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Creatine kinase in regulation of heart function and metabolism. I. Further evidence for compartmentation of adenine nucleotides in cardiac myofibrillar and sarcolemmal coupled ATPase-creatine kinase systems
254
Biochimica et Btop/~vstca Acta, 803 (1984) 254 264 Elsevier
BBA 11268
CREATINE KINASE IN REGULATION OF HEART FUNCTION AND METABOLISM I. FURTHER EVIDENCE FOR COMPARTMENTATION OF ADENINE NUCLEOTIDES IN CARDIAC MYOFIBRILLAR AND SARCOLEMMAL COUPLED ATPase-CREATINE KINASE SYSTEMS V.A. SAKS a,*, R. V E N T U R A - C L A P I E R b, Z.A. H U C H U A a, A.N. P R E O B R A Z H E N S K Y a and I.V. EMELIN ~ " Laboratory of Cardiac Bioenergetics, U.S.S.R. Cardiology Research Center, 3 Cherepkovskaya Street 15, Moscow 121552 ( U. S. S. R.) and Laboratoire de Physiologie Cellulaire Cardiaque, Universitb de Paris - Sud, Orst
Key words: Compartmentation; Adenine nucleotide; Creatine kinase," A TPase; (Heart)
In isolated and purified cardiac myofibriilar and sarcolemmal preparations, the route of movement of ADP produced in the Mg2+-ATPase reactions was studied by investigating the efficiency of competition between the endogenous creatine kinase and exogenous pyruvate kinase reactions. In the homogeneous control system composed of hexokinase and glucose as ATPase, soluble creatine kinase rapidly rephosphorylated ADP produced in the presence of 1 mM ATP, but the addition of pyruvate kinase in an increasing amount inhibited the reaction of creatine release from phosphocreatine and symmetrically increased the rate of pyrnvate prduction from phosphoenolpyruvate. At a pyruvate-kinase/creatine-kinase activity ratio (PK/CK) of 50, all ADP was used by the pyruvate kinase. In myofibrillar and sarcolemmai preparations containing particulate creatine kinase, the creatine kinase reaction was much less efficiently suppressed by pyruvate kinase, and at P K / C K = 50 half-maximal release of creatine was still observed. The rate of immediate myofibrillar MgADP rephosphorylation in the endogenous creatine-kinase reaction was observed to be governed by the concentration of phosphocreatine in accordance with the kinetics of this enzyme. The physiological significance of these findings is discussed.
Introduction An increasing amount of biochemical evidence supports the concept of energy channelling by the phosphocreatine-creatine shuttle in muscle cells [1-5], which explains many recent observations on cardiac muscle physiology under ischemic and hypodynamic conditions [3,6-8]. The importance of the phosphocreatine-creatine shuttle and the potential regulatory role of this system in cardiac muscle contraction is based on the assumed phe* To whom correspondence should be addressed. Abbreviations: CK, creatine kinase; PK, pyruvate kinase; EGTA, ethylene glycol bis(fl-aminoethylether)-N,N'-tetraacetate; IU, international units of enzyme activity, t~mol/min. 0167-4889/84/$03.00 © 1984 Elsevier Science Publishers B.V.
nomenon of compartmentation of adenine nucleotides inside the highly organized cellular structures, where only the amount of ATP which is located immediately in myofibrils is considered to be rapidly used for contraction and is replenished at the expense of phosphocreatine due to the creatine-kinase reaction [2,3]. In mitochondria, creatine-kinase reaction is coupled to oxidative phosphorylation via adenine nucleotide translocase and participates in compartmentation of adenine nucleotides in these stuctures [3,5,6,9-13]. The present joint study was undertaken to investigate the question whether close interaction of creatine kinase MM isoenzyme with ATPases, in myofibrils and on the sarcolemma, can provide efficient func-
255
Materials and Methods
buffered with 20 mM imidazole (pH 7.3). The gradients were centrifuged overnight in a Beckman SW-50 rotor at 35 000 rpm. The fraction at the 20 mM imidazole and 8.7% dextran 15 interface was collected and vesicles were recovered by sedimentation at 40000 rpm for 1.5 h. The pellet was resuspended in a small volume of buffer I and used as a sarcolemmal preparation.
Isolation of myofibrils
Reaction medium
Myofibrils were isolated from Wistar-rat hearts by a method described earlier in a paper from this laboratory [14] with the exception that the first part of step B - treatment in a solution of 0.1 M potassium phosphate and 0.1 M KC1 - was omitted. The final preparation was suspended in 60 mM K C I / 3 0 mM imidazole/2 mM MgC12/2 mM dithiothreitol (pH 7.2).
In experiments with myofibrils, in which the rates of creatine release from phosphocreatine and pyruvate release from phosphoenolpyruvate in the competing creatine-kinase and pyruvate-kinase reactions were determined, the reaction medium contained 25 mM Tris-HC1 (pH 7.4), 20 mM glucose, 5 mM MgCI 2, 1 mM ATP, 1 mM phosphoenolpyruvate, and usually 10 mM phosphocreatine. Dithiothreitol had to be omitted, since it was found to inhibit the colorimetric reaction of creatine assay. The reaction was run at 30°C and was started with addition of myofibrils to 1.0-1.5 m g / m l that gave 0.1-0.15 I U / m l of both creatine kinase and myofibrillar ATPase. Prior to the addition of myofibrils, pyruvate kinase was added to the medium to reach the P K / C K ratio required (from 0 to 100). The reaction was run for 2 and 4 min and stopped by mixing 1 ml of sample with 0.5 ml of 10% perchloric acid. After rapid neutralization with 2.5 M K2CO 3 the mixture was analyzed for free creatine, pyruvate and ADP. In control experiments hexokinase and soluble creatine kinase (rabbit skeletal muscle) of equal activity (0.1-0.15 I U / m l ) were added instead of myofibrils. The reaction was started with hexokinase.
tional compartmentation of adenine nucleotides in these systems. With this purpose the routes of ADP produced in the ATPase reactions were studied by using exogenous pyruvate kinase-phosphoenolpyruvate system, which competes with the creatine kinase reaction for ADP.
Isolation of sarcolemmal preparations Sarcolemmal preparation from rabbit heart was isolated by modification of the method recently described in Refs. 15 and 16. All isolation steps were carried out at 0-4°C. Rabbits were killed by cervical dislocation and hearts were placed in buffer I (20 mM imidazole/0.25 M sucrose (pH 7.3)). Ventricles were separated from atria and large blood vessels and were homogenized in buffer I (tissue weight/buffer volume, 1 : 5) using Virtis45 homogenizer at half-maximal speed for 1 min. The homogenate was filtered through cheesecloth, layered upon 1.1 M sucrose/20 mM imidazole (pH 7.3), and centrifuged at 26 000 rpm for 1 h in a Beckman SW-27 rotor. The supernatant was centrifuged at 40000 rpm for 1 h in a Beckman type 45 Ti rotor. The pellet was resuspended in 20 ml of buffer I and layered onto a sucrose-density step gradient consisting of 0.73 M sucrose (20 ml) and 1.78 M sucrose (7 ml), buffered with 20 mM imidazole (pH 7.3) (10 ml of homogenate/ centrifuge tube). The gradients were centrifuged in a Beckman SW-27 rotor at 26000 rpm for 2-3 h. Microsomal fraction was collected at the 0.25/0.73 M sucrose interface, diluted with buffer I and centrifuged at 40000 rpm for 1 h in a Beckman type 45 Ti rotor. The pellet was resuspended in 4 ml of 20 mM imidazole (pH 7.3) and layered on top of a discontinuous two-step gradient, containing 2 ml of 8.7% and 1 ml of 18% dextran 15,
Spectrophotometric determination of the rate of ADPase reaction This determination was carried out by using the coupled pyruvate kinase-lactate dehydrogenase system [17,18]. The reaction medium contained 0.30 M sucrose, 25 mM Tris-HC1 (pH 7.4), 5 mM magnesium acetate, 5 mM potassium acetate, 1 mM phosphoenolpyruvate, 1 mM ATP and 0.2 mM NADH, 0.3 mM dithiothreitol and 2 I U / m l lactate dehydrogenase. The reaction was started after stabilization of absorbance by addition of myofibrils (0.2-0.3 m g / m l ) or sarcolemmal pre-
256
paration (0.015-0.025 m g / m l ) . In the latter case. 100 mM Na + and 10 m M K + were present. The temperature was 30°C.
Assays Creatine concentration was determined by a colorimetric method [19]. A D P concentration as well as pyruvate concentration were determined enzymatically [20]. Pyruvate-kinase activity was determined in the medium described for spectrophotometric ATPase determination when the pyruvate kinase was a rate-limiting step. Creatinekinase activity was determined by the reverse reaction using a spectrophotometric-coupled enzyme hexokinase and glucose-6-phosphate dehydrogenase assay system [21]. Hexokinase activity was assayed under similar conditions. Protein concentration was determined by a biuret method
[]4]. Principles of the investigation of ADP-movement routes in myofibrillar and sarcolemmal preparation The principle of the method used is schematically illustrated in Fig. 1. This method takes into account two possible ways of A D P movement from the ATPase-active centers where it is produced at the constant steady-state rate, v0, under conditions of saturation of the ATPase with its substrate, MgATP. One way is a rapid dissociation of A D P into the medium, this giving rise to the free medium M g A D P concentration, [MgADP]m. The second way is a possible direct channelling of M g A D P to the active center of structurally bound creatine kinase without an intermediate release of A D P into the medium. To differentiate between these two possible ways of A D P movement, we
Pyruvate MgATP
'MgADP~_~:3~-MgAOPm
CK.MgAT~.~CK.MgADP Cr
PCr
Fig. 1. Schematic presentation of principles of a method used to study the route of movement of MgADP produced in the MgZ+-ATPase reactions in cardiac myofibrillar and sarcolemmal preparations. MgADP m, MgADP in the medium; PEP, phosphoenolpyruvate; Cr, creatine; PCr, phosphocreatine.
have studied the efficiency of competition of creatine kinase with the added exogenous pyruvatekinase system which utilized only A D P from the medium, MgADP m. In the homogeneous system, with the complete intermediate release of MgADP from the ATPase (hexokinase+ glucose), both creatine kinase and pyruvate kinase use the same pool of MgADP, MgADP m, and an increase in the P K / C K ratio should shift the pathway of ADP utilization from creatine-kinase (creatine release from phosphocreatine) to the pyruvate-kinase reaction (pyruvate release from phosphoenolpyruvate). In the second series of experiments, myofibrils or sarcolemmal preparations containing particulate-bound creatine kinase were used, and soluble exogenous pyruvate kinase was added to achieve the same P K / C K ratios, and to remove A D P from the medium. It should be expected that in the case of direct channeling of MgADP from ATPase to creatine kinase the latter reaction in myofibrils or in sarcolemmal preparation becomes less sensitive to the addition of pyruvate kinase, as compared with the homogeneous control system. A similar approach was earlier successfully used to study the mitochondrial creatine-kinase reaction
[131. Results
Competition between creatine kinase and pyruvate kinase for MgADP in the homogeneous system As shown in Fig. 2, in the reconstituted system consisting of hexokinase plus glucose as a sourve of MgADP, in the presence of 1 mM MgATP and soluble creatine kinase without pyruvate kinase, creatine is released from phosphocreatine in the creatine kinase reaction with a high rate, v0, equal to the rate of the ATP splitting by hexokinase, at the steady-state concentration of A D P in the medium of 0.1 + 0.03 mM. The addition of pyruvate kinase leads to a rapid shift of the reaction of A D P utilization from the creatine kinase to the pyruvate kinase reaction, as predicted. At the P K / C K ratio 50, all A D P producted at its very low steady-state concentration under these conditions (Fig. 2) is rapidly trapped by the pyruvate kinase, the rate of pyruvate release being equal to v0 at zero rate of creatine release. Mathematical simulation of the processes involved, using kinetic equa-
257
ADP,mM
V/Vo
V/~,
LADP,rnM
IO
).2 0.5
t
O.I
25 PK/CK
50
Fig. 2. The effect of increasing pyruvate-kinase/creatine-kinase activity ratio ( P K / C K ) on the rate of creatine and pyruvate release in the homogeneous system. The reaction mixture contained 0.1-0.15 I U / m l both of hexokinase and skeletal muscle creatine kinase, 1 m M M g A T P and 10 m M phosphocreatine, 1 m M phosphoenolpyruvate and different amounts of pyruvate kinase. Mean values for five experiments and standard deviations are shown. Solid lines (experimental results) and dotted lines (calculated dependences) obtained as described in Appendix. The reaction rates were determined by analysis of the creatine ( O ) and pyruvate (O) concentrations in the reaction mixture after termination of the reaction by HC10 4. A D P concentrations ( ~ ) were determined in the same samples, v0, the rate of creatine release without pyruvate kinase; v, the rate determined at given P K / C K ratio.
tions for pyruvate kinase and creatine kinase reactions (see Appendix), gives the theoretical dependence of reaction rates on the P K / C K ratio fitting with the experimental results within the range of experimental errors that conforms to the homogeneity of the system. Competition between endogenous creatine kinase and exogenous pyruoate kinase in myofibrillar system The same experiment as described in Fig. 2 was repeated with myofibrils instead of hexokinase and added creatine kinase. Purified cardiac myofibrils contain particulate creatine kinase (MM isoenzyme [14]) with activity (determined by the reaction of ATP production from ADP and phosphocreatine under standard condition with saturating substrate concentration) close to the MgATPase activity of the preparation (see Table I). It is apparent from Fig. 3 that in this case the external pyruvate kinase competes less effectively with the creatine kinase for MgADP. The steadystate concentration of the latter was found to be lower than in the homogeneous system, but the
0.5
25 PK/CK
50
Fig. 3. The effect of increasing pyruvate-kinase/creatine-kinase activity ratio ( P K / C K ) on the rate of creatine release in the myofibrillar creatine-kinase reaction. The reaction mixture contained 1.0-1.5 mg of myofibrillar protein per ml, 1 m M MgATP, 10 m M phosphocreatine, 1 m M phosphoenolpyruvate and different amounts of pyruvate kinase. Determinations were performed as described in the legend to Fig. 2 and symbols are as per Fig. 2.
rate of creatine release remained high, and at P K / C K ratio of 50 was still equal to half of v0. The rate of pyruvate release was significantly lower and reached correspondingly only 50% of o0 at PK/CK---50. Both curves reached a closely saturating level at this P K / C K . This result demonstrates clearly and directly that at least one-half of the MgADP produced by the Mg2+-ATPase is not available to the pyruvate-kinase reaction if the creatine-kinase reaction is imaximally activated (saturating concentrations Of phosphocreatine). Therefore, this part of MgADP is used by creatine kinase immediately in myofibrils without intermediate release into the medium. The rate of creatine release in these particulatecoupled reactions was found to be dependent on the phosphocreatine concentration, in accordance with the kinetics of creatine-kinase reaction (Fig. 4): the apparent K m for phosphocreatine was found to be 1.2 raM, which is very close to the K m for this substrate determined by a detailed kinetic analysis (1.67 mM [14]). With a decrease in phosphocreatine concentration, the rate of pyruvate release was simultaneously increased (Fig. 4) and the total rate of ADP consumption was constant. Therefore, the rate of immediate ADP rephosphorylation in myofibrillar space is governed by the kinetics of creatine kinase. Several factors have been found to affect the
258 A
B / /
o
,/: o
E 20 _4 in
0. I
A _ ~
""~.* PCr
PK/CK :5
D~Cr ~ tD_
0,05" :~
>-
D
/ V mox = 0,12
Km = I, 5 [~rJ ,mM
)0
-2
-I
0 I I/ [Per], mM
2
'OI JO.0
*PCr
app
I rain PCr ~
-PCr \ -PCr \
Fig. 4. (A) The dependence of the rate of creatine release in the myofibrillar creatine-kinase reaction on the phosphocreatine concentration in the presence of pyruvate kinase. O, creatine; t, pyruvate. (B) Linarization of the dependences in the double-reciprocal plots. PK/CK ratio was 5. Conditions as described in the legend to Fig. 3.
tight coupling described in Fig. 3. The ability of myofibrillar-coupled system to resist the high activity of exogenous pyruvate kinase was significantly lost when isolated myofibrils were kept in 50% glycerol for 1 week, or when myofibrillar preparation was vigorously homogenized in Virtis homogenizer at maximal speed for several minutes. The phenomenon observed seems to be shown only for intact myofibrillar structures. The second approach to detect the privileged access of A D P for myofibrillar particulate creatine kinase is a direct spectrophotometric determination of the rate of appearance of A D P in the medium by the coupled pyruvate kinase-lactate dehydrogenase system shown in Fig. 5. In this assay the added coupled-enzymatic reactions use up the M g A D P m released into the medium, and the slopes of recordings show the rate of appearance of M g A D P available for soluble pyruvate kinase. If there is some direct delivery of M g A D P from MgE+-ATPase to the myofibrillar creatine kinase, the activation of the latter reaction of M g A D P rephosphorylation in situ by addition of phosphocreatine into the medium should decrease the apparent rates of the coupled reactions. This is exactly what is observed in spectrophotometric experiments shown in Fig. 5. This figure reproduces the recordings for two P K / C K ratios, 5 and 20, and for 50 # M and 1.0 MgATP in the
Fig. 5. Spectrophotometric recordings of the rate of MgADP release in the myofibrillar Mg2+-ATPase reaction in the absence (-PCr) and in the presence (+ PCr) of 10 mM phosphocreatine at different PK/CK ratios and MgATP concentrations. (A) 50 #M MgATP; (B) 1.0 mM ATP; (C) 50 #M MgATP; (D) 1.0 mM ATP.
medium. In all cases, the decrease of the rate of M g A D P appearance in the medium in the presence of phosphocreatine is observed. Since in different cases the extent of inhibition of the reaction rate was different, it was of interest to record the dependences of the phosphocreatine effect described above, on the P K / C K ratio as well as on the MgATP concentration in the medium. Fig. 6 shows the dependences of the reaction rate recorded by using the pyruvate kinase-lactate dehydrogenase system when the Mg2+-ATPase-reaction rate in myofibrils was measured without (upper curve) and with (lower curve) 10 mM phosphocreatine at different P K / C K ratios, while MgATP concentration was kept constant at 1 mM and lactate dehydrogenase was added in high concentration in order not to limit the reaction rate. Since some commercial preparations of phosphocreatine were found to have inhibitory action on several enzymes, including pyruvate kinase [22,23], similar experiments were repeated for soluble enzyme system with hexokinase and soluble creatine kinase, added in amounts to fit the activities of MgE+-ATPase and creatine kinase in experiments with myofibrils (dotted curves). Both for myofibrillar MgE+-ATPase and hexokinase, dependences of reaction rates on the P K / C K ratio are very close and reach the maximal values at P K / C K higher than 20 (Fig. 6). The addition
259 V/Vo
f~l
' [[
0
k
•
50
e
I00
7/o/
PK / CK
Fig. 6. The effect of 10 mM phosphocreatine on the reaction of MgADP release from my©fibrils at different PK/CK ratios. Reaction rates are shown in Fig.5. ©, O, 0.2 mg/ml of myofibrillar protein was added in the absence (O) and in the presence (e) of 10 mM phosphocreatine, creatine-kinase activity was 0.016 IU/ml. A, IL the reaction mixture contained 0.015 IU/ml of hexokinase and 0.015 IU/ml of creatine kinase. A, without phosphocreatine; l, with phosphocreatine. MgATP concentration was 1.0 mM. %, maximal ATPase activity recorded in the absence of phosphocreatine.
of phosphocreatine has only very slight effect on the soluble (hexokinase plus creatine kinase) system seen at low P K / C K values, but remarkably decreases the reaction rate at any P K / C K ratio when myofibrillar Mg2+-ATPase was assayed (Fig. 6). These results completely agree with those of direct determination of tlae rates of creatine and pyruvate release (see Figs. 3 and 6) and support the conclusion that M g A D P produced in my©fibrils is used preferentially by the creatine-kinase reaction. In Fig. 7 the dependences of the Mg2÷-ATPase activity on the M g A T P concentration are shown in the double-reciprocal plots. As in several earlier works by other authors [24,25], the dependence has been found to give two straight lines in different ranges of M g A T P concentration. Two values of apparent K m ( M g A T P ) and of the maximal velocities were calculated: K m = 14 + 4.8 /~M and VmaX= 0.099 + 0.019 ~ m o l / m i n per mg for high apparent affinity site, and K m = 104 + 15 ffM and Vm,x = 0.19 + 0.016 / ~ m o l / m i n per mg for low-affinity site. The addition of E G T A significantly decreased the rates (increased l / v ) and a substrate inhibition of the reaction was observed as described earlier by G o o d n o et al. [25]. Phosphocreatine
////////X / / - I00
- 50
0
50
I00
200
ub~], r.M-' Fig. 7. The dependence of the myofibrillar Mg2+-ATPase activity on the MgATP concentration. The activities were determined spectrophotometrically. O, without phosphocreatine and added EGTA; A, without added EGTA and with 10 mM phosphocreatine; ©, with added EGTA (1 mM) in the absence of phosphocreatine; zx, with added EGTA (1 raM) and 10 mM phosphocreatine.
decreased the reaction rates both in the absence of E G T A and in its presence (correspondlingly, in the presence of Ca 2+ and in its absence). Therefore, for all types of myofibrillar ATPase (Ca 2+sensitive and residual, of high and low apparent affinity to M g A T P ) seem to show the functional coupling with the creatine kinase.
Competition between endogenous creatine kinase and exogenous pyruvate kinase in cardiac sarcolemmal preparations The enzymatic properties of the sarcolemmal preparation isolated from rabbit heart are shown in Table I. This preparation, like a similar one isolated from rat hearts [15], contains particulate creatine kinase of high activity close to that of digitoxigenin-sensitive (Na ÷ + K÷)-ATPase. This preparation was used to investigate the route of M g A D P m o v e m e n t from the ATPase-active centers by using the experimental protocol described in Fig. 6 for spectrophotometric assay of myofibrillar preparation. As seen from Fig. 8, the effect was qualitatively the same as observed for my©fibrils:
260
TABLE 1 THE ENZYMATIC PROPERTIES OF RAT HEART MYOFIBRILLAR AND RABBIT HEART SARCOLEMMAL PREPARATIONS Mean values for six experiments-+S.D. are given. PCr, phosphocreatine. Preparation
Enzyme
Additions
Activity (#mol.min l . m g 1)
Myofibril
ATPase
1 mM MgATP + 1 mM EGTA 10 mM PCr, 1 mM MgADP
0.077 _+0.024 0.016 _+0.004 0.09 _+0.03
Control: 100 mM Na ÷, 10 mM K +, 5 mM MgATP + 0.5 mM ouabain + 0.3 mM digitoxigenin + 1 mM EGTA + 2 t~g/ml oligomycin,2 p,g/ml rotenone 10 mM PCr, 1 mM MgADP
1.l 1,02 0.70 1.19 0.94 0.63
creatine kinase Sarcolemma
ATPase
creatine kinase
p h o s p h o c r e a t i n e in 10 m M c o n c e n t r a t i o n decreased the rate of M g A D P a p p e a r a n c e in the m e d i u m b y 50-30% d e p e n d i n g on the P K / C K ratio. At P K / C K ratio = 100, 35% i n h i b i t i o n was observed. O n l y at very high P K / C K ratios was the i n h i b i t o r y action lowered into the range of 5-10%. In contrast, p h o s p h o c r e a t i n e had only a small i n h i b i t o r y effect on the rates of A D P rephos-
V/Vo
0.5
' ////~//
y° 0
25
50 PK/CK
I00
Z50
Fig. 8. The effect of 10 mM phosphocreatine on the reaction of MgADP release from sarcolemmal (Na ÷ + K ÷ )ATPase at different PK/CK ratios. Reaction rates were recorded spectrophotometrically. Sarcolemmal preparations were added in concentrations of 10-20 #g/ml (0.01 IU of creatine kinase per ml). Reaction rates were recorded in the absence (I)) and in the presence (O) of 10 mM phosphoereatine. Control system: 0.01-0.02 IU/ml of hexokinase and 0.01-0.02 IU/ml of creatine kinase were present; (e), without phosphocreatine; (A), with 10 mM pbosphocratine, v0, maximal ATPase activity in the absence of phosphocreatine.
_+0.22 _+0.13 _+0.19 _+0.22 _+0.19 -+0.22
p h o r y l a t i o n by pyruvate kinase in a h o m o g e n e o u s system consisting of hexokinase and soluble creatine kinase. These results clearly show that creatine-kinase b i n d i n g to the sarcolemmal m e m b r a n e allows preferential r e p h o s p h o r y l a t i o n by this enzyme of A D P produced b y (Na ÷ + K ÷ ) - A T P a s e .
Discussion The results of this study show that the particulate creatine kinase in cardiac myofibrils as well as on the cardiac sarcolemma m a y function as a basis of c o m p a r t m e n t a t i o n of a d e n i n e nucleotides ( A T P a n d A D P ) in these structures, due to their close interaction with ATPases. They are able to remove significant part of A D P produced in the c o r r e s p o n d i n g A T P a s e reactions ( a c t o m y o s i n Mg 2÷-activated A T P a s e in myofibrils a n d ( N a ÷ + K ÷ ) - A T P a s e in sarcolemma) without its release i n t o the m e d i u m , a n d to rephosphorylate it into A T P to be used again by A T P a s e to support active c o n t r a c t i o n a n d ion transport. It may be supposed that rapid a n d direct removal of A D P by creatine kinases d e m o n s t r a t e d in this work is one of the most i m p o r t a n t reasons for localization of creatine kinase M M isoenzymes in these structures to prevent i n h i b i t i o n of ATPases by the reaction product, A D P , u n d e r c o n d i t i o n s of possibly limited diffusion of a d e n i n e nucleotides in the muscle cells [1-8,28,29]. The results of this study are in fact consistent with m a n y other investigations [30-35].
261
In a study with isolated myofibrils, Perry [30] succeeded in showing in 1954 that 5 mM phosphocreatine and 5-6/~M ADP caused the shortening of isolated myofibrils to the same extent as 100-110 ~M ATP. In in vitro systems composed of purified myosin and creatine kinase they were found to form complexes with high turnover of adenine nucleotides in the presence of phosphocreatine [31-34]. In a recent study from Bessman's laboratory the direct transfer of phosphoryl group from phosphocreatine into myofibrillar ATP has been documented by use of a radioisotopic technique [35] and recently in physiological experiments [36]. Preferential transfer of ADP from ( N a + + K+)-ATPase to the sarcolemmal creatine kinase observed is consistent with the data reported by Grosse et al. [37] which showed more active transport of Na + and K + across the vesicles when ATP was produced by the sarcolemmal creatine kinase than by added pyruvate kinase. However, these data were not confirmed by Philipson, who failed to find any difference between those two sources of ATP [38]. The contradiction is not understood, but may be related to the differences in the methods of preparing the vesicles and to the extreme easiness of losing the coupling during isolation. The results of these s.tudies have several important physiological implications. Physiological correlation between phosphocreatine content and tension have been noticed for a long time [39,40]. For example, during energetic deficiency such as hypoxia or ischemia, it is well known that tension decreases concomitantly with phosphocreatine concentration without early changes in adenine nucleotide pools [7,8,41-43]. In hypodynamic frog heart it has been shown that contractile activity is influenced by creatine and phosphocreatine concentrations [6,7]. It has been recognized also with skinned skeletal muscle fibers [44] or skinned heart bundles [45] that the presence of phosphocreatine is important to obtain fully relaxed fibers and high velocity of shortening, even with ATP concentrations as high as 2-5 mM. This leads to the hypothesis that a small ATP compartment with high turnover rates is immediately available for muscle
contraction as already proposed [2-8]. However, the exact way in which this 'ATP pool' influences contraction is not clear. A direct possible influence of the concentration of phosphocreatine on the contractile force is not generally taken into account, because ATP is the substrate for actomyosin interactions and its total cellular concentration during hypoxia is too high to influence myosin-ATPase activity. The results described here, together with those of Bessman et al. [35], provide evidence that myosin-ATPase activity is tightly coupled to creatine-kinase activity and that the immediate rephosphorylation of MgADP is regulated by phosphocreatine concentration in the millimolar range (Fig. 4). These results could be of great importance for understanding the mechanical failure during hypoxia or ischemia, giving evidence for a possible direct cause-and-effect relationship between the phosphocreatine and the contractile force. The nature of this ATP-ADP compartment, however, still remains unknown. Creatine kinase has been found to be bound mostly to the M-line in skeletal muscle [46,47]. For mammalian heart such an information is still absent. Formally, the effects described can be explained by enhancement of the local MgADP pool in some microcompartments in the vicinity of creatine kinase. The size of such a microcompartment depends on the closeness of creatine kinase and myosin heads in myofibrils. In sarcolemmal preparation there are no structures to create a physical barrier for MgADP diffusion, and for such a system direct channelling of MgADP to creatine kinase seems to be more plausible, as has been described for mitochondria [12,13]. However, since creatine kinase is known to establish rapid equilibrium between bound and medium MgADP [27,48], some limitations for dissociation of MgADP from immediate vicinity of creatine kinase should exist, or the reaction of phosphoryl-group transfer to MgADP should be considered to be more rapid than MgADP binding, and not a ratelimiting step for this particulate creatine kinase. These important questions require further detailed experimental studies.
262 Appendix
Mathematical simulation of homogeneous reaction system (creatine kinase, pyruvate kinase, hexokinase as A TPase) It was a s s u m e d that the system functions in s t e a d y - s t a t e with respect to a d e n i n e n u c l e o t i d e s . This a s s u m p t i o n leads to the e q u a t i o n : VATp = VCK "~- VpK
(|)
where VATP is the rate of ATP splitting into ADP and inorganic phosphate; vcK is the rate of ADP rephosphorylation into ATP in the creatine-kinase reaction from substrates in the medium; VpK is the rate of the pyruvate kinase reaction.The dependences of the rates of these reactions on the metabolic concentrations in the medium are described by the following kinetic equations [21,48-50]: [MgATP]/KATp VA*p="o 1+_+[MgATP] [MsADP] KATP
(2)
KADP
[MgADP][PCr I v(.~ = v 1
+ [Crl
[PCrl
[M~TPI
1 -~ib +~--~-id +
[MsATP][Cr] vl
KicK d
1+
Ki.
KiaK b
+ ~
(3)
1+ Kib
Kd
Kb
v2 [PEP][Ms 2+ ][MgADPI/KAK m /) PK
(KmPEP+[PEP])(I+ [Mgz+]K~+,(I+ I[MgZ+])[MgADP] ' Km + :( 1+ [ MKg -Z~+P]] ] [ A Ka D P ] ] ]+~( 1 + ~[ M g 2 + ] ) [MgATP]K,~ K~ TP
(4)
The dissociation constants used are described and given in Table II. Assuming that pH, concentration of total Mg, creatine, phosphocreatine, phosphoenolpyruvate (PEP). pyruvate and total nucleotides ATP + ADP are known, Eqns. 2 - 4 were introduced into Eqn. 1 and the TABLE II THE VALUES OF KINETIC CONSTANTS USED FOR MATHEMATICAL SIMULATION OF Mg2+-ATPase+CREATINE KINASE + PYRUVATE KINASE REACTIONS The kinetics of the reaction are described in Refs, 11, 27-29, 48-51. The constants for the creatine-kinase reaction were determined experimentally for the myofibrillar preparation used by a method described earlier [11,21,48]. Reaction
Constant
Mg 2+ -ATPase
KAT P KAD p
Creatine kinase
Kia Ka Kib Kb Ki~ K¢
Value (raM) 0.1 1.0 0.9 0.4 20 9 0.085 0.03
Reaction
Constant
Value (raM)
Creatine kinase (cont.)
Ka
0.4 1.04
Kid Pyruvate kinase
KA K~ K,~ K,1 K~
KPmF-p K1 K1ATP
0.015 0.047 0.047 0.08 0.25 0.14 2.1 0.14
263
quasi-steady-state concentrations of the nucleotides were computed by using numeral methods to fit Eqn. 1 for conditions given• From these calculations, individual reaction rates for each of these reactions were easily obtained. Calculations of free magnesium and Mg-complexes with substrates were performed as described earlier [21,51]. The results of calculations are shown in Fig. 2 (dotted line) and fit fairly well with the experimental data. That shows that the kinetics of a homogeneous enzyme system completely describe the behavior of these three enzymes and that the suppression of the creatine-kinase reaction by pyruvate kinase at high P K / C K ratios results from efficent removal of MgADP from the medium by this enzyme.
Acknowledgements The authors thank Professors V.N. Smirnov and H. Bricaud for making possible this cooperation under the joint France-U.S.S.R. Research Program and for their continuous interest to this study. The skillful technical assistance by Dr. G.B. Chernousova and L.A. Makhotina is gratefully recognized.
References 1 Seraydarian, M.W. (1982) Trends Pharmacol. Sic., October, 393-395 2 Gudbjarnason, S., Mathes, P. and Ravens, K.G. (1970) J. Mol. Cell Cardiol. 1,325-339 3 Saks, V.A. and Kupriyanov, V.V. (1982) in Advances in Myocardiology (Dhalla, N.S., Smirnov, V.N. and Chazov, E.I., eds.), vol. 3, pp. 475-497, Plenum Press, New York 4 Bessman, S.P. and Geiger, P.J. (1981) Science 211,488-492. 5 Jacobus, W.E. and Ingwall, J.S. (eds.) (1980) Heart Creatine Kinase, Williams & Wilkins, Baltimore 6 Saks, V.A., Rosenshtraukh, L.V., Smirnov, V.N. and Chazov, E.I. (1978) Can. J. Physiol. Pharmacol. 56, 691-706 7 Ventura-Clapier, R. and Vassort, G. (1980) J. Physiol. (Paris) 76, 583-589 8 Ventura-Clapier, R. and Vassort, G. (1980) J. Muscle Res. Cell Motility 1,429-444 9 Jacobus, W.E. and Lehninger, A.L. (1973) J. Biol. Chem., 248, 4803-4810 10 Saks, V.A., Kupriyanov, VN., Elizarova, G.V. and Jacobus, W.E. (1980) J. Biol. Chem. 255, 755-763 11 Jacobus, W.E., Saks, V.A. (1982) Arch. Biochem. Biophys., 219, 167-178 12 Moreadith, R.W. and Jacobus, W.E. (1982) J. Biol. Chem., 257, 899-905 13 Gellerich, F. and Saks, V.A. (1982) Biochem. Biophys. Res. Commun. 105, 1473-1481 14 Saks, V.A., Chernousova, G.B., Vetter, R., Smirnov, V.N. and Chazov, E.I. (1976) FEBS Lett., 62, 293-296 15 Kupriyanov, V.V., Preobrazhensky, A.N. and Saks, V.A. (1983) Biochim. Biophys. Acta 728, 239-253 17 Saks, V.A., Lipina, N.V., Sharov, V.G., Smirnov, V.N., Chazov, E.I. and Grosse, R. (1977) Biochim. Biophys. Acta 465, 550-558
18 Levitsky, D.O., Aliev, M.K., Kuzmin, A.V., Levchenko, T.S., Smirnov, V.N. and Chazov, E.I. (1976) Biochim. Biophys.Acta 443, 468-484 19 Eggleton, P., Elsden, S.R. and Gough, N. (1943) Biochem. J., 337, 526-529 20 Adam, H. (1965) in Methods of Enzymatic Analysis (Bergmeyer, H., ed.), pp. 573-577. Academic Press, New York 21 Saks, V.A., Chernousova, G.B., Gukovsky, D.E., Smirnov, V.N. and Chazov, E.I. (1975) Eur. J. Biochem., 57, 273-290 22 Fitch, C.D., Chevli, R. and Jellinek, M. (1979) J. Biol. Chem., 254, 11357-11359 23 Tornheim, K. and Lowenstein, J.M. (1979) J. Biol. Chem., 254, 10586-10587 24 Bendall, J.R. (1972) FEBS Lett. 22, 330-334 25 Goodno, C.C., Wall, C.M. and Perry, S.V. (1978) Biochem. J., 175, 813-821 26 Curtin, N.A. and Woledge, R.C. (1978) Physiol. Rev. 58, 690 761 27 Watts, D.C. (1973) in The Enzymes (Boyer, P.D., ed.), Vol. 8, pp. 384-431, Academic Press, New York 28 Borejdo, J. (1982) Biochemistry, 21, 234-241 29 Adelstein, R.S. and Eisenberg, E. (1980) Annu. Rev. Biochem., 49, 921-956 30 Perry, S.V. (1954) Biochem. J., 57, 427-434 31 Yagi, K. and Mase, R. (1962) J. Biol. Chem., 237, 397-403 32 Botts, J. and Stone, M.J. (1968) Biochemistry, 7, 2688-2696 33 Yagi, K. and Mase, R. (1965) in Molecular Biology of Muscular Contraction (Ebashi, S., Oosawa, F., Sekine, T. and Tonomura, Y., eds.), pp. 109-123, Elsevier, Amsterdam 34 Mani, R.S. and Kay, C.M. (1976) Biochim. Biophys. Acta 453, 391-399 35 Bessman, S.P., Yang, W.C.T., Geiger, P.J. and EricksonVitanen, S. (1980) Biochem. Biophys. Res. Commun. 96, 1414-1420 36 Savabi, F., Geiger, P.J. and Bessman, S.P. (1983) Biochem. Biophys. Res. Commun., 114, 785-790 37 Grosse, R., Spitzer, E., Kupriyanov, V.V., Saks, V.A. and Repke, K.R.H. (1980) Biochim. Biophys. Acta 603, 142-156 38 Philipson, K.D. (1983) J. Mol. Cell. Cardiol. 15, suppl. 1, p. 359. 39 Opie, L.H. (1969) Am. Heart J., 77, 383-410 40 Mommaerts, W.F.H.M. (1969) Physiol. Rev., 49, 427-508 41 Kubler, W. and Spieckenman, P.G. (1970) J. Mol. Cell. Cardiol., 1, 351-377 42 Dhalla, N.S., Yates, J.C., Waltz, D.A., McDonald, V.A. and Olson, R.E. (1972) Can. J. Physiol. Pharmacol., 50, 333-345
264 43 Neely, J.R., Rovetto, M.J., Whitmer, J.T. and Morgan, H.F,. (1983) Am. J. Physiol., 225, 651-658 44 Godt, R.E. (1974) J. Gen. Physiol., 63, 722 739 45 Maugham, D.W., Low, E.S. and Alpert. N.R. (1978) J. Gen. Physiol., 71,431-451 46 Turner, D,C., Walliman, T. and Eppenberger, H.M. (1973) Proc. Natl. Acad. Sci. U.S.A., 70, 702-705 47Walliman, T., Turner, D.C. and Eppenberger, N.M. (1977) J. Cell, Biol., 75, 297 317
48 Morrison, J.F. and James, E. (1965) Biochem. J., ~7, 37 52 49 Kupriyanov, V.V., Seppet, E.K., Emelin, l.V. and Silks. V.A. (1980) Biochem. Biophys. Res. Commun.. 592, 197~ 210 50 Kupriyanov, V.V., Seppet, E.K., Emelin, I.V. and Saks, V.A. (1979) Biokhimia, 44, 104-115 51 Saks, V.A., Katz, V.M., Vetter, R., Lyulina, N.V. and Shell, W. (1979) Biokhimiya 49, 1600 1613
Biochimica et Btop/~vstca Acta, 803 (1984) 254 264 Elsevier
BBA 11268
CREATINE KINASE IN REGULATION OF HEART FUNCTION AND METABOLISM I. FURTHER EVIDENCE FOR COMPARTMENTATION OF ADENINE NUCLEOTIDES IN CARDIAC MYOFIBRILLAR AND SARCOLEMMAL COUPLED ATPase-CREATINE KINASE SYSTEMS V.A. SAKS a,*, R. V E N T U R A - C L A P I E R b, Z.A. H U C H U A a, A.N. P R E O B R A Z H E N S K Y a and I.V. EMELIN ~ " Laboratory of Cardiac Bioenergetics, U.S.S.R. Cardiology Research Center, 3 Cherepkovskaya Street 15, Moscow 121552 ( U. S. S. R.) and Laboratoire de Physiologie Cellulaire Cardiaque, Universitb de Paris - Sud, Orst
Key words: Compartmentation; Adenine nucleotide; Creatine kinase," A TPase; (Heart)
In isolated and purified cardiac myofibriilar and sarcolemmal preparations, the route of movement of ADP produced in the Mg2+-ATPase reactions was studied by investigating the efficiency of competition between the endogenous creatine kinase and exogenous pyruvate kinase reactions. In the homogeneous control system composed of hexokinase and glucose as ATPase, soluble creatine kinase rapidly rephosphorylated ADP produced in the presence of 1 mM ATP, but the addition of pyruvate kinase in an increasing amount inhibited the reaction of creatine release from phosphocreatine and symmetrically increased the rate of pyrnvate prduction from phosphoenolpyruvate. At a pyruvate-kinase/creatine-kinase activity ratio (PK/CK) of 50, all ADP was used by the pyruvate kinase. In myofibrillar and sarcolemmai preparations containing particulate creatine kinase, the creatine kinase reaction was much less efficiently suppressed by pyruvate kinase, and at P K / C K = 50 half-maximal release of creatine was still observed. The rate of immediate myofibrillar MgADP rephosphorylation in the endogenous creatine-kinase reaction was observed to be governed by the concentration of phosphocreatine in accordance with the kinetics of this enzyme. The physiological significance of these findings is discussed.
Introduction An increasing amount of biochemical evidence supports the concept of energy channelling by the phosphocreatine-creatine shuttle in muscle cells [1-5], which explains many recent observations on cardiac muscle physiology under ischemic and hypodynamic conditions [3,6-8]. The importance of the phosphocreatine-creatine shuttle and the potential regulatory role of this system in cardiac muscle contraction is based on the assumed phe* To whom correspondence should be addressed. Abbreviations: CK, creatine kinase; PK, pyruvate kinase; EGTA, ethylene glycol bis(fl-aminoethylether)-N,N'-tetraacetate; IU, international units of enzyme activity, t~mol/min. 0167-4889/84/$03.00 © 1984 Elsevier Science Publishers B.V.
nomenon of compartmentation of adenine nucleotides inside the highly organized cellular structures, where only the amount of ATP which is located immediately in myofibrils is considered to be rapidly used for contraction and is replenished at the expense of phosphocreatine due to the creatine-kinase reaction [2,3]. In mitochondria, creatine-kinase reaction is coupled to oxidative phosphorylation via adenine nucleotide translocase and participates in compartmentation of adenine nucleotides in these stuctures [3,5,6,9-13]. The present joint study was undertaken to investigate the question whether close interaction of creatine kinase MM isoenzyme with ATPases, in myofibrils and on the sarcolemma, can provide efficient func-
255
Materials and Methods
buffered with 20 mM imidazole (pH 7.3). The gradients were centrifuged overnight in a Beckman SW-50 rotor at 35 000 rpm. The fraction at the 20 mM imidazole and 8.7% dextran 15 interface was collected and vesicles were recovered by sedimentation at 40000 rpm for 1.5 h. The pellet was resuspended in a small volume of buffer I and used as a sarcolemmal preparation.
Isolation of myofibrils
Reaction medium
Myofibrils were isolated from Wistar-rat hearts by a method described earlier in a paper from this laboratory [14] with the exception that the first part of step B - treatment in a solution of 0.1 M potassium phosphate and 0.1 M KC1 - was omitted. The final preparation was suspended in 60 mM K C I / 3 0 mM imidazole/2 mM MgC12/2 mM dithiothreitol (pH 7.2).
In experiments with myofibrils, in which the rates of creatine release from phosphocreatine and pyruvate release from phosphoenolpyruvate in the competing creatine-kinase and pyruvate-kinase reactions were determined, the reaction medium contained 25 mM Tris-HC1 (pH 7.4), 20 mM glucose, 5 mM MgCI 2, 1 mM ATP, 1 mM phosphoenolpyruvate, and usually 10 mM phosphocreatine. Dithiothreitol had to be omitted, since it was found to inhibit the colorimetric reaction of creatine assay. The reaction was run at 30°C and was started with addition of myofibrils to 1.0-1.5 m g / m l that gave 0.1-0.15 I U / m l of both creatine kinase and myofibrillar ATPase. Prior to the addition of myofibrils, pyruvate kinase was added to the medium to reach the P K / C K ratio required (from 0 to 100). The reaction was run for 2 and 4 min and stopped by mixing 1 ml of sample with 0.5 ml of 10% perchloric acid. After rapid neutralization with 2.5 M K2CO 3 the mixture was analyzed for free creatine, pyruvate and ADP. In control experiments hexokinase and soluble creatine kinase (rabbit skeletal muscle) of equal activity (0.1-0.15 I U / m l ) were added instead of myofibrils. The reaction was started with hexokinase.
tional compartmentation of adenine nucleotides in these systems. With this purpose the routes of ADP produced in the ATPase reactions were studied by using exogenous pyruvate kinase-phosphoenolpyruvate system, which competes with the creatine kinase reaction for ADP.
Isolation of sarcolemmal preparations Sarcolemmal preparation from rabbit heart was isolated by modification of the method recently described in Refs. 15 and 16. All isolation steps were carried out at 0-4°C. Rabbits were killed by cervical dislocation and hearts were placed in buffer I (20 mM imidazole/0.25 M sucrose (pH 7.3)). Ventricles were separated from atria and large blood vessels and were homogenized in buffer I (tissue weight/buffer volume, 1 : 5) using Virtis45 homogenizer at half-maximal speed for 1 min. The homogenate was filtered through cheesecloth, layered upon 1.1 M sucrose/20 mM imidazole (pH 7.3), and centrifuged at 26 000 rpm for 1 h in a Beckman SW-27 rotor. The supernatant was centrifuged at 40000 rpm for 1 h in a Beckman type 45 Ti rotor. The pellet was resuspended in 20 ml of buffer I and layered onto a sucrose-density step gradient consisting of 0.73 M sucrose (20 ml) and 1.78 M sucrose (7 ml), buffered with 20 mM imidazole (pH 7.3) (10 ml of homogenate/ centrifuge tube). The gradients were centrifuged in a Beckman SW-27 rotor at 26000 rpm for 2-3 h. Microsomal fraction was collected at the 0.25/0.73 M sucrose interface, diluted with buffer I and centrifuged at 40000 rpm for 1 h in a Beckman type 45 Ti rotor. The pellet was resuspended in 4 ml of 20 mM imidazole (pH 7.3) and layered on top of a discontinuous two-step gradient, containing 2 ml of 8.7% and 1 ml of 18% dextran 15,
Spectrophotometric determination of the rate of ADPase reaction This determination was carried out by using the coupled pyruvate kinase-lactate dehydrogenase system [17,18]. The reaction medium contained 0.30 M sucrose, 25 mM Tris-HC1 (pH 7.4), 5 mM magnesium acetate, 5 mM potassium acetate, 1 mM phosphoenolpyruvate, 1 mM ATP and 0.2 mM NADH, 0.3 mM dithiothreitol and 2 I U / m l lactate dehydrogenase. The reaction was started after stabilization of absorbance by addition of myofibrils (0.2-0.3 m g / m l ) or sarcolemmal pre-
256
paration (0.015-0.025 m g / m l ) . In the latter case. 100 mM Na + and 10 m M K + were present. The temperature was 30°C.
Assays Creatine concentration was determined by a colorimetric method [19]. A D P concentration as well as pyruvate concentration were determined enzymatically [20]. Pyruvate-kinase activity was determined in the medium described for spectrophotometric ATPase determination when the pyruvate kinase was a rate-limiting step. Creatinekinase activity was determined by the reverse reaction using a spectrophotometric-coupled enzyme hexokinase and glucose-6-phosphate dehydrogenase assay system [21]. Hexokinase activity was assayed under similar conditions. Protein concentration was determined by a biuret method
[]4]. Principles of the investigation of ADP-movement routes in myofibrillar and sarcolemmal preparation The principle of the method used is schematically illustrated in Fig. 1. This method takes into account two possible ways of A D P movement from the ATPase-active centers where it is produced at the constant steady-state rate, v0, under conditions of saturation of the ATPase with its substrate, MgATP. One way is a rapid dissociation of A D P into the medium, this giving rise to the free medium M g A D P concentration, [MgADP]m. The second way is a possible direct channelling of M g A D P to the active center of structurally bound creatine kinase without an intermediate release of A D P into the medium. To differentiate between these two possible ways of A D P movement, we
Pyruvate MgATP
'MgADP~_~:3~-MgAOPm
CK.MgAT~.~CK.MgADP Cr
PCr
Fig. 1. Schematic presentation of principles of a method used to study the route of movement of MgADP produced in the MgZ+-ATPase reactions in cardiac myofibrillar and sarcolemmal preparations. MgADP m, MgADP in the medium; PEP, phosphoenolpyruvate; Cr, creatine; PCr, phosphocreatine.
have studied the efficiency of competition of creatine kinase with the added exogenous pyruvatekinase system which utilized only A D P from the medium, MgADP m. In the homogeneous system, with the complete intermediate release of MgADP from the ATPase (hexokinase+ glucose), both creatine kinase and pyruvate kinase use the same pool of MgADP, MgADP m, and an increase in the P K / C K ratio should shift the pathway of ADP utilization from creatine-kinase (creatine release from phosphocreatine) to the pyruvate-kinase reaction (pyruvate release from phosphoenolpyruvate). In the second series of experiments, myofibrils or sarcolemmal preparations containing particulate-bound creatine kinase were used, and soluble exogenous pyruvate kinase was added to achieve the same P K / C K ratios, and to remove A D P from the medium. It should be expected that in the case of direct channeling of MgADP from ATPase to creatine kinase the latter reaction in myofibrils or in sarcolemmal preparation becomes less sensitive to the addition of pyruvate kinase, as compared with the homogeneous control system. A similar approach was earlier successfully used to study the mitochondrial creatine-kinase reaction
[131. Results
Competition between creatine kinase and pyruvate kinase for MgADP in the homogeneous system As shown in Fig. 2, in the reconstituted system consisting of hexokinase plus glucose as a sourve of MgADP, in the presence of 1 mM MgATP and soluble creatine kinase without pyruvate kinase, creatine is released from phosphocreatine in the creatine kinase reaction with a high rate, v0, equal to the rate of the ATP splitting by hexokinase, at the steady-state concentration of A D P in the medium of 0.1 + 0.03 mM. The addition of pyruvate kinase leads to a rapid shift of the reaction of A D P utilization from the creatine kinase to the pyruvate kinase reaction, as predicted. At the P K / C K ratio 50, all A D P producted at its very low steady-state concentration under these conditions (Fig. 2) is rapidly trapped by the pyruvate kinase, the rate of pyruvate release being equal to v0 at zero rate of creatine release. Mathematical simulation of the processes involved, using kinetic equa-
257
ADP,mM
V/Vo
V/~,
LADP,rnM
IO
).2 0.5
t
O.I
25 PK/CK
50
Fig. 2. The effect of increasing pyruvate-kinase/creatine-kinase activity ratio ( P K / C K ) on the rate of creatine and pyruvate release in the homogeneous system. The reaction mixture contained 0.1-0.15 I U / m l both of hexokinase and skeletal muscle creatine kinase, 1 m M M g A T P and 10 m M phosphocreatine, 1 m M phosphoenolpyruvate and different amounts of pyruvate kinase. Mean values for five experiments and standard deviations are shown. Solid lines (experimental results) and dotted lines (calculated dependences) obtained as described in Appendix. The reaction rates were determined by analysis of the creatine ( O ) and pyruvate (O) concentrations in the reaction mixture after termination of the reaction by HC10 4. A D P concentrations ( ~ ) were determined in the same samples, v0, the rate of creatine release without pyruvate kinase; v, the rate determined at given P K / C K ratio.
tions for pyruvate kinase and creatine kinase reactions (see Appendix), gives the theoretical dependence of reaction rates on the P K / C K ratio fitting with the experimental results within the range of experimental errors that conforms to the homogeneity of the system. Competition between endogenous creatine kinase and exogenous pyruoate kinase in myofibrillar system The same experiment as described in Fig. 2 was repeated with myofibrils instead of hexokinase and added creatine kinase. Purified cardiac myofibrils contain particulate creatine kinase (MM isoenzyme [14]) with activity (determined by the reaction of ATP production from ADP and phosphocreatine under standard condition with saturating substrate concentration) close to the MgATPase activity of the preparation (see Table I). It is apparent from Fig. 3 that in this case the external pyruvate kinase competes less effectively with the creatine kinase for MgADP. The steadystate concentration of the latter was found to be lower than in the homogeneous system, but the
0.5
25 PK/CK
50
Fig. 3. The effect of increasing pyruvate-kinase/creatine-kinase activity ratio ( P K / C K ) on the rate of creatine release in the myofibrillar creatine-kinase reaction. The reaction mixture contained 1.0-1.5 mg of myofibrillar protein per ml, 1 m M MgATP, 10 m M phosphocreatine, 1 m M phosphoenolpyruvate and different amounts of pyruvate kinase. Determinations were performed as described in the legend to Fig. 2 and symbols are as per Fig. 2.
rate of creatine release remained high, and at P K / C K ratio of 50 was still equal to half of v0. The rate of pyruvate release was significantly lower and reached correspondingly only 50% of o0 at PK/CK---50. Both curves reached a closely saturating level at this P K / C K . This result demonstrates clearly and directly that at least one-half of the MgADP produced by the Mg2+-ATPase is not available to the pyruvate-kinase reaction if the creatine-kinase reaction is imaximally activated (saturating concentrations Of phosphocreatine). Therefore, this part of MgADP is used by creatine kinase immediately in myofibrils without intermediate release into the medium. The rate of creatine release in these particulatecoupled reactions was found to be dependent on the phosphocreatine concentration, in accordance with the kinetics of creatine-kinase reaction (Fig. 4): the apparent K m for phosphocreatine was found to be 1.2 raM, which is very close to the K m for this substrate determined by a detailed kinetic analysis (1.67 mM [14]). With a decrease in phosphocreatine concentration, the rate of pyruvate release was simultaneously increased (Fig. 4) and the total rate of ADP consumption was constant. Therefore, the rate of immediate ADP rephosphorylation in myofibrillar space is governed by the kinetics of creatine kinase. Several factors have been found to affect the
258 A
B / /
o
,/: o
E 20 _4 in
0. I
A _ ~
""~.* PCr
PK/CK :5
D~Cr ~ tD_
0,05" :~
>-
D
/ V mox = 0,12
Km = I, 5 [~rJ ,mM
)0
-2
-I
0 I I/ [Per], mM
2
'OI JO.0
*PCr
app
I rain PCr ~
-PCr \ -PCr \
Fig. 4. (A) The dependence of the rate of creatine release in the myofibrillar creatine-kinase reaction on the phosphocreatine concentration in the presence of pyruvate kinase. O, creatine; t, pyruvate. (B) Linarization of the dependences in the double-reciprocal plots. PK/CK ratio was 5. Conditions as described in the legend to Fig. 3.
tight coupling described in Fig. 3. The ability of myofibrillar-coupled system to resist the high activity of exogenous pyruvate kinase was significantly lost when isolated myofibrils were kept in 50% glycerol for 1 week, or when myofibrillar preparation was vigorously homogenized in Virtis homogenizer at maximal speed for several minutes. The phenomenon observed seems to be shown only for intact myofibrillar structures. The second approach to detect the privileged access of A D P for myofibrillar particulate creatine kinase is a direct spectrophotometric determination of the rate of appearance of A D P in the medium by the coupled pyruvate kinase-lactate dehydrogenase system shown in Fig. 5. In this assay the added coupled-enzymatic reactions use up the M g A D P m released into the medium, and the slopes of recordings show the rate of appearance of M g A D P available for soluble pyruvate kinase. If there is some direct delivery of M g A D P from MgE+-ATPase to the myofibrillar creatine kinase, the activation of the latter reaction of M g A D P rephosphorylation in situ by addition of phosphocreatine into the medium should decrease the apparent rates of the coupled reactions. This is exactly what is observed in spectrophotometric experiments shown in Fig. 5. This figure reproduces the recordings for two P K / C K ratios, 5 and 20, and for 50 # M and 1.0 MgATP in the
Fig. 5. Spectrophotometric recordings of the rate of MgADP release in the myofibrillar Mg2+-ATPase reaction in the absence (-PCr) and in the presence (+ PCr) of 10 mM phosphocreatine at different PK/CK ratios and MgATP concentrations. (A) 50 #M MgATP; (B) 1.0 mM ATP; (C) 50 #M MgATP; (D) 1.0 mM ATP.
medium. In all cases, the decrease of the rate of M g A D P appearance in the medium in the presence of phosphocreatine is observed. Since in different cases the extent of inhibition of the reaction rate was different, it was of interest to record the dependences of the phosphocreatine effect described above, on the P K / C K ratio as well as on the MgATP concentration in the medium. Fig. 6 shows the dependences of the reaction rate recorded by using the pyruvate kinase-lactate dehydrogenase system when the Mg2+-ATPase-reaction rate in myofibrils was measured without (upper curve) and with (lower curve) 10 mM phosphocreatine at different P K / C K ratios, while MgATP concentration was kept constant at 1 mM and lactate dehydrogenase was added in high concentration in order not to limit the reaction rate. Since some commercial preparations of phosphocreatine were found to have inhibitory action on several enzymes, including pyruvate kinase [22,23], similar experiments were repeated for soluble enzyme system with hexokinase and soluble creatine kinase, added in amounts to fit the activities of MgE+-ATPase and creatine kinase in experiments with myofibrils (dotted curves). Both for myofibrillar MgE+-ATPase and hexokinase, dependences of reaction rates on the P K / C K ratio are very close and reach the maximal values at P K / C K higher than 20 (Fig. 6). The addition
259 V/Vo
f~l
' [[
0
k
•
50
e
I00
7/o/
PK / CK
Fig. 6. The effect of 10 mM phosphocreatine on the reaction of MgADP release from my©fibrils at different PK/CK ratios. Reaction rates are shown in Fig.5. ©, O, 0.2 mg/ml of myofibrillar protein was added in the absence (O) and in the presence (e) of 10 mM phosphocreatine, creatine-kinase activity was 0.016 IU/ml. A, IL the reaction mixture contained 0.015 IU/ml of hexokinase and 0.015 IU/ml of creatine kinase. A, without phosphocreatine; l, with phosphocreatine. MgATP concentration was 1.0 mM. %, maximal ATPase activity recorded in the absence of phosphocreatine.
of phosphocreatine has only very slight effect on the soluble (hexokinase plus creatine kinase) system seen at low P K / C K values, but remarkably decreases the reaction rate at any P K / C K ratio when myofibrillar Mg2+-ATPase was assayed (Fig. 6). These results completely agree with those of direct determination of tlae rates of creatine and pyruvate release (see Figs. 3 and 6) and support the conclusion that M g A D P produced in my©fibrils is used preferentially by the creatine-kinase reaction. In Fig. 7 the dependences of the Mg2÷-ATPase activity on the M g A T P concentration are shown in the double-reciprocal plots. As in several earlier works by other authors [24,25], the dependence has been found to give two straight lines in different ranges of M g A T P concentration. Two values of apparent K m ( M g A T P ) and of the maximal velocities were calculated: K m = 14 + 4.8 /~M and VmaX= 0.099 + 0.019 ~ m o l / m i n per mg for high apparent affinity site, and K m = 104 + 15 ffM and Vm,x = 0.19 + 0.016 / ~ m o l / m i n per mg for low-affinity site. The addition of E G T A significantly decreased the rates (increased l / v ) and a substrate inhibition of the reaction was observed as described earlier by G o o d n o et al. [25]. Phosphocreatine
////////X / / - I00
- 50
0
50
I00
200
ub~], r.M-' Fig. 7. The dependence of the myofibrillar Mg2+-ATPase activity on the MgATP concentration. The activities were determined spectrophotometrically. O, without phosphocreatine and added EGTA; A, without added EGTA and with 10 mM phosphocreatine; ©, with added EGTA (1 mM) in the absence of phosphocreatine; zx, with added EGTA (1 raM) and 10 mM phosphocreatine.
decreased the reaction rates both in the absence of E G T A and in its presence (correspondlingly, in the presence of Ca 2+ and in its absence). Therefore, for all types of myofibrillar ATPase (Ca 2+sensitive and residual, of high and low apparent affinity to M g A T P ) seem to show the functional coupling with the creatine kinase.
Competition between endogenous creatine kinase and exogenous pyruvate kinase in cardiac sarcolemmal preparations The enzymatic properties of the sarcolemmal preparation isolated from rabbit heart are shown in Table I. This preparation, like a similar one isolated from rat hearts [15], contains particulate creatine kinase of high activity close to that of digitoxigenin-sensitive (Na ÷ + K÷)-ATPase. This preparation was used to investigate the route of M g A D P m o v e m e n t from the ATPase-active centers by using the experimental protocol described in Fig. 6 for spectrophotometric assay of myofibrillar preparation. As seen from Fig. 8, the effect was qualitatively the same as observed for my©fibrils:
260
TABLE 1 THE ENZYMATIC PROPERTIES OF RAT HEART MYOFIBRILLAR AND RABBIT HEART SARCOLEMMAL PREPARATIONS Mean values for six experiments-+S.D. are given. PCr, phosphocreatine. Preparation
Enzyme
Additions
Activity (#mol.min l . m g 1)
Myofibril
ATPase
1 mM MgATP + 1 mM EGTA 10 mM PCr, 1 mM MgADP
0.077 _+0.024 0.016 _+0.004 0.09 _+0.03
Control: 100 mM Na ÷, 10 mM K +, 5 mM MgATP + 0.5 mM ouabain + 0.3 mM digitoxigenin + 1 mM EGTA + 2 t~g/ml oligomycin,2 p,g/ml rotenone 10 mM PCr, 1 mM MgADP
1.l 1,02 0.70 1.19 0.94 0.63
creatine kinase Sarcolemma
ATPase
creatine kinase
p h o s p h o c r e a t i n e in 10 m M c o n c e n t r a t i o n decreased the rate of M g A D P a p p e a r a n c e in the m e d i u m b y 50-30% d e p e n d i n g on the P K / C K ratio. At P K / C K ratio = 100, 35% i n h i b i t i o n was observed. O n l y at very high P K / C K ratios was the i n h i b i t o r y action lowered into the range of 5-10%. In contrast, p h o s p h o c r e a t i n e had only a small i n h i b i t o r y effect on the rates of A D P rephos-
V/Vo
0.5
' ////~//
y° 0
25
50 PK/CK
I00
Z50
Fig. 8. The effect of 10 mM phosphocreatine on the reaction of MgADP release from sarcolemmal (Na ÷ + K ÷ )ATPase at different PK/CK ratios. Reaction rates were recorded spectrophotometrically. Sarcolemmal preparations were added in concentrations of 10-20 #g/ml (0.01 IU of creatine kinase per ml). Reaction rates were recorded in the absence (I)) and in the presence (O) of 10 mM phosphoereatine. Control system: 0.01-0.02 IU/ml of hexokinase and 0.01-0.02 IU/ml of creatine kinase were present; (e), without phosphocreatine; (A), with 10 mM pbosphocratine, v0, maximal ATPase activity in the absence of phosphocreatine.
_+0.22 _+0.13 _+0.19 _+0.22 _+0.19 -+0.22
p h o r y l a t i o n by pyruvate kinase in a h o m o g e n e o u s system consisting of hexokinase and soluble creatine kinase. These results clearly show that creatine-kinase b i n d i n g to the sarcolemmal m e m b r a n e allows preferential r e p h o s p h o r y l a t i o n by this enzyme of A D P produced b y (Na ÷ + K ÷ ) - A T P a s e .
Discussion The results of this study show that the particulate creatine kinase in cardiac myofibrils as well as on the cardiac sarcolemma m a y function as a basis of c o m p a r t m e n t a t i o n of a d e n i n e nucleotides ( A T P a n d A D P ) in these structures, due to their close interaction with ATPases. They are able to remove significant part of A D P produced in the c o r r e s p o n d i n g A T P a s e reactions ( a c t o m y o s i n Mg 2÷-activated A T P a s e in myofibrils a n d ( N a ÷ + K ÷ ) - A T P a s e in sarcolemma) without its release i n t o the m e d i u m , a n d to rephosphorylate it into A T P to be used again by A T P a s e to support active c o n t r a c t i o n a n d ion transport. It may be supposed that rapid a n d direct removal of A D P by creatine kinases d e m o n s t r a t e d in this work is one of the most i m p o r t a n t reasons for localization of creatine kinase M M isoenzymes in these structures to prevent i n h i b i t i o n of ATPases by the reaction product, A D P , u n d e r c o n d i t i o n s of possibly limited diffusion of a d e n i n e nucleotides in the muscle cells [1-8,28,29]. The results of this study are in fact consistent with m a n y other investigations [30-35].
261
In a study with isolated myofibrils, Perry [30] succeeded in showing in 1954 that 5 mM phosphocreatine and 5-6/~M ADP caused the shortening of isolated myofibrils to the same extent as 100-110 ~M ATP. In in vitro systems composed of purified myosin and creatine kinase they were found to form complexes with high turnover of adenine nucleotides in the presence of phosphocreatine [31-34]. In a recent study from Bessman's laboratory the direct transfer of phosphoryl group from phosphocreatine into myofibrillar ATP has been documented by use of a radioisotopic technique [35] and recently in physiological experiments [36]. Preferential transfer of ADP from ( N a + + K+)-ATPase to the sarcolemmal creatine kinase observed is consistent with the data reported by Grosse et al. [37] which showed more active transport of Na + and K + across the vesicles when ATP was produced by the sarcolemmal creatine kinase than by added pyruvate kinase. However, these data were not confirmed by Philipson, who failed to find any difference between those two sources of ATP [38]. The contradiction is not understood, but may be related to the differences in the methods of preparing the vesicles and to the extreme easiness of losing the coupling during isolation. The results of these s.tudies have several important physiological implications. Physiological correlation between phosphocreatine content and tension have been noticed for a long time [39,40]. For example, during energetic deficiency such as hypoxia or ischemia, it is well known that tension decreases concomitantly with phosphocreatine concentration without early changes in adenine nucleotide pools [7,8,41-43]. In hypodynamic frog heart it has been shown that contractile activity is influenced by creatine and phosphocreatine concentrations [6,7]. It has been recognized also with skinned skeletal muscle fibers [44] or skinned heart bundles [45] that the presence of phosphocreatine is important to obtain fully relaxed fibers and high velocity of shortening, even with ATP concentrations as high as 2-5 mM. This leads to the hypothesis that a small ATP compartment with high turnover rates is immediately available for muscle
contraction as already proposed [2-8]. However, the exact way in which this 'ATP pool' influences contraction is not clear. A direct possible influence of the concentration of phosphocreatine on the contractile force is not generally taken into account, because ATP is the substrate for actomyosin interactions and its total cellular concentration during hypoxia is too high to influence myosin-ATPase activity. The results described here, together with those of Bessman et al. [35], provide evidence that myosin-ATPase activity is tightly coupled to creatine-kinase activity and that the immediate rephosphorylation of MgADP is regulated by phosphocreatine concentration in the millimolar range (Fig. 4). These results could be of great importance for understanding the mechanical failure during hypoxia or ischemia, giving evidence for a possible direct cause-and-effect relationship between the phosphocreatine and the contractile force. The nature of this ATP-ADP compartment, however, still remains unknown. Creatine kinase has been found to be bound mostly to the M-line in skeletal muscle [46,47]. For mammalian heart such an information is still absent. Formally, the effects described can be explained by enhancement of the local MgADP pool in some microcompartments in the vicinity of creatine kinase. The size of such a microcompartment depends on the closeness of creatine kinase and myosin heads in myofibrils. In sarcolemmal preparation there are no structures to create a physical barrier for MgADP diffusion, and for such a system direct channelling of MgADP to creatine kinase seems to be more plausible, as has been described for mitochondria [12,13]. However, since creatine kinase is known to establish rapid equilibrium between bound and medium MgADP [27,48], some limitations for dissociation of MgADP from immediate vicinity of creatine kinase should exist, or the reaction of phosphoryl-group transfer to MgADP should be considered to be more rapid than MgADP binding, and not a ratelimiting step for this particulate creatine kinase. These important questions require further detailed experimental studies.
262 Appendix
Mathematical simulation of homogeneous reaction system (creatine kinase, pyruvate kinase, hexokinase as A TPase) It was a s s u m e d that the system functions in s t e a d y - s t a t e with respect to a d e n i n e n u c l e o t i d e s . This a s s u m p t i o n leads to the e q u a t i o n : VATp = VCK "~- VpK
(|)
where VATP is the rate of ATP splitting into ADP and inorganic phosphate; vcK is the rate of ADP rephosphorylation into ATP in the creatine-kinase reaction from substrates in the medium; VpK is the rate of the pyruvate kinase reaction.The dependences of the rates of these reactions on the metabolic concentrations in the medium are described by the following kinetic equations [21,48-50]: [MgATP]/KATp VA*p="o 1+_+[MgATP] [MsADP] KATP
(2)
KADP
[MgADP][PCr I v(.~ = v 1
+ [Crl
[PCrl
[M~TPI
1 -~ib +~--~-id +
[MsATP][Cr] vl
KicK d
1+
Ki.
KiaK b
+ ~
(3)
1+ Kib
Kd
Kb
v2 [PEP][Ms 2+ ][MgADPI/KAK m /) PK
(KmPEP+[PEP])(I+ [Mgz+]K~+,(I+ I[MgZ+])[MgADP] ' Km + :( 1+ [ MKg -Z~+P]] ] [ A Ka D P ] ] ]+~( 1 + ~[ M g 2 + ] ) [MgATP]K,~ K~ TP
(4)
The dissociation constants used are described and given in Table II. Assuming that pH, concentration of total Mg, creatine, phosphocreatine, phosphoenolpyruvate (PEP). pyruvate and total nucleotides ATP + ADP are known, Eqns. 2 - 4 were introduced into Eqn. 1 and the TABLE II THE VALUES OF KINETIC CONSTANTS USED FOR MATHEMATICAL SIMULATION OF Mg2+-ATPase+CREATINE KINASE + PYRUVATE KINASE REACTIONS The kinetics of the reaction are described in Refs, 11, 27-29, 48-51. The constants for the creatine-kinase reaction were determined experimentally for the myofibrillar preparation used by a method described earlier [11,21,48]. Reaction
Constant
Mg 2+ -ATPase
KAT P KAD p
Creatine kinase
Kia Ka Kib Kb Ki~ K¢
Value (raM) 0.1 1.0 0.9 0.4 20 9 0.085 0.03
Reaction
Constant
Value (raM)
Creatine kinase (cont.)
Ka
0.4 1.04
Kid Pyruvate kinase
KA K~ K,~ K,1 K~
KPmF-p K1 K1ATP
0.015 0.047 0.047 0.08 0.25 0.14 2.1 0.14
263
quasi-steady-state concentrations of the nucleotides were computed by using numeral methods to fit Eqn. 1 for conditions given• From these calculations, individual reaction rates for each of these reactions were easily obtained. Calculations of free magnesium and Mg-complexes with substrates were performed as described earlier [21,51]. The results of calculations are shown in Fig. 2 (dotted line) and fit fairly well with the experimental data. That shows that the kinetics of a homogeneous enzyme system completely describe the behavior of these three enzymes and that the suppression of the creatine-kinase reaction by pyruvate kinase at high P K / C K ratios results from efficent removal of MgADP from the medium by this enzyme.
Acknowledgements The authors thank Professors V.N. Smirnov and H. Bricaud for making possible this cooperation under the joint France-U.S.S.R. Research Program and for their continuous interest to this study. The skillful technical assistance by Dr. G.B. Chernousova and L.A. Makhotina is gratefully recognized.
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