein
practically complete at 50% saturation.
This fraction showed certain properties which
suggested that it might consist, in part, of the
globulin X complex. According to Weber & Meyer
(1933) globulin X is precipitated at ionic strength
< 0-005 and there is evidence that in living muscle
S. V. PERRY
onlypart is in solution inthesarcoplasm. Dialysis of
the soluble fraction against distilled water adjusted
to pH 7, or against phosphate buffers containing
equimolar amounts of KH2PO4 and K2HPO4 and
ranging in total ionic strength from 0 001 to 0-1, did
not precipitate any significant amount of protein.
On this evidence it was concluded that the soluble
fraction did not contain globulin X.
Adeno8ine triphosphata8e activity of 8oluble frac-
tion. No adenosinetriphosphatase (ATPase) activity
was shown by the soluble fraction in the presence of
calcium ions at pH's 7-4 and 9-0, nor in the presence
of magnesium ions at pH 7-4. For example, when
12-14% of the total protein had leached out of the
myofibril, the whole suspension
activity which was entirely
localized in the readily centrifuged myofibrillar
residue. These facts indicate that myosin was not
a component of the soluble fraction.
Ab8ence of F-actin. Precipitation of the soluble
fraction with O-OlM-acetate buffer, pH 5-1, and
dissolution in a small volume ofdistilled water with
sufficient 0-1M-sodium bicarbonate to bring the pH
back to 7, gave a very viscous solution. This was not
F-actin as it did not form actomyosin on addition
to a solution of freshly prepared myosin in 0-5m-
potassium chloride, i.e. ATP did not produce a drop
in viscosity when added to the combined solutions.
Tropomyosin and the 8olublefraction. Addition of
potassium chloride to the soluble fraction, as
obtained fromthemyofibrils bycentrifugation, orto
the concentrated solution obtained after isoelectric
precipitation, gave a marked drop in viscosity
(Fig. 2). This salt effect was reversible and strongly
suggested the presence of tropomyosin, the only
protein so far isolated from muscle which exhibits
these properties and which is presumed to be myo-
fibrillar in origin on the basis of the procedure
employed for its extraction from washed muscle
mince (Bailey, 1948).
As usually obtained, the soluble fraction con-
tained 1-5-2-0 mg. protein/ml., and concentration
was necessary before electrophoretic investigation
could be undertaken. This was accomplished by
suspending the solution (containing 0-05M-phos-
phate buffer, pH 7-0) in a dialysis bag in front ofan
electric fan. When this process was carried out inthe
coldroom at 4° thevolume was reduced to one-sixth
in 36 hr. without any significant amount of de-
Examination of this concentrated
solution in the Tiselius electrophoresis apparatus
revealed two main peaks (Fig.
invariably seen, but in some preparations another
component, moving more slowly than the two main
peaks, and present in much smaller amount, could
be detected. The slower ofthetwomain components
was estimated to be present in twice the amount of
the faster. Addition of a sample of recrystallized
3). These were
tropomyosin to the soluble fraction did not result in
the appearance of a new peak but, as Fig. 3 clearly
shows, the fast component increased in amount
relative to the slower. This was considered as good
evidence for the identity ofthe fast component with
tropomyosin. In Table 1 are presented the mobili-
ties of the two main components of the soluble
KCI concentration (M)
Fig. 2. Effect of KCI on the viscosity of a solution of the
soluble fraction in 0-078m-borate buffer, pH 7-1. Protein
concentration approx. 0-5 mg./ml.
Fig. 3. Electrophoresis diagrams of the soluble fraction
extracted firom rabbit myofibrils, after 270min.
Soluble fraction; (b) soluble fraction with added tropo-
pH 7-1; 0-25M-NaCl.
On addition of potassium chloride to a final con-
centration of 0-5m the viscosity of the soluble
fraction dropped to a very low level (Fig. 2), and in
view ofthe electrophoretic evidence it was assumed
that at low salt concentrations the viscosity of the
extract was almost entirely due to the tropomyosin
present and that the main component contributed
PROTEINS OF THE MYOFIBRIL
little. On this asumption a value for the tropo-
myosin concentration of the soluble fraction was
obtained by comparing the reduction of viscosity
obtained on addition of 0-5M-potassium chloride
with that produced under identical conditions with
Table 1. Electrophoretic mobilitiea of component8
of the solublefraction of rabbitmyofibrils
(Electrophoresis carried out at 00
phosphate buffer, pH 6-5, and 0-22M-KCI. Values are the
average of two independent determinations.)
in 0 05M-sodium
a sample ofpure tropomyosin. Table 2, showing the
results of such determinations on several prepara-
tions, indicates that about 30% of the soluble
fraction consists of tropomyosin. This proportion
Phosphorus content of the solublefractwon. Hamoir
(1951) has shown that tropomyosin from carp and
rabbit muscle can form a crystallizable complex
with nucleic acid, but it is not yet clear whether this
protein exists in situ as such a complex or whether
the nucleic acid is picked up during the extraction
Consequently, it was of interest to
determine the amountofnucleic-acidphosphorus in
the soluble fraction, for under the conditions of
extraction tropomyosin is removed from the myo-
fibril without exposure to contamination by the
nucleic acid of intracellular components such as
nuclei and sarcosomes.
The soluble fraction contained variable amounts
of free inorganic phosphate which presumably had
leached out of the myofibril. Total nucleic-acid
phosphorus values also showed some variation, the
highest value obtained being 0-081 % (see Table 3).
If, in the extreme case, which is rather unlikely, all
this phosphorus occurred as nucleic acid associated
with tropomyosin in the soluble fraction, the com-
plex would contain 2-5-3-0% nucleic acid. Nucleo-
tropomyosin isolated by Hamoir (1951) from carp
Table 2. Tropomyosin extractedfrommyofibril8stored in 0-078M-borate buffer, pH 7-1
(as % soluble
stayed fairly constant during the extraction of the
soluble fraction, and the maximum amount of
tropomyosin extracted represented 4-0 % of the
Table 3. Nucleic-acid phosphorus content of
(All fractions were extracted with cold trichloroacetic
acid and fat solvents according to the procedure ofSchmidt
& Thannhauser (1945). The P figure represents the total
amount of ribonucleic and deoxyribonucleic acids and
phosphoprotein in the fraction analysed.)
Whole myofibrils after
removal of the soluble
total proteins of the myofibril. On the basis of his
organic solvent extraction method Bailey (1948)
estimated tropomyosin to account for 2 6% of the
total proteins of rabbit muscle, i.e. 4-0% of the
myofibril if the latter contains 65% of the total
protein of muscle.
muscle contained considerably more nucleic acid
Effect of low concentrations of potassium chloride
on the extraction ofthe solublefraction. To investigate
theeffect oflowconicentrations ofpotassiumchloride
on the extraction ofthe soluble fraction, samples of
Table 4. Protein passing into solution on storing
myofibrils in media of low ionic strength
Composition of medium
addition of TCA
to the supernatant
a freshly prepared myofibril, suspensionwere left for
15 days in various solutions, the compositions of
which are shown in Table 4. At the end of this
period, the suspensions were centrifugedfor 10 min.
at 10000 g and a sample ofeach supernatant added
S. V. PERRY
to an equal volume of 10% TCA. The relative
obtained, which represent
mately the relative amounts of protein which had
gone into solution, are shown in Table 4. It was
readily apparent that the minimum amount of
protein had leached out when the myofibrils were
stored in a solution containing 0-039M-borate
buffer, pH 7-1, and 0-025M-potassium chloride.
Fig. 4. Electronmicrographofrabbit myofibrils afterextrac-
tion for 17 days at 4° with 0-078M-borate buffer, pH 7-1.
Grold-palladium shadowed. Magnification, x 9250.
More precise investigation indicated that only 4%
ofthe total myofibrillar protein passed into solution
during storage for 15 days in this medium. This
solution was adopted for routine preparations when
it was necessary to reduce the leaching out ofmyo-
fibrillar protein to a minimum. It was noted that
myofibrils prepared in this manner sedimented
faster and formed a more compact mass in the
centrifuge tube than those prepared in 0-078M-
borate, and it was more difficult to free the prepara-
tions from nuclei and other rapidly sedimenting
cell components. During preparation in 0-078M-
borate the myofibrils became increasingly difficult
to centrifuge down at the speeds used (Perry, 1952)
and presumably in this buffer some hydration and
Myofibrils from which the soluble fraction had
been extracted were similar in general microscopic
appearance to the freshly prepared material.
methylene blue was used as an aid for more careful
observation, the contrast between the heavily
stained bands (which frequently appeared as a
double band, see below) and the lightly stained
bands was greater in the stored than in the fresh
preparations. This appearance ofincreased contrast
was possibly due to the fact that, comparedwith the
fresh material, myofibrils from which the soluble
fraction had been extracted stained very lightly
indeed in the less dense part of the structure.
Fig. 5. Electron micrograph of rabbit myofibrils after
30 days extraction with 0-078M-borate buffer, pH 7-1.
Gold-palladium shadowed. Magnification, x7600.
Electron-microscope examination revealed that
although there was evidence of longitudinal fila-
structures within the myofibril,
details could no longer be recognized. A character-
istic feature of these extracted myofibrils was the
appearance of regular dense bands (Figs. 4, 5). Two
ofthese bands occurred in each sarcomere, although
sometimes a pair was close enough together to
appear as one broader band. These bands are con-
sidered to correspond to the outer edges of the A
band or perhaps to the A substance which has
moved into the I band, for myofibrils isolated by the
method described are usually slightly contracted
(Perry, 1952). Features of the A band such as the
M line and H sub-lines (Perry & Home, 1952) were
never seen in electron microscope examinations of
myofibrils stored long enough to extract the soluble
fraction. Further work needs to be done to decide
the precise location within the myofibril of the
proteins of the soluble fraction.
Double refraction of the myofibril. Examination of
a number of myofibril preparations was kindly
carried out with the polarizing microscope by