Cell Fractionation Techniques

Beaufay, Henri; Amar-Costesec, A

doi:10.1007/978-1-4757-5817-7_1

Cited by 77 publications

(49 citation statements)

References 162 publications

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“…Distribution patterns of enzymes and protein after differential centrifugation [31] and density equilibration [32] are presented as previously described. Since density boundaries are not strictly reproducible, the histograms were normalized over 15 fractions as explained in Fig.…”

Section: Presentation Of Resultsmentioning

confidence: 99%

“…Acid proteinase as well as a-mannosidase behave in a way characteristic of lysosomes in other organisms [32]. Both enzymes show considerable structural latency in cytoplasmic extracts.…”

Section: Acid Proteinase Acid Rnase and A-mannosidasementioning

confidence: 99%

“…Density equilibration of the post-large-granule extract was performed in a linear sucrose gradient using the E-40 automatic zonal rotor described by Beaufay and Amar-Costesec [32]. Loading of the rotor was effected by the successive injection of 10 ml of a post-large-granule extract in sucrose/imidazole/EGTA medium containing a total of 40-80 mg protein, 32 ml of a linear sucrose gradient (e = 1.10-1.26 g/ cm3) buffered as sucrose/imidazole/EGTA, and a 6-ml cushion of sucrose with a density of 1.34 g/cm3.…”

Section: Cell Fractionationmentioning

confidence: 99%

“…Activities of various 'classical' eukaryotic marker enzymes (see [28,32] for reviews) could not be found in our material with a detection limit of 0.01 nmol x min-' x mg-I, notably (a) 5'-nucleotidase, NAD nucleosidase, alkaline phosphatase and alkaline phosphodiesterase (all representative of the plasma membrane) ; (b) 'true' glucose-6-phosphatase and sulphatase C (endoplasmic reticulum); nor (c) lysozyme, trehalase, 0-D-fucosidase, fl-glucuronidase, cc-N-acetylgalactosaminidase, a-N-glucosaminidase, p-glucosidase, cathepsin BI and sulphatases A, B (lysosomes).…”

Section: Enzyme Screeningmentioning

confidence: 99%

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Subcellular Fractionation of Trypanosoma brucei Bloodstream Forms with Special Reference to Hydrolases

1980

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Bloodstream forms of Trypanosoma brucei have been screened for the presence of enzymes that could serve as markers for the plasma membrane, flagellar pocket, lysosomes, endoplasmic reticulum and Golgi apparatus in order to study the subcellular organization of the digestive system of the parasite.Acetylesterase, acid DNase, acid phosphatase, acid phosphodiesterase, acid proteinase, acid RNase, alanine aminotransferase, galactosyl transferase, a-glucosidase, inosine diphosphatase and a-mannosidase were partially characterized and their assays optimized for pH-dependent activity, linearity of reaction with respect to incubation time and enzyme concentration, and the effect of inhibitors and activators. The association of these enzymes with particulate material and the presence of structural latency were investigated.Acid proteinase and a-mannosidase are particle-bound and latent in cytoplasmic extracts ; they can be activated and solubilized in part by Triton X-100. Similar results were obtained for acid phosphatase, acid phosphodiesterase and inosine diphosphatase. Neutral a-glucosidase, though partly sedimentable, does not show latency and is readily solubilized by the detergent. Galactosyl transferase is firmly membrane-bound even in the presence of 0.1 % Triton X-100, Cell fractionation by differential centrifugation and density equilibration on sucrose gradients revealed that both a-mannosidase and acid proteinase are associated with organelles that band at a density of about 1.20 g/cm3. Inosine diphosphatase, galactosyl transferase, acid phosphatase and acid phosphodiesterase sediment predominantly as microsomal constituents equilibrating at densities between 1.13 and 1.15 g/cm3. In addition, inosine diphosphatase and galactosyl transferase exhibit considerable activity at higher densities (1.18 -1.25 g/cm3). Neutral a-glucosidase is mainly recovered in the nuclear and microsomal fraction; its particulate part equilibrates as a single band at e = 1.22 g/cm3. Acetylesterase and acid DNase are largely soluble, whereas acid RNase does not produce distinct sedimentation and banding profiles.In intact cells, neutral a-glucosidase and acid phosphatase appear to be highly accessible to their substrates.It is tentatively concluded that (a) acid proteinase and a-mannosidase are lysosomal enzymes, (b) acid phosphatase and acid phosphodiesterase are associated with the flagellar pocket and part of the former enzyme probably with the endoplasmic reticulum, (c) galactosyl transferase is a constituent of the Golgi apparatus, and (d) a-glucosidase may serve as a marker for the plasma membrane. Inosine diphosphatase may also be derived from the latter structure.

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Section: Presentation Of Resultsmentioning

confidence: 99%

“…Acid proteinase as well as a-mannosidase behave in a way characteristic of lysosomes in other organisms [32]. Both enzymes show considerable structural latency in cytoplasmic extracts.…”

Section: Acid Proteinase Acid Rnase and A-mannosidasementioning

confidence: 99%

Section: Cell Fractionationmentioning

confidence: 99%

Section: Enzyme Screeningmentioning

confidence: 99%

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Subcellular Fractionation of Trypanosoma brucei Bloodstream Forms with Special Reference to Hydrolases

1980

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“…Fractionation of electric tissue homogenates by differential centrifugation was carried out as described for other tissues (Beaufay and Amar-Costesec, 1976;Amar-Costesec et al, 1985) Fig. 2.…”

Section: Microcrystalline Silica In Electric Organsmentioning

confidence: 99%

The Growth of Chalcedony (Nanocrystalline Silica) in Electric Organs from Living Marine Fish

Figueroa¹

2012

Advances in Crystallization Processes

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Subcellular Fractionation of Tissue Culture Cells

Aniento

Grüenberg

2003

CP Protein Science

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Cell fractionation techniques include some of the most important and widely used analytical tools in cell and molecular biology, and are essential for the development of cell-free assays that reconstitute complicated cellular processes. In addition to simple gradient systems, this unit discusses the immuno-purification of organelles, in particular endosomes. As antigens, purification can be achieved using endogenous or ectopically expressed proteins, provided that appropriate antibodies are available. Alternatively, tagged proteins can be used, when combined with anti-tag antibodies. Now that sequencing of the genomes of several organisms has been completed, biochemical strategies, and in particular fractionation and in vitro transport assays, are more necessary than ever to study the numerous protein and protein complexes that are being discovered.

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Cell Fractionation Techniques

Cited by 77 publications

References 162 publications

Subcellular Fractionation of Trypanosoma brucei Bloodstream Forms with Special Reference to Hydrolases

Subcellular Fractionation of Trypanosoma brucei Bloodstream Forms with Special Reference to Hydrolases

The Growth of Chalcedony (Nanocrystalline Silica) in Electric Organs from Living Marine Fish

Subcellular Fractionation of Tissue Culture Cells

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