scholarly journals Evidence that peroxisomal acyl-CoA synthetase is located at the cytoplasmic side of the peroxisomal membrane

1982 ◽  
Vol 204 (1) ◽  
pp. 17-23 ◽  
Author(s):  
G P Mannaerts ◽  
P Van Veldhoven ◽  
A Van Broekhoven ◽  
G Vandebroek ◽  
L J Debeer
Keyword(s):  
Density Gradient Centrifugation ◽  
Adenine Nucleotides ◽  
Gradient Centrifugation ◽  
Enzyme Analysis ◽  
Marker Enzyme ◽  
Peroxisomal Membrane ◽  
Mechanical Disruption ◽  
Percoll Gradients ◽  
Membrane Bound ◽  
Cytoplasmic Side

1. Subfractionation by isopycnic density-gradient centrifugation in self-generating Percoll gradients of peroxisome-rich fractions prepared by differential centrifugation confirmed the presence of acyl-CoA synthetase in peroxisomes. Peroxisomes did not contain nicotinamide or adenine nucleotides other than CoA. 2. The gradient fractions most enriched in peroxisomes were pooled and the peroxisomes sedimented by centrifugation, resulting in a 50-fold-purified peroxisomal preparation as revealed by marker enzyme analysis. 3. Palmitate oxidation by intact purified peroxisomes was CoA-dependent, whereas palmitoyl-CoA oxidation was not, demonstrating that the peroxisomal CoA was available for the thiolase reaction, located in the peroxisomal matrix, but not for acyl-CoA synthetase. This suggests that the latter enzyme is located at the cytoplasmic side of the peroxisomal membrane. 4. Additional evidence for this location of peroxisomal acyl-CoA synthetase was as follows. Mechanical disruption of purified peroxisomes resulted in the release of catalase from the broken organelles, but not of acyl-CoA synthetase, indicating that the enzyme was membrane-bound. Acyl-CoA synthetase was not latent, despite the fact that at least one of its substrates appears to have a limited membrane permeability, as evidenced by the presence of CoA in purified peroxisomes. Finally, Pronase, a proteinase that does not penetrate the peroxisomal membrane, almost completely inactivated the acyl-CoA synthetase of intact peroxisomes.

Separation and characterization of Leydig cells and macrophages from rat testes

1991 ◽  
Vol 130 (3) ◽  
pp. 357-365 ◽  
Author(s):  
G. Dirami ◽  
L. W. Poulter ◽  
B. A. Cooke
Keyword(s):  
Monoclonal Antibody ◽  
Density Gradient ◽  
Leydig Cells ◽  
Magnetic Beads ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sedimentation Velocity ◽  
Average Density ◽  
Centrifugal Elutriation ◽  
Percoll Gradients

ABSTRACT A method involving centrifugal elutriation followed by density gradient centrifugation and incubation with a macrophage monoclonal antibody has been investigated to separate and characterize Leydig cells and macrophages from adult rat testes. After dispersion of the testes with collagenase, the isolated interstitial cells were found to contain 18% Leydig cells and 12% macrophages. These cells were then separated by centrifugal elutriation into eight fractions (F1–F8) (9 to 74 ml/min at 386 g). Each of these fractions was then further purified by density gradient centrifugation on 0–90% Percoll gradients. After centrifugal elutriation, the macrophages were mainly eluted in the first three fractions (F1–F3), whereas the Leydig cell percentage increased in each fraction with increasing flow rate. After further purification of each fraction on Percoll gradients, high percentages of macrophages (11–20%) were found in fractions F1–F3 (average density 1·045 g/ml), containing 11–37% Leydig cells. Less than 3% of the cells in fraction F4–F8 (average density 1 ·075 g/ml) were macrophages and more than 95% were Leydig cells. Heterogeneity of Leydig cells with respect to sedimentation velocities and function was found. Leydig cells from elutriated-and Percoll-purified fractions F4–F8 were heterogeneous with respect to testosterone and cyclic AMP (cAMP) production but showed a similar binding capacity for 125I-labelled human chorionic gonadotrophin. Leydig cells with the highest sedimentation velocity (35·7 mm/h-g) from fractions F7 and F8 were approximately twofold more responsive to LH (3·3 nmol/l) with respect to testosterone and cAMP production compared with Leydig cells with the lowest sedimentation velocity (20·7 mm/h-g). The elutriated and Percoll-purified cells (corresponding to fractions F4–F8) were further purified by incubation with magnetic beads coated with a macrophage monoclonal antibody; this yielded very pure Leydig cells containing <0·3% macrophages. The incubation temperature (room temperature or 4 °C) during the purification with magnetic beads did not affect the degree of purity or the responsiveness of the Leydig cells to LH. The removal of the remaining macrophages with magnetic beads did not have any significant effect on the Leydig cell responsiveness to LH. It was concluded that Leydig cells purified by elutriation and density gradient centrifugation are heterogeneous with respect to their sedimentation velocities and responses to LH; the higher the sedimentation velocity, the higher is their capacity to respond to LH. Leydig cells free from macrophages can be prepared by further purification using magnetic beads coated with a macrophage monoclonal antibody. Journal of Endocrinology (1991) 130, 357–365

scholarly journals Two fractions of rough endoplasmic reticulum from rat liver. I. Recovery of rapidly sedimenting endoplasmic reticulum in association with mitochondria.

1977 ◽  
Vol 72 (3) ◽  
pp. 714-725 ◽  
Author(s):  
G C Shore ◽  
J R Tata
Keyword(s):  
Endoplasmic Reticulum ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Rat Liver ◽  
Rough Endoplasmic Reticulum ◽  
Structural Damage ◽  
Gradient Centrifugation ◽  
Mitochondrial Protein ◽  
Enzyme Analysis ◽  
Marker Enzyme

Low-speed centrifugation (640 g) of rat liver homogenates, prepared with a standard ionic medium, yielded a pellet from which a rapidly sedimenting fraction of rough endoplasmic reticulum (RSER) was recovered free of nuclei. This fraction contained 20-25% of cellular RNA and approximately 30% of total glucose-6-phosphatase (ER marker) activity. A major portion of total cytochrome c oxidase (mitochondrial marker) activity was also recovered in this fraction, with the remainder sedimenting between 640 and 6,000 g. Evidence is provided which indicates that RSER may be intimately associated with mitochondria. Complete dissociation of ER from mitochondria in the RSER fraction required very harsh conditions. Sucrose density gradient centrifugation analysis revealed that 95% dissociation could be achieved when the RSER fraction was first resuspended in buffer containing 500 mM KCl and 20 mM EDTA, and subjected to shearing. Excluding KCl, EDTA, or shearing from the procedure resulted in incomplete separation. Both electron microscopy and marker enzyme analysis of mitochondria purified by this procedure indicated that some structural damage and leakage of proteins from matrix and intermembrane compartments had occurred. Nevertheless, when mitochondria from RSER and postnuclear 6,000-g pellet fractions were purified in this way fromanimals injected with [35S]methionine +/- cycloheximide, mitochondria from the postnuclear 6,000-g pellet were found to incorporate approximately two times more cytoplasmically synthesized radioactive protein per milligram mitochondrial protein (or per unit cytochrome c oxidase activity) than did mitochondria from the RSER fraction. Mitochondria-RSER associations, therefore, do not appear to facilitate enhanced incorporation of mitochondrial proteins which are newly synthesized in the cytoplasm.

Membrane-bound carbonic anhydrase in the heart

1992 ◽  
Vol 262 (2) ◽  
pp. H577-H584 ◽  
Author(s):  
W. Bruns ◽  
G. Gros
Keyword(s):  
Carbonic Anhydrase ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Integral Membrane Protein ◽  
Rabbit Heart ◽  
Microsomal Membranes ◽  
Membrane Bound ◽  
The One ◽  
Triton X

Microsomal membranes from bovine heart homogenates were subfractionated by density gradient centrifugation. Fractions with high levels of a sarcolemmal (SL) marker are enriched in specific carbonic anhydrase (CA) activity up to ninefold compared with the microsomes. Fractions with high levels of a sarcoplasmic reticulum marker and a mitochondrial marker, respectively, exhibit specific CA activities that are similar to the one found in the microsomes. Determination of cytosolic markers reveals that the CA activity in the SL fraction is not due to contamination by cytosolic CA, and it is shown by Triton X-114 phase separation that the CA activity is due to an integral membrane protein. In cryosections from rabbit heart the SL region of cardiomyocytes is stained by the fluorescent CA inhibitor dansylsulfonamide. Intracellular staining occurs also, with a pattern suggesting the presence of CA associated with intracellular membranes. Although it cannot be excluded that there is a contribution by endothelial membranes, it appears likely that most CA of the heart is bound to the SL. The possible involvement of the enzyme in extracellular proton buffering is discussed.

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.

Changes in Hepatic Enzymes and Organelles in Alcoholic Liver Disease

1978 ◽  
Vol 55 (4) ◽  
pp. 383-389 ◽  
Author(s):  
Carol A. Seymour ◽  
T. J. Peters
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Liver Disease ◽  
Fatty Liver ◽  
Density Gradient ◽  
Alcoholic Liver Disease ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Equilibrium Density ◽  
Marker Enzyme

1. Liver biopsy specimens obtained from patients with alcoholic liver disease of varying severity were assayed for lysosomal and microsomal enzyme activities, the results being compared with values previously obtained in control subjects. 2. Analytical subcellular fractionation by sucrose-density-gradient centrifugation was performed on extracts of the biopsies and the properties of the lysosomes, plasma membrane, biliary canaliculi and endoplasmic reticulum membranes were determined. Increased activities of plasma membrane marker enzymes, particularly γ-glutamyl transpeptidase believed to be localized to the biliary canalicular membrane, were demonstrated. These findings were most marked in alcoholic cirrhosis. The centrifugation studies revealed no abnormalities in the properties of these membranes. 3. Although the total activities of the endoplasmic reticulum marker enzyme neutral α-glucosidase were unaltered in alcoholic liver disease, centrifugation studies showed a decrease in the density distribution of the membrane-bound enzyme in cirrhosis indicating an increase in the proportion of smooth endoplasmic reticulum membranes. 4. Apart from a small decrease in activity of certain acid hydrolases in fatty liver and in cirrhosis the activities of the lysosomal enzymes were unaffected by alcoholic liver disease. 5. Measurements of lysosomal integrity and density-gradient-centrifugation studies revealed no significant abnormalities in the various patient groups apart from increased stability and reduced equilibrium density of certain lysosomes in fatty liver. It is concluded that lysosomal disruption is not implicated in the pathogenesis of alcoholic liver disease.

scholarly journals Isolation of Spherosomes with Lysosome Characteristics from Seedlings

1965 ◽  
Vol 20 (7) ◽  
pp. 693-698 ◽  
Author(s):  
Ph. Matile ◽  
J. P. Balz ◽  
E. Semadeni ◽  
M. Jost
Keyword(s):  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Free Form ◽  
Acid Hydrolases ◽  
Animal Cells ◽  
Higher Plant ◽  
Tobacco Seedlings ◽  
Membrane Bound ◽  
Cytoplasmic Structures

Fractionation of cell free extracts from corn and tobacco seedlings by density gradient centrifugation resulted in the isolation of two fractions containing acid hydrolases: protease, phosphatase, esterase and ribonuclease. In one fraction prospherosomes were the predominant cytoplasmic structures. The other fraction contained spherosomes in either free form or enclosed together with other structured material in membrane-bound vacuoles. These forms of isolated spherosomes are identified with corresponding structures observed in situ. It is concluded that the spherosomes represent the lysosome equivalent of higher plant cells. The vacuoles containing spherosomes, mitochondria and other structured elements are interpreted as digestion vacuoles corresponding to the cytolysomes of animal cells.

Purification of gibberellic acid-induced lysosomes from wheat aleurone cells

1976 ◽  
Vol 22 (2) ◽  
pp. 413-425
Author(s):  
R.A. Gibson ◽  
L.G. Paleg
Keyword(s):  
Endoplasmic Reticulum ◽  
Gibberellic Acid ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Mechanisms Of Action ◽  
Marker Enzyme ◽  
Cell Organelles ◽  
Aleurone Cells ◽  
Ficoll Gradient ◽  
Single Region

Using isopycnic density gradient centrifugation, lysosomes were concentrated in a single region of a sucrose-Ficoll gradient (p = 1–10 g cm-3), well separated from most other cell organelles. Gibberellic acid-induced lysosomes were found to be rich in alpha-amylase and protease but not ribonuclease. The lysosomal band also contained a majority of the NADH2-cytochrome c reductase, a marker enzyme for endoplasmic reticulum, found in the gradient. Examination of electron micrographs revealed that a purified band of lyosomes contained at least 3 vesicle types, ranging in size from 0-1 to 0-5 mum. The significance of these findings to proposed mechanisms of action of gibberellic acid is discussed.

Separation of viable microfilariae free of blood cells on Percoll gradients

1984 ◽  
Vol 58 (1) ◽  
pp. 69-70 ◽  
Author(s):  
R. Chandrashekar ◽  
U. R. Rao ◽  
G. R. Rajasekariah ◽  
D. Subrahmanyam
Keyword(s):  
Density Gradient ◽  
Blood Cells ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Brugia Pahangi ◽  
Percoll Gradients ◽  
Reproducible Method ◽  
Dipetalonema Viteae

AbstractA consistent and reproducible method is described for isolating pure populations of microfilariae of Litomosoides carinii, Brugia pahangi, B. malayi and Dipetalonema viteae, free of cells, from blood, by density gradient centrifugation on Percoll in 0.25M sucrose. The recovery of the microfilariae was 85 to 97%.

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