Acetylcholinesterase in membrane fractions derived from sarcotubular system of skeletal muscle: presence of monomeric acetylcholinesterase in sarcoplasmic reticulum and transverse tubule membranes

Microsomes were isolated from white rabbit muscle and separated into several fractions by centrifugation in a discontinuous sucrose density gradient. Four membrane fractions were obtained namely surface membrane, light, intermediate and heavy sarcoplasmic reticulum. The origin of these microsomal vesicles was investigated by studying biochemical markers of sarcoplasmic reticulum and surface and T-tubular membranes. The transverse tubule derived membranes were further purified by using a discontinuous sucrose density gradient after loading contaminating light sarcoplasmic reticulum vesicles with calcium phosphate in the presence of ATP. All membrane preparations displayed acetylcholinesterase activity (AChE, EC 3.1.1.7), this being relatively more concentrated in T-tubule membranes than in those derived from sarcoplasmic reticulum. The membrane-bound AChE of unfractioned microsomes notably increased its activity by aging, treatment with detergents and low trypsin concentrations indicating that the enzyme is probably attached to the membrane in an occluded form, the unconstrained enzyme displaying higher activity than the vesicular acetylcholinesterase.Sedimentation analysis of Triton-solubilized AChE from different membrane fractions revealed enzymic multiple forms of 13.5S, 9–10S and 4.5–4.8S, the lightest form being the predominant one in all membrane preparations. Therefore, in both sarcoplasmic reticulum and T-tubule membrane the major component of AChE appears to be a membrane-bound component, probably a G₁ form.     

Halothane-induced functional and structural modifications in sarcoplasmic reticulum membranes from pig skeletal muscle

We investigated the effect of halothane on lipid and protein components of sarcoplasmic reticulum membranes isolated from pig trapezius muscle. We studied the relationships between the (Ca2(+)-Mg2+)-ATPase activity and the interaction of the anesthetic with lipid and protein moieties by means of EPR and fluorescence spectroscopic techniques. Our results clearly show that below 5 mumol per mg protein, halothane interacts mainly with the lipid components of the membrane. This interaction is shown to be localized in the central core of the phospholipid bilayer and to induce an increase of the membrane calcium permeability. The interaction with protein components only occurs at higher halothane concentrations and affects its conformational and functional states. These results are discussed with respect to new insights into diethylether-SR membrane interaction and to malignant hyperthermia syndrome in the pig.     

Calsequestrin binds to monomeric and complexed forms of key calcium-handling proteins in native sarcoplasmic reticulum membranes from rabbit skeletal muscle.

Ca(2+)-handling proteins are important regulators of the excitation-contraction-relaxation cycle in skeletal muscle fibres. Although domain binding studies suggest protein coupling between various Ca(2+)-regulatory elements of triad junctions, no direct biochemical evidence exists demonstrating high-molecular-mass complex formation in native microsomal membranes. Calsequestrin represents the protein backbone of the luminal Ca(2+) reservoir and thereby occupies a central position in Ca(2+) homeostasis; we therefore used calsequestrin blot overlay assays in order to determine complex formation between sarcoplasmic reticulum components. Peroxidase-conjugated calsequestrin clearly labelled four major protein bands in one-dimensional (1D) and 2D electrophoretically separated membrane preparations from adult skeletal muscle. Immunoblotting identified the calsequestrin-binding proteins of approximately 26, 63, 94 and 560 kDa as junctin, calsequestrin itself, triadin and the ryanodine receptor, respectively. Protein-protein coupling could be modified by ionic detergents, non-ionic detergents, changes in Ca(2+) concentration, as well as antibody and purified calsequestrin binding. Importantly, complex formation as determined by blot overlay assays was confirmed by differential co-immunoprecipitation experiments and chemical crosslinking analysis. Hence, the key Ca(2+)-regulatory membrane components of skeletal muscle form a supramolecular membrane assembly. The formation of this tightly associated junctional sarcoplasmic reticulum complex seems to underlie the physiological regulation of skeletal muscle contraction and relaxation, which supports the biochemical concept that Ca(2+) homeostasis is regulated by direct protein-protein interactions.     

Ca2+ release from sarcoplasmic reticulum vesicles derived from longitudinal reticulum and terminal cisternae of frog skeletal muscle

Fragmented sarcoplasmic reticulum (FSR) of bullfrog skeletal muscle was fractionated into light and heavy sarcoplasmic reticulum (LSR and HSR) by sucrose density gradient centrifugation. Morphological and biochemical studies revealed that large parts of LSR and HSR were derived from longitudinal reticulum and terminal cisternae of SR, respectively. The Ca2+ uptake ability and ATPase activity of LSR were higher than those of HSR. Ca2+ release from Ca2+ preloaded SR vesicles by changing the medium from K-gluconate to KCl was suppressed by addition of 0.3 M sucrose or glucose; there was no correlation between Ca2+ release and membrane potential change either in LSR or HSR vesicles. Dantrolene sodium (DAN, 20 microM) had no effect on Ca2+ release. It is concluded that ion-induced Ca2+ release from SR (both HSR and LSR) in the isolated system is due to an osmotic effect.     

Thyroxine: structural transformations in the membranes of rabbit skeletal muscle sarcoplasmic reticulum

The structure of the membranes of sarcoplasmic reticulum fragments (SRF) normally and in thyrotoxicosis was studied by the spin-label and spin-probe methods and by chemifluorescence. The curves of temperature dependence of the regularity parameter show a typical break for the spin probe at 20 degrees C shifted by 4 degrees C to sower temperatures for thyrotoxins. The same shift was observed with temperature dependence for the correlation period of the spin label covalently bound to the thiol groups of Ca2+ dependent ATPase of sarcoplasmic reticulum. The latent period of thyrotoxins was reduced and the chemifluorescence intensity increased. The results obtained suggest the occurrence of considerable changes in the structure of SRF membranes in thyrotoxicosis.     

Differential effects of ethanol on membrane-bound and soluble acetylcholinesterase from sarcoplasmic reticulum membranes

The action of ethanol on the activity of membrane-bound and soluble acetylcholinesterase (AChE) in sarcoplasmic reticulum of skeletal muscle has been studied. Treatment of membranes with 2.5–12.5% v/v ethanol produced a slight stimulation of the AChE activity and inhibition at higher concentration. The enzyme remained associated with the membranes after these treatments. The enzyme solubilized with Triton X-100 was inhibited by ethanol in a time-independent manner. Isolated 16 S (A₁₂), 10.5 S (G₄) and 4.5 S (G₁) forms of AChE were inhibited by ethanol to a similar extent. Samples were reversibly inhibited by ethanol, up to 12.5% v/v, and irreversibly at higher concentrations. Kinetic studies performed with isolated forms in the presence of 5–12.5% v/v ethanol showed that the solvent behaved as a competitive inhibitor of the asymmetric form but as a mixed inhibitor of the tetrameric and monomeric forms. The results show that the solvent interacts with active and/or regulatory sites of AChE from muscle microsomes.     

Drug-induced calcium release from heavy sarcoplasmic reticulum of skeletal muscle

Calcium release from isolated heavy sarcoplasmic reticulum of rabbit skeletal muscle by several calmodulin antagonistic drugs was measured spectrophotometrically with arsenazo III and compared with the properties of the caffeine-induced calcium release. Trifluoperazine and W7 (about 500 microM) released all actively accumulated calcium (half-maximum release at 129 microM and 98 microM, respectively) in the presence 0.5 mM MgCl2 and 1 mg/ml sarcoplasmic reticulum protein; calmidazolium (100 microM) and compound 48/80 (70 micrograms/ml) released maximally 30-40% calcium, whilst bepridil (100 microM) and felodipin (50 microM) with calmodulin antagonistic strength similar to trifluoperazine (determined by inhibition of the calcium, calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum) did not cause a detectable calcium release, indicating that this drug-induced calcium release is not due to the calmodulin antagonistic properties of the tested drugs. Calcium release of trifluoperazine, W7 and compound 48/80 and that of caffeine was inhibited by similar concentrations of magnesium (half-inhibition 1.4-4.2 mM compared with 0.97 mM for caffeine) and ruthenium red (half-inhibition for trifluoperazine, W7 and compound 48/80 was 0.22 microM, 0.08 microM and 0.63 micrograms/ml, respectively, compared with 0.13 microM for caffeine), suggesting that this drug-induced calcium release occurs via the calcium-gated calcium channel of sarcoplasmic reticulum stimulated by caffeine or channels with similar properties.     

Mechanism of anthraquinone-induced calcium release from skeletal muscle sarcoplasmic reticulum

The anthraquinones, doxorubicin, mitoxantrone, daunorubicin and rubidazone are shown to be potent stimulators of Ca2+ release from skeletal muscle sarcoplasmic reticulum (SR) vesicles and to trigger transient contractions in chemically skinned psoas muscle fibers. These effects of anthraquinones are the direct consequence of their specific interaction with the [3H] ryanodine receptor complex, which constitutes the Ca2+ release channel from the triadic junction. In the presence of adenine nucleotides and physiological Mg2+ concentrations (approximately 1.0 mM), channel activation by doxorubicin and daunorubicin exhibits a sharp dependence on submicromolar Ca2+ which is accompanied by a selective, dose-dependent increase in the apparent affinity of the ryanodine binding sites for Ca2+, in a manner similar to that previously reported with caffeine. Unlike caffeine, however, anthraquinones increase [3H]ryanodine receptor occupancy to the level observed in the presence of adenine nucleotides. A strong interaction between the anthraquinone and the caffeine binding sites on the Ca2+ release channel is also observed when monitoring Ca2+ fluxes across the SR. Millimolar caffeine both inhibits anthraquinone-stimulated Ca2+ release, and reduces anthraquinone-stimulated [3H]ryanodine receptor occupancy, without changing the effective binding constant of the anthraquinone for its binding site. The degree of cooperativity for daunorubicin activation of Ca2+ release from SR also increases in the presence of caffeine. These results demonstrate that the mechanism by which anthraquinones stimulate Ca2+ release is caused by a direct interaction with the [3H]ryanodine receptor complex, and by sensitization of the Ca2+ activator site for Ca2+.     

Isolation of two proteolipids from rabbit skeletal muscle sarcoplasmic reticulum

We have isolated two proteolipids from rabbit skeletal muscle sarcoplasmic reticulum by chromatography on columns of Sepharose CL-6B and Sephadex LH-60. One, PL-II, is identical to the proteolipid previously obtained by others using organic solvent extraction. The other, PL-I, has an amino acid composition very similar to those of proteolipids we previously isolated from canine cardiac SR and lamb kidney (Na,K)-ATPase.     

Ca-release by phosphoinositides from sarcoplasmic reticulum of frog skeletal muscle

To clarify the biological role of phosphoinositides including inositol trisphosphate (IP3) in the skeletal muscle, we examined the Ca-releasing action on the heavy fraction of sarcoplasmic reticulum (HFSR) from bullfrog skeletal muscle of IP3, phosphatidylinositol monophosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2), and glycerophosphoinositol 4,5-bisphosphate (GPIP2). Only PIP2 caused dose-dependent Ca release. IP3 (up to 55 microM), PIP (up to 37 microM), and GPIP2 (up to 33 microM) were ineffective. The PIP2-induced Ca release is due to the direct action of PIP2, but not its metabolite(s). The properties of the PIP2-induced Ca release are unique and cannot be accounted for by the Ca release mechanisms already reported, such as Ca2+-induced, ionic substitution-induced, or IP3-induced Ca release. The rate of the PIP2-induced Ca release, however, is so slow that it may have no physiological relevance unless stimulating factors or agents exist.     

Synthesis of polyphosphoinositides in transverse tubule and sarcoplasmic reticulum membranes of frog skeletal muscle.

Synthesis of polyphosphoinositides has been studied in transverse (T-) tubule and sarcoplasmic reticulum (SR) membrane fractions of frog skeletal muscle, following 32P-labeling with [gamma-32P]ATP. Purified SR and T-tubule fractions respectively synthesize 9.4 +/- 0.8 and 71.9 +/- 9.8 pmol PtdInsP/mg per min, indicating nearly 8-fold higher activity of PtdIns kinase in the T-tubules than in the SR. The activity of this enzyme in both membrane systems is maximum at pH 7 and pCa 6. PtdInsP2 is synthesized from the endogenous PtdInsP, only in T-tubule membranes by the action of PtdInsP kinase. This lipid is the most intensely 32P-labeled phosphoinositide (181.7 +/- 9.2 pmol/mg per min) in these membranes. PtdIns kinase in the T-tubule and SR membranes, and PtdInsP kinase in the former are modulated by the free [Mg2+]. Loss of radiolabel from transiently maximal 32P-incorporation in polyphosphoinositides in T-tubule membranes, concomitant with a decrease in the ATP concentration in the incubation buffer, shows the occurrence of phosphoinositidases in these membranes. Under the conditions used, no such activities were evident in SR membranes. Compound 48/80, a mixture of condensation products of N-methyl-p-methoxyphenethylamine with formaldehyde, known to block phosphoinositidase C and phospholipase A2, causes a dose-dependent increase in the 32P-label of PtdInsP, in T-tubule membranes. The synthesis of lyso PtdInsP2, a deacylated form of PtdInsP2 which occurs in nearly equal quantities in both T-tubule and SR membranes, may result from a mechanism independent of phospholipase A2.     

Fatty acid composition of phospholipids from rabbit and crayfish skeletal muscle sarcolemma and sarcoplasmic reticulum membranes]

A comparative analysis of fatty acid composition of lipid components from skeletal muscle sarcolemma and sarcoplasmic reticulum membranes of rabbits and crayfishes Astacus fluviatilis and A. leptodactylus has been made by means of gas-liquid chromatography of methyl esters of fatty acids. There are slight differences between external and internal membranes in fatty acids composition of lipids in the same animal. Considerable differences were found, however, when lipids in corresponding membranes of different species were compared. There are more unsaturated fatty acids in the crayfish than in the rabbit membranes. The most expressive differences in fatty acid composition between the two animals concern the content of linoleic acid type. Polyunsaturated acids in the crayfish are mostly of a 3 type and those in the rabbit of a 6 type. During biochemical evolution the following changes seem to take place, a decrease in the amount of unsaturated fatty acids as well as of polyunsaturated ones of a 3 type and an increase of those of the 6 type. These changes very probably reflect the transition from an aquatic to a terrestrial habitat.     

Microarray profiling of skeletal muscle sarcoplasmic reticulum proteins

Microarrays were developed to profile the level of proteins associated with calcium regulation in sarcoplasmic reticulum (SR) isolated from porcine Longissimus muscle. The microarrays consisted of SR preparations printed onto to glass slides and probed with monoclonal antibodies to 7 target proteins. Proteins investigated included: ryanodine receptor, (RyR), dihydropyridine receptor, (DHPR), triadin (TRI), calsequestrin (CSQ), 90 kDa junctional protein (JSR90), and fast-twitch and slow-twitch SR calcium ATPases (SERCA1 and SERCA2). Signal from a fluorescently-labeled detection antibody was measured and quantitated using a slide reader. The microarray developed was also employed to profile Longissimus muscle SR proteins from halothane genotyped animals. Significant (P<0.05) reductions in levels of several proteins were found including: RyR, CSQ, TRI, DHPR and SERCA2 in SR samples from halothane positive animals. The results illustrate the potential of microarrays as a tool for profiling SR proteins and aiding investigations of calcium regulation.     

Calcium-permeable channel in sarcoplasmic reticulum of rabbit skeletal muscle

Membrane vesicles from sarcoplasmic reticulum of rabbit skeletal muscle were incorporated into a bilayer lipid membrane. With this system, single current fluctuation was observed in the presence of 50 mM Ba-gluconate. This channel activity was observed only in vesicles from terminal cisternae. The single channel conductance was 14.1 pS, and the channel state was almost wholly open. The open-close transition of the channel obeyed simple two-state kinetics and was voltage-independent. The ionic selectivity was also studied, and the channel showed no selectivity among Ba, Ca, Mn, and Mg. On the other hand, it was less permeable to Cs than to Ba. Based on these results, the relation of the Ca channel to excitation-contraction coupling is discussed.     

A comparison of vesicles derived from terminal cisternae and longitudinal tubules of sarcoplasmic reticulum isolated from rabbit skeletal muscle

Sarcoplasmic reticulum vesicles were separated into heavy (derived from terminal cisternae) and light (derived from longitudinal tubules) fractions, according to Meissner [Biochim. Biophys. Acta, 389, 51-68 (1975)]. The similar Ca2+ sensitivities of phosphoprotein formation, ATPase activity and calcium uptake, and the similar phosphoprotein turnover rates (ATPase/phosphoprotein formation) of both fractions indicate that the same ATPase enzyme is present in the terminal cisternae and longitudinal sarcoplaxmic reticulum. The higher V for Ca2+-activated ATPase activity and calcium uptake in the light fraction correlated with the higher concentration of ATPase enzyme per mg of membrane protein in this fraction. In both the presence and absence of calcium-precipitating anions, the light fraction stored more calcium than the heavy. The Ca2+ dependence of calcium release after addition of EGTA appeared similar in both fractions, but the rate of calcium release was more rapid in the light fraction. These findings suggest that calcium release may occur more rapidly from longitudinal than terminal cisternae portions of the sarcoplasmic reticulum and that calcium release, like calcium uptake, may be mediated by the ATPase enzyme in the sarcoplasmic reticulum membrane. Although the activation energies for Ca2+-activated ATPase activity above and below the transition temperature were significantly different for the heavy and light fractions, their transition temperatures were similar. Partial purification of the ATpase enzyme by deoxycholate treatment modified the activation energies of the light but not the heavy fraction and caused the activation energies to become similar. The phosphoprotein levels of heavy and light vesicles did not become similar after deoxycholate treatment, although gel electrophoretograms indicated both samples contained > 90% ATPase protein. These results indicate the protein-lipid associations in these two fractions may be different.     

Calcium release from two fractions of sarcoplasmic reticulum from rabbit skeletal muscle

Calcium release from sarcoplasmic reticulum vesicles presumably derived from longitudinal tubules (LSR) and terminal cisternae (HSR) of rabbit skeletal muscle was investigated by dual wavelength spectrophotometry using the calcium-indicator antipyrylazo III. In 120 mM KCl, 5 mM MgCl2, 30 microM, CaCl2, 50 microM MgATP, 100 microM antipyrylazo III, 40 mM histidine (pH 6.8, 25 degrees C), LSR and HSR sequestered approx. 115 nmol calcium/mg, and then spontaneously released calcium. Analysis of ATP hydrolysis and phosphoenzyme level during LSR and HSR calcium sequestration indicated that this calcium release process was passive, occurring in the virtual absence of ATP and phosphoenzyme. Moreover, subsequent addition of ATP reinitiated the calcium sequestration-release sequence. Calcium release by HSR was more than 4-times faster than that by LSR. Analysis of the calcium release phase demonstrated a biexponential decay for both LSR (0.10 and 0.63 min-1) and HSR (0.26 and 1.65 min-1), suggestive of heterogeneity within each fraction. Replacement of 120 mM KCl with either 120 mM choline chloride, 240 mM sucrose, or H2O reduced maximal calcium sequestration by LSR, but had less effect on LSR calcium release rate constants. In the case of HSR, these changes in the ionic composition of the medium drastically reduced calcium release rate constants with little effect on calcium content. These marked differences between LSR and HSR are consistent with the hypothesis that the calcium permeability of the terminal cisternae is greater and more sensitive to the ionic environment than is that of the longitudinal tubules of sarcoplasmic reticulum.