Light Sarcoplasmic Reticulum Vesicles Research Articles

Ca 2+ effluxes from the sarcoplasmic reticulum vesicles of frog muscle: effects of cyclopiazonic acid and thapsigargin

Call efflux from frog muscle sarcoplasmic reticulum (SR) vesicles was studied by measuring external free [Ca 2+] using Fluo-3 fluorescence. Light SR vesicles were preloaded with Ca 2+ in the presence of ATP and inorganic phosphate (P i). Calcium pump reversal was activated by either depletion of the medium ATP by apyrase in the presence of 20 mM P i, or resuspending preloaded vesicles in an ATP-free solution containing 1 mM ADP and 20 mM P i. Cyclopiazonic acid (CPA) and thapsigargin (TG), at concentrations of 2.5 μM, which completely inhibit Ca 2+ uptake, both inhibited the pump reversal efflux almost completely. When active Ca 2+ uptake was stopped by either ATP-depletion or addition of CPA, a leak efflux of 6–7 nmole/mg/min was recorded. TG (2.5 μM) reduced this leak by over 50%, suggesting that TG, but not CPA, can slow the passage of calcium ions through the Ca 2+-ATPase passive channel.

Thermal uncoupling of the Ca(2+)-transporting ATPase in sarcoplasmic reticulum. Changes in surface properties of light vesicles.

It is known that the light fraction of rabbit skeletal muscle sarcoplasmic reticulum vesicles can release Ca2+ from the intravesicular space, although the Ca(2+)-conductive channels are present only in the heavy fraction of sarcoplasmic reticulum vesicles. To study the possible pathways of the Ca2+ leakage from light vesicles we have used a short-term treatment for 4.5 min at 45 degrees C which quickly decreases the efficiency of Ca(2+)-transporting ATPase operation without any visible effects on the hydrolytic activity of the Ca(2+)-ATPase in the membranes. The treatment of the vesicles decreased the negative membrane surface potential created by the Ca(2+)-ATPase. Comparative titration of control and heat-treated preparations of light sarcoplasmic reticulum vesicles by K+, Na+, Mg2+, and Ca2+ revealed clear differences in their surface properties. The short-term heating resulted in release of Ca2+ from the vesicles previously loaded with 45Ca2+, which indicates an increase in passive membrane permeability to Ca2+. Study of Ca(2+)-ATPase protein arrangement in the membrane indicated that the heat treatment induced protein oligomerization and some of the Ca(2+)-ATPase molecules acquired intermolecular and intramolecular covalent bonds. From these data, we have concluded that the changes in the surface and structure properties of the vesicle membranes after the short-term heat treatment were the result of clustering of the Ca(2+)-ATPase molecules. This protein rearrangement may create channels for calcium leakage from light sarcoplasmic reticulum vesicles.

Blockage of a pump‐related calcium‐efflux pathway in light sarcoplasmic reticulum vesicles by Mops

Mops, used as a proton buffer, specifically enhances the accumulation of calcium or strontium by light sarcoplasmic reticulum vesicles driven by ATP or dinitrophenylphosphate as energy-yielding substrates when calcium-precipitating agents are absent. The enhancement of ion uptake by Mops is much greater for strontium than for calcium and is further increased when potassium is replaced by sodium as the dominant monovalent cation. Mops affects neither the activity of the calcium- or strontium-activated transport enzyme nor the active accumulation of calcium in the presence of oxalate, i.e. when the pump runs unidirectionally forward. Passive calcium and strontium efflux rates of approximately 40-50 nmol.mg-1.min-1 are considerably reduced when histidine/glycerophosphate or Tris/maleate are exchanged for Mops. The observed passive efflux rates and their modulation by Mops are too small, in relation to the rate of ion influx, to account for either the relatively small calcium and strontium load in the absence of precipitating agents or for its modulation by Mops. The results imply that the pump itself mediates ion efflux dependent on pump activity and the different degree of saturation of lumenal ion-binding sites by calcium and strontium, as well as their susceptibility to Mops.

Monoclonal Antibody Characterization of the 1,4‐Dihydropyridine Receptor of Rabbit Skeletal Muscle

The 1,4-dihydropyridine receptor (DHPR) of the voltage-dependent Ca channel has been purified from transverse tubular membranes of skeletal muscle. Curtis and Catterall have shown that it consists of three polypeptides of 160,000 daltons, 50,000 Da, and 32,000 Da and that under reducing conditions the apparent molecular weight of the 160,000 Da subunit shifted to 130,000. Borsotto et al. have identified three polypeptides of 142,000 Da, 33,000 Da, and 32,000 Da in their preparation of the dihydropyridine receptor. Furthermore, they have shown by immunoblotting with polyclonal antibodies that the 142,000 Da and 32,000 Da subunits are produced by the reduction of a 170,000 Da polypeptide. We report here the identification and characterization with monoclonal antibodies (MAb) of an additional high molecular weight subunit of the DHPR which is distinct from that described by Curtis and Catterrall and Borsotto et al. Monoclonal antibodies capable of specifically immunoprecipitating the [H]PN200-110-labeled DHPR of rabbit skeletal muscle were produced by immunizing BALB/c mice initially with skeletal muscle triad vesicles followed by booster immunizations with purified DHPR from skeletal muscle triads. Hybridoma supernatants were screened for the production of anti-DHPR antibodies with an immunodot assay. Supernatants that reacted positively against the partially purified DHPR were then tested for their ability to immunoprecipitate the [H]PN200-110-labeled receptor from digitonin-solubilized triads (see TABLE 1). All three MAbs (IIC12, IIF7, IIID5) that were capable of immunoprecipitating the DHPR recognized a protein with an Mr of 170,000 on nitrocellulose transfers of skeletal muscle triads and transverse tubular membranes separated on sodium dodecyi sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions (FIGURE la). Wheat-germ agglutinin (WGA) peroxidase stained a 175,000 Da protein on similar nitrocellulose transfers. Neither the 170,000 Da polypeptide nor the 175,000 Da polypeptide was detected in light sarcoplasmic reticulum vesicles, a preparation devoid of DHPR. Under reducing conditions, the Mr of the 170,000 Da

Identification and characterization of the high affinity [3H]ryanodine receptor of the junctional sarcoplasmic reticulum Ca2+ release channel.

The high affinity ryanodine receptor of the Ca2+ release channel from junctional sarcoplasmic reticulum of rabbit skeletal muscle has been identified and characterized using monoclonal antibodies. Anti-ryanodine receptor monoclonal antibody XA7 specifically immunoprecipitated [3H]ryanodine-labeled receptor from digitonin-solubilized triads in a dose-dependent manner. [3H]Ryanodine binding to the immunoprecipitated receptor from unlabeled digitonin-solubilized triads was specific, Ca2+-dependent, stimulated by millimolar ATP, and inhibited by micromolar ruthenium red. Indirect immunoperoxidase staining of nitrocellulose blots of various skeletal muscle membrane fractions has demonstrated that anti-ryanodine receptor monoclonal antibody XA7 recognizes a high molecular weight protein (approximately 350,000 Da) which is enriched in isolated triads but absent from light sarcoplasmic reticulum vesicles and transverse tubular membrane vesicles. Thus, our results demonstrate that monoclonal antibodies to the approximately 350,000-Da junctional sarcoplasmic reticulum protein immunoprecipitated the ryanodine receptor with properties identical to those expected for the ryanodine receptor of the Ca2+ release channel.

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 G(1) form.

Immunological and biochemical properties of transverse tubule membranes isolated from rabbit skeletal muscle.

A new method for isolating transverse tubule membranes from rabbit skeletal muscle has been developed. This procedure has the advantage of being mild, fast, and producing with good yields a purified membrane fraction. The transverse tubule membranes are purified by a discontinuous sucrose density centrifugation after loading contaminating light sarcoplasmic reticulum vesicles with calcium phosphate in the presence of ATP. Immunofluorescence staining of cryostat sections of rabbit psoas muscle with purified goat antibodies directed against the purified membranes shows that the reacting antigens are distributed at the boundary of the A and I bands of the myofibrils where transverse tubules are localized in mammalian muscle. The purified antibodies showed no cross-reactivity with sarcoplasmic reticulum, nor did they show any fluorescence staining of the muscle plasma membrane, indicating that the isolated membranes indeed originate from the transverse tubules. The transverse tubule fraction has a characteristic protein composition distinguishable from that of sarcoplasmic reticulum, a much higher cholesterol content than that of the crude microsomes, plasma membrane, and sarcoplasmic reticulum, and a phospholipid content about twice as high as that of sarcoplasmic reticulum and plasma membrane. The purified transverse tubule membrane has a distinct phospholipid composition with high contents of sphingomyelin and phosphatidylserine. A Mg2+-activated ATPase characteristic of the transverse tubule fraction undergoes a 20-30-fold increase in specific activity during purification. The levels of Ca2+-ATPase activity present in the purified transverse tubule fraction remain comparable to those of sarcoplasmic reticulum even after extensive removal of the latter.

Further characterization of light and heavy sarcoplasmic reticulum vesicles. Identification of the ‘sarcoplasmic reticulum feet’ associated with heavy sarcoplasmic reticulum vesicles

Light and heavy sarcoplasmic reticulum vesicles were isolated from rabbit leg muscle using a combination of differential centrifugation and isophycnic zonal ultracentrifugation. Light sarcoplasmic reticulum vesicles obtained from the 30–32.5% and heavy sarcoplasmic reticulum vesicles obtained from the 38.5–42% sucrose regions of the linear sucrose gradient were determined to be free of surface and mitochondrial membrane contamination by marker enzyme analysis and electron microscopy. Thin sections of the light vesicles revealed empty vesicles of various sizes and shapes. Freeze-fracture replicas of the light vesicles showed an asymmetric distribution of intramembranous particles with the same orientation and distribution as the longitudinal sarcoplasmic reticulum in vivo. Heavy vesicles appeared as rounded vesicles of uniform size filled with electron dense material, similar to that seen in the terminal cisternae of the sarcoplasmic reticulum. The cytoplasmic surface of the membrane was decorated by membrane projections, closely resembling the ‘feet’ which join the sarcoplasmic reticulum to the transverse tubules in the intact muscle fiber. Freeze-fracture replicas of the heavy vesicles revealed an asymmetric distribution of particles which in some areas of the vesicle's surface are larger and less densely aggregated than those of the light vesicles. In the best quality replicas, some regions of the luminal leaflet were not smooth but showed evidence of pits. These structural details are characteristic of the area of sarcoplasmic reticulum membrane which is covered by the ‘feet’ in the intact muscle. Heavy vesicles contained greater than six times the calcium content of light vesicles, 54 vs. 9 nmol Ca 2+/μl of water space. After KCl washing both contained less than 4 nmol Ca 2+/μl of water space. Although they transported at the same rate and the same total amount of calcium, the rate of passive Ca 2+ efflux from the heavy vesicles was double that of light vesicles. The higher rate of calcium efflux from the heavy vesicles was inhibited by dantrolene, an inhibitor of Ca 2+ release. High resolution sodium dodecyl sulfate gel electrophoresis showed that the light vesicles contained predominantly Ca 2+-ATPase along with several approx. 55 000-dalton proteins and a 5000-dalton proteolipid, while the heavy vesicles contained Ca 2+-ATPase and calsequestrin along with several approx. 55 000-dalton proteins, extrinsic 34 000- and 38 000-dalton proteins, intrinsic 30 000- and 33 000-dalton proteins and two proteolipids of 5000 and 9000 daltons. KCl washing of the heavy vesicles removed both the approx. 34 000- and 38 000-dalton proteins, and the ‘sarcoplasmic reticulum feet’ were no longer seen on the heavy vesicles. The KCl supernatant was enriched in the 34 000- and 38 000-dalton proteins, indicating that these proteins are possible components of the sarcoplasmic reticulum feet. The biochemical and morphological data strongly support the view that the light vesicles are derived from the longitudinal sarcoplasmic reticulum and that the heavy vesicles are derived from the terminal cisternae containing junctional sarcoplasmic reticulum membrane with the intact ‘sarcoplasmic reticulum feet’.