scholarly journals Use of a novel rapid preparation of fat-cell plasma membranes employing Percoll to investigate the effects of insulin and adrenaline on membrane protein phosphorylation within intact fat-cells

1980 ◽  
Vol 192 (2) ◽  
pp. 457-467 ◽  
Author(s):  
G J Belsham ◽  
R M Denton ◽  
M J A Tanner
Keyword(s):  
Plasma Membranes ◽  
Microsomal Fraction ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sodium Dodecyl ◽  
Fat Cells ◽  
Fat Cell ◽  
Rapid Preparation ◽  
Cell Plasma ◽  
Rat Fat Cells

1. A rapid method was developed for the preparation of plasma membranes from either isolated rat fat-cells or intact epididymal fat-pads with the use of density-gradient centrifugation in the presence of Percoll. On the basis of 5′-nucleotidase activity, the yield of plasma membranes was about 50% and purification over 10-fold. Activities of marker enzymes indicated that contamination by mitochondria and microsomal fraction was small. 2. Incorporation of 32Pi into proteins associated with plasma membranes within isolated fat-cells was investigated. Four major bands of labelled phosphoproteins were separated by sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis; the apparent subunit mol.wts. were 67 000, 61 000, 26 000 and 20 000. None of these phosphoprotein bands corresponded to periodate/Schiff-staining glycoproteins. The extent of phosphorylation of the 61 000 mol.wt phosphoprotein band was increased by about 30 and 60% after exposure of fat-cells for 15 min to insulin or adrenaline respectively.

scholarly journals A rapid immunological procedure for the isolation of hormonally sensitive rat fat-cell plasma membrane

1976 ◽  
Vol 154 (1) ◽  
pp. 11-21 ◽  
Author(s):  
J P Luzio ◽  
A C Newby ◽  
C N Hales
Keyword(s):  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
Plasma Membranes ◽  
Membrane Preparation ◽  
Fat Cells ◽  
Fat Cell ◽  
Cell Plasma ◽  
Rat Fat Cells ◽  
Plasma Membrane Preparation ◽  
Isolated Rat

1. A rapid method for the isolation of hormonally sensitive rat fat-cell plasma membranes was developed by using immunological techniques. 2. Rabbit anti-(rat erythrocyte) sera were raised and shown to cross-react with isolated rat fat-cells. 3. Isolated rat fat-cells were coated with rabbit anti-(rat erythrocyte) antibodies, homogenized and the homogenate made to react with an immunoadsorbent prepared by covalently coupling donkey anti-(rabbit globulin) antibodies to aminocellulose. Uptake of plasma membrane on to the immunoadsorbent was monitored by assaying the enzymes adenylate cyclase and 5′-nucleotidase and an immunological marker consisting of a 125I-labelled anti-(immunoglobulin G)-anti-cell antibody complex bound to the cells before fractionation. Contamination of the plasma-membrane preparation by other subcellular fractions was also investigated. 4. By using this technique, a method was developed allowing 25-40% recovery of plasma membrane from fat-cell homogenates within 30 min of homogenization. 5. Adenylate cyclase in the isolated plasma-membrane preparation was stimulated by 5 μm-adrenaline.

scholarly journals Insulin-like actions of nickel and other transition-metal ions in rat fat-cells

1976 ◽  
Vol 154 (2) ◽  
pp. 349-357 ◽  
Author(s):  
E. D Saggerson ◽  
S R. Sooranna ◽  
C J. Evans
Keyword(s):  
Transition Metal ◽  
Metal Ions ◽  
Glucose Utilization ◽  
Transition Metal Ions ◽  
Fat Cells ◽  
Fat Cell ◽  
Cell Plasma Membrane ◽  
Cell Plasma ◽  
Glucose Incorporation ◽  
Rat Fat Cells

NiCl2 (1-6mM) decreased adrenaline and glucagon-stimulated lipolysis in rat fat-cells, and also considerably stimulated [U-14C]glucose incorporation into fat-cell lipids. 2. These insulin-like effects were also observed with CuCl, CuCl2, CoCl2 and (to a lesser extent) with MnCl2. 3. NiCl2 was less effective in mimicking insulin effects on [U-14C]fructose metabolism than on glucose utilization. 4. It is tentatively suggested that these transition-metal ions may mimic actions of insulin at the fat-cell plasma membrane which decrease lipolysis and stimulate glucose transport, but do not mimic certain other effects of the hormone on intracellular metabolic processes. 5. These results are discussed with reference to suggestions that redistributions of cellular Ca2+ are associated with insulin action in fat-cells.

scholarly journals Insulin-induced rapid decrease of a major protein in fat cell plasma membranes.

1984 ◽  
Vol 259 (19) ◽  
pp. 12112-12116
Author(s):  
E J Schoenle ◽  
L D Adams ◽  
D W Sammons
Keyword(s):  
Plasma Membranes ◽  
Rapid Decrease ◽  
Major Protein ◽  
Fat Cell ◽  
Cell Plasma

Regional variation in HDL metabolism in human fat cells: effect of cell size

1987 ◽  
Vol 252 (5) ◽  
pp. E654-E659 ◽  
Author(s):  
J. P. Despres ◽  
B. S. Fong ◽  
P. Julien ◽  
J. Jimenez ◽  
A. Angel
Keyword(s):  
Cell Size ◽  
Cellular Uptake ◽  
Plasma Membranes ◽  
Cholesterol Content ◽  
Cholesterol Concentration ◽  
Fat Cells ◽  
Obese Patients ◽  
Adipocyte Size ◽  
Fat Cell ◽  
Omental Fat

Abdominal obesity is related to reduced plasma high-density lipoprotein (HDL) cholesterol, and both are associated with cardiovascular disease risk. We have observed that plasma membranes from abdominal subcutaneous adipocytes have a greater HDL binding capacity than omental fat cell plasma membranes. The present study examined whether these binding characteristics could be due to differences in fat cell size or cholesterol concentration between the two adipose depots. Abdominal subcutaneous and deep omental fat were obtained from massively obese patients at surgery. Subcutaneous abdominal fat cells were significantly larger and their cellular cholesterol content greater than omental adipocytes. The uptake of HDL by collagenase-isolated fat cells was studied by incubating the cells for 2 h at 37 degrees C with 10 micrograms/ml 125I-HDL2 or 125I-HDL3. In both depots, the cellular uptake of 125I-HDL2 and 125I-HDL3 was specifically inhibited by addition of 25-fold excess unlabeled HDL and a close correlation was observed between the cellular uptake of 125I-HDL2 and 125I-HDL3. In obese patients, the uptake of 125I-HDL was higher in subcutaneous cells than in omental cells [5.85 +/- 0.53 vs. 2.74 +/- 0.30 pmol X 2 h-1. (10(6) cells)-1]. The cellular 125I-HDL uptake was significantly correlated with adipocyte size and fat cell cholesterol content but not with adipocyte cholesterol concentration. These results suggest that the higher HDL uptake observed in subcutaneous cells compared with omental cells in obesity is the result of differences in adipocyte size rather than differences in the cholesterol concentration (cholesterol-to-triglyceride ratio).(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Coypu insulin. Primary structure, conformation and biological properties of a hystricomorph rodent insulin

1986 ◽  
Vol 238 (2) ◽  
pp. 345-351 ◽  
Author(s):  
M Bajaj ◽  
T L Blundell ◽  
R Horuk ◽  
J E Pitts ◽  
S P Wood ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Model Building ◽  
Spatial Arrangement ◽  
Biological Properties ◽  
Bovine Insulin ◽  
Fat Cells ◽  
C Terminus ◽  
Biological Potency ◽  
A Chain ◽  
Rat Fat Cells

Insulin from a hystricomorph rodent, coypu (Myocaster coypus), was isolated and purified to near homogeneity. Like the other insulins that have been characterized in this Suborder of Rodentia, coypu insulin also exhibits a very low (3%) biological potency, relative to pig insulin, on lipogenesis in isolated rat fat-cells. The receptor-binding affinity is significantly higher (5-8%) in rat fat-cells, in rat liver plasma membranes and in pig liver cells, indicating that the efficacy of coypu insulin on receptors is about 2-fold lower than that of pig insulin. The primary structures of the oxidized A- and B-chains were determined, and our sequence analysis confirms a previous report [Smith (1972) Diabetes 21, Suppl. 2, 457-460] that the C-terminus of the A-chain is extended by a single residue (i.e. aspartate-A22), in contrast with most other insulin sequences, which terminate at residue A21. In spite of a large number of amino acid substitutions (relative to mammalian insulins), computer-graphics model-building studies suggest a similar spatial arrangement for coypu insulin to that for pig insulin. The substitution of the zinc-co-ordinating site (B10-His----Gln) along with various substitutions on the intermolecular surfaces involved in the formation of higher aggregates are consistent with the observation that this insulin is predominantly ‘monomeric’ in nature. The c.d. spectrum of coypu insulin is relatively similar to those of casiragua insulin and of bovine insulin at low concentration.

scholarly journals Isolation of the intercellular glycoproteins of desmosomes.

1981 ◽  
Vol 90 (1) ◽  
pp. 243-248 ◽  
Author(s):  
G Gorbsky ◽  
M S Steinberg
Keyword(s):  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Insoluble Residue ◽  
Differential Centrifugation ◽  
Electron Microscopic ◽  
Nonionic Detergent ◽  
Cell Layers ◽  
Membrane Attachment

To characterize the desmosome components that mediate intercellular adhesion and cytoskeletal-plasma membrane attachment, we prepared whole desmosomes and isolated desmosomal intercellular regions (desmosomal "cores") from the living cell layers of bovine muzzle epidermis. The tissue was disrupted in a nonionic detergent at low pH, sonicated, and the insoluble residue fractionated by differential centrifugation and metrizamide gradient centrifugation. Transmission electron microscopic analyses reveal that a fraction obtained after differential centrifugation is greatly enriched in whole desmosomes that possess intracellular plaques. Metrizamide gradient centrifugation removes most of the plaque material, leaving the intercellular components and the adjoining plasma membranes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis coupled with methods that reveal carbohydrate-containing moieties on gels demonstrate that certain proteins present in whole desmosomes are glycosylated. These glycoproteins are specifically and greatly enriched in the desmosome cores of which they are the principal protein constituents, and thus may function as the intercellular adhesive of the desmosome.

scholarly journals Magnesium ion exerts a central role in the regulation of inhibitory adenosine receptors

1985 ◽  
Vol 229 (1) ◽  
pp. 91-100 ◽  
Author(s):  
S M Yeung ◽  
L H Fossom ◽  
D L Gill ◽  
D M Cooper
Keyword(s):  
Dissociation Constant ◽  
Plasma Membranes ◽  
Alkylating Agents ◽  
Regulatory Protein ◽  
Agonist Binding ◽  
Fat Cell ◽  
Cation Site ◽  
Bivalent Cation ◽  
Cell Plasma ◽  
Highly Sensitive

Guanine nucleotides and Mg2+ differentially regulate agonist binding to adenosine (Ri) receptors in fat-cell plasma membranes. GTP alone decreases binding of the agonist ligand [3H]N6-cyclohexyladenosine (CHA) by increasing the dissociation constant (Kd). Mg2+ alone also decreases [3H]CHA binding, which is associated with a decrease in the number of receptors and in the dissociation constant. In the presence of Mg2+, the effect of GTP is to increase [3H]CHA binding by increasing the total number of receptors. It thus appears that Mg2+ acts specifically at a bivalent-cation site which, with GTP, regulates agonist binding. This putative Mg site is highly sensitive to alkylating agents. Mild treatment with N-ethylmaleimide (NEM) abolishes the characteristic GTP effect on agonist binding in the presence of Mg2+. In addition, the effect of Mg2+ alone is also eliminated. The effect of GTP alone is largely unaltered. Studies of the adenylate cyclase activity indicate that this NEM treatment also abolishes the inhibition of basal activity by adenosine analogues, whereas guanylyl imidodiphosphate inhibition of forskolin-stimulated activity is only slightly impaired at this NEM concentration. These observations indicate that a Mg2+ ‘site’ or ‘component’ is required for the integration of receptor (Ri) occupancy with regulation of catalytic activity (C). The regulatory role of Mg2+ is more demonstrable in receptor-GTP-regulatory-protein (Ri-Ni) interactions than in GTP-regulatory-protein-catalytic-unit (Ni-C) interactions.

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