Active Transport of Potassium Ion in
Heart Mitochondria
By Brian Safer, 6.A., and Arnold Schwartz, Ph.D.
ABSTRACT
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Several factors affecting K* transport by rabbit heart mitochondria were
examined, using a K+-sensitive electrode. The histone fractions f2a and j3-7
produced an energy-dependent efflux of K*. Inorganic phosphate was required
for optimal activity; Km for phosphate was 60 /AM. Both rate and extent of K+
efflux decreased as K+ concentration in the reaction medium was increased.
The direction of valinomycin-induced K* movements was shown to depend on
the net resultant of an active transport mechanism and increased membrane
permeability. The detergent triton X-100 produced a nonspecific increase in
membrane permeability that led to a rapid efflux of K+. Evidence is presented
for competition between ion transport and ATP formation for some common
energy intermediate. Possible mechanisms of action of histones and other
agents affecting heart mitochondrial K* transport are considered.
ADDITIONAL KEY WORDS
mitochondrial respiration
histone
• In recent years specific emphasis has been
directed toward elucidation of mitochondrial
activity in normal, hyper- and hypofunctioning heart muscle (1-3). Recognition of the
importance of mitochondria has received added impetus with the discovery of functions
other than oxidative phosphorylation. These
include reversal of electron transport associated with reductive transphosphorylation (4), DNA and RNA metabolism (5, 6)
and active cation accumulation and extrusion
(7-10). The last two phenomena may be
intimately associated with cell metabolism (11).
Previous studies from this laboratory have
described an energy-dependent histone-inFrom the Department of Pharmacology, Baylor
University College of Medicine, Houston, Texas
77025.
These studies were supported by grants HE 07906
and HE 05435 from the National Heart Institute,
Pharmacology Training Grant GM-670 and Grant
FR-00259 from the U. S. Public Health Service;
and by grants from the Texas Heart Association,
the Houston Heart Association, and the Liberty
Foundation.
Dr. Schwartz is recipient of a Career Research
Development Award from the U. S. Public Health
Service, National Heart Institute.
Accepted for publication May 4, 1967.
Circulation Reseafch, Vol. XXI, Juh 1967
common "high-energy" intermediate
valinomycin
rabbit
duced net efflux of K+ from liver and heart mitochondria (12). The effect of valinomycin, a
toxic antibiotic that is a specific inducer of
K+ transport by mitochondria, has been discussed in relation to two basic actions: (1)
stimulation of the carrier mechanism responsible for active translocation of K+ and (2)
induction of increased "permeability" of mitochondria to K+ (9). Depending upon the
energy state of the mitochondria, valinomycin
can produce either a net K+ influx or efflux.
Since both direction and rate of valinomycininduced K+ transport is thought to be a function of extramitochondrial K+ concentration, a
similar effect of K+ concentration upon histone-induced K* transport would be of importance with respect to the energy requirement for the histone effects.
In view of the possible importance of K+
transport in heart mitochondria,' the present
study was designed to investigate, in depth,
factors influencing this phenomenon.
Methods
New Zealand white rabbits (2- to 3-kg males)
were killed by a sharp blow on the back of the
neck. Their hearts were immediately excised and
placed in ice-cold isolation medium for rapid
chilling. The isolation medium contained 125
25
26
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n w choline chloride, 20 HIM tris (hydroxymethyl)
aminomethane (tris), 10 mM ethylenediaminetetraacetate (EDTA), and 1% bovine serum albumin, fraction V, and was adjusted to pH 7.4.
Choline chloride was chosen because it produces
no known alteration of mitochondrial function
and is not detected by the K* electrode. EDTA
was included to chelate Ca2+, which would otherwise produce uncoupling of oxidative phosphorylation and swelling (13). Bovine serum albumin
also prevents uncoupling that would be induced
by free fatty acids (13) in the tissue homogenate. The isolation medium described is a modification of that used by Von Korff (14).
The hearts were examined for any signs of
pathologic changes such as necrotic areas or excessive fat. If none was noted, both atria and the
adipose tissue sometimes located along the coronary arteries were removed and discarded. The
ventricles were then cut into small pieces, rinsed
free of blood, and weighed. The tissue was finely
minced, with sharp scissors, in a small amount of
isolation medium. Additional medium was then
added to produce a 5% tissue homogenate. The
suspension of finely minced heart tissue was homogenized in small portions using a glass PotterElvejhem homogenizer (A. C. Thomas, type C).
Only four complete passes were made with a
loosely fitting Teflon pestle driven at 500 rpm to
minimize mitochondrial damage. Microscopic examination of the homogenate revealed a small
residue of undisrupted myofibers (<10%). In some
experiments a tissue homogenate was obtained
by use of the Polytron tissue processor (Brinkmann Instruments). The pooled homogenates
were transferred to 50-ml Teflon tubes and centrifuged at 600 X g for 10 min. The supernate was
centrifuged at 6,000 X g for 10 min. The mitochondrial pellet was collected and washed, using
a glass Potter-Elvejhem homogenizer (A. C.
Thomas, type A) and Teflon pestle for resuspension, in K the volume of isolation media used to
homogenize the tissue. The suspension of mitochondria was recentrifuged at 6,000 X g for 10
min. The above procedure is a modification of the
method of Plaut and Plaut (15).
The mitochondria were suspended to a final
concentration of > 40 mg mitochondrial protein/
ml (about 0.2 ml/g heart tissue) to minimize loss
of intramitochondrial electrolytes and substrates,
and were stored at 0°C. The suspension medium
contained 0.25 M sucrose, 10 mM tris'HCl, and
1% bovine serum albumin, fraction V, adjusted to
pH 7.4. In some experiments, the mitochondria
were suspended in the isolation medium.
The concentration of K+ (extramitochondrial)
in the reaction medium is indicated on the ordinates of Figures 1 through 4. Movements of K+
were determined by means of a Beckman cationic
SAFER, SCHWARTZ
electrode (no. 39137), which was connected to a
Sargent recorder (Model XV) via a Beckman
Research pH meter. Mitochondrial respiration
was measured with a vibrating platinum electrode assembly (Oxygraph, Gilson Medical Electronics). Respiratory control ratios of 16 to 18
were consistently observed, and mitochondria were
used within 1 hour of preparation. Emphasis is
placed on high respiratory control ratios in considering metabolic functions of mitochondria. We
suggest that much lower values indicate damage
during isolation and make interpretation of results difficult.
The histone fractions f2a and /3-7 were isolated
from ox thymus and characterized by the procedure of Hnilica and Busch (16) and Busch
and Mauritzen (17), respectively. The fraction
/?-7 is electrophoretically homogeneous. Triton
X-100 was obtained from Rohm and Hass. Tris
salts of various anions used in these experiments
were prepared by passage through a Dowex-50
(H+) column. Nucleotide diphosphates were purchased from Nutritional Biochemicals Corporation, and amounts utilized were determined spectrophotometrically. The sources of other chemicals
used in these experiments are listed in a previous paper (18). Deionized glass-distilled water
was used throughout.
Results
Energetics of Histone and ValinomycinInduced K* Transport—The histone-fractions
f2a and /3-7 produce a net K+ efflux from rabbit heart mitochondria suspended in K+-free
media. These results are similar to those reported previously for liver mitochondria (12).
The K+ movement is supported either by
electron transport supplied by NADMinked
substrates or by succinate when endogenous
NADMinked substrate utilization is blocked
by rotenone (13). When electron transport
is completely blocked (antimycin A or cyanide), ATP effectively supports histone-induced K+ efflux. Inorganic phosphate is required for optimal activity, Km for phosphate being 60 fiM, which is significantly lower than the Km for oxidative phosphorylation
(19). The histone (2^ stimulates respiration
maximally at 8.3 ju-g/mg mitochondrial protein. The threshold concentration is about 1
/^g/mg mitochondrial protein. Respiration is
inhibited at higher values, presumably by
inhibition of the acidic protein cytochrome
oxidase (20). Valinomycin under identical
Circulation Research, Vol. XXI, July 1967
MITOCHONDRIA!. K+ TRANSPORT
27
conditions induces a net K+ efflux. However,
there is no requirement for energy or inorganic phosphate.
Effect of K* Concentration.—Increasing concentrations of K+ in the reaction medium reduces both the rate and extent of f2a-induced
K* efflux (Fig. 1). When the external concentration of K+ is zero, the initial rate of K*
efflux induced by the histone is 42 /J-Eq/g
mitochondrial protein per min. Under similar
conditions, valinomycin effects a K* loss at a
rate of 120 ftEq/g per min. As the external
K* concentration is raised, valinomycin-inDownloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
VAL
HISTONE
t)
0
1
TRITON
IO5O
duced K+ transport changes from a net efflux which is energy independent (data not
shown for heart mitochondria; see reference
12 for liver mitochondria) to a net uptake of
K+, which is energy dependent (8, 9 ) . The
latter requires a minimal external K+ concentration of 200 fiM. Although the rate of histone-induced K* efflux decreases significantly
as the external K* concentration is raised, no
net K+ accumulation was observed. Concentrations of K+ greater than 1 mM, however,
could not be monitored in our system. Consequently, any possible transition from efflux
to influx, at an external K* concentration of 1
mM or higher, could not be determined.
In contrast to f2a, the detergent triton X100 in concentrations as low as 0.001& produces a K+ loss which is completely independent of energy, i.e., inhibition of electron
transport does not alter the rate or extent of
2
TIME (MINUTES)
FIGURE 1
Effect of external K* concentration on K* transport
by rabbit heart mitochondria. Each experiment was
carried out in 8 ml of medium containing 125 MIN
choline chloride, 20 mtu tris-chloride (pH 7.4), 2.5
nun tris-glutamate, 2.5 mm tris-phosphate, and varying
concentrations of added KCl as indicated (from 0 to
1 IUM). Three groups of experiments, each containing
four different KCl concentrations, contained 1.6 ng
valinomycin, 40 y.g fta, and 250 \ig triton. Addition
of 6 mg mitochondrial protein initiated the K* movements, which were measured by means of a Beckman
electrode (see text). The point on the ordinate at
which each of the 12 curves begins represents the
concentration of K* in the medium before addition
of valinomycin, histone, or triton. The rate of K*
efflux was obtained using the maximum slope of nM.
K*/min.
Circulation Research, Vol. XXI, July 1967
FIGURE 2
Inhibition of the effects of histone by ADP. For curves
A and B, the medium consisted of 0.25 M sucrose, 10
mm tris-chloride (pH 7.4), 5 MM tris-phosphate, 1 nun
tris-glutamate, and 17 mg mitochondrial protein in a
final volume of 10.0 ml. The additions for curve A
were 500 I*M tris-ADP and 250 /ig /3-7 histone. The
additions for curve B were 500 /AN tris-ADP, 250 ng
fi-7 histone, and 25 \xg oligomycin. Curves A and B
denote the efflux of K* from the mitochondria. For
curves C and D, the medium, was the same as for
curves A and B except that the final volume was 2.0
ml and 4.3 mg of mitochondrial proteins were added.
The additions for curve C were 500 PIN tris-ADP and
50 fig p-7 histone. The additions for curve D were
500 HM tris-ADP, 50 y.g $-7 histone, and 10 pg
oligomycin. Curves C and D represent the uptake of
Ot by the mitochondria.
SAFER, SCHWARTZ
28
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K+ efflux (data not shown). Furthermore, increasing the external K+ concentration up to
1 HIM does not retard the triton-induced K*
loss. Microscopic examination did not reveal
any alteration of mitochondrial structure at
the low triton concentration.
ADP and K* Transport.—Mitochondria suspended in K*-free medium and respiring in
state 3 (presence of excess substrate and
ADP) maintain the same slow efflux of K+
observed in state 4 (presence of excess substrate and low ADP) and are unresponsive
to histone. However, when ATP formation is
blocked by the addition of oligomycin, the
histone f2a induces a rapid K+ efflux (Fig. 2).
Under the identical conditions, valinomycin
effects a K+ efflux whether the mitochondria
are in state 3 or state 4. At concentrations of
K* greater than 200 /UM, after maximal uptake
of K* induced by valinomycin, the addition
of ADP causes an immediate loss of K+ (Figs.
3 and 4). The duration of the efflux corresponds to the time required to complete a
transition from state 3 to state 4. When a
transition is completed, K* reenters to the
extent present before the addition of ADP.
Both NADMinked substrates and succinate
support the ADP-induced K* efflux. Addition
of larger amounts of ADP proportionately delays the recovery of K+, but the extent of K+
loss is not increased (Fig. 4). These cyclic
MITOCHONDRIA
.
SUCC.
560 •
y
A
/ ADP
/
A
\
VAL.
i
ROT.
r
\
\
/ADP
ATP\
J
OLIGOMYCIN
/t
/ANTIMYCIN A
Vi
MALONATE
0
2
4
6
8
TIME (MINUTES)
10
12
14
FIGURE 3
Energetics of valinomycin-induced K* transport.
The medium was the same as in Figure 1 and contained 500 HM KCl. The successive additions were 8
mg mitochondrial protein, 1.6 pg valinomycin, 70 HM
ADP, 2 ng rotenone, 2.5 VIM succinate, 70 HM ADP,
2.5 mM malonate, 2 ng antimycin A, 2 nut ATP and
2 fig oligomycin.
-i 250
4
TIME
6
8
(MINUTES)
FIGURE 4
Competition between ADP phosphorylation and
valinomycin-induced ion transport for a common
"energy-rich intermediate." Conditions for ion transport were identical to those in Figure 3. Similar conditions were used for mitochondrial respiration, but
valinomycin was not included. The successive additions were 7.6 mitochondrial protein, 1.6 \ig valinomycin, 70 I±M ADP, 140 FIM ADP, 350 TUN ADP, 2
fig oligomycin, and 70 y.M ADP.
K* changes may be repeated numerous times
upon successive additions of ADP and are
prevented by atractyloside or oligomycin, antibiotics which respectively inhibit ADP transport and phosphorylation (21, 22). Other
nucleoside 5'-diphosphates (GDP, UDP, IDP,
and CDP) in similar concentrations have no
effect on K+ transport. Uncoupling of mitochondria produced by aging in 0.25 M sucrose
at 0°C for 30 hours increasingly delays the
reentry (both rate and extent) of K\ Addition
of oligomycin, antimycin A, cyanide, octylguanidine or 2,4-dinitrophenol, after valinomycin-induced K+ uptake, causes a K" loss
similar to that observed when mitochondria
are treated with 0.001% triton X-100 (Fig. 3).
In the presence of NADMinked substrates,
addition of rotenone also yields similar results; the K+ reenters the mitochondria upon
addition of succinate. While oligomycin appears to have little or no effect on substratesupported ion transport, the antibiotic completely prevents ATP-supported K+ movements
(Figs. 2, 4). Octylguanidine and 2,4-dinitrophenol do not inhibit ATP-supported valinomycin-induced K* uptake (data not shown).
Circulation Research, Vol. XXI, July 1967
29
MITOCHONDRIA!. K+ TRANSPORT
Discussion
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Factors Affecting K* Transport.—The results of these experiments are basically in
agreement with recent conceptual models
proposed for movement of cations across the
inner mitochondrial membrane (9, 10, 23).
The direction of net K+ movement has been
shown to depend on a number of factors such
as the energy state of the carrier molecule,
existence of a chemical or electrical gradient
or both, presence of permeable counterions,
and membrane permeability. Recent studies,
based on the assumption that the total K+
within the mitochondria is in solution, estimate minimal intramitochondrial K+ concentration to be about 90 DIM (24). Under the
present experimental conditions of low external K+ and the presence of a permeable
anion (phosphate), net accumulation of K+
by mitochondria is opposed primarily by the
presence of a chemical potential gradient
directed from the matrix to the external medium. Under our experimental conditions,
net accumulation of K induced by valinomycin in rabbit heart proceeds only at or
above 200 JUM K*. Below this concentration
of K*, addition of valinomycin produces an
energy-independent K efflux. The fact that
rate and extent of efflux increased as energy
source was diminished confirms the suggestion
that valinomycin possesses two actions, one
on "permeability" (efflux) and the other on
active carrier-linked transport (influx). Rate of
histone-induced K efflux also responded to
external K* concentration, suggesting a mechanism similar to the valinomycin-induced K
influx. It is important to note that tritoninduced K movements were completely unresponsive to alteration of K concentration
or to a complete inhibition of energy. The
primary effect of triton is, therefore, thought
to be due to a nonspecific increase in mitochondrial permeability. This is distinct from
the effects of histone-induced K* efflux, which
are strictly energy dependent.
Mechanism of Histone-induced K* Transport.—The ability of a theoretical carrier molecule of cation transport to produce countergradient K* translocation depends on both
Circulation Research, Vol. XXI, July 1967
the slope of the gradient and energy level of
the carrier (23). The energy level of the carrier would in turn regulate the binding of
variable amounts of specific cation to the carrier. Agents affecting ion transport could conceivably act by influencing the degree of cation binding to the carrier at a specific energy
level. The ability of an agent to increase or
decrease carrier binding may determine the
direction of net cation movement. The relative
availability of the cation for combination
with the carrier molecule would also specify
the direction of net cation transport. The
primary action of histones and other basic
proteins influencing ion transport, therefore,
might be to alter the binding affinity of the
carrier molecule (possibly an acidic protein)
for K*, without producing an accompanying
"detergent-like" increase in membrane permeability. However, until a net accumulation
of K+ can be observed, such a proposed
mechanism is merely speculative. A previous
suggestion that histone exerts its effect
via some energy-dependent conformational
change of the mitochondrial membranes is
consistent with the present data (12).
Another possible interpretation of the data
is that histone uptake might require energy.
The basic molecule may exchange for K+; the
latter is then extruded via an energy-independent mechanism. The recent report of
Chappell and Crofts (25), describing an
amine-induced efflux of K+, is relevant.
Possible "Competition" between Ion Transport and ATP Formation.—While the mechanism of oxidative-phosphorylation is unknown, current evidence suggests the presence
of some intermediate energetic state prior
to the formation of ATP (13). Several recent
publications further hypothesize that K+ transport in mitochondria and oxidative phosphorylation are linked through common intermediate energetic state(s) (9, 10, 23).
ELECTRON TRANSPORT
ION
TRANSPORT•
INTERMEDIATE ENERGY STATE
ATP
30
SAFER, SCHWARTZ
The present data support these conclusions,
although no interpretation may be made concerning presence or absence of specific chemical intermediates.
Addition of ADP to substrate-supplemented
mitochondria in the presence of inorganic
phosphate appears to shift the flow of energy
toward the formation of ATP, diminishing
the amount of energy available for K+ transport. The latter is restored when all of the
ADP is esterified (see Fig. 4). Therefore, it
would seem that both ion transport processes
and at least part of the ATP-producing mechanism compete for the available energy.
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Acknowledgments
We are indebted to Drs. C. M. Mauritzen and
W. C. Starbuck for providing all histone fractions
used in this study. Valinomycin was generously provided by Dr. B. C. Pressman, Johnson Research
Foundation, University of Pennsylvania. Potassium
ion transport data were programmed and processed
by the Computational Research Center of Baylor
University of Medicine.'
9. HARRIS, E. J., COCKRELL,
11. RASMUSSEN, H., AND OGATA, E.: Cation flux
across the mitochondrial membrane as a possible pacemaker of tissue metabolism. In Control of Energy Metabolism, edited by B.
Chance, R. W. Estabrook, and J. R. Williamson. New York, Academic Press, 1965.
12. JOHNSON, C. L., SAFER, B., AND SCHWARTZ, A.:
Effect of histones and other polycations on
cellular energetics: IV. Further studies on
the stimulation of mitochondrial oxygen consumption. J. Biol. Chem. 241: 4513, 1966.
13. LEHNINGER, A. L.: Respiratory chain phosphorylation: experimental observations. In The
Mitochondrion. New York, W. A. Benjamin,
Inc., 1965.
14. VON KORFF, R. W.: Metabolic characteristics of
isolated rabbit heart mitochondria. J. Biol.
Chem. 240: 1351, 1965.
15. PLAUT, G. W. E., AND PLAUT, K. A.: Oxidative
metabolism of heart mitochondria. J. Biol.
Chem. 199: 141, 1952.
References
1.
WOLLENBERGER, A., K L E I T K E , B . , AND R A A B E ,
G.:
Some metabolic characteristics of mitochondria from chronically overloaded, hypertrophied hearts. Exptl. Mol. Pathol. 2: 251,
1963.
2. MEEBSON, F. Z.: Compensatory hyperfunction
of the heart. Circulation Res. 10: 250, 1962.
3. SCHWARTZ, A., AND LEE, K. S.: Study of heart
mitochondria and glycolytic metabolism in
experimentally induced cardiac failure. Circulation Res. 10: 321, 1962.
4.
CHANCE,
B.,
AND HOLLUNCER,
R., AND PRESSMAN,
B. C : Induced and spontaneous movements
of potassium ions into mitochondria. Biochem.
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10. RASMUSSEN, H.: Mitochondrial ion transport:
mechanism and physiological significance. Federation Proc. 25: 903, 1966.
G.:
16.
the histones of the Walker 256 carcinosarcoma
by combined chemical and chromatographic
techniques. J. Biol. Chem. 238: 918, 1963.
17. BUSCH,
18.
7. BARTLEY, W., AND DAVIES, R. E.: Active trans-
port of ions by subcellular particles. Biochem. J. 57: 37, 1954.
8. PRESSMAN, B. C.: Induced active transport of
ions in mitochondria. Proc. Natl. Acad. Sci.
U.S.A. 53: 1076, 1965.
C. M.:
Nuclear
SCHWARTZ, A., JOHNSON, C. L., AND STARBUCK,
W. C: Effect of histones and other polycations on cellular energetics: III. Swelling-contraction cycle of mitochondria. J. Biol. Chem.
241: 4505, 1966.
19. CHANCE, B., AND HACIHARA, B.: Direct spec-
troscopic measurements of interaction of components of the respiratory chain with ATP,
ADP, phosphate and uncoupling agents. In
Proceedings of the Fifth International Congress of Biochemistry, vol. 5, edited by E. C.
Slater. New York, Macmillan Co., 1963.
6. DIETHEH, N., AND HELGE, H.: Studies on nuc-
leotide incorporation into mitochondrial RNA.
Biochem. Biophys. Res. Commun. 4: 600,
1965.
H., AND MAURITZEN,
proteins. In Methods in Cancer Research, vol.
3, edited by H. Busch. New York, Academic
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General
properties and nature of the products of succinate-linked reduction of pyridine nucleotide. J. Biol. Chem. 236: 1534, 1961.
5. KALF, G. F.: Deoxyribonucleic acid in mitochondria and its role in protein synthesis.
Biochemistry 3: 1702, 1964.
HNILICA, L. S., AND BTJSCH, H.: Fractionation of
20.
PERSON, P., MORA, P. T., AND FINE, A. S.: Mac-
roion interactions involving cytochrome system components. J. Biol. Chem. 238: 4103,
1963.
21. BRUNI, A.: Mechanism of action of atractyloside.
In Regulation of Metabolic Processes in Mitochondria, edited by J. M. Tager, S. Papa, E.
Quagliariello, and E. C. Slater. Amsterdam,
Elsevier Publishing Company, 1966.
Circulation Research, Vol. XXI, July 1967
MITOCHONDRIAL K+ TRANSPORT
22. LARDY, H. A., JOHNSON, D., AND MCMURRAY,
31
chondrial electrolytes. Am. J. Physiol. 210:
W. C : Antibiotics as tools for metabolic
studies. Arch. Biochem. Biophys. 78: 587,
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25. CHAPPELL, J. B., AND CHOFTS, A. R.: Ion transport and reversible volume changes of iso-
23. COCKREIX, R., HARRIS, E., AND PRESSMAN, B.:
lated mitochondria. In Regulation of Metabol-
Energetics of potassium transport in mitochondria induced by valinomycin. Biochemistry 5: 2326, 1966.
ic Processes in Mitochondria, edited by J. M.
Tager, S. Papa, E. Quagliariello, and E. C.
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24. GAMBLE, J. L., JR., AND HESS, R. C , JR.: Mito-
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Circulation Research, Vol. XXI, July 1967
pany, 1966.
Classic Pages
No. 363(1. O C T . 14,
NAT
15W
resembles the protein of the yellow enzyme,
which when separated from its prosthetic group
is also rapidly inactivated at 3S° (Theorell'j.
Evidently in the intact tissue of the warm-blooded
iinimal (all experiments were performed on rabbit
miwcles), some conditions must exist which stabilize
t-he myosine against the jvction of temperature. A
marked stabilizing effect on the adenosinetriphosphatase activity seems to be produced by the adenylic
nucleotide itself. As can be seen from the accompanying graph, in the presence of adenosinetriphosphate
the liberation of phosphate proceeds at 37° over a
considerable period (Curves I, la and 16), whereas
the same myosine solution warmed alone to 37° for
10-15 min. shows on subsequent addition of adenosinetriphosphate an insignificant or no mineralization
whatever.
la
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i
i
Ib
p
_ _, -—
Myosine and Adenosinetriphosphatase
ORDINARY aqueous or potassium chloride extracts
of muscle exhibit but a slight capacity to mineralize
adenosinetriphosphate. Even this - slight liberation
of phosphate is mainly due, not to direct hydrolysis
of adenosinetriphosphate, but to a process of secondary, indirect mineralization, accompanying the trap*,
fer of phosphate from the adenylic system to creatine,
the corresponding enzymes (for which the name,
'phosphopherases' is suggested) being readily soluble.
In contrast to this lack of adenosinetriphosphatase
in the soluble fraction, a high adenosinetriphosphatase
activity is associated with the 'water-insoluble proteins
of muscle. This enzymatic activity is easily brought
into solution by all the buffer and concentrated salt
solutions usually employed for the extraction of
myosine. On precipitation of myosine from such
extracts, the adenosinetriphosphatase activity is
always found in the myosine fraction, whichever
mode of precipitation be used : dialysis, dilution,
cautious acidification, Baiting out. On repeated
reprecipitations of myoaine, the activity per mgm.
nitrogen attains a fairly constant level, unless denaturation of myosine takes place. Under the
conditions of our experiments (optimal conditions
have not been determined) the activity of myosine
preparations ranged in different experiments from
350 to 600 microgram phosphorus liberated per mgm.
nitrogen in 5 min. at 37°. Expressed as
ngm. P/3I x 22-4 N
mgm. N x 6-25 x hour/
this gives values of 500-850.
Acidification to pH below 4, which is known to
bring about the denaturation of myosine', rapidly
destroys the adenosinetriphosphatase activity. .Most
remarkable is the extreme thermolability of tho
adenoginetriphosphatase of muscle : the enzymntic
activity shown by myosine solutions is completely lost
after 10 min. exposure to 37". 'i'liis corresponds
with
the well-known thermolability of myosine1. In respect
of its high thermolability mlcnnsinetriphosplialitso
Qp(
:
/
in
£0
Time (min.)
::u
Crude buffer extracts accomplish a quantitative
hydrolysis of the labile phosphate groups of
ndenosinetriphosphate ; myosine, reprecipitated three
times, liberates but 50 per cent of the theoretical
amount of phosphorus (see figure). It acts as true
adenosine-(rt-phosphatase and- yields adenosinediphosphate, which is not further dephosphorylated
and has been isolated in substance. This may serve
as a convenient way of preparing adenosinediphosphate, instead of using crayfish muscle'. The
adenosinediphosphata.se is thus associated with the
more soluble proteins, occupying an intermediate
position between adenosinetriphosphatase and the
most readily soluble phosphopherases.
Under no conditions tested could we obtain a
separation of adenosinetriphosphatase from myosine.
Kit her the activity was found in the myosine prelipitate or else it was absent from the precipitates
and from the remaining solution. This disappearance
of the enzymatic activity we regard as the result of
l lie start of denaturation of the very unstable myosine.
We are led to conclude that the adenosinetriphoHphatase activity is to be ascribed to myosine or,
at least, to a protein very closely related to and at
present not distinguishable from myosine. Thus the
mineralization of adenosinetriphosphate, often reiirded aa the primary exothermic reaction in muscle
from W. A.
ENCELHARDT AND M. N. LJUBIMOWA
MYOSINE AND ADENOSINETBIPHOSPHATASE
NATURE, VOL. 144, PP. 668-669, 1939
Demonstration that the adenosinetriphosphatase (ATPase) activity of muscle is mainly associated with,
and not separable from, myosin itself.
Active Transport of Potassium Ion in Heart Mitochondria
BRIAN SAFER and ARNOLD SCHWARTZ
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Circ Res. 1967;21:25-31
doi: 10.1161/01.RES.21.1.25
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