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.

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 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.

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 Adenosine triphosphatase and adenosine diphosphate/adenosine triphosphate isotope-exchange activities of the chromaffin-granule membrane

1977 ◽  
Vol 167 (1) ◽  
pp. 297-300 ◽  
Author(s):  
D K Apps ◽  
G A Reid
Keyword(s):  
Isotope Exchange ◽  
Adenosine Triphosphatase ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Adenosine Diphosphate ◽  
Sucrose Density Gradient ◽  
Chromaffin Granule ◽  
Sucrose Density Gradient Centrifugation ◽  
Granule Membrane ◽  
Differential Sedimentation

The association of adenosine triphosphatase and ADP/ATP isotope-exchange activities with chromaffin-granule membranes was shown by sucrose-density-gradient centrifugation. The two activities were solubilized, and separated by differential sedimentation.

scholarly journals Separation of Adenosine Triphosphatase of HK and LK Sheep Red Cell Membranes by Density Gradient Centrifugation

1965 ◽  
Vol 48 (6) ◽  
pp. 1125-1143 ◽  
Author(s):  
D. C. Tosteson ◽  
P. Cook ◽  
R. Blount
Keyword(s):  
Specific Gravity ◽  
Atpase Activity ◽  
Density Gradient ◽  
Adenosine Triphosphatase ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Red Cell ◽  
High Potassium ◽  
Red Cell Membrane ◽  
Red Cell Membranes

Membrane fragments from high potassium (HK) and low potassium (LK) sheep red cells were separated by density gradient centrifugation. Three preparations were studied: (1) HK membranes sonicated for 20 minutes, (2) HK membranes sonicated for 3 minutes, and (3) LK membranes sonicated for 3 minutes. The adenosine triphosphatase (ATPase) activity in the maximally disrupted preparation (1) was not sensitive to Na + K and was recovered in relatively small but heavy (specific gravity 1.19) fragments which made up no more than 8 per cent of the total membrane. Both Na + K-sensitive (S) and Na + K-insensitive (I) ATPase activity were found in the more gently broken up preparations (2) and (3) but the ratio of S- to I-ATPase was much greater in HK than in LK membrane fragments. S-ATPase activity in preparation (2) was about 50 per cent that observed in HK membranes prior to sonication. S-ATPase activity was recovered from the density gradient in relatively large but light (specific gravity 1.10) fragments. As was the case with the maximally disrupted preparation (1), I-ATPase activity in both preparations (2) and (3) was recovered in small but heavy (specific gravity > 1.20) fragments. The possibility that sensitivity of sheep red cell membrane ATPase to Na + K depends on the association between units containing the enzyme(s) and large, light, phospholipid-containing components is discussed.

Association of Myxoviruses with an Adenosine Diphosphatase / Adenosine Triphosphatase / as Revealed by Chromatography on DEAE-Cellulose and by Density Gradient Centrifugation

1963 ◽  
Vol 18 (12) ◽  
pp. 1050-1052 ◽  
Author(s):  
A. Robert Neurath ◽  
František Sokol
Keyword(s):  
Density Gradient ◽  
Newcastle Disease ◽  
Adenosine Triphosphatase ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Deae Cellulose

Die Assoziation von einem, schon früher beschriebenen Adenosindiphosphat spaltenden Enzym mit den Teilchen von Myxoviren wurde mittels Chromatographie an DEAE-Cellulose und Dichte-Gradient-Zentrifugation von vorgereinigten Präparaten von Newcastle Disease, Sendai und A 2- Influenza-Viren sowie von dem gewebseigenen Enzym bestätigt. Das Enzym ist fähig, auch Adenosintriphosphat zu spalten.

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.

Sign in / Sign up

Export Citation Format

Share Document