Life Sciences, Vol . 21, pp . Printed in the U .S .A .
1171-1178
Pergamon Press
INHIBITION OF CARDIAC CAEATINE PHOSPHOKINASE BY FLUORODINITROBENZENE William C.T . Yang and Michael Dubick Department of Pharmacology University of Southern California School of Medicine, Los Angeles, CA . 90033 (Received in final form September 9, 1977) Summary: With electrophoretic evidence for a mitochondrial isoenzyme of creatine phosphokinase (CPK), we feel that functional studies are necessary to help further elucidate the properties of this isoenzyme. As one approach, fluorodinitrobenzene (FDNB) was used to examine its effect on mitochondria CPK. In both polarographic studies and direct enzymatic studies, 10 M FDNB was shown to almost completely inhibit the enzyme activity, as has been shown in skeletal muscle . In addition it was observed that the mitochondrial CPK was just as susceptible to the inhibitory effect of FDNB as the cytoplasmic isoenzyme. Jacobs, et al . (1) gave evidence for a mitochondrial isoenzyme of creative kinase in pigeon skeletal and heart muscle, and in rat skeletal and heart muscle and brain. In 1972, Sobel, et al . (2) clearly distinguished rabbit heart mitochondrial creatine phosphokinase (CPK) as different from the known isoenzymes isolated from the cytosol of brain, skeletal muscle, and heart (BB, MM, and MB as diners) . This group also reported that the rabbit heart mitochondrial CPR is relatively insensitive to the action of the specific CPK inhibitor, 1-fluoro, 2,4-dinitrobenzene (FDNB) (3,4), at a concentration of 10-2 M. In the past few years, FDNB has been used as a tool to further elucidate the role of creatine, phosphocreatine (PC), and CPK in the energy metab olism of cardiac muscle (5,6) . With this in mind, our group carried out studies of isolated rabbit heart mitochondria to elucidate the effectiveness of this inhibitor on the mitochondrial CPK in an attempt to further characterize this isoenzyme. Materials and Methods Male New Zealand albino rabbits were sacrificed by a sharp blow on the head and the heart was removed and washed of blood. The ventricles were placed in ice cold homogenization medium containing 0.35 M mannitol, 0.1 mM EDTA, and 10 mM Tris-buffer, pH 7 .2 . The tissue was cut into small pieces and run through a Harvard tissue press. This now fine tissue was hand homogenized in a Dounce-type homogenizer. The heart homogenate was then centrifuged for 10 minutes at SOOg in an International Refrigerated Centrifuge . Mitochondria were isolated from the above supernatant by centrifugation at 8000g for 10 minutes . The mitochondria were washed once or several times . They were then suspended in a medium containing the homogenization medium,0 .5% (w/v) dialyzed albumin, and 0.1 M Tris-phosphate buffer, pH 7 .2 . Polarographic studies were done to examine the effect of FDNB on mitochondrial CPK in both the forward (ATP + creatine + ADP + PC) and the reverse direction (ADP + PC + ATP + creative) . A GME Model KM Oxygraph with a standard 1171
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FDNB Inhibition of Cardiac CPK
Vol . 21, No . 8,
1977
Clark electrode was used for this purpose. The reaction medium for these studies consisted of 0 .25 M mannitol, 10 mM KC1, 10 mM Tris-Cl buffer, pH 7 .2, 0 .1 mM EDTA, and 5 mM phosphate buffer, pH 7 .2 . The reactions were run at a temperature of 28°C . Other substrates used included 0 .30 mM ADP, 3 .4 mM a-ketoglutarate, and 1 .4 mM MgC1 2 . Final concentrations for creatine, PC, and FDNB are as stated in the results . Direct assay of the heart mitochondrial CPK activity was done spectrophotometrically at 520 nm according to the Colorimetrical Determination procedure (#520) of the Sigma Chemical Company (7) . Protein determination was by either the Biuret
(8) or Folin methods (9) .
Results Effect of FDNB on the stimulation of mitochondrial oxygen uptake by ATP . When creatine was present in the reaction medium, ATP stimulated the 0 uptake of heart and skeletal muscle mitochondria (2,10,11,12) . The forward reaction, as catalyzed by mitochondrial CPK, yielded a continuous supply of ADP which stimulated the rate of mitochondrial 02 uptake . As illustrated in Fig . 1B, in the presence of 10 mM creatine, the addition of 0 .3 mM ADP initiated the state 3 respiration, with a return to the state 4 respiration after thh completion of the phosphorylation of the ADP. The addition of 1 .4 mM Mg resulted in a continuous high rate of 02 uptake (234 mu atoms oxygen/min/mg mitochondrial protein) . In the absence of creatine (Fig . 1A), the addition of Mg ++ stimulated the 02 uptake to a much lesser degree (121 my atoms oxygen/ + min/mg protein) . This confirms the statement of Sobel, et al . (2) that Mg is a necessary cofactor for the CPR reaction in the isolated mitochondrial system . ++ The effect of different concentrations of FDNB was studied on the Mg stimulated rgte of 02 uptake in the presence of creatine . As summarized in Table I, 10 M FDNB inhibited the increased rate of 02 uptake by 38%, whereas concentrations of 10-5 and above inhibited this increased rate of respiIn FU . 1C, in the presence of 10 mM creatine and rat on almost completely . 10 M FDNB, the addition of Mg no longer produced the continuous high rate of 02 uptake . TABLE I Effect of FDNB on Rate of ATP, Mg-Stimulated Mitochondrial Respiration Creatine*
+ + + + + +
FDNB, 0 0 10 6 5 3x10 10 10 4 3x10 4
(M)
% Control t Rate 100% 2 196.1±5 .1 164 .3 148.6 109.2±3 .9 105.3±2 .5 107.2
% Inhibition 1
(5) (2) (2) (5) (3) (2)
0.0 38 .2 53 .6 91 .0±3 .4 94 .8±2 .6 93 .2
* Creatine concentration-10 mM lMean±S.E . 2Control rate of respiration 105 my atoms oxygen/min/mg mitochondrial protein Numbers in parentheses indicate number of experiments
Vol. 21, No . 8, 1977
FDNB Inhibition of Cardiac CPR
1173
FIG. 1 Effect of creative a FDNB on rate of mitochondrial respiration A) Control; B) 10 mM creative ; C) 10 mM creative + 10-5 M FDNB Numbers represent mp atoms oxygen/min/mg mitochondrial protein. Effect of FDNB on the decreased 02 uptake stimulated by ADP in the presence of PC . As illustrated in Fig. 2B, when 1 mm PC was present in the reaction medium, the ADP (0 .3 ") stimulated 0Z uptake was significantly reduced in the presence of 1 .4 mm Mg , confirming the results of Vial and Gautheron (10) . The addition of Mg++ was again found necessary for the activity of the CPK . The reverse reaction catalyzed by mitochondrial CPR converted part of the ADP to ATP, thus reducing the amount of ADP available as the acceptor for oxidative phhosphorylation . The competition between the CPR catalyzed reaction and oxidative phosphorylation for ADP is illustrated in Table II . As higher concentrations of PC were employed, higher inhibition of mitochondrial 02 uptake as stimulated by ADP was observed . PC at a 1 mM concentration pr6duced a marked change in the 02 uptake stimulated by ADP in the presence of Mg , and so was used in subsequent experiments with FDNB .
FDNB Inhibition of Cardiac CPR
1174
Vol . 21, No . 8, 1977
ADP
FIG. 2 Effect of PC and FDNB on mitochondrial oen uptake A) Control ; B) 1 MM PCs C) 1 MM PC + 10-9 M FDNB Numbers represent mu atoms oxygen/ml TABLE II PC inhibition of ADP-Stimulated Mitochondrial Respiration PC Concentration, mM 0 0.0
0.01
0.03
0.10
0.30
1.0
3 .0
10 .0
4 .7± 2.4 1 (3)
7 .3± 4 .7 (3)
14 .3± 2.7 (3)
23 .0± 4 .4 (3)
36 .4± 1 .5 (23)
39 .0± 1 .9 (4)
50 .4± 3 .0 (7)
1All values are mean±S .E . as percent of inhibition Numbers in parentheses indicate number of experiments 10-6 to 3x104 produced an As shown in Table III, FDNB concentrations from increase in 02 uptake toward control values . At a concentration of 10-5 M, 0 uptake was found to be 951 of control as further illustrated in a comparison Ae tween Fig . 2C and 2A . This implies that an inhibition of CPR activity resulted Similar efin more ADP available as acceptor for oxidative phosphorylation . fects of FDNB were found when the PC concentration was raised to 3 mK and 10 mM (Fig . 3) .
Vol . 21, No . 8, 1977
FDNB Inhibition of Cardiac CPR
1175
TABLE III Effect of FDNB on PC* Inhibition of ADP-Stimulated Mitochondrial Respiration FIM,
PC* -+ + + + + + +
(M)
'k Control
0 0-6 10-6 3x10 10 5 5 3x10 10 4 3x10 4
1
100% 2 63 .6±1 .5(23) 60 .2±3 .9(4) 69 .7±7 .1(3) 95 .0±1 .8(11) 94 .6±1 .7(5) 93 .7±2 .6(5) 98 .0 (2)
* PC concentration 1 mM 1 Mean±S .E . 2 Control 02 uptake 87 .0 my atoms oxygen/ml Number in parentheses indicates number of experiments
60
40
20
2
4
6
e
10
PC [-MI FIG. 3 Effect of FDRB on PC inhibition of ADP-stimulated mitochondrial respiration . o - control e - 1-3x10 5 M FDNB Numbers in parentheses indicate number of experiments Effect of FDNB on the Spectrophotometric Assay of Mitochondrial and Cytoplasmic CPIC . As shown in Table IV, direct assay of nitochon~rial CPR activity showed more marked inhibition of CPR by FDNB . FDNB at 3x10 M produced over a 70% inhibition of enzyme activity. However, it required FDNB at 10-5 M or higher to produce a 90% inhibition . FDNB produced similar inhibition of the
1176
Vol . 21, No . 8,
FDNB Inhibition of Cardiac CPK
1977
cytoplasmic CPK activity . Statistical evaluation (Student's "t" test) indicated that FDNB was equally effective in the inhibition of both mitochondrial and cytoplasmic CPK activity . TABLE IV Percent Inhibition of CPK Activity by FDNB FDNB, 0 3x10-6 10 3x105 103x10 5
(M)
Mitcchondrial CPK*
Cytoplasmic CPK*
0.0 (2) 71 76 .7±7 .6(6) 83 .4±3 .9(8) 92 .0±2 .0(7) 94 .3±1 .7(4)
0.0 73 (2) 76 .8±7 .2(6) 90 .4±1 .5(7) 93 .5±2 .2(6) 96 .8±1 .3(4)
Mean±S .E . Numbers in parentheses indicate number of experiments Since FDNB was dissolved in propylene glycol, the effects of this solvent on mitochondrial respiration were studied both with and without PC and creatine . It also had no effect on the It was found to have no significant effect . direct assay of mitochondrial and cytoplasmic CPK activity . In addition FDNB had no effect on mitochondrial respiration in the absence of PC or creatine . Discussion our results indicate that FDNB inhibits both the forward and reverse reactions catalyzed by CPK in intact cardiac mitochondria, and botg reactions Lower were almost completely inhibited at an FDNB concentration of 10- M. concentrations produced less inhibition . Indications were that the inhibition is immediate, as no incubation time was required to achieve the results . These findings on rabbit heart agree nicely with the work of Baba, et al . (13) . They found, through histochemical techniques, that all CPK activity in the rat heart was inhibited by 30 LM FDNB . In addition our direct assay technique has indicated that this concentration of FDNB will completely inhibit mitochondrial CPK activity as effectively as the activity of the CPK isoenzyme located in the heart supernatant fraction (cytoplasmic CPK) . Therefore we conclude that FMB is an effective inhibitor of CPR activity, and that CPK from different sources appears to be equally sensitive to the action of FDNB . References 1. 2. 3. 4. 5. 6. 7. 8. 9.
H. JACOBS, H. HELDT, and M. KLINGENBERG, Biochem. Biophys . Res . Ccmmun . 16, 516-521 (1964) . B. SOBEL, W. SHELL and M. KLEIN, J. Molec . & Cell Cardiol. _4, 367-380, (1972) . S. KUBY and T. MAHOWALD, Fed. Proc . 18, 267 (1959) . D. CAIN and R. DAVIES, Biochem. Biophys. Res . Commun . _8, 361-366 (1962) . M. SERAYDARIAN, E. SATO and I. HARARY, Arch . Biochem. Biophys. _138, 233-238 (1970) . M. SERAYDARIAN and L. ARTAZA, J. Molec . & Cell Cardiol. _8, 669-678 (1976) . Sigma Technical Bulletin #520 (1976) . A. GORNALL, C . BARDAWILL and M. DAVID, J. Biol . Chem . 177, 751-766 (1949) . J. Biol . Chem . 193, O. LOWRY, N. ROSEBROUGH, A. PARR and R. RANDALL, 265-275 (1951) .
Vol . 21, No . 8, 1977
10 .
FDNB Inhibition of Cardiac CPR
1177
C . VIAL and D . GAUTBERON, Recent Advances in Studies of Myocardial Structure and Function , Vol . 3, pg . 81, Univ . Park Press, Baltimore (1973) . 11 . i9 . JACOBUS and A . IMTINGER, J . Biol . Chem . 21, 4803-4810 (1973) . 12 . S . BESSMAN and A . FONYO, Biochem . Biophys . Res. Commun . 22, 597-602 (1966) 13 . J N . BABA, S . KIM and E . FARRELL , . Molec . & Cell Cardiol.8, 599-617, (1976) .
1171-1178
Pergamon Press
INHIBITION OF CARDIAC CAEATINE PHOSPHOKINASE BY FLUORODINITROBENZENE William C.T . Yang and Michael Dubick Department of Pharmacology University of Southern California School of Medicine, Los Angeles, CA . 90033 (Received in final form September 9, 1977) Summary: With electrophoretic evidence for a mitochondrial isoenzyme of creatine phosphokinase (CPK), we feel that functional studies are necessary to help further elucidate the properties of this isoenzyme. As one approach, fluorodinitrobenzene (FDNB) was used to examine its effect on mitochondria CPK. In both polarographic studies and direct enzymatic studies, 10 M FDNB was shown to almost completely inhibit the enzyme activity, as has been shown in skeletal muscle . In addition it was observed that the mitochondrial CPK was just as susceptible to the inhibitory effect of FDNB as the cytoplasmic isoenzyme. Jacobs, et al . (1) gave evidence for a mitochondrial isoenzyme of creative kinase in pigeon skeletal and heart muscle, and in rat skeletal and heart muscle and brain. In 1972, Sobel, et al . (2) clearly distinguished rabbit heart mitochondrial creatine phosphokinase (CPK) as different from the known isoenzymes isolated from the cytosol of brain, skeletal muscle, and heart (BB, MM, and MB as diners) . This group also reported that the rabbit heart mitochondrial CPR is relatively insensitive to the action of the specific CPK inhibitor, 1-fluoro, 2,4-dinitrobenzene (FDNB) (3,4), at a concentration of 10-2 M. In the past few years, FDNB has been used as a tool to further elucidate the role of creatine, phosphocreatine (PC), and CPK in the energy metab olism of cardiac muscle (5,6) . With this in mind, our group carried out studies of isolated rabbit heart mitochondria to elucidate the effectiveness of this inhibitor on the mitochondrial CPK in an attempt to further characterize this isoenzyme. Materials and Methods Male New Zealand albino rabbits were sacrificed by a sharp blow on the head and the heart was removed and washed of blood. The ventricles were placed in ice cold homogenization medium containing 0.35 M mannitol, 0.1 mM EDTA, and 10 mM Tris-buffer, pH 7 .2 . The tissue was cut into small pieces and run through a Harvard tissue press. This now fine tissue was hand homogenized in a Dounce-type homogenizer. The heart homogenate was then centrifuged for 10 minutes at SOOg in an International Refrigerated Centrifuge . Mitochondria were isolated from the above supernatant by centrifugation at 8000g for 10 minutes . The mitochondria were washed once or several times . They were then suspended in a medium containing the homogenization medium,0 .5% (w/v) dialyzed albumin, and 0.1 M Tris-phosphate buffer, pH 7 .2 . Polarographic studies were done to examine the effect of FDNB on mitochondrial CPK in both the forward (ATP + creatine + ADP + PC) and the reverse direction (ADP + PC + ATP + creative) . A GME Model KM Oxygraph with a standard 1171
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FDNB Inhibition of Cardiac CPK
Vol . 21, No . 8,
1977
Clark electrode was used for this purpose. The reaction medium for these studies consisted of 0 .25 M mannitol, 10 mM KC1, 10 mM Tris-Cl buffer, pH 7 .2, 0 .1 mM EDTA, and 5 mM phosphate buffer, pH 7 .2 . The reactions were run at a temperature of 28°C . Other substrates used included 0 .30 mM ADP, 3 .4 mM a-ketoglutarate, and 1 .4 mM MgC1 2 . Final concentrations for creatine, PC, and FDNB are as stated in the results . Direct assay of the heart mitochondrial CPK activity was done spectrophotometrically at 520 nm according to the Colorimetrical Determination procedure (#520) of the Sigma Chemical Company (7) . Protein determination was by either the Biuret
(8) or Folin methods (9) .
Results Effect of FDNB on the stimulation of mitochondrial oxygen uptake by ATP . When creatine was present in the reaction medium, ATP stimulated the 0 uptake of heart and skeletal muscle mitochondria (2,10,11,12) . The forward reaction, as catalyzed by mitochondrial CPK, yielded a continuous supply of ADP which stimulated the rate of mitochondrial 02 uptake . As illustrated in Fig . 1B, in the presence of 10 mM creatine, the addition of 0 .3 mM ADP initiated the state 3 respiration, with a return to the state 4 respiration after thh completion of the phosphorylation of the ADP. The addition of 1 .4 mM Mg resulted in a continuous high rate of 02 uptake (234 mu atoms oxygen/min/mg mitochondrial protein) . In the absence of creatine (Fig . 1A), the addition of Mg ++ stimulated the 02 uptake to a much lesser degree (121 my atoms oxygen/ + min/mg protein) . This confirms the statement of Sobel, et al . (2) that Mg is a necessary cofactor for the CPR reaction in the isolated mitochondrial system . ++ The effect of different concentrations of FDNB was studied on the Mg stimulated rgte of 02 uptake in the presence of creatine . As summarized in Table I, 10 M FDNB inhibited the increased rate of 02 uptake by 38%, whereas concentrations of 10-5 and above inhibited this increased rate of respiIn FU . 1C, in the presence of 10 mM creatine and rat on almost completely . 10 M FDNB, the addition of Mg no longer produced the continuous high rate of 02 uptake . TABLE I Effect of FDNB on Rate of ATP, Mg-Stimulated Mitochondrial Respiration Creatine*
+ + + + + +
FDNB, 0 0 10 6 5 3x10 10 10 4 3x10 4
(M)
% Control t Rate 100% 2 196.1±5 .1 164 .3 148.6 109.2±3 .9 105.3±2 .5 107.2
% Inhibition 1
(5) (2) (2) (5) (3) (2)
0.0 38 .2 53 .6 91 .0±3 .4 94 .8±2 .6 93 .2
* Creatine concentration-10 mM lMean±S.E . 2Control rate of respiration 105 my atoms oxygen/min/mg mitochondrial protein Numbers in parentheses indicate number of experiments
Vol. 21, No . 8, 1977
FDNB Inhibition of Cardiac CPR
1173
FIG. 1 Effect of creative a FDNB on rate of mitochondrial respiration A) Control; B) 10 mM creative ; C) 10 mM creative + 10-5 M FDNB Numbers represent mp atoms oxygen/min/mg mitochondrial protein. Effect of FDNB on the decreased 02 uptake stimulated by ADP in the presence of PC . As illustrated in Fig. 2B, when 1 mm PC was present in the reaction medium, the ADP (0 .3 ") stimulated 0Z uptake was significantly reduced in the presence of 1 .4 mm Mg , confirming the results of Vial and Gautheron (10) . The addition of Mg++ was again found necessary for the activity of the CPK . The reverse reaction catalyzed by mitochondrial CPR converted part of the ADP to ATP, thus reducing the amount of ADP available as the acceptor for oxidative phhosphorylation . The competition between the CPR catalyzed reaction and oxidative phosphorylation for ADP is illustrated in Table II . As higher concentrations of PC were employed, higher inhibition of mitochondrial 02 uptake as stimulated by ADP was observed . PC at a 1 mM concentration pr6duced a marked change in the 02 uptake stimulated by ADP in the presence of Mg , and so was used in subsequent experiments with FDNB .
FDNB Inhibition of Cardiac CPR
1174
Vol . 21, No . 8, 1977
ADP
FIG. 2 Effect of PC and FDNB on mitochondrial oen uptake A) Control ; B) 1 MM PCs C) 1 MM PC + 10-9 M FDNB Numbers represent mu atoms oxygen/ml TABLE II PC inhibition of ADP-Stimulated Mitochondrial Respiration PC Concentration, mM 0 0.0
0.01
0.03
0.10
0.30
1.0
3 .0
10 .0
4 .7± 2.4 1 (3)
7 .3± 4 .7 (3)
14 .3± 2.7 (3)
23 .0± 4 .4 (3)
36 .4± 1 .5 (23)
39 .0± 1 .9 (4)
50 .4± 3 .0 (7)
1All values are mean±S .E . as percent of inhibition Numbers in parentheses indicate number of experiments 10-6 to 3x104 produced an As shown in Table III, FDNB concentrations from increase in 02 uptake toward control values . At a concentration of 10-5 M, 0 uptake was found to be 951 of control as further illustrated in a comparison Ae tween Fig . 2C and 2A . This implies that an inhibition of CPR activity resulted Similar efin more ADP available as acceptor for oxidative phosphorylation . fects of FDNB were found when the PC concentration was raised to 3 mK and 10 mM (Fig . 3) .
Vol . 21, No . 8, 1977
FDNB Inhibition of Cardiac CPR
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TABLE III Effect of FDNB on PC* Inhibition of ADP-Stimulated Mitochondrial Respiration FIM,
PC* -+ + + + + + +
(M)
'k Control
0 0-6 10-6 3x10 10 5 5 3x10 10 4 3x10 4
1
100% 2 63 .6±1 .5(23) 60 .2±3 .9(4) 69 .7±7 .1(3) 95 .0±1 .8(11) 94 .6±1 .7(5) 93 .7±2 .6(5) 98 .0 (2)
* PC concentration 1 mM 1 Mean±S .E . 2 Control 02 uptake 87 .0 my atoms oxygen/ml Number in parentheses indicates number of experiments
60
40
20
2
4
6
e
10
PC [-MI FIG. 3 Effect of FDRB on PC inhibition of ADP-stimulated mitochondrial respiration . o - control e - 1-3x10 5 M FDNB Numbers in parentheses indicate number of experiments Effect of FDNB on the Spectrophotometric Assay of Mitochondrial and Cytoplasmic CPIC . As shown in Table IV, direct assay of nitochon~rial CPR activity showed more marked inhibition of CPR by FDNB . FDNB at 3x10 M produced over a 70% inhibition of enzyme activity. However, it required FDNB at 10-5 M or higher to produce a 90% inhibition . FDNB produced similar inhibition of the
1176
Vol . 21, No . 8,
FDNB Inhibition of Cardiac CPK
1977
cytoplasmic CPK activity . Statistical evaluation (Student's "t" test) indicated that FDNB was equally effective in the inhibition of both mitochondrial and cytoplasmic CPK activity . TABLE IV Percent Inhibition of CPK Activity by FDNB FDNB, 0 3x10-6 10 3x105 103x10 5
(M)
Mitcchondrial CPK*
Cytoplasmic CPK*
0.0 (2) 71 76 .7±7 .6(6) 83 .4±3 .9(8) 92 .0±2 .0(7) 94 .3±1 .7(4)
0.0 73 (2) 76 .8±7 .2(6) 90 .4±1 .5(7) 93 .5±2 .2(6) 96 .8±1 .3(4)
Mean±S .E . Numbers in parentheses indicate number of experiments Since FDNB was dissolved in propylene glycol, the effects of this solvent on mitochondrial respiration were studied both with and without PC and creatine . It also had no effect on the It was found to have no significant effect . direct assay of mitochondrial and cytoplasmic CPK activity . In addition FDNB had no effect on mitochondrial respiration in the absence of PC or creatine . Discussion our results indicate that FDNB inhibits both the forward and reverse reactions catalyzed by CPK in intact cardiac mitochondria, and botg reactions Lower were almost completely inhibited at an FDNB concentration of 10- M. concentrations produced less inhibition . Indications were that the inhibition is immediate, as no incubation time was required to achieve the results . These findings on rabbit heart agree nicely with the work of Baba, et al . (13) . They found, through histochemical techniques, that all CPK activity in the rat heart was inhibited by 30 LM FDNB . In addition our direct assay technique has indicated that this concentration of FDNB will completely inhibit mitochondrial CPK activity as effectively as the activity of the CPK isoenzyme located in the heart supernatant fraction (cytoplasmic CPK) . Therefore we conclude that FMB is an effective inhibitor of CPR activity, and that CPK from different sources appears to be equally sensitive to the action of FDNB . References 1. 2. 3. 4. 5. 6. 7. 8. 9.
H. JACOBS, H. HELDT, and M. KLINGENBERG, Biochem. Biophys . Res . Ccmmun . 16, 516-521 (1964) . B. SOBEL, W. SHELL and M. KLEIN, J. Molec . & Cell Cardiol. _4, 367-380, (1972) . S. KUBY and T. MAHOWALD, Fed. Proc . 18, 267 (1959) . D. CAIN and R. DAVIES, Biochem. Biophys. Res . Commun . _8, 361-366 (1962) . M. SERAYDARIAN, E. SATO and I. HARARY, Arch . Biochem. Biophys. _138, 233-238 (1970) . M. SERAYDARIAN and L. ARTAZA, J. Molec . & Cell Cardiol. _8, 669-678 (1976) . Sigma Technical Bulletin #520 (1976) . A. GORNALL, C . BARDAWILL and M. DAVID, J. Biol . Chem . 177, 751-766 (1949) . J. Biol . Chem . 193, O. LOWRY, N. ROSEBROUGH, A. PARR and R. RANDALL, 265-275 (1951) .
Vol . 21, No . 8, 1977
10 .
FDNB Inhibition of Cardiac CPR
1177
C . VIAL and D . GAUTBERON, Recent Advances in Studies of Myocardial Structure and Function , Vol . 3, pg . 81, Univ . Park Press, Baltimore (1973) . 11 . i9 . JACOBUS and A . IMTINGER, J . Biol . Chem . 21, 4803-4810 (1973) . 12 . S . BESSMAN and A . FONYO, Biochem . Biophys . Res. Commun . 22, 597-602 (1966) 13 . J N . BABA, S . KIM and E . FARRELL , . Molec . & Cell Cardiol.8, 599-617, (1976) .
