scholarly journals Cystine storage in cultured myotubes from patients with nephropathic cystinosis

1987 ◽  
Vol 243 (3) ◽  
pp. 841-845 ◽  
Author(s):  
G S Harper ◽  
I Bernardini ◽  
O Hurko ◽  
J Zuurveld ◽  
W A Gahl
Keyword(s):  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Muscle Cells ◽  
Subcellular Fractionation ◽  
Nephropathic Cystinosis ◽  
Lysosomal Storage ◽  
Lysosomal Compartment ◽  
Cell Functions ◽  
Unique System ◽  
Cultured Myotubes

Sorted muscle cells, cultured from a patient with nephropathic cystinosis, stored 100 times normal amounts of cystine. Subcellular fractionation and density-gradient centrifugation confirmed that the cystine was located in a lysosomal compartment. 2. Myoblasts from cystinotic patients in culture underwent fusion to myotubes in a normal fashion. 3. The free thiol cysteamine effectively depleted cystinotic-muscle cells of cystine. 4. Cultured myoblast and myotubes offered a unique system for investigating the effects of lysosomal storage on differentiated cell functions.

scholarly journals Butyrate formation from glucose by the rumen protozoon Dasytricha ruminantium

1985 ◽  
Vol 228 (1) ◽  
pp. 187-192 ◽  
Author(s):  
N Yarlett ◽  
D Lloyd ◽  
A G Williams
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Subcellular Fractionation ◽  
Small Granule ◽  
Metabolic Reactions ◽  
Pyruvate:Ferredoxin Oxidoreductase ◽  
Butyrate Kinase ◽  
Coa Reductase ◽  
Butyrate Production

Production of butyrate by the holotrich protozoon Dasytricha ruminantium involves the enzymes of glycolysis, pyruvate:ferredoxin oxidoreductase, acetyl-CoA:acetyl-CoA C-acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, 3-hydroxyacyl-CoA hydro-lyase, 3-hydroxyacyl-CoA reductase, phosphate butyryltransferase and butyrate kinase. Subcellular fractionation by differential and density-gradient centrifugation on sucrose gradients indicated that all those enzymes except pyruvate:ferredoxin oxidoreductase were non-sedimentable at 6 × 10(6) g-min. Butyrate kinase and phosphate butyryltransferase were associated with the large- and small-granule fractions. Thus, although metabolic reactions necessary for butyrate production proceed predominantly in the cytosol, hydrogenosomes play a key role in the conversion of pyruvate into acetyl-CoA.

Subcellular Distribution of Radio-Labelled Iron during Intestinal Absorption in Guinea-Pig Enterocytes with Special Reference to the Mitochondrial Localization of the Iron

1979 ◽  
Vol 56 (2) ◽  
pp. 179-188 ◽  
Author(s):  
J. M. P. Hopkins ◽  
T. J. Peters
Keyword(s):  
Guinea Pig ◽  
Density Gradient Centrifugation ◽  
Early Stage ◽  
Gradient Centrifugation ◽  
Intestinal Transport ◽  
Subcellular Fractionation ◽  
Mitochondrial Fraction ◽  
Mitochondrial Localization ◽  
The Matrix ◽  
Pig Jejunum

1. 59Fe-labelled ferric chloride was introduced into tied loops of guinea-pig jejunum. 2. After 5–30 min the loop was removed, the enterocytes were isolated, homogenized and subjected to analytical subcellular fractionation. 3. Uptake of 59Fe was extremely rapid and after 5 min 45% of the radioactivity sedimented in the mitochondrial fraction. 4. Density gradient centrifugation indicated that approximately 80% of this radioactivity was associated with the mitochondria themselves; the remainder was in brush-border fragments. 5. Selective disruption of the mitochondria demonstrates that the iron is localized to the matrix and the inner membrane, indicating transport of the absorbed iron into the organelle. 6. It is suggested that mitochondria are actively implicated at an early stage in the intestinal transport of iron.

scholarly journals The post-synthetic sorting of endogenous membrane proteins examined by the simultaneous purification of apical and basolateral plasma membrane fractions from Caco-2 cells

1992 ◽  
Vol 283 (2) ◽  
pp. 553-560 ◽  
Author(s):  
J A Ellis ◽  
M R Jackman ◽  
J P Luzio
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Membrane Fraction ◽  
Apical Membrane ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Basolateral Membrane ◽  
Subcellular Fractionation ◽  
Integral Membrane Proteins ◽  
Basolateral Plasma Membrane

A subcellular fractionation method to isolate simultaneously apical and basolateral plasma membrane fractions from the human adenocarcinoma cell line Caco-2, grown on filter supports, is described. The method employs sucrose-density-gradient centrifugation and differential precipitation. The apical membrane fraction was enriched 14-fold in sucrase-isomaltase and 21-fold in 5′-nucleotidase compared with the homogenate. The basolateral membrane fraction was enriched 20-fold relative to the homogenate in K(+)-stimulated p-nitrophenylphosphatase. Alkaline phosphatase was enriched 15-fold in the apical membrane fraction and 3-fold in the basolateral membrane fraction. Analytical density-gradient centrifugation showed that this enzyme was a true constituent of both fractions, and experiments measuring alkaline phosphatase release following treatment with phosphatidylinositol-specific phospholipase C showed that in both membrane fractions the enzyme was glycosyl-phosphatidylinositol-linked. There was very little contamination of either membrane fraction by marker enzymes of the Golgi complex, mitochondria or lysosomes. Both membrane fractions were greater than 10-fold purified with respect to the endoplasmic reticulum marker enzyme alpha-glucosidase. Protein composition analysis of purified plasma membrane fractions together with domain-specific cell surface biotinylation experiments revealed the presence of both common and unique integral membrane proteins in each plasma membrane domain. The post-synthetic transport of endogenous integral plasma membrane proteins was examined using the devised subcellular fractionation procedure in conjunction with pulse-chase labelling experiments and immunoprecipitation. Five common integral membrane proteins immunoprecipitated by an antiserum raised against a detergent extract of the apical plasma membrane fraction were delivered with the same time course to each cell-surface domain.

Analytical Subcellular Fractionation of Guinea-Pig Myocardium

1977 ◽  
Vol 53 (1) ◽  
pp. 63-74
Author(s):  
F. J. Bloomfield ◽  
G. Wells ◽  
E. Welman ◽  
T. J. Peters
Keyword(s):  
Guinea Pig ◽  
Adenosine Triphosphatase ◽  
Small Volume ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Subcellular Fractionation ◽  
Marker Enzyme ◽  
Subcellular Organelles ◽  
Dual Localization ◽  
Glutamyl Transpeptidase

1. Homogenates of guinea-pig left ventricle were fractionated by differential pelleting and by centrifugation on continuous sucrose density gradients. 2. The principal subcellular organelles of myocardium, characterized by their marker enzyme content, were resolved by density gradient centrifugation in a small-volume zonal rotor. The equilibrium densities (p) of the principal organelles are (with marker enzymes in parentheses): sarcolemma, 1·12 (5′-nucleotidase); lysosomes, 1·16 (N-acetyl-β-glucosaminidase); mitochondria, 1·17 (cytochrome oxidase); peroxisomes, 1·18 (catalase); cytosol (lactate dehydrogenase). 3. The subcellular distribution of various adenosine triphosphatase activities and previously unassigned enzymes was determined. Leucyl-β-naphthylamidase and γ-glutamyl transpeptidase showed both cytosol and sarcolemma components. Ca2+-dependent adenosine triphosphatase showed dual localization to the mitochondria and to the sarcolemma.

The Organelle Pathology and Demonstration of Mitochondrial Superoxide Dismutase Deficiency in Two Patients with Dubin–Johnson–Sprinz Syndrome

1978 ◽  
Vol 54 (5) ◽  
pp. 549-553
Author(s):  
T. J. Peters ◽  
Carol A. Seymour
Keyword(s):  
Superoxide Dismutase ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Subcellular Fractionation ◽  
Sucrose Density Gradient Centrifugation ◽  
Mitochondrial Superoxide ◽  
Liver Biopsies ◽  
Mitochondrial Superoxide Dismutase

1. Liver biopsies from two patients with the Dubin—Johnson—Sprinz syndrome were subjected to analytical subcellular fractionation by sucrose-density-gradient centrifugation and enzymic micro-assays. 2. A selective deficiency of mitochondrial superoxide dismutase has been demonstrated in these patients. 3. The significance of this enzyme deficiency is discussed in relation to the organelle pathology of the syndrome.

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