Comparison of the (Ca2+ + Mg2+)-ATPase proteins from normal and dystrophic chicken sarcoplasmic reticulum

144

Biochimica et Biophysica Acta, 5 4 0 ( 1 9 7 8 ) ! 4 4 - - i 5 0 © Elsevier/North-Holland Biomedical Press

BBA 28515

COMPARISON OF THE (Ca 2+ + Mg2÷)-ATPase PROTEINS FROM NORMAL AND DYSTROPHIC CHICKEN SARCOPLASMIC RETICULUM

STEPHEN D. HANNA * and RONALD J. BASKIN Department of Zoology, University of California at Davis, Davis, Calif. 95616 (U.S.A.)

(Received July 22nd, 1977)

Summary The involvement of membrane protein in dystrophic chicken fragmented sarcoplasmie retieulum alterations has been examined. A purified preparation of the (Ca 2+ + Mg2+)-ATPase protein from dystrophic fragmented sarcopiasmic reticulum was found to have a reduced calcium-sensitive ATPase activity and phosphoenzyme level, in agreement with alterations found in dystrophic chicken fragmented sarcoplasmic reticulum. An amino acid analysis of the ATPase preparations showed no difference in the normal and dystrophic (Ca2+ + Mg2+)-ATPase. The (Ca2+ + Mg2+)-ATPase was investigated further by isoelectric focusing and proteolytie digestion of the fragmented sarcoptasmic reticulum. Neither of these methods indicated any alteration in the composition of the dystrophic (Ca2+ + Mg2+)-ATPase. We have concluded that t h e alterations observed in dystrophic fragmented sarcoplasmic reticulum are not due to increased amounts of non-(Ca 2+ + Mg2÷)-ATPase protein, and that the normal and dystrophic (Ca2÷ + Mg2+)-ATPase protein are not detectably different.

Introduction Biochemical alterations have been found in fragmented sarcoplasmic reticulum isolated from dystrophic human, mouse a n d chicken muscle [1--5], Of these animals, the dystrophic chicken has been investigated in the greatest detail. Alterations in calcium transport [4,61, ATP hydrolysis [41 and phosphoenzyme formation [4] have been reported. Dystrophic chicken fragmented sarcoplasmic reticulum also has an increased lipid-to-protein ratio [7], and

* Current address: Department of Physiology, University of California Medical Center, Los Angeles, Calif. 90024, U.S.A. Abbreviation: SDS, sodium dodecyl sulfate.

145 freeze-fracture electron microscopy indicates a reduced density of 80-A particles in dystrophic chicken fragmented sarcoplasmic reticulum vesicles [5,8]. We have previOusly summarized the possible alterations that could cause the abnormalities found in dystrophic chicken fragmented sarcoplasmic reticulum [4]. The possibilities are: 1. Dystrophic fragmented sarcoplasmic reticulum contains more non-(Ca2+ + Mg2+)-ATPase protein than the normal, causing a misleading interpretation of biochemical data, which are expressed per mg of fragmented sarcoplasmic reticulum protein. 2o The 100 000-dalton (Ca 2÷ + Mg2+)-ATPase protein of dystrophic fragmented sarcoplasmic reticulum is altered in composition. 3. The lipid composition of dystrophic fragmented sarcoplasmic reticulum is altered. 4. Both the (Ca ~÷ + Mg2+)-ATPase and the lipid c o m p o n e n t of the dystrophic membranes are altered. It has been previously demonstrated [7] that dystrophic chicken fragmented sarcoplasmic reticulum has an altered lipid composition. Therefore, we have investigated the first and third possibilities listed above. We have found that an elevated level of non-(Ca 2÷ + Mg2+)-ATPase protein does not explain the observed biochemical alterations, and that the (Ca 2+ + Mg2÷)-ATPase itself is n o t detectably altered in dystrophic fragmented sarcoplasmic reticulum. Materials and Methods Fragmented sarcoplasmic reticulum from normal and dystrophic chicken pectoralis m u s c l e was isolated as described by us [4], without sucrose gradient purification. The (Ca 2÷ +Mg2+)-ATPase enzyme was purified according to Warren et al. [9]. Protein was determined by a variation of the Lowry m e t h o d [10], Calcium-sensitive ATPase activity and phosphoenzyme formation were measured as described elsewhere [4]. Total amino acid analysis was performed after extraction of fragmented satcoplasmic reticulum with a double volume of ethanol/acetone (1 : 1). The prorein was pelleted in a clinical centrifuge and evaporated to dryness under a stream of dry nitrogen. The samples were then hydrolyzed in 1 ml of 6 M HC1 and 10 mg phenol for 48 h at 110°C, dissolved in 0.2 N sodium citrate buffer, pH 2.2, and analyzed on a Durrum Model D-500 Amino Acid Analyzer. Polyacrylamide gel electrophoresis was carried out on slab gels using the Tris/glycine system of Laemmli [11], as described by Weber and Osborn [ 12]. Digestion of fragmented sarcoplasmic reticulum with papain and chymotrypsin was performed in the presence of 0°5% SDS according to Cleveland et al. [13], at a fragmented sarcoplasmic reticulum concentration of 2 mg/ml and a proteolytic enzyme concentration of 0.01 mg/ml. For digestion of the (Ca 2÷ + Mg2+) ATPase, 160 pg of fragmented sarcoplasmic reticulum protein were run on a 25-slot, 7.5% acrylamide slab gel, with an SDS : protein ratio of at least 3 : 1, as suggested by Weber and Osborn [12]. The (Ca2+ + Mg2÷)-ATPase band was stained, destained, and cut o u t as described by Cleveland et al. [ 13]. The bands were then inserted into th~ sample wells of a 12 slot 15% acrylamide gel, overlayered with 0.01 ml of 20% sucrose, 0.125 M Tris • HC1, pH 6.8, 0.1% SDS

146 and 1 mM EDTA. Papain or chymotrypsin, in 10% glycerol, 0 . t 2 5 M Tris o HC1, pH 6.8, 0.1% SDS, and 1 mM EDTA, was then added to the sample well, in amounts of 0.2 and 2.0 gg, respectively. The gels were ~"an at a current of 25 mA until the tracking dye reached the top of the running gel, the current was shut off for 30 rain and the gels were then run to completion at a current of 25--30 mA. The best results were achieved when the 15% gel contained 1 mM EDTA, as suggested by Cleveland et al. [i3]o Isoelectric focusing in polyacrylamide gels was performed according to Madeira [14], at 10°C. The gels were fixed for 2 h at 55°C in 50% methanol and 10% acetic acid, stained overnight in 0.05% Coomassie blue i%/50% methanol/10% acetic acid, and destained in 5% methanol/10% acetic acid, until the background stain was removed. Second-dimension SDS gel electrophoresis was performed according to Madeira [ 14]. Results and Discussion A TPase preparation A purified ATPase preparation was made from both normal and dystrophic fragmented sarcoplasmic reticulum for two reasons: (1) Both types of fragmented sarcoplasmic reticulum contain a substantial amount of non-(Ca 2÷ + Mg2+)-ATPase protein. This may affect the interpretation of biochemical results, as such results are expressed per milligram of fragmented sarcoplasmic reticulum protein. (2) We wished to perform a total amino acid analysis on the (Ca2+ + Mg2+)-ATPase, and the ATPase preparation yields large amounts of relatively pure (Ca 2+ + Mg2+)-ATPase. Polyacrylamide gels of the ATPase preparations, as well as gels of normal and dystrophic fragmented sarcoplasmic reticulum, are presented in Fig. 1. T~e ATPase preparations contain little non-(Ca2+ + Mg2+)-ATPase protein, when compared with the fragmented sarcoplasmic reticulum preparation, and the (Ca2+ + Mg2+)-ATPase band is more intense in the ATPase preparations. An

0 C 8 A

F i g . 1. P o l y a c r y l a / n i d e gels o f f r a g m e n t e d s a r c o p l a s m i c r e t i c u l u m a n d A T P a s e p r e p a r a t i o n s . Sa~mples o f 2 0 ~Lg w e r e r u n o n a 1 2 - s l o t 7 . 5 % a c r y l a m i d e s l a b gel, as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . A. n o r m a l f r a g m e n t e d s a x c o p l a s m i c r e t i c u l u m ; B, d y s t r o p h i c f r a g m e n t e d s a r c a P l a s m i c r e t i c u l u m : C, n o r m a l A T P a s e : D , d y s t r o p h i c A T P a s e . T h e a r r o w p o i n t s t o t h e 1 0 6 0 0 0 - d a l t o n ( C a 2+ + M g 2 + ) - A T P a s e . T h e c a t h o d e is a t the left-hand side of the figure,

147

-t-

pH

9

8

7

6

5

4

Fig. ~I. Isoelectric focusing of fragmented saeroplasmic reticulum on p o l y a c r y l a m i d e gels (pH range was 3.5--10). A, n o r m a l fragrnented sacroplasmic r e t i c u l u m ; B, dystrophic fragmented sacroplasmic re t i c ul um; C, lobster fragmented sarcoplasrnic reticulum.

analysis of gel scans indicate that more than 85% of the protein is the (Ca 2+ + Mg2+)-ATPase protein in both the normal and dystrophic ATPase. (Both contain the same a m o u n t of (Ca 2+ + Mg2+)-ATPase protein per mg total protein.) However, the calcium-sensitive ATPase activity and phosphoenzyme levels of the dystrophic ATPase preparation are significantly reduced relative to the normal. The calcium sensitive ATPase activity in the purified normal (Ca 2+ + Mg2÷)-ATPase protein was 0.90-+ 0.09 pmol Ca2÷/mg per min whereas the purified dystrophic protein was 0.44-+ 0.07 pmol Ca2+/mg per min. Of even more significance, the p h o s p h o e n z y m e formation in the normal preparation was 4.83 + 0.75 nmol/mg and 1.84 + 0.30 nmol/mg in the dystrophic (the t-test gave P ~ 0.01 for each value). This is the same general difference observed between purified normal and dystrophic fragmented sarcoplasmic reticulum [4], and implies that the differences are n o t merely due to the presence of differing amounts of non-(Ca 2+ + Mg2+)-ATPase protein. The reduction in activity observed in the dystrophic ATPase preparation is n o t necessarily due to an alteration in the (Ca2+ + Mg2+)-ATPase protein, because the ATPase preparation,~ are made using the original fragmented sarcoplasmic reticulum lipids. Therefore, lipid alterations could also explain the differences observed. In fact, an ~mino acid analysis of the ATPase preparations reveals no difference in prorein composition (Table I). This result is in agreement with that of Hsu and Kaldor [15], who performed amino acid analysis on normal and dystrophic chicken fragmented sarcoplasmic reticulum. In order to obtain more precise information about the dystrophic (Ca 2+ + Mg2+)-ATPase, we have utilized the techniques of isoelectric focusing and proteolytic digestion.

Isoelectric focusing An isoelectric focusing m e t h o d has recently been developed by Madeira [ 14] for examining the (Ca 2+ + Mg2+)-ATPase of fragmented sarcoplasmic reticulum, using Triton X-100 solubilization of the membranes. We have used this m e t h o d to compare the isoelectric focusing patterns of the normal and dystrophic (Ca2÷ + Mg2+)-ATPase. The isoelectric focusing patterns produced by normal and dystrophic fragmented sarcoplasmic reticulum are very similar (Fig. 2), whilE; that of lobster is somewhat different. This indicates both that the technique is sensitive enough to detect species differences in the (Ca2+ + Mg2+) -

148 TABLE I A M I N O A C I D A N A L Y S I S O F (Ca 2+ + Mg2+)-ATPase P I ~ O T E I N V ~ u e s are p e r c e n t a g e s . Amino

acid

Lysine Histidine Arginine Aspa~tate Threonh~e Serine Glutamate Proline Glyeine Alanine Valine Isoleueine Leueine Tyrosine Phenylalanine

Normal

Dystrophic

5,77 1.81 5,04 8.99 5,49 4.52 10.67 5,05 8.00 I0.11 8.94 6.28 9.59 2.64 4.32

6,12 2,46 5.09 8.86 5.59 4.81 10.69 4.74 7.67 9.87 8.77 6.30 10.12 2.87 4.60

ATPase, and t h a t the normal and dystrophic (Ca 2+ * Mg2+)-ATPases are more similar to each other than they are to the lobster protein. The three major bands observed in chicken fragmented sarcoplasmie retieulum and the two bands observed in lobster fragmented sarcoptasmie reticulum are within the pH range 5.0--7°0, and second dimension polyacrylamide gel eleetrophoresis indicates that these bands correspond to the (Ca 2+ + Mg2+)-ATPase. Both of these observations are in general agreement with the observations of Madeira [14] on rabbit fragmented sareoplasmic retieulum.

Proteoly tic digestion A m e t h o d for the analysis of partial hydrolysis products of proteins on polyacrylamide gels has been developed by Clzveland et al. [13], involving digestion of proteins by papain or chymotrypsin in the presence of SDS. This technique has been shown to give reproducible banding patterns on polyacrylamide gels, and is ideally suited for comparative studies of protein structure. We have utilized this m e t h o d by digesting briefly normal and dystrophic chicken fragmented sarcoplasmic reticulum with papam or chymotrypsin. Digestion of normal and dystrophic fragmented sarcoplasmic reticulum with these enzymes shows a great similarity in the molecular weight of the digestion products (Fig. 3). The lobster fragmented sareoplasmic retieulum produces a different digestion pattern, indicating that this technique, like isoelectrie focusing, is sensitive enough to detect species differences in protein structure~ All fragmented sarcoplasmic reticulum digestions were performed in the presence of 0.5% SDS, which solubilized the membranes, so surface exposure of the (Ca2+ + Mg2+}ATPase is not a limiting factor° The new bands observed upon digestion presumably arise from breakdown of the (Ca 2+ + Mg2+)-ATPase, since this protein is the major constituent of the fragmented sarcoplasmic reticulum. However. there are substantial amounts of lower molecular weight protein bands observable in gels of both types of fragmented sarcopla~mic re~iculum before diges-

149

AB C D E F

GH

A

B

C

D

Fig. 3. D i g e s t i o n o f f r a g m e n t e d s a r c o p l a s z n i c r e t i c u l u m w i t h p a p a i n o r c h y m o t r y p s i n . S a m p l e s o f 8 0 # g w e r e r u n o n a 2 5 - s l o t 1 5 % a c r y l a m i d c slab gel d e s c r i b e d in M e t h o d s . A - - C , p a p a i n d i g e s t i o n ; D - - F , c h y r n o t r y p s i n d i g e s t i o n . A a n d D , n o r m a l f r a g m e n t e d s a r c o p l a s m i c r e t i c u l u r n ; B a n d E, d y s t r o p h i c f r a g m e n t e d s a r c o p l a s r n i c r e t i c u l u m ; C a n d F, l o b s t e r f r a g m e n t e d s a r c o p l a s m i c r e t i c u l m n . G e l s G a n d H are n o r m a l a n d dystrophic fragmented sarcoplasrnic reticulum respectively, without digestion. The arrow points to the p o s i t i o n o f t h e ( C a 2+ + M g 2 + ) - A T P a s e . Fig. 4. D i g e s t i o n o f n o r m a l a n d d y s t r o p h i c ( C a 2+ + M g 2 + ) - A T P a s e . T h e ( C a 2+ + M g 2 + ) - A T P a s e b a n d w a s c u t o u t o f t h e i n i t i a l gel, i n s e r t e d i n t o a s l o t o f a s e c o n d gel, a n d d i g e s t e d as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . A , n o r m a l f r a g m e n t e d s a r c o p l a m i c r e t i c u l u m + p a p a l n ; B, d y s t r o p h i c f r a g m e n t e d s a r c o p l a s m i c reticttl'mm + p a p a i n ; C, n o r m a l f r a g m e n t e d s a r c o p l a s m i c r e t i c u l u r n + c h y m o t r y p s i n ; D, d y s t r o p h i c fragr n e n t e d s a r c o p l a s r n i c r e t i c u l u m + c h y r n o t r y p s i n . Gel A is s o m e w h a t s h o r t e r t h a n t h e o t h e r gels, b e c a u s e t h a t s a m p l e w a s r u n o n a s e p a r a t e slab gel f o r a slightly s h o r t e r t i m e .

tion. As a result, we have obtained a more precise digestion of the (Ca2+ + Mg2÷)-ATPase by cutting out the (Ca 2÷ + Mg2+)-ATPase from one gel, inserting it into slots on a second gel, overlayering the bands with papain or chymotrypsin, and allowing digestion to occur in the stacking gel layer. These results are presented in Fig. 4. Digestion of the (Ca 2÷ + Mg~+)-ATPase produces m a n y lower' molecular weight bands on the gels, and the pattern produced by digestion of the dystrophic (Ca 2÷ + Mg~+)-ATPase is identical to that produced by digestion of the normal (Ca 2÷ + Mg2+)-ATPase. These studies indicate that the biochemical alterations found in dystrophic fragmented sarcoplasmic reticulum are n o t due to an increased a m o u n t of non(Ca2+ + Mg2+)-ATPase protein. The amino acid analysis, isoelectric focusing, and proteolytic digestion methods all indicate that no detectable difference exists between the normal and the dystrophic (Ca 2+ + Mg2÷)-ATPase proteins. Lobster fragmented sarcoplasmic reticulum exhibits a different isoelectric focusing and proteolytic digestion pattern, indicating that these two methods are eminently suitable for detecting differences in protein composition. It is

150 conceivable that slight modifications in a critical portion of the protein, such as the active site of the enzyme, could alter activity and not be detectable by the methods we have used. However, it seems more likely that the biochemical alterations observed in dystrophic fragmented sarcoplasmic reticulum and in the dystrophic ATPase preparation are due to alterations in the lipid environment of the membrane. This hypothesis is currently being investigated; hewever, our work is at present incomplete. The (Ca 2+ + Mg2+)-ATPase is known to be very sensitive to changes in its lipid environment [9~, particularly to changes in cholesterol levels [16,17]. The cholesterol content of dystrophic fragmented sarcoplasmic reticulum is elevated (ref. 7 and Hanna~ S.Do, Deamer~ D.W. and Baskin~ l~.j., unpublished), and this could prove to be a major factor in the altered biochemical activity of dystrophic fragmented sarcoplasmic reticulum.

Acknowledgement A portion of this work was done during the tenure of a Research Fellowship of Muscular Dystrophy Associations of America.

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