scholarly journals IDENTIFICATION OF CHLOROPLAST COUPLING FACTOR BY FREEZE-ETCHING AND NEGATIVE-STAINING TECHNIQUES

1974 ◽  
Vol 63 (1) ◽  
pp. 24-34 ◽  
Author(s):  
Melvin P. Garber ◽  
Peter L. Steponkus
Keyword(s):  
Outer Surface ◽  
Complete Removal ◽  
Coupling Factor ◽  
Negative Staining ◽  
Chloroplast Coupling Factor ◽  
Freeze Etching ◽  
Staining Techniques ◽  
Spinach Leaves

Identification of chloroplast coupling factor particles, by the freeze-etching and negative-staining techniques, was made utilizing chloroplast thylakoids isolated from spinach leaves. Complete removal of particles, comparable in diameter to purified coupling factor particles, from the outer surface of freeze-etched thylakoids was achieved by treatment with 0.8% silicotungstate. Reappearance of particles, comparable in diameter to purified coupling factor particles, on the outer surface of freeze-etched thylakoids was demonstrated by combining silicotungstate-treated thylakoids with purified chloroplast coupling factor. Negative-staining results were in agreement with the freeze-etch data. The results demonstrate that the chloroplast coupling factor particles are exposed on the outer surface.

Mobility of Chloroplast Coupling Factor 1 (CF1) at the Thylakoid Surface as Revealed by Freeze-Etching after Antibody Labelling

1974 ◽  
Vol 29 (11-12) ◽  
pp. 694-699 ◽  
Author(s):  
Richard Berzborn ◽  
Friedrich Kopp ◽  
Kurt Mühlethaler
Keyword(s):  
Outer Surface ◽  
Coupling Factor ◽  
Freeze Fracturing ◽  
Deep Etching ◽  
Chloroplast Coupling Factor ◽  
Freeze Etching ◽  
Chloroplast Coupling Factor 1 ◽  
Number Of Particles ◽  
Antibody Labelling

Abstract Freeze-Etching Freeze-fracturing and 60 sec deep-etching of isolated chloroplast thylakoid systems exposed large areas of the outer surface (matrix side) of the thylakoids. If the thylakoid systems were first treated with antisera against chloroplast coupling factor 1 (CF1), the 14 nm particles at the outer surface appeared aggregated. Between clusters these particles were absent. Since there is no change in the number of particles/area after treatment with antibodies, it is concluded that the 14 nm particles are mobile within the surface of the thylakoid. The antisera contained only anti­bodies against CF1 ; therefore the 14 nm particles at the outer surface are identified to be CF1 . The implication of a mobile ATP-synthetase (CF1) for the mechanisms of photophosphorylation is discussed.

scholarly journals A COMPARISON OF CHLOROPLAST MEMBRANE SURFACES VISUALIZED BY FREEZE-ETCH AND NEGATIVE STAINING TECHNIQUES; AND ULTRASTRUCTURAL CHARACTERIZATION OF MEMBRANE FRACTIONS OBTAINED FROM DIGITONIN-TREATED SPINACH CHLOROPLASTS

1969 ◽  
Vol 43 (1) ◽  
pp. 16-31 ◽  
Author(s):  
C. J. Arntzen ◽  
R. A. Dilley ◽  
F. L. Crane
Keyword(s):  
Freeze Fracture ◽  
Internal Surface ◽  
Coupling Factor ◽  
Negative Staining ◽  
Fracture Plane ◽  
Chloroplast Membranes ◽  
Chloroplast Membrane ◽  
Membrane Surfaces ◽  
Large Particles ◽  
Freeze Etching

Spinach chloroplast lamellae were washed free of negatively staining surface particles (carboxydismutase and coupling factor protein) and the resulting smooth-surfaced lamellae still showed the usual large (175 A) and small (110 A) particles seen by freeze-etching. Therefore, the freeze-fracture plane probably occurs along an internal surface of the chloroplast membrane. Fractions obtained by differential centrifugation of digitonin-treated chloroplast membranes were studied by negative staining, thin sectioning, and freeze-etching techniques for electron microscopy. The material sedimenting between 1,000 g and 10,000 g, enriched in photosystem II activity, was shown to consist of membrane fragments. These freeze-etched membrane fragments were found to have large particles on most of the exposed fracture faces. The large particles had the same size and distribution pattern as the 175 A particles seen in intact chloroplast membranes. The material sedimenting between 50,000 g and 144,000 g, which had only photosystem I activity, was found to consist of particles in various degrees of aggregation. Freeze-etching of this fraction revealed only small particles corresponding to the 110 A particles seen in intact chloroplasts. A model is presented suggesting that chloroplast lamellar membranes have a binary structure, which digitonin splits into two components. The two membrane fragments have different structures, revealed by freeze-etching, and different photochemical and biochemical functions.

Polymer Morphology Revealed by Staining Techniques

1981 ◽  
Vol 39 ◽  
pp. 340-341
Author(s):  
A. C. Reimschuessel ◽  
V. Kramer
Keyword(s):  
Polymer Melt ◽  
Nylon 6 ◽  
Morphological Features ◽  
Negative Staining ◽  
Polymer Morphology ◽  
Crystallizable Polymer ◽  
Staining Techniques ◽  
Long Chain

Staining techniques can be used for either the identification of different polymers or for the differentiation of specific morphological domains within a given polymer. To reveal morphological features in nylon 6, we choose a technique based upon diffusion of the staining agent into accessible regions of the polymer.When a crystallizable polymer - such as nylon 6 - is cooled from the melt, lamellae form by chainfolding of the crystallizing long chain macromolecules. The regions between adjacent lamellae represent the less ordered amorphous domains into which stain can diffuse. In this process the lamellae will be “outlined” by the dense stain, giving rise to contrast comparable to that obtained by “negative” staining techniques.If the cooling of the polymer melt proceeds relatively slowly - as in molding operations - the lamellae are usually arranged in a radial manner. This morphology is referred to as spherulitic.

Phosphate Binding to Isolated Chloroplast Coupling Factor (CF1)

1988 ◽  
Vol 43 (3-4) ◽  
pp. 213-218 ◽  
Author(s):  
Bernhard Huchzermeyer
Keyword(s):  
Atpase Activity ◽  
Binding Site ◽  
Inorganic Phosphate ◽  
Coupling Factor ◽  
Binding Domain ◽  
Atp Binding ◽  
Phosphate Binding ◽  
Chloroplast Coupling Factor ◽  
Single Binding Site ◽  
Site Binding

A single binding site for phosphate was found on isolated chloroplast coupling factor in the absence of nucleotides. In our experiments the phosphate binding site showed a Kd of 170 μᴍ. We did not observe any differences whether the ATPase activity of CF] had been activated or not. If the enzyme was incubated with [γ-32P]ATP the amount of 32P bound per CF1 depended on the pretreatment of the enzyme: In the presence of ADP no ATP or phosphate was bound to CF,. After activation of ATPase activity one mol of ATP per mol CF, was rapidly bound and hydrolyzed while there was a slowly occurring binding of another phosphate without concomitant nucleotide binding. We conclude that there are two different types of phosphate binding observed in our experiments: 1) Inorganic phosphate can be bound by one catalytic site per mol of CF1 2) The γ-phosphate of ATP is able to bind to an ATP binding domain of the enzyme if this domain can exchange substrates with the incubation medium. This ATP binding domain appears to differ from the site binding inorganic phosphate, because at least a portion of the coupling factor contains more than one labelled phosphate during our ATPase tests.

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