D-Glucose uptake and insulin binding by the human adipose cell plasma membrane as a function of its polypeptide composition

1977 ◽  
Vol 55 (2) ◽  
pp. 126-133 ◽  
Author(s):  
Bluma G. Brenner ◽  
Shiro Ozaki ◽  
Norman Kalant ◽  
Arthur Kahlenberg
Keyword(s):  
Plasma Membrane ◽  
Glucose Uptake ◽  
Plasma Membranes ◽  
Binding Capacity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Insulin Binding ◽  
Partial Purification ◽  
Scatchard Analysis ◽  
Molecular Weight Range

A preparation of plasma membranes isolated from human omental lipocytes is composed of about 15 major polypeptide components including three major glycoproteins with an apparent molecular weight range from 100 000 to 23 000, as determined by sodium dodecyl sulfate – polyacrylamide gel electrophoresis. Extraction of this membrane preparation with sodium iodide or 2,3-dimethylmaleic anhydride solubilized 50 and 70% of the membrane protein, respectively, resulting from the extensive extraction of protein from all but two of the major membrane polypeptide components. This removal of protein did not affect the membrane's stereospecific D-glucose-uptake activity but did reduce its total specific [l25I]insulin-binding activity by 46–67%. The binding of [125I]insulin to its specific receptor on lipocyte plasma membranes was detected at physiologic concentrations of the hormone and could be competitively displaced by increasing concentrations of native insulin. The kinetic behaviour of this reaction was approximated by Scatchard analysis, and both the affinity and binding capacity of the plasma membrane for insulin were increased at lower temperatures.These results suggest that D-glucose transport in human adipose tissue is mediated by an intrinsic component of the hydrophobic structure of the lipocyte plasma membrane, and represent a partial purification of this component. In addition, these studies demonstrate and characterize the binding of insulin to the plasma membrane isolated from human lipocytes. A quantitative study of this binding reaction may provide further understanding of the mechanisms underlying the decreased insulin responsiveness characteristic of human diabetes.

Partial purification and properties of the TSH receptors from human thyroid plasma membranes

1983 ◽  
Vol 103 (2) ◽  
pp. 198-204 ◽  
Author(s):  
Yasuhiro Iida ◽  
Junji Konishi ◽  
Kanji Kasagi ◽  
Keigo Endo ◽  
Takashi Misaki ◽  
...  
Keyword(s):  
Sodium Acetate ◽  
Plasma Membranes ◽  
Binding Capacity ◽  
Human Thyroid ◽  
Partial Purification ◽  
Affinity Column ◽  
Scatchard Analysis ◽  
Dependent Manner ◽  
Con A ◽  
Triton X

Abstract. Human thyroid plasma membranes were solubilized with 0.5% Triton X-100 and TSH receptors were purified by using DEAE-Sephadex, Con A and TSH affinity chromatography. A TSH binding activity was bound to DEAE-Sephadex equilibrated with 0.05 m sodium acetate, pH 6.3, 0.2% Triton X-100 and was eluted by a linear gradient of 0.1 m to 1.0 m ammonium acetate, pH 6.3. Eighty-five per cent of the activity was absorbed to Con A Sepharose and was eluted with 0.5 m α-methyl-D-mannoside, 0.05 m sodium acetate, pH 6.0. Seventy-five per cent of the TSH binding capacity could be absorbed to TSH-affinity column and was eluted with 0.1 m glycine-HCl, pH 3.0. By sequential application of the above procedures, more than 100-fold purification of the receptor activity was attainable. [125I]TSH binding of this fraction was inhibited by addition of unlabelled TSH in a dose-dependent manner. Scatchard analysis gave a curvilinear plot with a high affinity association constant of 0.72 × 109 m−1. By using Ultrogel AcA 34 gel filtration, the molecular size of the hormonereceptor complex was estimated to be 180 000.

Stimulation of glucose transport by thyroid hormone in 3T3-L1 adipocytes: increased abundance of GLUT1 and GLUT4 glucose transporter proteins

2000 ◽  
Vol 164 (2) ◽  
pp. 187-195 ◽  
Author(s):  
R Romero ◽  
B Casanova ◽  
N Pulido ◽  
AI Suarez ◽  
E Rodriguez ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Glucose Uptake ◽  
Glucose Transport ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Fold Increase ◽  
Glucose Transporters ◽  
Insulin Binding ◽  
Total Protein Content ◽  
Glucose Transporter Proteins

In 3T3-L1 adipocytes we have examined the effect of tri-iodothyronine (T(3)) on glucose transport, total protein content and subcellular distribution of GLUT1 and GLUT4 glucose transporters. Cells incubated in T(3)-depleted serum were used as controls. Cells treated with T(3) (50 nM) for three days had a 3.6-fold increase in glucose uptake (P<0.05), and also presented a higher insulin sensitivity, without changes in insulin binding. The two glucose carriers, GLUT1 and GLUT4, increased by 87% (P<0.05) and 90% (P<0. 05), respectively, in cells treated with T(3). Under non-insulin-stimulated conditions, plasma membrane fractions obtained from cells exposed to T(3) were enriched with both GLUT1 (3. 29+/-0.69 vs 1.20+/-0.29 arbitrary units (A.U.)/5 microg protein, P<0.05) and GLUT4 (3.50+/-1.16 vs 0.82+/-0.28 A.U./5 microg protein, P<0.03). The incubation of cells with insulin produced the translocation of both glucose transporters to plasma membranes, and again cells treated with T(3) presented a higher amount of GLUT1 and GLUT4 in the plasma membrane fractions (P<0.05 and P<0.03 respectively). These data indicate that T(3) has a direct stimulatory effect on glucose transport in 3T3-L1 adipocytes due to an increase in GLUT1 and GLUT4, and by favouring their partitioning to plasma membranes. The effect of T(3) on glucose uptake induced by insulin can also be explained by the high expression of both glucose transporters.

Downregulation of surface sodium pumps by endocytosis during meiotic maturation of Xenopus laevis oocytes

1990 ◽  
Vol 258 (1) ◽  
pp. C179-C184 ◽  
Author(s):  
G. Schmalzing ◽  
P. Eckard ◽  
S. Kroner ◽  
H. Passow
Keyword(s):  
Plasma Membrane ◽  
Xenopus Laevis ◽  
Sucrose Gradient ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sodium Dodecyl ◽  
Meiotic Maturation ◽  
Xenopus Laevis Oocytes ◽  
Sodium Pumps

During meiotic maturation, plasma membranes of Xenopus laevis oocytes completely lose the capacity to transport Na and K and to bind ouabain. To explore whether the downregulation might be due to an internalization of the sodium pump molecules, the intracellular binding of ouabain was determined. Selective permeabilization of the plasma membrane of mature oocytes (eggs) by digitonin almost failed to disclose ouabain binding sites. However, when the eggs were additionally treated with 0.02% sodium dodecyl sulfate (SDS) to permeabilize inner membranes, all sodium pumps present before maturation were recovered. Phosphorylation by [gamma-32P]ATP combined with SDS-polyacrylamide gel electrophoresis (PAGE) and autoradiography showed that sodium pumps were greatly reduced in isolated plasma membranes of eggs. According to sucrose gradient fractionation, maturation induced a shift of sodium pumps from the plasma membrane fraction to membranes of lower buoyant density with a protein composition different from that of the plasma membrane. Endocytosed sodium pumps identified on the sucrose gradient from [3H]ouabain bound to the cell surface before maturation could be phosphorylated with inorganic [32P]phosphate. The findings suggest that downregulation of sodium pumps during maturation is brought about by translocation of surface sodium pumps to an intracellular compartment, presumably endosomes. This contrasts the mechanism of downregulation of Na-dependent cotransport systems, the activities of which are reduced as a consequence of a maturation-induced depolarization of the membrane without a removal of the corresponding transporter from the plasma membrane.

scholarly journals Nonidet P-40 extraction of lymphocyte plasma membrane. Characterization of the insoluble residue

1984 ◽  
Vol 219 (1) ◽  
pp. 301-308 ◽  
Author(s):  
A A Davies ◽  
N M Wigglesworth ◽  
D Allan ◽  
R J Owens ◽  
M J Crumpton
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fold Increase ◽  
Insoluble Fraction ◽  
Insoluble Residue ◽  
Protein Ratio

Purified preparations of lymphocyte plasma membrane were extracted exhaustively with Nonidet P-40 in Dulbecco's phosphate-buffered saline medium. The insoluble fraction, as defined by sedimentation at 10(6) g-min, contained about 10% of the membrane protein as well as cholesterol and phospholipid. The lipid/protein ratio, cholesterol/phospholipid ratio and sphingomyelin content were increased in the residue. Density-gradient centrifugation suggested that the lipid and protein form a common entity. As judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the Nonidet P-40-insoluble fractions of the plasma membranes of human B lymphoblastoid cells and pig mesenteric lymph-node lymphocytes possessed similar qualitative polypeptide compositions but differed quantitatively. Both residues comprised major polypeptides of Mr 28 000, 33 000, 45 000 and 68 000, together with a prominent band of Mr 120 000 in the human and of Mr 200 000 in the pig. The polypeptides of Mr 28 000, 33 000, 68 000 and 120 000 were probably located exclusively in the Nonidet P-40-insoluble residue, which also possessed a 4-fold increase in 5′-nucleotidase specific activity. The results indicate that a reproducible fraction of lymphocyte plasma membrane is insoluble in non-ionic detergents and that this fraction possesses a unique polypeptide composition. By analogy with similar studies with erythrocyte ghosts, it appears likely that the polypeptides are located on the plasma membrane's cytoplasmic face.

scholarly journals Isolation and electrophoretic characterization of the plasma membrane of sea-urchin sperm

1983 ◽  
Vol 59 (1) ◽  
pp. 13-25
Author(s):  
N.L. Cross
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Blue Staining ◽  
Coomassie Blue Staining ◽  
Electrophoretic Patterns ◽  
Sperm Plasma Membrane

A subcellular fraction containing plasma membranes was isolated from flagella of the sperm of Strongylocentrotus purpuratus by differential centrifugation, and analysed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. Coomassie Blue staining revealed nine major bands and 14 minor species. Five bands of apparent molecular weights approximately 200 X 10(3), 149 X 10(3), 120 X 10(3), 75 X 10(3) and 59 X 10(3) also stained with periodic acid-Schiff's reagent and so are probably glycoproteins. These five components are externally exposed, as determined by lactoperoxidase-catalysed radio-iodination. Isolation of membranes from radio-iodinated sperm results in an enrichment of about tenfold in the specific activity of 125I. Comparison of the electrophoretic patterns of labelled sperm and of the membranes isolated from 125I-labelled sperm suggests that no major labelled proteins are lost during the isolation procedure, and so to this extent the membrane fraction is representative of the entire sperm plasma membrane.

scholarly journals Isolation of microvillus plasma membranes from suckling-rat intestine. The influence of premature induction of digestive enzymes by injection of cortisol acetate

1974 ◽  
Vol 144 (2) ◽  
pp. 293-302 ◽  
Author(s):  
G Galand ◽  
G G Forstner
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Hydrolytic Enzymes ◽  
Rat Plasma ◽  
Suckling Rats ◽  
Induction Of Enzymes ◽  
Old Rats ◽  
Membrane Changes

1. Cortisone administration to suckling rats leads prematurely to induction of enzymes of the intestinal microvillus plasma membrane and lengthening of the intestinal microvilli. To investigate the membrane changes that might be involved, a method for the isolation of a fraction enriched with microvillus plasma membrane was developed in suckling rats. Plasma-membrane fractions were compared from 13-day-old control rats and from 13-day-old rats given cortisol acetate by subcutaneous injection for 3 days. 2. After cortisol injection, the activity of maltase, trehalase, sucrase and leucyl β-naphthylamidase increased markedly, and to the same extent, in intestinal homogenates and plasma-membrane preparations. Purification, and recovery of five marker enzymes with respect to homogenate activity, and recovery of protein, were similar for both membrane preparations, particularly after correction for non-membrane activity, which was high in suckling rats and affected by cortisol. 3. In material released from the plasma membrane by digestion with papain, maltase protein was increased after cortisol injection at least as much as maltase activity. Sucrase activity increased at least 200-fold, and this increase was associated with the appearance of a new sucrase band on polyacrylamide-gel electrophoresis. 4. Sodium dodecyl sulphate electrophoresis of plasma-membrane proteins revealed at least four additional macromolecules after cortisol injection. Concurrently several proteins disappeared from the plasma membrane. The added proteins appeared in the main to be removed from the plasma membrane by papain, whereas the deleted proteins were in the papain-resistant fraction. 5. Enzymic stimulation induced by cortisol acetate in the suckling-rat plasma membrane therefore appears to involve the addition of new proteins, rather than activation of proteins in situ. Deletion of proteins from the membrane during induction of hydrolytic enzymes may reflect other phenomena such as protein reorganization associated with the change in microvillus shape.

Characterization of a common VIP-PACAP receptor in human small intestinal epithelium

1993 ◽  
Vol 264 (2) ◽  
pp. E294-E300 ◽  
Author(s):  
R. Salomon ◽  
A. Couvineau ◽  
C. Rouyer-Fessard ◽  
T. Voisin ◽  
D. Lavallee ◽  
...  
Keyword(s):  
Adenylyl Cyclase ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Growth ◽  
Receptor Site ◽  
Cyclase Activity ◽  
Scatchard Analysis ◽  
Adenylyl Cyclase Activity ◽  
Vip Receptors

Vasoactive intestinal peptide (VIP) receptors were characterized in epithelial plasma membranes from human small intestine. Native VIP inhibited the binding of 125I-labeled VIP to jejunal membranes, and Scatchard analysis of these data was consistent with the existence of one class of receptor with a dissociation constant of 42 pM and a maximal binding of 256 fmol/mg membrane protein. VIP stimulated adenylyl cyclase activity in human jejunal membranes in the 0.01 nM-1 microM range [half-maximal effective dose = 0.7 nM]. Coupling of VIP receptors with a Gs protein was further assessed by the ability of GTP (10(-8) to 10(-3) M) to inhibit 125I-VIP binding to membranes. 125I-VIP binding was seven to eight times higher in villus cells than in crypt cells. Finally, 125I-VIP binding was detectable throughout the small and large intestines with the highest binding in jejunum. Among the natural peptides structurally related to VIP, some inhibited 125I-VIP binding with the following order of potency: VIP = pituitary adenylate cyclase-activating peptide (PACAP)-27 = PACAP-38 > helodermin >> peptide histidine methionineamide = human growth hormone-releasing factor > secretin. The same order of potency of peptides for inhibiting 125I-VIP or 125I-labeled PACAP was observed, supporting that the two tracers bound to a common VIP-PACAP receptor site. This order of potency was also observed for the stimulation of adenylyl cyclase activity by these peptides. 125I-VIP was cross-linked to membranes using disuccinimidyl suberate. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, one single band of 70,000 mol wt was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Reconstitution of D-glucose transporter from human placental microvillous plasma membranes

1982 ◽  
Vol 242 (3) ◽  
pp. C166-C171 ◽  
Author(s):  
J. M. Bissonnette ◽  
J. A. Black ◽  
K. L. Thornburg ◽  
K. M. Acott ◽  
P. L. Koch
Keyword(s):  
Glucose Uptake ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Cytochalasin B ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Extraction Procedure ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Human Trophoblast

Proteins from microvillous plasma membrane vesicles of the maternal surface of human trophoblast were solubilized with octyl beta-D-glucoside. After incorporation of the soluble protein into phospholipid liposomes D-glucose uptake exceeded that of L-glucose. The reconstituted system showed that D-glucose uptake was not sodium dependent and was inhibited by cytochalasin B. Efflux of D-glucose from the proteoliposomes was retarded by cytochalasin B. D-Glucose uptake, but not L-glucose, was proportional to the amount of protein used in the reconstitution procedure. Membrane protein was also solubilized with octylglucoside from vesicles that had been extracted previously by dimethylmaleic anhydride. Proteoliposomes prepared from these latter proteins showed D-glucose uptake threefold greater than that from octylglucoside solubilization alone, but sodium dodecyl sulfate polyacrylamide gel electrophoresis of the extracted protein showed no clear difference between the double extraction procedure and the pattern obtained with the single detergent.

scholarly journals Molecular Characterization of the Plasma Membrane H+-ATPase, an Antifungal Target inCryptococcus neoformans

2000 ◽  
Vol 44 (9) ◽  
pp. 2349-2355 ◽  
Author(s):  
Patricia Soteropoulos ◽  
Tanya Vaz ◽  
Rosaria Santangelo ◽  
Padmaja Paderu ◽  
David Y. Huang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Molecular Mass ◽  
Plasma Membranes ◽  
Atp Hydrolysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Proton Pumps ◽  
Gene Encoding ◽  
Reading Frame ◽  
Whole Cells

ABSTRACT The Cryptococcus neoformans PMA1 gene, encoding a plasma membrane H+-ATPase, was isolated from a genomic DNA library of serotype A strain ATCC 6352. An open reading frame of 3,380 nucleotides contains six introns and encodes a predicted protein consisting of 998 amino acids with a molecular mass of approximately 108 kDa. Plasma membranes were isolated, and the H+-ATPase was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be slightly larger than the S. cerevisiaeH+-ATPase, consistent with its predicted molecular mass. The plasma membrane-bound enzyme exhibited a pH 6.5 optimum for ATP hydrolysis, Km and V maxvalues of 0.5 mM and 3.1 μmol mg−1 min−1, respectively, and an apparent Ki for vanadate inhibition of 1.6 μM. ATP hydrolysis in plasma membranes and medium acidification by whole cells were inhibited by ebselen, a nonspecific H+-ATPase antagonist which was also fungicidal. The predicted C. neoformans protein is 35% identical to proton pumps of both pathogenic and nonpathogenic fungi but exhibits more than 50% identity to PMA1 genes from plants. Collectively, this study provides the basis for establishing the CryptococcusH+-ATPase as a viable target for antifungal drug discovery.

scholarly journals Studies on rat liver plasma membrane. Altered protein and phospholipid metabolism after injection of d-galactosamine

1977 ◽  
Vol 166 (3) ◽  
pp. 455-462 ◽  
Author(s):  
W Bachmann ◽  
E Harms ◽  
B Hassels ◽  
H Henninger ◽  
W Reuitter
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Composition ◽  
Phospholipid Metabolism ◽  
Morphological Alterations ◽  
Galactosamine Hepatitis

1. The metabolism of protein and phospholipid in rat liver plasma membranes isolated by the method of Neville [(1960) J. Biophys. Biochem. Cytol. 8, 413-422] was investigated 3 and 6 h after the injection of D-galactosamine in vivo. During this time, all the biochemical and morphological alterations associated with hepatitis developed. 2. After the injection of D-galactosamine the concentration of sphingomyelin in the plasma membrane decreased to below 60% of the control values. 3. The activity of 5′-nucleotidase (EC 3.1.3.5), which has been purified as a sphingomyelin-protein complex, decreased in the total homogenate as well as in the plasma-membrane fraction of livers of rats treated with galactosamine, to about 60% of the control values. 4. Protein synthesis, as measured by the incorporation of [14C]leucine into plasma membranes, was decreased to 45% of that of the controls. However, only small differences were observed in the amino acid composition of the plasma membrane after D-galactosamine treatment. 5. The protein composition of the plasma membranes was determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The results showed a change from low- to high-molecular-weight proteins after the injection of galactosamine. 6. These results demonstrate different metabolic processes of the plasma membrane altered during the induction of galactosamine hepatitis.

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