(Received for publication, November 2, 1994; and in revised form, January 9, 1995)
From the
We previously suggested that an importance of adenylate kinase
(AdK) in skeletal muscle is to function as a high energy phosphoryl
transfer system regulating ATP generation in correspondence with its
consumption by specific cellular processes. The present experiments are
intended to define the ATP-generating system coupled to and regulated
by AdK-catalyzed phosphotransfer in skeletal muscle and also to examine
the relationship between AdK- and creatine kinase (CK)-catalyzed
phosphotransfer. Rates of phosphoryl transfer catalyzed by AdK were
assessed in intact, isolated rat diaphragm by determining rates of AMP
phosphorylation with endogenously generated
[-
O]ATP under conditions of altered
anaerobic and aerobic ATP production. AdK-catalyzed phosphoryl transfer
rates accelerated incrementally up to 12-fold in direct proportion to
stimulated contractile frequency in parallel with equivalent increases
in rates of ATP generation by lactate producing glycolysis.
Stoichiometric equivalent increases of AdK-catalyzed phosphotransfer
and anaerobic ATP production also occurred up to more than 20-fold when
oxidative phosphorylation was impaired by either O
deprivation or treatment with KCN or p-(trifluoromethoxy)-phenylhydrazone. These enhanced rates of
AMP phosphorylation were balanced by virtually identically increased
rates of AdK-catalyzed generation of AMP. This AMP was traced to arise
from AdK-catalyzed phosphotransfer involving ADP generated by a muscle
ATPase. Increased AdK-catalyzed phosphotransfer paired with the
apparent compensatory increase in ATP generation by anaerobic
glycolysis in oxygen-deprived muscle occurred coincident with
diminished rates of CK-catalyzed phosphoryl transfer indicative of a
pairing between oxidatively produced ATP and CK-catalyzed
phosphotransfer. A metabolic model consistent with these results and
conforming to the Mitchell general principle of vectorial ligand
conduction is suggested.
We have recently obtained the first information about the
kinetics and subcellular compartmental properties of AdK ()in the intact muscle cell(1, 2) . The
characteristics of AdK behavior in the intact cell provides a much
different perspective from which to define its biological importance
than has been possible from several decades of assessing its catalytic
properties by conventional cell-free analyses(3) . In the
intact muscle cells comprising the rat diaphragm, the net AdK-catalyzed
transfer of phosphoryls was found to be restricted to a limited number
of discrete subcellular compartments that increase in size and/or
number with increasing frequency of contractions(1) . This
increased rate of AdK-catalyzed phosphotransfer, associated with muscle
contraction, occurred with minimal or no measurable change in the
steady state levels of AMP, ADP, and ATP. The greatly restricted
velocity of AdK in the intact muscle compared to its much greater
activity measurable by cell-free analysis indicates that AdK-catalyzed
phosphotransfer is not merely involved in equilibrating cellular
adenine nucleotides according to the traditional view of its metabolic
importance. If contraction-associated bursts of AdK-catalyzed
phosphotransfer were only for this purpose, increases in AMP
concentration should have accompanied each contraction and most likely
would have been detected following the almost 1000 contractions that
occur during 4 min of contraction at 4 Hz. Instead of an accumulation
of AMP, the most prominent metabolic characteristic of AdK-catalyzed
phosphotransfer associated with contraction was the apparently very
rapid phosphorylation of AMP. To measure rates of AdK-catalyzed
phosphorylation of AMP in intact muscle, a procedure of
O-phosphoryl labeling was used that detects only net AMP
phosphorylation by anabolically generated ATP; it does not detect
reversals of AdK catalysis whereby ATP formed in the forward reaction
serves as the phosphoryl donor in the reverse reaction. Therefore, this
previously undetected AMP metabolic flux appeared to represent a
functional link with characteristic stoichiometry between contractile
and metabolic processes.
These results suggested a model of metabolic regulation (1) that provides a function for AdK, similar to that suggested by Bessman and Carpenter (4) and also by Dzeja et al. (5), of transferring high energy phosphoryls from a metabolic process generating ATP to cellular components consuming ATP in functionally definable subcellular locales. Also suggested from preliminary observations not documented experimentally in our previous report (1) was that in rat diaphragm muscle the source of the newly synthesized ATP involved in the AdK-catalyzed phosphotransfer derives from glycolysis resulting in lactate formation.
The results described here provide experimental evidence that in rat diaphragm a major function of AdK is to catalyze the transfer of high energy phosphoryls generated by anaerobic glycolysis in close correspondence with ADP produced by a contraction-related event. They also show an interrelationship between AdK- and creatine kinase (CK)-catalyzed phosphotransfer which is manifest as reciprocal changes in rate when oxidative ATP production is impaired. Oxygen deprivation resulted in a decrease in phosphotransfer catalyzed by CK and a shift to greater rates of phosphotransfer catalyzed by AdK paralleling compensatory increases in anaerobic glycolytic flux. A model of metabolic regulation consistent with these experimental results is presented whereby AdK-catalyzed transfer of nucleotide phosphoryls can rapidly regulate and coordinate the rate of ATP generation by anaerobic glycolysis with the rate of ATP utilization by specific cellular events supported by ATP deriving from this glycolytic process. It also shows an interrelated coupling of the AdK and CK phosphotransfer systems to anaerobic glycolytic and oxidative ATP production, respectively.
Figure 2:
Kinetics of O-labeled
phosphoryl appearance in
-ATP,
-ATP, and
-ADP in
non-contracting rat diaphragm incubated in oxygen- or
nitrogen-equilibrated medium. Diaphragm muscles were preincubated for
10 min in [
O]water-containing medium that was
equilibrated by continuous bubbling with 95% O
, 5% CO
or 95% N
, 5% CO
. The tissues were then
transferred to [
O]water-containing medium
equilibrated with the same gas mixture and incubated for 1, 2, or 8
min. The percentage (not computer-modeled estimates of velocities) of
-ATP,
-ATP, and
-ADP containing 1-3 atoms of
O is shown. The data are corrected for small variations in
the atom percent enrichment of [
O]water in the
medium of different incubations. This correction also takes into
account the theoretical maximum abundance of each
[
O]phosphoryl species (i.e.
O
,
O
,
O
) so that the maximum value for the ordinate is 100%. Each value represents the mean of triplicate
samples that varied by less than 7%. This is a representative
experiment of three that yielded virtually identical
results.
Adenylate kinase-catalyzed phosphoryl transfer proceeds at an
average rate of 2.1 ± 0.7 nmol mg protein
min
(n = 4) in
non-contracting rat diaphragm muscle (Fig. 1A). In
muscle electrically stimulated to contract, AdK catalysis was
previously found (1) to accelerate with increasing contractile
frequency. Shown in Fig. 1A are the results from a
representative experiment in which rat diaphragms were incubated for 4
min in [
O]water-enriched medium without
stimulation to allow a relatively large proportion (
80%) of the
-ATP to undergo labeling with
O while minimal
labeling of
-ADP and
-ATP occurred. Muscles were then
incubated for an additional 2 or 4 min without or with stimulation (1,
2, or 4 Hz). Shown is the rate of AdK-catalyzed phosphorylation of AMP
occurring during the second 4-min incubation period. The AdK-catalyzed
phosphoryl transfer rate increased 12-fold from 2.1 ± 0.7 to 24
± 1.2 nmol
mg protein
min
, with stimulation at 4 Hz (the rate per 4 min is
shown). At 4 Hz this phosphotransfer rate accounts for about 15% of the
total ATP metabolized in terms of the ADP processed by way of AdK
catalysis.
Figure 1:
The relationship of AdK catalytic rate
and the rate of lactate production to frequency of contraction in rat
diaphragm muscle. Intact rat diaphragms were isolated and incubated in
medium enriched 30-35% in [O]water without
stimulation for 4 min(1) . During a subsequent incubation in
the same medium, the diaphragms were subjected to no stimulation (0 Hz)
or electrically stimulated at 1, 2, or 4 Hz. At precisely 4 min after
the start of the second incubation period, the diaphragms were rapidly
frozen by immersion in liquid nitrogen, and the tissue content of AMP,
ADP, ATP, and lactate was determined as described under
``Experimental Procedures.'' The amount of lactate released
into the incubation medium was also determined. The rate of
AdK-catalyzed phosphoryl transfer was determined for the duration of
the second 4-min incubation by the rate of AMP phosphorylation as
described previously (1) from the rate of appearance of
O-labeled phosphoryls of
-ATP and
-ADP. Each
value represents the mean of three to four animals analyzed in
duplicate or triplicate. The standard errors ranged from less than
1-8% and are presented in the text under ``Results and
Discussion'' because most are smaller than the symbols. This
experiment is representative of three from which very similar results
were obtained. Panel A shows the rate per 4 min of AMP
phosphorylation by AdK (designated AMP flux) with and without
electrically stimulated contraction at the three frequencies tested
compared to the tissue concentrations of AMP, ADP, and ATP. The control
value represents the tissue adenine nucleotide level after the 4-min
preincubation. The 0 Hz value represents the concentration after the
additional 4-min incubation without stimulation. In panel B,
rates of AdK catalysis are compared with the rate of lactate production
at the contractile frequencies (Hz) shown in parentheses.
These increases in the rate of AdK-catalyzed phosphoryl
transfer manifest as rapid phosphorylation of AMP occur with little
measurable change in the concentration of muscle ATP, ADP, or AMP (Fig. 1A). In the experiment shown, the cellular
concentration of ATP was determined to be (mean ± S.E., n = 3-4) 36.9 ± 0.7, 37.9 ± 1.6, and 35.4
± 2.4 nmol mg protein
after a 4-min
period of stimulation at 1, 2, or 4 Hz, respectively, compared to a
value of 38.8 ± 1.0 nmol
mg protein
following a 4-min preincubation of the diaphragm at 0 Hz. The
maximum change is an 8.7% decrease in ATP concentration with
stimulation at 4 Hz. Similarly, the maximal change in the concentration
of cellular ADP was a 35% increase (from 7.36 ± 0.14 to 9.92
± 0.20 nmol
mg protein
) after 4 min of
stimulating the muscle at 4 Hz while AMP concentration increased 27%
(from 1.1 ± 0.04 to 1.4 ± 0.1 nmol
mg
protein
). The ability to detect small changes in the
levels of the adenine nucleotides over even a protracted period of
vigorous contraction does not eliminate the possibility that larger
changes occurred during each contractile event. These results, which
are consistent with other reports (9, 10) showing
little or no alteration in these nucleotide levels with muscle
contraction, nevertheless, indicate that any change that may have
occurred in ATP, ADP, or AMP concentration must have been very
transient and/or very small and restoration to the basal level very
quickly followed each contractile event.
A unique feature of the results shown in Fig. 1A is that although there is no relationship of stimulated muscle contraction with cellular concentrations of adenine nucleotides, there is a close correspondence between contractile frequency and the rate of AdK-catalyzed phosphoryl transfer which is manifest as increases in the rate of AMP metabolic turnover. From the relative constancy of the cellular AMP concentration, it is evident that the enhanced rate of AMP phosphorylation is very closely balanced by and probably coupled to an equivalent rate of AMP generation.
The next step in defining how this system
operates to produce the observed increases in AMP metabolic flux is to
identify the source of the ADP from which AMP is generated by
AdK-catalyzed phosphotransfer. Identifying this catalytic process can
be aided by exploiting a unique feature of the O-phosphoryl-labeling procedure which is that it detects
only net flux, represented by newly phosphorylated AMP accumulating as
[
-
O]ADP and
[
-
O]ATP. This analytical procedure cannot
detect the total number of AdK-catalyzed phosphotransfer events if
multiple reversals were to occur because this would not result in a net
increase in
-
O-labeled nucleotide. The major
reactions within eukaryotic cells generating ADP as a product are
ATPases and ATP-dependent kinases. Since the rate of this AMP
generation closely corresponds to the frequency of stimulated muscle
contraction it is most likely that the ADP derives from
contraction-related ATPase catalyzed hydrolysis of ATP. This could be
designated as the ``AMP flux generating'' reaction.
An
additional consideration stemming from the analytical characteristic of
the O-phosphoryl-labeling procedure to detect only the net
synthesis of newly generated [
-
O]ADP and
[
-
O]ATP is that ATP serving as the
phosphoryl donor also cannot derive from an AdK reversal reaction. It
must also arise from a process resulting in net production of ATP such
as glycolysis or oxidative phosphorylation.
This stoichiometric
equivalence between glycolytic flux and rate of AMP phosphorylation
(and AMP generation) occurs over the entire range of increased
catalytic rates exhibited by each of these metabolic pathways with
increasing frequency of contraction. Since lactate production is a
measure of anaerobic glycolytic rate, these results indicate a
fundamental metabolic relationship between ATP produced by anaerobic
glycolysis and AdK-catalyzed transfer of a -phosphoryl of ATP to
AMP. Further, the net production of this AMP was traced in the
preceding section to ADP generated by a muscle ATPase that had
undergone a loss of its
-phosphoryl as a result of AdK-catalyzed
phosphotransfer. Combined, this information demonstrates a functional
link by way of AdK-catalyzed phosphotransfer between ATP consumption by
an ATPase and ATP generation by anaerobic glycolysis. There is abundant
evidence that the paradoxical generation of lactate in fully oxygenated
tissues (11) represents a constitutive process for generating
ATP utilized selectively by specific cellular
components(12, 13) . This view is also consistent with
the recent observation using
C NMR (14) that two
pyruvate pools exist in muscle cells, one of which is associated with
nonoxidative metabolism (i.e. lactate formation).
In the oxygen-deprived diaphragms, the rate of O-labeled phosphoryl appearance in
-ADP and
-ATP
was greatly increased compared to fully oxygenated controls (compare Fig. 2, A and B). This increase occurred with
virtually no change in the rate of [
O]phosphoryl
appearance in
-ATP. This indicates that the overall rate of ATP
metabolism with respect to its energy-rich
-phosphoryl was not
impaired. These results also demonstrate that the large increase in the
rate of [
O]phosphoryl appearance in
-ADP
and
-ATP is the outcome of an increase in the rate of AdK
catalysis rather than a change in
O enrichment of the
-ATP phosphoryl donor.
Suppression of oxidative phosphorylation
by each of these experimental procedures produced the predicted effect
of enhancing anaerobic glycolytic flux; the rate of lactate generation
was increased in the range of 8-26-fold (Fig. 3). Another
outcome of impaired oxidative metabolism was an enhancement in the rate
of net AdK-catalyzed phosphorylation of AMP which increased by
approximately the same magnitude as the rate of anaerobic glycolysis (Fig. 3). The basal rate of AdK-catalyzed phosphoryl transfer
resulting in AMP phosphorylation increased approximately 20-, 25-, and
8-fold with N, FCCP and KCN, respectively. These net
increases in AdK-catalyzed phosphotransfer coincided with nearly
equivalent increases of about 16-, 26-, and 8-fold in anaerobic
glycolytic flux for N
, FCCP, and KCN, respectively (Fig. 3).
Figure 3:
The
relationship of suppressed oxidative phosphorylation to the rates of
AdK catalysis and lactate production. Non-stimulated rat diaphragms
were incubated with no addition to the medium or with addition of an
agent that suppresses mitochondrial oxidative phosphorylation. Tissues
receiving KCN (0.5 mM) and FCCP (10 µM) were
exposed to the inhibitors for 5 min prior to their transfer to
[O]water-containing medium. The tissues
incubated in N
-equilibrated medium were treated as
described in Fig. 2. The AdK velocities and lactate production
rates were calculated from determinations at 1, 2, and 4 min of
incubations in the [
O]water-enriched medium. The
values shown represent the mean rates ± S.E. determined from at
least four diaphragms. Lactate present in tissue incubated for only the
5-min preincubation period was subtracted from the total lactate in
tissues subjected to the subsequent incubation. Lactate present in the
[
O]water-containing medium was included in the
total. Tissue adenine nucleotide concentrations were also determined in
diaphragms incubated for 2 and 4 min in
[
O]water-containing medium, and the mean values
± S.E. are presented in the text.
In each of these experimentally induced states of
suppressed oxidative phosphorylation, the increases in anaerobic
glycolytic generation of ATP must have compensated reasonably well for
the diminished rate of ATP production oxidatively because the total ATP
utilization rate determined from O incorporation into
metabolic intermediates (2) was reduced (19%) only after
treatment with KCN (not shown). In contrast to the near equivalent
relationship uncovered between AMP metabolic flux and anaerobic
glycolytic rate (Fig. 3), changes that occurred in the cellular
concentrations of adenine nucleotides under these different conditions
of impaired oxidative ATP production showed little correspondence with
the magnitude of enhanced glycolysis. The correspondence sought in this
instance was a change in a metabolite effector (i.e. ATP, ADP,
and/or AMP) concentration that would elicit an 8-26-fold
acceleration in the velocity of a rate-controlling glycolytic enzyme.
For example, according to predictions of allosteric regulation (9, 15) a change of 400% in metabolite effector
concentration is required to produce a 9-fold increase (i.e. the smallest increase observed in gycolytic flux) in enzyme
velocity. The total cellular concentration of ATP was decreased from a
control value of 46.4 ± 4.6 nmol
mg protein
(n = 4) by 5.4, 20.4, and 14.5% with
N
, FCCP, or KCN treatment, respectively. The concentration
of ADP (control value 12.7 ± 3.0 nmol
mg
protein
, n = 4) increased by 26, 71,
and 31.6% with N
, FCCP, or KCN treatment, respectively. The
cellular concentration of AMP (control value 2.2 ± 0.9 nmol
mg protein
, n = 4) increased
by 27.5, 126, and 92% with N
, FCCP, or KCN, respectively.
Changes of similar magnitude in adenine nucleotide concentrations have
been observed with comparable treatments that result in suppression of
muscle oxidative ATP generation(16) . If or how these altered
levels in adenine nucleotides may contribute to the observed
accelerated glycolytic flux is not readily apparent from the magnitude
of their changes relative to those of glycolytic flux. For example, the
decrease in the concentration of ATP and increases in ADP and AMP
concentrations were greater with KCN treatment than with N
replacement, but the increase in gycolytic flux was more than two
times greater with N
than KCN. The possibility that
localized changes in nucleotide concentration represents a determinant
of the observed increases in glycolytic flux has not been eliminated by
these results.
On the other hand, the action of each treatment that suppressed oxidative ATP generation resulted in an accelerated rate of AMP metabolic flux that exhibited a very high fidelity with anaerobic glycolytic flux. This correspondence was manifest as a stoichiometry whereby each molecule of ATP generated as a result of the catabolism of triose phosphate to lactate coincides with an almost equivalent phosphorylation of an AMP molecule. This striking fidelity of AdK-catalyzed AMP phosphorylation to anaerobic glycolysis is, therefore, demonstrable in the nearly physiological state with intermittent stimulation of muscle contraction (Fig. 1, A and B) and also under experimental conditions whereby oxidative production of ATP is impaired continuously (Fig. 3).
The metabolic behavior of creatine kinase in
intact muscle is assessable from the rate of O appearance
in the phosphoryl of CrP. This results when the phosphoryl of
[
-
O]ATP is transferred to creatine to
generate [
O]CrP. When examined by this
analytical approach it was very evident that the kinetic behavior of
creatine kinase in intact muscle deprived of oxygen exhibited
reciprocal behavior relative to that of AdK. For example, when the
muscle was deprived of oxygen and AdK-catalyzed phosphotransfer
increased about 20-fold (Fig. 3), there was a decrease in the
rate of appearance of [
O]CrP by about 65% (Fig. 4A). Contributing to this diminished velocity of
CK catalysis was an approximately 60% reduction in the concentration of
cellular CrP when oxidative generation of ATP was impaired by N
equilibration of the incubation medium (Fig. 4, B and C). This reduction in the level of CrP occurred
during the 4-min preincubation in nitrogen-equilibrated medium and
reflects a new steady state that was sustained during the subsequent
incubations of 1, 2, or 8 min in nitrogen-equilibrated
[
O]water-enriched medium. As shown in Fig. 4C, the reduced concentration of CrP in the
oxygen-deprived muscle undergoes metabolic turnover (i.e. acquires
O-labeled phosphoryls) at an apparently
pseudo-first-order rate nearly identical to that of the approximately
two times greater CrP concentration in the oxygenated tissue (t
= 3.83 and 3.90 min in oxygenated and
N
-equilibrated tissue, respectively). The metabolic flux of
CrP, which is equal to the turnover rate times its cellular
concentration, is reduced by approximately the same magnitude as the
cellular concentration of CrP.
Figure 4:
Effect of oxygen deprivation on creatine
kinase-catalyzed generation of [O]creatine
phosphate and the cellular concentration and metabolic turnover of
creatine phosphate. Creatine phosphate was purified as described
previously (7) from the same tissues used to obtain the data
shown in Fig. 2, and the
O content of its
phosphoryl was determined after its enzymic transfer to glycerol (i.e. glycerol 3-phosphate) using CK and glycerol kinase. The
values shown are means from three diaphragms. Values from replicate
tissues varied by less than 10%. Panel A shows the relative
rate of CK-catalyzed phosphoryl transfer (i.e. appearance of
[
O]creatine phosphate) in fully oxygenated
tissues (
) and in tissues incubated in medium depleted of oxygen
(
). The ordinate represents the product of the tissue
concentration of creatine phosphate times the fraction of the total
cellular creatine phosphate with a phosphoryl containing one to three
atoms of
O (corrected for the enrichment of
[
O]water). Panels B and C show
the tissue concentrations of creatine phosphate (striped
portion) and the fraction containing an
O-labeled
phosphoryl (filled portion) in diaphragms incubated in fully
oxygenated medium (panel B) or nitrogen-equilibrated medium (panel C).
This decrease in CrP concentration is
most likely a consequence of the diminished rate of ATP generation by
oxidative phosphorylation apparently linked to CK-catalyzed
phosphorylation of creatine. Since the cellular ATP concentration is
sustained at very nearly the control level (i.e. 46.4 ±
4.6 versus 43.9 ± 2.8 nmol mg of
protein
, n = 4), and the ATP
metabolic flux is not compromised in the oxygen-deprived muscle (Fig. 2, A and B), both are probably
maintained in large measure by the compensatory increase observed in
anaerobic glycolytic ATP generation. This increased rate of anaerobic
ATP generation indicated by the enhanced rate of lactate production
occurs as was pointed out above (Fig. 3) in conjunction with an
almost equivalent increase in the net rate of AdK-catalyzed
phosphotransfer. This increase in AdK-catalyzed phosphotransfer could,
therefore, be viewed as compensatory phosphotransfer for the diminished
rate of CK-catalyzed phosphoryl transfer coinciding with the reduced
rate of oxidatively generated ATP in the oxygen-deprived muscle.
The concept that CrP is simply a reservoir of energy-rich phosphoryls used to maintain or ``buffer'' the cellular ATP concentration when ATP production does not keep pace with its utilization could not explain the results reported here. A constantly maintained cellular concentration of CrP exhibits a relatively rapid rate of turnover under basal conditions and continues to exhibit virtually the same turnover even in the oxygen-deprived state when the cellular CrP concentration is markedly compromised, but maintained at a relatively constant diminished steady state level.
These results support the conclusion that in skeletal muscle AdK-catalyzed phosphotransfer is linked to anaerobic glycolytically produced ATP and that catalyzed by CK involves oxidatively produced ATP. These results also indicate that the AdK and CK phosphotransfer systems share a similar metabolic function, and although they can operate independently of one another their functions are interrelated to the extent that AdK may replace the phosphotransfer function of CK.
Figure 5:
Suggested functional arrangement of the
proposed adenylate kinase and the creatine kinase-catalyzed high energy
phosphoryl transfer systems. The scheme shows a proposed mechanism of
communication between flux-generating (ATPases) and flux-responding
(glycolysis or oxidative phosphorylation) processes by way of
sequential transfer of phosphoryls catalyzed by a linear sequence of
adenylate kinase (AdK) (designated A) or creatine
kinase (CK) (designated B) enzyme molecules
comprising an energy transfer and regulation network in the cellular
cytosol. The proposed model, which incorporates the previously
suggested ``PCr circuit'' construct(19) , emphasizes
relaying the displacement of equilibrium along a corresponding chain of
enzyme molecules according to the Mitchell (20, 21) principle of vectorial ligand conduction.
Incoming ligands ``push'' others to create a
``pressure'' that is propagated through the network of
enzymes catalyzing rapid equilibration among ligands; the molecules
arriving at the distal sites of this bidirectional system represent the
equivalent rather than the specific molecule generated at the
originating site. A mechanism similar in principle has been proposed
for proton conduction pathways (e.g. ``proton
wires'') across biological membranes(25) . The reaction
sequence designated A shows how AdK could function at the site
of an ATPase to restore a molecule of ATP and simultaneously generate a
signal in the form of a molecule of AMP to promote glycolytic
production of a new molecule of ATP to replace the molecule of ATP
consumed. This AMP signal can enter the AdK-catalyzed transfer system
and push monophosphate molecules distally along a series of rapidly
equilibrating enzymes responding to the disequilibrium created at the
ATPase site. This AMP promotes the emergence of an equivalent at the
end of the transfer system distal to the flux generating ATPase and in
close proximity with an as yet unidentified glycolytic site. It can
promote glycolytic production of an ATP molecule by serving as
phosphoryl acceptor of a -phosphoryl from an ATP molecule acting
as an inhibitor of this control site in glycolysis. The reaction
sequence designated B shows how the previously proposed PCr
circuit (19) would be integrated according to the experimental
results described here with the operation of the AdK ligand conduction
system. The high energy phosphoryl represented by the
-phosphoryl
of an ATP molecule generated by oxidative phosphorylation is ordinarily
conducted through a series of phosphotransfers in the form of CrP or
ATP. However, if oxidative phosphorylation becomes impaired, viz.B
, the ADP from the ATPase ordinarily
rephosphorylated by CrP through CK catalysis becomes a reactant in the
AdK-catalyzed phosphotransfer system. This results in the generation of
an AMP signal that promotes anaerobic glycolytic flux as a compensatory
means of generating ATP along with a commensurably greater rate of
AdK-catalyzed transfer of this ATP.
The evidence in support of this
proposed scheme is as follows. The initiating or ``flux
generating'' reaction is designated as a localized cellular
ATPase. This was concluded because the AMP-generated and undergoing
phosphorylation was traced to an ADP reactant in an AdK-catalyzed
phosphotransfer reaction. Furthermore, this ADP was determined to
derive from a cellular reaction generating net ADP in close
correspondence with a contraction-related process very likely
represented by an ATPase activity. ATP generation from anaerobic
glycolysis is designated as the source of net ATP used for AMP
phosphorylation because this process exhibited equivalent stoichiometry
with that of AMP phosphorylation. Additionally, inhibition of oxidative
phosphorylation resulted in markedly enhanced rates of anaerobic
glycolysis coinciding, quantitatively, with AdK-catalyzed
phosphotransfer. Consistent with this conclusion is that AdK and
glycolytic enzymes are colocalized in muscle
fibers(26, 27, 28) . One of the two molecules
of ADP generated at this glycolytic site by AdK-catalyzed
phosphotransfer is envisaged to represent a substrate and/or allosteric
effector that can promote glycolytic flux and generation of another ATP
molecule. The other molecule of ADP is considered to represent a high
energy component due to its energy-rich -phosphoryl which is
transferable exclusively by the catalytic action of AdK along the
equilibrating sequence of AdK-catalyzed reactions as the
- or
-phosphoryl of ADP or ATP to the site of the ATPase where it can
regenerate a consumable molecule of ATP.
The suggestion that this
anaerobic glycolytically produced ATP may serve as a suppressor of
glycolysis is based on the logic that ATP should regulate its own
synthesis. In addition, there are at least three identifiable sites in
the glycolytic pathway, phosphofructokinase, aldolase, and
glyceraldehyde phosphate dehydrogenase, that are inhibitable (i.e. allosterically) by ATP(29, 30, 31) .
There is evidence that AdK can interact with phosphofructokinase (32) and also with aldolase, phosphoglycerate kinase, and
enolase which copurify with AdK on a Sepharose-Ap5A column. ()Recently, it was found that in Escherichia coli AdK is associated with glyceraldehyde phosphate dehydrogenase. (
)
Under consideration are two possible mechanisms by which this putative glycolytic inhibitory action of ATP could be reversed. First, the ATP acting as glycolytic suppressor could be bound to an ATP-inhibitable site and in rapid equilibrium with a localized pool of ATP. The concentration of ATP in this locale would become decreased by the AdK-catalyzed conversion of this ATP to ADP after arrival of an equivalent AMP.
A second possibility is that AdK may
itself act as an effector protein by analogy with G-proteins (e.g. ras p21) with which it shares structural
homology(33, 34) . AdK, like G-proteins undergoes very
marked conformational changes upon binding their triphosphate
ligand(34, 35) . In the case of adenylate kinase,
there is sufficient cytosolic ATP to maintain AdK in the
triphosphate-liganded state which may represent a conformation
imparting a suppressive influence on the glycolytic enzyme activity in
question. Upon delivery of AMP and conversion of ATP to ADP, resulting
from catalytic phosphotransfer, transformation from an inhibitory to a
non-inhibitory conformational state would occur. The magnitude of
deinhibition would, therefore, depend on the frequency of AMP
generation. This mode of regulation would be much more rapid than can
be accomplished with G-proteins which exhibit ``on/off'' time
constants of several seconds due to their inherently slow
phosphohydrolase activity(36) . This would compare to less than
a millisecond time constant required for the adenylate kinase catalytic
cycle. A precedent for this type of action for AdK can be inferred from
results (37) showing that a mutant form of AdK imparts
thermolability to another enzyme (e.g.sn-glycerol-3-phosphate acyltransferase) while the
``normal'' AdK is required for a stimulatory effect of ATP on
this enzyme activity. A recently uncovered AdK-catalyzed AMP metabolic
dynamic similar to that described here but with high fidelity to the
opening and closing of an ATP-inhibitable K channel in
insulin secreting transformed pancreatic
-cells (38) would
also be consistent with a regulatory role for the AdK enzyme protein in
the function of K
channel regulated by apparent ATP/ADP
liganding(39) .
The possibility of a series of AdK-catalyzed
equilibrium reactions is suggested by comparing the present results
with a report of the velocity of AdK-catalyzed phosphotransfer measured
by P NMR saturation transfer(40) . The rate
determined by the latter procedure is approximately 20-fold greater
than that determined by the procedure of
[
O]phosphoryl labeling in muscle and in red
blood cells(1, 41) . The
P NMR method
theoretically detects virtually all AdK-catalyzed phosphotransfers
occurring during the monitoring period, while the latter only detects
net AMP phosphorylation by newly synthesized ATP. The simple
interpretation of these apparently conflicting results is that each
newly generated ADP by AdK catalysis undergoes approximately 20
sequential phosphotransfers in a ligand conduction-type of system.
That oxidatively generated ATP is transferred primarily by the
CK-catalyzed phosphotransfer system is strongly suggested by the
decreased rate of [O]CrP generation accompanying
suppression of oxidative ATP production. This decreased oxidative
generation of ATP paralleled by diminished CK-catalyzed phosphotransfer
occurred in conjunction with a markedly enhanced rate of AdK-catalyzed
phosphotransfer and a correspondingly increased rate of anaerobic
glycolytic generation of ATP. Relevant to this issue is a subsequent
observation, which will be the subject of a separate report, that the
net rate of CK-catalyzed phosphotransfer in non-contracting rat
diaphragm muscle is equal to about 85% of the total ATP metabolic
turnover. Under the same conditions AdK-catalyzed phosphotransfer
accounts for less than 10%. However, when CK activity is progressively
suppressed in the intact muscle by increasing dinitrofluorobenzene
concentrations the activity of the AdK phosphotransfer system
progressively increases to the extent that when CK activity is 98%
inhibited, AdK accounts for the transfer of virtually 100% of the ATP
produced(42) . Under these circumstances over 80% of the ATP
continues to be generated by oxidative metabolism. This indicates that
AdK can also transfer oxidatively produced energy-rich phosphoryls when
CK-phosphotransfer system is directly impaired. The previously reported
coexistence of AdK and CK in discrete subcellular locales (26, 43) is consistent with this shift from one
phosphoryl transfer system to the other.
The CK- and AdK-catalyzed phosphotransfer systems therefore appear to share a similar metabolic function with respect to energy-rich phosphoryl transfer. Under certain physiological conditions such as at high muscle workloads when CrP concentration undergoes significant reduction (44, 45) AdK could be predicted to take over a substantial portion of this function when it cannot be carried out by a markedly constrained CK phosphotransfer system.