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Decreased mitochondrial creatine kinase activity in dystrophic chicken breast muscle alters creatine-linked respiratory coupling
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 240, No. 1, July, pp. 380-391, 1985
Decreased Mitochondrial Creatine Kinase Activity in Dystrophic Chicken Breast Muscle Alters Creatine-Linked Respiratory Coupling’ VICKIE
D. BENNETT, AND
NORMAN CLARENCE
Department of Biochemistq, Michigan *Departments of Medicine and Chemistry, Received
October
HALL,* MARLENE H. WELTER’
State University, East University of California,
29, 1984, and in revised
form
DELUCA,*
Lansing, Michigan 48824 and the San Diego, La Jolla, California %?O%’ February
27, 1985
Dystrophic chicken breast muscle mitochondria contain significantly less mitochondrial creatine kinase than normal breast muscle mitochondria. Breast muscle mitochondria from normal 16- to 40-day-old chickens contain approximately 80 units of mitochondrial creatine kinase per unit of succinate:INT (p-iodonitrotetrazolium violet) reductase, a mitochondrial marker, while dystrophic chicken breast muscle mitochondria contain 36-44 units. Normal chicken heart muscle mitochondria contain about 10% of the mitochondrial creatine kinase per unit of succinate:INT reductase as normal breast muscle mitochondria. The levels in heart muscle mitochondria from dystrophic chickens are not affected significantly. Evidence is presented which shows that the reduced level of mitochondrial creatine kinase in dystrophic breast muscle mitochondria is responsible for an altered creatine linked respiration. First, both normal and dystrophic breast muscle mitochondria respire with the same state 3 and state 4 respiration. Second, the post-ADP state 4 rate of respiration of normal breast muscle mitochondria in the presence of 20 mM creatine continues at the state 3 rate. However, the state 4 rate of dystrophic breast muscle mitochondria and mitochondria from other muscle types with a low level of mitochondrial creatine kinase, such as heart muscle and 5-day-old chicken breast muscle, is slower than the state 3 rate. Third, dystrophic breast mitochondria synthesize ATP at the same rate as normal breast muscle mitochondria but rates of creatine phosphate synthesis in 20-50 mM Pi are reduced significantly. Finally, increasing concentrations of Pi displace mitochondrial creatine kinase from mitoplasts of normal and dystrophic breast muscle mitochondria with the same apparent K,, indicating that the outer surface of the inner mitochondrial membrane and the mitochondrial creatine kinase from dystrophic muscle are not altered. 0 1985 Academic press, I”~.
Creatine kinase (adenosine-5’-triphosphate creatine phosphotransferase; EC 2.7.3.2) exists in several isozymic forms.
Eppenberger et al. (1) first reported three cytoplasmic forms composed of the M (muscle-type)3 or B (brain-type) subunits. These subunits dimerize to form MM, BB, and hybrid MB creatine kinase isozymes. The MM creatine kinase predominates in
i This work is supported by United States Public Health Service Grants GM 20716 (CHS) and HL 17682 (MD) and by the Michigan Agricultural Experiment Station, Journal Article No. 11117. The cost of this article was defrayed in part by the payment of page charges. This article must therefore be hereby marked “Advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact. a To whom correspondence should be addressed. 0003-9861185 $3.00 Copyright 0 1985 by Academic Press. Inc. All rights of reproduction in any form reserved.
3Abbreviations used: B, brain type subunit of creatine kinase; ereatine-P, creatine phosphate; glucose-6-P, glucose-6-phosphate; Pi, inorganic phosphate; INT, piodonitrotetrazolium violet; M, muscle type subunit of creatine kinase; RCR, respiratory control ratio; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid. 380
MITOCHONDRIAL
CREATINE
KINASE
mature skeletal muscle of birds and mammals and mammalian myocardium whereas BB; creatine kinase predominates in brain, neural tissue, embryonic skeletal muscle of mammals, and myocardial muscle of birds. Hybrid MB creatine kinase also appears in mammalian heart and skeletal mnscle. [See Ref. (2) for review of localization of various forms.] Jacobs et al. (3) reported an additional creatine kinase isozyme located on the outer surface of the inner mitochondrial membrane (4, 5). This mitochondrial isozyme differs from the cytoplasmic isozymes in amino acid compos.ition, electrophoretic mobility, and immunological properties; it does not hybridize with the cytoplasmic isozyme subunits (6, 7). In addition to these apparent structural differences between mitoehondrial ereatine kinase and the cytoplasmic forms, intracellular compartmentation creates functional differences. Jacobus and Lehninger (5) attribute an increase in the postADP state 4 respiration rate of rat heart mitochondria in the presence of creatine to mitochon.drial creatine kinase activity. In the presence of creatine, the mitochondrial isozyme regenerates ADP from the ATP produced during oxidative phosphorylation. Moreadith and Jacobus (8) and Gellerich and Saks (9) report additional evidence to support a functional coupling between mitochondrial creatine kinase and the adenine nucleotide translocase (5, lo14). Bessman and Geiger (15) discuss these results in terms of a creatine phosphate (creatine-P) shuttle where creatine plays an important regulatory role in intracellular energy transport of muscle. Based on the involvement of mitochondrial creatine kinase in the creatine-P shuttle and thus its importance in intracellular energy transport, we initiated a study of this creatine kinase isozyme and its function in mitochondria from normal and dystrophic avian pectoralis muscle. Previously, Mahler (16) reported a progressive decrease of mitochondrial creatine kinase activity per milligram of mitochondrial protein in the skeletal muscle of dystrophic chickens with increased age compared to normal controls.
ACTIVITY
IN
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MUSCLE
381
We confirm Mahler’s observation using genetically related, age-matched normal and dystrophic chickens; lines 412 and 413 (1’7), respectively. In addition, we report a loss of regulation by creatine on the respiration of mitochondria from dystrophic chicken breast muscle. MATERIALS
AND
METHODS
Materials. All biochemicals, enzymes, and creatine kinase assay kits were obtained from Sigma Chemical Company (St. Louis, MO.) unless otherwise specified. Creatine and rabbit muscle creatine kinase were purchased from Calbiochem-Behring Corporation (La Jolla, Calif.). Sephraphore III cellulose acetate electrophoresis strips were obtained from Gelman Sciences, Inc. (Ann Arbor, Mich.). Millipore filters were purchased from Millipore Corporation (Bedford, Mass.). Common laboratory chemicals were reagent grade or better. Solutions were prepared in distilled deionized water. Normal (line 412) and dystrophie (line 413) chickens were obtained from the Department of Avian Sciences, University of California, Davis, California. Isolation of breast and leg muscle mitochondria. Preparation of mitochondria from the pectoralis and the primarily red fiber leg muscles of normal and dystrophic chickens involved the basic procedure described by Lee et al. (18) substituting Sigma protease Type VII (1 mg/g muscle) for Nagarse (3 mg/g muscle). Mitochondrial yields were based on the recovery of succinate:p-iodonitrotetrazolium reductase (succinate:INT reductase), a mitochondrial marker, in the final mitochondrial suspension as compared to the activity in the initial crude homogenate. Yields ranged from 15 to 30% for mitochondria isolated from chickens between the ages of 12 and 20 days ex ova and 10% for mitochondria isolated from chickens 5 days ez ova Respiratory control ratios (RCR) at 25’C for normal and dystrophie breast muscle mitochondria from chickens 12-20 days ex ouo were 3.4 and 2.4, respectively, and ADP:O ratios were 1.5 and 1.2, respectively, using succinate as substrate in the presence of rotenone. RCR values at 25°C for normal and dystrophic breast muscle mitochondria from chickens 5 days ex ova were 1.5 and 1.2, respectively, and ADP:O ratios were 1.4 and 1.3, respectively. Isolation of heart mitochondria. Normal and dystrophic chicken heart muscle mitochondria were isolated by differential centrifugation as previously described (19). Mitochondrial yields based on the recovery of succinate:INT reductase in the final mitochondrial suspension compared to the initial crude homogenate ranged from 20 to 40%. Respiratory control ratios for normal and dystrophic heart mitochondria at 25°C were 1.8 and 1.6, respectively,
382
BENNETT
using succinate as substrate in the presence of rotenone. ADP:O ratios were 1.1 and 1.2, respectively. Enzyme assays and protein determination Total creatine kinase activity in the protease-treated heart, breast, and leg muscle mitochondria was solubilized by suitably diluting a portion of the final mitochondrial suspension into 0.1 M sodium phosphate, pH 6.5, 25 mM P-mercaptoethanol, 1 mM EDTA, and 1% Triton X-100 to give a final activity of 1-4 IU/ml. Creatine kinase activity was determined spectrophotometrically at 340 nm and 30°C using the creatine kinase assay mix from Sigma (20, 21). The mitochondrial marker, succinate:INT reductase, was assayed as described previously (22). Correcting for yield of mitochondria gave the units of mitochondrial creatine kinase per gram of muscle. Mitochondrial pellet protein was determined by the Lowry method (23) following a 1:l solubilization in 1 N NaOH for 1 h. Bovine serum albumin was used as a standard. Cellulose acetate electrophoresis. Creatine kinase isozymes were separated by cellulose polyacetate electrophoresis as previously described (24). Triton X-100 was added to the electrophoresis buffer (final concentration, 1%) to allow creatine kinase associated with mitochondria to migrate from the origin. Following electrophoresis, the strips were stained for creatine kinase isozymes as previously described (25) and scanned on a Gelman ACD-18 automatic computing densitometer. Isozyme positions were identified by comparing sample mobilities with the migration of purified fractions of MM, MB, BB, or mitochondrial creatine kinase on independent electrophoretic strips. In control electrophoretic strips, creatine-P was omitted from the assay mix to check for interference by adenylate kinase. Mitochondrial creatine kinuse. The proportion of mitochondrial creatine kinase isozyme in the final mitochondrial suspensions was determined from densitometer scans of the electrophoretically separated isozymes. Of the total creatine kinase in a mitochondrial preparation, mt-creatine kinase accounted for 79 + 17% in 27 normal leg and breast muscle preparations, 71 + 17% in 28 dystrophic preparations, and 100% in 28 heart preparations. Since the creatine kinase assay mix is optimized for MM creatine kinase and these conditions may vary somewhat for mitochondrial ereatine kinase, there may be some error in the value for the final mitochondrial creatine kinase activity. Mitochondrial creatine kinase activity was then normalized to the amount of mitochondria in each sample by dividing by the activity of succinate:INT reductase in the sample. Mitochwndrial oxygen consumption Mitochondrial respiration was monitored at 25°C on a Gilson Model K-IC oxygraph equipped with a Yellow Springs Instruments electrode and a high-sensitivity Teflon membrane (Yellow Springs Instruments). Standard skeletal muscle mitochondria buffer contained 150
ET
AL,
mM sucrose, 1 mM EDTA, 2 mg/ml bovine serum albumin, and 25 mM Tris-HCl, pH 7.5. Standard heart muscle mitochondria buffer contained 0.21 M mannitol, 70 mM sucrose, 1 mM EDTA, 2 mg/ml bovine serum albumin, and 10 mM Tris-HCl, pH 7.4. Breast and leg muscle mitochondria (50 ~1 of 20-30 mg protein/ml) were incubated in 1.75 ml of medium containing standard muscle mitochondria buffer, 5 mM succinate, 10 pM rotenone, and 5 mM MgS04 in addition to 2.5, 5, 10, 20, 50, 75, or 98 mM potassium phosphate and 0 or 20 mM creatine. Heart mitochondria were incubated under the same conditions except in medium containing standard heart mitochondria buffer and 0.5, 1, 2.5, 5, or 10 mM potassium phosphate. Adding 20 pl 10 mM ADP to each incubation mixture initiated state 3 respiration. The solubility of oxygen in buffered medium at 25°C was assumed to be 240 nmol &Jml (26). Rate of mitochondrial meat&e-P synthesis. Breast muscle mitochondria (125 ~1 of 20-30 mg protein/ ml) were incubated at 25°C in 5-ml reaction mixtures containing standard skeletal muscle mitochondria buffer, 5 mM succinate, 10 pM rotenone, 5 mM MgSOI, 20 mM creatine, and 2.5, 5, 10, 20, 50 or 98 mM potassium phosphate. Heart mitochondria were incubated under the same conditions except in medium containing standard heart mitochondria buffer and 0.5, 1, 2.5, 5, 10, or 20 mM phosphate (Pi). Samples (0.5 ml) were withdrawn from the reaction chamber at 1-min time intervals after addition of 50 ~1 10 mM ADP and immediately added to 0.5 ml of 30 mg/ml activated charcoal to remove ATP. The charcoal suspension was filtered through Millipore filters (GSWP, 0.22-pm pore size) as quickly as possible. A portion (200 ~1) from each filtered sample was added to each of two tubes containing 1 ml of 1.2 mM ADP, 3.7 mM AMP, 14 mM glucose, 9 mM magnesium acetate, 1.8 mM NADP, 4 mM dithiothreitol, 1.7 units/ml glucose-6-phosphate dehydrogenase, and 1.7 units/ml hexokinase in 20 mM Hepes, pH 7.4. Rabbit muscle creatine kinase (10 pl of 2 mg/ml) was added to one of the two tubes for each sample. All tubes were incubated at room temperature for 60 min and the absorbance was read at 340 nm. Rate of mitochondrial ATP synthesis Breast muscle mitochondria (125 ~1 of 20-30 mg protein/ml) were incubated at 25°C in 5-ml reaction mixtures containing standard skeletal muscle mitochondria buffer, 5 mM succinate, 10 pM rotenone, 5 mM MgSO,, 20 mM creatine, 5 mM glucose, 60 units hexokinase, and 5 or 50 mM potassium phosphate. Samples (0.5 ml) were withdrawn from the reaction chamber at lmin time intervals after addition of 50 pl of 10 mM ADP and immediately added to 0.5 ml of 30 mg/ml activated charcoal to remove ADP. The charcoal suspension was filtered through Millipore filters (GSWP, 0.22-pm pore size) as quickly as possible. The concentration of ATP in each sample (glucose
MITOCHONDRIAL 6-phosphate essentially creatine-P
CREATINE
KINASE
plus creatine phosphate) was determined as described in the previous section under synthesis. hfitop!mt proparotimz Mitoplasts from normal and dystrophic brleast muscle were prepared by first swelling the outer mitochondrial membrane in a hypotonic solution of 10 mM Tris-HCl, pH 7.5, at 0°C for 5 min. Adding a hypertonic solution (1.75 M sucrose, 2 mM ATP, and 2 mM MgSO& to the previous suspension at 0°C for 5 min allowed removal of the outer membrane by mild sonication (Branson Sonifier at 3 A for 20 s). Mitoplasts were then sedimented at 20,OOOg for 20 min. Efect of inorganic phosphate on the interaction of mitochondrial creatine kinase with mitoplasts. Mitoplasts (30-~1 aliquots) were incubated with shaking in a 750-p] solution containing 0.21 M mannitol, 70 mM sucrose, 0.1 mM EDTA, and 10 mM Tris-HCl, pH 7.4, and increasing concentrations of potassium phosphate at pH 7.4 for 2 h at 6°C. The mitoplast suspensions were then centrifuged in an Eppendorf centrifuge for 10 min and the supernatants were assayed for creatine kinase activity to determine the percentage of total mitochondrial creatine kinase released from the mitoplast membrane.
ACTIVITY
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383
differences are not significant at Days 37. Third, normal and dystrophic heart mitochondrial creatine kinase activities per unit of succinate:INT reductase remain relatively constant (in the three age groups), whereas the levels in normal and dystrophic leg and breast muscle are low at early ages but increase with age. Comparing the levels of succinate:INT reductase activity per gram of muscle for each muscle type (Panel B of Table I) reveals no significant differences between normal and dystrophic muscle of each type. There tends to be a slight increase in succinate:INT reductase in dystrophic breast muscle at 12-19 and 25-40 days, but the differences are not significant. The differences in the levels of mitochondrial creatine kinase per unit succinate:INT reductase between normal and dystrophic muscles therefore reflect differences in the mitochondrial creatine kinase activity and not differences in the number of mitochondria. In addition, the levels of miRESULTS tochondrial creatine kinase per gram of muscle (Panel C of Table I) basically Levels of Mitochondrial Creatine Kinase confirm these conclusions. Specifically, in Normal and Dystrophic Muscle there are no differences in units of mitoTable I contains a summary of the chondrial creatine kinase per gram of levels of mitochondrial creatine kinase both normal and dystrophic heart and leg and succinate:INT reductase (a mitochonmuscle at 25-40 days ex ouo. Mitochondrial drial marker enzyme) as a function of age creatine kinase per gram of dystrophic for normal and dystrophic chicken heart, breast muscle is slightly more than half leg, and breast muscle. The data in Panel that found in normal muscle but the difA of this table reveal several basic trends ferences are not significant at the 95% concerning the level of mitochondrial cre- confidence level. atine kinase per unit of succinate:INT Comparing differences between the reductase in the three muscle types from Days 3-7 age group and the days 12-19 both normal and dystrophic chickens. age group shows that mitochondrial creFirst, normal breast muscle mitochondria atine kinase concentrations in the breast contain the highest levels of mitochondrial muscle are low at hatch. Both units of creatine kinase per unit succinate:INT re- mitochondrial creatine kinase per unit of ductase. The levels in normal heart muscle succinate:INT reductase and per gram of mitochondria are roughly one-tenth those muscle are significantly lower in the Days in normal breast muscle mitochondria 3-7 age group compared to the Days 12while leg muscle mitochondria contain 19 age group. Because the adult levels in one-half to one-third those in breast the dystrophic breast muscle are demuscle mitochondria. Second, dystrophic pressed considerably, the differences bebreast muscle mitochondria contain half tween the above age groups are not sigto less than half as much mitochondrial nificant in these muscles. Units of succicreatine kinase per unit of succinate:INT nate:INT reductase per gram of muscle reductase as normal breast muscle mitoare also not significantly different when chondria at. Days 12-19 and 25-40. The these two age groups are compared.
f
f f P < 86 rt
27 84
27
(2)
(4)
15 (7) 26 (7) 0.001 21 (5)
8.4 (6)
16.1 27 f 10 -
8.1 f 1.7 (3) 6.0 + 3.7 (7) ns. 8.5 f 1.6 (7)
Normal
-
A. Mt-creatine
CREATINE
36
19 44
f 10 + 17 n.s. + 6
f 3.0 -t 3 < P < + 6.5
(7)
(6) (6)
(3) (4) 0.005 (6)
2.7 (6)
7.0 +
9.4 22 0.001 28
1.3 (7)
4.6 2.4 f
(2)
(succ:INT)
Dystrophic
kinase
OF MITOCHONDRIAL
n.s.
n.s.
n.s.’
P”
unit-‘)
AND
P < 0.001
Normal
reductase muscle)
B. Succ:INT (units g-i
0.9 f 0.5 (7) 0.5 f 0.2 (7) n.s. 0.5 f 0.1 (5)
2.0 f 0.6 (6)
2.6 (2) 1.0 + 0.3 (4)
1.0 + 0.4 (6) 0.8 + 0.6 (3) n.s. 0.7 f 0.4 (7)
2.3 f 0.8 (3) 1.2 f 0.1 (4) n.s. 1.6 f 0.3 (6)
2.4 4.6 f 1.0 (7) 3.1 * 1.0 (7)
Dystrophic
IN VARIOUS
REDUCTASE
3.1 f 0.4 (3) 3.7 f 1.4 (7) n.s. 2.4 ix 0.9 (7)
SUCCINATE:INT
n.s. 0.005 < P < 0.01
(unit
KINASE
I
ns.
n.s. n.s.
n.s.
n.s.
-
n.s.
n.s.
P”
NORMAL
DYSTROPHIC
22 50 0.001 46
7 (5)
kinase
MUXLES
f 9 (7) f 15 (7) < P < 0.005 k 12 (5)
43 f
44 (2) 27 + 10 (4) -
25 f 8 (3) 21 * 13 (7) n.s. 20 IF 8 (7)
Normal
C. Mt-creatine
AND
7 (5)
8 (7)
17 + 9 (6) 31 f 12 (6) n.s. 26 f 14 (7)
23 f 15 (3) 25 f 5 (4) n.s. 46 f 18 (6)
22 f
10.7 11 +
muscle)
(2)
g-i
Dystrophic
(unit
P”
Note. Creatine kinase in the protease-treated heart, leg, and breast muscle mitochondria was solubilized and assayed spectrophotometrically at 30°C as indicated under Materials and Methods. The mitochondrial marker enzyme, succinate:INT reductase, was assayed by an endpoint assay following lo- to 12min incubations using conditions described previously. Data are the means f SE for the number of mitochondrial samples given in parentheses. Each sample is the composite of mitochondria from at least two birds. P values were determined by Student’s t test for paired values. a P values for compared normal and dystrophic data at this age. b P values for compared data at ages 3-7 and 12-19 days. ’ n.s. not significant at the 95% confidence level.
Breast 3-7 12-19 Pb 25-40
3-7 12-19 Pb 25-40
Leg
Heart 3-7 11-19 Pb 25-40
(days)
Muscle age
LEVELS
TABLE
MITOCHONDRIAL
CREATINE
KINASE
Table II contains a summary of the initial state 4, state 3, and post-ADP state 4 rates of respiration for various muscle mitochondria from both normal and dystrophic chickens in the absence and presence of 20 mM creatine at various phosphate concentrations. The initial state 4 rates of both normal and dystrophic breast muscle mitochondria are not affected by either 20 mM creatine or 2.5 to 50 mM Pi. However, 20 mM creatine affects the postADP state 4 rates of normal breast muscle mitochondria by causing them to continue at the state 3 rate (Fig. 1). Specifically, in the absence of creatine, the post-ADP state 4 rates of normal breast muscle mitochondria range from 40 to 50% of the state 3 rate at all concentrations of Pi. In the presence of creatine, the post-ADP state 4 rates are statistically identical to the state 3 rate (Table II). Dystrophic breast muscle mitochondria respire differently under these same conditions (Fig. 1). First, the initial state 4 rates are nearly identical to those observed with normal breast muscle mitochondria (P’ > 0.5 at 2.5 to 10 mM Pi and 0.1 < P < 0.2 for 20 to 98 mM Pi). Second, the initial state 4 respiration rates of dystrophic breast muscle mitochondria are not affected by creatine. Third, the postADP state 4 rate of dystrophic breast muscle mitochondria in the absence of creatine is approximately 60% of the state 3 rate as Pi is increased from 2.5 to 98 mM. However, in the presence of creatine, the post-ADP state 4 rate of dystrophic breast muscle mitochondria, in contrast to the normal breast muscle mitochondria, is no longer identical to the state 3 rate as Pi increases. At 2.5 to 10 mM Pi, the post-ADP state 4 rate remains unchanged, but as Pi increases from 20 to 98 mM, the post-ADP state 4 rate becomes statistically different than the state 3 rate (Table II). An identical experiment reveals that creatine has no effect on the post-ADP state 4 rates of mitochondria from normal and dystrophic hearts (Table II). In this case, the post-ADP state 4 rates of both normal and dystrophic heart mitochondria in the prese.nce of 20 mM creatine remain
ACTIVITY
IN
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385
MUSCLE
at about 80% of the state 3 rate at all Pi concentrations ranging from 2.5 to 20 mM. Similarly, breast muscle mitochondria from 5-day-old normal and dystrophic chickens behave like the normal and dystrophic heart mitochondria (Table II). Therefore, in the presence of creatine, both normal and dystrophic heart mitochondria, as well as both normal and dystrophic breast muscle mitochondria from 5-day-old chickens, function like dystrophic breast muscle mitochondria from 16- to 18-day-old chickens at phosphate concentrations greater than 5 mM. Creatine Phosphate Production Res@ing Mitochondria
in
The effects of creatine and Pi on the respiration of normal and dystrophic heart and breast muscle mitochondria as discussed above are also reflected in their rates of synthesis of creatine-P. Figure 2 shows rates of synthesis of creatine-P (pm01 creatine-P produced min-’ [IU succinate:INT reductasel-‘) at various Pi concentrations for respiring breast and heart muscle mitochondria from both normal and dystrophic chickens. The inset in Fig. 2 shows creatine-P concentration as a function of time after initiation of mitochondrial respiration for dystrophic breast muscle mitochondria at various Pi concentrations. Slopes of these lines for each Pi concentration (after the initial lag period) determine the rates of creatine-P synthesis. Normal breast muscle mitochondria maintain high, nearly constant rates at all Pi concentrations from 5 to 98 mM. This high rate is expected because the post-ADP state 4 rate continues at the state 3 rate in the presence of creatine at all Pi concentrations up to 98 mM Pi. In contrast to normal breast muscle mitochondria, dystrophic breast muscle mitochondria exhibit a sharp decline in the rate of creatine-P synthesis after 10 mM Pi. This decline in the rate of creatine-P synthesis parallels oxygen consumption measurements in the presence of creatine (Table II); Pi concentrations from 20 to 98 mM allow return of a distinct postADP state 4 rate, indicating less conver-
II
D D
N D
N
16 16
16 16
5
5
Breast Breast
Heart Heart
Breast
Breast
6d
6d
17* 17*
12* lo*
9* 12*
12
5-50
5-50
0.5-20 0.5-20
2.5-10 20-98
2.5-10” 20-98
[Phosphate] bM)
0.23 _+ 0.03
0.22 f 0.07
0.45 * 0.15 0.24 + 0.05
0.53 f 0.05 0.61 + 0.14
0.52 f 0.13 0.52 + 0.12
-
Initial +
4
O./unit
0.22 f 0.02
0.22 + 0.04
0.46 t 0.05 0.26 f 0.06
0.55 f 0.12 0.57 f 0.09
0.57 f 0.07 0.54 f 0.07
state
pmol
0.87 + 0.07
0.72 f 0.03
1.48 f 0.31 0.80 f 0.10
2.38 f 0.55 2.65 I 0.30
+
(+20
0.86 f 0.10
0.64 * 0.05
0.57 * 0.03
0.54 -c 0.08
0.81 f 0.10 0.73 f 0.05
1.43 * 0.35 1.54 * 0.38 0.90 Ik 0.19
0.92 + 0.25 0.97 k 0.25
-
Post-ADP
mM creatine)
2.33 + 0.37 2.66 k 0.37 1.43 f 0.23
2.50 f 0.22 2.34 + 0.24
3
reductase State
2.58 f 0.19 2.22 f 0.25
-
succinate:INT
+
4
0.65 k 0.07
0.57 * 0.05
0.66 + 0.05
2.14 k 0.48 2.02 f 0.30 1.12 + 0.19
2.47 k 0.20 2.26 f 0.27
state
0.001 < P < 0.005
P < 0.001
P < 0.001
0.2 < P < 0.4 P < 0.001 P < 0.001
P > 0.5 0.4 < P < 0.5
P
Note. Mitochondrial respiration was monitored at 25°C. Mitochondria were incubated in their respective mitochondrial buffer, 5 mM succinate, 10 fiM rotenone, 5 mre MgS04, and Pi + 20 mM creatine as indicated. See Materials and Methods for details. The addition of 20 pl of 10 mM ADP initiated state 3 respiration. Data are the means f SE for the number of measurements given in parentheses. P values were determined by Student’s t test for paired values comparing state 3 and post-ADP state 4 rates in the presence of creatine. a Muscle (Normal, N; Dystrophic, D) (n, number of measurements). * Each sample contains the mitochondria in breast muscle from two birds. “Respiratory rates are the composite results of several different concentrations of Pi spanning the range indicated as detailed under Materials and Methods. d Each sample contains the mitochondria in breast muscle from three birds.
D
N N
16 16
5w
Breast Breast
Age (days)
Muscle”
SUMMARYOFOXYGENRESPIRATIONEXPERIMENTSWITHVARIOUSMUSCLEMITOCHONDRIAFROMNORMALANDDYSTROPHICCHICKENS
TABLE
F
2
2 3
MITOCHONDRIAL
mtor
KINASE
20 mhd phosphate
ACTIVITY
IN
DYSTROPHIC
387
MUSCLE
of the rates of glucose-6-P and creatineP when normal and dystrophic breast muscle mitochondria were allowed to respire in the presence of excess hexokinase and glucose and either 5 or 50 mM Pi. The data in this table indicate little effect of 5 to 50 mM Pi on the total rate of ATP synthesis in both normal and dystrophic
Mn0l
No Creotine 3s nlmln 02 L
CREATINE
20 mM Creattns 20 mM phosphate
I min
\
B.
No Creatine
20 tnh4 Creatine 20 r&t Phosphate
FIG. 1. Sample oxygen consumption traces for normal (A) and dystrophie (B) breast muscle mitochondria from chickens 19 days old. Mitochondria were added to standard breast muscle mitochondria buffer containing 20 mM phosphate and either 0 or 20 mM creatine at 25°C. Addition of ADP initiated state 3 respiration. The numbers in parentheses represent nanomoles Oa consumed per minute.
sion of ATP to creatine-P. Heart muscle mitochondria from both normal and dystrophic chickens have significantly lower rates of creatine-P synthesis than normal or dystrophic breast muscle mitochondria. These very low rates are consistent with the lack of either a creatine or Pi effect on oxygen consumption during respiration of normal and dystrophic heart mitochondria (Table II). Thus, the decreased rates of creatine-P synthesis in respiring mitochondria from various muscle types are consistent with decreased concentrations of mitochondrial creatine kinase activity in these mitochondria.
Rates of Al’P Synthesis in Normal and Dystrophic Breast Muscle Mitochcmdria The data in Table III show the rates of ATP synthesis calculated from the sum
00
IO
20
30
40 bw-4
50
60
70
80
90
fM
FIG. 2. Rates of creatine phosphate synthesis in normal and dystrophic breast and heart muscle mitochondria as a function of inorganic phosphate. Mitochondria were incubated with various concentrations of phosphate and 20 mM creatine at 25°C as described under Materials and Methods. The figure inset shows nanomoles creatine phosphate formed by dystrophic breast muscle mitochondria as a function of time for the indicated phosphate concentration. The slopes of each line in the inset are the rates of synthesis of creatine phosphate plotted in the main figure as a function of phosphate concentration. Rates of synthesis are expressed as micromoles creatine phosphate produced per minute per unit of succinate:INT reductase. The error bars for the breast muscle mitochondria data represent standard errors of the mean for the number of samples indicated by the number in parentheses. Each mitochondrial sample contained the composite of mitochondria from two birds. Heart data are averages obtained from two mitochondrial samples containing the composite of mitoehondria from two birds.
388
BENNETT
ET
TABLE RATE
OF SYNTHESIS
AL.
III
OF CREATINE-P AND ATP (CREATINE-P PLUS AND DYSTROPHIC BREAST MUSCLE
BY NORMAL
Type of mitochondria
[Phosphate] hM)
(pm01
mini
ATP [succ:INT]-‘)
Glc-6-P
WITH MITOCHONDRIA
ADDED
(firno
HEXOKINASE)
Creatine-P mini [succ:INT]-‘)
Normal
5 50
5.21 f 0.45 (2) 5.05 f 0.22 (2)
3.16 f 0.38 (7) 2.59 f 0.40 (6)
Dystrophic
5 50
6.22 zk 0.35 (2) 5.26 5~ 0.45 (2)
2.33 f 0.78 (3)” 0.74 + 0.12 (3)”
Note. The rates of synthesis of creatine-P and ATP by respiring normal and dystrophic breast muscle mitochondria at 25°C in mixtures containing breast muscle mitochondrial buffer, 5 mM succinate, 10 j.rM rotenone, 5 mM MgS04, 20 mM creatine, 5 mM glucose, 60 units hexokinase, and 5 or 50 mM potassium phosphate were determined as described in Materials and Methods. Data are the mean i SE for the number of samples given in brackets. “Because creatine does not cause a continuation of the state 3 rate throughout the incubation period of dystrophic breast muscle mitochondria, ADP concentrations become rate-limiting and thus these values are not maximum rates of creatine-P synthesis.
breast muscle mitochondria. Furthermore, mitochondria from dystrophic muscle produce ATP as effectively as those from normal muscle (Table III). Efect of Inorganic Phosphate on the Interactim of Mitochtmdrial Creatine Kinase with Normal and Dystrophic Breast Muscle Mitoplasts The effect of Pi on the interaction of mitochondrial creatine kinase with normal and dystrophic breast muscle mitoplasts is shown in Fig. 3. Increasing concentrations of Pi release increasing amounts of mitochondrial creatine kinase into the incubation medium until 50 mM, when 85% of the total mitochondrial creatine kinase is released. K0,5’s (concentration of phosphate required to release 50% of total mitochondrial creatine kinase) are 26.5 and 30 mM Pi for normal and dystrophic breast muscle mitoplasts, respectively. Identical results are obtained if the mitoplasts are incubated with increasing concentrations of inorganic phosphate for 10 min instead of 2 h at 6°C (S. P. J. Brooks, unpublished observations). The shape of the curves are very similar to the phosphate solubilization curve for mitochondrial creatine kinase reported by Vial et al. (27) for pig heart mitochondria, but the KO,, values are higher for chicken breast muscle mitochondria.
DISCUSSION
Comparing mitochondrial creatine kinase concentrations in various muscle types from normal and dystrophic chickens (Table I) basically confirms Mahler’s observation (16) that mitochondrial creatine kinase activity is decreased in dystrophic breast muscle compared to normal age-matched controls. Since Mahler used chickens at 44, 91, 200, and 566 days ex ova, when the dystrophic condition is in its advanced stages, we felt the need to examine levels of mitochondrial creatine kinase early in the onset of the dystrophic symptoms. The onset of dystrophy determined by the flip test becomes apparent at 9 days ex (yuo in the 413 line of chickens (17). The data in Table I show that this event occurs around the time when dystrophic breast muscle mitochondria contain half the normal level of mitochondrial creatine kinase. In addition, we express the levels of mitochondrial creatine kinase in terms of a specific mitochondrial marker enzyme, succinate:INT reductase, rather than per milligram of mitochondrial protein. This difference in reporting procedure is important when comparing normal and dystrophic mitochondria since the dystrophic mitochondrial pellet may contain extraneous protein because of the increased connective tissue in dystrophic muscle [see Ref. (28) for review].
MITOCHONDRIAL
CREATINE
KINASE
Of particular interest in this study is the relativlely high level of mitochondrial creatine kinase per unit of succinate:INT reductase in normal chicken breast muscle compared to chicken heart and leg muscle (Table I). In fact, Hall and DeLuca’s (29) survey of the level of mitochondrial creatine kinase per unit of succinate:INT reductase in rat skeletal muscle, liver, and heart; beef heart and liver; rabbit skeletal muscle, heart and brain; and chicken heart and breast muscle reveals that chicken breast muscle contains the highest concentration of this creatine kinase isozyme. The low level of mitochondrial creatine kinase in chicken heart muscle mitochondria contrasts with the generally accepted view that heart muscle mitochondria contain significant amounts of this isozyme (5). The data in Table I also show that normal chicken heart contains approximately one-half the mitochondrial creatine kinase as normal chicken breast muscle whlen based on muscle mass. The possible physiological effects of reduced levels of mitochondrial creatine kinase are discussed in terms of the creatine-P shuttle [for review see Ref. (15)]. This shuttle involves the mitochondrial and muscle isozymes of creatine kinase and evolveld in recent years basically from the initial work of Bessman and colleagues (10, 15, 30) and Jacobus and Lehninger (5). The mitochondrial isozyme is bound to the out.er surface of the inner mitochondrial membrane (4, 5), presumably funetional1.y coupled to the adenine nucleotide translocase (5, 8-14). Apparently, bound mitochondrial creatine kinase preferentially utilizes the ATP produced during oxidative phosphorylation to phosphorylate creatine and regenerate ADP. When breast muscle mitochondria contain their full complement of mitochondrial creatine kinase (after approximately 11 days ez o~o), creatine maintains the post-ADP state 4 rate equivalent to the state 3 rate at Pi concentrations ranging from 5 to 98 mM (Table II). However, when muscle mitochondria contain 30 to 40 units of mitochondrial creatine kinase per unit of succinate:INT reductase, the post-ADP state 4 rate is only 80% of the state 3 rate in the presence of 20 mM
ACTIVITY
IN
DYSTROPHIC
MUSCLE
389
creatine and Pi concentrations between 20 and 98 mM (Table II). At 2.5 to 10 mM Pi, the post-ADP state 4 rate in the presence of creatine is nearly equivalent to the state 3 rate. In addition, 20 mM creatine has little effect on the post-ADP state 4 rate of 5-day-old normal chicken breast muscle mitochondria since the muscle at this age has not yet reached its full complement of mitochondrial creatine kinase (Table II). Furthermore, heart mitochondria, containing around 8 units of mitochondrial creatine kinase per unit of succinate:INT reductase (Table I), behave like breast muscle mitochondria from 16- to 19-day-old dystrophic chickens and mitochondria from 5-day-old normal and dystrophic chickens. Again, 20 mM creatine, in the presence of 2.5 to 10 mM Pi, has little effect on the post-ADP state 4 rate of both normal and dystrophic heart muscle mitochondria (Table II). Thus, irregardless of whether the mitochondria are obtained from normal or dystrophic muscle, when the level of mitochondrial creatine kinase is below 40 units per unit of succinate:INT reductase 20 mM creatine in the presence of 20 to 98 mM Pi is unable to maintain the state 4 rate equivalent to the state 3 rate. The rates of creatine-P synthesis in respiring mitochondria isolated from heart and breast muscle (Fig. 2) strengthen the argument that the level of mitochondrial creatine kinase in normal and dystrophic breast muscle accounts for the differences of creatine and Pi on oxygen consumption measurements with these two mitochondria. Normal breast muscle mitochondria maintain a high, nearly constant rate of production of creatine-P at all Pi concentrations, consistent with the presence of large amounts of mitochondrial creatine kinase. The sharp decline in the rate of creatine-P production at Pi concentrations between 20 and 98 mM in dystrophic breast muscle mitochondria is consistent with the lower levels of mitochondrial creatine kinase aetivity and the drop in post-ADP state 4 rates of oxygen consumption at similar Pi concentrations. Heart muscle mitochondria produce creatine-P at significantly lower rates than normal and dystrophic breast
390
BENNETT
FIG. 3. Effect of inorganic phosphate on the soluhilization of mitochondrial creatine kinase (mt-CK) from normal and dystrophic breast muscle mitoplasts. Aliquots (30 ~1) of isolated mitoplasts (See Materials and Methods) were incubated at 6°C for 2 h in 750 ~1 standard breast muscle mitochondria buffer (See Materials and Methods) and the indicated concentrations of potassium phosphate. After centrifugation, the supernatants were assayed for creatine kinase activity. The percentage of total mt-CK in the supernatant, expressed as a percentage of soluble mt-CK, as a function of phosphate concentration is shown for normal (0) and dystrophic (X) breast muscle mitoplasts. Each point represents the average of data from four independent determinations + standard deviation.
muscle mitochondria, again consistent with the very low levels of mitochondrial creatine kinase activity and the lack of an appreciable effect of creatine on the post-ADP state 4 rate. The results in Fig. 3 indicate that mitochondrial creatine kinase and the outer surface of the inner mitochondrial membrane are not altered in dystrophic muscle mitochondria; the concentration of Pi required to release mitochondrial creatine kinase from the mitoplast membrane is the same for both normal and dystrophic mitochondria. Furthermore, the creatine phosphate production rates for each of the mitochondrial types shown in Fig. 2 indicates that 50 mM Pi has no insignificant effect on the rate of mitochondrial ATP synthesis in normal and dystrophic breast mitochondria. In addition, mitochondria from dystrophic muscle function
ET
AL.
as effectively as normal muscle mitochondria in synthesizing ATP (Table III). We do not suggest that the decreased levels of mitochondrial creatine kinase found in dystrophic breast muscle is the primary lesion in muscular dystrophy, but rather that this decrease may be of primary importance in causing the loss of function of white fiber muscle by decreasing the efficiency of trapping available mitochondrial ATP as creatine-P, the high-energy storage molecule for muscle contraction energy. It is now of interest to know whether only muscles with high levels of mitochondrial creatine kinase per unit of succinate:INT reductase are most severely affected by dystrophy. ACKNOWLEDGMENTS We gratefully acknowledge the assistance of the Department of Animal Science, Michigan State University, and particularly Ms. Bridget Grala, Dr. Robert Ringer and Dr. Donald Polin; the Department of Avian Sciences, University of California at Davis, and particularly Mr. Fayne Lantz; and Dr. Shelagh Ferguson-Miller, Department of Biochemistry, Michigan State University, for the use of her Gilson oxygraph. REFERENCES 1. EPPENBERGER,
H.
N. 0. 2. NEUMEIER,
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(1967)
M., DAWSON,
D. (1981) (Lang, H., New York.
zymes Verlag, 3. JACOBS,
H.,
HELDT,
(1964) Biochem. 516-521.
D.,
H. W.,
Res.
ADDIS,
P.,
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16,
WITPEETERS, 291, 764-
Acta
A. L. (1973)
DELUCA,
18, 1745-1751. R., AND GRACE, A. M. 255, 2870-2877.
M.
Commun
P. J., AND
Biophys.
773. 5. JACOBUS, W. E., AND LEHNINGER, Biol Chem. 248, 4803-4810. N.,
KAPLAN,
ANDKLINGENBERG,
Biophys.
4. SCHOLTE, H. R., WEIGERS, E. M. (1973) B&him
6. HALL,
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Cfiem 242,204-209. in Creatine Kinase Isoened.), pp. 85-109, Springer-
M.
J.
(1979)
Biochemistry 7. ROBERTS,
Chem. 8. MOREADITH,
Biol.
Chem
R. W.,
AND
JACOBUS,
(1980) W.
J. Biol.
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257,899-905.
F., AND SAKS, V. A. (1982) Bio&,em. Res. Ccnnmun 105,1473-1481. 10. BESSMAN, S. P., AND FONYO, A. (1966) Biochem Biophys. Res. Commun 22, 597-602. 11. SAKS, V. A., LIPINA, N. V., SMIRNOV, V. N., AND CHAZOV, E. I. (1976) Arch Biochem Biophys. 173,34-41. 9. GELLERICH,
Biophys.
MITOCHONDRIAL
CREATINE
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12. SAKS, V. A., KUPRIYANOV, V. V., ELIZAROUA, G. V., AND JACOBUS, W. E. (1980) J. Biol. Chem. 255, 755-763. 13. YANG, W. C. T., GEIGER, P. J., BESSMAN, S. P., AND BORREBAEK, B. (1977) B&hem Biophys. Res. Commun. 76, 882-887. 14. DEFURIA, R. A., INGWALL, J. S., FOSSEL, E. T., AND D’IGERT, M. K. (1980) in Heart Creatine Kinase: The Integration of Isozymes for Energy Distribution (Jacobus, W. E., and Ingwall, J. S., eds.), pp. 135-141, Williams and Wilkins, Baltimmore. 15. BESSMAN, S. P., AND GEIGER, P. J. (1981) Science (Washington, D. C.) 211, 448-452. 16. MAHLER, M. (1979) Biochem. Biophys. Res. Cornmun. 8#8, 895-906. 17. WILSON, B. W., RANDALL, W. R., PATTERSON, G. T., ~LND ENTRIKEN, R. R. (1979) Ann. N. E: Acad Sci. 317, 224246. 18. LEE, C. F’., MARTENS, M. E., JANKULOVSKA, L. AND NIEYMARK, M. A. (1979) Muscle Nerve 2, 340-348. 19. PANDE, S. V., AND BLANCHAER, M. C. (1971) J. Biol. Chem. 246, 402-411.
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MUSCLE
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20. OLIVER, I. T. (1955) Biochem. J. 61, 116-122. 21. ROSALKI, S. B. (1967) J. Lob. CZin. Med 69, 696705. 22. BAXTER, J. H., AND SUELTER, C. H. (1983) Muscle Nerve 6, 187-194. 23. LOWRY, P. H., ROSEBROUGH, N. J., FARR, A. O., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 24. HALL, N., ADDIS, P., AND DELUCA, M. (1977) B&hem. Biophys. Res. Commun 76,950-956. 25. HALL, N., AND DELUCA, M. (1976) Anal. Biochem. 76, 561-567. 26. CHAPPELL, J. B. (1964) Bicdem. J. 90, 225-237. 27. VIAL, C., FONT, B., GOLDSCHMIDT, D., AND GAUTHERON, D. C. (1979) B&hem. Biophys. Res. Cummun. 88, 1352-1359. 28. SWEENY, P. R., AND BROWN, R. G. (1981) Camp. B&hem. Physiol B 70, 27-33. 29. HALL, N., AND DELUCA, M. (1985) in Proceedings 2nd International Congress on Myocardial and Cellular Bioenergetics and Compartmentation, Los Angeles, Calif., 1984, in press. 30. BESSMAN, S. P. (1966) Amer. J. Med 40, 740
Decreased Mitochondrial Creatine Kinase Activity in Dystrophic Chicken Breast Muscle Alters Creatine-Linked Respiratory Coupling’ VICKIE
D. BENNETT, AND
NORMAN CLARENCE
Department of Biochemistq, Michigan *Departments of Medicine and Chemistry, Received
October
HALL,* MARLENE H. WELTER’
State University, East University of California,
29, 1984, and in revised
form
DELUCA,*
Lansing, Michigan 48824 and the San Diego, La Jolla, California %?O%’ February
27, 1985
Dystrophic chicken breast muscle mitochondria contain significantly less mitochondrial creatine kinase than normal breast muscle mitochondria. Breast muscle mitochondria from normal 16- to 40-day-old chickens contain approximately 80 units of mitochondrial creatine kinase per unit of succinate:INT (p-iodonitrotetrazolium violet) reductase, a mitochondrial marker, while dystrophic chicken breast muscle mitochondria contain 36-44 units. Normal chicken heart muscle mitochondria contain about 10% of the mitochondrial creatine kinase per unit of succinate:INT reductase as normal breast muscle mitochondria. The levels in heart muscle mitochondria from dystrophic chickens are not affected significantly. Evidence is presented which shows that the reduced level of mitochondrial creatine kinase in dystrophic breast muscle mitochondria is responsible for an altered creatine linked respiration. First, both normal and dystrophic breast muscle mitochondria respire with the same state 3 and state 4 respiration. Second, the post-ADP state 4 rate of respiration of normal breast muscle mitochondria in the presence of 20 mM creatine continues at the state 3 rate. However, the state 4 rate of dystrophic breast muscle mitochondria and mitochondria from other muscle types with a low level of mitochondrial creatine kinase, such as heart muscle and 5-day-old chicken breast muscle, is slower than the state 3 rate. Third, dystrophic breast mitochondria synthesize ATP at the same rate as normal breast muscle mitochondria but rates of creatine phosphate synthesis in 20-50 mM Pi are reduced significantly. Finally, increasing concentrations of Pi displace mitochondrial creatine kinase from mitoplasts of normal and dystrophic breast muscle mitochondria with the same apparent K,, indicating that the outer surface of the inner mitochondrial membrane and the mitochondrial creatine kinase from dystrophic muscle are not altered. 0 1985 Academic press, I”~.
Creatine kinase (adenosine-5’-triphosphate creatine phosphotransferase; EC 2.7.3.2) exists in several isozymic forms.
Eppenberger et al. (1) first reported three cytoplasmic forms composed of the M (muscle-type)3 or B (brain-type) subunits. These subunits dimerize to form MM, BB, and hybrid MB creatine kinase isozymes. The MM creatine kinase predominates in
i This work is supported by United States Public Health Service Grants GM 20716 (CHS) and HL 17682 (MD) and by the Michigan Agricultural Experiment Station, Journal Article No. 11117. The cost of this article was defrayed in part by the payment of page charges. This article must therefore be hereby marked “Advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact. a To whom correspondence should be addressed. 0003-9861185 $3.00 Copyright 0 1985 by Academic Press. Inc. All rights of reproduction in any form reserved.
3Abbreviations used: B, brain type subunit of creatine kinase; ereatine-P, creatine phosphate; glucose-6-P, glucose-6-phosphate; Pi, inorganic phosphate; INT, piodonitrotetrazolium violet; M, muscle type subunit of creatine kinase; RCR, respiratory control ratio; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid. 380
MITOCHONDRIAL
CREATINE
KINASE
mature skeletal muscle of birds and mammals and mammalian myocardium whereas BB; creatine kinase predominates in brain, neural tissue, embryonic skeletal muscle of mammals, and myocardial muscle of birds. Hybrid MB creatine kinase also appears in mammalian heart and skeletal mnscle. [See Ref. (2) for review of localization of various forms.] Jacobs et al. (3) reported an additional creatine kinase isozyme located on the outer surface of the inner mitochondrial membrane (4, 5). This mitochondrial isozyme differs from the cytoplasmic isozymes in amino acid compos.ition, electrophoretic mobility, and immunological properties; it does not hybridize with the cytoplasmic isozyme subunits (6, 7). In addition to these apparent structural differences between mitoehondrial ereatine kinase and the cytoplasmic forms, intracellular compartmentation creates functional differences. Jacobus and Lehninger (5) attribute an increase in the postADP state 4 respiration rate of rat heart mitochondria in the presence of creatine to mitochon.drial creatine kinase activity. In the presence of creatine, the mitochondrial isozyme regenerates ADP from the ATP produced during oxidative phosphorylation. Moreadith and Jacobus (8) and Gellerich and Saks (9) report additional evidence to support a functional coupling between mitochondrial creatine kinase and the adenine nucleotide translocase (5, lo14). Bessman and Geiger (15) discuss these results in terms of a creatine phosphate (creatine-P) shuttle where creatine plays an important regulatory role in intracellular energy transport of muscle. Based on the involvement of mitochondrial creatine kinase in the creatine-P shuttle and thus its importance in intracellular energy transport, we initiated a study of this creatine kinase isozyme and its function in mitochondria from normal and dystrophic avian pectoralis muscle. Previously, Mahler (16) reported a progressive decrease of mitochondrial creatine kinase activity per milligram of mitochondrial protein in the skeletal muscle of dystrophic chickens with increased age compared to normal controls.
ACTIVITY
IN
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MUSCLE
381
We confirm Mahler’s observation using genetically related, age-matched normal and dystrophic chickens; lines 412 and 413 (1’7), respectively. In addition, we report a loss of regulation by creatine on the respiration of mitochondria from dystrophic chicken breast muscle. MATERIALS
AND
METHODS
Materials. All biochemicals, enzymes, and creatine kinase assay kits were obtained from Sigma Chemical Company (St. Louis, MO.) unless otherwise specified. Creatine and rabbit muscle creatine kinase were purchased from Calbiochem-Behring Corporation (La Jolla, Calif.). Sephraphore III cellulose acetate electrophoresis strips were obtained from Gelman Sciences, Inc. (Ann Arbor, Mich.). Millipore filters were purchased from Millipore Corporation (Bedford, Mass.). Common laboratory chemicals were reagent grade or better. Solutions were prepared in distilled deionized water. Normal (line 412) and dystrophie (line 413) chickens were obtained from the Department of Avian Sciences, University of California, Davis, California. Isolation of breast and leg muscle mitochondria. Preparation of mitochondria from the pectoralis and the primarily red fiber leg muscles of normal and dystrophic chickens involved the basic procedure described by Lee et al. (18) substituting Sigma protease Type VII (1 mg/g muscle) for Nagarse (3 mg/g muscle). Mitochondrial yields were based on the recovery of succinate:p-iodonitrotetrazolium reductase (succinate:INT reductase), a mitochondrial marker, in the final mitochondrial suspension as compared to the activity in the initial crude homogenate. Yields ranged from 15 to 30% for mitochondria isolated from chickens between the ages of 12 and 20 days ex ova and 10% for mitochondria isolated from chickens 5 days ez ova Respiratory control ratios (RCR) at 25’C for normal and dystrophie breast muscle mitochondria from chickens 12-20 days ex ouo were 3.4 and 2.4, respectively, and ADP:O ratios were 1.5 and 1.2, respectively, using succinate as substrate in the presence of rotenone. RCR values at 25°C for normal and dystrophic breast muscle mitochondria from chickens 5 days ex ova were 1.5 and 1.2, respectively, and ADP:O ratios were 1.4 and 1.3, respectively. Isolation of heart mitochondria. Normal and dystrophic chicken heart muscle mitochondria were isolated by differential centrifugation as previously described (19). Mitochondrial yields based on the recovery of succinate:INT reductase in the final mitochondrial suspension compared to the initial crude homogenate ranged from 20 to 40%. Respiratory control ratios for normal and dystrophic heart mitochondria at 25°C were 1.8 and 1.6, respectively,
382
BENNETT
using succinate as substrate in the presence of rotenone. ADP:O ratios were 1.1 and 1.2, respectively. Enzyme assays and protein determination Total creatine kinase activity in the protease-treated heart, breast, and leg muscle mitochondria was solubilized by suitably diluting a portion of the final mitochondrial suspension into 0.1 M sodium phosphate, pH 6.5, 25 mM P-mercaptoethanol, 1 mM EDTA, and 1% Triton X-100 to give a final activity of 1-4 IU/ml. Creatine kinase activity was determined spectrophotometrically at 340 nm and 30°C using the creatine kinase assay mix from Sigma (20, 21). The mitochondrial marker, succinate:INT reductase, was assayed as described previously (22). Correcting for yield of mitochondria gave the units of mitochondrial creatine kinase per gram of muscle. Mitochondrial pellet protein was determined by the Lowry method (23) following a 1:l solubilization in 1 N NaOH for 1 h. Bovine serum albumin was used as a standard. Cellulose acetate electrophoresis. Creatine kinase isozymes were separated by cellulose polyacetate electrophoresis as previously described (24). Triton X-100 was added to the electrophoresis buffer (final concentration, 1%) to allow creatine kinase associated with mitochondria to migrate from the origin. Following electrophoresis, the strips were stained for creatine kinase isozymes as previously described (25) and scanned on a Gelman ACD-18 automatic computing densitometer. Isozyme positions were identified by comparing sample mobilities with the migration of purified fractions of MM, MB, BB, or mitochondrial creatine kinase on independent electrophoretic strips. In control electrophoretic strips, creatine-P was omitted from the assay mix to check for interference by adenylate kinase. Mitochondrial creatine kinuse. The proportion of mitochondrial creatine kinase isozyme in the final mitochondrial suspensions was determined from densitometer scans of the electrophoretically separated isozymes. Of the total creatine kinase in a mitochondrial preparation, mt-creatine kinase accounted for 79 + 17% in 27 normal leg and breast muscle preparations, 71 + 17% in 28 dystrophic preparations, and 100% in 28 heart preparations. Since the creatine kinase assay mix is optimized for MM creatine kinase and these conditions may vary somewhat for mitochondrial ereatine kinase, there may be some error in the value for the final mitochondrial creatine kinase activity. Mitochondrial creatine kinase activity was then normalized to the amount of mitochondria in each sample by dividing by the activity of succinate:INT reductase in the sample. Mitochwndrial oxygen consumption Mitochondrial respiration was monitored at 25°C on a Gilson Model K-IC oxygraph equipped with a Yellow Springs Instruments electrode and a high-sensitivity Teflon membrane (Yellow Springs Instruments). Standard skeletal muscle mitochondria buffer contained 150
ET
AL,
mM sucrose, 1 mM EDTA, 2 mg/ml bovine serum albumin, and 25 mM Tris-HCl, pH 7.5. Standard heart muscle mitochondria buffer contained 0.21 M mannitol, 70 mM sucrose, 1 mM EDTA, 2 mg/ml bovine serum albumin, and 10 mM Tris-HCl, pH 7.4. Breast and leg muscle mitochondria (50 ~1 of 20-30 mg protein/ml) were incubated in 1.75 ml of medium containing standard muscle mitochondria buffer, 5 mM succinate, 10 pM rotenone, and 5 mM MgS04 in addition to 2.5, 5, 10, 20, 50, 75, or 98 mM potassium phosphate and 0 or 20 mM creatine. Heart mitochondria were incubated under the same conditions except in medium containing standard heart mitochondria buffer and 0.5, 1, 2.5, 5, or 10 mM potassium phosphate. Adding 20 pl 10 mM ADP to each incubation mixture initiated state 3 respiration. The solubility of oxygen in buffered medium at 25°C was assumed to be 240 nmol &Jml (26). Rate of mitochondrial meat&e-P synthesis. Breast muscle mitochondria (125 ~1 of 20-30 mg protein/ ml) were incubated at 25°C in 5-ml reaction mixtures containing standard skeletal muscle mitochondria buffer, 5 mM succinate, 10 pM rotenone, 5 mM MgSOI, 20 mM creatine, and 2.5, 5, 10, 20, 50 or 98 mM potassium phosphate. Heart mitochondria were incubated under the same conditions except in medium containing standard heart mitochondria buffer and 0.5, 1, 2.5, 5, 10, or 20 mM phosphate (Pi). Samples (0.5 ml) were withdrawn from the reaction chamber at 1-min time intervals after addition of 50 ~1 10 mM ADP and immediately added to 0.5 ml of 30 mg/ml activated charcoal to remove ATP. The charcoal suspension was filtered through Millipore filters (GSWP, 0.22-pm pore size) as quickly as possible. A portion (200 ~1) from each filtered sample was added to each of two tubes containing 1 ml of 1.2 mM ADP, 3.7 mM AMP, 14 mM glucose, 9 mM magnesium acetate, 1.8 mM NADP, 4 mM dithiothreitol, 1.7 units/ml glucose-6-phosphate dehydrogenase, and 1.7 units/ml hexokinase in 20 mM Hepes, pH 7.4. Rabbit muscle creatine kinase (10 pl of 2 mg/ml) was added to one of the two tubes for each sample. All tubes were incubated at room temperature for 60 min and the absorbance was read at 340 nm. Rate of mitochondrial ATP synthesis Breast muscle mitochondria (125 ~1 of 20-30 mg protein/ml) were incubated at 25°C in 5-ml reaction mixtures containing standard skeletal muscle mitochondria buffer, 5 mM succinate, 10 pM rotenone, 5 mM MgSO,, 20 mM creatine, 5 mM glucose, 60 units hexokinase, and 5 or 50 mM potassium phosphate. Samples (0.5 ml) were withdrawn from the reaction chamber at lmin time intervals after addition of 50 pl of 10 mM ADP and immediately added to 0.5 ml of 30 mg/ml activated charcoal to remove ADP. The charcoal suspension was filtered through Millipore filters (GSWP, 0.22-pm pore size) as quickly as possible. The concentration of ATP in each sample (glucose
MITOCHONDRIAL 6-phosphate essentially creatine-P
CREATINE
KINASE
plus creatine phosphate) was determined as described in the previous section under synthesis. hfitop!mt proparotimz Mitoplasts from normal and dystrophic brleast muscle were prepared by first swelling the outer mitochondrial membrane in a hypotonic solution of 10 mM Tris-HCl, pH 7.5, at 0°C for 5 min. Adding a hypertonic solution (1.75 M sucrose, 2 mM ATP, and 2 mM MgSO& to the previous suspension at 0°C for 5 min allowed removal of the outer membrane by mild sonication (Branson Sonifier at 3 A for 20 s). Mitoplasts were then sedimented at 20,OOOg for 20 min. Efect of inorganic phosphate on the interaction of mitochondrial creatine kinase with mitoplasts. Mitoplasts (30-~1 aliquots) were incubated with shaking in a 750-p] solution containing 0.21 M mannitol, 70 mM sucrose, 0.1 mM EDTA, and 10 mM Tris-HCl, pH 7.4, and increasing concentrations of potassium phosphate at pH 7.4 for 2 h at 6°C. The mitoplast suspensions were then centrifuged in an Eppendorf centrifuge for 10 min and the supernatants were assayed for creatine kinase activity to determine the percentage of total mitochondrial creatine kinase released from the mitoplast membrane.
ACTIVITY
IN
DYSTROPHIC
MUSCLE
383
differences are not significant at Days 37. Third, normal and dystrophic heart mitochondrial creatine kinase activities per unit of succinate:INT reductase remain relatively constant (in the three age groups), whereas the levels in normal and dystrophic leg and breast muscle are low at early ages but increase with age. Comparing the levels of succinate:INT reductase activity per gram of muscle for each muscle type (Panel B of Table I) reveals no significant differences between normal and dystrophic muscle of each type. There tends to be a slight increase in succinate:INT reductase in dystrophic breast muscle at 12-19 and 25-40 days, but the differences are not significant. The differences in the levels of mitochondrial creatine kinase per unit succinate:INT reductase between normal and dystrophic muscles therefore reflect differences in the mitochondrial creatine kinase activity and not differences in the number of mitochondria. In addition, the levels of miRESULTS tochondrial creatine kinase per gram of muscle (Panel C of Table I) basically Levels of Mitochondrial Creatine Kinase confirm these conclusions. Specifically, in Normal and Dystrophic Muscle there are no differences in units of mitoTable I contains a summary of the chondrial creatine kinase per gram of levels of mitochondrial creatine kinase both normal and dystrophic heart and leg and succinate:INT reductase (a mitochonmuscle at 25-40 days ex ouo. Mitochondrial drial marker enzyme) as a function of age creatine kinase per gram of dystrophic for normal and dystrophic chicken heart, breast muscle is slightly more than half leg, and breast muscle. The data in Panel that found in normal muscle but the difA of this table reveal several basic trends ferences are not significant at the 95% concerning the level of mitochondrial cre- confidence level. atine kinase per unit of succinate:INT Comparing differences between the reductase in the three muscle types from Days 3-7 age group and the days 12-19 both normal and dystrophic chickens. age group shows that mitochondrial creFirst, normal breast muscle mitochondria atine kinase concentrations in the breast contain the highest levels of mitochondrial muscle are low at hatch. Both units of creatine kinase per unit succinate:INT re- mitochondrial creatine kinase per unit of ductase. The levels in normal heart muscle succinate:INT reductase and per gram of mitochondria are roughly one-tenth those muscle are significantly lower in the Days in normal breast muscle mitochondria 3-7 age group compared to the Days 12while leg muscle mitochondria contain 19 age group. Because the adult levels in one-half to one-third those in breast the dystrophic breast muscle are demuscle mitochondria. Second, dystrophic pressed considerably, the differences bebreast muscle mitochondria contain half tween the above age groups are not sigto less than half as much mitochondrial nificant in these muscles. Units of succicreatine kinase per unit of succinate:INT nate:INT reductase per gram of muscle reductase as normal breast muscle mitoare also not significantly different when chondria at. Days 12-19 and 25-40. The these two age groups are compared.
f
f f P < 86 rt
27 84
27
(2)
(4)
15 (7) 26 (7) 0.001 21 (5)
8.4 (6)
16.1 27 f 10 -
8.1 f 1.7 (3) 6.0 + 3.7 (7) ns. 8.5 f 1.6 (7)
Normal
-
A. Mt-creatine
CREATINE
36
19 44
f 10 + 17 n.s. + 6
f 3.0 -t 3 < P < + 6.5
(7)
(6) (6)
(3) (4) 0.005 (6)
2.7 (6)
7.0 +
9.4 22 0.001 28
1.3 (7)
4.6 2.4 f
(2)
(succ:INT)
Dystrophic
kinase
OF MITOCHONDRIAL
n.s.
n.s.
n.s.’
P”
unit-‘)
AND
P < 0.001
Normal
reductase muscle)
B. Succ:INT (units g-i
0.9 f 0.5 (7) 0.5 f 0.2 (7) n.s. 0.5 f 0.1 (5)
2.0 f 0.6 (6)
2.6 (2) 1.0 + 0.3 (4)
1.0 + 0.4 (6) 0.8 + 0.6 (3) n.s. 0.7 f 0.4 (7)
2.3 f 0.8 (3) 1.2 f 0.1 (4) n.s. 1.6 f 0.3 (6)
2.4 4.6 f 1.0 (7) 3.1 * 1.0 (7)
Dystrophic
IN VARIOUS
REDUCTASE
3.1 f 0.4 (3) 3.7 f 1.4 (7) n.s. 2.4 ix 0.9 (7)
SUCCINATE:INT
n.s. 0.005 < P < 0.01
(unit
KINASE
I
ns.
n.s. n.s.
n.s.
n.s.
-
n.s.
n.s.
P”
NORMAL
DYSTROPHIC
22 50 0.001 46
7 (5)
kinase
MUXLES
f 9 (7) f 15 (7) < P < 0.005 k 12 (5)
43 f
44 (2) 27 + 10 (4) -
25 f 8 (3) 21 * 13 (7) n.s. 20 IF 8 (7)
Normal
C. Mt-creatine
AND
7 (5)
8 (7)
17 + 9 (6) 31 f 12 (6) n.s. 26 f 14 (7)
23 f 15 (3) 25 f 5 (4) n.s. 46 f 18 (6)
22 f
10.7 11 +
muscle)
(2)
g-i
Dystrophic
(unit
P”
Note. Creatine kinase in the protease-treated heart, leg, and breast muscle mitochondria was solubilized and assayed spectrophotometrically at 30°C as indicated under Materials and Methods. The mitochondrial marker enzyme, succinate:INT reductase, was assayed by an endpoint assay following lo- to 12min incubations using conditions described previously. Data are the means f SE for the number of mitochondrial samples given in parentheses. Each sample is the composite of mitochondria from at least two birds. P values were determined by Student’s t test for paired values. a P values for compared normal and dystrophic data at this age. b P values for compared data at ages 3-7 and 12-19 days. ’ n.s. not significant at the 95% confidence level.
Breast 3-7 12-19 Pb 25-40
3-7 12-19 Pb 25-40
Leg
Heart 3-7 11-19 Pb 25-40
(days)
Muscle age
LEVELS
TABLE
MITOCHONDRIAL
CREATINE
KINASE
Table II contains a summary of the initial state 4, state 3, and post-ADP state 4 rates of respiration for various muscle mitochondria from both normal and dystrophic chickens in the absence and presence of 20 mM creatine at various phosphate concentrations. The initial state 4 rates of both normal and dystrophic breast muscle mitochondria are not affected by either 20 mM creatine or 2.5 to 50 mM Pi. However, 20 mM creatine affects the postADP state 4 rates of normal breast muscle mitochondria by causing them to continue at the state 3 rate (Fig. 1). Specifically, in the absence of creatine, the post-ADP state 4 rates of normal breast muscle mitochondria range from 40 to 50% of the state 3 rate at all concentrations of Pi. In the presence of creatine, the post-ADP state 4 rates are statistically identical to the state 3 rate (Table II). Dystrophic breast muscle mitochondria respire differently under these same conditions (Fig. 1). First, the initial state 4 rates are nearly identical to those observed with normal breast muscle mitochondria (P’ > 0.5 at 2.5 to 10 mM Pi and 0.1 < P < 0.2 for 20 to 98 mM Pi). Second, the initial state 4 respiration rates of dystrophic breast muscle mitochondria are not affected by creatine. Third, the postADP state 4 rate of dystrophic breast muscle mitochondria in the absence of creatine is approximately 60% of the state 3 rate as Pi is increased from 2.5 to 98 mM. However, in the presence of creatine, the post-ADP state 4 rate of dystrophic breast muscle mitochondria, in contrast to the normal breast muscle mitochondria, is no longer identical to the state 3 rate as Pi increases. At 2.5 to 10 mM Pi, the post-ADP state 4 rate remains unchanged, but as Pi increases from 20 to 98 mM, the post-ADP state 4 rate becomes statistically different than the state 3 rate (Table II). An identical experiment reveals that creatine has no effect on the post-ADP state 4 rates of mitochondria from normal and dystrophic hearts (Table II). In this case, the post-ADP state 4 rates of both normal and dystrophic heart mitochondria in the prese.nce of 20 mM creatine remain
ACTIVITY
IN
DYSTROPHIC
385
MUSCLE
at about 80% of the state 3 rate at all Pi concentrations ranging from 2.5 to 20 mM. Similarly, breast muscle mitochondria from 5-day-old normal and dystrophic chickens behave like the normal and dystrophic heart mitochondria (Table II). Therefore, in the presence of creatine, both normal and dystrophic heart mitochondria, as well as both normal and dystrophic breast muscle mitochondria from 5-day-old chickens, function like dystrophic breast muscle mitochondria from 16- to 18-day-old chickens at phosphate concentrations greater than 5 mM. Creatine Phosphate Production Res@ing Mitochondria
in
The effects of creatine and Pi on the respiration of normal and dystrophic heart and breast muscle mitochondria as discussed above are also reflected in their rates of synthesis of creatine-P. Figure 2 shows rates of synthesis of creatine-P (pm01 creatine-P produced min-’ [IU succinate:INT reductasel-‘) at various Pi concentrations for respiring breast and heart muscle mitochondria from both normal and dystrophic chickens. The inset in Fig. 2 shows creatine-P concentration as a function of time after initiation of mitochondrial respiration for dystrophic breast muscle mitochondria at various Pi concentrations. Slopes of these lines for each Pi concentration (after the initial lag period) determine the rates of creatine-P synthesis. Normal breast muscle mitochondria maintain high, nearly constant rates at all Pi concentrations from 5 to 98 mM. This high rate is expected because the post-ADP state 4 rate continues at the state 3 rate in the presence of creatine at all Pi concentrations up to 98 mM Pi. In contrast to normal breast muscle mitochondria, dystrophic breast muscle mitochondria exhibit a sharp decline in the rate of creatine-P synthesis after 10 mM Pi. This decline in the rate of creatine-P synthesis parallels oxygen consumption measurements in the presence of creatine (Table II); Pi concentrations from 20 to 98 mM allow return of a distinct postADP state 4 rate, indicating less conver-
II
D D
N D
N
16 16
16 16
5
5
Breast Breast
Heart Heart
Breast
Breast
6d
6d
17* 17*
12* lo*
9* 12*
12
5-50
5-50
0.5-20 0.5-20
2.5-10 20-98
2.5-10” 20-98
[Phosphate] bM)
0.23 _+ 0.03
0.22 f 0.07
0.45 * 0.15 0.24 + 0.05
0.53 f 0.05 0.61 + 0.14
0.52 f 0.13 0.52 + 0.12
-
Initial +
4
O./unit
0.22 f 0.02
0.22 + 0.04
0.46 t 0.05 0.26 f 0.06
0.55 f 0.12 0.57 f 0.09
0.57 f 0.07 0.54 f 0.07
state
pmol
0.87 + 0.07
0.72 f 0.03
1.48 f 0.31 0.80 f 0.10
2.38 f 0.55 2.65 I 0.30
+
(+20
0.86 f 0.10
0.64 * 0.05
0.57 * 0.03
0.54 -c 0.08
0.81 f 0.10 0.73 f 0.05
1.43 * 0.35 1.54 * 0.38 0.90 Ik 0.19
0.92 + 0.25 0.97 k 0.25
-
Post-ADP
mM creatine)
2.33 + 0.37 2.66 k 0.37 1.43 f 0.23
2.50 f 0.22 2.34 + 0.24
3
reductase State
2.58 f 0.19 2.22 f 0.25
-
succinate:INT
+
4
0.65 k 0.07
0.57 * 0.05
0.66 + 0.05
2.14 k 0.48 2.02 f 0.30 1.12 + 0.19
2.47 k 0.20 2.26 f 0.27
state
0.001 < P < 0.005
P < 0.001
P < 0.001
0.2 < P < 0.4 P < 0.001 P < 0.001
P > 0.5 0.4 < P < 0.5
P
Note. Mitochondrial respiration was monitored at 25°C. Mitochondria were incubated in their respective mitochondrial buffer, 5 mM succinate, 10 fiM rotenone, 5 mre MgS04, and Pi + 20 mM creatine as indicated. See Materials and Methods for details. The addition of 20 pl of 10 mM ADP initiated state 3 respiration. Data are the means f SE for the number of measurements given in parentheses. P values were determined by Student’s t test for paired values comparing state 3 and post-ADP state 4 rates in the presence of creatine. a Muscle (Normal, N; Dystrophic, D) (n, number of measurements). * Each sample contains the mitochondria in breast muscle from two birds. “Respiratory rates are the composite results of several different concentrations of Pi spanning the range indicated as detailed under Materials and Methods. d Each sample contains the mitochondria in breast muscle from three birds.
D
N N
16 16
5w
Breast Breast
Age (days)
Muscle”
SUMMARYOFOXYGENRESPIRATIONEXPERIMENTSWITHVARIOUSMUSCLEMITOCHONDRIAFROMNORMALANDDYSTROPHICCHICKENS
TABLE
F
2
2 3
MITOCHONDRIAL
mtor
KINASE
20 mhd phosphate
ACTIVITY
IN
DYSTROPHIC
387
MUSCLE
of the rates of glucose-6-P and creatineP when normal and dystrophic breast muscle mitochondria were allowed to respire in the presence of excess hexokinase and glucose and either 5 or 50 mM Pi. The data in this table indicate little effect of 5 to 50 mM Pi on the total rate of ATP synthesis in both normal and dystrophic
Mn0l
No Creotine 3s nlmln 02 L
CREATINE
20 mM Creattns 20 mM phosphate
I min
\
B.
No Creatine
20 tnh4 Creatine 20 r&t Phosphate
FIG. 1. Sample oxygen consumption traces for normal (A) and dystrophie (B) breast muscle mitochondria from chickens 19 days old. Mitochondria were added to standard breast muscle mitochondria buffer containing 20 mM phosphate and either 0 or 20 mM creatine at 25°C. Addition of ADP initiated state 3 respiration. The numbers in parentheses represent nanomoles Oa consumed per minute.
sion of ATP to creatine-P. Heart muscle mitochondria from both normal and dystrophic chickens have significantly lower rates of creatine-P synthesis than normal or dystrophic breast muscle mitochondria. These very low rates are consistent with the lack of either a creatine or Pi effect on oxygen consumption during respiration of normal and dystrophic heart mitochondria (Table II). Thus, the decreased rates of creatine-P synthesis in respiring mitochondria from various muscle types are consistent with decreased concentrations of mitochondrial creatine kinase activity in these mitochondria.
Rates of Al’P Synthesis in Normal and Dystrophic Breast Muscle Mitochcmdria The data in Table III show the rates of ATP synthesis calculated from the sum
00
IO
20
30
40 bw-4
50
60
70
80
90
fM
FIG. 2. Rates of creatine phosphate synthesis in normal and dystrophic breast and heart muscle mitochondria as a function of inorganic phosphate. Mitochondria were incubated with various concentrations of phosphate and 20 mM creatine at 25°C as described under Materials and Methods. The figure inset shows nanomoles creatine phosphate formed by dystrophic breast muscle mitochondria as a function of time for the indicated phosphate concentration. The slopes of each line in the inset are the rates of synthesis of creatine phosphate plotted in the main figure as a function of phosphate concentration. Rates of synthesis are expressed as micromoles creatine phosphate produced per minute per unit of succinate:INT reductase. The error bars for the breast muscle mitochondria data represent standard errors of the mean for the number of samples indicated by the number in parentheses. Each mitochondrial sample contained the composite of mitochondria from two birds. Heart data are averages obtained from two mitochondrial samples containing the composite of mitoehondria from two birds.
388
BENNETT
ET
TABLE RATE
OF SYNTHESIS
AL.
III
OF CREATINE-P AND ATP (CREATINE-P PLUS AND DYSTROPHIC BREAST MUSCLE
BY NORMAL
Type of mitochondria
[Phosphate] hM)
(pm01
mini
ATP [succ:INT]-‘)
Glc-6-P
WITH MITOCHONDRIA
ADDED
(firno
HEXOKINASE)
Creatine-P mini [succ:INT]-‘)
Normal
5 50
5.21 f 0.45 (2) 5.05 f 0.22 (2)
3.16 f 0.38 (7) 2.59 f 0.40 (6)
Dystrophic
5 50
6.22 zk 0.35 (2) 5.26 5~ 0.45 (2)
2.33 f 0.78 (3)” 0.74 + 0.12 (3)”
Note. The rates of synthesis of creatine-P and ATP by respiring normal and dystrophic breast muscle mitochondria at 25°C in mixtures containing breast muscle mitochondrial buffer, 5 mM succinate, 10 j.rM rotenone, 5 mM MgS04, 20 mM creatine, 5 mM glucose, 60 units hexokinase, and 5 or 50 mM potassium phosphate were determined as described in Materials and Methods. Data are the mean i SE for the number of samples given in brackets. “Because creatine does not cause a continuation of the state 3 rate throughout the incubation period of dystrophic breast muscle mitochondria, ADP concentrations become rate-limiting and thus these values are not maximum rates of creatine-P synthesis.
breast muscle mitochondria. Furthermore, mitochondria from dystrophic muscle produce ATP as effectively as those from normal muscle (Table III). Efect of Inorganic Phosphate on the Interactim of Mitochtmdrial Creatine Kinase with Normal and Dystrophic Breast Muscle Mitoplasts The effect of Pi on the interaction of mitochondrial creatine kinase with normal and dystrophic breast muscle mitoplasts is shown in Fig. 3. Increasing concentrations of Pi release increasing amounts of mitochondrial creatine kinase into the incubation medium until 50 mM, when 85% of the total mitochondrial creatine kinase is released. K0,5’s (concentration of phosphate required to release 50% of total mitochondrial creatine kinase) are 26.5 and 30 mM Pi for normal and dystrophic breast muscle mitoplasts, respectively. Identical results are obtained if the mitoplasts are incubated with increasing concentrations of inorganic phosphate for 10 min instead of 2 h at 6°C (S. P. J. Brooks, unpublished observations). The shape of the curves are very similar to the phosphate solubilization curve for mitochondrial creatine kinase reported by Vial et al. (27) for pig heart mitochondria, but the KO,, values are higher for chicken breast muscle mitochondria.
DISCUSSION
Comparing mitochondrial creatine kinase concentrations in various muscle types from normal and dystrophic chickens (Table I) basically confirms Mahler’s observation (16) that mitochondrial creatine kinase activity is decreased in dystrophic breast muscle compared to normal age-matched controls. Since Mahler used chickens at 44, 91, 200, and 566 days ex ova, when the dystrophic condition is in its advanced stages, we felt the need to examine levels of mitochondrial creatine kinase early in the onset of the dystrophic symptoms. The onset of dystrophy determined by the flip test becomes apparent at 9 days ex (yuo in the 413 line of chickens (17). The data in Table I show that this event occurs around the time when dystrophic breast muscle mitochondria contain half the normal level of mitochondrial creatine kinase. In addition, we express the levels of mitochondrial creatine kinase in terms of a specific mitochondrial marker enzyme, succinate:INT reductase, rather than per milligram of mitochondrial protein. This difference in reporting procedure is important when comparing normal and dystrophic mitochondria since the dystrophic mitochondrial pellet may contain extraneous protein because of the increased connective tissue in dystrophic muscle [see Ref. (28) for review].
MITOCHONDRIAL
CREATINE
KINASE
Of particular interest in this study is the relativlely high level of mitochondrial creatine kinase per unit of succinate:INT reductase in normal chicken breast muscle compared to chicken heart and leg muscle (Table I). In fact, Hall and DeLuca’s (29) survey of the level of mitochondrial creatine kinase per unit of succinate:INT reductase in rat skeletal muscle, liver, and heart; beef heart and liver; rabbit skeletal muscle, heart and brain; and chicken heart and breast muscle reveals that chicken breast muscle contains the highest concentration of this creatine kinase isozyme. The low level of mitochondrial creatine kinase in chicken heart muscle mitochondria contrasts with the generally accepted view that heart muscle mitochondria contain significant amounts of this isozyme (5). The data in Table I also show that normal chicken heart contains approximately one-half the mitochondrial creatine kinase as normal chicken breast muscle whlen based on muscle mass. The possible physiological effects of reduced levels of mitochondrial creatine kinase are discussed in terms of the creatine-P shuttle [for review see Ref. (15)]. This shuttle involves the mitochondrial and muscle isozymes of creatine kinase and evolveld in recent years basically from the initial work of Bessman and colleagues (10, 15, 30) and Jacobus and Lehninger (5). The mitochondrial isozyme is bound to the out.er surface of the inner mitochondrial membrane (4, 5), presumably funetional1.y coupled to the adenine nucleotide translocase (5, 8-14). Apparently, bound mitochondrial creatine kinase preferentially utilizes the ATP produced during oxidative phosphorylation to phosphorylate creatine and regenerate ADP. When breast muscle mitochondria contain their full complement of mitochondrial creatine kinase (after approximately 11 days ez o~o), creatine maintains the post-ADP state 4 rate equivalent to the state 3 rate at Pi concentrations ranging from 5 to 98 mM (Table II). However, when muscle mitochondria contain 30 to 40 units of mitochondrial creatine kinase per unit of succinate:INT reductase, the post-ADP state 4 rate is only 80% of the state 3 rate in the presence of 20 mM
ACTIVITY
IN
DYSTROPHIC
MUSCLE
389
creatine and Pi concentrations between 20 and 98 mM (Table II). At 2.5 to 10 mM Pi, the post-ADP state 4 rate in the presence of creatine is nearly equivalent to the state 3 rate. In addition, 20 mM creatine has little effect on the post-ADP state 4 rate of 5-day-old normal chicken breast muscle mitochondria since the muscle at this age has not yet reached its full complement of mitochondrial creatine kinase (Table II). Furthermore, heart mitochondria, containing around 8 units of mitochondrial creatine kinase per unit of succinate:INT reductase (Table I), behave like breast muscle mitochondria from 16- to 19-day-old dystrophic chickens and mitochondria from 5-day-old normal and dystrophic chickens. Again, 20 mM creatine, in the presence of 2.5 to 10 mM Pi, has little effect on the post-ADP state 4 rate of both normal and dystrophic heart muscle mitochondria (Table II). Thus, irregardless of whether the mitochondria are obtained from normal or dystrophic muscle, when the level of mitochondrial creatine kinase is below 40 units per unit of succinate:INT reductase 20 mM creatine in the presence of 20 to 98 mM Pi is unable to maintain the state 4 rate equivalent to the state 3 rate. The rates of creatine-P synthesis in respiring mitochondria isolated from heart and breast muscle (Fig. 2) strengthen the argument that the level of mitochondrial creatine kinase in normal and dystrophic breast muscle accounts for the differences of creatine and Pi on oxygen consumption measurements with these two mitochondria. Normal breast muscle mitochondria maintain a high, nearly constant rate of production of creatine-P at all Pi concentrations, consistent with the presence of large amounts of mitochondrial creatine kinase. The sharp decline in the rate of creatine-P production at Pi concentrations between 20 and 98 mM in dystrophic breast muscle mitochondria is consistent with the lower levels of mitochondrial creatine kinase aetivity and the drop in post-ADP state 4 rates of oxygen consumption at similar Pi concentrations. Heart muscle mitochondria produce creatine-P at significantly lower rates than normal and dystrophic breast
390
BENNETT
FIG. 3. Effect of inorganic phosphate on the soluhilization of mitochondrial creatine kinase (mt-CK) from normal and dystrophic breast muscle mitoplasts. Aliquots (30 ~1) of isolated mitoplasts (See Materials and Methods) were incubated at 6°C for 2 h in 750 ~1 standard breast muscle mitochondria buffer (See Materials and Methods) and the indicated concentrations of potassium phosphate. After centrifugation, the supernatants were assayed for creatine kinase activity. The percentage of total mt-CK in the supernatant, expressed as a percentage of soluble mt-CK, as a function of phosphate concentration is shown for normal (0) and dystrophic (X) breast muscle mitoplasts. Each point represents the average of data from four independent determinations + standard deviation.
muscle mitochondria, again consistent with the very low levels of mitochondrial creatine kinase activity and the lack of an appreciable effect of creatine on the post-ADP state 4 rate. The results in Fig. 3 indicate that mitochondrial creatine kinase and the outer surface of the inner mitochondrial membrane are not altered in dystrophic muscle mitochondria; the concentration of Pi required to release mitochondrial creatine kinase from the mitoplast membrane is the same for both normal and dystrophic mitochondria. Furthermore, the creatine phosphate production rates for each of the mitochondrial types shown in Fig. 2 indicates that 50 mM Pi has no insignificant effect on the rate of mitochondrial ATP synthesis in normal and dystrophic breast mitochondria. In addition, mitochondria from dystrophic muscle function
ET
AL.
as effectively as normal muscle mitochondria in synthesizing ATP (Table III). We do not suggest that the decreased levels of mitochondrial creatine kinase found in dystrophic breast muscle is the primary lesion in muscular dystrophy, but rather that this decrease may be of primary importance in causing the loss of function of white fiber muscle by decreasing the efficiency of trapping available mitochondrial ATP as creatine-P, the high-energy storage molecule for muscle contraction energy. It is now of interest to know whether only muscles with high levels of mitochondrial creatine kinase per unit of succinate:INT reductase are most severely affected by dystrophy. ACKNOWLEDGMENTS We gratefully acknowledge the assistance of the Department of Animal Science, Michigan State University, and particularly Ms. Bridget Grala, Dr. Robert Ringer and Dr. Donald Polin; the Department of Avian Sciences, University of California at Davis, and particularly Mr. Fayne Lantz; and Dr. Shelagh Ferguson-Miller, Department of Biochemistry, Michigan State University, for the use of her Gilson oxygraph. REFERENCES 1. EPPENBERGER,
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