scholarly journals Transbilayer distribution of lipids in a population of sarcoplasmic-reticulum vesicles sealed with their cytoplasmic side outwards

1981 ◽  
Vol 195 (1) ◽  
pp. 287-295 ◽  
Author(s):  
K A McGill ◽  
J P Bennett ◽  
G A Smith ◽  
R W Plumb ◽  
G B Warren
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Calcium Oxalate ◽  
Calcium Transport ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Exchange Reactions ◽  
Lipid Exchange ◽  
Sarcoplasmic Reticulum Membrane ◽  
Transverse Movement ◽  
Cytoplasmic Side

A population of sarcoplasmic-reticulum vesicles, all of which were sealed with their cytoplasmic side outwards, was obtained by density-gradient centrifugation after loading the vesicles with calcium oxalate. The calcium oxalate could subsequently be removed from the vesicles by the reverse action of the calcium-transport system. Measurements of the catalysed exchange of the phosphatidylcholine in the sarcoplasmic-reticulum cytoplasmic monolayer with an exogenous phosphatidylcholine pool suggested that phosphatidylcholine is symmetrically distributed across the sarcoplasmic-reticulum membrane. A similar result was obtained for phosphatidylethanolamine when sarcoplasmic-reticulum lipids were labelled with trinitrobenzenesulphonic acid. Further catalysed lipid-exchange reactions showed that the transverse movement of phosphatidylcholine across the membrane was exceedingly slow (t 1/2 greater than 15 days).

scholarly journals ULTRASTRUCTURE OF SARCOPLASMIC RETICULUM PREPARATIONS

1969 ◽  
Vol 42 (1) ◽  
pp. 296-307 ◽  
Author(s):  
D. W. Deamer ◽  
R. J. Baskin
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Calcium Oxalate ◽  
Calcium Transport ◽  
Osmium Tetroxide ◽  
Positive Staining ◽  
Rabbit Muscle ◽  
Calcium Oxalate Crystals ◽  
Electron Microscopic ◽  
Staining Techniques ◽  
Microscopic Techniques

Fragmented sarcoplasmic reticulum (FSR) from rabbit muscle was examined by positive staining, negative staining, and freeze-etch electron microscopic techniques in the absence and presence of calcium transport conditions. The existence of 30–40 A particles covering the outer surface of FSR vesicles was confirmed by two different negative stains in unfixed, glutaraldehyde-fixed and osmium tetroxide-fixed material. Freeze-etch microscopy revealed a second type of particle, 80–90 A in diameter, on the fractured surfaces of FSR vesicles. Following calcium oxalate accumulation, negative and positive staining techniques provided evidence for large nodular deposits within FSR vesicles which probably correspond to calcium oxalate crystals and are responsible for increments in turbidity during calcium oxalate accumulation. The most probable configuration of FSR vesicles in solution is spherical. "Tadpole" or tubular configurations were not seen by freeze-etch microscopy, positive staining, or in prefixed negatively stained material.

scholarly journals Evidence that peroxisomal acyl-CoA synthetase is located at the cytoplasmic side of the peroxisomal membrane

1982 ◽  
Vol 204 (1) ◽  
pp. 17-23 ◽  
Author(s):  
G P Mannaerts ◽  
P Van Veldhoven ◽  
A Van Broekhoven ◽  
G Vandebroek ◽  
L J Debeer
Keyword(s):  
Density Gradient Centrifugation ◽  
Adenine Nucleotides ◽  
Gradient Centrifugation ◽  
Enzyme Analysis ◽  
Marker Enzyme ◽  
Peroxisomal Membrane ◽  
Mechanical Disruption ◽  
Percoll Gradients ◽  
Membrane Bound ◽  
Cytoplasmic Side

1. Subfractionation by isopycnic density-gradient centrifugation in self-generating Percoll gradients of peroxisome-rich fractions prepared by differential centrifugation confirmed the presence of acyl-CoA synthetase in peroxisomes. Peroxisomes did not contain nicotinamide or adenine nucleotides other than CoA. 2. The gradient fractions most enriched in peroxisomes were pooled and the peroxisomes sedimented by centrifugation, resulting in a 50-fold-purified peroxisomal preparation as revealed by marker enzyme analysis. 3. Palmitate oxidation by intact purified peroxisomes was CoA-dependent, whereas palmitoyl-CoA oxidation was not, demonstrating that the peroxisomal CoA was available for the thiolase reaction, located in the peroxisomal matrix, but not for acyl-CoA synthetase. This suggests that the latter enzyme is located at the cytoplasmic side of the peroxisomal membrane. 4. Additional evidence for this location of peroxisomal acyl-CoA synthetase was as follows. Mechanical disruption of purified peroxisomes resulted in the release of catalase from the broken organelles, but not of acyl-CoA synthetase, indicating that the enzyme was membrane-bound. Acyl-CoA synthetase was not latent, despite the fact that at least one of its substrates appears to have a limited membrane permeability, as evidenced by the presence of CoA in purified peroxisomes. Finally, Pronase, a proteinase that does not penetrate the peroxisomal membrane, almost completely inactivated the acyl-CoA synthetase of intact peroxisomes.

scholarly journals Localization of the N-terminal and C-terminal ends of triadin with respect to the sarcoplasmic reticulum membrane of rabbit skeletal muscle

1995 ◽  
Vol 307 (3) ◽  
pp. 769-774 ◽  
Author(s):  
I Marty ◽  
M Robert ◽  
M Ronjat ◽  
I Bally ◽  
G Arlaud ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Rabbit Skeletal Muscle ◽  
Cytoplasmic Localization ◽  
Western Blots ◽  
Sarcoplasmic Reticulum Membrane ◽  
C Terminus ◽  
Luminal Side ◽  
Triad Junction ◽  
Cytoplasmic Side

Antibodies were raised against synthetic peptides corresponding to the N-terminal (residues 2-17) and C-terminal (residues 691-706) ends of rabbit skeletal muscle triadin, a 95 kDa protein located in the sarcoplasmic reticulum membrane at the triad junction. The specificity of the antibodies generated was tested by ELISA and Western blot analysis. These tests demonstrated the ability of the antibodies to react specifically with the proteins. The anti-N-terminus antibodies bound to sarcoplasmic reticulum vesicles, indicating that the N-terminal end of the membrane-embedded triadin is exposed on the cytoplasmic side of the vesicles. In contrast, the anti-C-terminus antibodies were able to react with sarcoplasmic reticulum vesicles only after permeabilization of the vesicles with a detergent, indicating that the C-terminal end is exposed on the luminal side of the vesicles. These immunological data were complemented by proteolysis experiments using carboxypeptidases and endoproteinase Arg C. A mixture of carboxypeptidases A, B and Y was used to induce degradation of the C-terminal end of triadin in sarcoplasmic reticulum vesicles. This degradation, and a concomitant loss of reactivity of the anti-C-terminus antibodies in Western blots, was observed only when the vesicles were permeabilized, providing further evidence for the luminal localization of the C-terminal end of triadin. Treatment of sarcoplasmic reticulum vesicles with endoproteinase Arg C resulted in the removal of the N-terminal end of triadin, probably due to cleavage after Arg-34. This is a further indication of the cytoplasmic localization of the N-terminal end of triadin (and of its first 34 amino acids). When the proteolysis with endoproteinase Arg C was carried out with permeabilized vesicles, the cleavage occurred after Arg-141 or Arg-157, indicating that at least one of these residues is luminal.

Uranyl Acetate and Rotary Shadowing to Increase the Contrast of Small Aggregated Virus Particles

1969 ◽  
Vol 27 ◽  
pp. 424-425
Author(s):  
Lee F. Ellis ◽  
Richard M. Van Frank ◽  
Walter J. Kleinschmidt
Keyword(s):  
Electron Microscopy ◽  
Tissue Culture ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Uranyl Acetate ◽  
Interferon Inducer ◽  
Virus Particles ◽  
Staining Methods ◽  
Rotary Shadowing

The extract from Penicillum stoliniferum, known as statolon, has been purified by density gradient centrifugation. These centrifuge fractions contained virus particles that are an interferon inducer in mice or in tissue culture. Highly purified preparations of these particles are difficult to enumerate by electron microscopy because of aggregation. Therefore a study of staining methods was undertaken.

The Combining Ability of Glycoproteins IIb, IIIa and Ca2+ in EDTA-Treated Nonaggregable Platelets

1983 ◽  
Vol 50 (04) ◽  
pp. 848-851 ◽  
Author(s):  
Marjorie B Zucker ◽  
David Varon ◽  
Nicholas C Masiello ◽  
Simon Karpatkin
Keyword(s):  
Density Gradient ◽  
Combining Ability ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Electrophoretic Pattern ◽  
Sucrose Density Gradient ◽  
Irreversible Loss ◽  
Sucrose Density Gradient Centrifugation ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

SummaryPlatelets deprived of calcium and incubated at 37° C for 10 min lose their ability to bind fibrinogen or aggregate with ADP when adequate concentrations of calcium are restored. Since the calcium complex of glycoproteins (GP) IIb and IIIa is the presumed receptor for fibrinogen, it seemed appropriate to examine the behavior of these glycoproteins in incubated non-aggregable platelets. No differences were noted in the electrophoretic pattern of nonaggregable EDTA-treated and aggregable control CaEDTA-treated platelets when SDS gels of Triton X- 114 fractions were stained with silver. GP IIb and IIIa were extracted from either nonaggregable EDTA-treated platelets or aggregable control platelets with calcium-Tris-Triton buffer and subjected to sucrose density gradient centrifugation or crossed immunoelectrophoresis. With both types of platelets, these glycoproteins formed a complex in the presence of calcium. If the glycoproteins were extracted with EDTA-Tris-Triton buffer, or if Triton-solubilized platelet membranes were incubated with EGTA at 37° C for 30 min, GP IIb and IIIa were unable to form a complex in the presence of calcium. We conclude that inability of extracted GP IIb and IIIa to combine in the presence of calcium is not responsible for the irreversible loss of aggregability that occurs when whole platelets are incubated with EDTA at 37° C.

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