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Cardiac myofibrillar creatine kinase Km is not influenced by contractile protein binding
Life Sciences, Vol. Printed in the U S A
50, pp. 1551-1559
Pergamon Press
CARDIAC MYOFIBRILLAR CREATINE KINASE KIa IS NOT INFLUENCED BY CONTRACTILE PROTEIN BINDING Russell T. Dowell and May C Fu
Tobacco & Health Research Institute and Department of Physiology & Biophysics University of Kentucky Lexington, KY 40.5464r236 (Received in final form March 6, 1992)
Summary Subcellular microcompartmentation underlies the proposed phosphorylcreatine shuttle mechanism in mammalian cardiac tissue. In mitochondria, CK coupling to oxidative phosphorylation via adenine nucleotide translocase decreases the Km for ATP and suggests both a functional and physical integration. In the present studies, substrate Km of myofibrillar CK was unaltered when determined in the intact, native state or after removal from the myofibril. In contrast to mitochondria, close spatial proximity between cardiac myofibrillar CK and ATPase is sufficient to establish phosphorylcreatine shuttle microcompartmentation. The phosphorylcreatine shuttle mechanism, proposed by Bessman and Geiger (1), envisioned conjoined energy producing and energy utilizing systems functioning to help regulate striated muscle contraction. Creatine kinase (CK) [ATP:creatine Nphosphotransferase,{EC 2.7 3.2} ] was recognized as a primary enzyme responsible for shuttle function and distinct CK isoenzyme forms have been identified. Muscle cytosolic M-isoform exists along with a specific mitochonddal CK-isoform. In adult, mammalian cardiac muscle, a portion of the M-CK is localized at myofibrillar sites of high ATP demand (2,3). Mitochondrial CK is associated with the outer surface of the inner mitochondrial membrane (4,5). Enzyme kinetic studies have been conducted to understand how these compartmentalized energy producing and energy utilizing systems may be regnlatecL Substrate preference for ADP and ATP has been shown for the M-OK and mitochondrial CK isoenzymes; however, Km differences do not provide a satisfactory regulatory explanation. Interestingly, substrate kinetic parameters of mitochondrial CK are dramatically altered when the enzyme is linked with the adenine nucleotide translocator and in the presence of oxidative phosphorylation (4,6,7). Coupling phosphorylcreatine production to oxidative phosphorylation via adenine nucleotide translocase significantly decreases the Km for ATP and suggests a functional integration due to submitochondrial localization. Because myofibril M-CK is spatially associated with myofibrillar ATPase, the present studies were conducted to ascertain whether and how this association establishes a kinetically favorable, coupled energy reaction. Copyright
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All rights reserved.
1552
Cardiac Myofibrillar Creatine Kinase
Vol. 50, No. 20, 1992
Methods and Material~ Adult rats were killed by cervical dislocation and the heart was rapidly excised. Purified left ventricular myofibdls were isolated and treated with Triton X-100 (8) to assure removal of contaminating membrane~ Protein concentration in all myofibdllar preparations was adjusted to 5 mg/ml prior to further treatment. For CK extraction, approximately 25 nag of myofibrillar protein was centrifuged at 1,500 rpm for 10 minutes in a refrigerated IEC centrifuge. Pelleted myofibrils were then extracted by stirring with 3 ml of 5 mM Tris, pH 7.4 containing 1 mM dithiothreitol. After centrifugation at 1,500 rpm for 10 minutes, extraction was twice repeated and the CK containing supernatants combined. MyofibrUs and extracted myofibrillar supernatant were analyzed at 30"C for activity in the "reverse reaction" using a coupled enzyme assay system containing: 100 mM imidazole, pH 6.9, 10 mM magnesium acetate, 20 mM glucose, 10 mM AMP, 0.7 mM NADP, 1 IU/ml hexokinase and 1 IU/ml glucose-6-phosphate dehydrogenase, 50 /~M diadenosine pentaphosphate, and 10 mM N-acetylcysteine in an assay volume of 1.0 ml. Michaelis constants were derived using ADP and creatine phosphate as substrates. In the presence of 2 mM ADP, creatine phosphate was varied from 1 mM to 30 mM. In the presence of 30 mM creatine phosphate, ADP was varied from 0.1 mM to 2 mM. All myofibrillar assays were conducted using 25 ~g protein. All creatine kinase extract assays were conducted using 1.5 ~g protein. Changes in optical density at 340 nm were recorded after substrate addition. ATPase was present at approximately 250 nmole/mg protein/rain in myofibrillar preparations However, there was no detectable interference with measured creatine kinase due to (a) the small amount of myofibrillar protein used in creatine kinase assay, (b) the relatively high ATP Km for ATPase versus the coupling enzyme, hexokinase, and (c) finding expected creatine kinase activity results in the presence of 0.05 mM to 1.0 mM exogenous ATP added to the myofibfillar reaction mixture (data not presented). Double reciprocal plots, (1/V vs 1/[S]), of enzyme activities (nmole/mg protein/min) were analyzed by linear regression to ascertain Vmaxand Km values. In some mynfibril and extract preparations, CK activation energy was determined by measuring enzyme activity at 25"C and 35"C (9). Selected myofibril and extract preparations were utilized for CK isoenzyme evaluation by agarose gel electrophoresis. Results
The present studies were designed to examine cardiac myofibrillar CK kinetic properties both in the enzyme's normal, structural configuration within the myofibril and after having been extracted from the myofibril. Methods utilized for extracting CK from cardiac myofibrils were quantitatively effective and reproducible as evidenced by 85 ± 5 percent extraction in the preparations studied with a range from 76-96 percent. Homogeneous CKMM isoenzyme was present in both purified myofibrils and extract as shown by the electrophoretic profiles and densitometric scans in Figure 1. CK enzyme activity and double reciprocal plots for myofibrils are illustrated in Figure 2 and Figure 3. Figure 2 presents enzyme results in the presence of a f ' ~ t , saturating concentration of creatine phosphate while ADP substrate is increased. Figure 3 presents enzyme results in the presence of a f'~ed, saturating concentration of ADP while creatine phosphate substrate concentration is increased. An appropriate
Vol. 50, No. 20, 1992
Cardiac Myofibrillar Creatine Kinase
1553
CONTROL ~~.~
II
|
I
MMMB BB MYOFiBRIL
!
EXTRACT
!
FIG. 1 Electrophoretic Profiles and Densitometric Scans of Purified Myofibril and Myofibrillar Extract. Electrophoresis was Carried out on Agaruse Gel, Stained for CK Isoenzymes, and Scanned Densitometrically. augmentation in enzyme activity is noted for both increasing substrate~ Enzyme activity plateaus at the higher substrate levels for both creatine phosphate and ADP. Similar enzyme activity and double reciprocal plots for extracted CK are illustrated in Figure 4 and Figure 5. As was the case for myofibrils, extracted CK activity increases appropriately to a demonstrable plateau value in the presence of a f'vcecl,saturating concentration of creatine phosphate while ADP concentration is increased (Figure 4) and in the presence of a fixed, saturating concentration of ADP while creatine phosphate concentration is increased (Figure
5). The enzyme specific activity observed in extracted CK is approximately two orders of magnitude greater than specific activity measured in myofibriis, thus reflecting the enzyme purification and concentration achieved by the extraction process. Enzyme kinetic constants for myofibril and extracted CK are given in Table I. Maximum enzyme velocity (Vmax) is consistent for either substrate, i.e. ADP and creatine phosphate; however, extracted CK exhibits a significantly higher Vma x value. The
1554
Cardiac Myofibrillar Creatine Kinase
Vol. 50, No. 20, 1992
CK, nmole/mg/min x 10 2
I
o.,.,
I
.~,
~
[*o,]. ,,,
1IV
I 6
-8
110
,,is]
FIG. 2 Myofibrillar CK Activity and Double Reciprocal Plots with Increasing Concentrations of ADP as Substrate.
CK, nmole/nql/mln • 10 2
$
O0
3
10
2
S2 1/Y
24
8
,/ f
I 1
,,[.]
I I
I 3
I 4
FIG. 3 MyofibriUar CK Activity and Double Reciprocal Plots with Increasing Concentrations of Creatine Phosphate as Substrate.
Vol.
50,
No.
20,
Cardiac Myofibrillar Creatlne Kinase
1992
I
4~K, nmole/mS/mbu • 504 1
Oo.;.;
.~
' , t'+~','].,,,,u
t/V
I
y
.8
/
i s
o
I 1o
''Is] FIG. 4
Extract CK Activity and Double Reciprocal Plots ~ t h lncreashlg Concentrations of ADP as Substrate.
OK. nlllOll/llll/mllll • 10 4
°o
[c,]. i 1/V
4
8
i i -1
o
1
l
8
I 4
1/Is] FIG.
5
Extract CK Acfi~fity and Double Reciprocal Plots with Increasing Concentrations of Creafine Phosphate as Substrate.
1555
1556
C a r d i a c M y o f l b r i l l a r Creatine Kinase
Vol. 50, No. 20, 1992
myofibrillar CK Michaelis constant (Km) for ADP is an order of magnitude lower than that for creatine phosphate. This reduced K m for ADP is also present in e l ( extracted from the myofibril as previously reported by Saks et al. (10) despite differences in extraction conditions Nearly identical Michaelis constants for both ADP and creatine phosphate are present in myofibril and extracted enzyme. Secondary Michaelis-Menten plots produced nearly identical Km values in myofibril and extracted enzyme (data not presented).
TABLE I Creatine Kinase K= and V w for ADP and CP in Myofibril and MyofibrU Extract
I<.__ ADP
CP
ADP
CP
505 48
521 46
.205 .005
2.35 .17
Myofibril (7) Mean SE Extract (9) Mean SE
25,427* 3,239
25.539* 2,671
.220 .020
2.53 .23
Values shown are mean and standard error (SE). Number of preparations given in parentheses. K~ = mM. Vmx = nmole/mg protein/min. * P _< 0.05 versus myofibril.
In addition to the enzyme kinetic characteristics described above, CK activation energy was determined in myofibril and extracted enzyme. As shown in Table II, nearly identical activation energy values were obtained for CK whether measured in myofibrils or myofibrillar extract. Discussion Subcellular microcompartmentation within mitochondria and myofibril underlies the proposed phosphorylcreatine shuttle mechanism in mammalian cardiac tissue (1,11,12,13). At the level of cardiac contractile protein, functional interaction occurs between myofibrillar CK and myosin ATPase (11,12,14). Removal of myofibrillar CK bound to the M-line results in loss of ATP regenerating potential (3) indicating that ATP is rapidly and preferentially redistributed from CK to myosin ATPase (11,12,13). However, the above information provides little insight regarding the means by which myofibrillar microcompartmentalization
Vol. 50, No. 20, 1992
C a r d i a c M y o f i b r i l l a r Creatine Kinase
1557
TABLE II Creatine Kinase Activation Energy in Myofibril and Myofibril Extract Mvofibril
Extract
36_5 0.8
34.9 4.4
Activation energy (6) Mean SE
Values shown are mean and standard error (SE). Number of preparations given in parenthese~ Activation energy = kJ/mole and was calculated from enzyme activities measured at 25" C and 35" C. is established. In the present studies, CK enzyme substrate K. and activation energy were unaltered when determined in the native state or after removal from the myofibril. Thus, a multi-enzyme complex involving CK and myosin ATPase is unlikely. According to Jacobus et al. (15), compartmentation can be established by (a) semipermeable limiting membranes, (b) multi-enzyme complexes with covalent substrate transport, and (c) enzyme-enzymeproximity effects without direct substrate transport. There is strong evidence for cardiac mitochondrial microcompach~entation due to limiting membranes and multi-enzyme complexes which lead to kinetic enhancement of enzymatic reactions. In the case of mitochondrial CK, the forward reaction in intact mitochondria was found to be accelerated when ATP was provided by oxidative phosphorylation. Enzyme V , x is not altered by the substrate source; however,Kin for ATP is reduced by more than an order of magnitude (6,7). These kinetic data lead to the proposal that CK localized on the outer surface of the inner mitochondrial membrane was functionally coupled to oxidative phosphorylation via adenine nucleotide translocase. Close spacial proximity and direct substrate channeling were further demonstrated by the preference of adenine nucleotide translocase for ADP produced by mitochondrial CK and, subsequently, transported into the mitochondrial matrix for rephosphorylation. Thus, kinetic studies supported the suggestion that both the nucleotide substrate and product of creatine phosphate synthesis are preferentially delivered to (4,16) and removed from (16,17,18) mitochondrial CK due m protein-protein coupling between membrane-bound CK and adenine nucleotide translocase. Recent physico-chemical evidence provides additional information regarding mitochondrial compartmentalization. Cardiac mitochondrial CK demonstrates interconvertible dimeric and octameric forms (19). The predominant octameric enzyme (M r 360,000) possesse~ a cube-like structure with a central cavity capable of channeling highenergy phosphate out of the mitochondrion (20). Octameric CK binds preferentially to the inner mitochondrial membrane and is functionally, and perhaps physically, coupled to adenine nucleotide translocase (5~0). Moreover, octameric mitochondrial CK is capable of simultaneously interacting with two opposing mitochondrial membrane interface~
1558
Cardiac Myofibrillar Creatine Kinase
Vol. 50, No. 20, 1992
Topological joining of inner and outer mitochondrial membranes has been identified at contact sites where mitochondrial CK is in close proximity, or direct contact, with pore protein in the outer mitochondrial membrane and adenine nucleotide transferase of the inner mitochondrial membrane (21,22,2324). Fusion of inner and outer mitochondrial membrane via mitochondrial CK provides a distinct kinetic advantage for transport of energy to the cytoplasm of the cardiac muscle cell. For cardiac myofibrils, an enzyme complex involving CK and myosin ATPase is unlikely. Membrane diffusion barriers are minimal within the myofibril. Therefore, close spatial proximity between enzymes appears to be sufficient to establish myofibrillar phosphorylcreatine shuttle microcompartmentalization.
ao du ul m This work was supported, in part, by research grants HL 33767, KTRB 541078, and UKMC 204941. References 1. Sa°. BESSMAN and P.J. GEIGER, Science. 211 448-452 (1981). 2. R. VENTURA-CLAPIER, V.A. SAKS, G. VASSORT, C. LAUER and G.V. ELIZAROVA, A m j . Physiol. 2 ~ C444-C455 (1987). 3. T. WALLIMANN, T. SCHLOSSER and H.M. EPPENBERGER, J. Biol. Chem. 259
5238-5246 (1984). 4. V.A. SAKS, A.V. KURZNETSOV, V.V. KUPRIYANOV, M.V. MICELI and W E . JACOBUS, J. Biol. Chem. 260 7757-7764 (19853. 5. J. SCHLEGEL, M. WYSS, H.M. EPPENBERGER and T. WAI.I.IMANN, J. Biol. Chem. 265 9221-9227 (1990). 6. V.A. SAKS, G.B. CHERNOUSOVA, D.E. GUKOVSKY, V.N. SMIRNOV and E.I. CHAZOV, Eur. J. Biochem. 57 273-290 (1975). 7. V.A. SAKS, V.V. KUPRIYANOV, G.V. ELIZAROVA and W.E. JACOBUS, J. Biol. Chem. 255 755-763 (1980). 8. R.T. DOWELL, Biochem. Med. Metab. Biol. 37 374-384 (1987). 9. R.T. DOWELL and A.F. MARTIN, Can. J. Physiol. Pharmacol. 63 627-629 (1985). 10. V.A. SAKS, G.B. CI-IERNOUSOVA, R. VETrER, VaN. SMIRNOV, and E.I. CHAZOV, FEBS Lett. 62 293-296 (1976). 11. F. SAVABI, PJ. GEIGER and S.P. BESSMAN, Am. J. Physiol. 247 C424-C432 (1984). 12. S.P. BESSMAN, W.C.T. YANG, PJ. GEIGER and S. ERICKSON-VIITANEN, Biochem. Biophys. Res. Comm. 96 1414-1420 (1980). 13. F. SAVABI, P J . GEIGER and SJ'. BESSMAN, Biochem. Biophys. Res. Comm. 114 785-790 (1983). 14. G. MCCLELLAN, A. WEISBERG and S. WINEGRAD, Am. J. Physiol. 245 C423-C427 (1983). 15. W.E. JACOBUS, R.W. MOREADITH and K.M. VANDEGAER, Ann. N.Y. Acad. Sci. 414 73-78 (1984). 16. R . L BARBOUR, J. RIBAUDO and S.H.P. CHAN, J. Biol. Chem. 259 8246-8251 (1984). 17. R.W. MOREADITH and W.E. JACOBUS, J. Biol. Chem. 257 899-905 (1982).
Vol. 50, No. 20, 1992
Cardiac Myofibr£11ar Creat£ne Kinase
1559
18. F. GELLERICH and V.A. SAKS, Biochem. Biophys. Res. Comm. 105 1473-1481 (1982). 19. J. SCHLEGEL, B. ZURBRIGGEN, G. WEGMANN, M. WYSS, H.M. EPPENBERGER and T. WALLIMANN, J. Biol. Chem. 263 16942-16953 (1988). 20. T. SCHNYDER, A. ENGEL, A. LUSTIG and T. WALLIMANN, J. Biol. Chem. 263 16954-16962 (1988). 21. M. KOTrKE, V. ADAMS, T. WALLIMANN, V.K. NALAM and D. BRDICTJ(A, Biochim. Biophys. Acta. 1061 215-225 (1991). 22. W. BIERMANS, A. BAKKER and W. JACOB, Biochim. Biophys. Acta. 1018 225-228 (1990). 23. K. NICOLAY, M. ROJO, T. WALLIMANN, R. DEMEL and R. HOVIUS, Bioehim. Biophys. Aeta. 1018 229-233 (1990). 24. D. BRIDCZKA, K. BUCHELER, M. KOTTKE, V. ADAMS and V.K. NALAM, Bioehim. Biophys. Aeta. 1018 234-238 (1990).
50, pp. 1551-1559
Pergamon Press
CARDIAC MYOFIBRILLAR CREATINE KINASE KIa IS NOT INFLUENCED BY CONTRACTILE PROTEIN BINDING Russell T. Dowell and May C Fu
Tobacco & Health Research Institute and Department of Physiology & Biophysics University of Kentucky Lexington, KY 40.5464r236 (Received in final form March 6, 1992)
Summary Subcellular microcompartmentation underlies the proposed phosphorylcreatine shuttle mechanism in mammalian cardiac tissue. In mitochondria, CK coupling to oxidative phosphorylation via adenine nucleotide translocase decreases the Km for ATP and suggests both a functional and physical integration. In the present studies, substrate Km of myofibrillar CK was unaltered when determined in the intact, native state or after removal from the myofibril. In contrast to mitochondria, close spatial proximity between cardiac myofibrillar CK and ATPase is sufficient to establish phosphorylcreatine shuttle microcompartmentation. The phosphorylcreatine shuttle mechanism, proposed by Bessman and Geiger (1), envisioned conjoined energy producing and energy utilizing systems functioning to help regulate striated muscle contraction. Creatine kinase (CK) [ATP:creatine Nphosphotransferase,{EC 2.7 3.2} ] was recognized as a primary enzyme responsible for shuttle function and distinct CK isoenzyme forms have been identified. Muscle cytosolic M-isoform exists along with a specific mitochonddal CK-isoform. In adult, mammalian cardiac muscle, a portion of the M-CK is localized at myofibrillar sites of high ATP demand (2,3). Mitochondrial CK is associated with the outer surface of the inner mitochondrial membrane (4,5). Enzyme kinetic studies have been conducted to understand how these compartmentalized energy producing and energy utilizing systems may be regnlatecL Substrate preference for ADP and ATP has been shown for the M-OK and mitochondrial CK isoenzymes; however, Km differences do not provide a satisfactory regulatory explanation. Interestingly, substrate kinetic parameters of mitochondrial CK are dramatically altered when the enzyme is linked with the adenine nucleotide translocator and in the presence of oxidative phosphorylation (4,6,7). Coupling phosphorylcreatine production to oxidative phosphorylation via adenine nucleotide translocase significantly decreases the Km for ATP and suggests a functional integration due to submitochondrial localization. Because myofibril M-CK is spatially associated with myofibrillar ATPase, the present studies were conducted to ascertain whether and how this association establishes a kinetically favorable, coupled energy reaction. Copyright
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All rights reserved.
1552
Cardiac Myofibrillar Creatine Kinase
Vol. 50, No. 20, 1992
Methods and Material~ Adult rats were killed by cervical dislocation and the heart was rapidly excised. Purified left ventricular myofibdls were isolated and treated with Triton X-100 (8) to assure removal of contaminating membrane~ Protein concentration in all myofibdllar preparations was adjusted to 5 mg/ml prior to further treatment. For CK extraction, approximately 25 nag of myofibrillar protein was centrifuged at 1,500 rpm for 10 minutes in a refrigerated IEC centrifuge. Pelleted myofibrils were then extracted by stirring with 3 ml of 5 mM Tris, pH 7.4 containing 1 mM dithiothreitol. After centrifugation at 1,500 rpm for 10 minutes, extraction was twice repeated and the CK containing supernatants combined. MyofibrUs and extracted myofibrillar supernatant were analyzed at 30"C for activity in the "reverse reaction" using a coupled enzyme assay system containing: 100 mM imidazole, pH 6.9, 10 mM magnesium acetate, 20 mM glucose, 10 mM AMP, 0.7 mM NADP, 1 IU/ml hexokinase and 1 IU/ml glucose-6-phosphate dehydrogenase, 50 /~M diadenosine pentaphosphate, and 10 mM N-acetylcysteine in an assay volume of 1.0 ml. Michaelis constants were derived using ADP and creatine phosphate as substrates. In the presence of 2 mM ADP, creatine phosphate was varied from 1 mM to 30 mM. In the presence of 30 mM creatine phosphate, ADP was varied from 0.1 mM to 2 mM. All myofibrillar assays were conducted using 25 ~g protein. All creatine kinase extract assays were conducted using 1.5 ~g protein. Changes in optical density at 340 nm were recorded after substrate addition. ATPase was present at approximately 250 nmole/mg protein/rain in myofibrillar preparations However, there was no detectable interference with measured creatine kinase due to (a) the small amount of myofibrillar protein used in creatine kinase assay, (b) the relatively high ATP Km for ATPase versus the coupling enzyme, hexokinase, and (c) finding expected creatine kinase activity results in the presence of 0.05 mM to 1.0 mM exogenous ATP added to the myofibfillar reaction mixture (data not presented). Double reciprocal plots, (1/V vs 1/[S]), of enzyme activities (nmole/mg protein/min) were analyzed by linear regression to ascertain Vmaxand Km values. In some mynfibril and extract preparations, CK activation energy was determined by measuring enzyme activity at 25"C and 35"C (9). Selected myofibril and extract preparations were utilized for CK isoenzyme evaluation by agarose gel electrophoresis. Results
The present studies were designed to examine cardiac myofibrillar CK kinetic properties both in the enzyme's normal, structural configuration within the myofibril and after having been extracted from the myofibril. Methods utilized for extracting CK from cardiac myofibrils were quantitatively effective and reproducible as evidenced by 85 ± 5 percent extraction in the preparations studied with a range from 76-96 percent. Homogeneous CKMM isoenzyme was present in both purified myofibrils and extract as shown by the electrophoretic profiles and densitometric scans in Figure 1. CK enzyme activity and double reciprocal plots for myofibrils are illustrated in Figure 2 and Figure 3. Figure 2 presents enzyme results in the presence of a f ' ~ t , saturating concentration of creatine phosphate while ADP substrate is increased. Figure 3 presents enzyme results in the presence of a f'~ed, saturating concentration of ADP while creatine phosphate substrate concentration is increased. An appropriate
Vol. 50, No. 20, 1992
Cardiac Myofibrillar Creatine Kinase
1553
CONTROL ~~.~
II
|
I
MMMB BB MYOFiBRIL
!
EXTRACT
!
FIG. 1 Electrophoretic Profiles and Densitometric Scans of Purified Myofibril and Myofibrillar Extract. Electrophoresis was Carried out on Agaruse Gel, Stained for CK Isoenzymes, and Scanned Densitometrically. augmentation in enzyme activity is noted for both increasing substrate~ Enzyme activity plateaus at the higher substrate levels for both creatine phosphate and ADP. Similar enzyme activity and double reciprocal plots for extracted CK are illustrated in Figure 4 and Figure 5. As was the case for myofibrils, extracted CK activity increases appropriately to a demonstrable plateau value in the presence of a f'vcecl,saturating concentration of creatine phosphate while ADP concentration is increased (Figure 4) and in the presence of a fixed, saturating concentration of ADP while creatine phosphate concentration is increased (Figure
5). The enzyme specific activity observed in extracted CK is approximately two orders of magnitude greater than specific activity measured in myofibriis, thus reflecting the enzyme purification and concentration achieved by the extraction process. Enzyme kinetic constants for myofibril and extracted CK are given in Table I. Maximum enzyme velocity (Vmax) is consistent for either substrate, i.e. ADP and creatine phosphate; however, extracted CK exhibits a significantly higher Vma x value. The
1554
Cardiac Myofibrillar Creatine Kinase
Vol. 50, No. 20, 1992
CK, nmole/mg/min x 10 2
I
o.,.,
I
.~,
~
[*o,]. ,,,
1IV
I 6
-8
110
,,is]
FIG. 2 Myofibrillar CK Activity and Double Reciprocal Plots with Increasing Concentrations of ADP as Substrate.
CK, nmole/nql/mln • 10 2
$
O0
3
10
2
S2 1/Y
24
8
,/ f
I 1
,,[.]
I I
I 3
I 4
FIG. 3 MyofibriUar CK Activity and Double Reciprocal Plots with Increasing Concentrations of Creatine Phosphate as Substrate.
Vol.
50,
No.
20,
Cardiac Myofibrillar Creatlne Kinase
1992
I
4~K, nmole/mS/mbu • 504 1
Oo.;.;
.~
' , t'+~','].,,,,u
t/V
I
y
.8
/
i s
o
I 1o
''Is] FIG. 4
Extract CK Activity and Double Reciprocal Plots ~ t h lncreashlg Concentrations of ADP as Substrate.
OK. nlllOll/llll/mllll • 10 4
°o
[c,]. i 1/V
4
8
i i -1
o
1
l
8
I 4
1/Is] FIG.
5
Extract CK Acfi~fity and Double Reciprocal Plots with Increasing Concentrations of Creafine Phosphate as Substrate.
1555
1556
C a r d i a c M y o f l b r i l l a r Creatine Kinase
Vol. 50, No. 20, 1992
myofibrillar CK Michaelis constant (Km) for ADP is an order of magnitude lower than that for creatine phosphate. This reduced K m for ADP is also present in e l ( extracted from the myofibril as previously reported by Saks et al. (10) despite differences in extraction conditions Nearly identical Michaelis constants for both ADP and creatine phosphate are present in myofibril and extracted enzyme. Secondary Michaelis-Menten plots produced nearly identical Km values in myofibril and extracted enzyme (data not presented).
TABLE I Creatine Kinase K= and V w for ADP and CP in Myofibril and MyofibrU Extract
I<.__ ADP
CP
ADP
CP
505 48
521 46
.205 .005
2.35 .17
Myofibril (7) Mean SE Extract (9) Mean SE
25,427* 3,239
25.539* 2,671
.220 .020
2.53 .23
Values shown are mean and standard error (SE). Number of preparations given in parentheses. K~ = mM. Vmx = nmole/mg protein/min. * P _< 0.05 versus myofibril.
In addition to the enzyme kinetic characteristics described above, CK activation energy was determined in myofibril and extracted enzyme. As shown in Table II, nearly identical activation energy values were obtained for CK whether measured in myofibrils or myofibrillar extract. Discussion Subcellular microcompartmentation within mitochondria and myofibril underlies the proposed phosphorylcreatine shuttle mechanism in mammalian cardiac tissue (1,11,12,13). At the level of cardiac contractile protein, functional interaction occurs between myofibrillar CK and myosin ATPase (11,12,14). Removal of myofibrillar CK bound to the M-line results in loss of ATP regenerating potential (3) indicating that ATP is rapidly and preferentially redistributed from CK to myosin ATPase (11,12,13). However, the above information provides little insight regarding the means by which myofibrillar microcompartmentalization
Vol. 50, No. 20, 1992
C a r d i a c M y o f i b r i l l a r Creatine Kinase
1557
TABLE II Creatine Kinase Activation Energy in Myofibril and Myofibril Extract Mvofibril
Extract
36_5 0.8
34.9 4.4
Activation energy (6) Mean SE
Values shown are mean and standard error (SE). Number of preparations given in parenthese~ Activation energy = kJ/mole and was calculated from enzyme activities measured at 25" C and 35" C. is established. In the present studies, CK enzyme substrate K. and activation energy were unaltered when determined in the native state or after removal from the myofibril. Thus, a multi-enzyme complex involving CK and myosin ATPase is unlikely. According to Jacobus et al. (15), compartmentation can be established by (a) semipermeable limiting membranes, (b) multi-enzyme complexes with covalent substrate transport, and (c) enzyme-enzymeproximity effects without direct substrate transport. There is strong evidence for cardiac mitochondrial microcompach~entation due to limiting membranes and multi-enzyme complexes which lead to kinetic enhancement of enzymatic reactions. In the case of mitochondrial CK, the forward reaction in intact mitochondria was found to be accelerated when ATP was provided by oxidative phosphorylation. Enzyme V , x is not altered by the substrate source; however,Kin for ATP is reduced by more than an order of magnitude (6,7). These kinetic data lead to the proposal that CK localized on the outer surface of the inner mitochondrial membrane was functionally coupled to oxidative phosphorylation via adenine nucleotide translocase. Close spacial proximity and direct substrate channeling were further demonstrated by the preference of adenine nucleotide translocase for ADP produced by mitochondrial CK and, subsequently, transported into the mitochondrial matrix for rephosphorylation. Thus, kinetic studies supported the suggestion that both the nucleotide substrate and product of creatine phosphate synthesis are preferentially delivered to (4,16) and removed from (16,17,18) mitochondrial CK due m protein-protein coupling between membrane-bound CK and adenine nucleotide translocase. Recent physico-chemical evidence provides additional information regarding mitochondrial compartmentalization. Cardiac mitochondrial CK demonstrates interconvertible dimeric and octameric forms (19). The predominant octameric enzyme (M r 360,000) possesse~ a cube-like structure with a central cavity capable of channeling highenergy phosphate out of the mitochondrion (20). Octameric CK binds preferentially to the inner mitochondrial membrane and is functionally, and perhaps physically, coupled to adenine nucleotide translocase (5~0). Moreover, octameric mitochondrial CK is capable of simultaneously interacting with two opposing mitochondrial membrane interface~
1558
Cardiac Myofibrillar Creatine Kinase
Vol. 50, No. 20, 1992
Topological joining of inner and outer mitochondrial membranes has been identified at contact sites where mitochondrial CK is in close proximity, or direct contact, with pore protein in the outer mitochondrial membrane and adenine nucleotide transferase of the inner mitochondrial membrane (21,22,2324). Fusion of inner and outer mitochondrial membrane via mitochondrial CK provides a distinct kinetic advantage for transport of energy to the cytoplasm of the cardiac muscle cell. For cardiac myofibrils, an enzyme complex involving CK and myosin ATPase is unlikely. Membrane diffusion barriers are minimal within the myofibril. Therefore, close spatial proximity between enzymes appears to be sufficient to establish myofibrillar phosphorylcreatine shuttle microcompartmentalization.
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