In-vivo uptake of human growth hormone in male rat liver
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125I-Labelled human GH (hGH) was injected i.v. to male rats and its subcellular distribution in the hepatocyte was examined using fractionation techniques. Uptake into liver homogenates was maximal by 15 min after injection and represented 24% of the injected radioactivity; it was markedly inhibited by coinjection of native hGH. 125I-Labelled hGH taken up by the liver underwent a time-dependent translocation process. The peak of specific labelling of plasma membranes occurred at 3 min whereas later on the radioactivity was concentrated in low-density structures present in Golgi-endosome fractions. To characterize the ligand-associated structures better, endosome-enriched fractions were prepared from a microsomal fraction by isopycnic centrifugation in a sucrose gradient and a Nycodenz gradient. The radioactivity was in one peak with a median density of 1.096 g/cm3 in the Nycodenz gradient fractions. The peak of radioactivity was distinct from that of galactosyltransferase activity which appeared at a median density of 1.114 g/cm3. The labelled material eluted from the various subcellular fractions appeared as intact hGH. Upon in-vivo interaction with male rat hepatocytes, 125I-Labelled hGH was internalized with a sequential association with plasma membranes and endocytic structures distinct from Golgi elements.Keywords:
Cell fractionation
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Density gradient
Isopycnic
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Polystyrene
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Spheroplast
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Abstract We have studied the subcellular fractionation of a clonal line of glial cells from rat brain. In comparison to rat brain, more of the total protein is collected in the crude nuclear fraction following differential centrifugation. When a crude mitochondrial fraction is prepared and resolved on a density gradient, we find that like brain the glial cell crude mitochondrial fraction is also heterogenous. On a sucrose density gradient, such as that used for the preparation of synaptosomes, the glial cell crude mitochondrial fraction resolves into two major fractions. One of these bands sediments to the same isopycnic density as synaptosomes. This fraction contains 20% of the protein, and 50% of the Na‐K ATPase and acid phosphatase of the crude mitochondrial fraction, but virtually no cytochrome oxidase or choline esterase. The fraction consists of numerous membrane structures resembling plasma membranes when examined by electron microscopy. Because the sedimentation properties of the glial membranes are very similar to synaptosomes, we conclude that synaptosomes prepared on sucrose gradients are probably contaminated by these membrane fragments. In contrast to the separation achieved on sucrose gradients, glial membranes separate from synaptosomes on Ficoll‐sucrose gradients.
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Guanidine
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Isopycnic
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Buoyant density
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Preliminary results from differential centrifugation experiments, washing treatments, and enrichment in linear sucrose gradients at a density of 1.09 grams per cubic centimeter all indicated that β-glucosidase activity in corn root homogenates was associated with a membrane such as tonoplast. A subsequent sucrose density gradient centrifugation time course showed that the β-glucosidase was actually a soluble enzyme which moved into the gradients. The problem of soluble enzymes contaminating light density membranes in sucrose gradients and the question of centrifugation time necessary for membrane vesicles to reach isopycnic conditions are addressed.
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Human brain
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Free nerve ending
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Neurotransmitter receptor
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Differential centrifugation
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5'-nucleotidase
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This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and isopycnic density gradient centrifugation. The major advantage of this procedure is that it does not rely on the sole use of solubilizing detergents to obtain subcellular fractions. This makes it possible to separate the plasma membrane from other membrane-bound organelles of the cell. This procedure will facilitate the determination of protein localization in cells with a reproducible, scalable, and selective method. This method has been successfully used to isolate cytosolic, nuclear, mitochondrial, and plasma membrane proteins from the human monocyte cell line, U937. Although optimized for this cell line, this procedure may serve as a suitable starting point for the subcellular fractionation of other cell lines. Potential pitfalls of the procedure and how to avoid them are discussed as are alterations that may need to be considered for other cell lines.
Cell fractionation
Differential centrifugation
Isopycnic
U937 cell
Organelle
Density gradient
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This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and isopycnic density gradient centrifugation. The major advantage of this procedure is that it does not rely on the sole use of solubilizing detergents to obtain subcellular fractions. This makes it possible to separate the plasma membrane from other membrane-bound organelles of the cell. This procedure will facilitate the determination of protein localization in cells with a reproducible, scalable, and selective method. This method has been successfully used to isolate cytosolic, nuclear, mitochondrial, and plasma membrane proteins from the human monocyte cell line, U937. Although optimized for this cell line, this procedure may serve as a suitable starting point for the subcellular fractionation of other cell lines. Potential pitfalls of the procedure and how to avoid them are discussed as are alterations that may need to be considered for other cell lines.
Cell fractionation
Isopycnic
Differential centrifugation
Organelle
U937 cell
Density gradient
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