“WI 31934 EFFECT OF pH AND TEMPERATURE UPON CALCIUM ACCUMULATIDN AND RELEASE BY BEEF AND RABBIT SARCDPLASMIC RETICULUM AND MITOCHDNDRIA By Daren Paui Cornforth A DISSERTATION .Submitted to Michigan State University in partiai fuifiiiment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1978 6/49“” ABSTRACT EFFECT OF pH AND TEMPERATURE UPON CALCIUM ACCUMULATION AND RELEASE BY BEEF AND RABBIT SARCOPLASMIC RETICULUM AND MITOCHONDRIA By Daren Cornforth The objectives of this study were to determine the relative abilities of red and white muscle sarcoplasmic reticulum (SR) and mitochondria to accumulate and release Ca++ under conditions known to exist in cold shortened muscle. The SR and mitochondria were isolated immediately after slaughter from beef Sternomandibularis (red) and rabbit Longissimus dorsi (white) muscle. Isolation was accomplished by homogenization of the muscle followed by differential centrifugation. Further purification of the SR was achieved by sucrose density gradient centrifugation. The yield of SR was 62 i 8 ug/gram of beef muscle as compared to lBO i 40 ug/gram for rabbit muscle. The yield of mitochondria from the two muscles was similar. However, histochemical staining for NADH-tetrazolium reductase acti- vity showed that the beef muscle contained a much higher concentration of mitochondria. Transmission electron micros- copy and $08 gel electrophoresis showed that the SR prepara- tions were essentially free from contamination. Electron microscopy similarly showed that mitochondrial preparations Daren Cornforth consisted primarily of mitochondria, and manometric measure- ment of oxygen consumption demonstrated that they contained actively respiring mitochondria. Ca++ accumulation was determined by using radio- active calcium (45Ca++). After Millipore filtration, the quantity of Ca++ accumulated by the SR or mitochondria was determined by measuring the radioactivity remaining in the filtrate. Ca++ release from preloaded SR or mitochon- dria due to changes in pH, temperature or oxygen content of the incubation medium was determined by comparison of the Ca++ content of the membranes before and after imposing conditions causing Ca++ release. The SR from both beef and rabbit muscle accumulated in excess of 500 nmol CaTT/mg protein at pH 7.3 and 37°C. Mitochondria from both muscles accumulated more than 400 nmol of Ca++lmg protein under similar conditions. Ca++ accumulation by both SR and mitochondrial suspensions was markedly temperature dependent. At 0°C, preparations from red or white muscle accumulated less than 80 nmol Ca++lmg protein, regardless of the pH. However, chilled SR and mitochondria retained the ability to accumulate significant quantities of Ca++, if the medium was warmed to 37°C. Ca++ accumulation by SR and mitochondria from both red and white muscle was also observed to be sensitive to pH. Low pH values, in the range 5.0 to 5.5 reduced the Ca++ accumulating capacity of both SR and mitochondria to O to 40 nmol Ca++lmg protein. Mitochondria accumulated significant Daren Cornforth quantities of Ca++ at pH 6.2 and 37°C, but Ca++ accumumula- tion was maximal for both SR and mitochondria in the pH range 6.8 to 7.3 Rabbit mitochondria accumulated somewhat more Ca++ under anaerobic conditions than beef mitochondria, but both 4. under preparations accumulated significant amounts of Ca+ anaerobic conditions or in the presence of the uncoupling agent, 2,4-dinitrophenol. This suggested that some mito- chondrial Ca++ accumulation was supported by ATP hydrolysis. Preloaded mitochondria from both beef and rabbit muscle released only small amounts of Ca++ when nitrogen was bub- bled through the medium. Nevertheless, the quantities released were sufficient to initiate shortening in intact muscle. Chilling of SR and mitochondria from both beef and rabbit muscle also caused the release of small but physiolo- gically significant quantities of Ca++. On lowering the pH to 5.0, virtually all of the initial Ca++ load was released. Since SR and mitochondria from red and white muscles did not differ in their response to conditions promoting cold shortening, it was concluded that cold shortening is related to the quantities of SR and mitochondria present in the muscle. The relationship of the SR and mitochondria to cold shortening was discussed, and a mechanism of cold shortening was proposed describing how postmortem muscle pH, temperature and anaerobic conditions influence the phenomenon. ACKNOWLEDGEMENTS The author expresses his sincere appreciation to Dr. A.M. Pearson for guidance and advice throughout the course of graduate study and during the preparation of the thesis. He is also indebted to Dr. R.A. Merkel, Dr. G.R. Hooper, and Dr. R.B. Young for their assistance during the course of this investigation, and to Dr. R.A. Merkel and Dr. G.R. Hooper for serving as members of the author’s guidance committee. Thanks are also extended to Dr. J.R. Brunner and Dr. S.D. Aust for serving as members of the guidance committee. The author further wishes to acknowledge the assistance of Dr. M.A. Porzio with gel electrophoresis, and Mr. Toyoteru Kanda for his help with the preparation and characterization of the sarcoplasmic reticulum fractions used in the present study. ii TABLE OF CONTENTS Page LIST OF TABLES. . . . . . . . . . . . . . . . . . . . vi LIST OF FIGURES . . . . . . . . . . . . . . . . . . . vii LIST OF APPENDIX FIGURES. . . . . . . . . . . . . . . x c—l INTRODUCTION. LITERATURE REVIEW . COLD SHORTENING . . . . Cold Shortening and Rigor . . Possible Mechanisms of Cold Shortening. SARCOPLASMIC RETICULUM. . Model for the Organization of Intact Sarco- plasmic Reticulum . . . The Contraction- Relaxation Cycle. Characterization of Sarcoplasmic Reticulum Vesicles. . Protein Composition of the Sarcoplasmic Reti- culum Vesicles. . Characterization of the SR- ATPase Molecule. Characterization of Calsequestrin and High Affinity CaTT- -Binding Protein . Lipid Composition of the Sarcoplasmic Reticu- lum Vesicles. . Model of the Organization of the SR Vesicles. Role of the Phosphoprotein Intermediate in Ca TT Transport. . Mechanism of Ca TT Transport in Sarcoplasmic Reticulum Vesicles. . . . . Evidence for Two Functional States of the ATPase Protein. . . . CaTT Accumulation by Sarcoplasmic Reticulum Vesicles. . . . . . . . Ca TT Release by Sarcoplasmic. Reticulum Vesicles. . . . . . Effects of pH and Temperature on CaTT Trans- port by Sarcoplasmic Reticulum. . . . . CaTT Accumulation by Sarcoplasmic Reticulum Vesicles of Postmortem Muscle . . . . . N N ._.I _.| —J —-l .._a_.a ._a —l—l —a h N \O O \J 05 45-5 (A) NN O mN \Imwww N 01 MITOCHONDRIA . . . . . Comparison of Sarcoplasmic Reticulum from Red and White Muscles. . . . . . . . . . . Massive Loading of Ca::. . . . . . . . . . . Limited Loading of CaT . . . . . . . . . . ATP- Supported Ca TT Accumulation. . . . . Proton Movement During Ca++ Accumulation Comparison of Heart and Liver Mitochondria Mitochondrial CaT Release . . Identity of Mitochondrial CaTT Binding Sites. . . . CaTT Transport and the Mechanism of Oxida- tive Phosphorylation . . Regulation of CaT TT Movements by Mitochon- dr a . . . Intracellular Localization of Ca++ . MATERIALS AND METHODS. . . T . . . RESULTS Isolation of Sarcoplasmic Reticulum. Isolation of Mitochondria. . . . . . . Protein Determination. . . A Manometric Assay for Mitochondrial Succi- nate Oxidase . . . Measurement of Mitochondrial Respiration Determination of Ca TT Accumulation in Sarco- plasmic Reticulum. Determination of Ca++ Accumulation by Mito- chondria . . 4+. Determination of Ca Release in Sarcoplas- mic Reticulum Vesicles or Mitochondria . Determination of Radioactivity . . . SDS Gel Electrophoresis of Beef and. Rabbit Sarcoplasmic Reticulum . . Transmission Electron Microscopy Muscle Fiber Type Determination. AND DISCUSSION . . . Cold Shortening in Beef. and Rabbit Muscle. Purity and Characterization of Sarcoplasmic Reticulum Preparations . . Purity and Characterization of Mitochondrial Preparations . . . Yield of Sarcoplasmic Reticulum from Beef and Rabbit Muscle. . . Yield of Mitochondria from Beef and. Rabbit Muscle . . . + Accumulation of CaT by Sarcoplasmic Reti- culum Vesicles . . . . . . Effects of Temperature of CaTT Accumulation by Sarcoplasmic Reticulum Vesicles . . . iv Effects of Temperature on Ca+ Release by Sarcoplasmic Reticulum Vesicles. . . . . 85 Effects of pH on Ca Accumulation by Sarco- plasmic Reticulum Vesicles . . . . 88 Effects of pH on Ca++ Release by SarCoplas- mic Reticulum Vesicles . . . . . . . . . 90 Effects of Protein Concentration on Mito-. chondrial Ca++ Accumulation. . . 90 Role of ATP in Mitochondrial CaTT Accumula- tion . . . . . . . 92 Effects of Nitrogen, Air, or 2, 4- dinitro- phenol on Mitochondrial Ca TT Accumulation. 95 Effects of Nitrogen on Mitochondrial CaT Release. . . . . lOO Influence of Temperature on Mitochondrial Ca+ +Accumulation. . . . l04 Influence of Temperature on Mitochondrial Ca TT Release . . . l05 Influence of pH on Mitochondrial CaTT Accu- mulation . . . . . . 108 Influence of pH on Mitochondrial CaTT Release. . . . l09 Effects of pH on CaTT Accumulation by .Mito- chondria and Sarcoplasmic Reticulum. . . . 109 Effects of Temperature of CaTT Accumulation by Mitochondria and Sarcoplasmic Reticu- lum. . . lll Reversibility .of Cold Shortening . ll3 An Explanation of Cold Shortening. ll5 SUMMARY. . . . . . . . . . . . . . . . . . . . . . . ll9 REFERENCES . . . . . . . . . . . . . . . . . . . . . l23 APPENDICES . . . . . . . . . . . . . . . . . . . . . I36 -Table LIST OF TABLES CaTT Accumulation by Sarcoplasmic Reticulum Vesicles from Red, White and Cardiac Muscle. Manometric Assay for Mitochondrial Succinate Oxidase (nmol succinate oxidized/mg protein/ minute). . . . . . . . . . Manometric Assay for Mitochondrial Respiration (nmol 02 uptake/mg protein/minute) CaTT Accumulation and Release by Beef Sarcoplas- mic Reticulum Vesicles (nmol Ca++/mg protein). CaTT Accumulation and Release by Rabbit Sarco- plasmic Reticulum Vesicles (nmol CaTT /mg protein) . . . . . Ca++ Accumulation and Release by Beef Mitochon- dria (nmol Ca TT/mg protein). . . . . Ca++ Accumulation+ and Release by Rabbit Mito- chondria (nmol CaT T/mg protein). vi Page 27 7O 71 83 84 96 97 Figure 10 LIST OF FIGURES Three dimensional reconstruction of the sarcoplasmic reticulum (SR) of frog sar- torius muscle. . . . . . . . . . . A model for the arrangement of the protein and lipid components in sarcoplasmic reticulum vesicles . . . . . . . . . . Flow sheet of procedure used in the isolation of sarcoplasmic reticulum vesicles Flow sheet of procedure used in the isolation of mitochondria. Standard curve for the determination of pro- tein by the method of Lowry gt 31. (1951). Flow sheet of the procedure used for the determination of CaTT accumulation Standard plot of log molecular weight and relative mobility of 10% acrylamide SDS-gel with 0.10% crosslinker: 1. myosin - 200,000; 2. v-globulin (unreduced) - 160,000; 3. muscle C-protein - 140,000; 4. B-galactosidase - 130,000; 5. a-actinin - 102,000; 6. bovine serum albumin - 68,000; 7. catalase - 60,000; 8. ovalalbumin - 43,000; 9. glyceraldehyde dehydrogenase - 36,000; 10. myokinase - 21,500; 11. haemoglobin - 15,500 . Beef and rabbit muscle strips chilled to 15 or 0°C immediately postmortem. . . . . . . . . SDS gels of beef and rabbit myofibrils and sarcoplasmic reticulum. The gels were 10.0% acrylamide, with 0.10% crosslinker . . . . . Transmission electron micrograph of beef sarcoplasmic reticulum vesicles. 44,800 X . Page 15 43 47 49 53 58 64 65 67 Figure 11 12 13 14 15 16 17 18 19 20 21 22 23 Page Transmission electron micrograph of rabbit sarcoplasmic reticulum vesicles. 44,800 X. . 68 Transmission electron micrograph of beef mitochondria. 28,000 X . . . . . . . . . . . 73 Transmission electron micrograph of rabbit mitochondria. 28,000 X . . . . . . . . . . . 74 Light micrograph of beef Sternomandibularis muscle. 440 X. . . . . . . . . . . . . . . . 78 Light micrograph of rabbit Longissimus dorsi muscle. 440 X. . . . . . . . . . . . . . . . 79 CaTT accumulation by sarcoplasmic reticulum vesicles at pH 7.3 and 37°C as a function of protein concentration. The reaction was allowed to proceed for 4 minutes. . . . . . . 81 Ca++ accumulation and release by beef sarco- plasmic reticulum vesicles as affected by temperature and pH. . . . . . . . . . . . . . 86 Ca++ accumulation and release by rabbit sarcoplasmic reticulum vesicles as affected by temperature and pH . . . . . . . . . . . . 87 CaTT accumulation by mitochondria at pH 7.3 and 37°C as a function of protein concentra- tion. The reaction was allowed to proceed for 4 minutes . . . . . . . . . . . . . . . . 91 Ca++ accumulation by beef mitochondria as affected by ATP, 2,4-dinitrophenol and anaerobic conditions. Incubation was carried out at pH 7.3 and 37°C. . . . . . . . 93 CaTT accumulation by rabbit mitochondria as affected by ATP, 2,4-dinitrophenol and anaerobic conditions. Incubation was carried out at pH 7.3 and 37°C. . . . . . . . 94 Ca++ release by beef mitochondria at pH 7. 3 and 37°C in the presence of nitrogen. . . . . lOl Ca+ +release by rabbit mitochondria at pH 7. 3 and 37°C in the presence of nitrogen. . . . 102 viii Figure 24 25 26 27 Ca++ accumulation and release by beef mito- chondria as affected by temperature and pH. Ca++ accumulation and release by rabbit mitochondria as affected by temperature and pH. . . . . . . . . . . . Effect of pH on the Ca++ accumulating ability of sarcoplasmic reticulum vesicles and migochondria of beef and rabbit muscle at 37 C. Effect of temperature on the CaTT accumula- ting ability of sarcoplasmic reticulum vesicles and mitochondria of beef and rabbit muscle at pH 7.3. ix Page 106 107 110 112 Table LIST OF APPENDIX Schedule for the preparation dehyde fixative solution. Schedule for the preparation wash buffer . . . . . . Schedule for the preparation resin . . . . . . . . . . Schedule for the preparation citrate stain . . . . Schedule for the preparation tetroxide fixative solution Schedule for the preparation stain . . . . . . . . . . . of glutaraldehyde TABLES of 1.25% glutaral- of Epon-Araldite of Reynolds lead of a 1% osmium of uranyl acetate Page 136 136 137 137 138 138 INTRODUCTION Cold shortening routinely occurs when beef or lamb carcasses are chilled after slaughter, resulting in a signi- ficant increase in muscle toughness (Marsh gt _L., 1974). Meat from animals containing a high proportion of white mus- cles (rabbits and pigs) do not cold shorten appreciably (Locker and Hagyard, 1963; Marsh gt 11., 1972). Cold shor- tening may be avoided by delaying chilling until after the muscles have gone into rigor, usually about 16 to 24 hours postmortem. Locker and Daines (1976) showed that the toughness associated with cold shortened meat may be avoided by rewarming the carcass to 37°C during the last stages of rigor, but this procedure has not been widely accepted by the meat industry. Fat cattle are generally thought to pro- duce high quality, tender meat. It has been suggested that this may be due to inhibition of cold shortening as a result of the excessive fat covering insulating the carcass, thus delaying chilling until the muscles have passed into rigor. Better methods for avoiding cold shortening could lead to the production of high quality tender meat from thinner cattle, which can be produced more cheaply. Pearson gt _1. (1973) demonstrated that microinjec- tions of Ca++ resulted in massive shortening, which was accompanied by toughening. They postulated that both cold shortening and thaw rigor are due to the ineffectiveness of the sarcoplasmic reticulum in retaining Ca++ ions at cold temperatures. Davey and Gilbert (1974) also theorized that the sarcoplasmic reticulum membrane releases Ca++ when chilled to 0°C, and is responsible for cold shortening. Buege and Marsh (1975) found that oxygen inhibited cold shortening in chilled beef strips. They postulated that anaerobic conditions cause the release of Ca++ from mito- chondria, and at low temperatures the sarcoplasmic reticulum is unable to accumulate Ca++, thus causing shortening. Buege and Marsh (1975) pointed out that red muscles contain more mitochondria than white muscles, accounting for the observation that red muscles cold shorten more extensively than white (Locker and Hagyard, 1963). Many changes occur in cold shortened muscle, including decreases in pH, temperature, ATP and oxygen levels (Kasten- Schmidt, 1970). The ability 6f muscle mitochondria to accumulate Ca++ under these conditions has never been fully documented. Consequently, the present study was undertaken to determine the relative abilities of mitochondria and sarcoplasmic reticulum from red and white muscle to accumu- late or release Ca++ under the influence of pH and tempera- ture conditions known to exist in cold shortened muscle. LITERATURE REVIEW I; COLD SHORTENING Cold Shortening and Rigor Locker and Hagyard (1963) were the first to report the cold shortening phenomenon. They observed that beef Sterno- mandibularis muscle strips shortened up to 47% when chilled to 0°C immediately postmortem. They also found that beef Longissimus dorsi and Psoas major muscle strips shortened 43% and 39%, respectively, whereas the same muscle strips from rabbit shortened only 9% and 7%. Shortening was com- pleted by 15 to 24 hours. Minimum shortening (10% or less) was observed in the temperature range of—14o to 19°C. Locker and Hagyard (1963) further observed that high tempe- rature shortening occurred in beef Sternomandibularis muscle strips, which shortened by 26% at 37°C. At higher tempera- tures, shortening was observed to coincide with the onset of rigor mortis (rigor shortening), but cold shortening began almost immediately upon chilling (Locker and Hagyard, 1963). Locker and Hagyard (1963) also found that upon rewar- ming, the cold shortening effect was reversible. Recooling caused the muscle to contract again, but both effects were diminished as postmortem time increased. The ability of the muscle to cold shorten persisted up to the onset of rigor, although the speed and degree of shortening diminished with time (Locker and Hagyard, 1963). As pointed out by Davey and Gilbert (1975), muscle is living tissue until it enters rigor mortis. 'As such, it can be stimulated to contract by cold. 1. (1967) compared cold shortening with Busch 3;. rigor shortening in beef muscle. They found that cold shor- tening begins much sooner than rigor shortening; that more tension develops during cold shortening; that cold shor- tening occurs in the presence of 5-6 mM ATP, while rigor shortening occurs only after the ATP level drops to 1 mM or less; and that cold shortening begins while the muscle pH is above 6.0, whereas rigor shortening begins at muscle pH values below 6.0. Cold shortened muscle is significantly tougher than non-cold shortened muscle (Marsh ££._l-: 1974; Locker and Daines, 1976), apparently due to the sarcomere shortening (Marsh and Carse, 1974; Marsh 2L.il-: 1974). It is well established that meat tenderness is influenced by the degree of sarcomere shortening (Marsh and Leet, 1966; Marsh and Carse, 1974). After the muscle enters rigor, the sarcomere length remains unchanged (Busch gt 11., 1972). However, Locker and Daines (1976) observed that raising the tempera- ture to 37°C in the final stages of rigor completely nulli- fies the toughness seen in cold shortened meat, without affecting shortening. Dayton 35_ 1. (1976) suggested that the tenderness associated with aged meat is due to endogenous proteolytic myofibrillar degradationat the level of the Z- line. However, Locker and Daines (1976) observed that the Z-lines were intact in cold shortened samples held at 370 for 7 hours. These same investigators suggested that the 37°C temperature treatment may modify the actin-myosin bonds of the muscle, resulting in the improved tenderness effect. Extensive cold shortening has also been found to have a tenderizing effect on muscle (Marsh 33 31., 1974), apparently due to the presence of supercontracted areas alternating with extensively stretched and torn sarcomeres (Marsh gt_al., 1974; Weidemann gt 11., 1967; Hsieh gt_al,, 1978). Possible Mechanisms of Cold Shortening Ca++ has been shown to activate muscle contraction in living muscle (Podolsky and Constantin, 1964; Podolsky, 1975). Davey and Gilbert (1974) demonstrated that Chilling prerigor muscle from 15 to 0°C increased the concentration of Ca++ by thirty to forty fold in the myofibrillar region. They concluded that chilling caused the release of Ca++ from the sarcoplasmic reticulum, which is the membraneous system responsible for Ca++ accumulation and storage in living muscle (Peachey, 1970). Martonosi and Feretos (1964) previously had shown that lower temperatures substantially reduce the activity of the Ca++ pump of the sarcoplasmic reticulum. Davey and Gilbert (1974) cited evidence (Gurd, 1963; Caputo, 1968) that cell membrane phospholipids undergo distinct temperature dependent phase transitions, producing membranes of changed pore size and electrical properties. With increased porosity, the Ca++ pump would be less able to stem the flow of Ca++ from the leaky reticulum. Thus, Davey and Gilbert (1974) ascribed the variable response of dif- ferent muscles to chilling as being due to the extent of development of the sarcoplasmic reticulum. Cold shortening is less likely to occur in fast acting, white muscles that are rich in sarcoplasmic reticulum (Revel, 1964) as compared to the slow red muscles which contain less sarcoplasmic reticulum (Hasselbach, 1964). Buege and Marsh (1975) have proposed that the muscle mitochondria are involved in cold shortening. They pointed out that those muscles that are obviously red in color cold shorten extensively. Chilling of the pale muscles of the rat and rabbit provokes little or no shortening (Locker and Hagyard, 1963; Hill, 1972). Red muscles have a much higher mitochondrial content than white muscles (Gauthier, 1970). Buege and Marsh (1975) found that thin strips of beef Sternomandibularis muscle chilled to 2°C in a nitrogen atmosphere shortened an average of 22%, but strips held in an oxygen atmosphere shortened much less. They found that the oxygen supression of cold shortening could be overcome by mitochondrial uncoupling or respiration inhibiting agents, including dinitrophenol, ruthenium red, dicumarol, and carbonyl cyanide m-chlorophenylhydrazone. These reagents had no significant effect on the length of muscle strips held at 20°C, but at 2°C they caused up to 40% shortening. Buege and Marsh (1975) further observed that strips of rabbit £5913 muscle (a white muscle), shortened only 3% when chilled in a nitrogen atmosphere or in the presence of ruthenium red or dinitrophenol. Mitochondria are a significant reservoir of intracel- lular CaTT (Popescu _e_t 11., 1976), and isolated liver mito- chondria have a large capacity for respiration linked Ca++ accumulation (Carafoli, 1975). Isolated liver mitochondria release calcium slowly when incubated anaerobically, and more rapidly when treated with uncoupling agents (Lehninger gt 11., 1967). Drahota gt 31. (1965) have shown that liver mito- chondria release Ca++ in the presence of respiratory inhibi- tors, or in the absence of a respiratory substrate. On this basis, Buege and Marsh (1975) concluded that cold shortening is a consequence of anoxia-induced Ca++ release from muscle mitochondria at a temperature low enough to prevent compen- sating Ca++ uptake by the temperature sensitive sarcoplasmic reticulum. II. SARCOPLASMIC RETICULUM Model for the Organization of Intact Sarcoplasmic Reticulum The sarcoplasmic reticulum (SR) is an extensive intra- cellular membrane system of muscle cells that functions as the storage site for Ca++ in the resting muscle fiber (Peachey, 1970). Peachey (1970) further stated that the sarcoplasmic reticulum is the organelle responsible for the release of Ca++ during activation of muscle contraction, and . ++ . . lS responsible for Ca accumulation during muscle relaxation. Peachey (1970) proposed a model (Figure l) for the organization of the sarcoplasmic reticulum system. According to this model, the sarcoplasmic reticulum consists of the terminal cisternae, the longitudinal tubules and the fenestrated collar. Peachey (1970) pointed out that the T-tubule system, which is a branching membrane system con- tinuous with the cell membrane, is not a part of the SR membrane system. Peachey (1970) showed that in frog muscle, the T (transverse) tubules form regular parallel arrays that cross the myofibrils at the level of the Z band. According to the model proposed by Peachey (1970), the ter- minal cisternae of the SR are located on both sides of the T tubule, forming the "triad". The longitudinal tubules extend from the terminal cisternae along the myofibril and connect with adjacent longitudinal tubules in the region called the "fenestrated collar", which encircles each sarco- mere of the myofibril (Peachey, 1970). Peachey (1970) also pointed out that larger or more quickly contracting muscle cells contained more extensively developed SR and T tubule membrane systems. The Contraction-Relaxation Cycle Huxley (1964) first observed that frog muscles soaked in ferritin prior to electron microscopic observation con- tained ferritin within the T tubules. Since ferritin cannot penetrate intact membranes, Huxley (1964) concluded that the T tubules are continuous with the surface membrane, and are thus responsible for carrying nerve impulses to the interior Myofibrils T system SR cisternae Longitudinal tubules of SR T r / SR cisternae '. mun " T - ‘ l .0 o. - "F9 t n ' ': '.'. g ,1 n g 2.,— ".5136“ [g M BAND ‘ 'n‘ .533; 2:. . '1'. . pug! wur = ‘ ‘ '= -i . Longitudinal tubules of SR SR cisternae T System TRIAD P—SARCONERE—d '-—A BAND—d Fig. 1. Three dimensional reconstruction of the sarco las ‘ . mic reticulum (SR) of frog sartorius muscle. p (PePChey. 1970) 10 of the muscle cell. Although the SR and the T tubules are closely coupled in the region of the triad, the membranes are not continuous, and a signal must be transmitted between them at specific junctional sites (Franzini-Armstrong, 1975). Depolarization of the T tubules does cause release of Ca++ from the SR system, by an unknown mechanism (Podolsky, 1975). Small quantities of added Ca++ also cause a release of Ca?+ from the SR, and it has been postulated that the release of small quantities of Ca++ from the T tubules during depolarization result in a more massive release of Ca++ from the SR (Podol- sky, 1975; Endo, 1977). After Ca++ is released into the myofibrillar region, Ca++ is bound to the protein troponin in the myofibrillar structure (Ebashi £3.ll-: 1969), and releases constraints on the interaction between actin and myosin, permitting muscle contraction (MacLennan, 1975). After nervous stimu- lation ceases, the sarcoplasmic reticulum accumulates Ca++ again through the action of its' Ca++ transport ATPase enzyme (MacLennan, 1975). MacLennan (1975) pointed out that the Ca++-troponin complex has a dissociation constant of 3 on, but the ATPase of SR has a Km for CaTT of 0.3 uM (Hasselbach, 1964). Therefore, Ca++ is drawn into the SR, and the muscle relaxes (MacLennan, 1975). Characterization of Sarcoplasmic Reticulum Vesicles Marsh (1951; 1952) was the first to recognize that a soluble fraction of a muscle homogenate could cause an 11 increase in the volume of myofibril preparations, which was in effect a relaxation process. This fraction was termed the "relaxation factor" and was later shown to contain sarco- plasmic reticulum vesicles (Muscatello gt 31., 1961; Ebashi and Lipmann, 1962). The vesicles could accumulate Ca++ in the presence of ATP (Hasselbach and Makinose, 1961; Ebashi and Lipmann, 1962). Weber 33 11. (1963) found that lowering the Ca++ concentration of the sarcoplasm to levels below 10'7 M caused muscle relaxation. These observations made it clear that the vesicles were derived from the muscle mem- brane responsible for 1 vivo muscle relaxation. The vesicles can be isolated by differential and sucrose gradient centrifugation. Meissner (1975) isolated the vesicles on a 25-45% (w/w) linear sucrose gradient. Starting with 2000 g of rabbit muscle, he obtained about 125 mg of light SR vesicles in the 28-32% sucrose fraction, 750 mg of intermediate density vesicles in 32-39% sucrose, and 150 mg of heavy vesicles in the 39-43% sucrose fraction. The differences in density of the vesicles were due to dif- fering phospholipid to protein ratios, the light and heavy vesicles containing 45 and 30% phospholipid, respectively. Some 90% of the protein of the light vesicles was due to the SR-ATPase protein, as determined by $05 gel electrophoresis. The heavy vesicles contained a Ca++ binding protein (MW 65,000) and a M 5 protein (MW 55,000) which accounted for 5 25 and 5% of the protein of the vesicles, respectively. Electron micrographs showed dense material in the heavy 12 vesicles, but not in the light vesicles. Similar micrographs of intact SR showed dense material in the terminal cisternae, but not in the longitudinal tubules. Meissner (1975) concluded that light and heavy vesicles are derived from the longitudinal tubules and the terminal cisternae of the sarcoplasmic reticulum, respectively. Protein Composition of the Sarcoplasmic Reticulum Vesicles Using sodium dodecyl sulfate (SDS) gel electrophoresis, MacLennan (1975) showed that ATPase (102,000 daltons) was the predominant protein in rabbit SR vesicles. Also present were protein bands of 55,000 daltons (the high affinity CaTT- binding protein), 44,000 daltons (calsequestrin), small bands with molecular weights of 30,000 and 20,000 and a pro- teolipid with a mobility equivalent to a molecular weight of 6,000. Characterization of the SR-ATPase molecule Incubation of the vesicles with trypsin cleaved the ATPase peptide to yield fragments of 55,000 and 45,000 dal- tons (MacLennan, 1975). The ATPase active site was identi- fied by phosphorylation with radioactive (gamma-32F) ATP; only the 55,000 dalton fragment was labelled (MacLennan, 1975). If tryptic digestion of the vesicles was continued for 30 minutes, fragments of 30,000 and 20,000 daltons were produced. The phosphorus label could be detected only in the 30,000 dalton fragment, indicating that this fragment contained the ATP hydrolytic site (MacLennan, 1975). From 13 amino acid analysis of the tryptic fragments and electron microscopic studies of the vesicles, MacLennan (1975) con- cluded that the 55,000 dalton fragment had a polar amino acid composition, contained the site for ATP hydrolysis, and was located on the exterior surface of the vesicles. The 45,000 dalton fragment was more hydrophobic, and was appar- ently buried in the membrane. Shamoo gt 11. (1976) obtained similar fragments upon tryptic digestion of SR vesicles. Only the 20,000 dalton fragment had Ca++ selective ionophore activity in artificial lipid bilayers, measured as conductance changes across the lipid membrane with Ca++ carrying the current. The 20,000 dalton fragment was further degraded with CNBr, and fragments of less than 2,000 daltons had ionophore activity (Shamoo gt ll-: 1976). They concluded that the ATPase and Ca++iono- phore sites were located in different parts of the 102,000 dalton peptide. Characterization of Calsequestrin and High Affinity Ca++- Binding Protein Calsequestrin and the high affinity Ca++-binding pro- tein can be isolated from SR vesicles using dilute deoxy- cholate and salt, and can be fractionated on DEAE-cellulose, using a salt gradient between 0.0-0.7 M (MacLennan, 1975). Calsequestrin is a very acidic protein, with 146 of its' 392 amino acid residues being either glutamic or aspartic acid (MacLennan, 1975). Calsequestrin binds up to 970mmfl Ca++/mg protein, with a dissociation constant of 50 DM (MacLennan, 14 1975). Both proteins are located on the inside of the SR vesicle membrane, since treatment of intact vesicles with proteases does not affect them (MacLennan, 1975). Lipid Composition of the Sarcoplasmic Reticulum Vesicles LeMaire gt _1. (1976) reported that the lipid fraction of the SR vesicles was composed largely of phospholipid, of which 66% was lecithin. Small quantities of cholesterol and triglyceride were also present. Several investigators (LeMaire gt 11., 1976; Scales and Inesi, 1976; MacLennan, 1975) have reported that the SR-ATPase polypeptide is tightly complexed with 20-30 phospholipid molecules, which are necessary for ATPase activity. LeMaire 33 11. (1976) found that deoxycholate removed the phospholipid from the ATPase polypeptide, but inactivated the ATPase. They further reported that the Ca++-ATPase is an oligomer in the native membrane, with a molecular weight of 400,000. Model of the Organization of the SR Vesicles MacLennan (1972) described a model for the organization of the proteins in the SR membrane, which is shown in Figure 2. According to this model, the proteolipid and the ATPase are tightly bound proteins associated with the membrane phospholipid bilayer. The ATPase appears to have an amphi- pathic character. A portion, perhaps half of the molecule, is relatively nonpolar and is buried in the bilayer region of the membrane and may contain the ionophoric site. The other portion of the molecule is more polar and is located 15 O - ATPase - - proteolipid X - phospholipid ( . calsequestrin l - 54,000 o - acidic proteins Fig. 2. A model for the arrangement of the protein and lipid components in sarcoplasmic reticulum vesicles. (MacLennan gt 11., 1972) 16 on the exterior of the membrane, and is the site of ATP hydrolysis. The interaction of the site for ATP hydrolysis and the ionophoric site controls the transport of Ca++ across the membrane. The acidic proteins, calsequestrin and the high affinity Ca++ binding protein are only loosely bound to the membrane, perhaps through divalent salt bridges to the membrane phospholipids, and are located on the inner surface of the vesicles. They apparently function by binding the Ca++ that is transported inside the vesicles by the ATPase protein. Role of the Phosphoprotein Intermediate in Ca++ Transport Hasselbach and Makinose (1962) concluded that SR vesicles catalyze a transphosphorylation reaction between nucleoside triphosphates and nucleoside diphosphates. The rate of ATP-ADP exchange is dependent on the free Ca++ con- centration, as is SR ATPase activity and calcium transport. They suggested that the ATP-ADP exchange is a partial reac- tion of ATP hydrolysis. Yamamoto and Tonomura (1967) dis- covered that there is a phosphoprotein intermediate in the reaction, whereas, Martonosi (1972) observed that this may be the connecting link among the previous observations on ATP-ADP exchange, ATPase activity, and Ca++ transport. The phosphoprotein intermediate was demonstrated after 32 32 incubation of vesicles with ATP- P or acetylphosphate- P, 32F radioactivity that was retained yielding protein bound even after washing the vesicles repeatedly with trichloro- acetic acid solution (Pucell and Martonosi, 1971). In a 17 review, Martonosi (1972) pointed out that the rate of phos- phoprotein formation is markedly dependent on the Ca++ 7 5 concentration of the medium in the range of 10' to 10- M Ca++. This is also the range where the hydrolysis of ATP and ATP-ADP exchange are markedly activated (Yamamoto and Tonomura, 1967), if 5 mM MgCl2 is also present. In the absence of Mg++, about 100 times greater Ca++ concentra- tions (1-5 mM) are required to produce a similar increase in the steady state concentration of phosphoprotein (Mar- tonosi, 1967). MacLennan (1975) also observed that the SR ATPase activity has a very precise requirement for both Mg++ and Ca++. In the presence of an optimum concentration of Mg++ (5 mM), he observed that there was no ATPase activity unless about 0.3 uM Ca++ was also added. In the presence of 0.3 UM free Ca++, there was no activity until MgClz was added. Mechanism of Ca++ Transport in Sarcoplasmic Reticulum Vesicles Yates and Duance (1976) used the flow dialysis method to measure the kinetics of substrate binding to the SR ATPase enzyme. They concluded that the binding of MgATP and Ca++ may occur in a random manner, with neither sUbstrate influ- encing the affinity of the enzyme for the other. The inde- pendence of Ca++ binding and phosphoprotein formation sug- gested that phosphorylation of the ATPase initiates a conformational change that leads to translocation of the CaTT previously bound to specific and chemically distinct sites (Martonosi, 1972). 18 The ATPase activity of SR vesicles can be divided into ' two steps, phosphorylation and dephosphorylation of the ATPase protein (MacLennan, 1975). The substrate for phos- phorylation is MgATP. The phosphorylation reaction has an absolute requirement for 0.3 uM free Ca++ (MacLennan, 1975). After phosphorylation, the affinity of the enzyme for Ca++ decreases (Berman g; 31., 1977) but the ATPase reaction goes to completion only after Mg++-dependent dephosphoryla- tion (Yamamoto, 1972; Garrahan g; _1, 1976). The SR ATPase protein of rabbit muscle contains one specific ATP site and two specific Ca++ sites per phosphorylation, resulting in the transport of two moles of Ca++ for each mole of ATP used during phosphorylation (Meissner, 1973). MacLennan (1975) postulated that Mg++ is the counterion for Ca++ transport. resulting in the release of Mg++, ADP and inorganic phos- phate on the membrane exterior, while Ca++ is released on the membrane interior. The SR membrane is freely permeable to MgTT, so the Ng++ content of the vesicles will not limit Ca++ uptake (Vale, 1975). Increasing the concentration of ADP above 1 mM pro- gressively inhibited ATPase activity and Ca++ transport by isolated SR vesicles (Makinose, 1969), but the phosphopro- tein concentration remained high (1.5 moles/10° g protein). Makinose (1969) concluded that ADP formed an unreactive com- plex with the ATPase phosphoprotein. 18 The ATPase activity of SR vesicles can be divided into ' two steps, phosphorylation and dephosphorylation of the ATPase protein (MacLennan, 1975). The substrate for phos- phorylation is MgATP. The phosphorylation reaction has an absolute requirement for 0.3 uM free Ca++ (MacLennan, 1975). After phosphorylation, the affinity of the enzyme for Ca++ decreases (Berman 33 31., 1977) but the ATPase reaction goes to completion only after Mg++-dependent dephosphoryla- tion (Yamamoto, 1972; Garrahan 33 _1, 1976). The SR ATPase protein of rabbit muscle contains one specific ATP site and two specific Ca++ sites per phosphorylation, resulting in the transport of two moles of Ca++ for each mole of ATP used during phosphorylation (Meissner, 1973). MacLennan (1975) postulated that Mg++ is the counterion for Ca++ transport, resulting in the release of Mg++, ADP and inorganic phos- phate on the membrane exterior, while Ca++ is released on the membrane interior. The SR membrane is freely permeable to Mg++, so the Mg++ content of the vesicles will not limit Ca++ uptake (Vale, 1975). Increasing the concentration of ADP above 1 mM pro- gressively inhibited ATPase activity and Ca++ transport by isolated SR vesicles (Makinose, 1969), but the phosphopro- tein concentration remained high (1.5 moles/106 g protein). Makinose (1969) concluded that ADP formed an unreactive com- plex with the ATPase phosphoprotein. 19 Evidence for Two Functional States of the'ATPase Protein In the absence of Ca++, rabbit SR vesicles have a low ATPase activity (basic activity), which greatly increases on addition of Ca++ to give the total activity (Hasselbach, 1964). The difference between the total and basic activities is known as the extra or Ca++-dependent ATPase, which is coupled to Ca++ transport (Hasselbach, 1964). Inesi 33 31. (1976) found that basic ATPase activity was predominant in the low density fraction of SR vesicles obtained by density gradient centrifugation. In all other fractions, the Ca++- dependent ATPase activity was predominant. In these frac- tions, the ratio of Ca++—dependent to basic ATPase activity was temperature dependent, being about 9.0 at 40°C and 0.5 at 4°C (Inesi 33 31., 1976). Conversion of ATPase from one state to another was postulated to involve changes in the conformation of the protein and/or its' membrane environment (Inesi 33 31., 1976). In both states, a phosphorylated intermediate of the ATPase was formed in the presence of Ca++. Hydrolysis of the phosphorylated intermediate occurred in state E2, which is coupled to Ca++ transport, whereas, in state E1 the ATPase catalyzed ATP hydrolysis (basic acti- vity) in the absence of Ca++ and independently of enzyme phosphorylation (Inesi t al., 1976). Ca++ Accumulation by Sarcoplasmic Reticulum Vesicles SR vesicles may accumulate Ca++ under both active and passive conditions. In the absence of ATP (passive 20 conditions), Ca++ is bound on the exterior of the membrane, but in the presence of ATP, Ca++ is transported to the interior of the vesicle (Vale _3 _1., 1976). Verjovski 33 31. (1977) concluded that SR vesicles contain low and high affinity sites for Ca++, and that both increase their affinity for Ca++ some 3-4 fold as the pH increases from 6.1 to 8.5. In the absence of membrane permeable Ca++ precipita- ting agents, such as oxalate or inorganic phosphate (Pi)’ the rate of Ca++ uptake by SR vesicles declines rapidly as the Ca++ concentration inside the vesicles increases, even if the levels of ATP and Ca++ in the medium are constant (Martonosi, 1972). After accumulation of TDD-zooimmTCaTT/mg protein, a steady state between Ca++ influx and Ca++ efflux is established. The steady state Ca++ flux is 50-100 times slower than the maximum initial rate of Ca++ uptake (Mar- tonosi, 1972). The activity of the Ca++ pump is inhibited if the intravesicular free Ca++ concentration exceeds 2 x 10"5 M (Makinose and Hasselbach, 1965). Oxalate or phos- phate, by decreasing the intravesicular free Ca++ concen- tration, permits Ca++ accumulation to proceed until 8,000- l0,000rmolCa++/mg protein are accumulated in the form of calcium oxalate or phosphate (Martonosi, 1972). However, the rate of Ca++ efflux from SR vesicles is greatly slowed in the presence of these agents (Sorenson and De Meis, 1977). 21, Several studies have reported that monovalent cations inhibit Ca++ accumulation by SR vesicles (De Meis and Hassel- bach, 1971; De Meis, 1971; De Meis and De Mello, 1973). However, Shigekawa and Pearl (1976) found that K+, Na+, RbT, NH4T, CsT, LiT, choiineT, and TrisT (in order of de- creasing effectiveness) stimulated Ca++ uptake and Ca++- dependent ATPase activity of SR vesicles, possibly due to an increased rate of decomposition of the phosphorylated ATPase intermediate in the presence of these ions. The most widely used technique for determining Ca++ 4°Ca++, and separating the vesicles accumulation is by using from the media by the Millipore filtration technique (Meissner and Fleisher, 1971; Kanda, 1975). With this method, Ca++ accumulation is measured over a period of 10- 20 minutes. As pointed out by Endo (1977), the Ca++ accu- mulating capacity of the SR measured by this method is suf- ficient to explain the relaxed state of muscle at steady state. However, the rate of uptake is too slow to explain the rate of relaxation in living muscle, possibly due to the nonphysiological state of the SR preparation (Endo, 1977). Much faster rates of Ca++ uptake have been observed using spectrophotometric methods for monitoring Ca++ move- ment, using Ca++ sensitive dyes such as murexide (Reed and ByGrave, 1975), ll = Protein concentration of sample (mg/m1) Determination of Ca++ Accumulation by Mitochondria CaTT accumulation was determined under "limiting loa- ding" conditions (Lehninger 33 31., 1967). The reaction nfixture contained 10 mM succinate, 3 mM ATP, 210nM mannitol, 53 Determination of Ca++ Accumulation Place 4 ml media in tube at desired temperature Add 40 or 100 pliters of 10 mM 45CaC12 Add SR or mitochondrial protein At intervals, remove .5 ml aliquots Filter (. 22 or .45 “meter pores) Add . 2 ml filtrate to 4 ml Aquasol Determine cpm by liquid scintillation (Ca++)Eontrol cpm - Sample Cpnj Control cpm Protein concentration Ca++ uptake = Fig. 6. Flgw sheet of the procedure used for the determina- tion of Ca accumulation. 54 70 mM sucrose and 10 mM Tris buffer, pH 7.4 (Jacobus 33 31., 1975; Carafoli, 1976). A stock solution of 10 mN 45 CaCl2 was prepared as described previously. After adjusting the pH, 4 ml of the reaction mixture was placed in a tube and equilibrated to 38, 15 or 0°C. 45 Twenty five uliters of the CaCl2 solution was added per m1 reaction mixture, so that the reaction mixture contained 45CaTT/ml, and approximately 20,000 cpm/m1. 250nmol The reaction was initiated by addition of 0.5-0.8 mg protein/ml reaction mixture. The reaction was terminated at various time intervals by filtration through a Millipore or Gelman filter with an average pore size of 0.45 uM (Jacobus 33 31., 1975). Ca++ accumulation was calculated as previously described (Fig. 6). Determination of Ca++ Release in Sarcoplasmic Reticulum Vesicles or Mitochondria Ca++ accumulation was allowed to proceed for 8 minutes. A portion of the mixture was then transferred to another tube. Calcium accumulation was measured in both tubes at 13 and 18 minutes. One tube served as a control. The second tube was subjected to any of several different conditions. Some tubes were placed in another water bath at the desired temperature. In some experiments, the pH of the mixture was lowered to pH 5.0 by addition of 0.1 N HCl, using a Corning model 12 pH meter to monitor pH. Nitrogen gas was bubbled through some tubes, using a disposable pipette as the hose 55 . + ~ . . tip. Ca + release was determined by comparison of the Ca++ accumulated at 8 minutes with the Ca++ accumulated under the changed conditions at 13 and 18 minutes. Determination of Radioactivity Aliquots of the filtrate (0.2 ml) were mixed with 4 ml of Aquasol-2, a scintillation liquid (New England Nuclear, Boston, Mass.). Sample radioactivity was determined using a Beckman liquid scintillation counter, model 3133P, using Chan- nel 8 set at 1.5% preset error. SOS Gel Electrophoresis of Beef and Rabbit Sarcoplasmic Reticulum Sodium dodecyl sulfate (SDS) electrophoresis was carried out using the method of Weber and Osborn (1969) as modified by Porzio and Pearson (1976). Sarcoplasmic reticulum pellets were dissolved in a solution composed of 1% SDS, 5 mM ethyl- enediaminetetraacetate (EDTA), 1 mM dithiothreitol and 50 mM Tris/glycine, pH 8.8. A stock solution of 2.0 M Tris/glycine (0.5 M Tris:l.5 M glycine, pH 8.8) was previously prepared. Samples were heated for 5 minutes at 100°C to aid in dissol- ving the proteins, then dialyzed overnight against 25 mM Tris-HCl (pH 7.4), 1 mM dithiothreitol and 0.2% SDS. Glycerol was added to a concentration of about 20%, and several drops of Pyronin Y tracking dye (0.25 mg/ml) were added to the sample immediately before loading onto the gel. For the 10% acrylamide (100:1) gel system, a stock solution was prepared by dissolving 25.0 9 acrylamide and 0.25 9 his in deionized water and bringing the final volume 56 to 100 ml. The solution was then passed through a 2 x 4 cm column of dry packed mixed bed deionizing resin before use. Stock solutions of 2.0 M Tris/glycine, 50% glycerol, 2.5% SOS/2.5 mM EDTA, 1% N, N, N', N'-tetramethylethylene- diamine (TEMED), and 1% ammonium persulfatevmre prepared. The casting solution consisted of stock solutions in the following proportions: 10 volumes acrylamide, 5 volumes Tris buffer, 2.5 volumes glycerol solution, 1 volume SOS/EDTA, 4.5 volumes water and 1 volume TEMED. The casting solution was deairated by application of vacuum, and 1 volume of the initiator, ammonium persulfate, was added immediately before casting. After the gels were loaded to within 5 mm of the top of the tube (8 cm x 5 mm ID tubes), a layering solution consisting of 400 mM Tris/glycine (pH 8.8), 0.04% TEMED, 0.1% SDS and 0.004% ammonium persulfate was layered on top of the acrylamide solution to form a smooth interface. After polymerization was completed (20 minutes), the layering solution was removed and the gel surface was relayered with a solution consisting of 400 mM Tris/glycine (pH 8.8), 5% glycerol and 0.1% SDS for an hour before use. Electrophoresis was conducted in a Buchler Polyanalyst unit. Each chamber contained 340 ml of chamber buffer consisting of 200 mM Tris/glycine (pH 8.8) and 0.1% SDS. A Heathkit IP-17 constant voltage power supply of 0-400 V, 100 mA capacity was employed. Protein samples (25-50 uL) were loaded on the gel and entry of the sample into the gel initiated at 0.5 mA per gel. After the dye had completely 57 entered the gel, the current was raised to 1.0 mA per tube (12 mA at 40 V for 12 gels). Migration continued until the dye front was within 5 mm of the bottom of the gel. The gels were removed from the tubes and the dye front was marked by insertion of a fine wire. The gels were fixed by soaking in a solution of 25% (v/v) isopropanol/10% (v/v) acetic acid for several hours. The samples were stained overnight in a solution consisting of 0.01% Coomassie Brilliant Blue R 250, 50% (v/v) methanol, and 7.5% (v/v) glacial acetic acid. The gels were destained in 10% (v/v) acetic acid/5% (v/v) methanol. A standard curve for molecular weight determination was prepared using the following proteins: myosin - 200,000; v-globulin (unreduced) - 160,000; muscle C-protein - 140,000; B-galactosidase - 130,000; o-actinin - 102,000; bovine serum albumin - 68,000; catalase - 60,000; ovalalbumin - 43,000; glyceraldehyde dehydrogenase - 36,000; myokinase 21,500; haemoglobin - 15,500. (Fig. 7). Transmission Electron Microscopy Small muscle samples, sarcoplasmic reticulum vesicles, or mitochondria obtained by centrifugation Were fixed using a modification of the procedure described by Sjostrand (1967). Samples were fixed for 2 hours in a buffer solution containing 1.25% glutaraldehyde, 0.048 M sodium phosphate (pH 7.4) and 0.043 M NaCl (415 milliosmolar solution). The tissue samples were washed for 1 hour in 2 changes of 58 5,50 5.25 5.00 .fTTZIZJ 4.75 log Molecular Weight 4.50 4.25 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Relative Mobility Fig. 7. Standard plot of log molecular weight and relative mobility on 10% acrylamide SOS—gel with 0.10% crosslinker. The protein standards and their molecular weights are as follows: (1) myosin - 200,000; (2) y-globulin (unreduced) - 160,000; (3) muscle C-protein - 140,000; (4) B-galactosidase - 130,000; (5) a-actinin - 102,000; (6) bovine serum albumin - 68,000; (7) catalase - 60,000; (8) ovalalbumin - 43,000; (9) glyceraldehyde dehydrogenase - 36,000; (10) myokinase - 21,500; (11) haemoglobin - 15,500. 59 0.094 M sodium phosphate and 0.043 M NaCl buffer solution at pH 7.4. The samples were then postfixed for 1 hour in 1% osmium tetroxide solution in veronal acetate buffer (pH 7.4), which was adjusted to 300 milliosmolar with NaCl, KCl, and CaClz. The composition of the buffer solutions and the fixative preparation schedules are shown in Appendix Table l - 6. After fixation, the samples were dehydrated for 15 minutes each in 25, 50, 75, and 95% ethanol. They were then placed in 2 changes of 100% ethanol for 30 minutes each. The dehydrated samples were transferred through 2 changes of propylene oxide for 30 minutes each, followed by 12 hours in a 1:1 mixture of propylene oxide andemon—araldite resin. The samples were embedded in pure epon-araldite resin using flat embedding molds (LKB Instruments, Inc.). The embedded samples were placed in a desiccator under slight vacuum for 12 hours, then placed in a 80°C oven for 36 hours to allow the blocks to harden. Epon-araldite embedded tissue blocks were trimmed by hand with a razor blade, and sectioned with either a diamond or glass knife to a thickness of 60 to 100 nM using an LKB 4801 ultramicrotome. Sections were picked up from the knife boat on uncoated 300 mesh copper grids. 'Staining of the tissue was accomplished by floating the grids for 30 minutes on a saturated solution of uranyl acetate, followed by thoroughly rinsing with distilled water, and then staining for 5 minutes in a solution of lead citrate (Abbott, 1976). 60 The sections were washed with 0.02 M NaOH, followed by dis- tilled water, and air dried. A Phillips EM-300 transmission electron microscope was used for observing the stained sections at an accelera- ting voltage of 60 KV. Photographs of each sample were taken using Kodak 8.25 x 10.16 cm sheet film. The film was developed for 4 minutes in Kodak D-l9 developer, washed for 1.5 minutes in running water, fixed 8 to 10 minutes in Kodak fixer, washed hirunning water for 1 minute, rinsed in Kodak Hypo-Clearing Agent, and washed for 10 minutes. The washed negatives were dipped in Kodak Photo-Flo solution and dried for 45 minutes with warmed air. All of the previous steps were performed using an Arkay nitrogen burst machine. Kodak Polycontrast Rapid Resin Coated Paper was ex- posed from the negatives using a Durst S-45-EM enlarger. The exposed paper was developed in Kodak Dektol developing solu- tion (stock solution diluted 1:2 with water) for 1.5 minutes. The prints were rinsed in Kodak Indicator Stop Bath for 5 seconds, and fixed for 2 minutes in a solution of Kodak Fixer. The prints were then washed 4 minutes in running water. The surface of the prints was blotted with a soft sponge to remove water droplets, and air dried at room temperature. All solu- tions were at 18-21°C during processing. Muscle Fiber Type Determination Immediately after slaughter muscle samples were removed and trimmed to small rectangular pieces approximately 3.5 x 61 3.5 x 5 mm, with the long (5 mm) dimension of the sample being cut parallel to the longitudinal direction of the muscle fibers. The samples were then wrapped in Saran wrap and aluminum foil, submerged in liquid nitrogen until frozen, and then placed in a previously chilled (-20°C) Slee-Pearse cryostat. The frozen samples were positioned on a microtome chuck using 0.C.T. compound (Miles Laboratories), so that cross sections of 10-12 WM in thickness could be obtained. The sections were placed on coverslips and allowed to dry at room temperature for 30 minutes. Fiber type was determined by the reduced diphospho- pyridine nucleotide-tetrazolium reductase method of Engel and Brooke (1965), which stains mitochondria. Red or oxida- tive fibers appear dark blue when viewed with a light micro- scope. White or glycolytic fibers remained unstained. The following schedule was used for staining the tissue sections: 1. Using a magnetic stirring bar, 20 mg of Nitro-blue Tetrazolium Chloride was dissolved in 20 ml of 0.2 M Tris buffer, pH 7.4. 2. After mixing, 16 mg of NADH (reduced) was added and the cover slips with the adherring tisSue sections were placed in the mixture. 3. The sections were incubated for 30 minutes at 36-38°C in a shaker water bath. 4. The sections were post-fixed for 3 minutes in 10% formaldehyde solution. 62 5. The tissue was rinsed in 3 changes of distilled water. 6. The coverslips were mounted on glass slides using Permount(Fjsher). Photographs of the stained sections were taken on a Zeiss Photomicroscope III, using Kodak Panatomic-X film. The film was developed at 20°C in a small lightproof tank using Kodak Microdol-X developing solution for 7 minutes. The film was rinsed for 30 seconds in Kodak Indicator Stop Bath solu- tion, fixed for 2-4 minutes in Kodak Fixer solution, washed for 20-30 minutes in running water (l8-24°C), and dipped in Kodak Photo-Flo solution before drying at room temperature. Enlargements were made using Kodak Polycontrast Resin Coated Paper as previously described. RESULTS AND DISCUSSION 3310 Shorteniflg i3 Beef and Rabbit Muscles Figure 8 shows beef muscle which has been subjected to cold shortening. Chilling beef Sternomandibularis muscle to 0°C resulted in 43% shortening, but at 15°C, very little shortening occurred. No appreciable shortening occurred in rabbit Longissim33 dorsi muscles at either 15 or 0°C. These results are similar to those reported by Locker and Hagyard (1963), and are included only to illustrate the extent to which cold shortening occurs in red muscles. Purity and Char3cterization of Sarcoplasmic Reticulum Pre- p3rations Working in this laboratory, Kanda (1975) established that SR prepared by the procedure of Meissner and Fleisher (1971) was essentially free of contamination from lysosomes, mitochondria, and plasma membrane. This was verified by measuring acid phosphatase activity, succinate-cytochrome c reductase activity, and 5'-nucleotidase activity, respecti- vely. SDS gels of beef and rabbit myofibrils and of SR pellets were prepared by the same procedure (Porzio and Pearson, 1977). A comparison of the gels for the myofibrils and the SR (Fig. 9) showed that gels from SR contained no 63 64 BeeF Sternomandibularis Rabbit Longissimus Jon. C O.C 5. 10cm Fig. 8. Beef and rabbit muscle strips chilled to 15 or 0°C immediately postmortem. 65 Fig. 9. SDS gels of beef and rabbit myofibrils and sarco- plasmic reticulum. The gels were 10.0% acrylamide, with 0.10% crosslinker. 66 myosin heavy chains (200,000 daltons) or actin (42,000 dal- tons), which are the predominant protein bands in SDS gels of myofibrils (Porzio and Pearson, 1977). Thus, SDS gel electrophoresis of both beef and rabbit SR preparations showed that the pellets were free of contamination from myofibrillar proteins. In the SDS gels of the beef and rabbit SR, SR-ATPase (102,000 daltons) was the predominant band. 505 gels of beef and rabbit myofibrils also contained a band with a molecular weight of 100,000, which was identified as o-actinin (Kanda 33 31., 1977b). In addition to the SR-ATPase protein, the SDS gels of the SR pellets contained bands with molecular weights of 33,000, 53,000, 60,000, 93,000 and 150,000 dal- tons. Kanda 33 31. (197nfi tentatively identified the 150,000 dalton band as an M-line protein, and the 93,000 dalton com- ponent as phosphorylase a. MacLennan (1975) reported 44,000 and 55,000 dalton bands on SOS gels of rabbit SR, which he identified as calsequestrin and the high affinity Ca++- binding protein, respectively. Meissner and Fleisher (1971) identified protein bands at 50,000 and 60,000 daltons in SDS gels of rabbit SR, which agrees well with the bands at 53,000 and 60,000 daltons found in the present study. Kanda t al. (1977b)also found two rabbit SR proteins with the same molecular weights. Yu 33_31. (1976) identified a 63,000 dalton component from rat SR as the high affinity Ca++ binding protein, which appears to correspond to the 60,000 dalton component in the present study. MacLennan (1975) 67 Fig. 10. Transmission electron micrograph of beef sarcoplas- mic reticulum vesicles. 44, 800 X. 68 Fig. 11. Transmission electron micrograph of rabbit sarco- plasmic reticulum vesicles. 44,800 . 69 identified a band at a molecular weight of 30,000 daltons in rabbit SR, which is in good agreement with the 33,000 dalton component observed in the present study. However, no function has been assigned to this protein. 'Transmission electron micrographs of the beef (Fig. 10) and rabbit SR pellets (Fig. 11) showed that the pellets contained membranous vesicles with little, if any, contami- nating particles. Preparations from beef and rabbit muscle were microscopically indistinguishable. Purity and Characterization of Mitochondrial Preparations Mitochondrial succinate oxidase activity was measured manometrically as a marker for the presence of mitochondrial membranes (King, 1967). Table 2 indicates that both beef and rabbit mitochondrial suspensions had about the same levels of succinate oxidase activity. Both suspensions oxidized about l9umolsuccinate/mg protein/minute over a one hour period. Thesedata indicates that the beef and rabbit mitochondrial preparations contained about the same amount of mitochondrial membrane per unit protein. Mitochondrial respiration was also measured manometri- cally (Ernster and Nordenbrand, 1967) to determine whether the mitochondrial suspensions contained intact mitochondria capable of respiration.. Table 3 shows that the beef mito- chondrial suspensions were respiring more actively than that of rabbit, indicating that the beef preparations contained more intact, respiring mitochondria per unit protein than 7O Table 2. Manometric Assay for Mitochondrial Succinate Oxidase (umol succinate oxidized/mg protein/minute) Time Beef Rabbit (min) mitochondria mitochondria 10 15.06 20.02 20 20.15 23.03 30 19.91 21.49 40 19.45 19.75 50 18.83 18.27 60 18.08 17.17 71 Table 3. Manometric Assay for Mitochondrial Respiration (umol 02 uptake/mg protein/minute) Time Beef Rabbit (min) mitochondria mitochondria 5 12.89 9.14 10 18.30 6.90 15 14.86 7.05 20 13.83 7.97 25 13.50 8.45 30 12.83 8.76 35 11.38 9.06 4D 11.00 9.19 that con: min con ml't bee di'f the act inn and dri tic inn sho nit Del tar am Pa ti me th 72 that from rabbit muscle. Beef mitochondrial suspensions consumed about 13umoloxygen/mg protein/minute over a 40 minute period. This may be compared to about 8umoloxygen consumed/mg protein/minute over the same period for rabbit mitochondrial suspensions. When viewed in the transmission electron microscope, beef and rabbit mitochondrial suspensions were strikingly different. Figure 12 shows that beef mitochondria were in the condensed conformation characteristic of state 3, i.e., actively respiring mitochondria (Lehninger, 1975). The inner mitochondrial membrane appeared dense, tightly folded and contorted. Figure 13 demonstrates that rabbit mitochon- dria were in the orthodox conformation, which is characteris- tic of state 4 or resting respiration. In this state, the inner membrane compartment completely fills the space bounded by the outer membrane (Lehninger, 1975). The micrographs show that the mitochondrial pellets consist predominantly of mitochondria, but some contaminating particles are present. Greaser 33 31. (1969) also observed that mitochondrial pellets isolated by differential centrifugation contain con- taminating particles. In spite of contamination, Ernster and Nordenbrand (1967) demonstrated that mitochondrial pre- parations isolated by differential centrifugation contained tightly coupled mitochondria suitable for use in the measure- ment of respiratory parameters. They further pointed out that mitochondria prepared with sucrose or other nonelectro- lytes in the homogenization medium are of inferior quality, 73 Fig. 12. Transmission electron micrograph of beef mitochon- dria. 28,000 X. i = inner mitochondrial membranes 0 = outer mitochondrial membranes P i drla 74 Fig. 13. Transmission electron micrograph of rabbit mitochon- dria. 28,000 X. ‘ i = inner mitochondrial membranes 0 = outer mitochondrial membranes i.e. acti work foli chon meas (Gre cent the the Prot rESp 097 42.3 musc 1501 that fold EELE Hari of H deVQ poin 1an 75 i.e., they have poor phosphorylating efficiency, high ATPase activity, and the yield of mitochondria is low. A number of workers (Greaser 33 31., 1969; Jacobus 33 31., 1975; Cara- foli, 1976; Kimura and Rasmussen, 1977) have isolated mito- chondria by differential centrifugation for subsequent measurements of Ca++ accumulation. In spite of contamination (Greaser 33 31., 1969; Jacobus 33 _1., 1975), differential centrifugation has been widely used because of the speed of the isolation procedure, the high mitochondrial yield, and the good quality of the isolated mitochondria. Yield_of Sarcoplasmig Reticulum from Beef and Rabbit Muscle In the present study, 62.2 t 8 and 180.3 s 40 ug of SR protein were isolated per gram of beef and rabbit muscle, respectively, using the procedure of Meissner and Fleisher (1971). Using the same procedure, Kanda (1975) obtained 42.3 t 8 and 380 i 40 ug SR protein/g tissue from the same muscles, respectively. Assuming that the method used for isolation of SR was quantitative, the present study indicates that the rabbit Longissim33 dorsi muscle contains about 3 fold greater amounts of SR membrane than beef Sternomandi- bularis muscle. This is in agreement with the results of Harigaya e3 31. (1968) and Schiaffino e331. (1970), both of whom concluded that the SR membrane system is less well developed in red muscle fibers. However, Kanda (1975) pointed out that the beef Sternomandibularis muscle contained large amounts of connective tissue, which may reduce the 76 yield of SR protein from this muscle. Martonosi (1972) reported that about 0.7 to 1.4 mg SR protein/g tissue could be isolated from red muscles, as compared to 2.5 to 4.0 mg SR protein/g of white muscle tis- sue. Martonosi (1972) further estimated that white muscles contain at least 5 mg SR protein/gram tissue. field of Mitocho3§ri3_from Beef and Rabbit Muscle In this study, 0.637 t 0.04 mg of mitochondrial protein were obtained per gram of beef 33ernomandibularis muscle, which compares with 0.590 t 0.07 mg of mitochondrial protein per gram of rabbit Longissimus dorsi muscle. Ernster and Nordenbrand (1967) obtained 2 to 3 mg mitochondrial protein/g rat skeletal muscle. Brand and Lehninger (1975) reported a yield of 4 mg mitochondrial protein/g rat heart, and 15 to 20 mg mitochondrial protein/g of rat liver. The low mitochon- drial yields obtained in this study may be due to the homo- genization method used. In view of the high connective tissue content of beef muscle, the minced muscle used in this study was homogenized with a Polytron homogenizer, instead of the all glass Potter-Elvehjem homogenizer, which was used by Ernster and Nordenbrand (1967). Polytron homo- genization appeared to disrupt the tissue so that the yield of intact mitochondria sedimenting at 10,000 g was reduced. Carafoli (1975) estimated that heart muscle contained 80 mg mitochondrial protein/g of heart tissue. Ernster and Nordenbrand (1967) obtained yields of 2 to 3 mg mitochondrial prot be c to 8 esti tiss mito clud much 11101‘8 ,— O J (I) 77 protein/g of rat skeletal muscle. On this basis, it may be concluded that rat skeletal muscle contains at least 6 to 8 mg mitochondrial protein/g of tissue. Martonosi (1972) estimated that white muscles contain about 5 mg SR protein/g tissue. Based on these estimates and the yield of SR and mitochondrial protein obtained in this Study, it may be con- cluded that both red and white muscles contain at least as much mitochondrial protein as SR protein. Gauthier (1970) pointed out that red muscles contain more mitochondria than white muscles. Muscle mitochondria may be histochemically located, using the NADH-tetrazolium reductase procedure, which specifically stains mitochondria (Engel and Brooke, 1965; Gauthier, 1970). Figures 14 and 15 show cross-sections of beef and rabbit muscle, respectively, stained by the NADH-tetrazolium reductase procedure. Red fibers, containing high mitochondrial concentrations, appear dark, while the white fibers are relatively unstained, indi- cating a low content of mitochondria (Engel and Brooke. 1965). A comparison of Figures 14 and 15 shows that rabbit tissue contains fewer red fibers and has a lower mitochon- drial concentration than beef muscle. Although the beef Sternomandibularis is a very red muscle when viewed with the unaided eye, Figure 14 shows that it should be more properly classified as a mixed muscle, containing red, white, and intermediate fiber types. Figure 15 shows that rabbit Longissimus dorsi contains some red fibers, but the great 78 Fig. 14. Light micrograph of beef Sternomandibularis muscle 440 X r = red fiber ' i = intermediate fiber w = white fiber Fig. 15. muscle. 440 x 79 Light micrograph of rabbit Longissimus dorsi red fibers white fibers 80 majority of the muscle is composed of white fibers. Accumualtion of CaTT by Sarcoplasmic Reticulum Vesicles Figure 16 shows that CaTT accumulation by beef and rabbit SR vesicles was approximately proportional to pro- tein concentration in a range from 0.03 to 0.3 mg of SR protein per ml of reaction mixture. Conseqdently subsequent experiments were conducted in this range, usually using 0.08 to 0.20 mg of SR protein per ml of reaction media. Tables 4 and 5 show that both beef and rabbit SR vesicles accumulated more than 500 nmol Ca++/mg protein/ 18 minutes, at pH 7.3 and 37°C. This corresponds to maxi- mum values of 250 and 40 nmol Ca++lmg protein/3 minutes reported by Sreter (1969) for SR vesicles from white and red rabbit muscle, respectively. Harigaya and Schwartz (1969) reported thatSR.vesiclestbm red rabbitnmscle accu- mulated only 58 nmol Ca” /mg protein, which agrees closely with the value of Sreter (1969). Sreter (1969) and Harigaya and Schwartz (1969) both concluded that SR vesicles from red muscles have a lower capacity for Ca++ accumulation than similar preparations from white muscles. The relatively high values for Ca++ accumulation reported in the current investivation for beef SR vesicles may be due to the fact that beef Sternomandibularis contains a significant number of intermediate and white fibers (Fig. 14). It is well known that white and intermediate fibers contain a well developed SR system (Peachey, 1970). Thus, SR vesicles ancn 81 60 o 50 Rabbit .. 40 E C E 30 .2 g Beef E :3 5’; 20 + +CU Q 10 w .l .2 .3 Protein Concentration ( mg/mll ++ Fig. 16. Ca accumulation by sarcoplasmic reticulum vesicles at pH 7.3 and 37°C as a function of protein con- centration. The reaction was allowed to proceed for 4 minutes. 82 derived from these fibers would be expected to have a high ++ capacity for Ca accumulation (Sreter, 1969), which is in agreement with results from the present study. Alternatively, the high capacities for Ca++ accu— mulation observed in the present study may be the result of a long incubation period in combination with an in- creasing concentration of inorganic phosphate (Pi) in the media, resulting from the release of Pi by the SR-ATPase. It is well known that Ca++-precipitating agents such as Pi and oxalate greatly increase the capacity of SR vesicles to accumulate Ca++ (Sreter, 1969; Martonosi, 1972). Sreter (1969) observed that in the presence of oxalate, rabbit SR vesicles could accumulate more than 6,000 nmol Ca++lmg protein/3 minutes. Effects of Temperature of Ca++ Accumulation by Sarcoplasmic Reticulum Vesicles Tables 4 and 5 show that beef and rabbit SR vesicles accumulated less than 70 nmol Ca++/mg protein/18 minutes, when incubated at 0°C and at pH 7.3. At 15°C, beef and rab- bit SR vesicles accumulated 89 and 113 nmol Ca++/mg protein/ 18 minutes, respectively, while at 37°C both preparations accumulated more than 500 nmol Ca++lmg protein/18 minutes. Figures 17 and 18 show that chilling the SR preparations does not inhibit subsequent Ca++ accumulation by the vesicles. 83 Table 4. CaTT Accumulation and Release by Beef Sarco- plasmic Reticulum Vesicles (nmol Ca +/mg protein) Temp Time P“ (0C.) (minutes) 7.3 6.8 6.2 5.5 5.0 37 1 77.5 70.8 73.7 -- O 37 2 90.8 119.8 54.6 69.6 8.5 37 4 190. 2 198.2 74.3 -- 14.3 37 8 360.1 306.0 84.9 81.7 23.8 37 13 505.8 401.6 94. 6 44.0 10.3 37 18 542.5 439.0 74.8 57.0 0 37 + 01 8 + 5 298.7 280.7 23. 3 9. 4 32.8 37 + 02 8 + 10 294.7 238.7 35. 4 47. 6 4.9 373 8 at pH 7.3 0 +10 at pH 5 0 15 1 -- 50.1 54.1 -- -- 15 2 72.2 47.5 53.3 57.1 4 9 15 4 78.3 16.7 63. 4 48. 7 -- 15 8 85.8 40.5 77.6 22. 9 0 15 13 80.4 54.6 67. 6 115.0 24.7 15 18 89.4 34.7 97.6 51.2 0 15 + o 8 + 5 60. 4 22.2 75.4 7. 6 o 15 + O 8 + 10 54.0 25.8 51.7 21.1 16.7 o 1 21.4 o -- -- .-- 0 2 27.6 4.5 36.7 0 0 0 4 42.7 o -- -- -- O 8 42.2 0 23.1 D O 0 13 49.3 o 37.7 9.8 o O 18 61.1 7.6 O 34.6 0 O + 37 8 + 5 267.4 180.0 118.5 34. 6 O O + 37 8 + 10 384.1 279.2 138.0 49.0 0 1Incubation at pH 7. 3 and 37 °Co for 8 minutes, followed by. incubation for 5 minutes at 00 C 2Incubation at pH 7. 3 and 37° C for 8 minutes, followed incubation for 10 minutes at 0° C 3Incubation at pH 7.3 and 37°C for 8 minutes, followed incubation for 10 minutes at pH 5.0 and 37° C 84 Table 5. Ca++ Accumulation and Release by Rabbit Sarco- plasmic Reticulum Vesicles (nmol Ca+ +/mg protein) Temp. Time pH (°C.) (minutes) 7.3 6.8 6.2 5.5 5.0 37 1 70.8 115.4 153.4 55.7 30.9 37 2 111.8 138.8 128.8 55.8 22.1 37 4 210.7 202.5 102.7 28.1 20.3 37 8 348.9 320.8 74.4 16.7 13.6 37 13 379.0 282.1 36.3 15.8 18.2 37 1 18 543.5 209.2 37.2 1.8 24.0 37 + 0 8 + 5 271.0 190.5 39.1 0.0 24.2 37 + 02 8 + 10 242.1 230.0 33.3 1.9 31.9 373 8 at pH 7.3 18.7 +10 at pH 5 0 15 1 72.7 76.3 115.3 25.0 14.2 15 2 71.4 87.4 116.9 25.1 14.9 15 4 79.8 93.9 129.3 36.1 30.1 15 8 92.3 100.5 130.6 38.0 52.1 i5 13 99.2 95.9 130.6 43.0 51 7 15 18 113.1 121.7 125.0 42.3 54.2 15 + o 8 + 5 78.7 87.9 91 9 30.0 -- 15 + 0 84+ 10 81.8 80.3 86 2 19.2 48.9 0 1 45.2 53.3 42.0 5.9 0 0 2 50.0 51.8 57.7 8.7 0 0 4 64.4 56.3 66.4 12.2 0 0 8 65.7 64.7 73.1 11.2 0 0 13 66.8 57.3 78.1 23.4 0 0 18 58.0 41.5 89.0 20.0 0 0 + 37 8 + 5 162.4 273.8 96.6 26.1 0 0 + 37 8 t 10 297.0 310.7 66.0 20.9 o 1Incubation incubation 2Incubation incubation 3Incubation incubation at pH 7. 3 and 37° Co for for 5 minutes at 0° C at pH 7. 3 and 37° C for for 10 minutes at 0° C at pH 7.3 and 37°C for for 10 minutes at pH 5. 8 minutes, followed by (1) minutes, followed by minutes, followed by and 37°C 000 85 When beef or rabbit SR vesicles were warmed to 37°C after being chilled at 0°C for 8 minutes, both preparations accu- mulated more than 300nmolCa++/mg protein in 10 minutes. These results show that Ca++ accumulation by SR vesicles is strongly temperature dependent. Several previous investiga- tions have reported similar results (Martonosi and Feretos, 1964; Sreter, 1969; La Court, 1971; Kanda, 1975). In con- trast, Sreter (1969) found that the Ca++ accumulating ability of rabbit SR vesicles increases with temperature over the range of 0 to 25°C, but at 35°C, the initial uptake was followed by release of Ca++. However, no explanation for + the release of Ca+ at 35°C was provided (Sreter, 1969). Effects of Tempgra3ure on Ca++ Release by Sarcop1gsmic Reticulum Vesicles Figures 17 and 18 show that after SR vesicles have accumulated Ca++ for 8 minUtes at pH 7.3 and 37°C, chilling the media to 0°C completely inhibits further accumulation. In fact, the drop in temperature from 37 to 0°C caused re- lease of some of the accumulated Ca++. After 10 minutes at 0°C, beef and rabbit SR vesicles released 66 and 106rmKH Ca++/mg protein (Tables 4 and 5, respectively). This cor- responds to 18 and 30 percent of the total accumulated Ca++ Chilling beef and rabbit SR vesicles from 15 to 0°C caused the release of 31 and linmoICaTT/mg protein/10 minutes, + corresponding to a release of 36 and 12% of the initial Ca+ load, respectively. 86 600 8er Sarcoplasmic Reticulum E 500 .23 E a. E” 400 +\ + (B Q "g 300 E . c: x. e 200 m ‘o '5 E 3 100 2 so... 00 + ‘ . Te pH 7. 315.0 L) o 5 10 15 20 TIME (minutes) Fig. 17. Ca++ accumulation and release by beef sarco« plasmic reticulum vesicles as affected by temperature and pH. 87 600 Rabbit Sarcoplasmic Reticulum § —‘— “:o 1 00 Ca++ Accumulation (nmol CaHImg protein) 8 pH 7. 315. o 5 10 15 20 TIME (minutes) Fig. 18. Ca++ accumulation and release by rabbit sarco- plasmic reticulum vesicles as affected by temperature and pH. 88 Kanda (1975) also observed the release of Ca++ from preloaded chilled SR vesicles. These findings support the conclusions of Pearson 33 _1. (1973) and Davey and Gilbert (1974), both of whom concluded that chilling the SR causes a release of Ca++, which in turn results in muscle cold shortening. It must be pointed out, however, that the frag- mented SR vesicles may be more susceptible to Ca++ leakage than the intact SR membrane system. Consequently, chilled SR vesicles may release more Ca++ than would be the case for the intact SR system. However, there is little doubt that chilling SR vesicles or intact muscle to 0°C virtually abolishes Ca++ accumulation. Furthermore, results of the present study suggest that Ca++ is released from chilled SR membranes, thus contributing to cold shortening. Effects of pH on Ca++ Accumulation pygSarcoplasmic Reticulum Vesicles Tables 4 and 5 show that beef and rabbit SR vesicles, respectively, accumulated 300-500nmolCa++/mg protein at pH 7.3 or pH 6.8 at 37°C. At pH 6.2 and 37°C, beef and rabbit SR vesicles accumulated a maximum of 94 and 153mmfl Ca++/mg protein, respectively. At pH 5.5 or below and at 37°C, neither SR preparation accumulated more than 82nmol CaTT/mg protein. At both 15 and 0°C, maximum Ca++ accumula- tion was generally less than 100nmolCa++/mg protein in both rabbit and beef SR vesicles, regardless of the pH. Even at these reduced temperatures, however, Ca++ accumulation by SR 89 vesicles was much less at pH 5.5 or 5.0 than at the higher pH values. The data indicate that at pH values of 6.2 or below, Ca++ accumulation by both beef and rabbit SR vesicles is markedly reduced. Several other investigators have also observed that Ca++ accumulation by SR vesicles is greatly reduced at pH 5.0 (Sreter, 1969; La Court, 1971; Kanda, 1., 1977). Berman 33_31, (1977) further 1975; Berman 33 reported that brief exposure of SR vesicles to pH values in the range of 5.5 to 6.0 caused rapid and irreversible inac- tivation of Ca++ accumulation. In contrast to the results of the present investigation and several other studies (La Court, 1971; Kanda, 1975; Ber- man 33 31., 1977), Sreter (1969) reported that optimum Ca++ accumulation by rabbit SR vesicles occurred in the pH range 5.6 to 6.5. Although Sreter (1969) observed an inhibition of Ca++ accumulation at pH 5.0, which agrees with the results of the present study, he concluded that at pH 7.0 Ca++ uptake was significantly decreased and at pH 7.4, Ca++ uptake was only 40% of maximum. This is surprising since Sreter's (1969) results indicate that Ca++ accumulation is minimum at physiological pH, and is maximum at pH values existing in muscle under rigor conditions. The results of the present study clearly show that low pH values in the range 5.0 to 5.5 inhibit Ca++ accumulation by SR vesicles. This suggests that white muscle may be less susceptible to cold shortening due to its rapid rate of post- mortemglycolysis, sometimes resulting in pH values of 5.5 90 or less in only 30 minutes postmorten (Kastenschmidt, 1970). .Effects of pH on Ca++ Release by Sarcoplasmic Reticulum Vesicles Figures 17 and 18 show that beef and rabbit SR vesicles preloaded with 300 to 375nmoICa++/mg protein released almost all of the accumulated Ca++ when the pH of the media was lowered to 5.0. The release of Ca++ was rapid, occurring in a 10 minute period. Thus, the results of the present study show that at low pH values in the range of 5.0 to 5.5, Ca++ accumulation by the SR vesicles is reduced, and significant amounts of Ca++ are released. In postmortem muscle, glycolysis lowers the muscle pH to values in the range of 5.0 to 6.0 (Kastenschmidt, 1970). Busch 33 l. (1967) reported that rigor shortening occurs at pH values below 6.0. 6011 33 _1. (1971) and La Court (1971) both concluded that postmortem proteolysis is responsible for inactivation of the SR membrane, causing Ca++ release and consequently rigor shortening. However, results of the present study indicate that the low postmortem pH alone is sufficient to inactivate the SR membrane and inhibit Ca++ accumulation. The results of Berman 33 a1. (1977) and Kanda (1975) support this conclusion. Effects of Protein Concentration on Mitochondrial Ca++ Agcumulation Figure 19 shows that mitochondrial Ca++ accumulation was not strictly proportional to protein concentration. 91 Beef 200 o o I: o E 150 T" o I: .2 £39. 100 Rabbit E D 8 A < + '36 50 C.) .4 . 8 1. 2 Protein Concentration ( mg/ml) Fig. 19. Ca++ accumulation by mitochondria at pH 7.3 and 37 C as a function of protein concentration. The reaction was allowed to proceed for 4 minutes. 92 Therefore, all subsequent experiments were conducted using 0.5 to 0.8 mg mitochondrial protein/m1 reaction mixture in order to eliminate the effects of protein concentration on Ca++ accumulation. Several investigators (Drahota t 1., 1965; Malstrom and Carafoli, 1975; Jacobus 33 31., 1975) used higher protein concentrations, ranging from 1.45 to 4.0 mg protein/ml, in assays for mitochondrial Ca++ accumu- lation. However, it was observed in the present study that at protein concentrations greater than 1.0 to 1.4 mg pro- tein/m1, the pores of the millipore filters were rapidly stopped. Consequently, a sufficient amount of filtrate could not be obtained for analysis. At protein concen- trations of 0.5 to 0.8 mg/ml, the solutions could be rapidly filtered, and mitochondrial Ca++ accumulation was signifi- cant (Fig. 19). Role of ATP in Mitochondrial Ca++ Accumulation In order to accumulate more than 60nmolca++/mg protein, beef (Fig. 20) and rabbit mitochondria (Fig. 21) required the presence of 3.0 mM ATP in the reaction media. This is in agreement with the results of Chance (1964), who reported that rat liver mitochondria had a limited capacity for Ca++ accumulation in the absence of ATP or Pi' He reported that under these conditions Ca++ accumulation did not exceed lOOnmol/mglnotein. 0n the other hand, Drahota 33 _1. (1965) observed that rat liver mitochondria in the presence of ATP could accumulate more than 500nmolCa++/mg protein, which 93 zoo BEEF MITOCHONDRIA .’ succinate ," +ATP+oir '0'. 9! *ATP '50 ' .. + ATP mm: " .ATP'PNz Cc" ACCUMULATION (nmol CaTT/m9 protein) u: C) 3 ® . ” *Nz - '7 '.~.~—-.A n + DNP \o', ‘ n . in? We .. «a. 5 10 15 20 TIME (minutes) Fig. 20. Ca” accumulation by beef mitochondria as affected by ATP, 2,4-dinitrophenol and anaerobic conditions. Incubation was carried out at pH 7.3 and 37°C. 200 150 “a 5'5: =2 <9- 100 -l :0 EE :\ 8:. < £2 0 us " so Fig.2]. Ca++ 94 RABBIT M ITOCHONDRIA succinct. +A‘I'P ” 'I'ATP" N2 ,." " +ATP+air " +ATP+DNP Ganglion-IIIIID ’9 * air a n: . O 0 fl -’ O O A A'AVAO ' o . A 1’ + P Av v " D" V \J' 5 IO 15 20 TIME (minutes) accumulation by rabbit mitochondria as affected by ATP, 2,4-dinitropheno] and anaerobic conditions. Incubation was carried out at pH 7.3 and 370C. 95 compares reasonably well with the values of over 400mmfl Ca++/ mg protein obtained in the present study (Table 6 and 7). There are two possible mechanisms by which ATP may sup- port mitochondrial Ca++ accumulation. First, the presence of ATP in the media may be necessary for the retention of Ca++ accumulated by a respiration-linked process, as was found to be the case for Ca++ accumulated by rat liver mito- chondria (Carafoli 33 _l., 1965; Kimura and Rasmussen, l977). Alternatively, Ca++ accumulation may actually be supported by ATP hydrolysis in the absence of mitochondrial respira- tion, as was demonstrated in rat liver mitochondria by Bielawski and Lehninger (l966) and Brand and Lehninger (l975). Unfortunately, the mechanism by which ATP supports Ca++ accumulation by muscle mitochondria was not conclusively determined by the present study. However, evidence to be presented later suggests that both mechanisms could possibly be important for Ca++ accumulation by muscle mitochondria. Effects of Nitrogen, Air, or 2,4-dinitrophenol on Mitochon- drial Ca++ Accumulation Buege and Marsh (l975) observed that chilled muscle strips cold shortened in the presence of mitochondrial uncoupling agents, such as 2,4-dinitrophenol, or in an anaero- bic atmosphere. However, aerobic conditions inhibited cold shortening. They postulated that these conditions influence cold shortening by affecting mitochondrial Ca++ accumulation or retention. 96 Table 6. Ca++ Accumulation and Release by Beef Mitochon- dria (nmol Ca++/mg protein) Temp. Time pH (00.) (minutes) 7.3 6.8 6.2 5.5 5.0 37 2 381.4 190.9 81.2 37.6 -- 37 4 374.7 278.5 164.7 32.0 28.2 37 8 337.2 294.0 196.1 26.1 26.2 37 13 390.2 302.1 256.1 37.6 21.8 37 1 18 259.2 305.5 280.1 70.2 19.1 37 + 0 8 + 5 290.2 289.5 327.2 -- -- 37 + 0 8 + 10 294.4 270.6 326.7 77.4 32.3 373 8 at pH 7.3 71.6 374 8 + 10 N2 gas 242.2 15 2 -- -- -- -- -- 15 4 179.7 186.2 80.7 54.4 0 15 8 249.4 190.3 99.8 21.1 0 15 13 260.7 233.4 118.1 33.9 8.7 15 18 311.2 244.9 159.5 19.9 0 15 8 + 5 at 0° -- 183.5 104.1 -- _- 15 8 + 10 at 0° 244.8 213.0 119.2 -- 0 0 2 44.1 36.1 12.3 0 0 0 4 162.1 38.7 14.4 5.8 0 0 8 134.0 50.2 15.5 8.3 0 0 13 65.4 38.5 35.6 0 0 0 18 59.0 59.2 55.4 0 0 o 8 + 5 at 37° -- 391.5 295.8 -- o 0 8 + 10 at 37° 298.5 390.7 286.3 36.5 0 1Incubation at pH 7.3 and 37°C for 8 minutes, followed by incubation'for 5 minutes at 0°c zIncubation at pH 7.3 and 37°C for 8 minutes, followed by incubation 3Incubation incubation 4Incubation pH 7.3 and 37°C for 10 minutes at 0°C at pH 7.3 and 37°C for for 10 minutes at pH 5 at pH 7.3 and 37°C for bubbling nitrogen gas through the 8 minutes, followed by .0 and 37°C 8 minutes, fo1lowed by medium for 10 minutes at T 97 able 7. Ca++ Accumulation and Release by Rabbit Mito- chondria (nmol Ca++/mg protein) Temp. Time pH (°C ) (minutes) 7.3 6.8 6. 5.5 5.0 37 2 147.4 75.6 78.5 95.4 _- 37 4 202.3 98.7 115.7 78.5 _- 37 8 282.6 169.5 164.6 65.1 32.7 37 13 371.0 180.7 139.7 52. 7 30. 7 37 18 454.7 231.1 137.2 73. 7 15.9 37 + 0; 8 + 5 -- 164.0 153.9 -- _- 37 + 0 8 + 10 234.4 218.4 196.3 93.6 10.2 373 8 + 10 at pH 5.0 69.7 374 8 + 10 N2 gas 264.0 15 2 90.0 105.9 -- -- 0 15 4 111.0 70.7 106.7 32.1 0 15 8 164.8 67.4 96.5 38.4 0 15 13 175.3 70.7 96.7 44.5 0 15 18 164.2 83.9 134.2 51.0 0 15 8 + 5 177.7 70.8 -- 42.1 0 15 8 + 10 164.6 76.8 118 1 35. 6 0 0 2 78.8 -- -- -- 0 0 4 88.2 51.7 45.1 10.1 0 0 8 80.5 43.3 56.8 11.7 0 0 13 76.7 65.7 -- -- 0 0 18 81.3 73.7 76.4 7.0 0 0 8 + 5 at 37° 266.7 168.0 -- -- -- 0 8 + 10 at 37° 359.2 177.2 176.0 53.2 -- 1Incubation at pH 7.3 and 37°C for 2 incubation for 5 minutes at 0°C Incubation at pH 7. 3 and 370 C for incubation for 10 minutes at 00 C 3Incubation at pH 7.3 and 37°C for 4 incubation for 10 minutes at pH 5 Incubation at pH 7. 3 and 370 C for bubbling nitrogen gas through the at pH 7. 3 and 37°C 8 minutes, 8 minutes, 8 minutes, .0 and 37°C 8 minutes, medium for followed by followed by followed by followed by 10 minutes 98 In the present study, direct measurements of Ca++ accumulation and release by muscle mitochondria were con- ducted, using conditions similar to those described by Buege and Marsh (1975). Figure 20 shows that Ca++ accumula- tion by beef mitochondria was somewhat reduced in the presence of nitrogen at incubation times of either 13 or 18 minutes. Under the same conditions, on the other hand, Ca++ accumulation by rabbit mitochondria (Fig. 21) was not significantly affected by the presence of nitrogen. Bubbling air through the mitochondrial preparations did not increase mitochondrial Ca++ accumulation as compared to preparations incubated in the absence of bubbling air or nitrogen gas (Fig. 20 and 21). The mitochondrial uncoup1er, 2.4-dinitrophenol, par- tially inhibited Ca++ accumulation by rabbit mitochondria (Fig. 21), but did not significantly inhibit Ca++ accumula- tion by beef mitochondria (Fig. 20). However, Figures 20 and 21 show that both beef and rabbit mitochondria accumu- lated significant amounts of Ca++, with values in excess of lOOrmnlCa++/mg protein/l8 minutes, when incubated in the presence of a mitochondrial uncoup1er (2,4-dinitrophenol) or an anaerobic environment (bubbling nitrogen gas). In contrast to the results of the present experiment, Carafoli (1976) stated that 2,4-dinitrophenol completely inhibited Ca++ accumulation and initiated rapid release of Ca++ from rat liver mitochondria. Sordahl (1974) also ob- served an inhibition of Ca++ accumulation by rabbit heart 99 mitochondria if the medium was allowed to become anaerobic. Thus, results of the present investigation show that muscle mitochondria do not respond to mitochondrial uncouplers or anaerobic conditions in the same manner as mitochondria from rat liver or rabbit heart. Furthermore, results obtained in the present study indicate that the response of muscle mitochondria to anaerobic conditions is not as pronounced as might be expected based on the hypothesis of Buege and Marsh (1975). The results of the present study suggest that at least a portion of the mitochondrial Ca++ uptake for both beef and rabbit mitochondria is accumulated in a respiration-indepen- dent process, perhaps supported by ATP hydrolysis. Ca++ accumulation supported by ATP hydrolysis would not be depen- dent upon mitochondrial respiration, and thus, anaerobic conditions would not lead to Ca++ release. This is suppor- ted by the results of Weber gt _1. (1966). They observed that mitochondrial inhibitors decreased Ca++ accumulation by only 10% for rabbit muscle mitochondria, while the same in— hibitors completely inhibited mitochondrial Ca++ accumulation in tissues other than muscle. Greaser gt ll- (1969) simi- larly observed that 5 mM sodium azide, which blocks mito- chondrial Ca++ accumulation in most tissues (Fanburg 33 al., 1955), inhibited only 5% of the Ca++ accumulating ability of pig muscle mitochondria. Greaser gt _1. (1969) further suggested that contami- nating SR fragments may play an important role in Ca++ 100 accumulation by muscle mitochondrial fractions. However, since muscle mitochondrial preparations (Tables 6 and 7) accumulate Ca++ at about the same level as pure SR prepara- tions (Table 4 and 5), it is improbable that SR contamination could account for the high levels of Ca++ accumulated by mitochondria in this study. Thus, the evidence points towards muscle mitochondrial accumulation of Ca++ supported by ATP hydrolysis. Effects of Nitrogen on Mitochondrial Ca++ Release Both beef and rabbit mitochondrial suspensions released a portion of their previously accumulated Ca++ when nitrogen gas was bubbled through the medium. Figure 22 shows that beef mitochondrial suspensions released about 95nmilCa++/mg protein/10 minutes as compared to only 18nmo§1Ca++ for rabbit mitochondrial suspensions (Fig. 23). Expressed on a percen- tage basis, the beef mitochondrial suspensions released 28% of their accumulated Ca++ as compared to a loss of only 6% from rabbit mitochondrial suspensions. The greater propor- tion of Ca++ released by beef muscle mitochondria may be due to the higher levels of Ca++ accumulated prior to imposing anaerobic conditions. Initially, beef mitochondria contained 337nmplCa++/mg protein as compared to 282 for rabbit mito- chondria. The greater loss of Ca++ by beef mitochondria is supported by the results of Jacobus gt 11. (1975), who con- cluded that the amount of Ca++ released is proportional to the initial Ca++ load of the mitochondria. Martonosi and 101 § 500 12 Beef Mitochondria E” 400 + + (U Q ' "g 300 E C o '13 200 35 E :3 8 100 <: + 4.. 5° 0 5 10 15 20 TIME (minutes) Fig. 22. Ca++ release by beef mitochondria at pH 7.3 and 37 C in the presence of nitrogen. 102 500 Rabbit Mitochondria 100 Ca++ Accumulation (nmol Ca++lmg protein) 8 5 10 15 20 TIME (minutes) Fig. 23. Ca++ release by rabbit mitochondria at pH 7.3 and 37°C in the presence of nitrogen. 103 Feretos (1964) had previously reached the same conclusion using SR vesicles. Alternatively, the greater proportion of Ca++ released by beef mitochondria may be due to the fact that more Ca++ is bound to beef mitochondria in a respiration linked manner. Electron micrographs showed that beef mitochondria were in r 1 an actively respiring state (Fig. 12), while rabbit mitochon- g dria were in a state of resting respiration (Fig. 13). i Manometric measurements also showed that beef mitochondrial suspensions (Table 2) were more actively respiring than rabbit mitochondria (Table 3). Figures 20 and 21 show that the presence of nitrogen reduced Ca++ accumulation by beef mitochondria, while having no effect on rabbit mitochondria. These results suggest that beef mitochondria accumulate Ca++ in arespiration linked manner, while rabbit mitochondria accumulate Ca++ supported by ATP hydrolysis. Consequently, beef mitochondria would be more susceptible to anaerobic conditions, thus releasing more Ca++ Sordahl (1974) also observed that anaerobic conditions initiated the release of Ca++ mitochondrial preparations. He found that rabbit heart mitochondria released 70% of the accumulated Ca++ when the medium became anaerobic. Drahota gt 31. (1965) reported that rat liver mitochondria released Ca++ in the absence of a respiratory substrate. These studies suggest that mitochondrial respiration is necessary for retention of Ca++, at least in rabbit heart and rat 104 liver mitochondria. Results of the present study also indi- cated that respiration may be necessary for retention of a part of the Ca++ accumulated by muscle mitochondria, espe- cially in the case of beef muscle mitochondria. It is unlikely that intact muscle mitochondria contain as much Ca++ as they are able to accumulate in an ifl_vltgg_ system. However, Carafoli (1975) pointed out that very small amounts of Ca++ are necessary to initiate muscle contraction. He stated that muscle contraction occurs when the Ca++ concentration in the myofibrillar region increases 7 to 10.5 from 10- M, which corresponds to an increase from 0.1 to 10.01mnlCa++/g muscle tissue. The results of this ifl.li££2 study show that anaerobic conditions, low pH and low temperature can initiate the release of more than enough Ca++ to cause cold shortening. Influence of Temperature on Mitochondrial Ca++ Accumulation Tables 6 and 7 show that mitochondrial Ca++ accumulation is temperature dependent. At 37°C and pH 7.3, both beef (Table 6) and rabbit mitochondrial suspensions (Table 7) accumulated more than 390nmolCa++/mg protein/18 minutes. At 15°C, beef and rabbit mitochondrial suspensions accumu- lated 311 and 163nm01Ca++/mg protein/18 minutes, while at 0°C the suspensions accumulated only 69 and 81nmolCa++/mg protein, respectively. Thus, muscle mitochondrial Ca++ accumulation is significantly reduced at low temperatures such as occur in conditions favoring cold shortening. 105 Reed and Bygrave (1975) also observed that mitochon- drial Ca++ accumulation was temperature dependent, and sug- gested that kinetic studies of mitochondrial Ca++ accumula- tion be conducted at reduced temperatures in order to obtain more accurate estimates of initial rate parameters. The results of the present investigation support the theory that low temperatures reduce Ca++ accumulation by mitochondria. Influence of Temperature on Mitochondrial Ca++ Release Figures 24 and 25 demonstrate that preloaded muscle mitochondrial preparations at 37°C and pH 7.3 released small amounts of Ca++ when the suspension was chilled to 0°C. Beef (Fig. 24) and rabbit mitochondrial suspensions (Fig. 25) released 43 and 48nmolCa++/mg protein/10 minutes, which corresponds to the release of 13 and 17% of the initial Ca++ load, respectively. However, the effects of chilling on the mitochondrial preparations were reversible by warming the suspensions to 37°C. Figures 24 and 25 also show that both beef and rabbit mitochondrial suspensions were able to accumulate more than 300nmolCa++/mg protein/10 minutes, when warmed to 37°C after previous chilling for 8 minutes at 0°C. This clearly demonstrates the reversible nature of Ca++ ‘ accumulation with temperature. Although the present study shows that chilled mito- chondria release Ca++, Drahota gt _1. (1965) found that rat liver mitochondria retained more Ca++ at 0 than at 30°C. However, they preloaded the mitochondria with Ca++ in the 106 500 Beef Mitochondria pH 7. 315. 0 0 *‘1l § Ca“ Accumulation (nmol CaHImg protein) 00 5 10 15 20 TIME (minutes) Fig. 24. Ca++ accumulation and release by beef mitochon- dria as affected by temperature and pH. 107 Rabbit Mitochondria 500 300 Q Q pH 7. 315. 0 ‘Q.@ . Ca++ Accumulation (nmol CaH/mg protein) 5 10 15 20 TIME (minutes) Fig. 25. Ca++ accumulation and release by rabbit mitochon- dria as affected by temperature and pH. 108 presence of Pi’ and then resuspended the mitochondria in a buffered medium lacking ATP or respiratory substrate. Slow Ca++ release would be expected under such conditions. Chilling the medium would slow the rate at which Ca++ was released from the phosphate precipitate in the mitochondria, thus explaining their results. The results of the present study show that chilled mitochondria release sufficient Ca++ to initiate cold shor- tening, as was previously shown for anoxic mitochondria and chilled SR preparations. These results further show that anoxic conditions are not a prerequisite for Ca++ release from mitochondrial suspensions, at least under in_vitrg_ conditions. Since both low temperatures and anoxic condi- tions are favorable for the development of cold shortening, it iS‘probable that both conditions may contribute to the initiation of this phenomenon. Influence of pH on Mitochondrial Ca++ Accumulation Tables 6 and 7 show that both beef and rabbit mito- chondria have a maximum Ca++ accumulating ability at pH 7.3 and 37°C, exceeding 390nmo.lCa++ mg/protein. At pH 6.8, both preparations accumulated in excess of 230nmolCa++/mg protein, and at pH 6.2 still retain a large capacity for Ca++ accumu- lation with values in excess of 160nmol. At pH 5.5 or 5.0, however, neither mitochondrial suspensions could accumulate more than 95nmolCa++, and in general, Ca++ accumulation was in the range of 20-30nmolCa++/mg protein. These results 109 show that low pH values, in the range 5.0 to 5.5, greatly reduce the Ca++ accumulating ability of mitochondrial prepa- rations, similar to the effects of low pH on SR preparations, which was previously shown herein. Influence of pH on Mitochondrial Ca++ Release Figures 24 and 25 show that beef and rabbit muscle mitochondria release almost all of the initial ta++ 1oad when the pH of the medium is lowered to pH 5.0. Both mitochon- drial preparations released more than 200nm010a++mg protein/ 10 minutes under these conditions. These results suggest that the low pH encountered in postmortem muscle is suffi- cient to cause release of Ca++ from mitochondria, as well as SR membranes, and could be a factor in rigor shortening. Effects of pH on Ca++ Accumulation by_Mitochondria and Sar- coplasmic Reticulum Mitochondrial and SR fractions of beef and rabbit muscle have much greater capacities for Ca++ accumulation at pH values of 7.3 to 6.8 than at lower values (Fig. 26). At pH 6.2, mitochondrial preparations have a significantly greater capacity for Ca++ accumulation than is the case for SR preparations. This suggests that muscles containing high levels of mitochondria may be more resistant to the inhibiting effects of low pH on Ca++ accumulation and reten- tion, and consequently may be more resistant to the develop- ment of rigor. It is well known that the time course of rigor development is much faster in white than in red 110 4.: any 600 A - Rabbit Sarcoplasmic Reticulum B " “Of n n i 500 C - Rabbit Mitochondria D " “Of n n 0000000000000 COOOOOOOOOOOOO Ca" ACCUMULAT ION (nmol Co” Img protein / 18 min.) ‘ u C) 0 Id 0 O 100 Fig. 26. Effect of pH on the Ca++ accumulating ability of‘sarco— plasmic reticulum vesicles and mitochondria of beef and rabbit muscle at 37°C. 111 muscles (Kastenschmidt, 1970). This has been attributed to the higher rate of postmorten glycolysis in white muscles, resulting in a faster drop in postmortem muscle pH to values at which rigor occurs (Kastenschmidt, 1970). The rate of postmortem glycolysis is undoubtedly an important factor in determining the time at which the muscle goes into rigor. Results of this study indicate that muscle mitochondria are more resistant to the effects of low pH (in the range of 6.2) than the SR membranes, which may be a contributing factor in the resistance of red muscles to rigor development. However, at pH values in the range of 5.5 to 5.0, both mitochondrial and SR membranes are inactivated with respect to Ca++ accumu- lation, and rigor ensues. These results suggest that muscle pH is important as a permissive, but not a causative factor for cold shortening. At pH values of 6.2, 6.8, or 7.3 red muscles retain the ability to accumulate Ca++. If Ca++ accumulation is temperature sensitive, cold shortening could occur. As the postmortem muscle pH is progressively lowered, the temperature sensitivity of the muscle diminishes as observed by Locker and Hagyard (1963). Then as the pH drops to 5.5 to 5.0, Ca++ accumulation is markedly reduced and rigor develops. As a consequence the muscle is no longer temperature sensitive. Effects of Temperature on Ca++ Accumulation by Mitochondria and Sarcoplasmic Reticulum Figure 27 shows that mitochondria and SR preparations both have a very significant capacity for Ca++ accumulation 112 600 j A 3 A -' Rabbit Sarcoplasmic Roticulum ’ B - Boot " ” C - Rabbit Mitochondria 5C“! D - Boot 1’ 400 Co" Accumuunon (nmol Ca**lmg protein/18min.) u o o 100 37° 1 5° 0° TEMPERATURE (°C) Fig.27. Effect of temperature on the Ca++ accumulating abil— ity of sarcoplasmic reticulum vesicles and mitochondria of beef and rabbit muscle.at pH 7.3. 113 at 37°C. At 15°C both preparations retain the ability to accumulate large quantities of Ca++, in the range of 100 nmolCak7mg protein or more. However, the mitochondrial preparations have a significantly greater capacity for Ca+f accumulation than the SR, usually in the range of 200nm01Ca++ or more. The ability of the mitochondrial membranes to accumulate large amounts of Ca++ at 15°C may contribute to the ability of muscle to avoid cold shortening at this tem- perature. At 0°C both mitochondrial and SR fractions retain only a very limited ability to accumulate Ca++. At this temperature, Ca++ is probably bound only to external sites on the membrane, in an energy or respiration independent manner (Martonosi, 1972; Lehninger 33 al., 1967). These results suggest that at the temperatures which favor development of cold shortening, the ability of both SR and mitochondria to accumulate Ca++ is greatly reduced. Very low Ca++ accumulation, in combination with the release of Ca++ from both SR and mitochondrial sites appears to pro- vide sufficient Ca++ in the myofibrillar region of the mus- cle to initiate cold shortening. Reversibility of Cold Shortening As pointed out earlier, Locker and Hagyard (1963) first observed that cold shortened muscle relaxed when returned to room temperature. However, the muscle was capa- ble of cold shortening again, if it was chilled to 0°c. The speed and degree of cold shortening decreased with time 114 postmortem. After the muscle passed into rigor, it was no longer temperature sensitive. Buege and Marsh (1975) proposed that cold shortening was the result of Ca++ released by anoxic mitochondria. How- ever, their proposal offers no explanation for the repeated ability of muscle to cold shorten after being relaxed by warming, i.e., the phenomenon of reversibility (Locker and Hagyard, 1963). It is unlikely that rewarming the muscle permits respiration dependent accumulation of Ca++ by already anoxic mitochondria. It is even more unlikely that the anoxic mitochondria would be the direct source of Ca++ initiating cold shortening when the muscle is chilled a second time. On this basis, it is evident that anoxic con- ditions alone cannot account for the cold shortening pheno- menon. It is proposed that cold shortening results from the release of Ca++ as a consequence of chilling the SR, the mitochondria or both. In support of this conclusion, the present study demonstrated that SR and mitochondrial prepa- rations from both beef and rabbit muscle release Ca++ as a direct result of chilling. Moreover, SR and mitochondrial preparations from both beef and rabbit muscles were capable of accumulating Ca++ upon warming, as would be necessary for relaxation of cold shortened muscle. The results of this study further suggest that it is the postmortem drop in muscle pH that is responsible for the diminished reversibility of cold shortening with 115 increasing time postmortem. Although decreasing ATP levels and postmortem proteolysis could conceivably contribute to the inactivation of Ca++ accumulation by the SR membranes in postmortem muscle (0011 £3 31., 1971), the present study clearly shows that low pH alone will inactivate Ca++ accumu- lation by SR and mitochondrial membranes, leading to rigor. An Explanation of Cold Shortening The results of the present study show that isolated membrane fractions from red muscle respond in essentially the same manner and same degree to temperature, pH and anoxic conditions as those of white muscle, yet the intact muscles respond very differently to cold. Apparently, the major dif- ference between red and white muscle is in the relative con- tents of the mitochondrial and SR membranes, and this dif- ference somehow leads to cold shortening in red muscles. Based on these observations, the following sequence of events is proposed to lead to cold shortening. After slaughter, the muscle rapidly becomes anoxic, and the mito- chondria will release Ca++ under anoxic conditions as proposed by Buege and Marsh (1975). Both red and white muscle mito- chondria release Ca++ under anoxic conditions, but a greater total quantity of Ca++ is released in red muscle, simply because red muscles contain more mitochondria. All of the Ca++ is accumulated by the SR membranes at room temperature, and no contraction occurs. However, the SR membranes of red muscle contain much higher concentrations of accumulated 116 Ca++at room temperature, due to the greater amounts of Ca++ released, and the smaller amount of SR membranes available to accumulate Ca++. When the muscles are chilled to 0°C, Ca++ accumulation in both muscles is greatly reduced. How- ever, the SR of red muscles is more susceptible to the effects of chilling, due to the high concentration of accu- mulated Ca++ present, and consequently releases greater quantities of Ca++ than that of white muscle, resulting in cold shortening of the red muscle. This is supported by the observation of Martonosi and Feretos (1964), who found that when the Ca++ accumulating ability of rat SR vesicles was inhibited, the subsequent release of Ca++ was much faster from vesicles having higher initial Ca++ concentrations. Upon rewarming the muscles, the SR membrane reaccumu- lates the Ca++, and if sufficient ATP is present, the muscle relaxes. Chilling the muscle a second time will again lead to greater release of Ca++ from the red muscle SR membranes by the same process, and the muscle again shortens. As the pH drops with increasing time postmortem, the SR membranes gradually lose their ability to reaccumulate Ca++, and con- sequently the ability to relax diminishes. When the muscle enters rigor, the SR membrane can no longer reaccumulate Ca++ upon warming, and the muscle is therefore temperature insensitive. Results of the present study suggested that the mito- chondria may also release Ca++ when chilled, and reaccumu- ++ . . late Ca upon warming. However, for th1s process to occur 117 in intact muscle, Ca++ accumulation would have to be suppor- ted by a respiration independent process, since the muscle is anaerobic. Isolated mitochondria have the capacity to accu- mulate Ca++ by a respiration independent process, i.e., ATP hydrolysis. It is unlikely, however, that the control mecha- nisms of intact mitochondria would permit the use of ATP for support of Ca++ accumulation. It is proposed, therefore, that mitochondria play no role in cold shortening other than providing the initial Ca++ to overload the SR membrane system of red muscle. According to this proposal, anoxic conditions may be thought of as the priming factor, leading to release of Ca++ from the mitochondria, and the resultant overloading of the SR membranes in red muscle with Ca++. Anoxic conditions alone are not sufficient to cause shortening, because the SR membranes can accumulate the Ca++ at room temperature. Chilling the musc1e to 0°c may be thought of as the initia- ting factor causing the release of Ca++ from the overloaded SR of red muscle, causing shortening. Chilling the muscle is the direct stimulus for cold shortening, and cold shor- tening may be reversed simply by removal of the stimulus. The muscle pH and possibly the muscle ATP levels may be thought of as permissive factors for cold shortening. At sufficiently high pH values and in the presence of adequate ATP, cold shortening may occur if the other conditions such as anoxia and chilling, exist. However, cold shortening is not permitted if the muscle pH and/or the muscle ATP levels 118 fall too low. Stated somewhat differently, high mitochondrial con- tent, anoxic conditions, high ATP levels and high pH values are all necessary for cold shortening to occur, but are not sufficient to initiate cold shortening, unless the muscle is also chilled. This is in contrast to micro injections of Ca++, which overloads the SR and produces shortening even in the absence of cold (Pearson 93 al., 1973). This proposal is similar to that of Buege and Marsh (1975), in that cold shortening is related to mitochondrial content of the muscle, and anoxic conditions ultimately lead to cold shortening. However, the present proposal further explains the phenomenon of reversibility of cold shortening, based on the response of the SR membrane to changes in tem- perature. The present study concurs with the previous conclusions of Pearson gt 31. (1973) and Davey and Gilbert (1974) that Ca++ release from SR membranes provides a source of Ca++ sufficient to initiate cold shortening. SUMMARY Ca++ accumulation and release by isolated SR and mitochondria of beef and rabbit muscle were determined at several pH values and temperatures. Isolation of SR and mitochondria was accomplished by homogenization of the muscle followed by differential centrifugation. Further purifica- tion of the SR was achieved by sucrose density gradient centrifugation The yield of SR from rabbit muscle was approximately 3-fold greater than that from beef, while the yield of mito- chondria from the two muscles was similar. Histochemical staining for NADH-tetrazolium reductase activity showed that beef muscle contained a much higher concentration of mito- chondria. Transmission electron microscopy and S05 gel electrophoresis revealed that the SR preparations were essentially free from contamination with myofibrillar proteins or other subcellular organelles. Electron microscopy demon- strated that the mitochondrial preparations were relatively pure. Mitochondrial succinate oxidase activity was similar in suspensions from beef and rabbit muscle, but manometric measurement of oxygen consumption showed that the prepara- tions from beef muscle contained more intact respiring mito- chondria. 119 120 The SR vesicles from both beef and rabbit muscle accu- mulated more than 500nmol Ca++/m9 protein at PH 7-3 and 37°C. Mitochondria from both muscles accumulated more than 400 nmol Ca++/mg protein under similar conditions. Ca++ accumulation by mitochondria and SR from both beef and rabbit muscle was markedly temperature dependent. At 15°C and pH 7.3, Ca++ accumulation was reduced, although the mitochondrial prepara- tions still accumulated in excess of 150nmol Ca*+/mg protein. At 0°C, Ca++ accumulation by both SR and mitochondria was reduced to below 85 mmfICa++/mg protein. It is probable that at 0°C most of the Ca++ was bound to the external side of the membranes in an energy independent process. In spite of the reduction in Ca++ accumulation at low temperatures, chilled SR and mitochondria still accumulated significant quantities of Ca++ upon warming to 37°C. Values for pH in the range 5.0 to 5.5 greatly reduced the capacity of SR and mitochondria to accumulate Ca++. At pH 6.2, Ca++ accumulation by SR vesicles was low, but mito- chondrial suspensions still accumulated more than l60rmml Ca++/mg protein. Ca++ accumulation for all preparations was maximal at pH 6.8 to 7.3 Rabbit mitochondria accumulated somewhat more Ca++ under anaerobic conditions than beef mitochondria. However, both rabbit and beef mitochondria accumulated significant + + . . . . amounts of Ca under anaerobic conditions or in the presence of the uncoupling agent,2,4~dinitrophenol. 'This suggested that 121 at least part of the mitochondrial Ca++ in vitro is accumu- lated by a respiration-independent process, such as ATP hydrolysis. Preloaded mitochondria released small quantities of Ca++ when nitrogen was bubbled through the medium. Never- theless, the quantities released were sufficient to initiate shortening in intact muscle. The data indicated that the F: mitochondrial preparations were sensitive to anaerobic con- ; ditions, and that mitochondrial respiration is necessary for retention of at least a portion of the accumulated Ca++. "' Chilling of SR and mitochondrial preparations from beef and rabbit muscle also caused the release of small but signifi- cant amounts of Ca++. 0n lowering the pH to 5.0, virtually all of the initial Ca++ load was released by SR and mito- chondria. Results demonstrated that SR and mitochondria from beef and rabbit muscle did not significantly differ in their response to conditions promoting cold shortening. It is, therefore, apparent that cold shortening is related to the relative concentrations of SR and mitochondria in muscle. 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Schedule for the preparation of Epon-Araldite resin Reagents Amount Epon 813 62 m1 Araldite 506 81 ml DDSA (hardener) 100 ml dibutyl phthalate 4-7 ml DMP-30 1.5-3.0% Mixing solution: Epon 812, Araldite 506, and DDSA are mixed, after which the dibutyl phthalate and the DMP-30 are added. After mixing, the resin is ready for embedding the tissue. Appendix Table 4. Schedule for the preparation of Reynolds lead citrate stain Reagents Amount Lead citrate 1.33 g Sodium citrate 1.76 g l N NaOH 8 ml H 0 (freshly boiled) make to 50 ml 2 138 Appendix Table 5. Schedule for the preparation of a 1% osmium tetroxide fixative solution Reagents Amount A. Stock Solution A Sodium acetate 9.714 g Veronal-sodium 14.714 9 Make to 500 m1 final volume with H 0 2 8. Stock Solution 8 Sodium chloride 40.25 9 Potassium chloride 2.10 9 Calcium chloride .90 9 Make to 500 ml final volume with H20 The solutions are mixed according to the following scheme: . Solution A 10.0 ml Solution B 3.4 m1 Dilute to 50 ml with H20 0.1 N HCl approx. 11 m1 Solutions A and B are measured out and made to a 50 m1 volume with distilled water. The pH is then adjusted to 7.2-7.4 with 0.1 N HCl. To this mixture 0.5 g of osmium tetroxide is added and stored in a brown glass stoppered bottle. Appendix Table 6. Schedule for the preparation of uranyl acetete stain Reagents Amount Uranyl acetate 8 9 H20 (glass distilled) 100 ml