Download Labeled Phosphate Distribution in Working and Nonworking

Labeled Phosphate Distribution in Working and
Nonworking Myocardium
Bi/ KENNETH K. TSUBOI, PH.D., NANCY M. BUCKLEY, M.D., AND
NORMAN J. ZEIG, M.D.
\T isolated dog heart preparation has been
. described,1 in which only the left ventricle performs external work. The coronary
system is maintained in such hearts with the
right ventricle performing no external work.
The heinodynamic determinants of the stroke
work of the left ventricle can be varied and
maintained at desired levels and over fixed
intervals. The continuous perfusion of both
ventricles by an adequate coronary blood supply and intimate distribution of added metabolites (including isotopically labeled substrates) throughout the myocardium is assured by maintaining the aortic pressure
above 70 mm. Hg.
The unique experimental advantages of a
single heart providing simultaneously both
"resting" (i.e., nonworking) and working
metabolic states have not been previously
duplicated in any biochemical investigation
into the work function of this organ. The results of an application of such preparations
will be described in the present report in
which the comparative metabolism of phosphate compounds in working and nonworking
myocardium was examined with the aid of
P a2 . Investigation of phosphate compounds
was prompted by the recognized obvious importance of certain members of this group
and a possible involvement of others in the
energy processes associated with the work
function of this organ. The intimate partiei-
A
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pation by nucleotides, creatine phosphate and
phosphorylated carbohydrates in muscle energetics is well recognized. On the other hand,
information concerning the role (if any) of
other tissue phosphates including the phospholipids and ribose nucleic acid (RNA) as
possible energy substrates is lacking. Investigation of phospholipid metabolism would appear to be especially relevant in view of the
demonstrated2'3 ready utilization of fatty
acids as an energy source by cardiac tissues.
RNA as a possible energy intermediate in
muscle metabolism has apparently not been
previously considered. An enzyme catalyzed
reaction leading to the synthesis of highly
polymerized ribonucleotide chains from micleoside diphosphates has been described.4
The reaction is currently viewed as a possible
mechanism of cellular RNA synthesis. The reaction has been found to be freely reversible5
and RNA by analogy could therefore conceivably represent a readily available biologic store of high-energy nucleoside polyphosphates.
Methods
Cardiac Preparation and its Perfusion
Left Ventricle Preparations
Six dog hearts prepared by procedures detailed
in a previous report0 were utilized for the present
series of studies. The perfusion circuit of the
functionally isolated left ventricle preparations
employed is illustrated dingrainmaticnlly in figure
1. The left ventricle (LV) was filled from either
of 2 inflow reservoirs: one (P) for perfusion
with the isotope-containing medium and the other
(W) with isotope-free medium. The hydrostatic
pressure available for diastolic filling was kept
constant, in either case, at a physiologic level.
The contracting left ventricle ejected blood into
the coronary circulation and an artificial peripheral
circuit which included an arterial resistance unit
From the Department of Pediatrics, Cornell University Medical College, and tlie Department of Physiology, Albert Einstein College of Medicine, New York,
N. Y.
Supported by grants from the PHS (No. H-3477),
Commonwealth Fund and the Life Insurance Medical
Research Fund (No. G-56-57).
Received for publication December 10, 3959.
Circulation Research, Volume VIII, July 1980
703
704
TSIJBOT, BUCKLEY, ZEIG
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Figure 1
Diagrammatic illustration of the external circuit
employed with the isolated left ventricle preparation (for details, consult text).
(A.R.) of the Starling type and an oxygenator
(Ox.) of the Kusserow type. Details concerning the
performance of the left ventricle perfused under
these conditions have been reported.1 The controllable hemodynamies assured a constant level
of left ventricular work and an optimal coronary
blood supply. In the present experiments, ventricular performance was evaluated from direct
measurements of intraventricular pressure and external work. Left ventricular pressure was registered by a Statham P23D transducer attached to
a short 16-gauge cannula in the ventricle and
recorded together with an electrocardiogram from
the screen of a Tektronix dual-trace oscilloscope.
Left ventricular work was calculated as the product of the collected output per minute and mean
arterial pressure. The latter was registered on a
damped mercury manometer. Right ventricular
external work remained at zero.
Perfusion Medium
Defibrinated whole dog blood or semisynthetie
media of low phosphate content were employed,
the latter in experiments of short duration where
a minimum dilution of added labeled phosphate
was desired in order to provide maximum tissue
incorporation of the isotope for analytical purposes. The composition of the various media is
shown in a footnote to table 1.
Isotope Perfusion
High specific activity P32-labeled Na 2 HP0 4 was
added directly to the circulating medium. Approximately 5 inc. of the isotope and between 200 and
300 ml. perfusion medium were used in all experiments.
h
Cardi ae ou tput
(ml./ min.l
Mean aorti
pressi re
(mm. Hg)
Work
(Gm.
70
72
60
63
85
60
Perfu
mediu
1
2
3
4
5
6
i
Heart
(Gm.
Heart
prep. no.
Table 1
Work Performance of Cardiac Preparations Employed
(A)
(B)
(B)
(A)
(A)
(G)
300
375
245
330
490
574
70
95
115
75
70
70
286t
484
383
2851
470
570
u
*(A) defibrinated whole blood; (B) washed erythrocytes in Kinger's containing 1-2 per cent PVP;
(C) washed erythrocytes in Feigen's medium" containing 1-2 per cent PVP. Dextrose (0.1 per cent)
and insulin (10 units/100 ml.) were also included in
all media. Synthetic salt media contained either 0.25
mg. per cent or no added NanPOi.
tDecreased to minimum at time of muscle sampling.
'Wash Procedure for Removal of Excess Isotope
Following circulation of the isotope for a desired period of time, excess isotope was washed
from the heart by changing to isotope-free medium
and continuing the perfusion for an additional
10 minutes. The wash procedure removed excess
isotope of extracellular origin, allowing for a
reliable measure of the S.A. (specific activity) of
the intracellular Pj (inorganic orthophosphate).
The level of isotope remaining in the circulatingfluid following isotope perfusion and subsequent
wash of a cardiac preparation is shown infigure2.
Extraction and Isolation of Phosphorylated
Components from Cardiac Muscle
Soluble Components
On termination of the perfusion procedure, muscle strips from the nonworking right and working
left ventricles were rapidly excised, weighed and
separately homogenized in 20 volumes of 60 per
cent acetone at room temperature. The aqueousacetone extraction was employed for the removal
of soluble small molecule phosphates (nucleotides,
sugar phosphates, etc.) rather than a conventional
perchloric or trichloracetic acid extraction, in
order to allow maximum preservation of labile
components. Nucleotide extraction with this procedure was only slightly less complete than that
obtained with cold perchloric acid.
Following removal of acetone in a flash evaporator, the nucleotides were separated free of other
Circulation Research, Volume VlIF, July i960
LABELED PHOSPHATE DISTRIBUTION
705
ATP
Developm
i,
GDP
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\Z/
GTP
10
PERFUSION TIME (MIN.)
20
Figure 2
Isotope perfusion and subsequent removal of excess isotope by further perfusion of a cardiac
preparation with isotope-free medium.
remaining soluble phosphates on norite A and
resolved by 2-diniensional paper chromatography,
yielding- products of high radioehemical purity.
The result of a typical fnietionation is illustrated
in the ra.dioautogrn.ph reproduced in figure 3. Details concerning the isolation technic and identification of individual components have been presented in an earlier report.7 A further fractionation
sequence was recently developed8 for the localization of labeled P in nucleoside polyphosphates
and has been applied in the present studies for
the determination of isotope distribution in cardiac
AMP, ADP and ATP. The fractionation is illustrated in the paired radioautographs reproduced
in figure 4. Following isolation of AMP, ADP
and ATP by an initial development in solvent 1,
the ADP and ATP were next partially degraded
in situ with acid and heat,8 to yield some AMP
from the ADP deposit and both AMP and ADP
from the ATP deposit. The resulting mixtures
were resolved by a second development (fig. 4
left). A further third development was required
(fig. 4 right) to remove derived Pj, which appears as a contaminant of the ADP deposit. The
S.A. of each of the individual P groups can now
be obtained from measurements on the isolated
radio-chemically pure products by procedures which
have been detailed in a pi'evious report.7 Radioehemical purity is established for each deposit by
in situ technics detailed in a later section.
Fractionation of the remaining soluble phosphates, following nueleotide removal on norite A,
Circulation Research, Volume VIII, July 1060
;
UOP
CD'
UDPAG
UOPG-*UMP
UTP
2nd Developmeni l+Xl
Figure 3
liadioautograph of P-1f-labeled dog cardiac nucleotides from heart preparation 5 resolved by 2dimensional paper chromatography. Broken Hues
represent compounds located by virtue of radioactivity only; solid lines, those com pounds located
in addition by absorption of 25-1-m/j, light. The
first development was for 20 hours in solvent 1
and the second for 56 hours in solvent 5.
involved a preliminary separation at pH S to 9
of calcium insoluble salts (P, and some fructose
diphosphate). Practically all of the remaining
phosphates were next isolated as barium salts in
the presence of 4 volumes of ethanol and following barium removal, resolved by 2-dimensional
paper chromatography. A typical fraetionation is
illustrated in the radioautograph reproduced in
figure 5. Specific details concerning the isolation
and identification of the various components will
be published elsewhere.
Phospholipids
The cardiac residue remaining after initial
extraction for soluble phosphates was next extracted 3 times with 10 volumes of acetone and
the extracts discarded. The residue was then
extracted with 10 volumes of a 2:1 chloroformmethanol mixture. The resulting phospholipid containing extract was concentrated 20-fold, and
thoroughly washed by repeated shaking with dilute
hydrochloric acid and finally dialyzed. A further
isolation of a-glycerophosphate from the phospholipid fraction was accomplished by the following
sequence: hydrolysis in 6X hydrochloric acid for
1 hour at 100 C, precipitation of the liberated
glycerophosphate as the barium salt with ethanol
706
TSUBOI, BUCKLEY, ZEIU
o
AMP-:
I
I
mm
o
•§'
S 6-P
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Tigure 4
Fmrtionation sequence for the localization and
measure of labeled P in individual phosphoryl
groups of adenine nucleotides. Adenine nucleotides were isolated from an initial complex mixture
of dog cardiac nucleotides (preparation 5) by
initial development for 20 hours in solvent 1.
A DP and ATP deposits were sprayed with dilute
HCl (.01 N in 90 per cent EtOH) and heated
(16 hours at 80 C). The resulting products were
resolved by a second development for 72 hours in
solvent, 5 (left). A third development for 18 hours
in solvent 3 ivas required to remove contaminating
Pi (right).
and final isolation in pure form by ehromatography in paper, using solvent 5 (for solvent composition, see later section).
UNA Nnclootides
Isolation of RNA from the cardiac residue
following phospholipid removal was essentially as
described in an earlier report.9 The residue was
dried and extracted twice with 5 times the original
tissue volume of 10 per cent N a d for 30 minute
periods at 100 C. To the combined extracts were
added 3 volumes of 95 per cent ethanol and the
precipitated nucleic acids removed after overnight
standing at -20 C. The washed nucleic acids were
dissolved and treated with norite A to insure
complete removal of possible contaminating free
nucleotides,10 then hydrolyzed for 45 minutes at
80 C. in 0.1N NaOH. RNA nucleotides (present
as 2' and 3' phosphate mixed isomers) resulting
from the alkaline hydrolysis were removed from
the digest on norite," following preliminary acidification and removal of insoluble DNA by centrifugation. The norite adsorbed nucleotides were
eluted7 and chromatographed on paper in 2
dimensions, using first solvent 1 then 3 (for
solvent compositions, see later section) yielding
isolated deposits of each in high radiochemical
purity (not illustrated). Details concerning the
0
2nd
Dtv.
O
(+X)
Figure 5
Radioautograph of PS2-labeled micleotide soluble phosphorylated components from dog heart
preparation 4 following resolution by 2-dimensional
paper chromatography. First development was for
48 hours in solvent 5j second development for
24 hours in solvent 1.
isolation and evaluation of the procedures will be
presented in conjunction with other work to be
published elsewhere.
Isolation, Detection and Measure of Labeled Cardiac
Phosphates Resolved by Paper Chromatography
Cliromatography
Final resolution of the numerous phosphorylated
components obtained from the cardiac tissues was
achieved in all cases (with the exception of phospholipids) by paper chromatographie technics.
The composition of the 3 solvent systems employed
in the separations were as follows: solvent 1:
66 vol. isobutyric acid, 33 water, and 1.5 cone.
XH 4 0H; 1 1 solvent 3: 60 vol. isopropyl alcohol,
30 glacial acetic acid, and 10 water;12 solvent 5:
60 vol. n-propanol, 30 cone. NH 4 0H, and 10
water.13 Whatman 3MM paper sheets of 18 X
22 inches, washed in 0.1N acetic acid, were employed for the chromatography. Chromatograms
were developed by descending technic in stainless
steel chromatocabs of appropriate dimension.
Detection and Measure
Ultraviolet fluoroscopes were prepared" for
visualization in chromatograms of purine and
pyrimidine containing deposits absorbing 254-m/x
light emitted from a Mineralight lamp. Deposits
containing labeled phosphate were visualized by
radioautography (Kodak, No-Screen x-ray film,
14 X 17 inches). Nonlabeled or weakly labeled
(e.g., a-glyeerophosphate from phospholipid), nonCirculation Research, Volume VU1, July I960
LABELED PHOSPHATE DISTEIBUTION
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nueleotide phosphate deposits were localized by
molybdate reduction.14 Quantitative measurements
of amount and radioactivity appearing in nueleotide deposits were curried out by procedures designed to provide explicit control of radiochemical
purity of the isolated products,7 involving a combination of technics including serial in situ measurements across deposits as well as conventional
measurements on the eluted material. The in situ
measurements were carried out with an ultraviolet
photon counter designed for chromatographic densitometry at 254-m/xlr> and a beta counter for
radioactivity, respectively. Measurements of nucleotides in solution, following elution of deposits
in 2 per cent NH 4 HC0 3 , were made with a Beckman DU ultraviolet spectrophotometer. Radioactivity was determined on aliquots of the eluted
nueleotide solutions.
Non-nueleotide phosphates following localization
on the chromatogram primarily by radioautography were measured by the following procedures :
Each localized deposit was cut out and the paper
weighed. Adjacent blank areas of the paper of
only approximately equivalent area were also taken
and weighed. The cut-out paper areas were transferred to heavy wall pyrex tubes and dry ashed
overnight at 400 to 500 C. The resulting minute
ashed residues were hydrolyzed in dilute acid and
aliquots removed for counting and phosphate determinations.10 Appropriate paper blank corrections (corrected on the basis of amount of molybdate reducing material contributed per unit weight
of paper) were essential to obtaining reliable
results.
Results and Discussion
Work Performance of Cardiac Preparations
Pertinent physiologic data of each preparation employed in these studies are summarized
in table 1. Cardiac output (ml./min.) was
calculated as the aortic outflow plus coronary
drainage. Mean aortic pressure was calculated
as the weighted average of systolic and diastolic pressures. Work per minute (Gm.M/
min.) was calculated as pressure-volume work,
the product, of cardiac output and mean
aortic pressure. A typical record of left ventricular pressure and electrocardiogram is
shown in figure 6. In the 2 hearts in which
work per minute decreased by the time of
sampling, the left ventricular pressure curve
then became characteristic of a hypodynamic
ventricle. The work performance of the left
ventricle and the analysis of intraveutricular
Circulation Research, Volume VIII, July 1900
707
LVP
(mm. Hg.)
120-iH
*)
h-2OO msec.
Figure 6
Left ventricular pressure (LVP) and electrocardiograph records from heart #3 (see table 1).
pressure curves is comparable to those reported previously for a larger group of dog
hearts.1 Iu every heart, right ventricular
work was zero.
Nucleotides in Working and Nonworking
Myocardium
Composition Studies
Nueleotide components appearing in extracts of dog myocardium have not, apparently, been previously thoroughly resolved. Resolution of dog cardiac nucleotides has been
accomplished by 2-dimensional paper chromatography and the results illustrated in the
radioautograph reproduced in figure 3. A preliminary examination of uucleotides in working and nonworking myocardium included
comparative analyses with respect to composition. The results of a representative set of
analyses have been summarized in table 2. In
addition to nucleotides, the analyses included
all other small-molecule purine and pyrimidine components appearing in the extracts.
From the results, it is apparent that only
minor differences in composition can be demonstrated with respect to these constituents.
Phoxphate-P" Incorporation Studies
Further comparative analyses of nucleotides in working and nonworking myocardium
included an investigation of the rate of phosphate renewal in adenine nucleotides using
P32-labeled phosphate. In these experiments,
left ventricles were subjected to various periods of work with the corresponding right ventricles serving as nonworking controls. Details concerning the administration of the isotope have been previously presented. The results of these studies have been summarized
in table 3. Differences in labeled phosphate incorporation could not be demonstrated in any
TSUBOI, BUCKLEY, ZBIG
708
Table 2
Nucleoticle and Derivatives in Working and Nonworking Dog Myocardium"
Compound
AMP
ADP
ATP
DPN
Inosine
Uridine
Hypoxantliine
Uridine +
As fraction of total concentration
Non-working (RV)
Working (LV)
1.8
2.7
18.6
65.0
20.3
62.1
5.8
1.0
1.1
6.0
1.2
0.9
tnice
trace
6.0
6.8
guanosinc
polyphosphates
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•Heart preparation no. 2 (see table 1) was employed for analyses. Components were resolved by 2-dimensioiiiil paper cliromatograpliy, with the exception
of the uridine and guanosine polyphosphates. These
latter compounds were resolved free of most other
niicleotkk's after a single development in solvent 1
and were subsequently measured as a mixture.
of the P groups of adenine nueleotides from
working as compared with nonworking myocardium following either very short (3 minutes), or long periods (40 minutes) of induced
cardiac work. It had been an initial objective
of these experiments to attempt a quantitative
correlation between cardiac work and highenergy phosphate turnover. It is obvious from
the results that a measure of phosphate turnover was not achieved in these experiments.
The extent of isotope incorporation in the
rapidly exchanged high-energy phosphate
groups is unfortunately limited in cardiac
muscle (as in striated muscle18) by a prior
rate-limiting step involving transfer of the
perfused isotope across the cell membrane.
Isotope on entry into the cell, by whatever
mechanism (see later), presumably equilibrates between the Pi and high-energy phosphate pools by reactions of considerably
greater rate. In this respect, isotope equilibration among the high-energy phosphate
groups of ADP and ATP occurred within the
shortest measured time interval (3 minutes).
The apparent lack of equilibration within this
period between the high-energy phosphates
and tissue Pi was attributed to the contaminating presence of metabolically inactive high-
specific activity. Pi of extracellular origin retained in the preparation due to the omission
of the wash procedure.
In view of the above considerations, investigations relating to nucleotide high-energy
phosphate turnover in cardiac tissue using
phosphate-P32 as a tracer cannot be considered
valid. In this respect, a recent report10 purporting to demonstrate differences in cardiac
ATP turnover between young and old rats on
the basis of P 32 incorporation rates cannot be
accepted. The data in fact appear to support
an altered rate of isotope passage into cardiac
cells of older rats. Further, a study has been
described20 in which an increased rate of phosphate-P82 incorporation into cardiac nucleotide high-energy phosphate groups has been
attributed to an increased turnover following
induced cardiac work associated with muscular exercise. These conclusions similarly cannot be accepted. In view of the limited samples taken for analyses in these studies (about
30 ing. obtained by biopsy) and the inadequate
fractionation technics necessarily employed,
the radiochemical purity of the measured
products would appear to be questionable.
Cardiac Non-nucleotide Soluble Phosphates
Composition
The remaining non-nucleotide phosphates
appearing in aqueous-acetone extracts of dog
myocardium have not been previously systematically examined. Fractionation of this
group of compounds Avas carried out, by 2dimensional paper chromatography. Resolution of components by these technics is illustrated in the radioautograph reproduced in
figure 5. The compound labeled K is a ketohexo.se which has not been further characterized. An unexpected finding was the presence
of a-glycerophosphate as a major component
in cardiac extracts. Details concerning the isolation and identification of the separated compounds are to be published elsewhere.
lJhosphate-P" Incorporation Studies
The rate of labeled phosphate incorporation
into the non-nucleotide soluble phospliateswas
examined as a function of cardiac work. Comparative isotope analyses on isolated compoCirculation Research, Voluvic VIII, July 1960
LABELED PHOSPHATE DLSTKIBUTIOX
Labeled 1' Distribution
Myocardium
Duration of
perfusion of isotope*
1. 3 minutes
(no wash)
AMP
ADP
ATP
10 minutes
(10-minutc wash )
AMP
ADP
ATP
a. 4.0 minutes
(10-minutc wash)
Table 3
Nucleotides of Working
in Adcninr
Isolated
adenine
nucleotide
709
and Nonworking
Dog
S.A. of P groups relative to p . t
Working (LV)
P.
PPi
nucleotide
Nonworking (RV)
Pl
nucleotide
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<0.01
<0.01
<0.01
0.37
0.37
<0.01
0.98
1.93
<0.01
<0.01
<0.01
0.98
0.95
0.01
0.97
1.98
0.01
0.01
0.01
0.97
0.99
AMP
ADP
ATP
—
0.40
<0.01
0.42
0.79
<0.01
<0.01
<0.01
0.42
0.37
0.98
<0.01
0.97
1.89
<0.01
<0.01
<0.0.1
0.97
0.94
0.92
0.01
0.96
2.01
0.01
0.01
0.01
—
<0.01
o.:i7
0.77
__
0.42
—
0.95
0.98
3.0.1.
0.97
*Cardiae preparations 3, 2 and 1 (see table 1) were employed for the 3-, 10- and 40-niinute
isotope perfusion experiments, respectively.
tTho summarized results represent averages obtained from at least duplicate determinations
and have been computed relative to the S.A. of Pi. P 1 refers to the carbon-bound P, P" to
tlie terminal (ADP) or middle P (ATP), and P" to the terminal P of ATP.
nents prepared from working left ventricles
and nonworking right ventricles failed to reveal differences in labeling rates which could
be attributed to the imposed work (for a representative experiment, see table 4). Differences in isotope incorporation which were encountered could be correlated with a corresponding inequality in distribution of the perfused labeled phosphate between the working and nonworking ventricles. Questions relating to the effect of contraction and work
on the distribution and transport, of the isofope in muscle tissue will not be considered in
this report.
Although information relevant to work
function was not obtained, the results summarized in table 4 provide evidence for a
phosphorylation mechanism (rather than a
simple diffusion mechanism) in the transport
of externally perfused labeled phosphate across
the cell membrane. The results suggested a
phosphorylation of hexose as an intermediate
reaction (e.g., glycogeu + P ( •* glucose-1-phosphate) in the entry of phosphate into the
cell. This conclusion is based on the consistent
finding that the hexose phosphate fractions
contained isotope in excess of that present
in the rapidly exchanged high-energy phosCirculation Research, Volume VIII, July 1960
phate groups of: creatine phosphate, ATP
and ADP. If we assume a near constant
isotope equilibrium between the intracellular Pi pool and high-energy phosphate groups
(for which indirect evidence has been previously presented), Pi cannot possibly represent the entering unit. A direct measure of
the isotope content of the intracellular P,
pool cannot, unfortunately, be obtained without prior removal of isotope of extracellular
origin. The wash procedure has been avoided
in these studies in order to preserve the existing isotope distribution pattern among the
various measured constituents. Further evidence relating to the postulated transport
mechanism has been obtained and will be
presented as a separate report.
Phospholipid Metabolism in Working and
Nonworking Myocardium
J'lioxphate-P" Incorporation Studies
In the event that phospholipids participate
in the recognized ready utilization of fatty
acids as an energy source in cardiac muscle,2' 8
it was considered relevant to examine the metabolism of this group of compounds as a
function of cardiac work using phosphate-P32
as a tracer. Incorporation of labeled phosphate into phospholipid components in a work-
TSUBOI, BUCKLEY, ZEIG
710
Table 4
Labeled P Incorporation in Soluble Organic Phosphates of Working and Nonworking Myocardium"
Isolated component!
Specific activity (c.p.m./m/imole)
Nonworking
Working
(LV)
(RV)
a-Glycerophosphate
Glucose-1-phosphato 4fructose-6-phosphsite
Glucose-6-phosphate
Ketohexose phosphate (K)
Fructose-l-6-diphosphate
Creatiiic phosphate
(ADP - P 2 )
(P.)t
77
75
118
110
97
106
83
112
102
95
97
84
(86)
(145)
(80)
(131)
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'Heart preparation 4 (see table 1) was employed
for analyses. Isotope perfusion lasted 25 minutes with
no subsequent wash.
tlsolation of components was by 2-dimensional
paper chroinatography (see fig. 5 for illustration of
separated components).
tPi fractions are contaminated by isotope of extracellular origin, due to omission of wash procedure in
this experiment.
ing left and nonworking right ventricle was
therefore examined following isotope perfusion of an isolated dog heart preparation. The
results of such a study have been summarized
in table 5. Labeled phosphate incorporation
into cardiac phospholipids differs little in
either the working or nonworking myocardium and proceeds at considerably lesser rates
than in nucleotide high-energy phosphate
groups or glycolytic intermediates. These results suggest either a lack of participation of
phospholipids in the energy metabolism of
cardiac tissue or the more likely alternative
that phosphate turnover is an inadequate
measure of an involvement restricted to the
nontagged fatty acid residues.
Phospholipid
cc-Glycerophosphate
An additional glycerophosphate containing
phospholipid, cardiolipin,21 has been found in
cardiac tissue. A further isolation of the glycerophosphate components of the phospholipid fractions was carried out for specific
examination. The isotope content of these
residues was determined following isolation
from acid digests. The glycerophosphate obtained by acid hydrolysis was a mixture of
Table 5
Effect of Work on Labeled P Incorporation into
Cardiac
Phospholipid^
Isolated
compound
Phospholipid
a-G-P
(P.)
Nonworking (RV)
S.A.
/imole/ (c.p.m./
Gm.
/imole)
15.0
—
—
1,300
2,280
300,000
Working (LV)
S.A.
ymole/ (c.p.m./
Gm.
jimole)
14.5
1,000
—
1,420
—
260,000
"Heart preparation o (sec table 1) was employed
for analyses. Isotope perfusion was for 40 minutes
followed by a 15-minute wash. For details concerning the isolation of components, see "Procedures."
both a- and /?-isomers with the former predominating. These results did not differ significantly from those obtained with the intact
phospholipid fractions.
Isolation of the a-glycerophosphate component was prompted in view of the limited
information available on the metabolism of
cardiolipin. In addition, it was considered of
interest to investigate a possible metabolic
relationship between the free a-glycerophosphate described earlier in cardiac extracts
with that combined as phospholipid. In view
of the finding of considerably less isotope in
the lipid bound a-glycerophosphate, it is apparent that this cannot be the source of the
soluble, free compound. The relative isotope
content of the free a-glycerophosphate is consistent with a metabolic relationship associated
with the glycolytic pathway.
Metabolism of ENA as a Function of Cardiac Work
Phosphate-P" Studies
The possibility of RNA participation in the
energetics of cardiac tissue was examined
using phosphate-P32 incorporation as a measure of relative turnover of individual nucleotide components. RNA as a possible intermediate in nucleotide high-energy phosphate
production or utilization in the course of
cardiac work might be expected to incorporate
labeled nucleotides at rates reflecting the work
load imposed. Evidence has been recently obtained for the in vivo incorporation of acidsoluble nucleoside 5'-mouophosphate units
(presumably as residues of the corresponding
Circulation Research, Volume VIII, July I960
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LABELED PHOSPHATE DISTRIBUTION
711
di- or tri-phosphates) into mammalian liver
RNA (to be published). Isotope present in
the carbon-bound P groups of the acid-soluble
nucleotides, therefore, represents the entering
label into the RNA molecule. Maximum incorporation of isotope into RNA cannot consequently exceed the level of incorporation in
the carbon-bound P groups of the acid-soluble
nucleotide pools.
The results of an experiment of phosphateP 32 incorporation into RNA nucleotides in
working and nonworking myocardium have
been summarized in table 6. The nucleotide
composition of RNA from the working left
and nonworking right ventricles has also been
presented. The results indicate little significant difference in composition or isotope incorporation in the RNA of working as compared with nonworking myocardium. These
results have been presented to document an
apparent lack of participation of RNA in the
work function of cardiac muscle.
Table 6
Effect of Work on Labeled P Incorporation into
Cardiac BNA*
Summary
Investigation of phosphate metabolism with
the aid of labeled phosphorus has been carried
out, using isolated heart preparations in
which only the left ventricles perform external work. The corresponding right ventricles in such preparations perform no external work with the unique experimental
advantage of a single heart, providing simultaneously both working and "resting" (nonworking) metabolic states.
Comparative incorporation of labeled phosphorus into nucleotide phosphate groups, nonnucleotide soluble phosphorylated components, phospholipids and ribose nucleic acid
was examined as a function of cardiac work.
Incorporation of the label into the rapidly
exchanged high-energy phosphate groups was
found to be restricted by a prior rate—limiting entry of the externally perfused isotope
into the cells, thereby precluding a measure
of turnover of these groups by this technic.
Differences could not be demonstrated in isotope incorporation in either the phospholipids
or RNA of working as compared to nonworking myocardium. It was concluded from
Circulation Research, Volume VIII, July I960
w a.
S.A. (c.p.m./
£
a
Working (LV)
Cone. rel.
to AMP
Cone. rel.
to AMP
Nucleotidet
Nonworking (RV)
AMP (2', 3')
GMP (2', 3')
CMP (2', 3')
—
560
—
360
1.06
480
1.12
380
1.28
470
1.43
380
UPM (2', 3')
0.70
790
0.77
700
—
300,000
—
260,000
(P.)
"Cardiac preparation no. 5 (see table 1) was employed for the RNA analyses. Isotope perfusion was
for 40 minutes, followed by a 15-minute wash.
tFor details concerning the isolation and specific
activity measure of RNA nucleotides, sec "Procedures. ' '
these results that RNA is clearly not involved
in energy reactions associated with cardiac
work function. Phospholipid participation,
on the other hand, could be clearly excluded
only insofar as the phosphate moiety of these
compounds is concerned.
Information relevant to a mechanism of
phosphate entry into cardiac cells was obtained in which a phosphorylation of hexose
appeared to be involved. The composition of
cardiac tissue with respect to nucleotide and
non-nucleotide soluble components was examined in working and nonworking myocardium. Differences in composition were not:
found with respect to these constituents. A
major component appearing in extracts of
cardiac tissue was found and subsequently
identified to be a-glycerophosphate. On the
basis of isotope incorporation data, it was concluded that its metabolic source was of carbohydrate rather than phospholipid origin. •
Summario in Interlingua
Con le adjuta de marcation a phosphoro radioactive,
le metabolismo de phosphato esseva investigate in
cordes isolate preparate de maniora que solmente le
ventriculos sinistre es externemente active. In till
preparatos, le correspondente ventriculo dextcrc es
TSUBOI, BUCKLEY, ZEIG
712
Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
externemente inactive. Isto resulta in le exquisite
avantage experimental quo le niesnie eorde provide
siniultaiieeniente active e inactive (i.e. "reposante")
statos metabolic.
Le incorporation del phosphoro radioactive in
gruppos de phospliato nucleotidic, in solubile componentes phospliorj'lnte non-nuclcotidie, in phospholipidos, e in acido ribonucleic esseva studiate comparntivemente como function del labor cardiac. Le
incorporation del marca in le rapidemente excambiate
gruppos phospliatic a alte energia se revelava como
rcstringite per le effecto inliibitori del previe entrata
del extornemente perfusionate isotopo a in le cellula.
Isto rendeva iinpossibile lo determination del rhythmo
metabolic do iste gruppos per medio del presentc
technica. Nullc differentias potova esser demonstrate
in le incorporation del isotopo in phospliolipidos o
in acido ribonucleic in le myocardio active in comparation con le myocardio in roposo. Super le base
de iste resultatos il esseva concludite quo acido ribonucleic es nottemente oxempte ab oiniic participation
in le reactiones do energia quo os associate con le
function de travalio del parte del corde. Del altero
latore, lc participation do phospliolipidos poteva esscr
excludite nettemente solmentc con rospecto al componente phospliatic dc isto coinpositos.
Infornintioncs do interessc eon rospecto al inechanismo del entrata de phosphato in le cellulas cardiac
esseva obtenite, suggerento que un phosphorylation
de liexosa occurre in le processo. Lo composition de
tissu cardiac con respecto a solubile componentes
nucleotidic o non-nucleotidic esseva examinate pro le
myocardio active e reposante. Nulle difforentias do
composition esseva constatate con respecto al constituontes nientionate. Essevn trovato un componente
major in extractos do tissu cardiac, le qual—subsequentementc—essova identificate como glycerophosphato alpha. Super lo base del datos relative al incorporation del isotopo, le conclusion esseva formulate
que le origine metabolic de i 1 le componente esseva
in liydratos de carbon plus tosto quo in phospliolipidos.
ties of a polynucleotide from adenosine polyphasphate. Nature 177: 790, 1956.
(i.
lateral ventricular failure in the isolated dog
heart. Am. J. Physiol. 197: 247, 1959.
7. TSUBOI, K. K., AND PRICE, T. D.: Isolation, de-
tection and measure of mierogram quantities
of labeled tissue nucleotides. Arch. Biochem.
81: 223, 1959.
8. —•: Labeled phosphorus distribution in nucleoside polyphosphatos: Adeniue nuelootides of
rat tissues. Arch. Biochem. 83: 445, 1959.
9. —, AND STOWELL, R. E.: Mouse liver nucleic
acids. I. Isolation and chemical characterization. Biochim. et biophys. acta 6: 192, 3950.
10. HERHERT, EDWARD:
Incorporation
of
adenine
nucleotides into ribonucleic acid of cell-free systems from liver. J. Biol. Chem. 231: 975, 1958.
11. MAGASANIK, B., YISCHER, E., DONINGER, R.,
ELSON, D., AND CHARGAFF, E.: Separation and
estimation of ribonuelcotides in minute quantities. J. Biol. Chem. 186: 37, 1950.
.12. MONTREUIL, J.,
AND BOULANGER, P.:
Analyse
Biochomiquo. Chroma tographie Quantitative
sur Papier des Ribonucleotidcs. Compt. rend.
231: 247, 1950.
13. HANES, C. S., AND ISHERWOOD, !F. A.: Separation
of the phosphoric esters on the filter paper
chromatogram. Nature 164: 1107, 1949.
J4.
BANDURSKI, R. S., AND AXELROD, B.: Chroma-
tographio identification of some biologically important phosphate esters. J. Biol. Chem. 193:
405, 1952.
:15. PRICE, T. D., AND HUDSON, P.
B.:
Specific
activity determination with beta and TJV-photon counter. Nucleonics 13: 54, 1955.
36. FISKE, C. H., AND SUBBAROW, Y.: Colorimetric
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J. Biol. Chem.
.17. FEIGEN, G. A., MATSUOKA, D. T., THIENES, C. H.,
SAUNDERS, P. R., AND SUTHERLAND, G. B.:
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Circulation Research, Volume VIII, July 1960
Labeled Phosphate Distribution in Working and Nonworking Myocardium
KENNETH K. TSUBOI, NANCY M. BUCKLEY and NORMAN J. ZEIG
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Circ Res. 1960;8:703-712
doi: 10.1161/01.RES.8.4.703
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