ESPEKIhIENTAL
NEUROLOGY
(Na+
49,
+ K+)-ATPase
TAKAFUMI
Newowmcular School
345-355
(1975)
in Subcellular Skeletal Muscle KAGATOMO
AND
JA~VES
Disease Rcsearcll Laboratories. of dfcdicinr, Los A-I~lgrlrs.
Recriwd
January
10, 19i.5’;
rczision
Fractions
B. PETER
of Rat
’
L:nizIersity of California, California 90024 rcc~civcd
Jmr
9, 197.5
Sodium and potassium-stimulated, magnesium-dependent, ouabain-sensitive ATPase has been used as an enzymic marker for muscle membrane (sarcolemma). However, the precise localization of this enzyme is still unknown. The enzyme activity was determined in muscle homogenates, sarcolemma, mitochondria, myosin B, and fragmented sarcoplasmic reticulum fractions isolated from frozen rat skeletal muscle. NaI-extracted fragmented sarcoplasmic reticulum isolated from frozen muscle ( NaI-extracted high-speed fraction) contained a much higher specific (Na+ + K+)-ATPase activity (102 nmoles P,/mg/min) than did a similar fraction isolated from fresh skeletal muscle (34 nmoles P,/mg/min). NaI-treated muscle homogenate, sarcolemma, myosin B, and mitochondria had lower (Na’ i- Ii’) -ATPase specific activity than the NaI-extracted high-speed fraction. The fragmented sarcoplasmic reticulum prepared from fresh or frozen muscle had the same calcium accumulating capacity; this capacity was almost aholished in the fragmented sarcoplasmic reticulum of both fresh and frozen muscle treated with NaI. The methods described may he useful in the study of muscle from humans or experimental animals where the amount of tissue available is limited. The results reemphasize the possihle hazard in determining precise subcellular localization of enzyme activities in frozen tissues, as is a necessarily routine procedure in histochemistry.
IT\‘TRODIJCTION (Na+ + K+) -stimulated, Mg++-dependent ATPase (EC3.6.1.3.) has been found in membrane preparations from various tissues (2, 8, 15, 21-23, 29). 1 This investigation was supported by USPHS Grant NS-07587. Dr. Nagatomo is a Postdoctoral Fellow of the Muscular Dystrophy Associations of America, Inc. The authors would like to thank Dr. Ronald RI. Andiman for his help in the preparation of this manuscript and Kerstin E. Stempel for excellent technical assistance. Reprint requests to James B. Peter, M.D., Ph.D., University of California, School of Medicine, Los Angeles, California 90024.
Copyright All rights
0
1975
by Academic
of reproduction
in any
Press, Inc.
form
reserved.
346
NAGATOMO
AND
PETER
In view of the critical role of cation transport in skeletal muscle function, the role of a “cation transport ATPase” characterized by Skou (24) is likely to be an important one. Several investigators have reported such a (Na’ + K+)-ATPase in muscle membrane preparations (1, 3, 6, 12, 25). The precise localization of this enzyme, however, is still unproved because the plasma membrane is not easily separated from other organelles. (Na’ + K+)-ATP ase activity can become manifest by treatment with detergents like sodium deoxycholate (DOC) (4, 19, 20) and high concentrations of salts such as sodium iodide (10, 11) and lithium bromide (1, 3, 25). Nakao et al. (10, 11) have prepared a (Na’ + K+) -ATPase of high specific activity from brain microsomes by treatment with 2 M NaI. In this study we have used a modification of Nakao’s method to produce a NaI-extracted high-speed fraction of frozen skeletal muscle which has a (Na+ + K+)-ATPase of higher specific activity than that of similarly treated whole rat skeletal muscle homogenate, sarcolemma, mitochondria, or myosin B. METHODS NaI-extracted Muscle Homogenate. Typically about l-2 g of fresh muscle, cleaned of connective tissue, fat, and nerve are frozen in liquid nitrogen, stored overnight at -70 C and sectioned transversely with a razor blade to about 2 mm thickness. The muscle slices are then suspended in ice-cold 50 mM CaCI, at 25% (w/v). The suspension is homogenized twice for 30 set each in Polytron homogenizer (Kinematica BMBH. Luzern-Schweiz, Switzerland) at setting 2. The resultant suspension is decanted from any unhomogenized residue, diluted with an equal volume of 50 XTIM CaCl* and spun at 120 g for 3 min. The supernatants are discarded and the pellets vigorously suspended in the same volume of a buffer containing 30 mM KHC03, 45 ItIM KCI, 2.5 mM histidine, and 2.5 mM Tris-HCI buffer at pH 7.8. This latter step is repeated three times followed by incubation in an equal volume of buffer at 37 C for 30 min in an oscillating water bath. The suspension is then spun at 270 g for 3 min. The resultant supernatant is decanted and the pellets suspended in an equal volume of 0.1 mM Tris-ATP solution. This suspension is treated with 2 M NaI solution (see details below). Sarcolemma, Fragmented Sarcoplamanic Reticulum, Mitochondria, Myosin B. Sarcolemma ( 12), fragmented sarcoplasmic reticulum ( 14)) mitochondria (13), and myosin B (5), are prepared as described previously. NaI-extracted sarcolemma, NaI-extracted mitochondria and NaIextracted myosin B were prepared by treatment of each of the purified fractions with 2 M NaI.
ATPASE
ACTIVITIES
347
NuI-extracted High-speed Fraction. Two grams of skeletal muscle from the hind legs of rats are used after being dissected free of nerve, fat and connective tissue. The muscle is frozen in liquid nitrogen and stored at -70 C overnight. The frozen muscle is thawed in a small volume of cold medium containing 50 mM TES, 100 mM KCl, 1 mM Na2ATP, 5 mM MgSOa, 1 mM EDTA and 50 mM phosphate buffer at pH 7.4 (low-speed medium). The muscle is finely minced with scissors and placed in homogenizing bottles on ice with a ratio of glass beads (1 mm diameter) to muscle of 7 : 1 (w/v) and a tissue concentration of 18% (w/v). These suspensions are homogenized in COz-cooled mechanical shaker (Bronwill Scientific, Inc., New York) at 4000 cycles/min for 30 set at 2 C. The suspensions are transferred to precooled 50 ml centrifuge tubes and spun at 270 g for 10 min. The residue is discarded and the supernatants spun at 15,000 g for 15 min. The pellet is resuspended in 10 ml high-speed medium (same as low-speed medium except without NazATP; cf. above) and spun again at 15,000 g for 15 min. The supernatants are mixed and spun at 40,000 g for 60 min. The pellets are resuspended in 0.1 mM Tris-ATP. Thus the fraction extracted was obtained between 15,000 g and 40,000 g, under conditions known to yield active fragmented sarcoplasmic reticulum (14). The purified fragmented sarcoplasmic reticulum fraction prepared from frozen muscles is then treated with 2 M NaI. NaI-treatment. Each fraction (muscle homogenate, sarcolemma, fragmented sarcoplasmic reticulum, mitochondria or myosin B) is placed in 10 ml of 0.1 mM Tris-ATP and mixed by gentle stirring at 4 C with an equal volume of a freshly prepared solution containing 4 M NaI, 10 rnM MgC12, 7.6 mM NazATP, 0.2 mM Na,EDTA, pH 7.2, 100 mM cysteine, and 80 mM Tris-HCl at pH 7.2. After 60 min the suspension is diluted with three volumes of double-distilled water to a final concentration of 0.5 M NaI and centrifuged at 25,000 g for 20 min. The supernatant is decanted and the pellets suspended in 0.1 mM Tris-ATP solution. This suspension is spun at 25,000 g for 20 min. This step is repeated one more time. The final pellets from each 25,000 g fraction are then suspended in 0.1 mM Tris-ATP solution followed by gentle homogenization with a teflon-glass homogenizer. ATPase Assays. In all cases, (Na’ + Ii’)-ATPase is measured at 37 C in 1 ml of a medium containing 3 mM Tris-ATP, 20 mM Tris-HCl (pH 7.4), 1 mM MgS04, 60 mM NaCI, and 5 mM KC1 at pH 7.4 to which 100 pg of protein is added. When Mg++-ATPase is measured, 65 mM Tris-HCl (pH 7.4) is substituted for the 60 mM NaCl and 5 mM KCI. The Ca++ATPase assay medium is identical to that of Mg++-ATPase except 1 mM CaClz is substituted for the MgS04. The reaction is terminated with 1 ml of 12% (w/v) trichloroacetic acid, followed by centrifugation and Pi was
6
2
3
1
fraction
mitochondria
Mitochondria
NaI-extracted fraction
Myosin
were
the same in all cases.
conditions
0 Assay
B
1
myosin
f 0.08
0.56
3.01
32.10
0.54
4.22 f
0.11
1.35 zk 0.29
0.54
zk 0.06
2.83 f
0.50
0.60 f
4.99
muscle)
IN FRACTIONS
Yield (mg/protein/g
+ K+)-ATPASE
NaI-extracted fraction
B fraction
3
high
NaI-extracted fraction
speed
7
sarcoplasmic fraction
13
Fragmented reticulum
sarcolemma
fraction
Sarcolemma
4
NaI-extracted fraction
homogenates
NaI-treated
NQ.
(Na+
1
14
74
99f
768
1.52 f
572 f
130 f
240 f
37f
33
10
114
11
22
5
Na+ Pi/min/mg
RAT SKELETAL
Mg ++-ATPase (nmoles
OF FROZEN
TABLE
13
0
29%
0
102 f
1-l f
72f
45f
41f
4
12
6.9
8
6
3
+ K+)-ATPase protein)
MUSCLES
39
0
16
0
138
40
36
27
20.5
Total Na+ + K+-ATPase (nmoles Pi/min/g muscle)
ATPASE
ACTIVITIES
349
determined in the supernatant by the method of Rockstein and Herron (17). In all cases less than 20%’ of the ATP present is hydrolyzed. Unless otherwise indicated (Na’ + K+)-ATPase refers to the ATPase activity (nmoles Pi/min/mg protein j in the presence of Mg++, lia’ and K+ minus that in the presence of Mg” alone. Protein determinations were done by the method of Lowry et al. (9) on the TCA precipitate after overnight solubilization in 0.5 N NaOH at 1 C. C‘(l++ U/&kc. Calcium uptake by fragmented sarcoplasmic reticulum is measured by the method of \Vorsfold and Peter (30). RESULTS The yields and the activities of the ATPases of the various fractions of frozen rat skeletal muscle are listed in Table 1. The fragmented sarcoplasmic reticulum, mitochondria and myosin B fractions had negligible (Na’ + K+)-ATPase activities. The protein yield and the basal Mg++-dependent ATPase activities of the sarcolemma, fragmented sarcoplasmic reticulum, mitochondria, and myosin B fractions were reduced after NaI-treatment and consequently the specific (Na’ + K+)-ATPase activity (see under ATPase Assays) of these fractions was higher than the corresponding fractions prepared according to the methods described by Peter (5, 12-14). The NaI-extracted high-speed fraction contained the highest specific (Na’ + K+)-ATPase activity (102 * 12 nmoles Pi/mg/min * SE) of all preparations. This activity was two to three times as high as that foutid in whole muscle homogenates treated with NaI. However, the NaI-extracted muscle homogenate yielded the highest total activity of all the fractions studied (Table 1). Table 2 shows the effect of NaI on the ATPase activities of the fragmented sarcoplasmic reticulum fraction prepared from fresh and frozen skeletal muscle. The Mg++-ATPase and Mg++-dependent ( Na+ -t K+)stimulated ATPase activities of fragmented sarcoplasmic reticulum from fresh skeletal muscle were slightly higher than those of frozen muscle but Ca++-ATPase was almost the same in both fractions. Treatment with NaI reduced the ATPase activities of both fresh and frozen muscles. The highest (Na’ + K+)-ATPase specific activity was obtained from NaI-extracted high-speed fraction of frozen skeletal muscle and this activity was very reproducible; much lower (Na’ + K+)-ATPase activity was obtained from the NaI-extracted fragmented sarcoplasmic reticulum fraction from fresh muscle. Inhibition of the (Na’ + K’) -ATPase by ouabain has been described as characteristic of the enzyme (29). The effect of ouabain (0.1 KIM) on the (Na+ + Ii+)-ATPase activity of the NaI-extracted high-speed fraction of
3.50
NAGATOMO
AND
PETER
TABLE ATPASE
ACTIVITIES FROM FRESH
OF NAI
NaI
2
OF FKA~~MENTED SARCOPLASMIC AND FROZEN SKELETAL MUSCLE: ON ATPASE
treatment
ACTIVITIES”
Fresh ~
muscle
1.850 1703 1789 86 676
a ATPase results are in nmoles Pi/mg * This is the NaI-extracted high-speed
Frozen -
+
~--_____
Yield of protein (mg/g wet weight) Mg++-ATPase (Mg++ + Na+ + K+)-ATPase (Mg++ + Na+ + I(+)-ATPase minus Mg++-ATPase Ca++-APTase
RETICULUM FRACTION THE EFFECT
muscle +*
0.400 128 162
2.439 623 645
0.307 240 386
31 124
22 594
146 129
protein/min. fraction of frozen
skeletal
muscle.
frozen skeletal muscle is shown in Table 3. The ouabain inhibition of this (Na+ + K+)-ATPase activity was 52% of the total (Na’ + K+)-ATPase activity. Fragmented sarcoplasmic reticulum has the capacity to accumulate Ca++. That this capacity was essentially the same for fractions prepared from both fresh and frozen muscle indicates the presence of intact fragmented sarcoplasmic reticulum vesicles (Figs. 1 and 2). Fragmented sarcoplasmic reticulum prepared from fresh muscle and then stored at either -70 C or 4 C for 24 hr had the same Ca++ accumulating capacity (Fig. 2). The NaIextracted high-speed fraction from either fresh or frozen muscle, on the other hand, showed a very low Cat+ accumulating capacity (Figs. 1 and 2). TABLE ATPASE
3
ACTIVITIES OF THE NAI-EXTRACTED FROM FROZEN RAT SKELETAL
ATPase
Mg++-ATPase (Mg++ + Na+ + K+)-ATPase (Mg++ + Na+ + K+)-ATPase + OuabainO Ca++-ATPase (Ca++ + Mg++)-ATPase (Mg++ + Na+ + K+)-ATPase minus Mg++-ATPase Ouabaina inhibitable (Na+ + K+)-ATPase Ouabaina $?Jeinhibition a Ouabain
concentration
0.1 mM.
HIGH-SPEED MUSCLE
FRACTION
Specific activity (nmoles/min/mg) 152 f 253 f 201 f 145 f 177 f 101 f 52 * 51.5 f
10 19 24 14 11 12 5 6.7
ATPASE
351
ACTIVITIES
TIME
(mnl
FIG. 1. Ca” accumulation by fragmented sarcoplasmic reticulum (FSR) prepared from frozen muscle and then treated or not treated with NaI after storage at 4 C for 24 hr.
DISCUSSION Sodium, potassium-stimulated. nlagnesiutn-depeiIdeIlt, ouabain-sensitive ATPase has been correlated with ATPase-dependent, ouabain-sensitive K+ influx and Na+ efflux across the membrane. Consequently, it has been used as an enzymic marker for muscle membrane (11: 3. 6. 12, 25). The major
l
Frozen
FSR
Fresh FSR A NoI extracted
J-L-. 2
4
1 6
8
10
l?
14
FSR
16
T M E : n. n ;
FIG. 2. Cat+ accumulation by fragmented sarcoplasmic reticulum (FSR) prepared from fresh muscle and then either stored at 4 C [fresh fragmented sarcoplasmic reticulum (FSR)] ; frozen at -70 C [f rozen fragmented sarcoplasmic reticulum (FSR)] for 24 hr; extracted with NaI after 24 hr at 4 C [NaI-extracted fragmented sarcoplasmic reticulum (FSR)].
3.52
KAGATOMO
AND
PETER
obstacles of muscle cell membrane isolation have been the difficulty of solubilization and extraction of the tissue’s high content of fibrillar contractile proteins without membrane disruption. In the present paper, NaI solution was used for the solubilization and extraction of the fibrillar contractile proteins. In each of the fractions studies, extraction with 2 M NaI increased the specific activity of the (Na’ + I(+)-ATPase and reduced the Mg*+-ATPase specific activity. Such treatment also reduced the protein yields of the fractions studied. The mechanism by which NaI enhances the activity of (Na+ + I(+)-ATPase is not clear but the data (Table 1) are consistent with the notion that pretreatment with NaI removed ouabain-insensitive ATPase activity and “extraneous” protein. The experimental results from our work with sarcolemma serve as a case in point. Sarcolemma isolated from rat skeletal muscle contain ouabaininhibitable (Na’ + K’) -ATPase, and this preparation produces relatively pure, readily recognizable sarcolemma as judged by phase contrast microscopy (12). The (N a+ + K+)-ATPase of sarcolemma appears to decrease with multiple washings in 0.01 mM EGTA, despite the fact that the purity of the preparation increase, as judged by the diminution of cytochrome oxidase activity and its phase contrast microscopic appearance (12). When sarcolemma isolated by our standard technique is treated with 2 M NaI, sarcolemmal tubes are no longer recognizable but the specific activity of (Na+ + Ii+)-ATPase is significantly increased owing to decreased protein. The total yield of (Na’ + K+)-ATPase activity remains about the same, suggesting a purification of this activity and not an “unmasking” of another (Na’ + K+) -ATPase (Table 1) . An ATPase was obtained from NaI-extracted high-speed fractions from frozen muscle. The specific activity of this enzyme is high compared to our other preparations and is much less variable (Table 1). Apparently, freezing the whole muscle is crucial to the appearance of (Na’ + K+)ATPase in the NaI-extracted high-speed fraction because identical preparations from fresh muscle show little or no such activity. The fact that the preparations require frozen muscle is not easily explained. Freezing is well known to cause damage to cells. The rate of freezing appears to be directly related to the (Na+ + K+)-ATPase activity obtained in other tissues (16, 27). This relationship has been attributed to alterations in protein-lipid interaction (26), as well as to changes in permeability of the substrates. Mitochondrial and myosin B fractions prepared from frozen muscles had negligible (Na’ + K+)-ATPase activities. After NaI-treatment the protein yield and the basal Mg++-ATPase activities of these fractions were reduced, but the specific activity of the (Na+ + K+)-ATPase in these fractions was significantly increased, and this activity was inhibited 60% by 0.1 mM ouabain. Possibly the increased (Na’ + K’) -ATPase activity
ATPAX
AC’TIVITIES
353
in these Nal-extracted fractions reflects contaminants from the sarcolemmal membrane fraction which remained after NaI-treatment (Table 1). The experimental data presented here provide some clues as to the subcellular origin of the KaI-extracted high-speed fraction. Fragmented sarcoplasmic reticulum is generally prepared from fresh muscle ( 14). However, if frozen muscle is used as the starting material the fragmented sarcoplasmic reticulum \-ekicles apparently remain intact as judged by their mialtered capacity to accumulate Ca++ ( Fig. 1). The method for preparing the NaI-extracted high-speed fraction is identical to that of the fragmented sarcoplasmic reticulum preparation derived from frozen muscle except for the NaI-extraction which destroys its Cat+ accumulating capacity. The NaI-extracted high-speed fraction then clearly contains remnants of fragmented sarcoplasmic reticulum. Although some investigators have shown that (Na’ + K+)-ATPase activity is present in “microsomal” fractions (7, 21)) the majority of workers using various tissues, including skeletal muscle, are of the view that this “transport ATPase” is a constituent of the cell membrane (28). Kogus, Price, and Zierler (18) described a relatively crude preparation of a (Xa’ + K+) -ATPase from rat skeletal muscle and suggested that the (Na’ + E;‘) -ATPase of muscle may reside in the sarcoplasmic reticulum and not in the sarcolemma. However, it is possible that in this impure preparation they have failed to separate the sarcoplasmic reticulum ATPase activity from that of the plasma membrane, the f-system, or both. It is likely that in our NnI-extracted high-speed fraction any ATPase activity is due to the presence of sarcolemmal constituents because (Na’ + K+)-ATPase activity was not found after NaI-extraction of fragmented sarcoplasmic reticulum from nonfrozen skeletal muscle. In addition, however, the results again point out the enormous changes in the composition of various subcellular fractions that can be induced by manipulation (in this case freezing) of the whole tissue. JVhether our results reflect the unmasking of an enzyme activity by freezing or a translocation of activity from one fraction to another is under study. The work reemphasizes the caution necessary in interpreting precise subcellular locations based on study of frozen muscle. These studies form the basis for future efforts designed to obtain an even more pure (Na’ + K+)-ATPase fractions, including those derived from sarcolemma. The technique for preparing the KaI-extracted high-speed fraction from rat skeletal muscle can be usefully applied to the investigation of the human skeletal disorders because : (a) only a small amount of muscle is required in this preparation : (b) the technique may be applied to muscle stored at -70 C, thereby obviating the necessity for doing the experimental work immediately after obtaining fresh tissue; and (c) the preparation technique is straightforward.
354
NAGATOMO
AND
PETEK
REFERENCES 1. ANDREW, C. J., and S. H. APPEL. 1973. Macromolecular characterization of muscle membranes. I. Proteins and sialic acid of normal and denervated muscle. J. Biol. Chm. 248: 5156-5163. 2. AUDITORE, J. V. 1962. Sodium-potassium activated G-strophanthin sensitive ATPase in cardiac muscle. Proc. Sot. Exp. Biol. Med. 110: 595597. 3. BOEGMAN, R. J., J. F. MANERY, and L. PINTERIC. 1970. The separation and partial purification of membrane-bound (Na’ + K’) -dependent Mg++-ATPase and (Na’ + K+)-independent Mg++-ATPase from frog skeletal muscle. Biochim Biophys. Acta 203 : 506-530. 4. BONTING, S. L., L. L. CARAVAGCIO, and N. M. HA\VI
NEUROLOGY
(Na+
49,
+ K+)-ATPase
TAKAFUMI
Newowmcular School
345-355
(1975)
in Subcellular Skeletal Muscle KAGATOMO
AND
JA~VES
Disease Rcsearcll Laboratories. of dfcdicinr, Los A-I~lgrlrs.
Recriwd
January
10, 19i.5’;
rczision
Fractions
B. PETER
of Rat
’
L:nizIersity of California, California 90024 rcc~civcd
Jmr
9, 197.5
Sodium and potassium-stimulated, magnesium-dependent, ouabain-sensitive ATPase has been used as an enzymic marker for muscle membrane (sarcolemma). However, the precise localization of this enzyme is still unknown. The enzyme activity was determined in muscle homogenates, sarcolemma, mitochondria, myosin B, and fragmented sarcoplasmic reticulum fractions isolated from frozen rat skeletal muscle. NaI-extracted fragmented sarcoplasmic reticulum isolated from frozen muscle ( NaI-extracted high-speed fraction) contained a much higher specific (Na+ + K+)-ATPase activity (102 nmoles P,/mg/min) than did a similar fraction isolated from fresh skeletal muscle (34 nmoles P,/mg/min). NaI-treated muscle homogenate, sarcolemma, myosin B, and mitochondria had lower (Na’ i- Ii’) -ATPase specific activity than the NaI-extracted high-speed fraction. The fragmented sarcoplasmic reticulum prepared from fresh or frozen muscle had the same calcium accumulating capacity; this capacity was almost aholished in the fragmented sarcoplasmic reticulum of both fresh and frozen muscle treated with NaI. The methods described may he useful in the study of muscle from humans or experimental animals where the amount of tissue available is limited. The results reemphasize the possihle hazard in determining precise subcellular localization of enzyme activities in frozen tissues, as is a necessarily routine procedure in histochemistry.
IT\‘TRODIJCTION (Na+ + K+) -stimulated, Mg++-dependent ATPase (EC3.6.1.3.) has been found in membrane preparations from various tissues (2, 8, 15, 21-23, 29). 1 This investigation was supported by USPHS Grant NS-07587. Dr. Nagatomo is a Postdoctoral Fellow of the Muscular Dystrophy Associations of America, Inc. The authors would like to thank Dr. Ronald RI. Andiman for his help in the preparation of this manuscript and Kerstin E. Stempel for excellent technical assistance. Reprint requests to James B. Peter, M.D., Ph.D., University of California, School of Medicine, Los Angeles, California 90024.
Copyright All rights
0
1975
by Academic
of reproduction
in any
Press, Inc.
form
reserved.
346
NAGATOMO
AND
PETER
In view of the critical role of cation transport in skeletal muscle function, the role of a “cation transport ATPase” characterized by Skou (24) is likely to be an important one. Several investigators have reported such a (Na’ + K+)-ATPase in muscle membrane preparations (1, 3, 6, 12, 25). The precise localization of this enzyme, however, is still unproved because the plasma membrane is not easily separated from other organelles. (Na’ + K+)-ATP ase activity can become manifest by treatment with detergents like sodium deoxycholate (DOC) (4, 19, 20) and high concentrations of salts such as sodium iodide (10, 11) and lithium bromide (1, 3, 25). Nakao et al. (10, 11) have prepared a (Na’ + K+) -ATPase of high specific activity from brain microsomes by treatment with 2 M NaI. In this study we have used a modification of Nakao’s method to produce a NaI-extracted high-speed fraction of frozen skeletal muscle which has a (Na+ + K+)-ATPase of higher specific activity than that of similarly treated whole rat skeletal muscle homogenate, sarcolemma, mitochondria, or myosin B. METHODS NaI-extracted Muscle Homogenate. Typically about l-2 g of fresh muscle, cleaned of connective tissue, fat, and nerve are frozen in liquid nitrogen, stored overnight at -70 C and sectioned transversely with a razor blade to about 2 mm thickness. The muscle slices are then suspended in ice-cold 50 mM CaCI, at 25% (w/v). The suspension is homogenized twice for 30 set each in Polytron homogenizer (Kinematica BMBH. Luzern-Schweiz, Switzerland) at setting 2. The resultant suspension is decanted from any unhomogenized residue, diluted with an equal volume of 50 XTIM CaCl* and spun at 120 g for 3 min. The supernatants are discarded and the pellets vigorously suspended in the same volume of a buffer containing 30 mM KHC03, 45 ItIM KCI, 2.5 mM histidine, and 2.5 mM Tris-HCI buffer at pH 7.8. This latter step is repeated three times followed by incubation in an equal volume of buffer at 37 C for 30 min in an oscillating water bath. The suspension is then spun at 270 g for 3 min. The resultant supernatant is decanted and the pellets suspended in an equal volume of 0.1 mM Tris-ATP solution. This suspension is treated with 2 M NaI solution (see details below). Sarcolemma, Fragmented Sarcoplamanic Reticulum, Mitochondria, Myosin B. Sarcolemma ( 12), fragmented sarcoplasmic reticulum ( 14)) mitochondria (13), and myosin B (5), are prepared as described previously. NaI-extracted sarcolemma, NaI-extracted mitochondria and NaIextracted myosin B were prepared by treatment of each of the purified fractions with 2 M NaI.
ATPASE
ACTIVITIES
347
NuI-extracted High-speed Fraction. Two grams of skeletal muscle from the hind legs of rats are used after being dissected free of nerve, fat and connective tissue. The muscle is frozen in liquid nitrogen and stored at -70 C overnight. The frozen muscle is thawed in a small volume of cold medium containing 50 mM TES, 100 mM KCl, 1 mM Na2ATP, 5 mM MgSOa, 1 mM EDTA and 50 mM phosphate buffer at pH 7.4 (low-speed medium). The muscle is finely minced with scissors and placed in homogenizing bottles on ice with a ratio of glass beads (1 mm diameter) to muscle of 7 : 1 (w/v) and a tissue concentration of 18% (w/v). These suspensions are homogenized in COz-cooled mechanical shaker (Bronwill Scientific, Inc., New York) at 4000 cycles/min for 30 set at 2 C. The suspensions are transferred to precooled 50 ml centrifuge tubes and spun at 270 g for 10 min. The residue is discarded and the supernatants spun at 15,000 g for 15 min. The pellet is resuspended in 10 ml high-speed medium (same as low-speed medium except without NazATP; cf. above) and spun again at 15,000 g for 15 min. The supernatants are mixed and spun at 40,000 g for 60 min. The pellets are resuspended in 0.1 mM Tris-ATP. Thus the fraction extracted was obtained between 15,000 g and 40,000 g, under conditions known to yield active fragmented sarcoplasmic reticulum (14). The purified fragmented sarcoplasmic reticulum fraction prepared from frozen muscles is then treated with 2 M NaI. NaI-treatment. Each fraction (muscle homogenate, sarcolemma, fragmented sarcoplasmic reticulum, mitochondria or myosin B) is placed in 10 ml of 0.1 mM Tris-ATP and mixed by gentle stirring at 4 C with an equal volume of a freshly prepared solution containing 4 M NaI, 10 rnM MgC12, 7.6 mM NazATP, 0.2 mM Na,EDTA, pH 7.2, 100 mM cysteine, and 80 mM Tris-HCl at pH 7.2. After 60 min the suspension is diluted with three volumes of double-distilled water to a final concentration of 0.5 M NaI and centrifuged at 25,000 g for 20 min. The supernatant is decanted and the pellets suspended in 0.1 mM Tris-ATP solution. This suspension is spun at 25,000 g for 20 min. This step is repeated one more time. The final pellets from each 25,000 g fraction are then suspended in 0.1 mM Tris-ATP solution followed by gentle homogenization with a teflon-glass homogenizer. ATPase Assays. In all cases, (Na’ + Ii’)-ATPase is measured at 37 C in 1 ml of a medium containing 3 mM Tris-ATP, 20 mM Tris-HCl (pH 7.4), 1 mM MgS04, 60 mM NaCI, and 5 mM KC1 at pH 7.4 to which 100 pg of protein is added. When Mg++-ATPase is measured, 65 mM Tris-HCl (pH 7.4) is substituted for the 60 mM NaCl and 5 mM KCI. The Ca++ATPase assay medium is identical to that of Mg++-ATPase except 1 mM CaClz is substituted for the MgS04. The reaction is terminated with 1 ml of 12% (w/v) trichloroacetic acid, followed by centrifugation and Pi was
6
2
3
1
fraction
mitochondria
Mitochondria
NaI-extracted fraction
Myosin
were
the same in all cases.
conditions
0 Assay
B
1
myosin
f 0.08
0.56
3.01
32.10
0.54
4.22 f
0.11
1.35 zk 0.29
0.54
zk 0.06
2.83 f
0.50
0.60 f
4.99
muscle)
IN FRACTIONS
Yield (mg/protein/g
+ K+)-ATPASE
NaI-extracted fraction
B fraction
3
high
NaI-extracted fraction
speed
7
sarcoplasmic fraction
13
Fragmented reticulum
sarcolemma
fraction
Sarcolemma
4
NaI-extracted fraction
homogenates
NaI-treated
NQ.
(Na+
1
14
74
99f
768
1.52 f
572 f
130 f
240 f
37f
33
10
114
11
22
5
Na+ Pi/min/mg
RAT SKELETAL
Mg ++-ATPase (nmoles
OF FROZEN
TABLE
13
0
29%
0
102 f
1-l f
72f
45f
41f
4
12
6.9
8
6
3
+ K+)-ATPase protein)
MUSCLES
39
0
16
0
138
40
36
27
20.5
Total Na+ + K+-ATPase (nmoles Pi/min/g muscle)
ATPASE
ACTIVITIES
349
determined in the supernatant by the method of Rockstein and Herron (17). In all cases less than 20%’ of the ATP present is hydrolyzed. Unless otherwise indicated (Na’ + K+)-ATPase refers to the ATPase activity (nmoles Pi/min/mg protein j in the presence of Mg++, lia’ and K+ minus that in the presence of Mg” alone. Protein determinations were done by the method of Lowry et al. (9) on the TCA precipitate after overnight solubilization in 0.5 N NaOH at 1 C. C‘(l++ U/&kc. Calcium uptake by fragmented sarcoplasmic reticulum is measured by the method of \Vorsfold and Peter (30). RESULTS The yields and the activities of the ATPases of the various fractions of frozen rat skeletal muscle are listed in Table 1. The fragmented sarcoplasmic reticulum, mitochondria and myosin B fractions had negligible (Na’ + K+)-ATPase activities. The protein yield and the basal Mg++-dependent ATPase activities of the sarcolemma, fragmented sarcoplasmic reticulum, mitochondria, and myosin B fractions were reduced after NaI-treatment and consequently the specific (Na’ + K+)-ATPase activity (see under ATPase Assays) of these fractions was higher than the corresponding fractions prepared according to the methods described by Peter (5, 12-14). The NaI-extracted high-speed fraction contained the highest specific (Na’ + K+)-ATPase activity (102 * 12 nmoles Pi/mg/min * SE) of all preparations. This activity was two to three times as high as that foutid in whole muscle homogenates treated with NaI. However, the NaI-extracted muscle homogenate yielded the highest total activity of all the fractions studied (Table 1). Table 2 shows the effect of NaI on the ATPase activities of the fragmented sarcoplasmic reticulum fraction prepared from fresh and frozen skeletal muscle. The Mg++-ATPase and Mg++-dependent ( Na+ -t K+)stimulated ATPase activities of fragmented sarcoplasmic reticulum from fresh skeletal muscle were slightly higher than those of frozen muscle but Ca++-ATPase was almost the same in both fractions. Treatment with NaI reduced the ATPase activities of both fresh and frozen muscles. The highest (Na’ + K+)-ATPase specific activity was obtained from NaI-extracted high-speed fraction of frozen skeletal muscle and this activity was very reproducible; much lower (Na’ + K+)-ATPase activity was obtained from the NaI-extracted fragmented sarcoplasmic reticulum fraction from fresh muscle. Inhibition of the (Na’ + K’) -ATPase by ouabain has been described as characteristic of the enzyme (29). The effect of ouabain (0.1 KIM) on the (Na+ + Ii+)-ATPase activity of the NaI-extracted high-speed fraction of
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TABLE ATPASE
ACTIVITIES FROM FRESH
OF NAI
NaI
2
OF FKA~~MENTED SARCOPLASMIC AND FROZEN SKELETAL MUSCLE: ON ATPASE
treatment
ACTIVITIES”
Fresh ~
muscle
1.850 1703 1789 86 676
a ATPase results are in nmoles Pi/mg * This is the NaI-extracted high-speed
Frozen -
+
~--_____
Yield of protein (mg/g wet weight) Mg++-ATPase (Mg++ + Na+ + K+)-ATPase (Mg++ + Na+ + I(+)-ATPase minus Mg++-ATPase Ca++-APTase
RETICULUM FRACTION THE EFFECT
muscle +*
0.400 128 162
2.439 623 645
0.307 240 386
31 124
22 594
146 129
protein/min. fraction of frozen
skeletal
muscle.
frozen skeletal muscle is shown in Table 3. The ouabain inhibition of this (Na+ + K+)-ATPase activity was 52% of the total (Na’ + K+)-ATPase activity. Fragmented sarcoplasmic reticulum has the capacity to accumulate Ca++. That this capacity was essentially the same for fractions prepared from both fresh and frozen muscle indicates the presence of intact fragmented sarcoplasmic reticulum vesicles (Figs. 1 and 2). Fragmented sarcoplasmic reticulum prepared from fresh muscle and then stored at either -70 C or 4 C for 24 hr had the same Ca++ accumulating capacity (Fig. 2). The NaIextracted high-speed fraction from either fresh or frozen muscle, on the other hand, showed a very low Cat+ accumulating capacity (Figs. 1 and 2). TABLE ATPASE
3
ACTIVITIES OF THE NAI-EXTRACTED FROM FROZEN RAT SKELETAL
ATPase
Mg++-ATPase (Mg++ + Na+ + K+)-ATPase (Mg++ + Na+ + K+)-ATPase + OuabainO Ca++-ATPase (Ca++ + Mg++)-ATPase (Mg++ + Na+ + K+)-ATPase minus Mg++-ATPase Ouabaina inhibitable (Na+ + K+)-ATPase Ouabaina $?Jeinhibition a Ouabain
concentration
0.1 mM.
HIGH-SPEED MUSCLE
FRACTION
Specific activity (nmoles/min/mg) 152 f 253 f 201 f 145 f 177 f 101 f 52 * 51.5 f
10 19 24 14 11 12 5 6.7
ATPASE
351
ACTIVITIES
TIME
(mnl
FIG. 1. Ca” accumulation by fragmented sarcoplasmic reticulum (FSR) prepared from frozen muscle and then treated or not treated with NaI after storage at 4 C for 24 hr.
DISCUSSION Sodium, potassium-stimulated. nlagnesiutn-depeiIdeIlt, ouabain-sensitive ATPase has been correlated with ATPase-dependent, ouabain-sensitive K+ influx and Na+ efflux across the membrane. Consequently, it has been used as an enzymic marker for muscle membrane (11: 3. 6. 12, 25). The major
l
Frozen
FSR
Fresh FSR A NoI extracted
J-L-. 2
4
1 6
8
10
l?
14
FSR
16
T M E : n. n ;
FIG. 2. Cat+ accumulation by fragmented sarcoplasmic reticulum (FSR) prepared from fresh muscle and then either stored at 4 C [fresh fragmented sarcoplasmic reticulum (FSR)] ; frozen at -70 C [f rozen fragmented sarcoplasmic reticulum (FSR)] for 24 hr; extracted with NaI after 24 hr at 4 C [NaI-extracted fragmented sarcoplasmic reticulum (FSR)].
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obstacles of muscle cell membrane isolation have been the difficulty of solubilization and extraction of the tissue’s high content of fibrillar contractile proteins without membrane disruption. In the present paper, NaI solution was used for the solubilization and extraction of the fibrillar contractile proteins. In each of the fractions studies, extraction with 2 M NaI increased the specific activity of the (Na’ + I(+)-ATPase and reduced the Mg*+-ATPase specific activity. Such treatment also reduced the protein yields of the fractions studied. The mechanism by which NaI enhances the activity of (Na+ + I(+)-ATPase is not clear but the data (Table 1) are consistent with the notion that pretreatment with NaI removed ouabain-insensitive ATPase activity and “extraneous” protein. The experimental results from our work with sarcolemma serve as a case in point. Sarcolemma isolated from rat skeletal muscle contain ouabaininhibitable (Na’ + K’) -ATPase, and this preparation produces relatively pure, readily recognizable sarcolemma as judged by phase contrast microscopy (12). The (N a+ + K+)-ATPase of sarcolemma appears to decrease with multiple washings in 0.01 mM EGTA, despite the fact that the purity of the preparation increase, as judged by the diminution of cytochrome oxidase activity and its phase contrast microscopic appearance (12). When sarcolemma isolated by our standard technique is treated with 2 M NaI, sarcolemmal tubes are no longer recognizable but the specific activity of (Na+ + Ii+)-ATPase is significantly increased owing to decreased protein. The total yield of (Na’ + K+)-ATPase activity remains about the same, suggesting a purification of this activity and not an “unmasking” of another (Na’ + K+) -ATPase (Table 1) . An ATPase was obtained from NaI-extracted high-speed fractions from frozen muscle. The specific activity of this enzyme is high compared to our other preparations and is much less variable (Table 1). Apparently, freezing the whole muscle is crucial to the appearance of (Na’ + K+)ATPase in the NaI-extracted high-speed fraction because identical preparations from fresh muscle show little or no such activity. The fact that the preparations require frozen muscle is not easily explained. Freezing is well known to cause damage to cells. The rate of freezing appears to be directly related to the (Na+ + K+)-ATPase activity obtained in other tissues (16, 27). This relationship has been attributed to alterations in protein-lipid interaction (26), as well as to changes in permeability of the substrates. Mitochondrial and myosin B fractions prepared from frozen muscles had negligible (Na’ + K+)-ATPase activities. After NaI-treatment the protein yield and the basal Mg++-ATPase activities of these fractions were reduced, but the specific activity of the (Na+ + K+)-ATPase in these fractions was significantly increased, and this activity was inhibited 60% by 0.1 mM ouabain. Possibly the increased (Na’ + K’) -ATPase activity
ATPAX
AC’TIVITIES
353
in these Nal-extracted fractions reflects contaminants from the sarcolemmal membrane fraction which remained after NaI-treatment (Table 1). The experimental data presented here provide some clues as to the subcellular origin of the KaI-extracted high-speed fraction. Fragmented sarcoplasmic reticulum is generally prepared from fresh muscle ( 14). However, if frozen muscle is used as the starting material the fragmented sarcoplasmic reticulum \-ekicles apparently remain intact as judged by their mialtered capacity to accumulate Ca++ ( Fig. 1). The method for preparing the NaI-extracted high-speed fraction is identical to that of the fragmented sarcoplasmic reticulum preparation derived from frozen muscle except for the NaI-extraction which destroys its Cat+ accumulating capacity. The NaI-extracted high-speed fraction then clearly contains remnants of fragmented sarcoplasmic reticulum. Although some investigators have shown that (Na’ + K+)-ATPase activity is present in “microsomal” fractions (7, 21)) the majority of workers using various tissues, including skeletal muscle, are of the view that this “transport ATPase” is a constituent of the cell membrane (28). Kogus, Price, and Zierler (18) described a relatively crude preparation of a (Xa’ + K+) -ATPase from rat skeletal muscle and suggested that the (Na’ + E;‘) -ATPase of muscle may reside in the sarcoplasmic reticulum and not in the sarcolemma. However, it is possible that in this impure preparation they have failed to separate the sarcoplasmic reticulum ATPase activity from that of the plasma membrane, the f-system, or both. It is likely that in our NnI-extracted high-speed fraction any ATPase activity is due to the presence of sarcolemmal constituents because (Na’ + K+)-ATPase activity was not found after NaI-extraction of fragmented sarcoplasmic reticulum from nonfrozen skeletal muscle. In addition, however, the results again point out the enormous changes in the composition of various subcellular fractions that can be induced by manipulation (in this case freezing) of the whole tissue. JVhether our results reflect the unmasking of an enzyme activity by freezing or a translocation of activity from one fraction to another is under study. The work reemphasizes the caution necessary in interpreting precise subcellular locations based on study of frozen muscle. These studies form the basis for future efforts designed to obtain an even more pure (Na’ + K+)-ATPase fractions, including those derived from sarcolemma. The technique for preparing the KaI-extracted high-speed fraction from rat skeletal muscle can be usefully applied to the investigation of the human skeletal disorders because : (a) only a small amount of muscle is required in this preparation : (b) the technique may be applied to muscle stored at -70 C, thereby obviating the necessity for doing the experimental work immediately after obtaining fresh tissue; and (c) the preparation technique is straightforward.
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REFERENCES 1. ANDREW, C. J., and S. H. APPEL. 1973. Macromolecular characterization of muscle membranes. I. Proteins and sialic acid of normal and denervated muscle. J. Biol. Chm. 248: 5156-5163. 2. AUDITORE, J. V. 1962. Sodium-potassium activated G-strophanthin sensitive ATPase in cardiac muscle. Proc. Sot. Exp. Biol. Med. 110: 595597. 3. BOEGMAN, R. J., J. F. MANERY, and L. PINTERIC. 1970. The separation and partial purification of membrane-bound (Na’ + K’) -dependent Mg++-ATPase and (Na’ + K+)-independent Mg++-ATPase from frog skeletal muscle. Biochim Biophys. Acta 203 : 506-530. 4. BONTING, S. L., L. L. CARAVAGCIO, and N. M. HA\VI
