scholarly journals Structural and functional reconstitution of inner dynein arms in Chlamydomonas flagellar axonemes.

1992 ◽  
Vol 117 (3) ◽  
pp. 573-581 ◽  
Author(s):  
E F Smith ◽  
W S Sale
Keyword(s):  
Electron Microscopy ◽  
Binding Sites ◽  
Polyacrylamide Gels ◽  
Active Conformation ◽  
Arm Position ◽  
Coomassie Blue ◽  
Tubulin Isoforms ◽  
Correct Location ◽  
Dynein Arms

The inner row of dynein arms contains three dynein subforms. Each is distinct in composition and location in flagellar axonemes. To begin investigating the specificity of inner dynein arm assembly, we assessed the capability of isolated inner arm dynein subforms to rebind to their appropriate positions on axonemal doublet microtubules by recombining them with either mutant or extracted axonemes missing some or all dyneins. Densitometry of Coomassie blue-stained polyacrylamide gels revealed that for each inner dynein arm subform, binding to axonemes was saturable and stoichiometric. Using structural markers of position and polarity, electron microscopy confirmed that subforms bound to the correct inner arm position. Inner arms did not bind to outer arm or inappropriate inner arm positions despite the availability of sites. These and previous observations implicate specialized tubulin isoforms or nontubulin proteins in designation of specific inner dynein arm binding sites. Further, microtubule sliding velocities were restored to dynein-depleted axonemes upon rebinding of the missing inner arm subtypes as evaluated by an ATP-induced microtubule sliding disintegration assay. Therefore, not only were the inner arm dynein subforms able to identify and bind to the correct location on doublet microtubules but they bound in a functionally active conformation.

Rebinding of Tetrahymena 13 S and 21 S dynein ATPases to extracted doublet microtubules. The inner row and outer row dynein arms

1985 ◽  
Vol 77 (1) ◽  
pp. 263-287 ◽  
Author(s):  
F.D. Warner ◽  
J.G. Perreault ◽  
J.H. McIlvain
Keyword(s):  
Atpase Activity ◽  
Binding Sites ◽  
Sodium Dodecyl ◽  
Sedimentation Velocity ◽  
Major Protein ◽  
Arm Position ◽  
Radial Spokes ◽  
Turbidimetric Assay ◽  
Dynein Arms ◽  
Protein Components

Ciliary axonemes from Tetrahymena contain a second salt-extractable ATPase distinguishable from outer arm 21 S dynein by sedimentation velocity (congruent to 13 S), electrophoretic mobility and substrate specificity. As characterized by turbidimetric assay, gel electrophoresis in the presence of sodium dodecyl sulphate, ATPase activity and electron microscopy, the 13 S dynein ATPase rebinds to extracted doublet microtubules. Compared to structural-side (ATP-insensitive) 21 S dynein binding, which is moderately specific for the 24 nm outer row arm position, rebinding of 13 S dynein is highly specific but for the inner row arm position. However, 13 S dynein rebinds to the A subfibre with a spacing that coincides with the triplet spacing of the radial spokes (24–32-40 nm periods; 96 nm repeat). All of the major protein components present in the 13 S or 21 S fractions rebind to extracted doublets under conditions that both restore and activate dynein ATPase activity. Unlike active-side (ATP-sensitive) rebound 21 S dynein, rebound 13 S dynein is completely insensitive to dissociation by ATP-vanadate and does not independently decorate the B subfibre. The saturation profile for rebinding of 13 S dynein exhibits a lack of cooperativity between binding events (h = 1.0) similar to structural-side rebinding of 21 S dynein. At low 21 S/doublet stoichiometry there is no measureable competition between the 13 S and 21 S dyneins for binding sites on the A subfibre lattice, although at saturating concentrations of 21 S dynein, rebinding of 13 S dynein is blocked completely.

Sites of Insulin Action Using Ferritin Labeling

1971 ◽  
Vol 29 ◽  
pp. 540-541
Author(s):  
Burton B. Silver ◽  
Ronald S. Nelson
Keyword(s):  
Electron Microscopy ◽  
Binding Sites ◽  
Anterior Pituitary ◽  
Insulin Action ◽  
Diabetic Rats ◽  
Insulin Antibody ◽  
Fat Pad ◽  
Target Organs ◽  
Uranium Salts ◽  
Sugar Levels

Some investigators feel that insulin does not enter cells but exerts its influence in some manner on the cell surface. Ferritin labeling of insulin and insulin antibody was used to determine if binding sites of insulin to specific target organs could be seen with electron microscopy.Alloxanized rats were considered diabetic if blood sugar levels were in excess of 300 mg %. Test reagents included ferritin, ferritin labeled insulin, and ferritin labeled insulin antibody. Target organs examined were were diaphragm, kidney, gastrocnemius, fat pad, liver and anterior pituitary. Reagents were administered through the left common carotid. Survival time was at least one hour in test animals. Tissue incubation studies were also done in normal as well as diabetic rats. Specimens were fixed in gluteraldehyde and osmium followed by staining with lead and uranium salts. Some tissues were not stained.

scholarly journals A simple and rapid method for the preparation of adenosine triphosphatase from submitochondrial particles

1975 ◽  
Vol 148 (3) ◽  
pp. 533-537 ◽  
Author(s):  
R B Beechey ◽  
S A Hubbard ◽  
P E Linnett ◽  
A D Mitchell ◽  
E A Munn
Keyword(s):  
Electron Microscopy ◽  
Rapid Method ◽  
Adenosine Triphosphatase ◽  
Pure Form ◽  
Heart Mitochondria ◽  
Polyacrylamide Gels ◽  
Submitochondrial Particles ◽  
Bovine Heart

An almost pure form of the bovine heart mitochondrial adenosine triphosphatase (ATPase) is released from the membrane by shaking submitochondrial particles with chloroform. Analyses on polyacrylamide gels and by electron microscopy, and also sensitivity to inhibitors, show that the chloroform-released enzyme is similar to other ATPase preparations from bovine heart mitochondria.

Quantification of Coomassie Blue stained proteins in polyacrylamide gels based on analyses of eluted dye

1975 ◽  
Vol 63 (2) ◽  
pp. 595-602 ◽  
Author(s):  
Claudia Fenner ◽  
Robert R. Traut ◽  
Dean T. Mason ◽  
Joan Wikman-Coffelt
Keyword(s):  
Polyacrylamide Gels ◽  
Coomassie Blue

Analytical electrofocusing and two-dimensional electrophoresis of proteins extracted from the mycelia of aggressive and nonaggressive strains of Ophiostoma ulmi

1986 ◽  
Vol 64 (9) ◽  
pp. 2073-2081 ◽  
Author(s):  
Robert S. Jeng
Keyword(s):  
Dutch Elm Disease ◽  
Two Dimensional ◽  
Polyacrylamide Gels ◽  
Ophiostoma Ulmi ◽  
Two Dimensional Electrophoresis ◽  
Coomassie Blue ◽  
Isoelectric Points ◽  
Additional Protein ◽  
Dimensional Electrophoresis ◽  
Electrophoresis Of Proteins

Soluble mycelial proteins from Ophiostoma ulmi (Buism.) Nannf., the causal agent of Dutch elm disease, were separated by analytical electrofocusing and two-dimensional electrophoresis in polyacrylamide gels. Results showed the aggressive and nonaggressive strains of this pathogen each had about 60 Coomassie blue stained bands having isoelectric points from 3 to 7. Both strains of this fungus had their own characteristic electrofocusing patterns. Nonaggressive isolate S116, for example, lacked two protein bands, one near the anode and one near the cathode, but it had five additional protein bands distributed from pH 4 to 6. Two-dimensional electrophoresis of total soluble proteins depicted that there were 36 proteins found to be specific for the nonaggressive isolate S116 and 12 proteins for the aggressive isolate RR2.

The fine structure and the protein composition of γ phage of Bacillus anthracis

1975 ◽  
Vol 21 (11) ◽  
pp. 1889-1892 ◽  
Author(s):  
Takashi Watanabe ◽  
Akinori Morimoto ◽  
Toshiro Shiomi
Keyword(s):  
Electron Microscopy ◽  
Fine Structure ◽  
Bacillus Anthracis ◽  
Protein Composition ◽  
Staining Technique ◽  
Negative Staining ◽  
Polyacrylamide Gels ◽  
Long Tail

The fine structure of γ phage of Bacillus anthracis was studied by electron microscopy with a negative-staining technique. The phage has a hexagonal head and a long tail without a sheath. By electrophoresis on polyacrylamide gels, the proteins of the phage particles are separate into 10 polypeptides with moleclar weights ranging from 140 000 to 12 000.

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