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Sarcoplasmic reticulum of the flight-muscles of Locusta migratoria. Purification of sarcoplasmic reticulum vesicles and properties of sarcoplasmic reticulum atpase
Comp. Biodwm. Ph.~siol.. VoL 60B. pp. 481 to 485. Pt,rqtlt~lotl Prt's.~ Ltd 1978. Printed ill Great Britain
0305-0491,78 0715-0481 $02.00/0
S A R C O P L A S M I C R E T I C U L U M O F THE F L I G H T - M U S C L E S O F L O C U S T A MIGRATORIA. PURIFICATION OF SARCOPLASMIC RETICULUM VESICLES A N D P R O P E R T I E S O F SARCOPLASMIC RETICULUM ATPASE HANS VOLMER Zoologisches Institut der Universit~it Mtinster, Lehrstuhl fiir Tierphysiologie, Hindenburgplatz 55. D-4400 Miinster, Federal Republic of Germany
(Received 26 September 1977) Abstract--l. Sarcoplasmic reticulum vesicles were prepared from the flight-muscles of Locusta mi9ratoria by cell fractionation and sucrose gradient fractionation. 2. Properties of the sarcoplasmic reticulum ATPase were studied. 3. The ATPase was strongly dependent on Ca z+, Mg 2+ and K ÷. 4. The ATPase exhibited a pH optimum at pH 7.5 and was inhibited completely by salyrgan. INTRODUCTION
Relaxation of skeletal muscle is elicited by a decrease in the concentration of Ca 2 + in the sarcoplasm. This change in the sarcoplasmic Ca 2 + level is caused by a protein system which is able to t r a n s p o r t C a 2+ against a high concentration gradient into a tubular n e t w o r k - - t h e sarcoplasmic reticulum (SR). The energy required for this active transport is provided by an ATPase located in the m e m b r a n e of the SR. This enzyme was first studied by Kielley & Meyerhof (1948) who succeeded in preparing purified sarcoplasmic reticulum for the first time. They found that the ATPase was activated by M g 2+ and inhibited by Ca 2 + in the millimolar range. Later on it was shown that the ATPase from sarcoplasmic reticulum could be activated by Ca 2+ in the micromolar range (Hasselbach & Makinose, 1961). Furthermore, it could be d e m o n s t r a t e d that C a 2÷ uptake and b r e a k d o w n of A T P were closely correlated. By splitting of 1 mole of ATP, 2 moles of Ca 2÷ are transported into the SR. Properties of the ATPase from sarcoplasmic reticulum were studied by Martinosi & Ferretos (1964), Martinosi (1968) and M a c L e n n a n (1970). M a c L e n n a n also succeeded in purifying the enzyme 6-fold. SR for studying the properties of the ATPase was preferentially prepared from vertebrate skeletal muscle. However, there is little information about sarcoplasmic reticulum ATPase from invertebrate muscles. Studies concerning Ca2+-uptake by the SR from muscles of various insect species have been carried out by Tsukamoto et al. (1966), St6ssel & Zebe (1968), Haakshorst (1974) and H u d d a r t et al. (1974), but so far there is no detailed information a b o u t sarcoplasmic reticulum ATPase of insect muscle. In this paper results on preparation of sarcoplasmic reticulum vesicles from the flight-muscles of Locusta migratoria and some properties of the ATPase are reported. MATERIALS A N D M E T H O D S
Preparation of sarcoplasmic reticulum vesicles Flight-muscles of 40-50 adult locusts (both sexes) were 481
homogenized in 300mM sucrose and 5 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) pH 7.4 with a glass Teflon homogenizer. Following homogenization the homogenate was centrifuged at 1_5000 (2 x 15min), 10,000y (2 x 30min) and 44,0000 for 1 hr. The 44,0000 pellet was suspended in 600mM KCI and 5 m M HEPES, pH 7.4 and stirred for 2 hr. After centrifugation at 44,000 0 the pellet was resuspended in the homogenization buffer and centrifuged at 50000 for 30min. 3-5ml of the 5000 g supernatant containing the sarcoplasmic reticulum vesicles were then placed on the top of a step gradient containing five layers with different percentages of sucrose as described by Meissner & Fleischer (1971). The gradient consisted of 4ml 37.2~., 3 ml 33.9°~,, 3 ml 31.6!!~i, 3 ml 29.1°/o and 4ml 26.4~, sucrose and 5raM HEPES, pH 7.4, respectively. After centrifugation at 75,0000 for 2.5 hr the bottom of the centrifugation tubes was pierced by a hypodermic syringe and 0.8 ml fractions were collected in graduated tubes.
Enzyme assays The incubation mixture for the ATPase assay contained l0 mM histidine pH 7.4, 100 mM KCI, 5 mM MgC12 and 5 mM Ca-EGTA in 2ml final volume. After addition of sarcoplasmic reticulum vesicles (20-50/ag protein) the mixture was preincubated at 32'C for 10min. The reaction was started by addition of 5 #moles ATP. After 10-30 min the reaction was stopped by addition of 100 ~d 500/,, trichloracetic acid. Inorganic phosphate was determined according to Yoda & Hokin (1970). Calcium uptake was assayed in an incubation mixture containing 10raM histidine pH 7.4, 100mM KCI, 5 m M MgCl2, 5 m M potassium oxalate, 0.05 mM 45CAC12 and 20-50 pg sarcoplasmic reticulum protein. The reaction was started by addition of 5/~moles ATP. Incubation was carried out at 22"C. After 10 rain a 0.4 ml sample was filtered through a Millipore ® filter (HAWP 025001. The filter discs were washed with l0 ml histidine buffer and dried. 45Ca in the sarcoplasmic reticulum vesicles trapped on the filter discs was counted in 10 ml of a scintillation fluid containing 0.5 g (l,4-Di[2-(5-phenyl-oxazolyl)] benzene and 5 g 2,5-diphenyloxazole per litre toluene-ethylene glycol monomethyl ether (9:1). Cytochrome oxidase activity was assayed as described by Wharton & Tzagaloff (1967). Protein was determined according to Lowry et al. (1951).
482
VOLMI~
Hays
Table l Ca 2* dependence (",, inhibition by EGTA)
Relative specific activity of cytochrome oxidase
Purification step 10,000 O fraction (mitochondria) 44,000g fraction after KC1 wash 5000 O supernatant Step gradient fraction 11 15
37
100 17 10.5
65 71
5.8
8,7
dependence of total ATPase on the concentration of Ca 2 + was tested in both fractions. Since sarcoplasmic reticulum ATPase is strongly dependent on Ca 2+ in contrast to rnitochondrial ATPase, a high inhibition of ATPase by the Ca z + chelating agent EGTA* may indicate the presence of highly purified vesicles. O n the other hand, low inhibition of total ATPase may be due to the presence of mitochondrial ATPase as well as sarcoplasmic reticulum ATPase. The ATPase of the 44,0000 .fraction was considerably inhibited (65')/o) by EGTA. However, ATPase of highly purified preparations of sarcoplasmic reticulum vesicles can be inhibited up to 85°,. This indicates the presence of other, less Ca 2 +-sensitive ATPases in the 44,000 0 fraction. These results suggest a contamination with mitochondrial fragments or other organelles exhibiting ATPase activity. Therefore, further purification was necessary. This was achieved by two additional purification steps: (I) centrifugation of the 44,000 O fraction at 50000; and (2) step gradient centrifugation. After the last purification step 27 fractions of 0.8 ml were obtained. In order to identify the fractions containing purified sarcoplasmic reticulum vesicles, calcium uptake capacity, ATPase activity and cyto-
RESULTS
1. Purification of sarcoplasmic reticulum ~;esicles Sarcoplasrnic reticulum vesicles are usually prepared by fractionating homogenates of muscle tissue by centrifugation at 600-15000, 8000-115000, and 38,(XK)-44,000 0. Mitochondria are sedimented at 8000-12,5000, whereas the sarcoplasrnic reticulum vesicles remain in the supernatant. The vesicles are then sedimented at 38,000-44,000 0. A homogenate of locust flight-muscles was fractionated in this way. Specific activity of the mitochondrial enzyme cytochrome oxidase was measured in both the 10,0000 and 44,000 0 fraction in order to detect the amount of contamination with mitochondria in the latter fraction. As can be seen in Table 1, specific activity of the enzyme in the 44,000 0 fraction amounts to approx 15°';; of the specific activity in the mitochondrial fraction, i.e. contamination with mitochondria or mitochondrial fragments in the 44,0000 fraction was considerable. Furthermore, the
* EGTA~thyleneglycol-bis-(fl-aminoethyl
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Fig. l. Upper graph: Calcium uptake capacity (0.... O) and ATPase activity (O - - 0 ) in the fractions of the step gradient. ATPase peaks are numbered I to V. Lower graph: Cytochrome oxidase activity in the fractions of the step gradient. 1 milliunit (munit) is defined as the amount which causes a change in absorbancy of 0.001/min at 25"C. All enzyme assays were carried out as described in Materials and Methods.
483
Sarcoplasmic reticulum of flight-muscles chrome oxidase activity was measured. The results are shown in Fig. 1. They may be briefly summarized as follows: (1) cytochrome oxidase activity could be detected in each fraction. The level was significantly increased in the first five fractions. (2) ATPase activity appeared in five peaks (denominated peak I - V in Fig. l). More t h a n 500o of total activity was recovered in fraction 11 15 (peak III). (3) Calcium uptake capacity was very high in the same fractions (approx 80"o of total capacity). Three minor peaks were detected, clearly correlated to the ATPase peaks II, IV and V. (4j Efficiency of calcium uptake, i.e. the ratio of calcium uptake to ATPase activity, was very high in the fraction 11 15 but much lower in the other fractions. These results suggest the occurrence of purified sarcoplasmic reticulum vesicles in the fraction 11 15. This assumption is confirmed by further data concerning dependence of ATPase on the C a : ÷ concentration. Only in these fractions (peak III) is ATPase inhibited by E G T A to 80°o, whereas in the other fractions inhibition is considerably lower (50°'o or less). Furthermore, specific cytochrome oxidase activity is very low in the fraction 11-15 (Table 1). Therefore it may be concluded that purified sarcoplasmic reticulum vesicles are concentrated mainly in the 31.6~o and 29.11'o sucrose layer (fraction 11 15) of the step gradient after centrifugation. These fractions were combined and used in further experiments.
2. Properties of the A TPase EfJect of mono- and divalent cations. ATPase from locust flight-muscles is strongly dependent on m o n o and divalent cations (Table 2). In the absence of any cation only m i n i m u m activity can be detected. If either Mg 2+ or Ca 2+ are added, activity increases nearly 3-fold. If b o t h cations are present in the incubation mixture, ATPase activity is further enhanced. M a x i m u m activity will be reached by addition of K + to the incubation mixture containing already Ca 2+ and Mg 2+. K + stimulates ATPase only in the presence of b o t h Ca 2 ÷ and Mg z+, whereas the activity of ATPase is slightly decreased after addition of K + to an incubation mixture containing either Ca 2+ or Mg 2 +. By studying the dependence of ATPase on the concentration of b o t h Ca 2 + and Mg 2÷ great changes of activity can be detected. At constant concentration
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Fig. 2. Effect of Ca/+ concentration on ATPase activity. The reaction mixture contained 10raM histidine pH 7.1. 5 m M MgC12, 100mM KCI, 25/~g protein and 2.5mM ATP. Concentration of free Ca 2÷ was adjusted with calcium buffers in the 1 0 - 9 - 1 0 - 5 M range, higher concentrations by addition of CaClv Incubation was carried out at 32cC for 30min. of Mg 2÷ and K ÷, m a x i m u m ATPase activity will be measured at 5 × 10 -6 M Ca 2+ (Fig. 2). If Ca 2+ concentration is enhanced to 10 -2 M, ATPase is inhibited to more t h a n 90~o. If concentration of Ca 2+ and K ÷ is held constant, ATPase is stimulated by 1 0 m M Mg 2÷ to a m a x i m u m extent (Fig. 3). Changes of K ÷ concentration will not cause such drastic effects on the ATPase activity as observed in the case of Ca 2+ and Mg 2+. Activity reaches a m a x i m u m level at 1 0 0 m M K ÷. If concentration of K ÷ is increased to 600 mM, activity of ATPase is reduced to approximately 50~o. ATPase activity can be enhanced not only by C a : +, Mg 2+ and K ÷, but also by other m o n o - and divalent cations. These cations do not stimulate activity in the absence of b o t h Ca 2÷ and Mg 2÷. However, if one of these cations is omitted ATPase activity can be
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Table 2 Ions added (mM) None Mg 2÷ (5) Ca 2+ (0.005) K + (100) Mg 2 + (5) + K ÷ (100) Ca 2 + (0.005) + K ÷ (100) Ca2 + (0.005) + Mg 2+ (5) Ca 2+ (0.005) + Mg 2. (5) + K ÷ (100)
Relative activity (° o of maximum activity) 13 37 35 13 23
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Fig. 3. Effect of Mg 2+ concentration on ATPase activity. The incubation mixture contained 10 mM histidine pH 7.4, 100mM KCI, 5 m M Ca-EGTA, 2-100mM MgCI2, 50pg protein and 2.5 mM ATP. Final volume: 2 ml. Incubation was carried out at 32°C for 20min.
484
HANS
VOLMER
.E
Table 3 Addition (mM) None EGTA(1) EGTA(1) + EGTA(1) + EGTA(1) + EGTA(1) + EGTA (1) +
®
Relative activity (~',i of control)
Q.
100 18 30 168 21 37 38
x c
Mg z+ (0.1) Sr z+ (0.1) Ba z+ 10.1) Mn 2+ (0.1) Co 2+ (0.1)
2--
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enhanced by various other divalent cations. These data are shown in Table 3 and Table 4. As can be seen, Mg 2÷ can be substituted by Mn 2÷ and Co 2÷. The decrease of ATPase activity caused by omission of Ca z÷ can be compensated by addition of Sr 2÷. Ba 2+ shows no effect on the ATPase. Rates of activation by various concentrations of Mn 2 + and Co 2 ÷ in the absence of Mg 2 +, and of Sr 2 ÷ in the absence of Ca 2 ÷ were studied. All these cations can fully restore activity reduced by omission of Ca 2 ÷ or Mg 2÷. Mn 2÷ and Co z+ will cause maximum activation at a concentration of 1 mM. Activation by Sr 2÷ reaches a maximum level at 5 × 10 -a M. An effect of monovalent cations on ATPase can only be demonstrated in the presence of both Mg 2÷ and Ca 2÷. If K ÷ is omitted, ATPase activity can be fully restored either by Na ÷ or by NH,~, whereas Li ÷ shows shows little effect. pH-dependence. ATPase from locust flight-muscle SR exhibits a pH optimum at 7.4-7.6 (Fig. 4). At pH 6.0 and 8.5, respectively, activity is reduced to 15-20%. Effect of caffeine, ouabain and salyroan. Caffeine and ouabain did not affect ATPase to a considerable extent. On the other hand, the enzyme was inhibited completely by salyrgan (mersalyl). The dependence of ATPase activity on the concentration of salyrgan is shown in Fig. 5. Half maximum inhibition was achieved at 10 - 6 M.
o aI-<
t
I
7.0
6.0
l
80
9.0
pH Fig. 4. Dependence of ATPase activity on the pH. In this experiment 0.1 M Tris-maleate buffers were used. Other reagents: 5raM MgCI2, 50pM CaCI2, 100mM KC1, 25/~g protein and 2.5 mM ATP. Final volume: 2ml. Incubation was carried out at 32°C for 30 min, The main problem in preparing sarcoplasmic reticulum vesicles from locust flight-muscles is the removal of mitochondrial fragments. This could be partially achieved by centrifugation at 5000 g and by sucrose gradient fractionation, The latter purification step was found to be very effective. Although the occurrence of cytochrome oxidase activity in all fractions of the gradient indicates contamination by mitochondrial fragments, the bulk of mitochondrial fragments is concentrated in the heaviest layer (37.2~o sucrose) of the gradient. O n the other hand, the vesicles are enriched in the middle layers (31, 6~o and 29.1% sucrose), as indicated by the high calcium uptake capacity. That calcium uptake measured in these fractions is caused by mitochondria can be excluded, for intact mitochondria solely able to concentrate calcium should not occur in the middle region of the gradient, but only in the 10,000 g fraction. Furthermore, calcium uptake in the fractions of
DISCUSSION c:
A preparation of sarcoplasmic reticulum vesicles mostly free from mitochondrial fragments has been prepared from the flight-muscles of Locusta mi#ratoria. Because of the low protein content of this preparation further purification could not be performed. However, the biochemical data suggest a grade of purity of the preparation sufficient for the studies intended, Table 4
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x
o
~.
o
E ~k >
Ion added (mM)
Relative activity (% of control)
None Mg 2+ (5) Ca 2 + (5) Sr 2 + (5) Mn 2 + (5) Co 2+ (5) Ba 2+ (5)
100 465 148 107 488 437 111
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I LO-9
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ice
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=~6
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Fig. 5. Effect of salyrgan on ATPase activity. The reaction mixture contained 10 mM histidine pH 7.4, 5 mM MgCI2, 5 mM Ca-EGTA, 100 mM K C 1 , 20/~g protein, 10-9-10-4M salyrgan and 2.5mM ATP. Final volume: 2 ml. Incubation was carried out at 32°C for 30 min.
485
Sarcoplasmic reticulum of flight-muscles the gradient with comparatively high content of mitochondrial fragments is negligible. Though a great deal of sarcoplasmic reticulum vesicles is concentrated in the middle region of the gradient, calcium uptake could also be measured in the other fractions. This calcium uptake is clearly correlated with ATPase activity. These results suggest the occurrence of vesicles in other zones of the gradient, also. The divergent sedimentation rate of these vesicles may be explained by their different size. Calcium uptake efficiency in these vesicles seems to be considerably lower. This may be caused by additional ATPase, perhaps from sarcolemma or mitochondria. This would result in increased ATPase activity, whereas calcium uptake is not increased. Another explanation may be as follows: Defect sarcoplasmic reticulum vesicles unable to accumulate calcium, but still capable of splitting ATP, are associated with intact vesicles. In both cases the ratio of calcium uptake to ATPase activity is changed by additional ATPase simulating lower calcium uptake efficiency. Distinct calcium uptake and ATPase activity was detected in the fraction corresponding to the top of the gradient (peak V), i.e. this ATP-splitting and calcium-accumulating material will not migrate at all into the gradient even at high g forces. This phenomenon cannot be explained at the present time. Further investigations should help to clarify this problem. Studies on biochemical properties of the ATPase were performed with vesicles purified by the procedure described. Purification of the ATPase from purified sarcoplasmic reticulum vesicles could not be carried out because of the small amount of the material available. MacLennan (1970) has shown that properties of ATPase of intact vesicles will not considerably differ from properties of the enzyme purified by solubilation and ammonium acetate fractionation. Therefore purification of the enzyme from the purified vesicles does not seem to be necessary for studying its properties. ATPase from locust flight-muscle SR shows no marked differences to the enzyme prepared from rabbit skeletal muscle with regard to pH-dependence and the effects of various cations on activity. Both ATPase are inhibited completely by salyrgan in the micromolar concentration range. Various cations have been shown to affect the ATPase from locust flight-muscle SR in vitro. However, under physiological conditions only C a 2+, Mg 2+ and K ÷ should affect ATPase, for the sarcoplasmic concentration of Mg 2÷ and K ÷ is widely constant. Furthermore, it is in a range in which the ATPase is activated to a maximum extent. Therefore other cations, i.e. Mn 2÷, Co 2+, Na + and N H ~ , could not affect the ATPase. Sarcoplasmic concentration of Ca/+, however, is changed drastically by the SR in contrast to the concentration of Mg 2÷ and K ÷. Minimum concentration of Ca 2 + in the sarcoplasm is at
10 -s M. Under these conditions, Sr 2+ could substitute for Ca 2+ in vitro. However, the concentration of Sr 2 ÷ in the sarcoplasm may not be high enough to show any effect on ATPase activity. So the effect of Sr 2+, Mn 2÷, Co 2÷, Na ÷ and N H 2 should be without importance under physiological conditions. It should be noticed, however, that Mn 2÷ and Co 2÷ which can substitute for Mg 2÷ are more effective in activating the ATPase than Mg 2÷, because these two cations stimulate the ATPase to a maximum extent at concentrations considerably lower than the optimum concentration of Mg 2÷. Acknowledgement I wish to thank Professor Dr G. Beinbrech for his valuable help in the preparation of this manuscript.
REFERENCES HAAKSHORSTR. (1974) Diplomarbeit, MOnster (FRG) HASSELBACH W. (~ MAKINOSE M. (1961) Die Calciumpumpe der "Erschlaffungsgrana" des Muskels und ihre Abh~ingigkeit yon der ATP-Spaltung. Biochem. Z. 333, 518-528. HUDDART H., GREENWOODM. t~ WILLIAMS A. J. (1974) The effect of some organophosphorus and organochlorine compounds on calcium uptake by sarcoplasmic reticulum isolated from insect and crustacean skeletal muscle. J. Comp. Physiol. 93, 139 150. KIELLEY W. W. (~ MEYERHOFO. (1948) Studies on adenosine triphosphatase of muscle. II. A new magnesium-activated adenosine triphosphatase. J. biol. Chem. 176, 591 601. LOWRY O. H., ROSEBROUGHN. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. MA('LENNAN D. H. (1970) Purification and properties of an adenosine triphosphatase from sarcoplasmic reticulum. J. biol. Chem. 245, 4508-4518. MARTINOSIA. (1968) Sarcoplasmic reticulum. IV. Solubilization of microsomal adenosine triphosphatase. J. biol. Chem. 243, 71-80. MARTINOSIA. & FERRETOSR. (1964) Sarcoplasmic reticulum. I1. Correlation between adenosine triphosphatase activity and Ca 2÷ uptake. J. biol. Chem. 239, 659-668. MEISSNER G. & FLEISCHER S. (1971) Characterization of sarcoplasmic reticulum from skeletal muscle. Biochim. biophys. Acta 241, 356 378. STi3SSELW. & ZEBE E. (1968) Zur intrazellul~iren Regulation der Kontraktionsaktivitht. PfliJgers Arch. ties. Physiol. 302, 38-56. TSUKAMOTO M., NAGAI Y., MARUVAMAK. & AKITA Y. (1966) The occurrence of relaxing granules in the muscle of the locust Locusta migratoria. Comp. Biochem. Physiol. 17, 569 581. WHARTON D. C. & TZAGALOFFA. (1967) Cytochrome oxidase from beef heart mitochondria. Methods in En:ymology, Vol. X, pp. 245 250. YODA A. & HOKIN L. E. (1970) On the reversibility of binding of cardiotonic steroids to a partially purified (Na-K)-activated adenosine triphosphatase from beef brain. Biochem. biophys. Res. Commun. 40, 880-886.
0305-0491,78 0715-0481 $02.00/0
S A R C O P L A S M I C R E T I C U L U M O F THE F L I G H T - M U S C L E S O F L O C U S T A MIGRATORIA. PURIFICATION OF SARCOPLASMIC RETICULUM VESICLES A N D P R O P E R T I E S O F SARCOPLASMIC RETICULUM ATPASE HANS VOLMER Zoologisches Institut der Universit~it Mtinster, Lehrstuhl fiir Tierphysiologie, Hindenburgplatz 55. D-4400 Miinster, Federal Republic of Germany
(Received 26 September 1977) Abstract--l. Sarcoplasmic reticulum vesicles were prepared from the flight-muscles of Locusta mi9ratoria by cell fractionation and sucrose gradient fractionation. 2. Properties of the sarcoplasmic reticulum ATPase were studied. 3. The ATPase was strongly dependent on Ca z+, Mg 2+ and K ÷. 4. The ATPase exhibited a pH optimum at pH 7.5 and was inhibited completely by salyrgan. INTRODUCTION
Relaxation of skeletal muscle is elicited by a decrease in the concentration of Ca 2 + in the sarcoplasm. This change in the sarcoplasmic Ca 2 + level is caused by a protein system which is able to t r a n s p o r t C a 2+ against a high concentration gradient into a tubular n e t w o r k - - t h e sarcoplasmic reticulum (SR). The energy required for this active transport is provided by an ATPase located in the m e m b r a n e of the SR. This enzyme was first studied by Kielley & Meyerhof (1948) who succeeded in preparing purified sarcoplasmic reticulum for the first time. They found that the ATPase was activated by M g 2+ and inhibited by Ca 2 + in the millimolar range. Later on it was shown that the ATPase from sarcoplasmic reticulum could be activated by Ca 2+ in the micromolar range (Hasselbach & Makinose, 1961). Furthermore, it could be d e m o n s t r a t e d that C a 2÷ uptake and b r e a k d o w n of A T P were closely correlated. By splitting of 1 mole of ATP, 2 moles of Ca 2÷ are transported into the SR. Properties of the ATPase from sarcoplasmic reticulum were studied by Martinosi & Ferretos (1964), Martinosi (1968) and M a c L e n n a n (1970). M a c L e n n a n also succeeded in purifying the enzyme 6-fold. SR for studying the properties of the ATPase was preferentially prepared from vertebrate skeletal muscle. However, there is little information about sarcoplasmic reticulum ATPase from invertebrate muscles. Studies concerning Ca2+-uptake by the SR from muscles of various insect species have been carried out by Tsukamoto et al. (1966), St6ssel & Zebe (1968), Haakshorst (1974) and H u d d a r t et al. (1974), but so far there is no detailed information a b o u t sarcoplasmic reticulum ATPase of insect muscle. In this paper results on preparation of sarcoplasmic reticulum vesicles from the flight-muscles of Locusta migratoria and some properties of the ATPase are reported. MATERIALS A N D M E T H O D S
Preparation of sarcoplasmic reticulum vesicles Flight-muscles of 40-50 adult locusts (both sexes) were 481
homogenized in 300mM sucrose and 5 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) pH 7.4 with a glass Teflon homogenizer. Following homogenization the homogenate was centrifuged at 1_5000 (2 x 15min), 10,000y (2 x 30min) and 44,0000 for 1 hr. The 44,0000 pellet was suspended in 600mM KCI and 5 m M HEPES, pH 7.4 and stirred for 2 hr. After centrifugation at 44,000 0 the pellet was resuspended in the homogenization buffer and centrifuged at 50000 for 30min. 3-5ml of the 5000 g supernatant containing the sarcoplasmic reticulum vesicles were then placed on the top of a step gradient containing five layers with different percentages of sucrose as described by Meissner & Fleischer (1971). The gradient consisted of 4ml 37.2~., 3 ml 33.9°~,, 3 ml 31.6!!~i, 3 ml 29.1°/o and 4ml 26.4~, sucrose and 5raM HEPES, pH 7.4, respectively. After centrifugation at 75,0000 for 2.5 hr the bottom of the centrifugation tubes was pierced by a hypodermic syringe and 0.8 ml fractions were collected in graduated tubes.
Enzyme assays The incubation mixture for the ATPase assay contained l0 mM histidine pH 7.4, 100 mM KCI, 5 mM MgC12 and 5 mM Ca-EGTA in 2ml final volume. After addition of sarcoplasmic reticulum vesicles (20-50/ag protein) the mixture was preincubated at 32'C for 10min. The reaction was started by addition of 5 #moles ATP. After 10-30 min the reaction was stopped by addition of 100 ~d 500/,, trichloracetic acid. Inorganic phosphate was determined according to Yoda & Hokin (1970). Calcium uptake was assayed in an incubation mixture containing 10raM histidine pH 7.4, 100mM KCI, 5 m M MgCl2, 5 m M potassium oxalate, 0.05 mM 45CAC12 and 20-50 pg sarcoplasmic reticulum protein. The reaction was started by addition of 5/~moles ATP. Incubation was carried out at 22"C. After 10 rain a 0.4 ml sample was filtered through a Millipore ® filter (HAWP 025001. The filter discs were washed with l0 ml histidine buffer and dried. 45Ca in the sarcoplasmic reticulum vesicles trapped on the filter discs was counted in 10 ml of a scintillation fluid containing 0.5 g (l,4-Di[2-(5-phenyl-oxazolyl)] benzene and 5 g 2,5-diphenyloxazole per litre toluene-ethylene glycol monomethyl ether (9:1). Cytochrome oxidase activity was assayed as described by Wharton & Tzagaloff (1967). Protein was determined according to Lowry et al. (1951).
482
VOLMI~
Hays
Table l Ca 2* dependence (",, inhibition by EGTA)
Relative specific activity of cytochrome oxidase
Purification step 10,000 O fraction (mitochondria) 44,000g fraction after KC1 wash 5000 O supernatant Step gradient fraction 11 15
37
100 17 10.5
65 71
5.8
8,7
dependence of total ATPase on the concentration of Ca 2 + was tested in both fractions. Since sarcoplasmic reticulum ATPase is strongly dependent on Ca 2+ in contrast to rnitochondrial ATPase, a high inhibition of ATPase by the Ca z + chelating agent EGTA* may indicate the presence of highly purified vesicles. O n the other hand, low inhibition of total ATPase may be due to the presence of mitochondrial ATPase as well as sarcoplasmic reticulum ATPase. The ATPase of the 44,0000 .fraction was considerably inhibited (65')/o) by EGTA. However, ATPase of highly purified preparations of sarcoplasmic reticulum vesicles can be inhibited up to 85°,. This indicates the presence of other, less Ca 2 +-sensitive ATPases in the 44,000 0 fraction. These results suggest a contamination with mitochondrial fragments or other organelles exhibiting ATPase activity. Therefore, further purification was necessary. This was achieved by two additional purification steps: (I) centrifugation of the 44,000 O fraction at 50000; and (2) step gradient centrifugation. After the last purification step 27 fractions of 0.8 ml were obtained. In order to identify the fractions containing purified sarcoplasmic reticulum vesicles, calcium uptake capacity, ATPase activity and cyto-
RESULTS
1. Purification of sarcoplasmic reticulum ~;esicles Sarcoplasrnic reticulum vesicles are usually prepared by fractionating homogenates of muscle tissue by centrifugation at 600-15000, 8000-115000, and 38,(XK)-44,000 0. Mitochondria are sedimented at 8000-12,5000, whereas the sarcoplasrnic reticulum vesicles remain in the supernatant. The vesicles are then sedimented at 38,000-44,000 0. A homogenate of locust flight-muscles was fractionated in this way. Specific activity of the mitochondrial enzyme cytochrome oxidase was measured in both the 10,0000 and 44,000 0 fraction in order to detect the amount of contamination with mitochondria in the latter fraction. As can be seen in Table 1, specific activity of the enzyme in the 44,000 0 fraction amounts to approx 15°';; of the specific activity in the mitochondrial fraction, i.e. contamination with mitochondria or mitochondrial fragments in the 44,0000 fraction was considerable. Furthermore, the
* EGTA~thyleneglycol-bis-(fl-aminoethyl
ether)N,N'-
tetraacetic acid.
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Fig. l. Upper graph: Calcium uptake capacity (0.... O) and ATPase activity (O - - 0 ) in the fractions of the step gradient. ATPase peaks are numbered I to V. Lower graph: Cytochrome oxidase activity in the fractions of the step gradient. 1 milliunit (munit) is defined as the amount which causes a change in absorbancy of 0.001/min at 25"C. All enzyme assays were carried out as described in Materials and Methods.
483
Sarcoplasmic reticulum of flight-muscles chrome oxidase activity was measured. The results are shown in Fig. 1. They may be briefly summarized as follows: (1) cytochrome oxidase activity could be detected in each fraction. The level was significantly increased in the first five fractions. (2) ATPase activity appeared in five peaks (denominated peak I - V in Fig. l). More t h a n 500o of total activity was recovered in fraction 11 15 (peak III). (3) Calcium uptake capacity was very high in the same fractions (approx 80"o of total capacity). Three minor peaks were detected, clearly correlated to the ATPase peaks II, IV and V. (4j Efficiency of calcium uptake, i.e. the ratio of calcium uptake to ATPase activity, was very high in the fraction 11 15 but much lower in the other fractions. These results suggest the occurrence of purified sarcoplasmic reticulum vesicles in the fraction 11 15. This assumption is confirmed by further data concerning dependence of ATPase on the C a : ÷ concentration. Only in these fractions (peak III) is ATPase inhibited by E G T A to 80°o, whereas in the other fractions inhibition is considerably lower (50°'o or less). Furthermore, specific cytochrome oxidase activity is very low in the fraction 11-15 (Table 1). Therefore it may be concluded that purified sarcoplasmic reticulum vesicles are concentrated mainly in the 31.6~o and 29.11'o sucrose layer (fraction 11 15) of the step gradient after centrifugation. These fractions were combined and used in further experiments.
2. Properties of the A TPase EfJect of mono- and divalent cations. ATPase from locust flight-muscles is strongly dependent on m o n o and divalent cations (Table 2). In the absence of any cation only m i n i m u m activity can be detected. If either Mg 2+ or Ca 2+ are added, activity increases nearly 3-fold. If b o t h cations are present in the incubation mixture, ATPase activity is further enhanced. M a x i m u m activity will be reached by addition of K + to the incubation mixture containing already Ca 2+ and Mg 2+. K + stimulates ATPase only in the presence of b o t h Ca 2 ÷ and Mg z+, whereas the activity of ATPase is slightly decreased after addition of K + to an incubation mixture containing either Ca 2+ or Mg 2 +. By studying the dependence of ATPase on the concentration of b o t h Ca 2 + and Mg 2÷ great changes of activity can be detected. At constant concentration
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Fig. 2. Effect of Ca/+ concentration on ATPase activity. The reaction mixture contained 10raM histidine pH 7.1. 5 m M MgC12, 100mM KCI, 25/~g protein and 2.5mM ATP. Concentration of free Ca 2÷ was adjusted with calcium buffers in the 1 0 - 9 - 1 0 - 5 M range, higher concentrations by addition of CaClv Incubation was carried out at 32cC for 30min. of Mg 2÷ and K ÷, m a x i m u m ATPase activity will be measured at 5 × 10 -6 M Ca 2+ (Fig. 2). If Ca 2+ concentration is enhanced to 10 -2 M, ATPase is inhibited to more t h a n 90~o. If concentration of Ca 2+ and K ÷ is held constant, ATPase is stimulated by 1 0 m M Mg 2÷ to a m a x i m u m extent (Fig. 3). Changes of K ÷ concentration will not cause such drastic effects on the ATPase activity as observed in the case of Ca 2+ and Mg 2+. Activity reaches a m a x i m u m level at 1 0 0 m M K ÷. If concentration of K ÷ is increased to 600 mM, activity of ATPase is reduced to approximately 50~o. ATPase activity can be enhanced not only by C a : +, Mg 2+ and K ÷, but also by other m o n o - and divalent cations. These cations do not stimulate activity in the absence of b o t h Ca 2÷ and Mg 2÷. However, if one of these cations is omitted ATPase activity can be
o 3--
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Table 2 Ions added (mM) None Mg 2÷ (5) Ca 2+ (0.005) K + (100) Mg 2 + (5) + K ÷ (100) Ca 2 + (0.005) + K ÷ (100) Ca2 + (0.005) + Mg 2+ (5) Ca 2+ (0.005) + Mg 2. (5) + K ÷ (100)
Relative activity (° o of maximum activity) 13 37 35 13 23
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2
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Fig. 3. Effect of Mg 2+ concentration on ATPase activity. The incubation mixture contained 10 mM histidine pH 7.4, 100mM KCI, 5 m M Ca-EGTA, 2-100mM MgCI2, 50pg protein and 2.5 mM ATP. Final volume: 2 ml. Incubation was carried out at 32°C for 20min.
484
HANS
VOLMER
.E
Table 3 Addition (mM) None EGTA(1) EGTA(1) + EGTA(1) + EGTA(1) + EGTA(1) + EGTA (1) +
®
Relative activity (~',i of control)
Q.
100 18 30 168 21 37 38
x c
Mg z+ (0.1) Sr z+ (0.1) Ba z+ 10.1) Mn 2+ (0.1) Co 2+ (0.1)
2--
o
enhanced by various other divalent cations. These data are shown in Table 3 and Table 4. As can be seen, Mg 2÷ can be substituted by Mn 2÷ and Co 2÷. The decrease of ATPase activity caused by omission of Ca z÷ can be compensated by addition of Sr 2÷. Ba 2+ shows no effect on the ATPase. Rates of activation by various concentrations of Mn 2 + and Co 2 ÷ in the absence of Mg 2 +, and of Sr 2 ÷ in the absence of Ca 2 ÷ were studied. All these cations can fully restore activity reduced by omission of Ca 2 ÷ or Mg 2÷. Mn 2÷ and Co z+ will cause maximum activation at a concentration of 1 mM. Activation by Sr 2÷ reaches a maximum level at 5 × 10 -a M. An effect of monovalent cations on ATPase can only be demonstrated in the presence of both Mg 2÷ and Ca 2÷. If K ÷ is omitted, ATPase activity can be fully restored either by Na ÷ or by NH,~, whereas Li ÷ shows shows little effect. pH-dependence. ATPase from locust flight-muscle SR exhibits a pH optimum at 7.4-7.6 (Fig. 4). At pH 6.0 and 8.5, respectively, activity is reduced to 15-20%. Effect of caffeine, ouabain and salyroan. Caffeine and ouabain did not affect ATPase to a considerable extent. On the other hand, the enzyme was inhibited completely by salyrgan (mersalyl). The dependence of ATPase activity on the concentration of salyrgan is shown in Fig. 5. Half maximum inhibition was achieved at 10 - 6 M.
o aI-<
t
I
7.0
6.0
l
80
9.0
pH Fig. 4. Dependence of ATPase activity on the pH. In this experiment 0.1 M Tris-maleate buffers were used. Other reagents: 5raM MgCI2, 50pM CaCI2, 100mM KC1, 25/~g protein and 2.5 mM ATP. Final volume: 2ml. Incubation was carried out at 32°C for 30 min, The main problem in preparing sarcoplasmic reticulum vesicles from locust flight-muscles is the removal of mitochondrial fragments. This could be partially achieved by centrifugation at 5000 g and by sucrose gradient fractionation, The latter purification step was found to be very effective. Although the occurrence of cytochrome oxidase activity in all fractions of the gradient indicates contamination by mitochondrial fragments, the bulk of mitochondrial fragments is concentrated in the heaviest layer (37.2~o sucrose) of the gradient. O n the other hand, the vesicles are enriched in the middle layers (31, 6~o and 29.1% sucrose), as indicated by the high calcium uptake capacity. That calcium uptake measured in these fractions is caused by mitochondria can be excluded, for intact mitochondria solely able to concentrate calcium should not occur in the middle region of the gradient, but only in the 10,000 g fraction. Furthermore, calcium uptake in the fractions of
DISCUSSION c:
A preparation of sarcoplasmic reticulum vesicles mostly free from mitochondrial fragments has been prepared from the flight-muscles of Locusta mi#ratoria. Because of the low protein content of this preparation further purification could not be performed. However, the biochemical data suggest a grade of purity of the preparation sufficient for the studies intended, Table 4
¢) o
x
o
~.
o
E ~k >
Ion added (mM)
Relative activity (% of control)
None Mg 2+ (5) Ca 2 + (5) Sr 2 + (5) Mn 2 + (5) Co 2+ (5) Ba 2+ (5)
100 465 148 107 488 437 111
g_
I LO-9
J
ice
i(r Concentrcltion,
=~6
id5
1~4
M
Fig. 5. Effect of salyrgan on ATPase activity. The reaction mixture contained 10 mM histidine pH 7.4, 5 mM MgCI2, 5 mM Ca-EGTA, 100 mM K C 1 , 20/~g protein, 10-9-10-4M salyrgan and 2.5mM ATP. Final volume: 2 ml. Incubation was carried out at 32°C for 30 min.
485
Sarcoplasmic reticulum of flight-muscles the gradient with comparatively high content of mitochondrial fragments is negligible. Though a great deal of sarcoplasmic reticulum vesicles is concentrated in the middle region of the gradient, calcium uptake could also be measured in the other fractions. This calcium uptake is clearly correlated with ATPase activity. These results suggest the occurrence of vesicles in other zones of the gradient, also. The divergent sedimentation rate of these vesicles may be explained by their different size. Calcium uptake efficiency in these vesicles seems to be considerably lower. This may be caused by additional ATPase, perhaps from sarcolemma or mitochondria. This would result in increased ATPase activity, whereas calcium uptake is not increased. Another explanation may be as follows: Defect sarcoplasmic reticulum vesicles unable to accumulate calcium, but still capable of splitting ATP, are associated with intact vesicles. In both cases the ratio of calcium uptake to ATPase activity is changed by additional ATPase simulating lower calcium uptake efficiency. Distinct calcium uptake and ATPase activity was detected in the fraction corresponding to the top of the gradient (peak V), i.e. this ATP-splitting and calcium-accumulating material will not migrate at all into the gradient even at high g forces. This phenomenon cannot be explained at the present time. Further investigations should help to clarify this problem. Studies on biochemical properties of the ATPase were performed with vesicles purified by the procedure described. Purification of the ATPase from purified sarcoplasmic reticulum vesicles could not be carried out because of the small amount of the material available. MacLennan (1970) has shown that properties of ATPase of intact vesicles will not considerably differ from properties of the enzyme purified by solubilation and ammonium acetate fractionation. Therefore purification of the enzyme from the purified vesicles does not seem to be necessary for studying its properties. ATPase from locust flight-muscle SR shows no marked differences to the enzyme prepared from rabbit skeletal muscle with regard to pH-dependence and the effects of various cations on activity. Both ATPase are inhibited completely by salyrgan in the micromolar concentration range. Various cations have been shown to affect the ATPase from locust flight-muscle SR in vitro. However, under physiological conditions only C a 2+, Mg 2+ and K ÷ should affect ATPase, for the sarcoplasmic concentration of Mg 2÷ and K ÷ is widely constant. Furthermore, it is in a range in which the ATPase is activated to a maximum extent. Therefore other cations, i.e. Mn 2÷, Co 2+, Na + and N H ~ , could not affect the ATPase. Sarcoplasmic concentration of Ca/+, however, is changed drastically by the SR in contrast to the concentration of Mg 2÷ and K ÷. Minimum concentration of Ca 2 + in the sarcoplasm is at
10 -s M. Under these conditions, Sr 2+ could substitute for Ca 2+ in vitro. However, the concentration of Sr 2 ÷ in the sarcoplasm may not be high enough to show any effect on ATPase activity. So the effect of Sr 2+, Mn 2÷, Co 2÷, Na ÷ and N H 2 should be without importance under physiological conditions. It should be noticed, however, that Mn 2÷ and Co 2÷ which can substitute for Mg 2÷ are more effective in activating the ATPase than Mg 2÷, because these two cations stimulate the ATPase to a maximum extent at concentrations considerably lower than the optimum concentration of Mg 2÷. Acknowledgement I wish to thank Professor Dr G. Beinbrech for his valuable help in the preparation of this manuscript.
REFERENCES HAAKSHORSTR. (1974) Diplomarbeit, MOnster (FRG) HASSELBACH W. (~ MAKINOSE M. (1961) Die Calciumpumpe der "Erschlaffungsgrana" des Muskels und ihre Abh~ingigkeit yon der ATP-Spaltung. Biochem. Z. 333, 518-528. HUDDART H., GREENWOODM. t~ WILLIAMS A. J. (1974) The effect of some organophosphorus and organochlorine compounds on calcium uptake by sarcoplasmic reticulum isolated from insect and crustacean skeletal muscle. J. Comp. Physiol. 93, 139 150. KIELLEY W. W. (~ MEYERHOFO. (1948) Studies on adenosine triphosphatase of muscle. II. A new magnesium-activated adenosine triphosphatase. J. biol. Chem. 176, 591 601. LOWRY O. H., ROSEBROUGHN. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. MA('LENNAN D. H. (1970) Purification and properties of an adenosine triphosphatase from sarcoplasmic reticulum. J. biol. Chem. 245, 4508-4518. MARTINOSIA. (1968) Sarcoplasmic reticulum. IV. Solubilization of microsomal adenosine triphosphatase. J. biol. Chem. 243, 71-80. MARTINOSIA. & FERRETOSR. (1964) Sarcoplasmic reticulum. I1. Correlation between adenosine triphosphatase activity and Ca 2÷ uptake. J. biol. Chem. 239, 659-668. MEISSNER G. & FLEISCHER S. (1971) Characterization of sarcoplasmic reticulum from skeletal muscle. Biochim. biophys. Acta 241, 356 378. STi3SSELW. & ZEBE E. (1968) Zur intrazellul~iren Regulation der Kontraktionsaktivitht. PfliJgers Arch. ties. Physiol. 302, 38-56. TSUKAMOTO M., NAGAI Y., MARUVAMAK. & AKITA Y. (1966) The occurrence of relaxing granules in the muscle of the locust Locusta migratoria. Comp. Biochem. Physiol. 17, 569 581. WHARTON D. C. & TZAGALOFFA. (1967) Cytochrome oxidase from beef heart mitochondria. Methods in En:ymology, Vol. X, pp. 245 250. YODA A. & HOKIN L. E. (1970) On the reversibility of binding of cardiotonic steroids to a partially purified (Na-K)-activated adenosine triphosphatase from beef brain. Biochem. biophys. Res. Commun. 40, 880-886.