Electron Microscopic Evidence for the Transmembrane Displa cement of Calcium ATPase

1984 ◽  
Vol 39 (1-2) ◽  
pp. 177-179 ◽  
Author(s):  
Donald J. Scales ◽  
Stefan R. Highsmith
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Atpase Activity ◽  
Freeze Fracture ◽  
Calcium Atpase ◽  
Two Dimensional ◽  
Electron Microscopic ◽  
Microscopic Evidence ◽  
Sarcoplasmic Reticulum Membrane ◽  
First Time

Abstract Incubation of the Ca2+-ATPase in vanadate solutions leads to the formation of two-dimensional arrays in the sarcoplasmic reticulum membrane. Electron micrographic freeze fracture replicas show depressions on the inner leaflet for the first time. This indicates that the ATPase has moved perpendicular to the plane of the membrane. Our results also suggest that aggregation of the Ca2+-ATPase into the two-dimensional arrays occurs before they move into the membrane. These phenomena were observed as soon as 15 minutes after vanadate was added. The effects of vanadate appear to be completely reversible. When SR was incubated in the vanadate solutions and was then diluted into a buffer containing Ca2+ and ATP, the ATPase activity was normal for up to several hours of incubation and only somewhat reduced after 3 days.

scholarly journals Biochemical heterogeneity of skeletal-muscle microsomal membranes. Membrane origin, membrane specificity and fibre types

1982 ◽  
Vol 202 (2) ◽  
pp. 289-301 ◽  
Author(s):  
Giovanni Salviati ◽  
Pompeo Volpe ◽  
Sergio Salvatori ◽  
Romeo Betto ◽  
Ernesto Damiani ◽  
...  
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Atpase Activity ◽  
Total Phospholipid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Freeze Fracture ◽  
Sucrose Density Gradient ◽  
Average Density ◽  
Slow Muscle ◽  
Fast And Slow Muscle ◽  
Dependent Atpase

1. Microsomes were isolated from rabbit fast-twitch and slow-twitch muscle and were separated into heavy and light fractions by centrifugation in a linear (0.3–2m) sucrose density gradient. The membrane origin of microsomal vesicles was investigated by studying biochemical markers of the sarcoplasmic-reticulum membranes and of surface and T-tubular membranes, as well as their freeze-fracture properties. 2. Polyacrylamide-gel electrophoresis showed differences in the Ca2+-dependent ATPase/calsequestrin ratio between heavy and light fractions, which were apparently consistent with their respective origin from cisternal and longitudinal sarcoplasmic reticulum, as well as unrelated differences, such as peptides specific to slow-muscle microsomes (mol.wts. 76000, 60000, 56000 and 45000). 3. Freeze-fracture electron microscopy of muscle microsomes demonstrated that vesicles truly derived from the sarcoplasmic reticulum, with an average density of 9nm particles on the concave face of about 3000/μm2 for both fast and slow muscle, were admixed with vesicles with particle densities below 1000/μm2. 4. As determined in the light fractions, the sarcoplasmic-reticulum vesicles accounted for 84% and 57% of the total number of microsomal vesicles, for fast and slow muscle respectively. These values agreed closely with the percentage values of Ca2+-dependent ATPase protein obtained by gel densitometry. 5. The T-tubular origin of vesicles with a smooth concave fracture face in slow-muscle microsomes is supported by their relative high content in total phospholipid and cholesterol, compared with the microsomes of fast muscle, and by other correlative data, such as the presence of (Na++K+)-dependent ATPase activity and of low amounts of Na+-dependent membrane phosphorylation. 6. Among intrinsic sarcoplasmic-reticulum membrane proteins, a proteolipid of mol.wt. 12000 is shown to be identical in the microsomes of both fast and slow muscle and the Ca2+-dependent ATPase to be antigenically and catalytically different, though electrophoretically homogeneous. 7. Basal Mg2+-activated ATPase activity was found to be high in light microsomes from slow muscle, but its identification with an enzyme different from the Ca2+-dependent ATPase is still not conclusive. 8. Enzyme proteins that are suggested to be specific to slow-muscle longitudinal sarcoplasmic reticulum are the flavoprotėin NADH:cytochrome b5 reductase (mol.wt. 32000), cytochrome b5 (mol.wt. 17000) and the stearoyl-CoA desaturase, though essentially by criteria of plausibility.

scholarly journals Membrane asymmetry and enhanced ultrastructural detail of sarcoplasmic reticulum revealed with use of tannic acid.

1978 ◽  
Vol 79 (3) ◽  
pp. 601-616 ◽  
Author(s):  
A Saito ◽  
C T Wang ◽  
S Fleischer
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Tannic Acid ◽  
Freeze Fracture ◽  
Membrane Vesicles ◽  
Thin Sections ◽  
Membrane Asymmetry ◽  
The Asymmetry ◽  
First Time ◽  
Negative Staining Electron Microscopy

Fixation of purified sarcoplasmic reticulum (SR) membrane vesicles, using glutaraldehyde supplemented with 1% tannic acid, reveals newly visualized ultrastructure in thin sections. The trilaminar appearance of the membrane is highly asymmetric; the outer electron-opaque layer is appreciably wider (70 A) than the inner layer (20 A). The asymmetry is not referable to lack of penetration of the tannic acid since: (a) SR vesicles made permeable with 1 mM EDTA, pH 8.5, show similar asymmetry; (b) treatment of SR with trypsin results in progressive loss in protein content and decrease in the thickness of the outer layer, until in the limit the trilayer has a symmetric appearance; (c) within the same muscle section, the SR membrane appears highly asymmetric whereas the sarcolemma has a more symmetric appearance; (d) reconstituted SR vesicles have a symmetric appearance with equally broad inner and outer layers (approximately 70 A); the symmetric structure is confirmed by freeze-fracture and negative staining electron microscopy. Heavy and light SR vesicles obtained by isopycnic density sedimentation of purified SR have the same asymmetric appearance of the membrane and seem to differ mainly in that the heavy vesicles contain internal contents consisting largely of Ca++-binding protein. The asymmetry of the SR membrane is referable mainly to the unidirectional alignment of the Ca++ pump protein, the major component (90% of the protein) of the membrane. The asymmetry of the SR membrane can be visualized now for the first time in situ in thin sections of muscle.

Genetic dissection of the final exocytosis steps in Paramecium tetraurelia cells: cytochemical determination of Ca2+-ATPase activity over performed exocytosis sites

1980 ◽  
Vol 46 (1) ◽  
pp. 17-40 ◽  
Author(s):  
H. Plattner ◽  
K. Reichel ◽  
H. Matt ◽  
J. Beisson ◽  
M. Lefort-Tran ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Freeze Fracture ◽  
Secretory Vesicles ◽  
Genetic Dissection ◽  
Paramecium Tetraurelia ◽  
Electron Microscopic ◽  
X Ray ◽  
Electron Microscopic Cytochemistry ◽  
Function Correlation

In different Paramecium tetraurelia strains the occurrence of a Ca2+-ATPase (or p-nitro-phenylphosphatase) activity at the preformed attachment and exocytosis sites of the secretory vesicles (trichocysts) was analysed by electron-microscopic cytochemistry and X-ray microanalysis. In conjunction with freeze-fracture studies it was found that only those strains, which contain rosette particles, display this Ca2+-ATPase activity (7S, K 40I, nd 9 (18 degrees C), while other strains (nd 6, nd 9 (27 degrees C), tam 38) are devoid of both these characteristics. The presence (absence) of rosette particles and of Ca2+-ATPase activity at the preformed exocytosis sites is correlated with the capability (incapability) of performing exocytosis in these strains. We discuss several possible interpretations of this structure-function correlation.

scholarly journals The effect of dantrolene on skeletal-muscle sarcoplasmic-reticulum function in malignant hyperpyrexia in pigs

1983 ◽  
Vol 212 (2) ◽  
pp. 399-405 ◽  
Author(s):  
M D White ◽  
J G Collins ◽  
M A Denborough
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Atpase Activity ◽  
Muscle Relaxant ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Malignant Hyperpyrexia ◽  
Dependent Atpase ◽  
Sarcoplasmic Reticulum Membrane

The effect of the muscle relaxant dantrolene on isolated sarcoplasmic reticulum was studied in control and malignant-hyperpyrexia-susceptible Landrace pigs. The membranes prepared from both sources showed similar Ca2+-dependent ATPase activities, had comparable phospholipid/protein ratios, and their sodium dodecyl sulphate/polyacrylamide-gel patterns were indistinguishable. Membranes from both sources appeared to bind similar amounts of dantrolene. The drug did not stimulate Ca2+-dependent ATPase activity in preparations from either source. The rates of Ca2+ exchange and Ca2+ efflux appeared to be similar in sarcoplasmic reticulum of control and malignant-hyperpyrexia-susceptible pigs. Dantrolene did not affect either the rates or the amount of Ca2+ lost from the vesicles. These results suggest that dantrolene does not directly affect the movement of Ca2+ across the sarcoplasmic-reticulum membrane.

scholarly journals Chemical modification of sarcoplasmic reticulum. Stimulation of Ca2+ release

1986 ◽  
Vol 240 (2) ◽  
pp. 509-517 ◽  
Author(s):  
V Shoshan-Barmatz
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Acetic Anhydride ◽  
Atpase Activity ◽  
Chemical Modification ◽  
Amino Groups ◽  
Reactive Group ◽  
Rapid Release ◽  
Sarcoplasmic Reticulum Membrane ◽  
Positively Charged ◽  
Stimulation Of

Pretreatment of sarcoplasmic membranes with acetic or maleic anhydrides, which interact principally with amino groups, resulted in an inhibition of Ca2+ accumulation and ATPase activity. The presence of ATP, ADP or adenosine 5′-[beta, gamma-imido]triphosphate in the modification medium selectively protected against the inactivation of ATPase activity by the anhydride but did not protect against the inhibition of Ca2+ accumulation. Acetic anhydride modification in the presence of ATP appeared to increase specifically the permeability of the sarcoplasmic reticulum membrane to Ca2+ but not to sucrose, Tris, Na+ or Pi. The chemical modification stimulated a rapid release of Ca2+ from sarcoplasmic reticulum vesicles passively or actively loaded with calcium, from liposomes reconstituted with the partially purified ATPase fraction but not from those reconstituted with the purified ATPase. The inactivation of Ca2+ accumulation by acetic anhydride (in the presence of ATP) was rapid and strongly pH-dependent with an estimated pK value above 8.3 for the reactive group(s). The negatively charged reagents pyridoxal 5-phosphate and trinitrobenzene-sulphonate, which also interact with amino groups, did not stimulate Ca2+ release. Since these reagents do not penetrate the sarcoplasmic reticulum membranes, it is proposed that Ca2+ release is promoted by modification of internally located, positively charged amino group(s).

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