Isolation of transverse tubules by fractionation of sarcoplasmic reticulum preparations in ion-free sucrose density gradients

A new method for the preparation of transverse tubules (T-tubules) from rabbit skeletal muscles is reported. When crude sarcoplasmic reticulum (SR) preparations were centrifuged on sucrose density gradients containing buffering ions (buffered gradients) 70-80% of the material sedimented as a single heavy band in the region of 43% sucrose. When this fraction (or crude SR) was recentrifuged on sucrose gradients prepared free of buffer or other ions (ion-free gradients) the heavy band dissociated into three fractions of different densities. The lightest fraction sedimented at 28% sucrose and was identified as T-tubules on the basis of its nitrendipine and ouabain binding properties. The enzymatic properties, cholesterol contents, and protein compositions of the fractions obtained when SR is centrifuged on buffered and ion-free sucrose density gradients were measured. The T-tubules were enriched in cholesterol and in marker enzymes for surface membranes while the other fractions were shown to be terminal cisternae and longitudinal cisternae on the basis of their (Ca2+,Mg2+)-ATPase activities and characteristic protein profiles.     

Isolation and characterization of two types of sarcoplasmic reticulum vesicles.

A purified preparation of sarcoplasmic reticulum from rabbit skeletal muscle has been found to consist of a heterogeneous population of vesicles. Isopycnic centrifugation was used to obtain "light" and "heavy" vesicles from the upper and lower ends of a 25 to 45% (w/w) linear sucrose gradient. Each fraction accounted for about 10 to 15% of the total vesicles. The remainder of the vesicles were of intermediate density and banded between the light and heavy fraction. Light vesicles were composed of about equal amounts of phospholipid and Ca-2+ pump protein which contained approx. 90% of the protein. Heavy vesicles contained in addition to the Ca-2+ pump protein (55-65% of the protein) two other major protein components, the Ca-2+ binding and M55 proteins which accounted for 20-25 and 5-7% of the protein of these vesicles, respectively. The sarcoplasmic reticulum subfractions had 32-P-labelled phosphoenzyme levels proportional to their Ca-2+ pump protein content and contained similar Ca-2+-stimulated ATPase activities. They were capable of accumulating Ca-2+ in the presence of ATP and of releasing the accumulated Ca-2+ when placed into a medium with a low Ca-2+ concentration. The vesicles differed significantly in that heavy vesicles had a greater number of non-specific Ca-2+ binding sites than light vesicles (approx. 220 vs 75 nmol of bound Ca-2+ per mg protein), in accordance with their high content of Ca-2+ binding protein. Electron dense material could be seen within the compartment of heavy but not light vesicles. Removal of Ca-2+ binding and M55 proteins from heavy vesicles resulted in empty membranous structures consisting mainly of Ca-2+ pump protein and phospholipid. Electron micrographs of sections of muscle showed dense material in terminal cisternae but not in longitudinal sections of sarcoplasmic reticulum. These experiments are consistent with the interpretation that (1) the electron dense material inside heavy vesicles may be referable to Ca-2+ binding and/or M55 proteins, and that (2) light and heavy vesicles may be derived from the longitudinal sections and terminal cisternae of sarcoplasmic reticulum, respectively.     

Identification of a new subpopulation of triad junctions isolated from skeletal muscle; Morphological correlations with intact muscle

Summary It has been previously recognized that a number of protocols may cause breakage of the triad junction and separation of the constituent organelles of skeletal muscle. We now describe a fraction of triad junctions which is refractory to the known protocols for disruption. Triads were passed through a French press and the dissociated organelles were separated on a sucrose density gradient, which was assayed for PN200-110, ouabain and ryanodine binding. Ryanodine binding showed a single peak at the density of heavy terminal cisternae. On the other hand, the PN200-110 and ouabain, which are external membrane ligands, bound in two peaks: one at the free transverse tubule region and the other at the light terminal cisternae. Similarly, a two peak pattern of PN200-110 and ouabain binding was observed when triad junctions were broken by the Ca²⁺-dependent protease, calpain, which selectively hydrolyzes the junctional foot protein. The light terminal cisternae vesicles were subjected to three different procedures of junctional breakage: French press, hypertonic salt treatment, and protease digestion using calpain or trypsin. The treated membranes were then centrifuged on density gradients. Only extensive trypsin digestion caused a partial shift of ouabain activity into the free transverse tubule region. These observations suggest that the triads are a composite mixture of breakage susceptible, weak, and breakage resistant, strong, triads. Scatchard analysis of PN200-110 suggests that the transverse tubules of strong triads contain a relatively high number of dihydropyridine receptors compared to those of weak triads. Thin section electron microscopic images of the strong triads comparable to those of intact muscle are presented.     

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 Ca²⁺/μl of water space. After KCl washing both contained less than 4 nmol Ca²⁺/μl of water space. Although they transported at the same rate and the same total amount of calcium, the rate of passive Ca²⁺ 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 Ca²⁺ release. High resolution sodium dodecyl sulfate gel electrophoresis showed that the light vesicles contained predominantly Ca²⁺-ATPase along with several approx. 55 000-dalton proteins and a 5000-dalton proteolipid, while the heavy vesicles contained Ca²⁺-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’.     

Intracellular Calcium Movements of Frog Skeletal Muscle during Recovery from Tetanus

Radioautographs of ⁴⁵Ca-labeled frog skeletal muscles have been prepared using freeze-dry and vapor fixation techniques to avoid displacement of the isotope during the preparation of the radioautographs. ⁴⁵Ca has been localized in resting muscles exposed to ⁴⁵Ca Ringer's for 5 min or 5 hr and in isotopically labeled muscles recovering from tetanic stimulation at room temperature or at 4°C. In muscles soaked at rest for 5 min ⁴⁵Ca was present almost exclusively in the terminal cisternae. In all other muscles there were three sites at which the isotope was concentrated: (a) the terminal cisternae, (b) the intermediate cisternae and the longitudinal tubules, and (c) the A band portion of the myofibrils. The terminal cisternae were labeled more rapidly than the myofibrils, but both exchanges were accelerated by electrical stimulation. The amount of ⁴⁵Ca in the longitudinal tubules and the intermediate cisternae decreased with time after a tetanus as the amount in the terminal cisternae increased. It is proposed that electrical stimulation releases calcium from the terminal cisternae and that relaxation occurs from the binding of the released calcium by the longitudinal tubules and the intermediate cisternae. Complete recovery from mechanical activity involves the transport of this bound calcium into the reticulum and its subsequent binding by the terminal cisternae. Resting exchange of calcium occurs primarily between the terminal cisternae and the transverse tubules.     

Penetration of horseradish peroxidase into the terminal cisternae of frog skeletal muscle fibers and blockade of caffeine contracture by Ca++ depletion

Summary Horseradish peroxidase, an extracellular marker, was given intravenously to frogs, and 40 min later the sartorius muscles were removed. The isolated muscles were exposed for an additional hour to Ringer solution containing peroxidase, then fixed with glutaraldehyde. Peroxidase activity was found in the T tubules, in some of the terminal cisternae (TC) of the SR, and occasionally in the longitudinal tubules of the SR. In transverse sections, the structures containing tracer formed a pattern of approximately parallel columns reaching to the cell surface; the statistical distribution of their spacing was nearly the same as that of the interdistances between the current-sensitive spots on the Z-line which triggered localized contraction (Huxley and Taylor, 1958). The caffeine contracture of frog sartorius muscles remained unchanged in isotonic Ringer solutions which were Ca⁺⁺-free or contained Mn⁺⁺ or La⁺⁺⁺; however, contracture was blocked by prior exposure of the muscles to the same solutions made 2 × hypertonic with sucrose (known to produce swelling of T tubules and (TC). Since Mn⁺⁺ and La⁺⁺⁺ are known to depress Ca⁺⁺ influx, these results suggest that washout of Ca⁺⁺ from the TC, and penetration of La⁺⁺⁺ or Mn⁺⁺ into it, occur more rapidly due to the swelling of T tubules and TC associated with hypertonicity. It is concluded that at least some of the terminal cisternae are open to the interstitial fluid via the T tubules. Thus, depolarization of the T tubules could readly depolarize the cisternae and lead to Ca⁺⁺ influx into the myoplasm.Supported by grants from the Public Health Service (HE-11155, HE-05815, and HE-10384) and from the American Heart Assocation. The authors are indebted to Mrs. Jan Redick for expert technical assistance.     

Penetration of horseradish peroxidase into the terminal cisternae of frog skeletal muscle fibers and blockade of caffeine contracture by Ca++ depletion

Summary Horseradish peroxidase, an extracellular marker, was given intravenously to frogs, and 40 min later the sartorius muscles were removed. The isolated muscles were exposed for an additional hour to Ringer solution containing peroxidase, then fixed with glutaraldehyde. Peroxidase activity was found in the T tubules, in some of the terminal cisternae (TC) of the SR, and occasionally in the longitudinal tubules of the SR. In transverse sections, the structures containing tracer formed a pattern of approximately parallel columns reaching to the cell surface; the statistical distribution of their spacing was nearly the same as that of the interdistances between the current-sensitive spots on the Z-line which triggered localized contraction (Huxley and Taylor, 1958). The caffeine contracture of frog sartorius muscles remained unchanged in isotonic Ringer solutions which were Ca⁺⁺-free or contained Mn⁺⁺ or La⁺⁺⁺; however, contracture was blocked by prior exposure of the muscles to the same solutions made 2 × hypertonic with sucrose (known to produce swelling of T tubules and (TC). Since Mn⁺⁺ and La⁺⁺⁺ are known to depress Ca⁺⁺ influx, these results suggest that washout of Ca⁺⁺ from the TC, and penetration of La⁺⁺⁺ or Mn⁺⁺ into it, occur more rapidly due to the swelling of T tubules and TC associated with hypertonicity. It is concluded that at least some of the terminal cisternae are open to the interstitial fluid via the T tubules. Thus, depolarization of the T tubules could readly depolarize the cisternae and lead to Ca⁺⁺ influx into the myoplasm.Supported by grants from the Public Health Service (HE-11155, HE-05815, and HE-10384) and from the American Heart Assocation. The authors are indebted to Mrs. Jan Redick for expert technical assistance.     

Identification of a constituent of the junctional feet linking terminal cisternae to transverse tubules in skeletal muscle

This study describes the biochemical composition of junctional feet in skeletal muscle utilizing a fraction of isolated triad junctions. [3H]Ouabain entrapment was employed as a specific marker for T-tubules. The integrity of the triad junction was assayed by the isopycnic density of [3H]ouabain activity (24-30% sucrose for free T-tubules, 38- 42% sucrose for intact triads). Trypsin, chymotrypsin, and pronase all caused separation of T-tubules from terminal cisternae, indicating that the junction is composed as least in part of protein. Trypsin and chymotrypsin hydrolyzed four proteins: the Ca2+ pump, a doublet 325,000, 300,000, and an 80,000 Mr protein. T-tubules which had been labeled covalently with 125I were joined to unlabeled terminal cisternae by treatment with K cacodylate. The reformed triads were separated from free T-tubules and then severed by passage through a French press. When terminal cisternae were separated from T-tubules, some 125I label was transferred from the labeled T-tubules to the unlabeled terminal cisternae. Gel electrophoresis showed that, although T-tubules were originally labeled in a large number of different proteins, only a single protein doublet was significantly labeled in the originally unlabeled terminal cisternae. This protein pair had molecular weights of 325,000 and 300,000 daltons. Transfer of label did not occur to a substantial degree without K cacodylate treatment. We propose that the transfer of 125I label from T-tubules to terminal cisternae during reformation and breakage of the triad junction is a property of the protein which spans the gap between T-tubules and terminal cisternae.     

Canine traceealis membrane fractionation and characterization

Canine trachealis was homogenized and the various membrane fractions isolated by differential centrifugation and discontinuous sucrose gradient centrifugation. A membrane fraction enriched in the plasma membrane marker enzymes 5′-nucleotidase (5-fold) and K⁺-activated ouabain sensitive p-nitrophenylphosphatase (3-fold) was obtained. The fraction contained very low levels of the inner mitochondrial marker succinate: cytochrome c oxidoreductase. These plasma membrane vesicles showed higher ATP-dependent Ca-uptake (20 μmoles/g protein) than any other submicrosomal fraction. The active Ca-uptake was enhanced by oxalate. The Ca taken up by the plasma membrane vesicles was released instantaneously by dilution in 5m$\text{M}$ EGTA and 10μ$\text{M}$ A23187 and more slowly by dilution only in 5m$\text{M}$ EGTA.     

Comparison of the catalytic and structural properties of Ca2+-ATPase in longitudinal tubules and terminal cisternae of the sarcoplasmic reticulum

The catalytic behavior and structural features of Ca2+-ATPase in the vesicles of longitudinal tubules and terminal cisternae of the sarcoplasmic reticulum isolated from rabbit skeletal muscles was analysed. pH measurements have shown under optimal conditions Ca2+-ATPase has similar catalytic behavior both in the fractions of longitudinal tubules and terminal cisternae. Under non-optimal conditions, the behavior similarity was not observed. The specific activity of the ATPase enzyme under optimal conditions was shown to be much higher in the fraction of longitudinal tubules than in the fraction of terminal cisternae. Caffeine added to both fractions had no effect on the catalytic behavior of Ca2+-ATPase. As judged from fluorescence analysis, the structure of Ca2+-ATPase of longitudinal tubules differs from that structure of terminal cisternae. In sarcoplasmic reticulum membrane, at least half of the tryptophan residues of Ca2+-ATPase was shown to be buried in the lipid bilayer. Our findings suggest that in terminal cisternae some of the Ca2+-ATPase molecules exist as an oligomeric protein and do not participate in ATP hydrolysis (named "silent" Ca2+-ATPase).     

THE SARCOPLASMIC RETICULUM AND TRANSVERSE TUBULES OF THE FROG''S SARTORIUS

The sarcoplasmic reticulum of the frog's sartorius muscle was examined by electron microscopy following sequential fixation in glutaraldehyde and osmium tetroxide and embedding in Epon. The earlier results of Porter and Palade on Ambystoma muscle were confirmed in the sartorius. In addition, the transverse tubules were observed to be continuous across the width of the fiber, a set of flat intermediate cisternae was seen to connect the terminal cisternae to the longitudinal tubules in the A band, and the continuous reticulum collar at the center of the A band was found to be perforated by circular and elongated pores (the fenestrated collar). The transverse tubules have a volume about 0.3 per cent of the fiber volume, and a surface area about 7 times the outer cylindrical surface area for a fiber 100 µ in diameter. The terminal cisternae, the intermediate cisternae, and the longitudinal tubules together with the fenestrated collar each have a volume of 4 to 5 per cent of the fiber volume and a surface area 40 to 50 times the outer surface area of a fiber 100 µ in diameter. Some evidence for continuity of the transverse tubules with the fiber surface is presented, but this is thought to be not so convincing as evidence presented by others. The results are discussed in terms of a possible mechanism for a role of the transverse tubules and sarcoplasmic reticulum in excitation-contraction coupling, as suggested by their morphology and a variety of physiological studies. In this scheme, the transverse tubules are thought to be electrically coupled to the terminal cisternae, so that depolarization of the fiber surface spreads inward along the transverse tubules and to the terminal cisternae, initiating the release of a contraction-activating substance.     

beta-adrenergic receptor and adenylate cyclase in transverse tubules of skeletal muscle.

Experiments were carried out to clarify the sites of action of beta-adrenergic agonists in skeletal muscle microsomes. Microsomes were fractionated into longitudinal reticulum, terminal cisternae, and isolated transverse tubules. Transverse tubules were selectively labeled and tracked with [3H]ouabain. beta-adrenergic receptor was identified by [3H]dihydroalprenolol binding. Assays of beta-adrenergic receptor, adenylate cyclase, and protein kinase-stimulated phosphorylation showed: 1) beta-adrenergic receptor was detected in transverse tubules with a receptor density of 0.61 pmol/mg of protein. No significant binding was detected in longitudinal reticulum or in terminal cisternae. 2) Isoproterenol-stimulated adenylate cyclase was present in microsomes but was similarly confined to the transverse tubular fraction. The activity of F- stimulated cyclase in transverse tubules was 2.3 nmol/mg of protein/min. 3) No phosphorylation of microsomes by cyclic AMP and protein kinase could be detected. We conclude that the action of epinephrine on skeletal muscle is mediated through receptors and adenylate cyclase in the external membrane.     

Preparation of synaptosomes and vesicles with sodium diatrizoate

Abstract— Synaptosomes were prepared from rat brain on a continuous gradient of sodium diatrizoate and compared with synaptosomes obtained on a discontinuous sucrose gradient. The yield of synaptosomal protein using diatrizoate was double that obtained with sucrose. No significant differences in quality between the two preparations were found based on measurement of: β-glucuronidase, RNA polymerase, 2′,3′-cyclic nucleotide 3′-phosphohydrolase, total and Na⁺, K⁺ -ATPase, acetylcholinesterase, lactic acid dehydrogenase, glucose utilization, and serotonin uptake. Electron microscopy showed the vesicles in the diatrizoate synaptosomes to be better preserved. Vesicles prepared on diatrizoate segregated into two distinct bands which differed in electron microscopic appearance and enzyme activity. The less dense vesicles were smaller, had much higher Mg²⁺ -ATPase activity, and a lower content of acetylcholinesterase than the more dense vesicles. The less dense vesicle preparation was very homogeneous morphologically, free of myelin and mitochondria, and contained occasional organelle fragments and double membrane structures.     

ISOLATION OF A GOLGI APPARATUS-RICH FRACTION FROM RAT LIVER : I. Method and Morphology

Golgi apparatus were released without fixatives from rat hepatocytes by gentle homogenization, concentrated by differential centrifugation, and purified by sucrose gradient centrifugation. Examination of sections of purified fractions by electron microscopy showed fields of morphologically intact units of Golgi apparatus consisting of stacks of parallel flattened cisternae, secretory vesicles, and small vesicular profiles. Negative staining of unfixed pellets revealed a complex network of anastomotic tubules continuous with platelike structures and secretory vesicles. These structures corresponded, respectively, to the small vesicular profiles and parallel flattened cisternae with attached secretory vesicles of sectioned material. Small fragments of granular endoplasmic reticulum were often closely associated with the peripheral tubules, suggesting sites of continuity in intact hepatocytes.     

Preparation and characterization of longitudinal tubules of sarcoplasmic reticulum from fast skeletal muscle

Sarcoplasmic reticulum (SR) serves a central role in calcium uptake and release, thereby regulating muscle relaxation and contraction, respectively. Recently, we have isolated fractions referable to longitudinal tubules (R2) and terminal cisternae (R4), the two major types of sarcoplasmic reticulum (A. Saito et al. (1984) J. Cell Biol. 99, 875-885). The terminal cisternae contain two types of membranes, the calcium pump membrane and the junctional face membrane. The terminal cisternae are filled with electron-opaque contents which serve as a Ca2+ reservoir. The longitudinal tubules consist mainly of the calcium pump membrane. In this study, we describe a new longitudinal tubule fraction (F2) and characterize it together with the R2 and R4 SR fractions. The calcium pump membrane of the longitudinal tubules is a highly specialized membrane consisting of about 90% calcium pump protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Extensive changes in morphology can be observed in the SR fractions referable to osmotic differences during the fixation conditions using either glutaraldehyde-tannic acid or osmium tetroxide fixatives. The changes include swelling or shrinkage and aggregation of the compartmental contents when the fixative contains calcium ions. The two types of SR have different osmotic permeability to the same medium, as indicated by differential swelling or shrinkage. Both longitudinal tubule and terminal cisternae vesicles of SR appear larger and are spherical vesicles when the glutaraldehyde-tannic acid fixative is isotonic as compared with the "standard" fixation method. We have previously reported that the ruthenium red-sensitive calcium release channels are localized to the terminal cisternae. The terminal cisternae as isolated are leaky to Ca2+ since these channels are in the "open state" (S. Fleischer et al. (1985) Proc. Natl. Acad. Sci USA 82, 7256-7259). Thus, the Ca2+, Mg2+-dependent ATPase (Ca2+ ATPase) rate is only slightly enhanced in the presence of a Ca2+ ionophore, which dissipates the Ca2+ gradient across the SR membrane. We now find that preincubation with ruthenium red restores the tight coupling of the Ca2+ ATPase activity to Ca2+ transport. That is to say, ATPase activity is reduced and the addition of ionophore stimulates the Ca2+ ATPase activity 4- to 7-fold. The Ca2+ ATPase activity in longitudinal tubules is already tightly coupled. It is minimal after a Ca2+ gradient has been generated, but can be stimulated 9- to 20-fold when the Ca2+ gradient is dissipated with ionophore. This finding suggests that the Ca2+ ATPase activity in SR is tightly coupled to Ca2+ transport in situ.     

Enrichment of Triadic and Terminal Cisternae Vesicles from Rabbit Skeletal Muscle

An enriched triad and terminal cisternae preparation was achieved from skeletal muscle through alterations of the differential centrifugation and muscle homogenization protocols. Both yield and specific activity (pmoles of radioligand binding per mg protein) were optimized for ³H-PN200-l10 (transverse tubule marker) and ³H-ryanodine (terminal cisternae marker) binding sites. By pelleting crude microsomes between 2,000 an 12,000 × g without any rehomogenizations, we improved both the yield and specific activity of transverse tubule and terminal cisternae markers in crude microsomes by approximately 4-fold to 1000–3000 pmoles binding sites (starting material: approximately 400 grams wet weight fast twitch skeletal muscle), with 10–15 pmoles/mg. Rehomogenization of the 1,000 × g pellet, which is typically discarded, allowed recovery of an additional 5000 pmoles PN200-110 binding sites and an additional 8000 pmoles ryanodine binding sites. Crude microsomes from the rehomogenized 1,000 × g pellets typically displayed specific activities of 20–25 pmoles binding/mg for both ³H-PN200-110 and ³H-ryanodine. Separation of crude microsomes on a sucrose gradient increased specific activity up to a maximum of 50 pmoles/mg in a specific fraction, a five- to ten-fold increase over standard triadic or terminal cisternae preparations. The mean specific activity for enriched triads was 30–40 pmoles/mg for both PN200-110 and ryanodine in pooled fractions, while pooled fractions of enriched terminal cisternae displayed low ³H-PN200-110 binding (3–5 pmoles/mg) and high ³H-ryanodine-specific activity (30–40 pmoles/mg).     

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.     

Plasma membrane vesicles prepared from unadhered monocytes: Characterization of calcium transport and the calcium ATPase

We have purified unadhered human monocytes in sufficient quantities to prepare monocyte plasma membrane vesicles and study vesicular calcium transport. Monocytes were isolated from plateletpheresis residues by counterflow centrifugal elutriation. By combining this source and procedure, 7 x 10(8) monocytes of over 90% purity were obtained. The membranes, isolated on a sucrose step gradient, had an 18-fold enrichment in Na,K-ATPase, a 29-fold diminution of succinate dehydrogenase activity and were vesicular on transmission electron micrographs. The membrane vesicles loaded with oxalate accumulated calcium only in the presence of Mg and ATP. Calcium uptake did not occur if ATP was replaced by any of five nucleotide phosphates or if Mg was omitted. Calcium transport had a maximal velocity of 4 pmoles calcium/micrograms vesicle protein/min and a Km for calcium of 0.53 microM. The ionophore A23187 completely inhibited calcium accumulation while 5 mM sodium cyanide and 10 microM ouabain had no effect. A calcium-activated ATPase was present in the same plasma membrane vesicles. The calcium ATPase had a maximal velocity of 18.0 pmoles calcium/micrograms vesicle protein/min and a Km for calcium of 0.60 microM. Calcium-activated ATPase activity was absent if Mg was omitted or if (gamma - 32P) GTP replaced (gamma - 32P) ATP. Monocyte plasma membranes that were stripped of endogenous calmodulin by EGTA treatment showed a reduced level of calcium uptake and calcium ATPase activity. The addition of exogenous calmodulin restored the transport activity to that of unstripped monocyte plasma membranes. Thus, monocyte plasma membrane vesicles contain a highly specific, ATP-dependent calcium transport system and a calcium-ATPase with similar high calcium affinities.     

Localization and partial characterization of the oligomeric disulfide-linked molecular weight 95,000 protein (triadin) which binds the ryanodine and dihydropyridine receptors in skeletal muscle triadic vesicles.

A monoclonal antibody, GE 4.90, has been produced following immunization of mice with the 95-kDa protein (triadin) of terminal cisternae of rabbit fast skeletal muscle isolated in nondenaturing detergent. The antibody binds to a protein of Mr95K in Western blots of microsomal vesicles electrophoresed in the presence of mercaptoethanol. The greatest intensity of the immunoblot reaction is to enriched terminal cisternae vesicles while little binding is seen to longitudinal reticulum and transverse tubules. The content of antigen in different microsomal subfractions has been estimated by immunoassay: terminal cisternae/triads contain 5.6 micrograms/mg of protein while heavy terminal cisternae contain 32 micrograms/mg. The molar content of triadin in vesicles is approximately the same as that of the ryanodine receptor. When Western blots of gels of terminal cisternae are run in nonreducing conditions, little protein of Mr95K is visible. A number of bands, however, forming a ladder of higher molecular weight are discerned, indicating that the 95-kDa protein forms a disulfide-linked homopolymer. A biotinylated aromatic disulfide reagent (biotin-HPDP) labels the 95-kDa protein, the junctional foot protein, and the Mr 106K protein described by others as a Ca(2+)-release channel (SG 106). This latter protein migrates in gel electrophoresis under nonreducing conditions at a molecular weight different from that of the 95-kDa protein. We did not detect any alteration of binding of the 95-kDa protein to the dihydropyridine receptor or junctional foot protein dependent on the state of oxidation of cysteine residues of either triadin or receptor protein used as the overlay probe.     

Isolation and nitrogen-fixing activity of Frankia sp. strain CpI1 vesicles.

Under N2-fixing conditions in aerobic culture and in symbiosis, frankiae produce spherical, multicellular structures that have been called vesicles. The vesicles have been proposed as the site of nitrogen fixation. We isolated vesicles by using density centrifugation in a single-step sucrose gradient. Vesicles migrated out of 50% (wt/vol) sucrose and banded at the 40 to 50% sucrose interface; they were intact, as assessed by transmission electron microscopy, and were free of hyphal contamination. Specific activities of nitrogenase in vesicles prepared anaerobically were up to 100-fold greater than the specific activity of the largely hyphal pellet, depending on the recovery of vesicles. All of the activity in the pellet could be accounted for by the number of vesicles present in the pellet. Glutamine synthetase activity in crude extracts of vesicles was extremely low.