scholarly journals Identification of functional insulin receptors on membranes from an insulin-producing cell line (RINm5F)

1985 ◽  
Vol 226 (3) ◽  
pp. 867-872 ◽  
Author(s):  
H Gazzano ◽  
P Halban ◽  
M Prentki ◽  
R Ballotti ◽  
D Brandenburg ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Cell Line ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Insulin Receptors ◽  
Target Cells ◽  
Pancreatic Cell ◽  
Rinm5f Cell ◽  
Receptor Complexes ◽  
Reducing Conditions

Insulin receptors on RINm5F cell membranes (an insulin-producing rat pancreatic cell line) were studied. To study the insulin receptor alpha-subunit, 125I-labelled photoreactive insulin was covalently bound to the membranes in the absence or presence of unlabelled insulin. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under reducing conditions showed specific labelling of an Mr 130 000 protein. The receptor beta-subunit was studied by using a cell-free phosphorylation assay. Analysis under reducing conditions showed a phosphoprotein of Mr 95 000 whose level of phosphorylation was selectively increased by insulin, and which was specifically immunoprecipitated by antibodies to the insulin receptor. Further, covalent hormone-receptor complexes purified with anti-insulin antibodies were able to undergo autophosphorylation, indicating the existence of operational receptor subunit arrangements. RINm5F cell insulin receptors (and, by analogy, possibly those of native B-cells) thus display structural and functional integrity comparable with those of conventional insulin target cells.

Effect of exercise on insulin receptor binding and kinase activity in skeletal muscle

1989 ◽  
Vol 256 (1) ◽  
pp. E138-E144 ◽  
Author(s):  
J. L. Treadway ◽  
D. E. James ◽  
E. Burcel ◽  
N. B. Ruderman
Keyword(s):  
Skeletal Muscle ◽  
Insulin Receptor ◽  
Insulin Action ◽  
White Muscle ◽  
Kinase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Similar Data

Insulin action in skeletal muscle is markedly enhanced for several hours after an acute bout of exercise. The purpose of this study was to examine the possible involvement of the intrinsic tyrosine kinase activity of the insulin receptor in mediating these effects. Red and white muscles were removed from rats either at rest or following a treadmill run (45 min at 18 m/min), and insulin receptors were isolated in partially purified form. Basal and insulin-stimulated receptor kinase activity was higher in red than in white muscle, in agreement with previous studies (J. Biol. Chem. 261: 14939-14944, 1986). There was no effect of exercise on insulin binding, basal and insulin-stimulated receptor autophosphorylation, or basal and insulin-stimulated exogenous kinase activity, in either red or white muscle. Similar data were obtained when phosphatase inhibitors were used during receptor isolation. The structure of insulin receptors isolated from the muscle of exercised and control rats was similar as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of affinity cross-linked insulin receptors. We conclude that enhanced insulin action in muscle during the postexercise state is not related to increased kinase activity of the insulin receptor.

Identification of the insulin receptor of cerebral microvessels

1985 ◽  
Vol 248 (1) ◽  
pp. E115-E125 ◽  
Author(s):  
J. F. Haskell ◽  
E. Meezan ◽  
D. J. Pillion
Keyword(s):  
Molecular Weight ◽  
Insulin Receptor ◽  
Sodium Dodecyl ◽  
Binding Constants ◽  
Insulin Receptors ◽  
Alpha Subunit ◽  
Scatchard Analysis ◽  
Cerebral Microvessels ◽  
Reducing Conditions ◽  
Binding Data

Cerebral microvessels are known to possess receptors for insulin and have recently been shown to respond to physiological levels of this hormone. Scatchard analysis of binding data obtained with isolated cerebral microvessels gave curvilinear plots and showed that neonatal porcine cerebral microvessels have a greater number of insulin receptors per unit of protein than adult bovine cerebral microvessels. The high-affinity form of the insulin receptors of both neonatal porcine and adult bovine cerebral microvessels have similar binding constants (dissociation constant = 0.3 X 10(-9) M). Dissociation of 125I-insulin from cerebral microvessels was accelerated by the presence of unlabeled insulin in preparations from both neonatal pigs and adult cows. 125I-insulin was covalently cross-linked to its receptor in cerebral microvessels with disuccinimidyl suberate, and the hormone-receptor complex was isolated on sodium dodecyl sulfate-polyacrylamide gels. Under reducing conditions, 125I-insulin was found associated with a polypeptide with a molecular weight of 130,000, which is indistinguishable from the alpha-subunit of the liver insulin receptor. In contrast, nonvascular cerebral cortical tissue contained an insulin receptor with an alpha-subunit that was lower in molecular weight than the form isolated from cerebral cortical microvessels.

scholarly journals Structure of covalent insulin-receptor complexes (I-S-S-R) in isolated rat adipocytes and human placental membranes

1985 ◽  
Vol 229 (2) ◽  
pp. 513-519 ◽  
Author(s):  
S Clark ◽  
L C Harrison
Keyword(s):  
Insulin Receptor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Single Species ◽  
Buffer System ◽  
Rat Adipocytes ◽  
Receptor Complexes ◽  
Placental Membranes ◽  
Triton X ◽  
Binding Subunit

The structure of naturally-formed covalent disulphide-linked complexes between insulin and its receptor was examined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. To prevent destabilization of disulphide bonds at alkaline pH the standard discontinuous electrophoresis conditions were changed to a continuous buffer system at pH 7.0. 125I-insulin was first bound to either rat adipocytes or human placental membranes for 10 min at 37 degrees C. After washing, non-dissociable radioactivity was extracted from cells or membranes in Triton X-100 and immunoprecipitated with an antiserum (B-2) to the insulin receptor. Electrophoresis of the immune precipitate revealed the two smaller of the three reported species of native insulin receptor (Mr values approx. 350 000, 290 000 and 260 000); in addition, a species of Mr 200 000 was also frequently observed in adipocytes. When non-dissociable 125I-insulin was chemically crosslinked to adipocytes or placental membranes, prior to solubilization and immunoprecipitation, all three species of the native receptor were labelled; after reduction, only a single species of Mr 130000 was observed. These findings indicate that disulphide exchange of insulin occurs with the Mr 130000 (alpha) binding subunit within partially reduced species of the native, oligomeric receptor. The degree of disulphide binding of insulin could therefore depend on the relative abundance of partially reduced receptor species and on the redox state of the cell membrane.

scholarly journals Fc gamma R-dependent mitogen-activated protein kinase activation in leukocytes: a common signal transduction event necessary for expression of TNF-alpha and early activation genes.

1996 ◽  
Vol 184 (3) ◽  
pp. 1027-1035 ◽  
Author(s):  
R Trotta ◽  
P Kanakaraj ◽  
B Perussia
Keyword(s):  
Nk Cells ◽  
Cell Line ◽  
Kinase Inhibitor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mitogen Activated Protein Kinase ◽  
Monocytic Cell ◽  
Fast Kinetics ◽  
Mitogen Activated Protein ◽  
Stimulation Of

Cross-linking the receptors for the Fc domain of IgG (Fc gamma R) on leukocytes induces activation of protein tyrosine kinases. The intermediary molecules that transduce to the nucleus the signals leading to induction of the diverse biological responses mediated by these receptors are not clearly identified. We have investigated whether mitogen-activated protein kinases (MAPK) are involved in transmembrane signaling via the three Fc gamma R present on monocytic, polymorphonuclear, and natural killer (NK) cells. Our results indicate that occupancy of Fc gamma RI and Fc gamma RII on the monocytic cell line THP-I and on polymorphonuclear leukocytes (PMN) induces, transiently and with fast kinetics, MAPK phosphorylation, as indicated by decreased electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and increased amounts of the proteins in antiphosphotyrosine antibody immunoprecipitates. This, associated with increased enzymatic activity, also occurs upon stimulation of the transmembrane isoform of CD16 (Fc gamma RIIIA) in NK cells and in a T cell line expressing transfected Fc gamma RIIIA alpha ligand-binding chain in association with zeta, but not upon stimulation of the glycosil-phosphatidylinositol-anchored Fc gamma RIIIB on PMN. Using the specific MAP kinase kinase inhibitor-PD 098059, we show that activation of MAPK is necessary for the Fc gamma R-dependent induction of c-fos and tumor necrosis factor alpha mRNA expression in monocytes and NK cells. These results underscore the role of MAPK as signal-transducing molecules controlling the expression of different genes relevant to leukocyte biology upon Fc gamma R stimulation.

scholarly journals Impaired secretion of mutant alpha 2-plasmin inhibitor (alpha 2 PI- Nara) from COS-7 and HepG2 cells: molecular and cellular basis for hereditary deficiency of alpha 2-plasmin inhibitor

Blood ◽  
1990 ◽  
Vol 75 (5) ◽  
pp. 1092-1096 ◽  
Author(s):  
O Miura ◽  
N Aoki
Keyword(s):  
Cell Line ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Hepatoma Cell ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Secretory Process ◽  
Hepatoma Cell Line ◽  
Pi Deficiency ◽  
Plasmin Inhibitor

Abstract The elongated mutant of alpha 2-plasmin inhibitor (alpha 2 PI) designated as alpha 2 PI-Nara is caused by a frameshift mutation found near the 3′ end of the coding region of the alpha 2 PI gene. To elucidate the mechanism by which this molecular abnormality leads to alpha 2 PI deficiency in plasma, we transfected an expression plasmid for alpha 2 PI-Nara into a monkey kidney cell line COS-7 or human hepatoma cell line HepG2 synthesizing alpha 2 PI, and analyzed the secretory process of the expressed alpha 2 PI-Nara by radioimmunoprecipitation followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography. The results obtained showed that the recombinant alpha 2 PI-Nara was retained within the cells for prolonged periods as an endoglycosidase H- sensitive precursor form, and only a small portion of the recombinant protein was secreted into the medium as a neuraminidase-sensitive mature form. These results suggest that instead of being secreted from the cells, most of the alpha 2 PI-Nara undergoes degradation within the cells while its transport is retarded in the intracellular secretory pathway; thus, alpha 2 PI-Nara should lead to the alpha 2 PI deficiency primarily by causing a block in the intracellular transport from the endoplasmic reticulum to the Golgi complex.

Atrial natriuretic peptide receptors in renal papilla of DOCA-salt hypertensive rats

1990 ◽  
Vol 259 (1) ◽  
pp. F130-F137
Author(s):  
E. Nuglozeh ◽  
G. Gauquelin ◽  
R. Garcia ◽  
J. Tremblay ◽  
E. L. Schiffrin
Keyword(s):  
Atrial Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Plasma Renin ◽  
Renal Papilla ◽  
Hypertensive Rats ◽  
Reducing Conditions ◽  
Anp Receptors ◽  
Atrial Natriuretic

The receptor for atrial natriuretic peptide (ANP) in the rat renal papilla was characterized pharmacologically. After solubilization and irreversible binding with disuccinimidylsuberate, it was shown on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) to be made of a single peptide of 125 kDa. The regulation of the renal papillary ANP receptor was studied in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. DOCA-salt rats had suppressed plasma renin activity and increased plasma ANP concentrations (408 +/- 35 vs. 133 +/- 12 pg/ml in uninephrectomized controls, P less than 0.01). The renal papilla was hypertrophied in DOCA-salt hypertensive rats (93 +/- 1 vs. 52 +/- 1 mg, P less than 0.01). The density of ANP sites in the papilla was significantly higher in DOCA-salt rats (141 +/- 31 fmol/papilla) than in controls (34 +/- 8 fmol/papilla, P less than 0.01). Affinity of sites in DOCA-salt rats and controls was similar. The production of guanosine 3',5'-cyclic monophosphate (cGMP) in renal papilla in response to ANP was significantly higher in DOCA-salt rats. In contrast to the renal papillary ANP receptor, acid-washed vascular and glomerular ANP sites were significantly decreased in density in DOCA-salt hypertensive rats. In blood vessels and glomeruli, both the high- and low-molecular mass receptor (as detected on SDS-PAGE under reducing conditions) was proportionately decreased in density in DOCA-salt hypertensive rats. The present results suggest that an increased number of ANP receptors and exaggerated cGMP response to ANP in the renal papilla may underlie the increased natriuretic responsiveness of the kidney to ANP in DOCA-salt hypertensive rats.

scholarly journals Biochemical characterization and purification of HILDA, a human lymphokine active on eosinophils and bone marrow cells

Blood ◽  
1988 ◽  
Vol 71 (6) ◽  
pp. 1618-1623 ◽  
Author(s):  
A Godard ◽  
H Gascan ◽  
J Naulet ◽  
MA Peyrat ◽  
Y Jacques ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Biochemical Characterization ◽  
Bone Marrow Cells ◽  
Cell Clone ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Pure Material ◽  
T Cell Clone ◽  
Sds Page

Abstract We previously described a lymphokine termed HILDA (for human interleukin DA) produced by T-lymphocyte alloreactive clones after antigenic stimulation. This factor sustains the growth of a murine IL3- sensitive cell line (DA2). In addition, HILDA is a potent activator of eosinophils and displays a burst-promoting activity on human bone marrow. In the present study, HILDA was purified to homogeneity from T- cell clone supernatant using successively sequential concentration, concanavalin A (ConA) affinity chromatography with differential elution (alpha-D glucopyranoside and alpha-D mannopyranoside), high-performance liquid chromatography (HPLC) gel filtration and reverse-phase HPLC. The pure material appeared as a 38-kd glycoprotein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or nonreducing conditions. Biologic activity could be recovered from SDS- PAGE gel slices corresponding to the 38-kd band. We conclude from the specificity of the DA-2 cell line and biochemical characteristics described that this lymphokine is different from other known factors produced by human T lymphocytes.

Glutathione-s-transferase is an important antigen in the eel nematode Anguillicola crassus

1997 ◽  
Vol 71 (4) ◽  
pp. 319-324 ◽  
Author(s):  
M.E. Nielsen ◽  
K. Buchmann
Keyword(s):  
Specific Antibody ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Anguilla Anguilla ◽  
European Eel ◽  
Glutathione S Transferase ◽  
Elisa System ◽  
Humoral Immune ◽  
Anguillicola Crassus ◽  
Reducing Conditions

AbstractDifferent organs and secretions/excretions of the swimbladder parasite, Anguillicola crassus (Nematoda), were tested for the presence of antigens to the humoral immune response previously detected in the European eel, Anguilla anguilla. Proteins from different fractions of Anguillicola crassus were separated using SDS–PAGE (sodium-dodecyl-sulphate polyacrylamide-gel electrophoresis) under reducing conditions and electroblotted onto nitrocellulose membranes. Infected eels showed a specific antibody response to a 43 kDa antigen in the cuticle and towards two gonad antigens around 34 and 43 kDa. In protein released from the worms, two secretory/excretory antigens of approximately 28 kDa were found. The secretion/excretion rate of protein from the parasite to the surroundings was determined. Subsequently, an ELISA system was established applying these antigens as the first layer of coating. Furthermore, antigens from Anguillicola crassus were examined for the presence of glutathione-s-transferase (GST) using a specific antibody against GST. The antigens were found to be subunits of GST.

scholarly journals Immunoprecipitation of the lutropin receptor. Loss of receptor molecules during down-regulation

1984 ◽  
Vol 224 (2) ◽  
pp. 467-471 ◽  
Author(s):  
M K Metsikkö ◽  
H J Rajaniemi
Keyword(s):  
Gamma Globulin ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Down Regulation ◽  
Gonadotropin Receptor ◽  
Reducing Conditions ◽  
Lutropin Receptor ◽  
Rat Ovary ◽  
Triton X ◽  
Receptor Antibodies

Specific anti-(lutropin receptor) antibodies were produced by immunizing rabbits with lutropin receptor purified from pseudopregnant rat ovary. The anti-receptor serum at 1:100 dilution together with anti-(rabbit gamma-globulin) serum immunoprecipitated 70% of 3H-labelled, purified lutropin receptor and 42% of 125I-chorio-gonadotropin-receptor complex. The antiserum inhibited hormone binding to rat ovarian particles. Pseudopregnant rat ovarian particles were labelled with periodate/NaB3H4 and solubilized with Triton X-100. The Triton X-100 extract was subjected to immunoprecipitation using the anti-receptor serum. When the immunoprecipitate was dissolved and analysed by polyacrylamide-gel electrophoresis in sodium dodecyl sulphate under reducing conditions followed by fluorography, a receptor polypeptide with an apparent Mr 95000 was detected. A receptor down-regulating dose of choriogonadotropin was injected into pseudopregnant rats and their ovaries were removed and homogenized 4 days later, and analysed for immunoprecipitable receptors as above. No receptor molecules were found. Accordingly, the lutropin receptor molecules actually disappear rather than merely become masked from hormone during homologous down-regulation.

Insulin Glulisine—A Comprehensive Preclinical Evaluation

2006 ◽  
Vol 25 (1) ◽  
pp. 25-33 ◽  
Author(s):  
Ingo Stammberger ◽  
Gerhard Seipke ◽  
Thomas Bartels
Keyword(s):  
Dna Synthesis ◽  
Insulin Receptor ◽  
Cell Line ◽  
Binding Affinity ◽  
Human Insulin ◽  
Receptor Binding ◽  
Insulin Receptors ◽  
Preclinical Evaluation ◽  
Receptor Binding Affinity ◽  
Insulin Glulisine

Receptor binding and signaling and the mitogenic potential of insulin glulisine (glulisine), regular human insulin (RHI), and Asp(B10) were compared in vivo and in vitro. Insulin and insulin-like growth factor 1 (IGF-1) receptor binding was studied with human insulin receptors (293HEK cells) and the human osteosarcoma-derived cell line B10. Insulin receptor–mediated signaling was assessed in rat-1 fibroblasts overexpressing insulin receptors. Activation of insulin receptor substrates 1 and 2 (IRS-1/IRS-2) was studied in rat and human myoblasts and rat cardiomyocytes. DNA synthesis induction was assessed by [3H] thymidine incorporation in the human epithelial breast cell line MCF10. Interaction with the IGF-1 receptor, DNA synthesis, and intracellular signal transduction were assessed in cardiac K6 myoblasts. Immunohistochemical examination of Sprague-Dawley rat tissue treated with glulisine for 6 months ( n = 40), and glulisine and RHI for 12 months ( n = 60), was performed. Steady-state insulin receptor binding affinity was slightly lower for glulisine versus RHI (~0.70). IGF-1 receptor binding affinity was lower (four-to fivefold) for glulisine, but significantly higher (four-fold) for Asp(B10) versus RHI. Glulisine, Asp(B10), and RHI showed similar insulin receptor–association kinetics; however, Asp(B10) revealed increased insulin receptor affinity. Glulisine and RHI showed similar insulin receptor–mediated phosphorylation and IRS-2 activation. Activation of IRS-1 was 6- to 10-fold lower with glulisine; glulisine was less potent and Asp(B10) slightly more potent in stimulating DNA synthesis versus RHI. Stimulation of DNA synthesis was comparable for glulisine and RHI in K6 myoblasts. At 12 months, there was no significant difference between glulisine and RHI in proliferative activity. This preclinical evaluation suggests that structural changes in glulisine versus RHI are not associated with any safety issues.

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