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 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 The properties and extracellular location of 5′-nucleotidase of the rat fat-cell plasma membrane

1975 ◽  
Vol 146 (3) ◽  
pp. 625-633 ◽  
Author(s):  
A C Newby ◽  
J P Luzio ◽  
C N Hales
Keyword(s):  
Plasma Membrane ◽  
Fat Cells ◽  
Isolated Fat Cells ◽  
Fat Cell ◽  
Cell Plasma Membrane ◽  
Intact Cells ◽  
Low Concentrations ◽  
Cell Plasma ◽  
Atp Analogues ◽  
Extracellular Location

1. A phosphohydrolase specific for 5′-nucleotides was characterized by using a particulate fraction from isolated fat-cells. 2. The activity of intact cells towards 5′-AMP was studied. 3. The activity in either situation had the same KM for AMP (45 muM) and was inhibited by low concentrations of ATP (less than 50 muM), but less potently by the ATP analogues AMP-P(CH2)P(adenylyl (β γ-methylene)diphosphonate) and AMP-P)NH)P (adenylylimidodiphosphate). 4. Homogenization of intact fat-cells caused no increase in activity and at least 85% of the activity was recovered in the particulate preparation. 5. The preparation of fat-cells used in this work was not freely permeable to AMP. 6. The ability of intact fat-cells to hydrolyse AMP implies that 5′-nucleotidase is an ectoenzyme in fat-cells. 7. Concentrations of ATP 100 times lower than intracellular concentrations inhibit the enzyme when added extracellularly to intact fat-cells, implying that this effect is also medicated at the extracellular face of the membrane. 8. Antibodies raised to whole liver cells and whole fat-cells inhibit 5′-nucleotidase in intact cells. 9. Incubation of intact fat-cells with adrenaline (1 mug/ml) or insulin (50 mui.u./ml) failed to alter the KM or Vmax. of the enzyme.

scholarly journals G-proteins of fat-cells Role in hormonal regulation of intracellular inositol 1,4,5-trisphosphate

1986 ◽  
Vol 240 (1) ◽  
pp. 35-40 ◽  
Author(s):  
P J Rapiejko ◽  
J K Northup ◽  
T Evans ◽  
J E Brown ◽  
C C Malbon
Keyword(s):  
Adenylate Cyclase ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Hormonal Regulation ◽  
Alpha Subunit ◽  
Fat Cells ◽  
Fat Cell ◽  
Alpha Subunits ◽  
Inositol 1,4,5 Trisphosphate ◽  
Rat Fat Cells

Pertussis toxin abolishes hormonal inhibition of adenylate cyclase, hormonal stimulation of inositol 1,4,5-trisphosphate accumulation in rat fat-cells, and catalyses the ADP-ribosylation of two peptides, of Mr 39,000 and 41,000 [Malbon, Rapiejko & Mangano (1985) J. Biol. Chem. 260, 2558-2564]. The 41,000-Mr peptide is the alpha-subunit of the G-protein, referred to as Gi, that is believed to mediate inhibitory control of adenylate cyclase by hormones. The nature of the 39,000-Mr substrate for pertussis toxin was investigated. The fat-cell 39,000-Mr peptide was compared structurally and immunologically with the alpha-subunits of two other G-proteins, Gt isolated from the rod outer segments of bovine retina and Go isolated from bovine brain. After radiolabelling in the presence of pertussis toxin and [32P]NAD+, the electrophoretic mobilities of the fat-cell 39,000-Mr peptide and the alpha-subunits of Go and Gt were nearly identical. Partial proteolysis of these ADP-ribosylated proteins generates peptide patterns that suggest the existence of a high degree of homology between the fat-cell 39,000-Mr peptide and the alpha-subunit of Go. Antisera raised against purified G-proteins and their subunits were used to probe immunoblots of purified Gt, Gi, Go, and fat-cell membrane proteins. Although recognizing the 36,000-Mr beta-subunit band of Gt, Gi, Go and a 36,000-Mr fat-cell peptide, antisera raised against Gt failed to recognize either the 39,000- or the 41,000-Mr peptides of fat-cells or the alpha-subunits of Go and Gi. Antisera raised against the alpha-subunit of Go, in contrast, recognized the 39,000-Mr peptide of rat fat-cells, but not the alpha-subunit of either Gi or Gt. These data establish the identity of Go, in addition to Gi, in fat-cell membranes and suggest the possibility that either Go or Gi alone, or both, may mediate hormonal regulation of adenylate cyclase and phospholipase C.

An improved procedure for the purification of plasma membranes from Dictyostelium discoideum

1977 ◽  
Vol 55 (12) ◽  
pp. 1233-1236 ◽  
Author(s):  
N. R. Gilkes ◽  
G. Weeks
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Dictyostelium Discoideum ◽  
Plasma Membranes ◽  
Membrane Preparation ◽  
Preparation Procedure ◽  
Contamination Level ◽  
Marker Enzymes ◽  
Plasma Membrane Preparation

A novel procedure was recently described for the purification of plasma membranes of Dictyostelium discoideum (Gilkes, N. R. &Weeks, G. (1977) Biochim. Biophys. Acta 464, 142–156). Considerable enrichment of plasma membrane marker enzymes was achieved, but since purified mitochondrial and endoplasmic reticulum fractions were unavailable, it was not possible to accurately assess the contamination level of these organelles. We have therefore slightly modified the plasma membrane preparation procedure, improving purification, and have prepared partially purified mitochondrial and endoplasmic reticulum fractions. The data suggest that the contamination of the plasma membranes by endoplasmic reticulum membranes is no greater than 10%, and probably considerably less. No mitochondrial contamination is detectable.

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 Isolation of rat hepatocyte plasma membranes. I. Presence of the three major domains.

1983 ◽  
Vol 96 (1) ◽  
pp. 217-229 ◽  
Author(s):  
A L Hubbard ◽  
D A Wall ◽  
A Ma
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Large Fraction ◽  
Membrane Preparation ◽  
Electron Microscope Autoradiography ◽  
Canalicular Membrane ◽  
Membrane Markers ◽  
Liver Plasma Membrane ◽  
Bile Canalicular Membrane ◽  
Plasma Membrane Preparation

A rat liver plasma membrane preparation was isolated and characterized both biochemically and morphologically. The isolation procedure was rapid, simple and effective in producing a membrane fraction with the following biochemical characteristics: approximately 40-fold enrichment in three plasma membrane markers, 5'-nucleotidase, alkaline phosphodiesterase I (both putative bile canalicular membrane enzymes), and the asialo-glycoprotein (ASGP) receptor (a membrane glycoprotein present along the sinusoidal front of hepatocytes); a yield of each of these plasma membrane markers that averaged approximately 16%; and minimal contamination by lysosomes, nuclei, and mitochondria, but persistent contamination by elements of the endoplasmic reticulum. Morphological analysis of the preparation revealed that all three major domains of the hepatocyte plasma membrane (sinusoidal, lateral, and bile canalicular) were present in substantial amounts. The identification of sinusoidal membrane was further confirmed when ASGP binding sites were localized predominantly to this membrane in the isolated PM using electron microscope autoradiography. By morphometry, the sinusoidal front membrane accounted for 47% of the total membrane in the preparation, whereas the lateral surface and bile canalicular membrane accounted for 6.8% and 23% respectively. This is the first report of such a large fraction of sinusoidal membrane in a liver plasma membrane preparation.

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