scholarly journals Sucrose Gradient Sedimentation Analysis of Mitochondrial Ribosomes

2020 ◽  
pp. 211-226
Author(s):  
Austin Choi ◽  
Antoni Barrientos
Keyword(s):  
Sucrose Gradient ◽  
Sedimentation Analysis ◽  
Mitochondrial Ribosomes

scholarly journals Acetylcholinesterase from Apis mellifera head. Evidence for amphiphilic and hydrophilic forms characterized by Triton X-114 phase separation

1988 ◽  
Vol 255 (2) ◽  
pp. 463-470 ◽  
Author(s):  
L P Belzunces ◽  
J P Toutant ◽  
M Bounias
Keyword(s):  
Phase Separation ◽  
Apis Mellifera ◽  
Aqueous Phase ◽  
Sucrose Gradient ◽  
Electrophoretic Analysis ◽  
High Salt ◽  
Sedimentation Analysis ◽  
Low Salt ◽  
S Peak ◽  
Triton X

The polymorphism of bee acetylcholinesterase was studied by sucrose-gradient-sedimentation analysis and non-denaturing electrophoretic analysis of fresh extracts. Lubrol-containing extracts exhibited only one form, which sedimented at 5 S when analysed on high-salt Lubrol-containing gradients and 6 S when analysed on low-salt Lubrol-containing gradients. The 5 S/6 S form aggregated upon removal of the detergent when sedimented on detergent-free gradients and was recovered in the detergent phase after Triton X-114 phase separation. Thus the 5 S/6 S enzyme corresponds to an amphiphilic acetylcholinesterase form. In detergent-free extracts three forms, whose apparent sedimentation coefficients are 14 S, 11 S and 7 S, were observed when sedimentations were performed on detergent-free gradients. Sedimentation analyses on detergent-containing gradients showed only a 5 S peak in high-salt detergent-free extracts and a 6 S peak, with a shoulder at about 7 S, in low-salt detergent-free extracts. Electrophoretic analysis in the presence of detergent demonstrated that the 14 S and 11 S peaks corresponded to aggregates of the 5 S/6 S form, whereas the 7 S peak corresponded to a hydrophilic acetylcholinesterase form which was recovered in the aqueous phase following Triton X-114 phase separation. The 5 S/6 S amphiphilic form could be converted into a 7.1 S hydrophilic form by phosphatidylinositol-specific phospholipase C digestion.

scholarly journals The sedimentation of rat skeletal-muscle ribosomes. Effect of hydrocortisone, insulin and diet

1968 ◽  
Vol 106 (4) ◽  
pp. 913-919 ◽  
Author(s):  
V. R. Young ◽  
S. C. Chen ◽  
Jane Macdonald
Keyword(s):  
Skeletal Muscle ◽  
Protein Synthesis ◽  
Sucrose Gradient ◽  
Muscle Protein ◽  
Muscle Protein Synthesis ◽  
Insulin Injection ◽  
Sedimentation Analysis ◽  
Rat Skeletal Muscle ◽  
Protein Free Diet ◽  
Post Mitochondrial Supernatant

1. The influence of hydrocortisone, insulin and diet on the size distribution of ribosomes in a post-mitochondrial supernatant prepared from rat skeletal muscle was studied by sedimentation analysis with a linear 15–40% (w/v) sucrose gradient. 2. Within 4hr. after the injection of 5mg. of hydrocortisone to well-nourished rats, a decrease in the yield per g. of muscle and proportion of total RNA due to polyribosomes was observed. Similar results were obtained in rats given a protein-free diet for 3 days before administration of the hormone. 3. Insulin injection increased the yield and proportion of polyribosomes within 2hr. and decreased the proportion of the lighter ribosomal aggregates. Similar results were noted in rats given a protein-free diet for 3 days before injection. A protein-free diet given for 3 days decreased the yield and proportion of polyribosomes. Insulin did not increase the yield of polyribosomes if rats were starved for 52hr. before injection, but decreased the yield and proportion of the lighter ribosome species. 4. A 52hr. period of starvation or 2,4-dinitrophenol (15mg./kg. body wt.) given 1hr. before the rats were killed resulted in a decreased yield and proportion of polyribosomes, and, within 6hr. of re-feeding the rats with protein-free diets, an increased concentration of polyribosomes was noted. 5. The effects of a protein-free diet, hydrocortisone and insulin on the sedimentation of muscle ribosomes were found to be in accord with their net effects on muscle protein synthesis.

scholarly journals Assembly of the Epithelial Na+Channel Evaluated Using Sucrose Gradient Sedimentation Analysis

1998 ◽  
Vol 273 (35) ◽  
pp. 22693-22700 ◽  
Author(s):  
Chun Cheng ◽  
Lawrence S. Prince ◽  
Peter M. Snyder ◽  
Michael J. Welsh
Keyword(s):  
Sucrose Gradient ◽  
Na Channel ◽  
Sedimentation Analysis ◽  
Epithelial Na Channel

scholarly journals Internalization of Echovirus 1 in Caveolae

2002 ◽  
Vol 76 (4) ◽  
pp. 1856-1865 ◽  
Author(s):  
Varpu Marjomäki ◽  
Vilja Pietiäinen ◽  
Heli Matilainen ◽  
Paula Upla ◽  
Johanna Ivaska ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Sucrose Gradient ◽  
Close Association ◽  
Dominant Negative ◽  
Endocytic Pathway ◽  
Sedimentation Analysis ◽  
Human Pathogen ◽  
Caveolin 1 ◽  
Α2β1 Integrin ◽  
Perinuclear Area

ABSTRACT Echovirus 1 (EV1) is a human pathogen which belongs to the Picornaviridae family of RNA viruses. We have analyzed the early events of infection after EV1 binding to its receptor α2β1 integrin and elucidated the route by which EV1 gains access to the host cell. EV1 binding onto the cell surface and subsequent entry resulted in conformational changes of the viral capsid as demonstrated by sucrose gradient sedimentation analysis. After 15 min to 2 h postinfection (p.i.) EV1 capsid proteins were seen in vesicular structures that were negative for markers of the clathrin-dependent endocytic pathway. In contrast, immunofluorescence confocal microscopy showed that EV1, α2β1 integrin, and caveolin-1 were internalized together in vesicular structures to the perinuclear area. Electron microscopy showed the presence of EV1 particles inside caveolae. Furthermore, infective EV1 could be isolated with anti-caveolin-1 beads 15 min p.i., confirming a close association with caveolin-1. Finally, the expression of dominant negative caveolin in cells markedly inhibited EV1 infection, indicating the importance of caveolae for the viral replication cycle of EV1.

ANALYSIS OF RADIOIMMUNOASSAY BY DISC ELECTROPHORESIS AND SUCROSE GRADIENT CENTRIFUGATION

1971 ◽  
Vol 66 (2) ◽  
pp. 257-265 ◽  
Author(s):  
Lubomir Valenta ◽  
Michel L. Aubert
Keyword(s):  
Sucrose Gradient ◽  
Solid Phase ◽  
Gradient Centrifugation ◽  
Human Growth ◽  
Disc Electrophoresis ◽  
Separate Analysis ◽  
Adrenocorticotrophic Hormone ◽  
Sucrose Gradient Centrifugation ◽  
Before And After ◽  
Polypeptide Hormones

ABSTRACT Radioiodine-labelled synthetic adrenocorticotrophic hormone (ACTH), human growth hormone (HGH), human chorionic somato-mammotrophin (HCS), and human (HTSH) and bovine (BTSH) thyroid stimulating hormones were studied by disc-electrophoresis and sucrose gradient centrifugation before and after incubation with corresponding antisera. All antisera contained 7 S antibodies. After incubation, soluble antigenantibody complexes besides a small amount of precipitate were observed in the incubation mixture, characteristic of each hormone. The complexes migrated like gamma globulins or more slowly on disc-electrophoresis. and on sucrose gradient centrifugation showed patterns dependent on the time of incubation. Light 7 or 9 S, or < 12 S complexes occurred mostly after incubation for several minutes (up to 30 min) before analysis. When incubation was prolonged to 24 h and more, these relatively light complexes disappeared or diminished in favour of heavier soluble or precipitating complexes. Reproducibly obtainable sedimentation patterns of the soluble complexes suggested some definite recombination of antigen molecules with 7 S antibodies. The complexes did not occur on incubation with other sera than an antiserum to a given hormone. They were not influenced by EDTA. Displacement of the radioactivity of the complexes into the free hormone peak was obtained by addition of a non-labelled hormone identical with the labelled one. Sucrose gradient centrifugation and disc-electrophoresis are recommended for the study of immunoreaction of diluted materials and for a separate analysis of different steps of the radioimmunoassay. Radioimmunoassay was introduced for the measurement of protein hormones by Yalow & Berson (1960). The method, described originally for insulin, was later adapted to the detection of a number of protein and polypeptide hormones. On incubation of the hormone with its antiserum, a soluble antigenantibody complex is formed, which is separated from an excess of the free hormone by various methods, e. g. chromatoelectrophoresis, precipitation with a second antibody, adsorption on a solid phase etc. (Hunter 1967). Sucrose gradient centrifugation and disc-electrophoresis were occasionally used to follow some isolated aspect of radioimmunoassay (Fitschen 1965; Monjardino et al. 1968). We are demonstrating that these methods made it possible to analyze the radioimmunoassay step by step and thus may be useful for practical purposes as well as in a study of the immunoreaction of diluted materials.

THE RELATIVE DISTRIBUTION OF THYROXINE, TRIIODOTHYRONINE AND 3,3′,5′-(REVERSE)-TRIIODOTHYRONINE IN VARIOUS FRACTIONS OF THYROGLOBULIN

1978 ◽  
Vol 88 (2) ◽  
pp. 298-305 ◽  
Author(s):  
Peter Laurberg
Keyword(s):  
Ionic Strength ◽  
Guinea Pig ◽  
Sucrose Gradient ◽  
Deae Cellulose ◽  
High Ionic Strength ◽  
Relative Distribution ◽  
Thyroid Extract ◽  
Reverse Triiodothyronine ◽  
Thyroid Secretion ◽  
Stable Iodine

ABSTRACT Thyroglobulin fractions rich and poor in new thyroglobulin were separated by means of DEAE-cellulose chromatography of dog thyroid extracts and by zonal ultracentrifugation in a sucrose gradient of guinea pig thyroid extract incubated at low temperature. The distribution of thyroxine, triiodothyronine and 3,3′,5′-(reverse)-triiodothyronine in hydrolysates of the different fractions was estimated by radioimmunoassays. Following DEAE-cellulose chromatography there was a small but statistically significant increase in the T4/T3 ratio in thyroglobulin fractions eluted at high ionic strength - that is fractions relatively rich in stable iodine but poor in fresh thyroglobulin. There were no differences in the T4/rT3 ratios between the different fractions. The ratios between iodothyronines were almost identical in the various thyroglobulin fractions following zonal ultracentrifugation in a sucrose gradient of cold treated guinea pig thyroid extract. These findings lend no support to the possibility that a relatively high content of triiodothyronines in freshly synthesized thyroglobulin modulates the thyroid secretion towards a preferential secretion of triiodothyronine and 3,3′,5′-(reverse)-triiodothyronine at the expense of the secretion of thyroxine.

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