Rabbit White Muscle Research Articles

Ca 2+ transport and heat production in vesicles derived from the sarcoplasmic reticulum terminal cisternae: Regulation by K +

In this work, we compared the effect of K + on vesicles derived from the longitudinal (LSR) and terminal cisternae (HSR) of rabbit white muscle. In HSR, K + was found to inhibit both the Ca 2+ accumulation and the heat released during ATP hydrolysis by the Ca 2+-ATPase (SERCA1). This was not observed in LSR. Valinomycin abolished the HSR Ca 2+-uptake inhibition promoted by physiological K + concentrations, but it did not modify the thermogenic activity of the Ca 2+ pump. The results with HSR are difficult to interpret, assuming that a single K + is binding to either the ryanodine channel or to the Ca 2+-ATPase. It is suggested that an increase of K + in the assay medium alters the interactions among the various proteins found in HSR, thus modifying the properties of both the ryanodine channel and SERCA1.

Influence of N-dodecyl- N,N-dimethylamine N-oxide on the activity of sarcoplasmic reticulum Ca 2+-transporting ATPase reconstituted into diacylphosphatidylcholine vesicles: Effects of bilayer physical parameters

Sarcoplasmic reticulum Ca-transporting ATPase (EC 3.6.1.38) was isolated from rabbit white muscle, purified and reconstituted into vesicles of synthetic diacylphosphatidylcholines with monounsaturated acyl chains using the cholate dilution method. In fluid bilayers at 37 °C, the specific activity of ATPase displays a maximum (31.5 ± 0.8 IU/mg) for dioleoylphosphatidylcholine (diC18 : 1PC) and decreases progressively for both shorter and longer acyl chain lengths. Besides the hydrophobic mismatch between protein and lipid bilayer, changes in the bilayer hydration and lateral interactions detected by small angle neutron scattering (SANS) can contribute to this acyl chain length dependence. When reconstituted into dierucoylphosphatidylcholine (diC22 : 1PC), the zwitterionic surfactant N-dodecyl- N,N-dimethylamine N-oxide (C12NO) stimulates the ATPase activity from 14.2 ± 0.6 to 32.5 ± 0.8 IU/mg in the range of molar ratios C12NO : diC22 : 1PC = 0 ÷ 1.2. In dilauroylphosphatidylcholines (diC12 : 0PC) and diC18 : 1PC, the effect of C12NO is twofold—the ATPase activity is stimulated at low and inhibited at high C12NO concentrations. In diC18 : 1PC, it is observed an increase of activity induced by C12NO in the range of molar ratios C12NO : diC18 : 1PC ≤ 1.3 in bilayers, where the bilayer thickness estimated by SANS decreases by 0.4 ± 0.1 nm. In this range, the 31P-NMR chemical shift anisotropy increases indicating an effect of C12NO on the orientation of the phosphatidylcholine dipole N +–P − accompanied by a variation of the local membrane dipole potential. A decrease of the ATPase activity is observed in the range of molar ratios C12NO : diC18 : 1PC = 1.3 ÷ 2.5, where mixed tubular micelles are detected by SANS in C12NO + diC18 : 1PC mixtures. It is concluded that besides hydrophobic thickness changes, the changes in dipole potential and curvature frustration of the bilayer could contribute as well to C12NO effects on Ca 2+-ATPase activity.

The Thermogenic Activity of Rat Brown Adipose Tissue and Rabbit White Muscle Ca2 + ‐ATPase

The Ca2+-ATPase (SERCA) found in vesicles derived from the sarco/endoplasmic reticulum vesicles of rats brown adipose tissue and rabbit white muscle were identified by gel electrophoresis, Western blot, electron microscopy and immunolabeling with gold particles. In both tissues, the isoform found was SERCA 1. The Ca2+ affinity of the fat SERCA 1 was different from the muscle isoform. The degree of uncoupling is estimated measuring the ratio between Ca2+ transport and ATP cleaved. In brown fat vesicles the degree of uncoupling varied depending on the Ca2+ concentration of the medium. This was not observed in vesicles derived from muscle. At all Ca2+ concentrations tested, the uncoupling was not related to Ca2+ leakage from the membrane and was far more pronounced in fat than in muscle vesicle. When a Ca2+ gradient was formed across the vesicles membrane the heat released during ATP hydrolysis varied between 22 and 26 Kcal/mol in both fat and muscle vesicles but in the absence of a gradient the heat released was 17 Kcal/mol in fat and 12 Kcal/mol in muscle. The data reported indicate that the SERCA 1 of brown adipocytes is far more thermogenic than the white muscle SERCA 1, and suggest that, in addition to storing Ca2+ inside the endoplasmic reticulum, the SERCA 1 may represent a source of heat production contributing to the thermogenic function of brown adipose tissue.

Recognition force microscopy/spectroscopy of ion channels: applications to the skeletal muscle Ca 2+ release channel (RYR1)

The skeletal muscle Ca 2+ release channel (ryanodine receptor 1, RYR1) plays an important role in the excitation–contraction coupling process. We purified ryanodine receptor type 1 from rabbit white muscle and adsorbed it to mica sheets with the cytoplasmic side facing up. Single receptors of uniformly distributed size and shape of 10–12 nm height and 40–50 nm width, and occasionally some aggregates were seen in contact mode AFM images. These immobilized RYR1 were specifically recognized by rabbit anti-RYR1 (antibody#8) with at least 30% efficiency, as measured by an enzyme immunoassay with goat-anti-rabbit. Single specific antibody–antigen recognition events were detected with AFM tips to which an antibody#8 was tethered. In linear scans, the occurrence of antibody–antigen binding showed significant lateral dependence, which allowed for the localization of binding sites with nm resolution. Variation of the loading rate in force spectroscopy experiments revealed a logarithmic dependence of the unbinding forces, ranging from 42 to 73 pN. From this dependence, a bond width of the binding pocket of L=0.2 nm and a kinetic off-rate of k off=12.7 s −1 was determined.

Recognition Force Microscospy of Single Ryanodine Receptor Type 1 (RyR1)

The skeletal muscle Ca2+ release channel (ryanodine receptor, RyR1) plays an important role in the excitation-contraction coupling process. We purified ryanodine receptor type1 from rabbit white muscle [1] and adsorbed it to mica sheets from a solution of 14 μg/ml RyR1 in Na-Pipes buffer, containing 1 mM Ca2+ , at pH 7.1. Single receptors and occasionally some aggregates were observed in contact mode AFM images (Fig. 1). The surface coverage was about 50 %. Specific binding events were detected in force-distance cycles (Fig. 2) with AFM tips to which an antibody (Ab#8) was coupled via a PEG spacer [2], indicating that the cytoplasmic side of the RyR was oriented towards the aqueous phase. The binding probability (i.e. the ratio of observed binding events to all measured force distance cycles, 32% on average) showed significant lateral dependence and allowed for the localization of binding sites. The mean unbinding force at a vertical scan frequency of 1 Hz was 70 pN. Specificity of antibody recognition was proven by blocking the antigenic sites with free Ab#8 which reduced the binding probability to 5%. In addition, variation of the force velocity revealed a linear dependence of the unbinding force in a halflogarithmic plot (an increase in the loading rate leads to an increase in the unbinding force). The method shows potential for the epitope mapping of ligand- or antibody-binding sites on ion channels.

Steroid hormone induced effects on membrane fluidity.

The classical mechanism of steroid hormone action involves the niolecule binding to high affinity intracellular receptors. This steroid-receptor complex then travels to the nucleus where it modulates gene transcription. Some steroid hormone actions, however, are not due to genoniic changes, e.g., modification of behaviour by progesterone [ l ] and altered neuronal firing activity after gonadal and adrenal steroid hormone action 121. These actions are almost immediate or of R short latency and are insensitive to inhibitors of RNA and protein synthesis. Steroid horniones in such cases may act directly on plasma membranes. Indeed, receptors for oestradiol, testosterone and progesterone have been identified in synaptic membranes [ 3 ] . Steroid hormones may also alter the dynamic properties of membrane bilayers. The structural requirements for maxiniising the interaction of sterols with the bilayer appear to be R 3-p-hydroxy group, an isocaproyl side chain and a flat cholestene skeleton 14). Given the structural nnalogy between cholesterol and steroid hormones it is possible that such hormones both alter membrane fluidity and influence membrane protein organisation and function. Deliconstantinos [5] has observed a change in synaptic membrane ordering after exposure to steroid hormones. The influences of steroid hormones on model membrane bilayer fluidity and on natural membrane ordering was therefore investigated. Measurements of membrane fluidity were performed using the steady-state fluorescence polarisation of 1,6-diphenyl 1,3,5hexatriene (DPH) according to the method described by Shinitzky and Barenholz 161. The data obtained are expressed in terms of the emission anisotropy (r) where r = (Iw I@I + 244 . and IvH being the emission intensities observed parall8 and perpendicular to the polarisation plane of the exciting light. Lipid bilayer membranes were prepared by dispersing egg yolk phosphatidylcholine in 0.2M Tris-C1 buffer pH 7.4. Cholesterol was added to some liposomes to give a molar ratio of cholesterol to phospholipid of 0.8, similar to the ratio in synaptic membranes. Liposomal membranes containing steroid hormones were prepared by co-dissolving the hormone together with the requisite amount of phosphatidylcholine in chloroformmethanol (2: 1 v/v). The solvent was then removed under a stream of nitrogen. 0.2M Tris-CI buffer pH 7.4 was added to the dry hormone-lipid mixture and unilamellar liposomes formed by sonication. The final concentration of hormone varied between 2 and 1OpM. This method could not be used with natural membranes. In this case, the steroid hormones were incorporated into dipalmitoyl phosphatidylcholine (DPPC) liposomes. The hormone-associated DPPC WRS added in a 1:l molar ratio to the lipid in the membranes, resulting in transference of hormone from the DPPC bilayers to the membrane lipid. The suspension was then centrifuged on a 20% (w/v) sucrose layer on a 60% sucrose (w/v) cushion for 2 hours at 30,000 xg to separate the biomembrane from excess DPPC and any unassociated hormone. Progesterone was found to decrease membrane fluidity. Testosterone, however, caused no change in the ordering of the membrane whereas its metabolite 17-P-oestradiol increased membrane fluidity as did 17-a-oestradiol and oestriol. In both liposomes and natural membranes containing cholesterol the steroid hormone effect on membrane fluidity was less pronounced. It has been proposed [7] that cholesterol limits steroid hormones intercalating between lipid bilayers. They consequently bind more to the surface of the membrane, explaining the lower change in anisotropy. Thus in natural membranes the steroid hormones have more influence on sarcoplasmic reticulum membranes from rabbit white muscle with a neglible cholesterol content than in synaptic membranes. Steroid hormones have been shown to alter the dynamic properties of bilayers. Further work will show if this influences the activity of membrane-bound enzymes. Rorlesenme Oeslradiol

Solubilization and partial characterization of acetylcholinesterase from the sarcotubular system of skeletal muscle.

Attempts were made to solubilize acetylcholinesterase (AChE) from microsomal membranes isolated from rabbit white muscle. The preparative procedure included a step in which the microsomes were incubated in a solution containing high salt concentration (0.6 M KCl). About 15% of the total enzyme activity could be solubilized with dilute buffer. Addition of EDTA (1 mM), EGTA (1 mM) or NaCl (0.5 and 1 M) to the extraction buffer did not improve the solubilization yield. Several non-ionic detergents and biliary salts were then used to bring the enzyme into solution. Triton X-100, C12E9 (dodecylnonaethylenglycol monoether) and biliary salt, above their critical micellar concentration, proved to be very effective as solubilizing agents. The occurrence of multiple molecular forms in detergent-soluble AChE was investigated by means of molecular sieving, centrifugation analysis, and slab gel electrophoresis. Experiments on gel filtration showed that, during the process, half of the enzyme was transformed into aggregates, the rest of the activity appearing as peaks with Stokes radii ranging from 3.7 to 7.9 nm. Both ionic strength and detergent nature modify the number and relative proportion of these peaks. Centrifugation analysis of Triton-saline-soluble AChE yielded molecular forms of 4.8S, 10-11S, and 13.5S, whereas deoxycholate extracts revealed species of 4.8S, 10S, and 15S, providing that gradients were prepared with 0.5 M NaCl. In the absence of salt, forms of 6.5-7.5S, 10S, and 15S were measured. The lightest species was always the predominant form. Slab gel electrophoresis showed several bands (68,000-445,000). The 4.8S component only yielded bands of 65,000-70,000.(ABSTRACT TRUNCATED AT 250 WORDS)

Spontaneous formation of a monolayer membrane from sarcoplasmic reticulum at an air-water interface.

Surface pressure was found to be produced spontaneously at the interface between air and a suspension containing fragmented sarcoplasmic reticulum (FSR) from rabbit white muscle. Large and stable surface pressure was formed only in a limited concentration range of FSR in the suspension and the pressure formation was proved to be an irreversible phenomenon, suggesting the formation of a monolayer membrane resulting from the disruption of FSR vesicles. Monolayer formation was directly confirmed by analyzing the components included in the membrane and by calculating the surface area occupied by these components. The monolayer included phospholipids, cholesterol and proteins, and appeared to originate from FSR vesicles since the molecular ratios of these components as well as the results of the SDS polyacrylamide gel electrophoresis were similar in both membranes. This phenomenon can be utilized as a method of monolayer preparation from biological membrane vesicles, and should be very useful for the reconstitution of planar biological membranes.

Acetylcholinesterase from sarcoplasmic reticulum of white muscle

1. 1. Two membrane fractions were separated from rabbit white muscle SR by discontinuous sucrose gradient. 2. 2. Both crude and membrane fractions were shown to contain AChE, Ca 2+-stimulated and Ca 2+ -independent ATPase activities. 3. 3. 1% w/v Triton X-100 solubilized most of the AChE and Ca 2+-stimulated ATPase but the Ca 2+ -independent ATPase was poorly solubilized. 4. 4. AChE was sensitive to BW284c51, non-sensitive to ethopropazine and presented inhibition by excess of the substrate, ATCh. 5. 5. Polyacrylamide gel electrophoresis from Triton-treated crude SR revealed several bands of AChE and ATPase activities. 6. 6. SDS-gel electrophoresis from crude SR showed two polypeptides specifically labelled with [ 3H]DFP.

Comparison of the Effects of Added Orthophosphate on Calcium Uptake and Release by Bovine and Rabbit Muscle Sarcoplasmic Reticulum

ABSTRACTIn respect of Ca accumulating capacity in the absence of added inorganic orthophosphate (Pi) and maximum rate of Ca uptake, sarcoplasmic reticulum (SR) prepared from beef sternomandibularis muscles is intermediate between SR from rabbit white muscles and SR from rabbit red muscles. In the presence of added Pi, the Ca accumulating capacity of SR, whatever the source, is increased and a constant rate of uptake is reached. This constant rate is greater the higher the Pi concentration. The cold‐induced release of Ca from beef muscle SR is only partly reduced by including up to 50 mM Pi in the medium, whereas the release from rabbit white muscle SR is completely prevented by only about 20 mM Pi. This last finding is discussed in terms of the possible importance of postmortem accumulation of Pi in relation to cold shortening.

Studies on the heterogeneity of sarcoplasmic reticulum vesicles

Isolated sarcoplasmic reticulum vesicles from rabbit white muscle were separated into a light (15–20% of total microsomes) and a heavy (80–85%) fraction by density gradient centrifugation. The ultrastructure, chemical composition, enzymic activities and localization of membrane components in the vesicles of both fractions were investigated. From the following results it was concluded that both fractions are derived from the membranes of the sarcoplasmic reticulum system of the muscle: 1. (i) The protein pattern of both fractions is essentially the same, except for different ratios of acidic, Ca 2+-binding proteins. 2. (ii) The 105 000 dalton protein of the light fraction cross-reacts immunologically with the Ca 2+-dependent ATPase of the heavy fraction. 3. (iii) Ca 2+-dependent ATPase, although of different specific activity, is found in both fractions. After rendering the vesicles leaky, specific activities in both fractions reach the same value. The light fraction was found to consist of “inside-out” vesicles by the following criteria: 1. (i) No Ca 2+ accumulation can be measured and the Ca 2+-dependent ATPase activity is low and variable. 2. (ii) The rate of trypsin digestion is lower and, compared to the heavy microsomes, a different ratio of degradation products is obtained. 3. (iii) The sarcoplasmic reticulum membrane has a highly asymmetrical lipid distribution. This distribution of aminophospholipids is opposite to that in vesicles of heavy fraction.

Separation of two HMM-S1 species from white muscle myosin

Heavy meromyosin subfragment 1 was resolved by chromatography on DEAE-cellulose into two fractions characterized by the nature of the alkali light chains present. It was shown that even in an HMM-S1 preparation with an extensive fragmentation of the heavy chain a polyacrylamide gel electrophoresis analysis differentiates alkali light chains among the light fragmentation components. A non-fragmented HMM-S1 was obtained from a papain digest of myofibrils and the chromatographic analysis supplied further evidence of the separation of the two species of HMM-S1 present in rabbit white muscle myosin.

Postmorten glycolysis in ground skeletal muscle as influenced by prerigor freezing and subsequent thawing.

Ground bovine longissimus and rabbit white muscles were frozen (-25 degrees C) in the prerigor state and subsequently thawed(+20 degrees C) to determine the combined effect of grinding and freezing on glycolytic metabolites and enzymes. Frozen ground muscle, when compared to unfrozen ground tissue, showed significantly lower hexose monophosphates and significantly greater levels of all metabolites from fructose diphosphate to phosphoenolpyruvate, indicating activation of phosphofructokinase and inhibition of pyruvate kinase during the freezing process. Thawing caused rapid glycolysis and the metabolites returned to near unfrozen levels by the time glycolysis ceased, due to strong activation of phosphorylase by Ca2+. Both muscle types had a similar pattern of changes.

Effect of heparin on the mitochondrial ATPase system of rabbit white muscles

Effect of heparin on the mitochondrial ATPase system of rabbit white muscles

Electrophoretic studies of muscle proteins. II. Complex formation between delta protein, myogen and myosin.

AbstractIn actin‐free extracts of rabbit white muscles complexes form between delta protein, myogen (mainly glycolytic enzymes) and myosin, detectable in electrophoretic patterns of both limbs. The complexes are concentration sensitive, nearly absent when total protein is below 5 mgm/ml, but increasing at higher concentrations. At 65 mgm/ml about 60% is bound at 1°C.Complex concentrations can be calculated by making Rayleigh fringe counts on two portions of the same solution, one more concentrated, the other diluted with buffer. A “fringe balance” can then be constructed which measures the forward movement, in the concentrated descending pattern, of those portions of myogen and myosin which have united with delta and moved with it in a fast position. Similar calculations are possible for the ascending limb.When commercial aldolase or lactic dehydrogenase solutions are mixed with concentrated extracts from which most free myogen has been removed the patterns show that portions of the added enzymes have complexed with myosin and delta and move with them in fast positions.Mixtures of pure aldolase and nearly pure myosin solutions also show com‐plexing, less well developed than when other enzymes are present. The glycolytic enzymes may have to unite with each other before they can readily complex with myosin and delta. There may be a lineal order of the glycolytic enzymes laid out along the fibrous proteins, permitting a rapid handling of their substrates.