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Calcium transport and phosphoenzyme formation in sarcoplasmic reticulum isolated from normal and dystrophic chickens
Calcium
Transport and Phosphoenzyme Formation Sarcoplasmic Reticulum isolated from Normal and Dystrophic Chickens STEPHEN
D. HANNA
Received
AND RONALD
November
2.
in
J. BASKIN
1976
INTRODUCTION The presence of an extensive membrane system in muscle cells, the sarcoplasmic reticulum (SR), has been known since the ultrastructural investigations of Porter and Palade (I), who observed the presence of a modified endoplasmic reticulum in muscle cells. According to current models of muscle contraction, this modified endoplasmic reticulum plays a major role in the regulation of contraction by releasing calcium ions to initiate contraction and sequestering calcium at the expense of ATP to cause muscle relaxation. Isolated SR is capable of calcium transport in the presence or absence of divalent anions, and these processes have become identified in the literature as calcium uptake and calcium binding, respectively. However. the term calcium binding is technically incorrect when used in this context, because binding refers to passive electrostatic binding and not to an active transport process. For the present work, the term “calcium transport’ ’ will be used in place of “calcium binding,” and the term “precipitant-anion calcium transport” will be used in place of “calcium uptake.” The properties of dystrophic SR have been investigated in a number of dystrophic animals, namely, humans, mice, hamster, and chickens. Transmission electon microscopy has revealed that the SR in most dystrophies is often swollen in appearance (2, 3). The dystrophic chicken is a suitable animal for examining biochemical abnormalities in muscular dystrophy for the following reasons: (i) The dystrophic animals can breed, allowing unequivocal identification of homozygous dystrophic animals; and (ii) the pectoralis muscles are drastically affected by the disease process. 300 Copyright ,411 right\
@ lY77 hy Academic Pre\\. Inc. of reproduction m any form rcwrved
I SSN W.K-1444
DYSTROPHIC
SARCOPLASMIC
301
RETICULUM
Hsu and Kaldor studied SR from normal and dystrophic chickens (4,5). They found a reduced precipitant-anion calcium transport ability in dystrophic SR and also an increased susceptibility of dystrophic SR to phospholipase c digestion (4). They also found that dystrophic SR has a higher 1ipid:protein ratio than does normal chicken SR (5). In 1970, Baskin (2) found an increased yield of SR from 6 to &week dystrophic chicken muscle and a reduction in dystrophic membrane-bound creatine phosphokinase activity. However, he found no difference in the amount of calcium accumulated or ATP hydrolyzed after 10 min of incubation. In 1973, Sylvester and Baskin (6) examined the kinetics of precipitant-anion calcium transport by normal and dystrophic 6- to 8week chickens and, in sharp contrast to other animal models, they found that the rate of transport was significantly increased in the dystrophic SR. The work presented here was stimulated by the finding that dystrophic chicken SR exhibits a higher rate of precipitant-anion calcium transport than does normal chicken SR (6), a result that is in contrast to results found in other dystrophic animal species. The present work consists of the sucrose density-gradient purification of normal and dystrophic SR, and the biochemical characterization of the purified SR.’ A portion of this work was presented at the APS meetings in 1975 (9). MATERIALS AND METHODS SR Isolation Normal (line 412) and dystrophic (line 413) chickens were obtained from the Department of Avian Sciences, University of California at Davis. The animals were from 6 to 8 weeks of age; a stage of pectoralis muscle hypertrophy in the dystrophic animals. The animals were killed by cervical dislocation, the pectoralis muscles were excised, and the SR was prepared according to Baskin (IO). The SR was purified further by ultracentrifugation at 51 ,OOOg for 90 min on a 20/30% discontinuous sucrose gradient, using a Beckman Model L ultracentrifuge. The following fractions were obtained: fraction 1, did not enter the 20% layer: fraction 2, between the 20 and 30% layers; and fraction 3, pellet. Protein was determined by a modification of the Lowry procedure (1 I), and all biochemical assays were performed within 24 hr of isolation. Biochemical
Assays
The ATPase activity of the SR preparations was measured in the absence of oxalate according to Warren et al. (12). All measurements were made at pH 7.0 and 23”, and the protein concentration was 8-16 pg ’ Since this investigation been published (7. 8).
was begun,
two reports
dealing
with dystrophic
chicken
SR have
302
HANNA
AND
BASKliL
of protein/ml. Measurements were made in the presence of 0. I mM EGTA (basal) and 0.1 mM CaCl, plus 0. I mM EGTA (total). The calciumsensitive ATPase activity was determined by subtraction of the basal ATPase activity from the total activity. Precipitant-anion calcium transport was measured using established methods ( IO), at a protein concentration of 0.03 mgiml. Calcium transport was measured in the presence of an ATP-regenerating system consisting of 1.5 IU of pyruvate kinaseiml and 4 mM PEP, in order to maintain a constant ATP concentration and prevent ADP accumulation. Protein concentration was 0.2 mg/ml. and samples were taken after I min of incubation. Phosphoprotein formation was measured at 4”, according to Panet and Selinger ( 13). The reaction was initiated by the addition of 32ATP and terminated after 30 sec. Polyacrylamide gels were prepared according to Fairbanks et (11. ( 14) and were run on a slab gel apparatus (Aquebogue Machine and Repair Shop, Aquebogue, N.Y.). Chemicals were obtained from Sigma Chemical Co., St. Louis, MO.
RESULTS Dystrophic muscle yields more SR protein per gram of tissue than a comparably aged normal muscle (Table 1). This increase in SR yield may be explained in one of three ways: (i) Dystrophic muscle may contain more SR per gram of muscle than does normal muscle. SR in dystrophic chicken pectoralis muscle has previously been described as enlarged or swollen (2, 3). (ii) Due to an altered structure of dystrophic muscle, the homogenization procedure may extract a greater percentage of the total SR from dystrophic muscle than from normal. (iii) The dystrophic SR pellet may contain more nonSR membrane contaminants than the normal SR pellet. TABLE YIELD Yield
I DATA
Normal
f Test
Dystrophic
Milligrams of SR per gram of muscle Fraction Iu
0.295 0.012
t f
0.024 0.002
0.446 0.029
+ 0.026 f 0.004
/J i 0.001 p < 0.01
Fraction Fraction
0.219 0.165
t 0.014 k 0.028
0.197 0.166
2 0.014 + 0.018
n.s.d.: n.s.d.
2 3
” The figures for fractions I. 2. and 3 are milligrams of SR applied to sucrose gradient. L. n.s.d., not significantly different.
in
the
units
of
milligrams
of
protein/total
DYSTROPHIC
SARCOPLASMIC
RETICULUM
When the SR was separated into different fractions on gradient, the total protein in each fraction was assayed. indicates, a difference in protein distribution occurs in fraction measurable difference occurs in fractions 2 or 3. (Fraction accounts for less than 10% of the total protein recovered.) Polyacrylamide
303
the sucrose As Table 1 I, while no 1, however.
Gels
The major protein observed on polyacrylamide gels is the 106,000dalton ATPase protein. As Fig. 1 illustrates, fraction 2 contains the most ATPase protein of all the sucrose fractions. This observation, plus the preliminary finding that fraction 2 contains the greatest calcium-sensitive ATPase activity, identified fraction 2 as the purified SR fraction. (Fraction 1 contains little ATPase protein and may correspond to fraction 1 of Bat-ret and Headon (15), found in density-gradient purified rabbit SR). Normal and dystrophic fraction 2 do not appear to differ significantly in their appearance on polyacrylamide gels. Purijica tion
Early in the course of this work, it was observed that both normal and dystrophic SR contained a high level of basal ATPase activity (in the presence of 0.1 mM EGTA, no CaCl,). One reason for attempting the
FIG. I. Polyacrylamide gels of sarcoplasmic reticulum fractionated on sucrose gradient. NSR is SR before gradient fractionation; N-l, N-2, and N-3 refer to the fractions described in Methods. N refers to normal chicken: D refers to dystrophic chicken. The arrow indicates the location of the 106,000-dalton ATPase protein. Ten micrograms of protein were applied to each gel.
purification on the sucrose density gradient was to see if this basal ATPase activity could be eliminated by eliminating nonSR membrane contaminants. The two pure SR preparations could then be compared with one another. Table 2 represents the results obtained by this procedure. Samples were assayed for total ATPase activity (+ 0. I mM CaCl, and 0.1 mM EGTA), basal ATPase activity (+ 0. I mM EGTA). maximal calcium-sensitive ATPase activity (+ 0.1 mM CaCl, and 0.1% Triton X-100), degree of mitochondrial contamination (+ 10 mM sodium azide). and phosphoenzyme levels. As indicated in Table 2, the basal ATPase level is still elevated in fraction 2, and is not significantly different from the basal ATPase level observed in the original SR. This may be interpreted in one of two ways: Either (i) the basal ATPase level is the result of a contaminating membrane fraction, which cannot be separated by the techniques used, or (ii) the basal ATPase level is a property of purified SR membranes from chicken skeletal muscle, having an unknown function. The total ATPase activity is increased in fraction 2, although this increase is not significantly different for dystrophic membranes. More importantly, the calcium-sensitive ATPase activity is increased in both normal and dystrophic fraction 2. This observation, plus the previous observation that the basal ATPase level is unchanged in fraction 2, indicates that the sucrose-gradient separation eliminates some contaminating protein from the SR fraction. The addition of 10 mM sodium azide causes a reduction of basal ATPase activities within all four fractions. Even though the ATPase levels are reduced, the azide ATPase activities exhibit the same trend as the ATPase activities without azide; total ATPase activity is significantly increased in fraction 2, while basal ATPase activity is in essence unchanged in fraction 2. The most important property, the calcium-sensitive ATPase activity, remains unchanged in all fractions in the presence of azide. The azide concentration is the same as that used by Katz and Repke (16) and Solar0 and Briggs ( 17), who were measuring the degree of mitochondrial contamination of cardiac SR preparations. The curious property of azide of reducing total and basal ATPase activity, while leaving calcium sensitivity unaffected, was noted previously on cardiac SR by Katz and Repke ( 16). ATPuse
Activity,
Frcrction
2
A comparison of the ATPase activity of normal versus dystrophic fraction 2 is shown in Table 3. The basal ATPase activity, as mentioned previously, is present at a relatively high level, and the basal ATPase activity is significantly higher in normal fraction 2. Not only is the basal ATPase activity higher in normal, but the total
DYSTROPHK
SARCOPLASMlC
RETlCULUM
305
306
HANN.4
.ANI)
BASKlic
I 4BI.l-. /+i( I I\‘1 I \
01
: f’K.%(‘I
ION
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--..-..~
Conditions Total (t Basal
Normal ATPase
3.36
0.1 mM CaCI,) ATPase
I + 0. I mM
Dyxtr-ophic
-in 0.44
: Ie41
I.72
+- 0.17
I’
0.01
2. I? _i- 0.2 I
I .7h
t- 0.09
,’
O.OI
EGTA) 2.62
-+ 0.18
2.14
i
+ 0. IQ triton Calcium-sensitive
1.23
-+ 0.29
0.47
2 0.11
p < 0.05
ATPase (total-basal) Precipitant-anion
0.28
?z 0.12
0.94
2 0.18
,I c: 0.05
0. I rnM
CaCl*
0.15
n.s.d.‘
calcium transport (~mimgimin) Calcium
transport
0.039
(I*molimg) Phosphorylated
3.26
2 0.005 f
0.44
0.037 1.45
? 0.004 2 0.27
n.s.d. p c: 0.01
intermediate (nmoleimg) : n.s.d..
not
significantly
different.
ATPase activity is significantly higher as well. This is reflected in the fact that the calcium-sensitive ATPase activity is significantly higher in normal fraction 2 than in dystrophic. This result suggests either that the normal preparation is substantially purified relative to the dystrophic, or that the normal ATPase protein is able to function at a higher rate than the dystrophic ATPase protein. The Triton X-loo-treated fraction 2 samples show no significant difference from one another. This indicates that perhaps the dystrophic ATPase protein has the same potential for ATP hydrolysis when solubilized, but is more restricted by its membrane environment than is the normal ATPase protein. Precipitant-Anion Calcium Transport, Calcium Transport. and Phosphoen~ytne Formation In order to fully characterize fraction 2, its capabilities for calcium transport, precipitant-anion calcium transport, and phosphoenzyme formation were measured (Table 3). The precipitant-anion calcium transport rate of dystrophic fraction 2 is greater than that of normal fraction 2. This indicates that the results of Sylvester and Baskin (6) were not merely due to contaminating membranes. However, no difference exists in the steady-state calcium transport ability of the two types of purified SR. These steady-state calcium transport values are relatively low when compared to skeletal SR from
DYSTROPHIC SARCOPLASMIC RETICULUM
307
other sources, which typically exhibit calcium-binding values between 0.1 and 0.2 pmol/mg of SR protein. In contrast to precipitant-anion calcium transport and calcium transport, the phosphorylated intermediate level of normal fraction 2 is significantly elevated over that of dystrophic fraction 3L. The overall picture from biochemical data is a complex one, with dystrophic fraction 2 having an increased precipitant-anion calcium transport rate, the same calcium transport steady-state level, a decreased calcium-sensitive ATPase rate, a decreased level of phosphoprotein formation, and an increased calcium-transported/ATP-hydrolyzed ratio relative to normal fraction 2. DISCUSSION The Basal ATPase
Acti\vity
The basal ATPase activi.ty observed in chicken SR is higher than the basal ATPase found in mammalian SR ( 18). The basal ATPase may be a Na+,K+-ATPase, since this is a widespread membrane-bound ATPase, and may be either a constituent of SR membranes or a membrane contaminant. The elimination of the basal ATPase activity by Triton X-100 solubilization supports the idea of a Na+,K+-ATPase, because Na+,K+ATPases are typically detergent labile (19). The question of the basal ATPase being’a contaminant or an endogenous SR ATPase is difficult to answer; evidence exists which supports both hypotheses. Forbes and Sperelakis (20) found that a Na+,K+-ATPase was present in skeletal muscle SR, observed by cytochemical techniques, which supports the idea of the basal ATPase being an endogenous SR ATPase. However, Schapira et al. (21) found that some plasma membrane was associated with mammalian SR even after sucrose density-gradient centrifugation, which supports the idea of the basal ATPase activity being a contaminant. Therefore, using available data, one cannot decide whether the basal ATPase activity is an endogenous SR ATPase, a contaminant, or a combination of the two. Calcium
Transport
Steady-state calcium transport levels of normal and dystrophic SR are similar, however, freeze-fracture electron micrographs suggest that dystrophic membranes have a greater surface area and vesicular volume per milligram of SR protein than do normal membranes. Steady-state levels of active transport may depend upon the concentration gradient; dystrophic membranes, which have established equivalent steady-state levels with normal membranes (per milligram of protein) may have actually developed less of a concentration gradient. Dystrophic membranes may have transported the same net amount of calcium as normal membranes,
308
HL\NNA
AND
RASkiK
but into a larger intravesicular volume. Under these circumstances, normal SR would transport more calcium per unit of membrane surface area. The calcium transport results differ from those of Sabbadini ef ul. (7) who found a decreased rate and steady-state level of calcium transport in dystrophic chicken SR. The reason for this discrepancy is not known, although it could be due to differences in the method of SR preparation. The steady-state levels reported by Yap and MacLennan (8) are relatively high. It is possible, however, that due to their IO-min incubation time liberated phosphate served to allow precipitant-anion transport. The reduction we observed in calcium-sensitive ATPase activity in dystrophic SR is also in contrast to the results of Sabbadini rt al. (71. However, the assay medium used by these investigators contained Triton X-100 and, as Table 3 shows. we also find no difference in ATPase activities in the presence of Triton X-100. Phosphoenzyme levels have never before been measured in dystrophic SR, so it is significant that dystrophic chicken SR exhibits a reduced level of phosphoenzyme formation. This indicates that dystrophic SR has either a reduced functional ability or a lesser degree of purification. The phosphoenzyme levels, the calcium-sensitive ATPase activity, and the steady-state calcium transport levels indicate that dystrophic SR has a decreased enzymatic activity. The precipitant-anion calcium transport rates do not correlate well with the rest of the data, and it may be that in chicken SR precipitant-anion transport is not a representative measure of calcium transport capability. The entry of precipitant anions into SR vesicles is a complex process not sufficiently explained by diffusion (221, and alterations in anion permeability could cause misleading results. If the alterations in dystrophic SR do represent in r~vo alterations, then these alterations could explain previously observed mechanical alterations in dystrophic muscle (23, 24). A reduced rate of calcium transport and/or an altered calcium release mechanism could explain the reduced tetanus tension, increased relaxation time, and reduced intensity and duration of the active state observed in dystrophic mouse muscle. The alterations found in dystrophic chicken SR could be due to one of four possibilities: (i) The arrangement of lipid and protein in the membrane is altered, causing an altered enzymatic function. (ii) The lipid composition of the membrane is altered. (iii) The ATPase protein is altered. (iv) Both the lipid component and the ATPase protein are altered. Studies on items (ii) and (iii) have been performed in the past (4, 3, but both the SR preparation techniques and the ages of the animals were different from the present investigation, precluding direct comparison.
DYSTROPHIC
SARCOPLASMIC
RETICULUM
309
SUMMARY Sarcoplasmic reticulum from normal and dystrophic pectoralis muscle was isolated and purified using a discontinuous sucrose gradient. Puritied SR from both normal and dystrophic sources showed an increased calcium-sensitive ATPase activity relative to unpurified SR. Calciumsensitive ATPase activity of unpurified and purified SR from both normal and dystrophic muscle showed no reduction in the presence of sodium azide, indicating little or no mitochondrial contamination. Purified dystrophic SR showed a reduced calcium-sensitive ATPase activity, an increased precipitant-anion calcium transport rate, no change in steady-state calcium transport level, and a reduced phosphoprotein level, relative to purified normal SR. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. IO. I I. I?. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 73. 24.
Porter, K., and Palade. G., J. Biopl?.vs. Biochem. Cytol. 3, 269 (1957). Baskin, R. J.. Lab. Invest. 23, 581 (1970). Van Breeman, V. L., Amer. J. Pathol. 37, 337 (1960). Hsu, Q.. and Kaldor. G., Proc. Sot. Exp. Biol. Med. 31, 1398 (1969). Hsu, Q.. and Kaldor. G.. Proc. Sot. Exp. Biol. Med. 38, 733 (1971). Sylvester, R., and Baskin. R. J., Biochem. Med. 8(Z), 213 (1973). Sabbadini, R.. Scales, D.. and Inesi. G., FERS Letters 54(l). 8 (1975). Yap, J.. and MacLennan, D., Can. J. Biochem. 54, 670 (1976). Baskin, R. J., and Hanna, S., Ph~~siologist 18, 132 (1975). Baskin, R. J., J. Ultrustruc. Res. 49, 348 (1974). Schacterle, G., and Pollack, R., An&. Biochem 51, 654 (1973). Warren, G., Toon. P., Birdsall. N., Lee, A., and Metcalfe. J., Proc. Nat. Acad. SC;. USA 71(3), 622 (1974). Panet, R., and Selinger, S., J. Biol. Chem. 246(23), 7349 (1971). Fairbanks, G.. Steck, T., and Wallach, D., Biochemistry lO( 13). 2606 (1971). Barrett, E., and Headon, D., FEBS Letters 51(l), I21 (1975). Katz, A., and Repke, D., Circ. Res. 21, I53 ( 1967). Solaro, R., and Briggs, F., Circ. Res. 34, 53 I ( 1974). Walter, H., and Hasselbach, W., Eur. J. Biochem. 36, I10 (1973). Dahl, J.. and Hokin, L., Ann. Rels. Biochem. 43, 327 (1974). Forbes, M., and Sperelakis, N., Z. Ze/&rsch 134, I (1972). Schapira, G., Boboy, I., Piau. J., and Delain, E.,Biochim. Biophys. Acfa 345,348 (1974). Hasselbach, W., and Weber, H., in ‘*Membrane Proteins in Transport and Phosphorylation” (G. F. Azzons, M. E. Klingenberg, E. Quaglianello. and N. Liliprandi. Eds.). p. 103. North Holland, Amsterdam (1974). Douglas, W.. Jr., and Baskin, R. J.. Amer. J. Physiol. 220(5). 1344 (1971). Sabbadini, R., and Baskin, R. J.. Amer. J. Physiol. 230(4), 1138 (1976).
Transport and Phosphoenzyme Formation Sarcoplasmic Reticulum isolated from Normal and Dystrophic Chickens STEPHEN
D. HANNA
Received
AND RONALD
November
2.
in
J. BASKIN
1976
INTRODUCTION The presence of an extensive membrane system in muscle cells, the sarcoplasmic reticulum (SR), has been known since the ultrastructural investigations of Porter and Palade (I), who observed the presence of a modified endoplasmic reticulum in muscle cells. According to current models of muscle contraction, this modified endoplasmic reticulum plays a major role in the regulation of contraction by releasing calcium ions to initiate contraction and sequestering calcium at the expense of ATP to cause muscle relaxation. Isolated SR is capable of calcium transport in the presence or absence of divalent anions, and these processes have become identified in the literature as calcium uptake and calcium binding, respectively. However. the term calcium binding is technically incorrect when used in this context, because binding refers to passive electrostatic binding and not to an active transport process. For the present work, the term “calcium transport’ ’ will be used in place of “calcium binding,” and the term “precipitant-anion calcium transport” will be used in place of “calcium uptake.” The properties of dystrophic SR have been investigated in a number of dystrophic animals, namely, humans, mice, hamster, and chickens. Transmission electon microscopy has revealed that the SR in most dystrophies is often swollen in appearance (2, 3). The dystrophic chicken is a suitable animal for examining biochemical abnormalities in muscular dystrophy for the following reasons: (i) The dystrophic animals can breed, allowing unequivocal identification of homozygous dystrophic animals; and (ii) the pectoralis muscles are drastically affected by the disease process. 300 Copyright ,411 right\
@ lY77 hy Academic Pre\\. Inc. of reproduction m any form rcwrved
I SSN W.K-1444
DYSTROPHIC
SARCOPLASMIC
301
RETICULUM
Hsu and Kaldor studied SR from normal and dystrophic chickens (4,5). They found a reduced precipitant-anion calcium transport ability in dystrophic SR and also an increased susceptibility of dystrophic SR to phospholipase c digestion (4). They also found that dystrophic SR has a higher 1ipid:protein ratio than does normal chicken SR (5). In 1970, Baskin (2) found an increased yield of SR from 6 to &week dystrophic chicken muscle and a reduction in dystrophic membrane-bound creatine phosphokinase activity. However, he found no difference in the amount of calcium accumulated or ATP hydrolyzed after 10 min of incubation. In 1973, Sylvester and Baskin (6) examined the kinetics of precipitant-anion calcium transport by normal and dystrophic 6- to 8week chickens and, in sharp contrast to other animal models, they found that the rate of transport was significantly increased in the dystrophic SR. The work presented here was stimulated by the finding that dystrophic chicken SR exhibits a higher rate of precipitant-anion calcium transport than does normal chicken SR (6), a result that is in contrast to results found in other dystrophic animal species. The present work consists of the sucrose density-gradient purification of normal and dystrophic SR, and the biochemical characterization of the purified SR.’ A portion of this work was presented at the APS meetings in 1975 (9). MATERIALS AND METHODS SR Isolation Normal (line 412) and dystrophic (line 413) chickens were obtained from the Department of Avian Sciences, University of California at Davis. The animals were from 6 to 8 weeks of age; a stage of pectoralis muscle hypertrophy in the dystrophic animals. The animals were killed by cervical dislocation, the pectoralis muscles were excised, and the SR was prepared according to Baskin (IO). The SR was purified further by ultracentrifugation at 51 ,OOOg for 90 min on a 20/30% discontinuous sucrose gradient, using a Beckman Model L ultracentrifuge. The following fractions were obtained: fraction 1, did not enter the 20% layer: fraction 2, between the 20 and 30% layers; and fraction 3, pellet. Protein was determined by a modification of the Lowry procedure (1 I), and all biochemical assays were performed within 24 hr of isolation. Biochemical
Assays
The ATPase activity of the SR preparations was measured in the absence of oxalate according to Warren et al. (12). All measurements were made at pH 7.0 and 23”, and the protein concentration was 8-16 pg ’ Since this investigation been published (7. 8).
was begun,
two reports
dealing
with dystrophic
chicken
SR have
302
HANNA
AND
BASKliL
of protein/ml. Measurements were made in the presence of 0. I mM EGTA (basal) and 0.1 mM CaCl, plus 0. I mM EGTA (total). The calciumsensitive ATPase activity was determined by subtraction of the basal ATPase activity from the total activity. Precipitant-anion calcium transport was measured using established methods ( IO), at a protein concentration of 0.03 mgiml. Calcium transport was measured in the presence of an ATP-regenerating system consisting of 1.5 IU of pyruvate kinaseiml and 4 mM PEP, in order to maintain a constant ATP concentration and prevent ADP accumulation. Protein concentration was 0.2 mg/ml. and samples were taken after I min of incubation. Phosphoprotein formation was measured at 4”, according to Panet and Selinger ( 13). The reaction was initiated by the addition of 32ATP and terminated after 30 sec. Polyacrylamide gels were prepared according to Fairbanks et (11. ( 14) and were run on a slab gel apparatus (Aquebogue Machine and Repair Shop, Aquebogue, N.Y.). Chemicals were obtained from Sigma Chemical Co., St. Louis, MO.
RESULTS Dystrophic muscle yields more SR protein per gram of tissue than a comparably aged normal muscle (Table 1). This increase in SR yield may be explained in one of three ways: (i) Dystrophic muscle may contain more SR per gram of muscle than does normal muscle. SR in dystrophic chicken pectoralis muscle has previously been described as enlarged or swollen (2, 3). (ii) Due to an altered structure of dystrophic muscle, the homogenization procedure may extract a greater percentage of the total SR from dystrophic muscle than from normal. (iii) The dystrophic SR pellet may contain more nonSR membrane contaminants than the normal SR pellet. TABLE YIELD Yield
I DATA
Normal
f Test
Dystrophic
Milligrams of SR per gram of muscle Fraction Iu
0.295 0.012
t f
0.024 0.002
0.446 0.029
+ 0.026 f 0.004
/J i 0.001 p < 0.01
Fraction Fraction
0.219 0.165
t 0.014 k 0.028
0.197 0.166
2 0.014 + 0.018
n.s.d.: n.s.d.
2 3
” The figures for fractions I. 2. and 3 are milligrams of SR applied to sucrose gradient. L. n.s.d., not significantly different.
in
the
units
of
milligrams
of
protein/total
DYSTROPHIC
SARCOPLASMIC
RETICULUM
When the SR was separated into different fractions on gradient, the total protein in each fraction was assayed. indicates, a difference in protein distribution occurs in fraction measurable difference occurs in fractions 2 or 3. (Fraction accounts for less than 10% of the total protein recovered.) Polyacrylamide
303
the sucrose As Table 1 I, while no 1, however.
Gels
The major protein observed on polyacrylamide gels is the 106,000dalton ATPase protein. As Fig. 1 illustrates, fraction 2 contains the most ATPase protein of all the sucrose fractions. This observation, plus the preliminary finding that fraction 2 contains the greatest calcium-sensitive ATPase activity, identified fraction 2 as the purified SR fraction. (Fraction 1 contains little ATPase protein and may correspond to fraction 1 of Bat-ret and Headon (15), found in density-gradient purified rabbit SR). Normal and dystrophic fraction 2 do not appear to differ significantly in their appearance on polyacrylamide gels. Purijica tion
Early in the course of this work, it was observed that both normal and dystrophic SR contained a high level of basal ATPase activity (in the presence of 0.1 mM EGTA, no CaCl,). One reason for attempting the
FIG. I. Polyacrylamide gels of sarcoplasmic reticulum fractionated on sucrose gradient. NSR is SR before gradient fractionation; N-l, N-2, and N-3 refer to the fractions described in Methods. N refers to normal chicken: D refers to dystrophic chicken. The arrow indicates the location of the 106,000-dalton ATPase protein. Ten micrograms of protein were applied to each gel.
purification on the sucrose density gradient was to see if this basal ATPase activity could be eliminated by eliminating nonSR membrane contaminants. The two pure SR preparations could then be compared with one another. Table 2 represents the results obtained by this procedure. Samples were assayed for total ATPase activity (+ 0. I mM CaCl, and 0.1 mM EGTA), basal ATPase activity (+ 0. I mM EGTA). maximal calcium-sensitive ATPase activity (+ 0.1 mM CaCl, and 0.1% Triton X-100), degree of mitochondrial contamination (+ 10 mM sodium azide). and phosphoenzyme levels. As indicated in Table 2, the basal ATPase level is still elevated in fraction 2, and is not significantly different from the basal ATPase level observed in the original SR. This may be interpreted in one of two ways: Either (i) the basal ATPase level is the result of a contaminating membrane fraction, which cannot be separated by the techniques used, or (ii) the basal ATPase level is a property of purified SR membranes from chicken skeletal muscle, having an unknown function. The total ATPase activity is increased in fraction 2, although this increase is not significantly different for dystrophic membranes. More importantly, the calcium-sensitive ATPase activity is increased in both normal and dystrophic fraction 2. This observation, plus the previous observation that the basal ATPase level is unchanged in fraction 2, indicates that the sucrose-gradient separation eliminates some contaminating protein from the SR fraction. The addition of 10 mM sodium azide causes a reduction of basal ATPase activities within all four fractions. Even though the ATPase levels are reduced, the azide ATPase activities exhibit the same trend as the ATPase activities without azide; total ATPase activity is significantly increased in fraction 2, while basal ATPase activity is in essence unchanged in fraction 2. The most important property, the calcium-sensitive ATPase activity, remains unchanged in all fractions in the presence of azide. The azide concentration is the same as that used by Katz and Repke (16) and Solar0 and Briggs ( 17), who were measuring the degree of mitochondrial contamination of cardiac SR preparations. The curious property of azide of reducing total and basal ATPase activity, while leaving calcium sensitivity unaffected, was noted previously on cardiac SR by Katz and Repke ( 16). ATPuse
Activity,
Frcrction
2
A comparison of the ATPase activity of normal versus dystrophic fraction 2 is shown in Table 3. The basal ATPase activity, as mentioned previously, is present at a relatively high level, and the basal ATPase activity is significantly higher in normal fraction 2. Not only is the basal ATPase activity higher in normal, but the total
DYSTROPHK
SARCOPLASMlC
RETlCULUM
305
306
HANN.4
.ANI)
BASKlic
I 4BI.l-. /+i( I I\‘1 I \
01
: f’K.%(‘I
ION
.1
--..-..~
Conditions Total (t Basal
Normal ATPase
3.36
0.1 mM CaCI,) ATPase
I + 0. I mM
Dyxtr-ophic
-in 0.44
: Ie41
I.72
+- 0.17
I’
0.01
2. I? _i- 0.2 I
I .7h
t- 0.09
,’
O.OI
EGTA) 2.62
-+ 0.18
2.14
i
+ 0. IQ triton Calcium-sensitive
1.23
-+ 0.29
0.47
2 0.11
p < 0.05
ATPase (total-basal) Precipitant-anion
0.28
?z 0.12
0.94
2 0.18
,I c: 0.05
0. I rnM
CaCl*
0.15
n.s.d.‘
calcium transport (~mimgimin) Calcium
transport
0.039
(I*molimg) Phosphorylated
3.26
2 0.005 f
0.44
0.037 1.45
? 0.004 2 0.27
n.s.d. p c: 0.01
intermediate (nmoleimg) : n.s.d..
not
significantly
different.
ATPase activity is significantly higher as well. This is reflected in the fact that the calcium-sensitive ATPase activity is significantly higher in normal fraction 2 than in dystrophic. This result suggests either that the normal preparation is substantially purified relative to the dystrophic, or that the normal ATPase protein is able to function at a higher rate than the dystrophic ATPase protein. The Triton X-loo-treated fraction 2 samples show no significant difference from one another. This indicates that perhaps the dystrophic ATPase protein has the same potential for ATP hydrolysis when solubilized, but is more restricted by its membrane environment than is the normal ATPase protein. Precipitant-Anion Calcium Transport, Calcium Transport. and Phosphoen~ytne Formation In order to fully characterize fraction 2, its capabilities for calcium transport, precipitant-anion calcium transport, and phosphoenzyme formation were measured (Table 3). The precipitant-anion calcium transport rate of dystrophic fraction 2 is greater than that of normal fraction 2. This indicates that the results of Sylvester and Baskin (6) were not merely due to contaminating membranes. However, no difference exists in the steady-state calcium transport ability of the two types of purified SR. These steady-state calcium transport values are relatively low when compared to skeletal SR from
DYSTROPHIC SARCOPLASMIC RETICULUM
307
other sources, which typically exhibit calcium-binding values between 0.1 and 0.2 pmol/mg of SR protein. In contrast to precipitant-anion calcium transport and calcium transport, the phosphorylated intermediate level of normal fraction 2 is significantly elevated over that of dystrophic fraction 3L. The overall picture from biochemical data is a complex one, with dystrophic fraction 2 having an increased precipitant-anion calcium transport rate, the same calcium transport steady-state level, a decreased calcium-sensitive ATPase rate, a decreased level of phosphoprotein formation, and an increased calcium-transported/ATP-hydrolyzed ratio relative to normal fraction 2. DISCUSSION The Basal ATPase
Acti\vity
The basal ATPase activi.ty observed in chicken SR is higher than the basal ATPase found in mammalian SR ( 18). The basal ATPase may be a Na+,K+-ATPase, since this is a widespread membrane-bound ATPase, and may be either a constituent of SR membranes or a membrane contaminant. The elimination of the basal ATPase activity by Triton X-100 solubilization supports the idea of a Na+,K+-ATPase, because Na+,K+ATPases are typically detergent labile (19). The question of the basal ATPase being’a contaminant or an endogenous SR ATPase is difficult to answer; evidence exists which supports both hypotheses. Forbes and Sperelakis (20) found that a Na+,K+-ATPase was present in skeletal muscle SR, observed by cytochemical techniques, which supports the idea of the basal ATPase being an endogenous SR ATPase. However, Schapira et al. (21) found that some plasma membrane was associated with mammalian SR even after sucrose density-gradient centrifugation, which supports the idea of the basal ATPase activity being a contaminant. Therefore, using available data, one cannot decide whether the basal ATPase activity is an endogenous SR ATPase, a contaminant, or a combination of the two. Calcium
Transport
Steady-state calcium transport levels of normal and dystrophic SR are similar, however, freeze-fracture electron micrographs suggest that dystrophic membranes have a greater surface area and vesicular volume per milligram of SR protein than do normal membranes. Steady-state levels of active transport may depend upon the concentration gradient; dystrophic membranes, which have established equivalent steady-state levels with normal membranes (per milligram of protein) may have actually developed less of a concentration gradient. Dystrophic membranes may have transported the same net amount of calcium as normal membranes,
308
HL\NNA
AND
RASkiK
but into a larger intravesicular volume. Under these circumstances, normal SR would transport more calcium per unit of membrane surface area. The calcium transport results differ from those of Sabbadini ef ul. (7) who found a decreased rate and steady-state level of calcium transport in dystrophic chicken SR. The reason for this discrepancy is not known, although it could be due to differences in the method of SR preparation. The steady-state levels reported by Yap and MacLennan (8) are relatively high. It is possible, however, that due to their IO-min incubation time liberated phosphate served to allow precipitant-anion transport. The reduction we observed in calcium-sensitive ATPase activity in dystrophic SR is also in contrast to the results of Sabbadini rt al. (71. However, the assay medium used by these investigators contained Triton X-100 and, as Table 3 shows. we also find no difference in ATPase activities in the presence of Triton X-100. Phosphoenzyme levels have never before been measured in dystrophic SR, so it is significant that dystrophic chicken SR exhibits a reduced level of phosphoenzyme formation. This indicates that dystrophic SR has either a reduced functional ability or a lesser degree of purification. The phosphoenzyme levels, the calcium-sensitive ATPase activity, and the steady-state calcium transport levels indicate that dystrophic SR has a decreased enzymatic activity. The precipitant-anion calcium transport rates do not correlate well with the rest of the data, and it may be that in chicken SR precipitant-anion transport is not a representative measure of calcium transport capability. The entry of precipitant anions into SR vesicles is a complex process not sufficiently explained by diffusion (221, and alterations in anion permeability could cause misleading results. If the alterations in dystrophic SR do represent in r~vo alterations, then these alterations could explain previously observed mechanical alterations in dystrophic muscle (23, 24). A reduced rate of calcium transport and/or an altered calcium release mechanism could explain the reduced tetanus tension, increased relaxation time, and reduced intensity and duration of the active state observed in dystrophic mouse muscle. The alterations found in dystrophic chicken SR could be due to one of four possibilities: (i) The arrangement of lipid and protein in the membrane is altered, causing an altered enzymatic function. (ii) The lipid composition of the membrane is altered. (iii) The ATPase protein is altered. (iv) Both the lipid component and the ATPase protein are altered. Studies on items (ii) and (iii) have been performed in the past (4, 3, but both the SR preparation techniques and the ages of the animals were different from the present investigation, precluding direct comparison.
DYSTROPHIC
SARCOPLASMIC
RETICULUM
309
SUMMARY Sarcoplasmic reticulum from normal and dystrophic pectoralis muscle was isolated and purified using a discontinuous sucrose gradient. Puritied SR from both normal and dystrophic sources showed an increased calcium-sensitive ATPase activity relative to unpurified SR. Calciumsensitive ATPase activity of unpurified and purified SR from both normal and dystrophic muscle showed no reduction in the presence of sodium azide, indicating little or no mitochondrial contamination. Purified dystrophic SR showed a reduced calcium-sensitive ATPase activity, an increased precipitant-anion calcium transport rate, no change in steady-state calcium transport level, and a reduced phosphoprotein level, relative to purified normal SR. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. IO. I I. I?. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 73. 24.
Porter, K., and Palade. G., J. Biopl?.vs. Biochem. Cytol. 3, 269 (1957). Baskin, R. J.. Lab. Invest. 23, 581 (1970). Van Breeman, V. L., Amer. J. Pathol. 37, 337 (1960). Hsu, Q.. and Kaldor. G., Proc. Sot. Exp. Biol. Med. 31, 1398 (1969). Hsu, Q.. and Kaldor. G.. Proc. Sot. Exp. Biol. Med. 38, 733 (1971). Sylvester, R., and Baskin. R. J., Biochem. Med. 8(Z), 213 (1973). Sabbadini, R.. Scales, D.. and Inesi. G., FERS Letters 54(l). 8 (1975). Yap, J.. and MacLennan, D., Can. J. Biochem. 54, 670 (1976). Baskin, R. J., and Hanna, S., Ph~~siologist 18, 132 (1975). Baskin, R. J., J. Ultrustruc. Res. 49, 348 (1974). Schacterle, G., and Pollack, R., An&. Biochem 51, 654 (1973). Warren, G., Toon. P., Birdsall. N., Lee, A., and Metcalfe. J., Proc. Nat. Acad. SC;. USA 71(3), 622 (1974). Panet, R., and Selinger, S., J. Biol. Chem. 246(23), 7349 (1971). Fairbanks, G.. Steck, T., and Wallach, D., Biochemistry lO( 13). 2606 (1971). Barrett, E., and Headon, D., FEBS Letters 51(l), I21 (1975). Katz, A., and Repke, D., Circ. Res. 21, I53 ( 1967). Solaro, R., and Briggs, F., Circ. Res. 34, 53 I ( 1974). Walter, H., and Hasselbach, W., Eur. J. Biochem. 36, I10 (1973). Dahl, J.. and Hokin, L., Ann. Rels. Biochem. 43, 327 (1974). Forbes, M., and Sperelakis, N., Z. Ze/&rsch 134, I (1972). Schapira, G., Boboy, I., Piau. J., and Delain, E.,Biochim. Biophys. Acfa 345,348 (1974). Hasselbach, W., and Weber, H., in ‘*Membrane Proteins in Transport and Phosphorylation” (G. F. Azzons, M. E. Klingenberg, E. Quaglianello. and N. Liliprandi. Eds.). p. 103. North Holland, Amsterdam (1974). Douglas, W.. Jr., and Baskin, R. J.. Amer. J. Physiol. 220(5). 1344 (1971). Sabbadini, R., and Baskin, R. J.. Amer. J. Physiol. 230(4), 1138 (1976).