scholarly journals Cell fractionation studies indicate that dystrophin is a protein of surface membranes of skeletal muscle

1989 ◽  
Vol 258 (3) ◽  
pp. 837-841 ◽  
Author(s):  
G Salviati ◽  
R Betto ◽  
S Ceoldo ◽  
E Biasia ◽  
E Bonilla ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Cholesterol Content ◽  
Binding Activity ◽  
Cell Fractionation ◽  
Ouabain Binding ◽  
Low Concentrations ◽  
Peripheral Membrane Protein ◽  
T Tubules ◽  
Triton X ◽  
Terminal Cisternae

We studied the subcellular localization of dystrophin in rabbit skeletal muscle. In Western-blot analysis of membrane preparations, dystrophin was associated with the sarcolemmal fraction, as indicated by cholesterol content and co-purification with ouabain-binding activity and beta-adrenergic receptor. Dystrophin was also found with junctional T-tubules, but not with ‘free’ T-tubules, longitudinal portions or terminal cisternae of the sarcoplasmic reticulum. Dystrophin was not solubilized by high salt solutions, but it was solubilized by low concentrations of detergents (Triton X-100 and deoxycholate), suggesting that it is a peripheral membrane protein.

A transmission delay and the effect of temperature at the triadic junction of skeletal muscle

1986 ◽  
Vol 229 (1254) ◽  
pp. 97-110 ◽  
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Effect Of Temperature ◽  
Muscle Fibres ◽  
Time Interval ◽  
Strong Temperature Dependence ◽  
Ca Channels ◽  
T Tubules ◽  
Skeletal Muscle Fibres ◽  
Terminal Cisternae

The coupling process at the triadic junctions in skeletal muscle fibres is characterized by a significant latency between the depolarization of the transverse tubular membrane and the release of Ca from the sarcoplasmic reticulum. This time interval, the triadic delay, is sufficiently long to allow for the participation of a chemical process. The strong temperature dependence of the triadic delay ( Q 10 near 2.7) suggests that a sequence of chemical steps may link the electical signal in the T-tubules to the opening of Ca channels in the terminal cisternae of the sarcoplasmic reticulum.

In situ localization of cholesterol in skeletal muscle by use of a monoclonal antibody

2000 ◽  
Vol 89 (2) ◽  
pp. 731-741 ◽  
Author(s):  
Mark S. F. Clarke ◽  
Charles R. Vanderburg ◽  
Marcas M. Bamman ◽  
Robert W. Caldwell ◽  
Daniel L. Feeback
Keyword(s):  
Skeletal Muscle ◽  
Monoclonal Antibody ◽  
Sarcoplasmic Reticulum ◽  
Gradient Centrifugation ◽  
Cholesterol Content ◽  
Cell Fractionation ◽  
Synthetic Membranes ◽  
Subcellular Membrane ◽  
The Relationship

A common perception is that cholesterol, the major structural lipid found in mammalian membranes, is localized nearly exclusively to the plasma membrane of living cells and that it is found in much smaller quantities in internal membranes. This perception is based almost exclusively on cell fractionation studies, in which density gradient centrifugation is used for purification of discrete subcellular membrane fractions. Here we describe a monoclonal antibody, MAb 2C5-6, previously reported to detect purified cholesterol in synthetic membranes (Swartz GM Jr, Gentry MK, Amende LM, Blanchette-Mackie EJ, and Alving CR. Proc Natl Acad Sci USA85: 1902–1906, 1988), that is capable of detecting cholesterol in situ in the membranes of skeletal muscle sections. Localization of cholesterol, the dihydropyridine receptor of the T tubule, and the Ca2+-ATPase of the sarcoplasmic reticulum (SERCA2) by means of double and triple immunostaining protocols clearly demonstrates that cholesterol is primarily localized to the sarcoplasmic reticulum membranes of skeletal muscle rather than the sarcolemmal or T tubule membranes. The availability of this reagent and its ability to spatially localize cholesterol in situ may provide a greater understanding of the relationship between membrane cholesterol content and transmembrane signaling in skeletal muscle.

scholarly journals Ultrastructural observations of isolated intact and fragmented junctions of skeletal muscle by use of tannic acid mordanting.

1982 ◽  
Vol 93 (3) ◽  
pp. 533-542 ◽  
Author(s):  
J P Brunschwig ◽  
N Brandt ◽  
A H Caswell ◽  
D S Lukeman
Keyword(s):  
Skeletal Muscle ◽  
Tannic Acid ◽  
Dense Matter ◽  
Transverse Tubules ◽  
Electron Dense Material ◽  
Junctional Region ◽  
Intact Muscle ◽  
Characteristic Features ◽  
Triton X ◽  
Terminal Cisternae

Tannic acid mordanting during fixation of isolated vesicles from skeletal muscle enhanced the resolution of the images. Isolated triadic junctions displayed two characteristic features not previously described: (a) a clear gap separated terminal cisternae from transverse tubules; (b) this gap was bridged by a separating array of structures which resembled the "feet" of intact muscle. When the triad was broken in a French press and subsequently reassembled by joining the two organelles, a similar gap was seen but the structure of the feet was less well defined. When the membrane of the triad was extracted by Triton X-100, the junctional region was retained and a similar gap between the two organelles could be discerned. The terminal cisternae characteristically displayed a thickening of the cytoplasmic leaflet of the membrane in select areas in which electron-dense material was apposed on the luminal leaflet. This thickened membrane was not observed in longitudinal reticulum or in terminal cisternae regions distal to the electron-dense matter. This thickened leaflet was not invariably associated with the junction, and some junctional regions did not display discernible thickening of the membrane. When the triad was treated with KCl, the electron-dense aggregate was dispersed and the thickened leaflet of the terminal cisternae dissipated, whereas the triadic junctional region with its feet remained unchanged. KCl treatment caused dissolution of three proteins of Mr = 77,000, 43,000, and 38,000. Treatment of Triton-resistant vesicles with KCl caused the loss of electron-dense aggregate but did not otherwise influence the appearance of the junction. A good degree of correlation both qualitatively and in quantitative parameters between the isolated vesicles and the intact muscle was observed.

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

1982 ◽  
Vol 93 (3) ◽  
pp. 543-550 ◽  
Author(s):  
J J Cadwell ◽  
A H Caswell
Keyword(s):  
Skeletal Muscle ◽  
Protein Pair ◽  
Specific Marker ◽  
Molecular Weights ◽  
Single Protein ◽  
T Tubules ◽  
Triad Junction ◽  
Terminal Cisternae ◽  
Substantial Degree ◽  
Triad Junctions

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.

scholarly journals The roles of phospholipase D and a GTP-binding protein in guanosine 5′-[γ-thio]triphosphate-stimulated hydrolysis of phosphatidylcholine in rat liver plasma membranes

1990 ◽  
Vol 272 (3) ◽  
pp. 749-753 ◽  
Author(s):  
K M Hurst ◽  
B P Hughes ◽  
G J Barritt
Keyword(s):  
Rat Liver ◽  
Phospholipase D ◽  
Plasma Membranes ◽  
Regulatory Protein ◽  
Gtp Binding ◽  
Liver Plasma Membranes ◽  
Low Concentrations ◽  
Rat Liver Plasma Membranes ◽  
Triton X ◽  
Hydrolysis Of

1. Guanosine 5′-[gamma-thio]triphosphate (GTP[S]) stimulated by 50% the rate of release of [3H]choline and [3H]phosphorylcholine in rat liver plasma membranes labelled with [3H]choline. About 70% of the radioactivity released in the presence of GTP[S] was [3H]choline and 30% was [3H]phosphorylcholine. 2. The hydrolysis of phosphorylcholine to choline and the conversion of choline to phosphorylcholine did not contribute to the formation of [3H]choline and [3H]phosphorylcholine respectively. 3. The release of [3H]choline from membranes was inhibited by low concentrations of SDS or Triton X-100. Considerably higher concentrations of the detergents were required to inhibit the release of [3H]phosphorylcholine. 4. Guanosine 5′-[beta gamma-imido]triphosphate and guanosine 5′-[alpha beta-methylene]triphosphate, but not adenosine 5′-[gamma-thio]-triphosphate, stimulated [3H]choline release to the same extent as did GTP[S]. The GTP[S]-stimulated [3H]choline release was inhibited by guanosine 5′-[beta-thio]diphosphate, GDP and GTP but not by GMP. 5. It is concluded that, in rat liver plasma membranes, (a) GTP[S]-stimulated hydrolysis of phosphatidylcholine is catalysed predominantly by phospholipase D with some contribution from phospholipase C, and (b) the stimulation of phosphatidylcholine hydrolysis by GTP[s] occurs via a GTP-binding regulatory protein.

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