AMP deaminase
binding
in contracting
rat skeletal muscle
KENNETH W. RUNDELL, PETER C. TULLSON, AND RONALD L. TERJUNG Department of Physiology, State University of New York Health Science Center at Syracuse, and Department of Health and Physical Education, Syracuse University, Syracuse, New York 13210 Rundell, Kenneth W., Peter C. Tullson, and Ronald L. Terjung. AMP deaminase binding in contracting rat skeletal muscle. Am. J. Physiol. 263 (Cell Physiol. 32): C287-C293, 1992. -AMP deaminase, which hydrolyses AMP to inosine Y-monophosphate (IMP) and NH,, at high rates during excessive energy demands in skeletal muscle, is activated when bound to myosin in vitro. We evaluated AMP deaminase binding in vivo during muscle contractions to assess whether binding 1) is inherent to deamination and found only with high rates of IMP production or simply coincident with the contractile process and 2) requires cellular acidosis. AMP deaminase activity (pm01 . min-l agg’) was measured in the supernatant (free) and 10*-g pellet (bound) homogenate fractions of muscle of anesthetized rats after in situ contractions to determine the percent bound. In resting muscle, nearly all (-90%) AMP deaminase is free (cytosolic). During contractions when energy balance was well maintained, binding did not significantly differ from resting values. However, during intense contraction conditions that lead to increased IMP concentration, binding increased to -60% (P < 0.001) in fasttwitch and -50% in slow-twitch muscle. Binding increased in an apparent first-order manner and preceded initiation of IMP formation. Further, binding rapidly declined within 1 min after cessation of intense stimulation, even though the cell remained extremely acidotic. Extensive binding during contractions was also evident without cellular acidosis (iodoacetic acid-treated muscle). Thus the in vivo AMP deaminase-myosin complex association/dissociation is not coupled to changes in cellular acidosis. Interestingly, binding remained elevated after contractions, if energy recovery was limited by ischemia. Our results are consistent with myosin binding having a role in AMP deaminase activation and subsequent IMP formation in contracting muscle. inosine 5’-monophosphate; deamination; contractile proteins; muscle fiber types
intense
contractions;
CONTRACTIONS can result in an extensive decrease in total adenine nucleotide content (AMP -t ADP -t ATP) in fast-twitch muscle, amounting to as much as -50% of the ATP pool (10, 11, 17-19). This decrease is accompanied by a production of ammonia and inosine 5’-monophosphate (IMP) by the nonequilibrium reaction catalyzed by AMP deaminase (11, 17-19)
INTENSE
AMP + HZ0 + IMP + NH3 The action of AMP deaminase serves to limit the increase in AMP concentration within the contracting muscle. This in turn pulls the myokinase reaction (ZADP ++ AMP + ATP) to the right, thereby tempering the elevation in ADP concentration. Thus the coordinate activities of the AMP deaminase and myokinase reactions help maintain a high ATP-to-ADP ratio, a potentially important role for AMP deaminase within the cell (14). Kinetic analysis of purified AMP deaminase has demonstrated that ATP, guanosine 5’-triphosphate (GTP), and orthophosphate inhibit the enzyme; the last factor is probably responsible for the effective inhibition of AMP deaminase in resting muscle (14, 30). Current ev0363-6143/92
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idence suggests that AMP, ADP, and H+, factors known to activate purified AMP deaminase (30), are also important in AMP deaminase activation in contracting muscle (11, 19). The available free concentration of AMP (AMPJ within muscle increases as energy turnover increases (12). Because the K,,, of AMP deaminase for AMP (0.5-1.0 mM AMP; Refs. 14, 20, 22, 26, 30) is much greater than the physiological AMPf, the rate of IMP production should be proportional to the increase in AMPI- within the cell. Further, AMP deaminase activity is enhanced by ADP, a modulator that also increases in active muscle (12). As pH decreases from 7.0 to 6.5, ADP becomes a more effective activator of AMP deaminase (30). Although cellular acidosis is not required for high rates of AMP deamination in vivo (11, 12), purified AMP deaminase activity is enhanced by acidosis in vitro, with an optimum pH of -6.2-6.5 (l4,2O, 22), values similar to those found in the myocyte during severe contractions (11). Although important factors for the in vitro regulation of AMP deaminase have been defined, our understanding of the influence of these modulators within contracting muscle is incomplete. Purified AMP deaminase binds reversibly to myosin and is activated by such binding (3, 4, 7, 25). Although the precise mechanism responsible for this enzyme activation has yet to be determined, it is thought that myosin binding releases inhibition (e.g., from GTP) by allosteric modification (3). Barshop and Frieden (7) found that decreases in pH, to an optimum of -6*5, enhanced the binding between AMP deaminase and myosin or its subfragments; however, this influence has not been confirmed (25). Further, Marquetant et al. (15) found that changes in ATP concentration, similar to those observed during intense contractions, modify binding of purified AMP deaminase to myosin. These findings suggest that binding of AMP deaminase by myosin may serve an important regulatory function and help explain the control of AMP deamination that occurs during muscle contraction. The potential physiological role of AMP deaminasemyosin binding has been bolstered by the recent work of Shiraki et al. (23). The fraction of total muscle AMP deaminase activity located in the myosin-containing pellet of rat muscle homogenate increased more than fivefold after intense stimulation (23). It is presently unclear, however, whether the binding of AMP deaminase by myosin is directly related to conditions of enzyme activation or simply occurs with muscle contractions. Further, it is unknown whether AMP deaminase-myosin binding in viva precedes or is a result of ATP depletion. The purpose of this study was to evaluate the potential role of AMP deaminase-myosin binding in different skeletal muscle fiber types in situ during contraction
0 1992 the American
Physiological
Society
C287
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C288
AMP
DEAMINASE
BINDING
conditions known to activate deamination. We determined whether binding of AMP deaminase to myosin is inherent to conditions where high rates of AMP deamination occur or simply coincident with muscle contractions, even in the absence of IMP formation. In addition, we determined whether cellular acidosis is essential for in vivo binding of AMP deaminase to myosin. Our results support the hypothesis that binding of AMP deaminase to myosin contributes to the control of IMP and ammonia production in vivo. METHODS
Animal care and stimulation procedures. Adult SpragueDawley rats (386 t 8.6 g; n = 76; Taconic Farms, Germantown, NY) were housed three per cage in a temperature-controlled room (ZO-21°C) with a 12:12 h light-dark cycle. Purina Lab Chow and water were provided ad libitum. Animals were anesthetized with pentobarbital sodium (50 mg/kg ip). The right gastrocnemius-plantaris-soleus muscle complex was prepared for in situ stimulation as done previously (18). The limb was secured with a pin through the distal shank of the femur, and the distal tendon was connected to a strain gauge mounted on an adjustable frame. During preparation, care was taken not to disturb blood flow to the lower limb. The tibia1 nerve was cut and placed across a shielded platinum bipolar electrode connected to a Grass S48 stimulator. Muscle length was adjusted to provide maximal tension and stimulated 1) with supramaximal pulses (6 V, 0.1 ms duration) of 0.5 or 5 Hz or 2) tetanically with supramaximal square-wave pulses (6 V, 0.1 ms duration) of lOO-ms trains at PO0 Hz at a frequency of 12 tetani/ min. The exposed muscles were kept moist and maintained at 37°C wit,h a heat lamp for the duration of each experiment. Mean aortic pressure was continuously monitored via a catheter inserted into the right carotid artery and was generally stable throughout the contraction period, averaging 110 t 5 mmHg (n = 60). Four series of conditions were used to evaluate in vivo binding of AMP deaminase. Series 1 evaluated the coincidence of binding with deamination. Contraction conditions that have previously demonstrated high rates of IMP formation in fast-twitch white (5 Hz, 3 min), fast-twitch red (5 Hz, ischemic, 3 min), and slow-twitch red (12 tetani/min, ischemic, 10 min) muscle fiber sections were established (1 I, 19, 31). Each fiber section required different contraction conditions to produce deamination, because of differences in the inherent oxidative and glycolytic capacities among muscle fiber types. For example, when stimulated at 5 Hz, fast-twitch white fibers of the rat deplete a large fraction of their ATP (-50%) and become acidotic, whereas fast-twitch red and slow-twitch red fibers demonstrate an excellent energy balance with little increase in IMP content and no significant acidosis (I 1). When the stimulated muscle is rendered ischemic by ligation of the femoral artery, contracting fast-twitch red fibers also display a high rate of deamination and acidosis similar to fast-twitch white fibers (11). Likewise, under extended ischemic stimulation at 12 tetani/min, slow-twitch red fibers incur an w-30-40% decrease in ATP content plus an increase in IMP (29, 31). At the appropriate time during stimulation, the superficial section of the medial gastrocnemius (predominately fast-twitch white fiber type), the deep red section of the lateral gastrocnemius (predominately fast-twitch red fiber type), and the soleus (predominately slow-twitch red fiber type) were clamp frozen with aluminum tongs cooled in liquid nitrogen. The corresponding muscle sections of the contralateral limb were frozen in exact Series 2 conditions were used to examine whether AMP deaminase binding was simply coincident with the contractile
IN MUSCLE
process and therefore not related to the cellular events leading to IMP production. Muscles were stimulated at frequencies where ATP was maintained with modest phosphocreatine hydrolysis and little or no increase in IMP (0.5 Hz and 5 min for fasttwitch white and 5 Hz and 3 min for fast-twitch red and slowtwitch red) (II, 19, 31). Series 3 was performed to assess the effects of cellular acidosis on in vivo binding of AMP deaminase by using iodoacetic acid (IAA) treatment to inhibit lactate formation and the consequent acidosis during contractions (9). Four minutes before and during ~-HZ stimulation, IAA was infused (1.4 pm01 . min-l .g body wt-‘) in the descending aorta via the contralateral femoral artery as done previously (1 I). Muscle samples of the right gastrocnemius-plantaris-soleus complex were clamp frozen after 30 s stimulation as described above. Control muscle samples were clamp frozen after 4 min IAA infusion with no stimulation. Series 4 was performed to 1) measure the correlation between AMP deaminase-myosin binding and IMP formation over time and 2) examine the persistence of binding during recovery after stimulation. Fast-twitch white muscle sections were clamp frozen after 15, 30, 60, and 180 s of ~-HZ contractions. Recovery samples were taken at 15, 30, 60, and 120 s after 60 s of stimulation. To further explore binding retention, ischemic fasttwitch white muscle sections were clamp frozen after 60 s of ~-HZ contractions and 30,60, and 120 s after 60 s of stimulation. Ischemia was established by ligation of the femoral artery IO s before stimulation and maintained through the 120-s recovery period. Enzyme assay. AMP deaminase activity (pm01 . min+ . g-l) was measured spectrophotometrically at 285 nm (extinction coefficient at 285 nm = 0.3) (26). The assay was performed at 30°C in 1 ml of medium containing 2 mM AMP, 150 mM KCl, and 50 mM imidazole HCl (pH 7.0). Specificity of the assay system was verified by the total elimination of activity by the presence of 1 X 10B6 M coformycin, a potent inhibitor of AMP deaminase (I), in the assay medium. Frozen tissue samples were crushed under liquid nitrogen and homogenized in 9 vol of 150 mM KCl, 2 mM EDTA, 10 mM glutathione, pH 7.0 for 20 s using a ground-glass Potter-Elvehjem tissue grinder. Myofilaments are insoluble in 150 mM KC1 and rapidly sediment during centrifugation (microcentrifuge at - 16,000 g for 1 min). Myosin-bound AMP deaminase is carried along, because at this concentration of KCl, AMP deaminase does not dissociate from myosin (3, 23). We verified this by altering the homogenizing medium to conditions known to modify AMP deaminase binding. Neither acidosis (pH 6.5 vs. pH 7.0) nor high (IO mM) orthophosphate concentration changed the recovery of bound AMP deaminase in the pellet. Thus, as done by Shiraki et al. (23), the activity in the supernatant was considered that of the free (unbound) enzyme. Assay of AMP deaminase bound to myosin in the muscle homogenate pellet was made difficult because of the presence of cell debris that cosediments. Therefore we assayed the bound fraction of AMP deaminase by first extracting the enzyme in a high-KC1 buffer. This was achieved by suspending the myosin-containing pellet in 9 vol of 500 mM KC1 and 50 mM Tris HCl, pH 7.55. Bound AMP deaminase was dissociated from myosin by incubation at 37°C for 30 min and then centrifuged at - 16,000 g for 1 min. Activity in the supernatant of the rehomogenized pellet was considered that of myosin-bound enzyme, because previous studies (3, 7) have shown that the AMP deaminase-myosin complex is completely dissociated in 500 mM KCl. We verified quantitative recovery of the formerly bound enzyme activity in the supernatant. No AMP deaminase activity remained in the particulate fraction of the 500 mM KC1 extract. Assays were performed immediately upon sample preparation, because there is some loss of enzyme activity over time (- 15% in 20 min).
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AMP
DEAMINASE
BINDING
However, there was verv little loss of activitv between duplicate samples (XUL?& l/s~-& 2 = 1.005 t 0.0657; rt = 2OOj. As a measure of reliabilitv, activity in the whole homogenate of each tissue sample was cbmpared‘with activity in the-150 mM KC1 supernatant plus activity in the 500 mM KC1 supernatant of the rehomogenized pellet. The sum of the activity of the component fractions was within 5% of the activity of the whole homogenate [i.e., (150 mM KC1 supernatant + 500 mM KC1 supernatant)/ whole homogenate = 1.05 t 0.036; n = 801. Therefore total muscle enzyme activity was obtained as the sum of the two fractions. Metabolic asscws. Frozen tissue samples from series 3 and series 4 were extracted in ethanolic perchloric acid and neutralized with KOH containing 0.5 M triethanolamine as routinely done (31). The perchloric acid extracts were assayed for phosphocreatine, creatine, and lactate as done previously (31). ATP, ADP, AMP, and IMP were quantified by reverse-phase highperformance liquid chromatography as done previously (29). Because the responses of IMP accumulation of series 1 and series 2 have been well characterized (I l), we have not repeated these determinations. Muscle water content was determined by drying ~0.2 g tissue sample at 80°C to a constant dry weight. All metabolites were corrected to the water content of the nonstimulated contralateral control muscle tissue, to account for any change in water content that occurred with muscle contractions (18). Statistical analyses. Statistical comparisons between and within fiber types were made by analysis of variance. Critical differences (P < 0.05) were determined by Tukey’s post hoc analysis. RESULTS
Total actiuity. The total enzyme activity (free plus bound) of the muscle is unchanged by contractions (Table 1, P > 0.05). However, the distribution of AMP deaminase is markedly shifted from a free to bound form during intense contractions that result in extensive AMP deamination. Values of total AMP deaminase activity given in Table 1 also demonstrate differences between each of the skeletal muscle fiber sections as reported previously (32). Series 1: AMP deaminase binding and deamination. In rested muscle, nearly all (-90%) AMP deaminase is free (not bound; Fig. 1A). During stimulation conditions (5 Hz, 3 min) where a ~50% depletion of ATP leads to large increases in IMP and acidosis (estimated pH 6.1) (1 I), binding increased sixfold over resting values (P < 0.001) in the fast-twitch white gastrocnemius muscle section. Similarly, during stimulation conditions that resulted in a significant increase in IMP content (-40% depletion of ATP) with a reduction in estimated cellular pH to C6.2 in the fast-twitch red gastrocnemius muscle section (ischemic contractions at 5 Hz) (1 l), AMP deaminase binding increased about fivefold over control values (Fig. lA, Table 1. Total muscle AMP deaminase activity Gastrocnemius Soleus White
section
28 Eontrol Stimulated
324t9.0 30729.1
Red
section
10
9
172&12.9
84.W4.9
184t28.0
75.31r3.4
Values are means t SE in prnoll’. min-’ .g; n, no. of muscles. Gastrocnemius is made up predominately of fast-twitch red and white fibers and soleus of predominately slow-twitch red fibers.
C289
IN MUSCLE I
A
Fig. 1. AMP deaminase activity as percent of total bound during contractions that result in high rates of AMP deamination (A) and during contractions that result in little AMP deamination (B). See RESULTS for specific conditions (n = 4-6 per tissue).
P < 0.001). Thus, during intense contractions, both fasttwitch fiber types exhibited increased AMP deaminase binding when IMP production concomitant with a decreased pH occurred. Binding also increased about fivefold in the slow-twitch red soleusafter 10 min of ischemic stimulation at 12 tetani/min (Fig. lA, P < O.OOl), shown previously to lead to significant deamination (m 1.25 ,umol/g IMP) (29,31). Thus binding of AMP deaminase is not unique to either fast-twitch or slow-twitch fibers but occurs during conditions that lead to high rates of AMP deamination. This would have to occur if binding of AMP deaminase to myosin is essential for enzyme activation. Series 2: coincidence of A*MP deaminase binding and contraction. In contrast to the response of muscle where high rates of AMP deamination occur, AMP deaminase binding was not increased during contractions that do not result in substantial IMP production. This was found for all three fiber type sections (Fig. IB). Although the specific stimulation conditions differed among fiber types (fast-twitch white gastrocnemius 0.5 Hz, fast-twitch red gastrocnemius and slow-twitch red soleus 5 Hz), little if any accumulation of IMP occurs (11,17,29,31). Thus the binding of A.MP deaminase does not occur as a simple consequence of muscle contractions. Series 3: effects of cellular acidosis on binding. Because acidosis has been identified as an important condition contributing to in vitro binding of AMP deaminase to myosin (7) and is coincident with deamination during contractions in vivo (1 l), we evaluated whether cellular acidosis is obligatory for AMP deaminase binding in vivo. After 30 s of stimulation at 5 Hz, the bound fraction of AMP deaminase from IAA-treated muscle significantly increased for fast-twitch white, fast-twitch red, and slowtwitch red muscle fiber sections (Fig. 2, P < 0.001). The fraction of bound enzyme from nonstimulated control tissue sampled after 4 min of IAA infusion (before stimulation) was not significantly different from that of rested muscle without IAA infused (P > 0.05). Lactate contents of IAA-treated rat fast-twitch white muscle (2.2 t- 0.22 pmol/g, n = 5) after stimulation did not differ from rested fast-twitch white muscle (2.0 t 0.60 pmol/g, P > 0.05); however, an accelerated depletion of ATP content
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c290
AMP
DEAMINASE
0
0.05). However, as binding of AMP deaminase proceeded to its peak response of an approximately sixfold increase over resting values, IMP formation became evident (Fig. 3). IMP formation accounted for -500% depletion of ATP while AMP deaminase remained maximally bound. When muscle stimulation ended at 60 s, no further ATP depletion or IMP accumulation occurred (Table Z), and AMP deaminase binding returned rapidly to resting values in nonischemic muscle tissue (Fig. 4). The extreme cellular lactic acidosis, characterized by a lactate content of -30 pmol/g (Table Z), did not sustain AMP deaminase binding in the absence of continued intense muscle stimulation (Fig. 4). However, the rapid dissociation of binding
no
0
I
30
=
I
60
Time
=,
-
90
I
120
-
I
150
-
1
L 0.0
180
(seconds)
Fig. 3. Time course of AMP deaminase binding and inosine Y-monophosphate (IMP) accumulation in fast-twitch white muscle samples during 180 s of contractions at 5 Hz. Note that significant binding (P < 0.001) precedes IMP formation at 15 s of contractions (n = 34 and 6 per point for control and stimulated, respectively). The 120-s IMP value was taken from Dudley et al. (12).
Considerable evidence demonstrates that AMP deaminase binds reversibly to purified myosin (3-5, 7, 13, 24). The demonstration by Shiraki et al. (23) that AMP deaminase binds to myosin during contractions suggests that binding may be an important factor in the regulation of deamination in situ. Our results extend the work of Shiraki et al. (23) to show that AMP deaminase binding occurs in all skeletal muscle fiber types, requires an excessively high energy demand during contractions, precedes IMP production, does not require extreme cellular acidosis, and is retained after contractions by ischemia. We interpret the activity of AMP deaminase found in the pellet after low-ionic-strength homogenization as the bound form of the enzyme caused by contractions in vivo. Myofilaments are insoluble in 150 mM KCl, and the myosin-bound AMP deaminase is carried to the pellet upon centrifugation. The unbound AMP deaminase remains in the supernatant. This separation is not an artifact, because homogenizing the quick-frozen tissue in a medium known to increase binding [pH 6.5 vs. 7.0 (7)] or decrease binding [high orthophosphate (7)] did not modify the bound activity. Rather, our results indicate that inducing an energy deficit within contracting muscle seems to be the major factor prompting AMP deaminase binding to myosin. AMP deaminase binding. Binding of AMP deaminase could be induced in all three muscle fiber type sections. The contraction conditions necessary to induce binding in each fiber also caused an extensive AMP deamination to IMP and ammonia. This occurs in fast-twitch muscle when the rate of ATP hydrolysis exceeds the rate of ADP rephosphorylation (11, 18). The contraction conditions necessary to create an energy imbalance can differ among fiber type sections, because each section possesses an inherently different oxidative capacity available to meet the energy demand (6). The energetic demands required to produce high rates of deamination were most easily created in the low-oxidative fast-twitch white section of the gastrocnemius muscle. Coincident with an extensive activation of AMP deaminase that results in an ~50% decrease in ATP content (11)) the bound fraction of AMP deaminase increased about sixfold (Fig. 1A). The occurrence of AMP deaminase binding was not simply a result of muscle contractions, because the high-oxidative fasttwitch red section of the same contracting muscle group did not demonstrate any increase in AMP deaminase binding (Fig. 1B). This muscle section has an extremely high aerobic capacity and is capable of meeting the energy needs of this contraction condition without a substantial rate of AMP deamination (11, 17). Consistent with this, an absence of binding during contractions
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AMP
Table 2. Fast-twitch
white gastrocnemius
DEAMINASE
metabolites
Stimulation,
Rested Control
BINDING
15
during
Poststimulation,
30
:TP IMP PCr Lactate
6 6.57t0.34 0.023t0.006 11.89kO.92 13.8t1.6
S-Hz stimulation
s 60
Blood 34 7.42t0.10 0.013t0.007 22.69t0.41 2.9t0.2
c291
IN MUSCLE
6 6.29t0.43 0.465kO.217 6.71kO.62 31.4k2.2
15
flow
30
s 60
120
4 5.55&O. 13 0.93620.2 12 7.76k3.11 32.0t6.5
4 5.15t0.19 1.072&O. 189 ll.lOt3.03 28.0t4.3
intact
6 5.26&O. 14 1.281k0.169 3.86t0.55 37.3t2.5
4 4.94t0.35 0.854t0.219 4.70t0.54 23.Ok1.8
4 5.56t0.22 0.864kO.127 7.18tl.11 31.4t2.9
Ischemic 16 iTP 7.60~10.15 IMP 0.005~0.001 PCr 20.80t0.78 Lactate 3.6t0.2 Values are means -t- SE in pmol/g;
4 4.78t1.03 1.214t0.199 4.04t1.60 57.226.0 IMP, inosine
n, no. of animals/group.
Non-ischemic ---0
-60
0
30
Time
d
60
90
120
(seconds)
Fig. 4. Time course of AMP deaminase binding in fast-twitch white muscle samples during recovery after 60 s of nonischemic and ischemic contractions at 5 Hz. Note significant differences (P < 0.05) between nonischemic and ischemic muscle at 30, 60, and 120 s of recovery (n = 4 per point).
could also be demonstrated in the white gastrocnemius section; however, a relatively low energy demand contraction condition (i.e., 0.5 Hz) is required to remain within the capacity of these low-oxidative muscle fibers (17). Therefore the absence of AMP deaminase binding in the red gastrocnemius section is not a unique feature of this muscle type. When the oxidative capacity of the fasttwitch red gastrocnemius muscle was compromised by ligation of the femoral artery during ~-HZ contractions, a high rate of AMP deamination similar to that occurring in fast-twitch white gastrocnemius section was achieved (11). Again, coincident with enzyme activation, AMP deaminase binding increased to about fivefold that of the nonischemic muscle section contracting at the same intensity (Fig. 1A). Thus AMP deaminase binding to myosin in contracting muscle is associated with contraction conditions that lead to substantial enzyme activation and high rates of IMP and ammonia formation Although the slow-twitch red fibers of the soleus muscle exhibit an inherent difference in the stimulation conditions required to activate AMP deaminase, compared with fast-twitch muscle (18, 29, 31), it is possible to induce extensive AMP deamination with prolonged lowfrequency stimulation (12 tetani/min) when blood flow is eliminated (29, 31). Coincident with extensive AMP deamination, amounting to 36% of the ATP pool (31),
5’-monophosphate;
4 4 5.36k0.33 4.65kO.23 1.55OkO.29 1 1.843kO.153 3.23t0.65 1.66~10.48 58.6k4.4 53.4k3.7 PCr, phosphocreatine.
4 5.24t0.56 1.506kO.328 3.29t0.68 49.2t5.6
this fiber section exhibited an increase in AMP deaminase binding of about fivefold (Fig. 1A). Again, if contractions were elicited without extensive AMP deamination, binding was unchanged from resting muscle (Fig. IB). Thus binding of AMP deaminase could be demonstrated in all three muscle fiber sections of the rat but only under conditions with an energy imbalance that leads to significant ATP depletion. This suggests that the circumstances leading to AMP deaminase binding are related to unique cellular conditions associated with enzyme activation resulting in extensive IMP production. The coincidence between AMP deaminase binding and ATP depletion observed in each of the muscle fiber sections during muscle stimulation in situ does not, of course, establish a cause-and-effect relationship. However, evidence obtained from our time-course evaluation (Fig. 3) suggests that AMP deaminase binding precedes AMP deamination. A significant approximately threefold elevation of AMP deaminase binding occurred before any accumulation of IMP was found (Table 2). Then, AMP deamination proceeded to increase muscle IMP content -7O-fold, while AMP deaminase binding progressed to and remained at its maximum of ---fold above that found in resting muscle (Fig. 3). Although the work of Shiraki et al. (23) demonstrated that AMP deaminase binding was rapid after the onset of contractions, the relationship between enzyme binding and activity could not be established because significant IMP accumulation was not evident. Binding of AMP deaminase to myosin has been shown to activate the enzyme, possibly by release of allosteric inhibition (7), and increase the rate of product formation at low AMP concentrations (21). Thus our findings strongly imply that binding of AMP deaminase by myosin plays a role in the control of AMP deamination in contracting muscle. Although the factors regulating AMP deaminase binding to myosin have not been well characterized, Marquetant et al. (15) found that raising ATP concentration to 4-5 mM induced elution of AMP deaminase off a myosin affinity column. This might suggest that decreasing ATP concentration within the cell could instigate myosin binding of AMP deaminase. It would appear, however, that muscle ATP concentration decreases after binding is initiated (Fig. 3, Table 2). Thus it seems unlikely that
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c292
AMP
DEAMINASE
alterations in ATP concentration play a major role in modifying myosin-AMP deaminase binding in vivo. It is also doubtful that the increase in orthophosphate concentration that occurs during contraction contributes to the binding, because the expected influence of orthophosphate is the opposite of that observed. Increases in orthophosphate, in the range found in contracting muscle, prompt dissociation between myosin and AMP deaminase in vitro (3). Acidosis (pH 6.2-6.5) may be an important factor contributing to the binding of purified AMP deaminase to myosin (7): although this finding has not been uniformly found (23). Our results with muscle that had glycolysis inhibited with IAA indicate that cellular acidosis is not required for extensive AMP deaminase binding or activation to occur. Further, extreme acidosis associated with high lactate content in normal muscle was incapable of sustaining AMP deaminase binding (Fig. 3). AMP deaminase binding returned rapidly toward resting values upon the cessation of contractions, even though lactate contents remained very high (Table 2). However, whether cellular acidosis contributes to AMP deaminase binding and deamination within each of the fiber types has yet to be fully assessed. A low cellular pH is usually (11, 29) but not always (8) found during conditions of significant AMP deamination during exercise in vivo. It may be that factors controlling binding are closely related to the energy state of the cell. In each stimulation condition where AMP deaminase binding occurred, an extensive energy imbalance was evident. Conversely, if an excellent balance was well maintained by keeping contraction conditions within the aerobic function of the muscle section, no increase in AMP deaminase binding occurred. The importance of energy balance is further supported by the rapid decline in AMP deaminase binding after the cessation of contractions, when the high rate of energy expenditure was no longer required. Consistent with this premise is the sustained high binding observed after contractions when the muscle is ischemic (Fig. 4). Isoforms and muscle fiber types. Muscle AMP deaminase exists in two isoforms: form A is the sole isoform present in the heart, and form B is the sole isoform present in fast-twitch white muscle (20). Each isoform demonstrates very different kinetic behavior with respect to cooperativity towards substrate (20, 27), with form B demonstrating a threefold greater affinity for AMP (20). Most relevant to the present results, in vitro studies show that the heart isozyme is insensitive to declines in pH within the range found in contracting muscle (20) and does not bind to purified myosin (24). The rat soleus muscle, which is comprised of predominantly slow-twitch red fibers (Z), contains both isoforms, with -2O-30% of the total as isoform A (20, 28). The extensive binding of isoform B of the fast-twitch white muscle section is self evident (Fig. 1A). On the other hand, if the isoform A found in rat soleus muscle manifests a lack of myosin binding characteristic of the heart isoform, a diminished fraction of total binding of AMP deaminase might be expected during contractions. If this is the case, then the -50% of total AMP deaminase bound in the soleus muscle (cf. Fig. 1) may reflect the extensive binding of isoform
BINDING
IN MUSCLE
B, typical of fast-twitch muscle. Control of AMP deamination. Orthophosphate has been implicated as the primary inhibitor of AMP deaminase in vivo (14, 30) with a Ki of l-2 mM (30). Orthophosphate concentration increases dramatically during intense muscle contractions (16), and its inhibition of AMP deaminase must be effectively overcome for AMP deamination to occur. This may be due, in part, to the increase in ADPf and H+ during contractions (11, 14). The increase in AMPf within the muscle must exert an important and possibly dominant influence to increase AMP deamination, because the enzyme is functioning well below its Km of -1 mM for AMP (11, 14). The results of the present study support the hypothesis that binding of AMP deaminase to myosin also contributes to the control of AMP deamination in vivo. In vitro studies have shown that myosin-bound AMP deaminase is more sensitive to regulation by nucleotides than is free AMP deaminase (7, 25). In addition, in our companion study (21)) we demonstrate that binding of AMP deaminase increases the rate of product formation at low physiological AMP concentrations and reduces inhibition by orthophosphate in the presence of ADP (11). Thus AMP deaminase binding to myosin could be an important means of regulating AMP deamination in vivo. In conclusion, our results indicate that under contraction conditions that result in high rates of IMP formation in rat muscle fibers, the fraction of bound AMP deaminase increases. Additional studies are needed to establish the intriguing hypothesis that AMP deaminase binding to myosin serves as an integral function in controlling enzyme activity in contracting muscle. The excellent technical assistance of David Barrett shour was gratefully appreciated. This study was supported by National Institute of Musculoskeletal and Skin Diseases grant AR-21617. Address for reprint requests: R. L. Terjung, Dept. SUNY Health Science Center Syracuse, 766 Irving Ave., 13210. Received
19 April
1991; accepted
in final
form
10 April
and Judy
Fre-
Arthritis
and
of Physiology, Syracuse, NY 1992.
REFERENCES 1. Agarwal, R. P., and R. E. Parks, Jr. Potent inhibition of muscle V-AMP deaminase by the nucleoside antibiotics coformytin and deoxycoformycin. Biochem. Pharmacol. 26: 663-666,1977. 2. Armstrong, R. B., and R. 0. Phelps. Muscle fiber type composition of the rat hindlimb. Am. J. Anat. 171: 259-272, 1984. 3. Ashby, B., and C. Frieden. Interaction of AMP-aminohydrolase with myosin and its subfragments. J. BioL. Chem. 252: 18691872, 1977. B., and C. Frieden. Adenylate deaminase. Kinetic and 4. Ashby, binding studies on the rabbit muscle enzyme. J. BioL. Chem. 253: 8728-8735, 1978. 5. Ashby, B., C. Frieden, and R. Bischoff. Immunofluorescent and histochemical localization of AMP deaminase in skeletal muscle. J. Cell BioZ. 81: 361-373, 1979. 6. Baldwin, K. M., G. H. Klinkerfuss, R. L. Terjung, P. A. MO&, and J. 0. Holloszy. Respiratory capacity of white, red, and intermediate muscle: adaptive response to exercise. Am. J. Physiol. 222: 373-378, 1972. 7 . Barshop, B. A., and C. Frieden. Analysis of the interaction of rabbit skeletal muscle adenylate deaminase with myosin subfragments. J. Biol. Chem. 259: 60-66, 1984. 8‘. Bromberg, S., and K. Sahlin. Adenine nucleotide degradation in human skeletal muscle during prolonged exercise. J. Appl. Physiol. 67: 116-122, 1989.
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AMP
DEAMINASE
9. Brumback, R. A. Iodoacetate inhibition of glyceraldehyde-3phosphate dehydrogenase as a model of human myophosphorylase deficiency (McArdle’s disease) and phosphofructokinase deficiency (Tarui’s disease). J. NeuroZ. Sci. 48: 383-398, 1980. 10. Dudley, G. A., and R. L. Terjung. Influence of aerobic metabolism on IMP accumulation in fast-twitch muscle. Am. J. Physiol. 248 (Cell Physiol. 17): C37-C42, 1985. 11. Dudley, G. A., and R. L. Terjung. Influence of acidosis on AMP deaminase activity in contracting fast-twitch muscle. Am. J. Physiol. 248 (Cell Physiol. 17): C43-C50, 1985. 12. Dudley, G. A., P. C. Tullson, and R. L. Terjung. Influence of mitochondrial content on the sensitivity of respiratory control. J. Biol. Chem. 262: 9109-9114, 1987. 13. Koretz, J. F. Structural studies of isolated native thick filaments from rabbit psoas muscle with AMP deaminase decoration. Proc. Nutl. Acad. Sci. USA 79: 6205-6209, 1982. 14. Lowenstein, J. M. The purine nucleotide cycle revised. Int. J. Sports Med. 11: 537-546, 1990. 15. Marquetant, R., R. L. Sabina, and E. W. Holmes. Identification of a noncatalytic domain in AMP deaminase that influences binding to myosin. Biochemistry 28: 8744-8749, 1989. 16. Meyer, R. A., T. R. Brown, B. L. Krilowicz, and M. J. Kushmerick. Phosphagen and intracellular pH changes during contraction of creatine-depleted rat muscle. Am. J. Physiol. 250 (Cell Physiol. 19): C264-C274, 1986. 17. Meyer, R. A., G. A. Dudley, and R. L. Terjung. Ammonia and IMP in different skeletal muscle fibers after exercise in rats. J. Appl. Physiol. 49: 1037-1041, 1980. 18. Meyer, R. A., and R. L. Terjung. Differences in ammonia and adenylate metabolism in contracting fast and slow twitch muscle. Am. J. Physiol. 237 (Cell Physiol. 6): Clll-C118, 1979. 19. Meyer, R. A., and R. L. Terjung. AMP deamination and IMP reamination in working skeletal muscle. Am. J. Physiol. 239 (Cell Physiol. 8): C32-C38, 1980. 20. Raggi, A., and M. Ranieri-Raggi. Regulatory properties of AMP deaminase isoenzymes from rabbit red muscle. Biochem. J. 242: 875-879, 1987. 21. Rundell, K. W., P. C. Tullson, and R. L. Terjung. Altered kinetics of AMP deaminase by myosin binding. Am. J. Physiol.
BINDING
IN MUSCLE
c293
263 (Cell Physiol. 32): C294-C299, 1992. 22. Setlow, B., and J. M. Lowenstein. Adenylate deaminase. II. Purification and some regulatory properties of the enzyme from calf brain. J. BioL. Chem. 242: 607-615, 1967. 23. Shiraki, H., S. Miyamoto, Y. Matsuda, E. Momose, and H. Nakagawa. Possible correlation between binding of muscle type and deaminase to myofibrils and ammoniagenesis in rat skeletal muscle on electrical stimulation. Biochem. Biophys. Res. Commun. 100: 1099-1103, 1981. 24. Shiraki, H., H. Ogawa, Y. Matsuda, and H. Nakagawa. Interaction of rat muscle AMP deaminase with myosin: I. Biochemical study of the interaction of AMP deaminase and myosin in rat muscle. Biochim. Biophys. Acta 566: 335-344, 1979. 25 Shiraki, H., H. Ogawa, Y. Matsuda, and H. Nakagawa. ’ Interaction of rat muscle AMP deaminase with myosin: II. Modification of the kinetic and regulatory properties of rat muscle AMP deaminase by myosin. Biochim. Biophys. Acta 566: 345-352, 1979. 26 Smiley, K. L., A. J. Berry, and C. H. Suelter. An improved ’ purification, crystallization, and some properties of rabbit muscle 5’-adenylic acid deaminase. J. Biol. Chem. 242: 2502-2506, 1967. 27 C., and C. J. Coffee. Differential response of AMP ’ Solano, deaminase isozymes to changes in the adenylate energy charge. Biochem. Biophys. Res. Commun. 85: 564-571, 1978. J. L., R. L. Sabina, and D. A. Riley. AMP28* Thompson, deaminase (AMP-da) isozymes in rat skeletal muscle (Abstract). Med. Sci. Sports Exercise 23: S341, 1991. P. C., D. A. Whitlock, and R. L. Terjung. Adenine 2g* Tullson, nucleotide degradation in slow-twitch red muscle. Am. J. Physiol. 258 (Cell Physiol. 27): C258X265, 1990. T. J., and J. M. Lowenstein. Adenylate deaminase 30. Wheeler, from rat muscle. J. Biol. Chem. 254: 8994-8999, 1979. 31. Whitlock, D., and R. L. Terjung. ATP depletion in slowtwitch red muscle of rat. Am. J. Physiol. 253 (Cell Physiol. 22): C426-C432, 1987. 32. Winder, W. W., R. L. Terjung, K. M. Baldwin, and J. 0. Holloszy. Effect of exercise on AMP deaminase and adenylosuccinase in rat skeletal muscle. Am. J. Physiol. 227: 1411-1414,1974.
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binding
in contracting
rat skeletal muscle
KENNETH W. RUNDELL, PETER C. TULLSON, AND RONALD L. TERJUNG Department of Physiology, State University of New York Health Science Center at Syracuse, and Department of Health and Physical Education, Syracuse University, Syracuse, New York 13210 Rundell, Kenneth W., Peter C. Tullson, and Ronald L. Terjung. AMP deaminase binding in contracting rat skeletal muscle. Am. J. Physiol. 263 (Cell Physiol. 32): C287-C293, 1992. -AMP deaminase, which hydrolyses AMP to inosine Y-monophosphate (IMP) and NH,, at high rates during excessive energy demands in skeletal muscle, is activated when bound to myosin in vitro. We evaluated AMP deaminase binding in vivo during muscle contractions to assess whether binding 1) is inherent to deamination and found only with high rates of IMP production or simply coincident with the contractile process and 2) requires cellular acidosis. AMP deaminase activity (pm01 . min-l agg’) was measured in the supernatant (free) and 10*-g pellet (bound) homogenate fractions of muscle of anesthetized rats after in situ contractions to determine the percent bound. In resting muscle, nearly all (-90%) AMP deaminase is free (cytosolic). During contractions when energy balance was well maintained, binding did not significantly differ from resting values. However, during intense contraction conditions that lead to increased IMP concentration, binding increased to -60% (P < 0.001) in fasttwitch and -50% in slow-twitch muscle. Binding increased in an apparent first-order manner and preceded initiation of IMP formation. Further, binding rapidly declined within 1 min after cessation of intense stimulation, even though the cell remained extremely acidotic. Extensive binding during contractions was also evident without cellular acidosis (iodoacetic acid-treated muscle). Thus the in vivo AMP deaminase-myosin complex association/dissociation is not coupled to changes in cellular acidosis. Interestingly, binding remained elevated after contractions, if energy recovery was limited by ischemia. Our results are consistent with myosin binding having a role in AMP deaminase activation and subsequent IMP formation in contracting muscle. inosine 5’-monophosphate; deamination; contractile proteins; muscle fiber types
intense
contractions;
CONTRACTIONS can result in an extensive decrease in total adenine nucleotide content (AMP -t ADP -t ATP) in fast-twitch muscle, amounting to as much as -50% of the ATP pool (10, 11, 17-19). This decrease is accompanied by a production of ammonia and inosine 5’-monophosphate (IMP) by the nonequilibrium reaction catalyzed by AMP deaminase (11, 17-19)
INTENSE
AMP + HZ0 + IMP + NH3 The action of AMP deaminase serves to limit the increase in AMP concentration within the contracting muscle. This in turn pulls the myokinase reaction (ZADP ++ AMP + ATP) to the right, thereby tempering the elevation in ADP concentration. Thus the coordinate activities of the AMP deaminase and myokinase reactions help maintain a high ATP-to-ADP ratio, a potentially important role for AMP deaminase within the cell (14). Kinetic analysis of purified AMP deaminase has demonstrated that ATP, guanosine 5’-triphosphate (GTP), and orthophosphate inhibit the enzyme; the last factor is probably responsible for the effective inhibition of AMP deaminase in resting muscle (14, 30). Current ev0363-6143/92
$2.00
Copyright
idence suggests that AMP, ADP, and H+, factors known to activate purified AMP deaminase (30), are also important in AMP deaminase activation in contracting muscle (11, 19). The available free concentration of AMP (AMPJ within muscle increases as energy turnover increases (12). Because the K,,, of AMP deaminase for AMP (0.5-1.0 mM AMP; Refs. 14, 20, 22, 26, 30) is much greater than the physiological AMPf, the rate of IMP production should be proportional to the increase in AMPI- within the cell. Further, AMP deaminase activity is enhanced by ADP, a modulator that also increases in active muscle (12). As pH decreases from 7.0 to 6.5, ADP becomes a more effective activator of AMP deaminase (30). Although cellular acidosis is not required for high rates of AMP deamination in vivo (11, 12), purified AMP deaminase activity is enhanced by acidosis in vitro, with an optimum pH of -6.2-6.5 (l4,2O, 22), values similar to those found in the myocyte during severe contractions (11). Although important factors for the in vitro regulation of AMP deaminase have been defined, our understanding of the influence of these modulators within contracting muscle is incomplete. Purified AMP deaminase binds reversibly to myosin and is activated by such binding (3, 4, 7, 25). Although the precise mechanism responsible for this enzyme activation has yet to be determined, it is thought that myosin binding releases inhibition (e.g., from GTP) by allosteric modification (3). Barshop and Frieden (7) found that decreases in pH, to an optimum of -6*5, enhanced the binding between AMP deaminase and myosin or its subfragments; however, this influence has not been confirmed (25). Further, Marquetant et al. (15) found that changes in ATP concentration, similar to those observed during intense contractions, modify binding of purified AMP deaminase to myosin. These findings suggest that binding of AMP deaminase by myosin may serve an important regulatory function and help explain the control of AMP deamination that occurs during muscle contraction. The potential physiological role of AMP deaminasemyosin binding has been bolstered by the recent work of Shiraki et al. (23). The fraction of total muscle AMP deaminase activity located in the myosin-containing pellet of rat muscle homogenate increased more than fivefold after intense stimulation (23). It is presently unclear, however, whether the binding of AMP deaminase by myosin is directly related to conditions of enzyme activation or simply occurs with muscle contractions. Further, it is unknown whether AMP deaminase-myosin binding in viva precedes or is a result of ATP depletion. The purpose of this study was to evaluate the potential role of AMP deaminase-myosin binding in different skeletal muscle fiber types in situ during contraction
0 1992 the American
Physiological
Society
C287
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C288
AMP
DEAMINASE
BINDING
conditions known to activate deamination. We determined whether binding of AMP deaminase to myosin is inherent to conditions where high rates of AMP deamination occur or simply coincident with muscle contractions, even in the absence of IMP formation. In addition, we determined whether cellular acidosis is essential for in vivo binding of AMP deaminase to myosin. Our results support the hypothesis that binding of AMP deaminase to myosin contributes to the control of IMP and ammonia production in vivo. METHODS
Animal care and stimulation procedures. Adult SpragueDawley rats (386 t 8.6 g; n = 76; Taconic Farms, Germantown, NY) were housed three per cage in a temperature-controlled room (ZO-21°C) with a 12:12 h light-dark cycle. Purina Lab Chow and water were provided ad libitum. Animals were anesthetized with pentobarbital sodium (50 mg/kg ip). The right gastrocnemius-plantaris-soleus muscle complex was prepared for in situ stimulation as done previously (18). The limb was secured with a pin through the distal shank of the femur, and the distal tendon was connected to a strain gauge mounted on an adjustable frame. During preparation, care was taken not to disturb blood flow to the lower limb. The tibia1 nerve was cut and placed across a shielded platinum bipolar electrode connected to a Grass S48 stimulator. Muscle length was adjusted to provide maximal tension and stimulated 1) with supramaximal pulses (6 V, 0.1 ms duration) of 0.5 or 5 Hz or 2) tetanically with supramaximal square-wave pulses (6 V, 0.1 ms duration) of lOO-ms trains at PO0 Hz at a frequency of 12 tetani/ min. The exposed muscles were kept moist and maintained at 37°C wit,h a heat lamp for the duration of each experiment. Mean aortic pressure was continuously monitored via a catheter inserted into the right carotid artery and was generally stable throughout the contraction period, averaging 110 t 5 mmHg (n = 60). Four series of conditions were used to evaluate in vivo binding of AMP deaminase. Series 1 evaluated the coincidence of binding with deamination. Contraction conditions that have previously demonstrated high rates of IMP formation in fast-twitch white (5 Hz, 3 min), fast-twitch red (5 Hz, ischemic, 3 min), and slow-twitch red (12 tetani/min, ischemic, 10 min) muscle fiber sections were established (1 I, 19, 31). Each fiber section required different contraction conditions to produce deamination, because of differences in the inherent oxidative and glycolytic capacities among muscle fiber types. For example, when stimulated at 5 Hz, fast-twitch white fibers of the rat deplete a large fraction of their ATP (-50%) and become acidotic, whereas fast-twitch red and slow-twitch red fibers demonstrate an excellent energy balance with little increase in IMP content and no significant acidosis (I 1). When the stimulated muscle is rendered ischemic by ligation of the femoral artery, contracting fast-twitch red fibers also display a high rate of deamination and acidosis similar to fast-twitch white fibers (11). Likewise, under extended ischemic stimulation at 12 tetani/min, slow-twitch red fibers incur an w-30-40% decrease in ATP content plus an increase in IMP (29, 31). At the appropriate time during stimulation, the superficial section of the medial gastrocnemius (predominately fast-twitch white fiber type), the deep red section of the lateral gastrocnemius (predominately fast-twitch red fiber type), and the soleus (predominately slow-twitch red fiber type) were clamp frozen with aluminum tongs cooled in liquid nitrogen. The corresponding muscle sections of the contralateral limb were frozen in exact Series 2 conditions were used to examine whether AMP deaminase binding was simply coincident with the contractile
IN MUSCLE
process and therefore not related to the cellular events leading to IMP production. Muscles were stimulated at frequencies where ATP was maintained with modest phosphocreatine hydrolysis and little or no increase in IMP (0.5 Hz and 5 min for fasttwitch white and 5 Hz and 3 min for fast-twitch red and slowtwitch red) (II, 19, 31). Series 3 was performed to assess the effects of cellular acidosis on in vivo binding of AMP deaminase by using iodoacetic acid (IAA) treatment to inhibit lactate formation and the consequent acidosis during contractions (9). Four minutes before and during ~-HZ stimulation, IAA was infused (1.4 pm01 . min-l .g body wt-‘) in the descending aorta via the contralateral femoral artery as done previously (1 I). Muscle samples of the right gastrocnemius-plantaris-soleus complex were clamp frozen after 30 s stimulation as described above. Control muscle samples were clamp frozen after 4 min IAA infusion with no stimulation. Series 4 was performed to 1) measure the correlation between AMP deaminase-myosin binding and IMP formation over time and 2) examine the persistence of binding during recovery after stimulation. Fast-twitch white muscle sections were clamp frozen after 15, 30, 60, and 180 s of ~-HZ contractions. Recovery samples were taken at 15, 30, 60, and 120 s after 60 s of stimulation. To further explore binding retention, ischemic fasttwitch white muscle sections were clamp frozen after 60 s of ~-HZ contractions and 30,60, and 120 s after 60 s of stimulation. Ischemia was established by ligation of the femoral artery IO s before stimulation and maintained through the 120-s recovery period. Enzyme assay. AMP deaminase activity (pm01 . min+ . g-l) was measured spectrophotometrically at 285 nm (extinction coefficient at 285 nm = 0.3) (26). The assay was performed at 30°C in 1 ml of medium containing 2 mM AMP, 150 mM KCl, and 50 mM imidazole HCl (pH 7.0). Specificity of the assay system was verified by the total elimination of activity by the presence of 1 X 10B6 M coformycin, a potent inhibitor of AMP deaminase (I), in the assay medium. Frozen tissue samples were crushed under liquid nitrogen and homogenized in 9 vol of 150 mM KCl, 2 mM EDTA, 10 mM glutathione, pH 7.0 for 20 s using a ground-glass Potter-Elvehjem tissue grinder. Myofilaments are insoluble in 150 mM KC1 and rapidly sediment during centrifugation (microcentrifuge at - 16,000 g for 1 min). Myosin-bound AMP deaminase is carried along, because at this concentration of KCl, AMP deaminase does not dissociate from myosin (3, 23). We verified this by altering the homogenizing medium to conditions known to modify AMP deaminase binding. Neither acidosis (pH 6.5 vs. pH 7.0) nor high (IO mM) orthophosphate concentration changed the recovery of bound AMP deaminase in the pellet. Thus, as done by Shiraki et al. (23), the activity in the supernatant was considered that of the free (unbound) enzyme. Assay of AMP deaminase bound to myosin in the muscle homogenate pellet was made difficult because of the presence of cell debris that cosediments. Therefore we assayed the bound fraction of AMP deaminase by first extracting the enzyme in a high-KC1 buffer. This was achieved by suspending the myosin-containing pellet in 9 vol of 500 mM KC1 and 50 mM Tris HCl, pH 7.55. Bound AMP deaminase was dissociated from myosin by incubation at 37°C for 30 min and then centrifuged at - 16,000 g for 1 min. Activity in the supernatant of the rehomogenized pellet was considered that of myosin-bound enzyme, because previous studies (3, 7) have shown that the AMP deaminase-myosin complex is completely dissociated in 500 mM KCl. We verified quantitative recovery of the formerly bound enzyme activity in the supernatant. No AMP deaminase activity remained in the particulate fraction of the 500 mM KC1 extract. Assays were performed immediately upon sample preparation, because there is some loss of enzyme activity over time (- 15% in 20 min).
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AMP
DEAMINASE
BINDING
However, there was verv little loss of activitv between duplicate samples (XUL?& l/s~-& 2 = 1.005 t 0.0657; rt = 2OOj. As a measure of reliabilitv, activity in the whole homogenate of each tissue sample was cbmpared‘with activity in the-150 mM KC1 supernatant plus activity in the 500 mM KC1 supernatant of the rehomogenized pellet. The sum of the activity of the component fractions was within 5% of the activity of the whole homogenate [i.e., (150 mM KC1 supernatant + 500 mM KC1 supernatant)/ whole homogenate = 1.05 t 0.036; n = 801. Therefore total muscle enzyme activity was obtained as the sum of the two fractions. Metabolic asscws. Frozen tissue samples from series 3 and series 4 were extracted in ethanolic perchloric acid and neutralized with KOH containing 0.5 M triethanolamine as routinely done (31). The perchloric acid extracts were assayed for phosphocreatine, creatine, and lactate as done previously (31). ATP, ADP, AMP, and IMP were quantified by reverse-phase highperformance liquid chromatography as done previously (29). Because the responses of IMP accumulation of series 1 and series 2 have been well characterized (I l), we have not repeated these determinations. Muscle water content was determined by drying ~0.2 g tissue sample at 80°C to a constant dry weight. All metabolites were corrected to the water content of the nonstimulated contralateral control muscle tissue, to account for any change in water content that occurred with muscle contractions (18). Statistical analyses. Statistical comparisons between and within fiber types were made by analysis of variance. Critical differences (P < 0.05) were determined by Tukey’s post hoc analysis. RESULTS
Total actiuity. The total enzyme activity (free plus bound) of the muscle is unchanged by contractions (Table 1, P > 0.05). However, the distribution of AMP deaminase is markedly shifted from a free to bound form during intense contractions that result in extensive AMP deamination. Values of total AMP deaminase activity given in Table 1 also demonstrate differences between each of the skeletal muscle fiber sections as reported previously (32). Series 1: AMP deaminase binding and deamination. In rested muscle, nearly all (-90%) AMP deaminase is free (not bound; Fig. 1A). During stimulation conditions (5 Hz, 3 min) where a ~50% depletion of ATP leads to large increases in IMP and acidosis (estimated pH 6.1) (1 I), binding increased sixfold over resting values (P < 0.001) in the fast-twitch white gastrocnemius muscle section. Similarly, during stimulation conditions that resulted in a significant increase in IMP content (-40% depletion of ATP) with a reduction in estimated cellular pH to C6.2 in the fast-twitch red gastrocnemius muscle section (ischemic contractions at 5 Hz) (1 l), AMP deaminase binding increased about fivefold over control values (Fig. lA, Table 1. Total muscle AMP deaminase activity Gastrocnemius Soleus White
section
28 Eontrol Stimulated
324t9.0 30729.1
Red
section
10
9
172&12.9
84.W4.9
184t28.0
75.31r3.4
Values are means t SE in prnoll’. min-’ .g; n, no. of muscles. Gastrocnemius is made up predominately of fast-twitch red and white fibers and soleus of predominately slow-twitch red fibers.
C289
IN MUSCLE I
A
Fig. 1. AMP deaminase activity as percent of total bound during contractions that result in high rates of AMP deamination (A) and during contractions that result in little AMP deamination (B). See RESULTS for specific conditions (n = 4-6 per tissue).
P < 0.001). Thus, during intense contractions, both fasttwitch fiber types exhibited increased AMP deaminase binding when IMP production concomitant with a decreased pH occurred. Binding also increased about fivefold in the slow-twitch red soleusafter 10 min of ischemic stimulation at 12 tetani/min (Fig. lA, P < O.OOl), shown previously to lead to significant deamination (m 1.25 ,umol/g IMP) (29,31). Thus binding of AMP deaminase is not unique to either fast-twitch or slow-twitch fibers but occurs during conditions that lead to high rates of AMP deamination. This would have to occur if binding of AMP deaminase to myosin is essential for enzyme activation. Series 2: coincidence of A*MP deaminase binding and contraction. In contrast to the response of muscle where high rates of AMP deamination occur, AMP deaminase binding was not increased during contractions that do not result in substantial IMP production. This was found for all three fiber type sections (Fig. IB). Although the specific stimulation conditions differed among fiber types (fast-twitch white gastrocnemius 0.5 Hz, fast-twitch red gastrocnemius and slow-twitch red soleus 5 Hz), little if any accumulation of IMP occurs (11,17,29,31). Thus the binding of A.MP deaminase does not occur as a simple consequence of muscle contractions. Series 3: effects of cellular acidosis on binding. Because acidosis has been identified as an important condition contributing to in vitro binding of AMP deaminase to myosin (7) and is coincident with deamination during contractions in vivo (1 l), we evaluated whether cellular acidosis is obligatory for AMP deaminase binding in vivo. After 30 s of stimulation at 5 Hz, the bound fraction of AMP deaminase from IAA-treated muscle significantly increased for fast-twitch white, fast-twitch red, and slowtwitch red muscle fiber sections (Fig. 2, P < 0.001). The fraction of bound enzyme from nonstimulated control tissue sampled after 4 min of IAA infusion (before stimulation) was not significantly different from that of rested muscle without IAA infused (P > 0.05). Lactate contents of IAA-treated rat fast-twitch white muscle (2.2 t- 0.22 pmol/g, n = 5) after stimulation did not differ from rested fast-twitch white muscle (2.0 t 0.60 pmol/g, P > 0.05); however, an accelerated depletion of ATP content
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c290
AMP
DEAMINASE
0
0.05). However, as binding of AMP deaminase proceeded to its peak response of an approximately sixfold increase over resting values, IMP formation became evident (Fig. 3). IMP formation accounted for -500% depletion of ATP while AMP deaminase remained maximally bound. When muscle stimulation ended at 60 s, no further ATP depletion or IMP accumulation occurred (Table Z), and AMP deaminase binding returned rapidly to resting values in nonischemic muscle tissue (Fig. 4). The extreme cellular lactic acidosis, characterized by a lactate content of -30 pmol/g (Table Z), did not sustain AMP deaminase binding in the absence of continued intense muscle stimulation (Fig. 4). However, the rapid dissociation of binding
no
0
I
30
=
I
60
Time
=,
-
90
I
120
-
I
150
-
1
L 0.0
180
(seconds)
Fig. 3. Time course of AMP deaminase binding and inosine Y-monophosphate (IMP) accumulation in fast-twitch white muscle samples during 180 s of contractions at 5 Hz. Note that significant binding (P < 0.001) precedes IMP formation at 15 s of contractions (n = 34 and 6 per point for control and stimulated, respectively). The 120-s IMP value was taken from Dudley et al. (12).
Considerable evidence demonstrates that AMP deaminase binds reversibly to purified myosin (3-5, 7, 13, 24). The demonstration by Shiraki et al. (23) that AMP deaminase binds to myosin during contractions suggests that binding may be an important factor in the regulation of deamination in situ. Our results extend the work of Shiraki et al. (23) to show that AMP deaminase binding occurs in all skeletal muscle fiber types, requires an excessively high energy demand during contractions, precedes IMP production, does not require extreme cellular acidosis, and is retained after contractions by ischemia. We interpret the activity of AMP deaminase found in the pellet after low-ionic-strength homogenization as the bound form of the enzyme caused by contractions in vivo. Myofilaments are insoluble in 150 mM KCl, and the myosin-bound AMP deaminase is carried to the pellet upon centrifugation. The unbound AMP deaminase remains in the supernatant. This separation is not an artifact, because homogenizing the quick-frozen tissue in a medium known to increase binding [pH 6.5 vs. 7.0 (7)] or decrease binding [high orthophosphate (7)] did not modify the bound activity. Rather, our results indicate that inducing an energy deficit within contracting muscle seems to be the major factor prompting AMP deaminase binding to myosin. AMP deaminase binding. Binding of AMP deaminase could be induced in all three muscle fiber type sections. The contraction conditions necessary to induce binding in each fiber also caused an extensive AMP deamination to IMP and ammonia. This occurs in fast-twitch muscle when the rate of ATP hydrolysis exceeds the rate of ADP rephosphorylation (11, 18). The contraction conditions necessary to create an energy imbalance can differ among fiber type sections, because each section possesses an inherently different oxidative capacity available to meet the energy demand (6). The energetic demands required to produce high rates of deamination were most easily created in the low-oxidative fast-twitch white section of the gastrocnemius muscle. Coincident with an extensive activation of AMP deaminase that results in an ~50% decrease in ATP content (11)) the bound fraction of AMP deaminase increased about sixfold (Fig. 1A). The occurrence of AMP deaminase binding was not simply a result of muscle contractions, because the high-oxidative fasttwitch red section of the same contracting muscle group did not demonstrate any increase in AMP deaminase binding (Fig. 1B). This muscle section has an extremely high aerobic capacity and is capable of meeting the energy needs of this contraction condition without a substantial rate of AMP deamination (11, 17). Consistent with this, an absence of binding during contractions
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AMP
Table 2. Fast-twitch
white gastrocnemius
DEAMINASE
metabolites
Stimulation,
Rested Control
BINDING
15
during
Poststimulation,
30
:TP IMP PCr Lactate
6 6.57t0.34 0.023t0.006 11.89kO.92 13.8t1.6
S-Hz stimulation
s 60
Blood 34 7.42t0.10 0.013t0.007 22.69t0.41 2.9t0.2
c291
IN MUSCLE
6 6.29t0.43 0.465kO.217 6.71kO.62 31.4k2.2
15
flow
30
s 60
120
4 5.55&O. 13 0.93620.2 12 7.76k3.11 32.0t6.5
4 5.15t0.19 1.072&O. 189 ll.lOt3.03 28.0t4.3
intact
6 5.26&O. 14 1.281k0.169 3.86t0.55 37.3t2.5
4 4.94t0.35 0.854t0.219 4.70t0.54 23.Ok1.8
4 5.56t0.22 0.864kO.127 7.18tl.11 31.4t2.9
Ischemic 16 iTP 7.60~10.15 IMP 0.005~0.001 PCr 20.80t0.78 Lactate 3.6t0.2 Values are means -t- SE in pmol/g;
4 4.78t1.03 1.214t0.199 4.04t1.60 57.226.0 IMP, inosine
n, no. of animals/group.
Non-ischemic ---0
-60
0
30
Time
d
60
90
120
(seconds)
Fig. 4. Time course of AMP deaminase binding in fast-twitch white muscle samples during recovery after 60 s of nonischemic and ischemic contractions at 5 Hz. Note significant differences (P < 0.05) between nonischemic and ischemic muscle at 30, 60, and 120 s of recovery (n = 4 per point).
could also be demonstrated in the white gastrocnemius section; however, a relatively low energy demand contraction condition (i.e., 0.5 Hz) is required to remain within the capacity of these low-oxidative muscle fibers (17). Therefore the absence of AMP deaminase binding in the red gastrocnemius section is not a unique feature of this muscle type. When the oxidative capacity of the fasttwitch red gastrocnemius muscle was compromised by ligation of the femoral artery during ~-HZ contractions, a high rate of AMP deamination similar to that occurring in fast-twitch white gastrocnemius section was achieved (11). Again, coincident with enzyme activation, AMP deaminase binding increased to about fivefold that of the nonischemic muscle section contracting at the same intensity (Fig. 1A). Thus AMP deaminase binding to myosin in contracting muscle is associated with contraction conditions that lead to substantial enzyme activation and high rates of IMP and ammonia formation Although the slow-twitch red fibers of the soleus muscle exhibit an inherent difference in the stimulation conditions required to activate AMP deaminase, compared with fast-twitch muscle (18, 29, 31), it is possible to induce extensive AMP deamination with prolonged lowfrequency stimulation (12 tetani/min) when blood flow is eliminated (29, 31). Coincident with extensive AMP deamination, amounting to 36% of the ATP pool (31),
5’-monophosphate;
4 4 5.36k0.33 4.65kO.23 1.55OkO.29 1 1.843kO.153 3.23t0.65 1.66~10.48 58.6k4.4 53.4k3.7 PCr, phosphocreatine.
4 5.24t0.56 1.506kO.328 3.29t0.68 49.2t5.6
this fiber section exhibited an increase in AMP deaminase binding of about fivefold (Fig. 1A). Again, if contractions were elicited without extensive AMP deamination, binding was unchanged from resting muscle (Fig. IB). Thus binding of AMP deaminase could be demonstrated in all three muscle fiber sections of the rat but only under conditions with an energy imbalance that leads to significant ATP depletion. This suggests that the circumstances leading to AMP deaminase binding are related to unique cellular conditions associated with enzyme activation resulting in extensive IMP production. The coincidence between AMP deaminase binding and ATP depletion observed in each of the muscle fiber sections during muscle stimulation in situ does not, of course, establish a cause-and-effect relationship. However, evidence obtained from our time-course evaluation (Fig. 3) suggests that AMP deaminase binding precedes AMP deamination. A significant approximately threefold elevation of AMP deaminase binding occurred before any accumulation of IMP was found (Table 2). Then, AMP deamination proceeded to increase muscle IMP content -7O-fold, while AMP deaminase binding progressed to and remained at its maximum of ---fold above that found in resting muscle (Fig. 3). Although the work of Shiraki et al. (23) demonstrated that AMP deaminase binding was rapid after the onset of contractions, the relationship between enzyme binding and activity could not be established because significant IMP accumulation was not evident. Binding of AMP deaminase to myosin has been shown to activate the enzyme, possibly by release of allosteric inhibition (7), and increase the rate of product formation at low AMP concentrations (21). Thus our findings strongly imply that binding of AMP deaminase by myosin plays a role in the control of AMP deamination in contracting muscle. Although the factors regulating AMP deaminase binding to myosin have not been well characterized, Marquetant et al. (15) found that raising ATP concentration to 4-5 mM induced elution of AMP deaminase off a myosin affinity column. This might suggest that decreasing ATP concentration within the cell could instigate myosin binding of AMP deaminase. It would appear, however, that muscle ATP concentration decreases after binding is initiated (Fig. 3, Table 2). Thus it seems unlikely that
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c292
AMP
DEAMINASE
alterations in ATP concentration play a major role in modifying myosin-AMP deaminase binding in vivo. It is also doubtful that the increase in orthophosphate concentration that occurs during contraction contributes to the binding, because the expected influence of orthophosphate is the opposite of that observed. Increases in orthophosphate, in the range found in contracting muscle, prompt dissociation between myosin and AMP deaminase in vitro (3). Acidosis (pH 6.2-6.5) may be an important factor contributing to the binding of purified AMP deaminase to myosin (7): although this finding has not been uniformly found (23). Our results with muscle that had glycolysis inhibited with IAA indicate that cellular acidosis is not required for extensive AMP deaminase binding or activation to occur. Further, extreme acidosis associated with high lactate content in normal muscle was incapable of sustaining AMP deaminase binding (Fig. 3). AMP deaminase binding returned rapidly toward resting values upon the cessation of contractions, even though lactate contents remained very high (Table 2). However, whether cellular acidosis contributes to AMP deaminase binding and deamination within each of the fiber types has yet to be fully assessed. A low cellular pH is usually (11, 29) but not always (8) found during conditions of significant AMP deamination during exercise in vivo. It may be that factors controlling binding are closely related to the energy state of the cell. In each stimulation condition where AMP deaminase binding occurred, an extensive energy imbalance was evident. Conversely, if an excellent balance was well maintained by keeping contraction conditions within the aerobic function of the muscle section, no increase in AMP deaminase binding occurred. The importance of energy balance is further supported by the rapid decline in AMP deaminase binding after the cessation of contractions, when the high rate of energy expenditure was no longer required. Consistent with this premise is the sustained high binding observed after contractions when the muscle is ischemic (Fig. 4). Isoforms and muscle fiber types. Muscle AMP deaminase exists in two isoforms: form A is the sole isoform present in the heart, and form B is the sole isoform present in fast-twitch white muscle (20). Each isoform demonstrates very different kinetic behavior with respect to cooperativity towards substrate (20, 27), with form B demonstrating a threefold greater affinity for AMP (20). Most relevant to the present results, in vitro studies show that the heart isozyme is insensitive to declines in pH within the range found in contracting muscle (20) and does not bind to purified myosin (24). The rat soleus muscle, which is comprised of predominantly slow-twitch red fibers (Z), contains both isoforms, with -2O-30% of the total as isoform A (20, 28). The extensive binding of isoform B of the fast-twitch white muscle section is self evident (Fig. 1A). On the other hand, if the isoform A found in rat soleus muscle manifests a lack of myosin binding characteristic of the heart isoform, a diminished fraction of total binding of AMP deaminase might be expected during contractions. If this is the case, then the -50% of total AMP deaminase bound in the soleus muscle (cf. Fig. 1) may reflect the extensive binding of isoform
BINDING
IN MUSCLE
B, typical of fast-twitch muscle. Control of AMP deamination. Orthophosphate has been implicated as the primary inhibitor of AMP deaminase in vivo (14, 30) with a Ki of l-2 mM (30). Orthophosphate concentration increases dramatically during intense muscle contractions (16), and its inhibition of AMP deaminase must be effectively overcome for AMP deamination to occur. This may be due, in part, to the increase in ADPf and H+ during contractions (11, 14). The increase in AMPf within the muscle must exert an important and possibly dominant influence to increase AMP deamination, because the enzyme is functioning well below its Km of -1 mM for AMP (11, 14). The results of the present study support the hypothesis that binding of AMP deaminase to myosin also contributes to the control of AMP deamination in vivo. In vitro studies have shown that myosin-bound AMP deaminase is more sensitive to regulation by nucleotides than is free AMP deaminase (7, 25). In addition, in our companion study (21)) we demonstrate that binding of AMP deaminase increases the rate of product formation at low physiological AMP concentrations and reduces inhibition by orthophosphate in the presence of ADP (11). Thus AMP deaminase binding to myosin could be an important means of regulating AMP deamination in vivo. In conclusion, our results indicate that under contraction conditions that result in high rates of IMP formation in rat muscle fibers, the fraction of bound AMP deaminase increases. Additional studies are needed to establish the intriguing hypothesis that AMP deaminase binding to myosin serves as an integral function in controlling enzyme activity in contracting muscle. The excellent technical assistance of David Barrett shour was gratefully appreciated. This study was supported by National Institute of Musculoskeletal and Skin Diseases grant AR-21617. Address for reprint requests: R. L. Terjung, Dept. SUNY Health Science Center Syracuse, 766 Irving Ave., 13210. Received
19 April
1991; accepted
in final
form
10 April
and Judy
Fre-
Arthritis
and
of Physiology, Syracuse, NY 1992.
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9. Brumback, R. A. Iodoacetate inhibition of glyceraldehyde-3phosphate dehydrogenase as a model of human myophosphorylase deficiency (McArdle’s disease) and phosphofructokinase deficiency (Tarui’s disease). J. NeuroZ. Sci. 48: 383-398, 1980. 10. Dudley, G. A., and R. L. Terjung. Influence of aerobic metabolism on IMP accumulation in fast-twitch muscle. Am. J. Physiol. 248 (Cell Physiol. 17): C37-C42, 1985. 11. Dudley, G. A., and R. L. Terjung. Influence of acidosis on AMP deaminase activity in contracting fast-twitch muscle. Am. J. Physiol. 248 (Cell Physiol. 17): C43-C50, 1985. 12. Dudley, G. A., P. C. Tullson, and R. L. Terjung. Influence of mitochondrial content on the sensitivity of respiratory control. J. Biol. Chem. 262: 9109-9114, 1987. 13. Koretz, J. F. Structural studies of isolated native thick filaments from rabbit psoas muscle with AMP deaminase decoration. Proc. Nutl. Acad. Sci. USA 79: 6205-6209, 1982. 14. Lowenstein, J. M. The purine nucleotide cycle revised. Int. J. Sports Med. 11: 537-546, 1990. 15. Marquetant, R., R. L. Sabina, and E. W. Holmes. Identification of a noncatalytic domain in AMP deaminase that influences binding to myosin. Biochemistry 28: 8744-8749, 1989. 16. Meyer, R. A., T. R. Brown, B. L. Krilowicz, and M. J. Kushmerick. Phosphagen and intracellular pH changes during contraction of creatine-depleted rat muscle. Am. J. Physiol. 250 (Cell Physiol. 19): C264-C274, 1986. 17. Meyer, R. A., G. A. Dudley, and R. L. Terjung. Ammonia and IMP in different skeletal muscle fibers after exercise in rats. J. Appl. Physiol. 49: 1037-1041, 1980. 18. Meyer, R. A., and R. L. Terjung. Differences in ammonia and adenylate metabolism in contracting fast and slow twitch muscle. Am. J. Physiol. 237 (Cell Physiol. 6): Clll-C118, 1979. 19. Meyer, R. A., and R. L. Terjung. AMP deamination and IMP reamination in working skeletal muscle. Am. J. Physiol. 239 (Cell Physiol. 8): C32-C38, 1980. 20. Raggi, A., and M. Ranieri-Raggi. Regulatory properties of AMP deaminase isoenzymes from rabbit red muscle. Biochem. J. 242: 875-879, 1987. 21. Rundell, K. W., P. C. Tullson, and R. L. Terjung. Altered kinetics of AMP deaminase by myosin binding. Am. J. Physiol.
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