The rat liver insulin receptor

Romovacek, Hana; Titus, Gail; Ridge, Kevin D.; Raffensperger, John A.; Finn, Frances M.

doi:10.1021/bi00397a028

Cited by 22 publications

(14 citation statements)

References 18 publications

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“…Na Bl_(6-biotinylamido)hexanoyl-Na.A1,B 9-bis(t-butyl-oxycarbonyl)insulin, succinoylavidin-Sepharose 4B, and wheat germ agglutinin-Sepharose 4B were prepared as described (2 ,uCi/mg (1 Ci = 37 GBq). An equal volume of NaDodSO4 (4%) in sodium phosphate buffer (50 mM) at pH 7.0 containing dithiothreitol (0.2 M) was added, and the solution was heated at 100°C for 2 min.…”

Section: Methodsmentioning

confidence: 99%

“…A wheat germ agglutinin eluate was prepared in the usual manner (2). The ligand NaB1_(6-biotinylamido)hexanoylNaA1,NEB -bis(t-butyloxycarbonyl)-insulin was used as the affinity ligand (2).…”

Section: Methodsmentioning

confidence: 99%

“…The binding of '25I-labeled insulin was determined by the polyethylene glycol assay (4) as described (5), and the results were subjected to Scatchard (6) (2). Samples were analyzed by NaDodSO4/PAGE as described by Laemmli (8) with a 5% stacking and a 7.5% resolving gel.…”

Section: Methodsmentioning

confidence: 99%

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ATP sensitizes the insulin receptor to insulin.

Ridge

Finn²

1988

Proc. Natl. Acad. Sci. U.S.A.

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Insulin receptor with high insulin binding and tyrosine kinase activities has been prepared from human placenta. Based on a molecular mass of 306 kDa for the receptor (the value obtained from the sum of the amino acid residues), this preparation is capable of binding 1.48 mol of insulin per mol of receptor. The receptor is free from phosphatase and ATPase activity and is not stimulated by sodium vanadate. Autophosphorylation is linear with respect to receptor concentration, and the 32p incorporated is stable even in the presence of a 100-fold excess of unlabeled ATP. The Km for ATP is 208 ,.M. N-Ethylmaleimide inhibits autophosphorylation. Alkylation with 3H-labeled N-ethylmaleimide results in the incorporation of 1.13 ± 0.37 mol of N-ethylmaleimide per mol of insulin binding activity exclusively into the f8 subunit of the receptor. The nonhydrolyzable ATP analog adenosine 5'-[fi,y-imido]triphosphate stimulates autophosphorylation of the receptor, an effect that is evident at ATP concentrations below 1 mM. The stimulatory effect of adenosine 5'-[j3,y-imido]triphosphate is the result of increasing the binding of insulin to the a subunit, and this reflects itself in a shift to the left of the insulin dose-response curve for autophosphorylation. The same is true for ATP. As a consequence, it is now possible to reconcile the concentration of insulin necessary for stimulating the autophosphorylation reaction with physiological levels and with the levels of insulin required for its classical biological effects.The tyrosine kinase activity of the human placental insulin receptor has been the subject of numerous investigations. The majority of these studies were performed with wheat germ agglutinin-purified receptor (wheat germ receptor) that contains only -0.3% of the insulin receptor (1). It appears that this preparation of the receptor was used rather than affinity-purified material because the latter, in spite of high insulin binding activity, exhibits very poor tyrosine kinase activity. However, the wheat germ receptor, which has relatively high tyrosine kinase activity, can be expected to contain phosphate-metabolizing enzymes such as ATPases and phosphatases, which are likely to interfere with accurate kinetic measurements. The disadvantages inherent in both of these preparations prompted the present investigation, which had as its primary goal the development of methodology for the preparation of highly purified receptor exhibiting tyrosine kinase activity comparable to that of the wheat germ receptor. This goal was achieved by the use of avidin-biotin technology and the development of an improved method for eluting the receptor from the affinity resin. The ensuing preparations, which exhibited a high level of insulin binding activity and a high degree of tyrosine kinase activity, appeared to be well suited for accurate kinetic measurements. Such studies are the subject of this paper. MATERIALS AND METHODSNa Bl_(6-biotinylamido)hexanoyl-Na.A1,B 9-bis(t-butyl-oxycarbonyl)insulin, succinoylavidin-Sepharose...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

ATP sensitizes the insulin receptor to insulin.

Ridge

Finn²

1988

Proc. Natl. Acad. Sci. U.S.A.

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“…For preparation of liver cell membranes the method of Hoffman et al (1987) was used. Livers were homogenized in Potter-Elvejhem homogenizer in TES buffer pH 7.4 (25 mM Tris, 1.25 mM EDTA, 0.25 M sucrose, with protease inhibitors cocktail) 1:4 (m:V) on 4°C.…”

Section: Insulin Binding Assaymentioning

confidence: 99%

Age-related changes of insulin receptors, plasma insulin and glucose level

Korićanac¹,

Vulovic²,

Radivojsa³

et al. 2004

Biogerontology

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The effects of aging on hepatic and erythrocyte insulin receptors have been investigated in 6, 12, 18 and 21-months-old compare to 3-months-old rats. Plasma insulin was elevated in 6, 12 and 18-months-old rats. Specific binding of insulin in liver was increased at the age of 8 months and accompanied with increase in concentration of low affinity binding sites, while specific binding to erythrocytes as well as concentration of both classes of binding sites was increased in 6-months-old rats. The protein and mRNA content of hepatic receptor were decreased only in the oldest animals. Plasma glucose was elevated starting from 12-months-old rats, while, after decrease in 6-months-old animals, citrulline was raised in the oldest group. The results demonstrating that specific binding of insulin in liver and erythrocytes and the concentration of binding sites in both tissues were not decreased during aging, as well as the absence of changes in affinity of insulin binding sites do not point out to occurrence of insulin resistance. However, the increase in insulinemia in the middle of lifespan, elevated plasma glucose and citrulline as well as decrease of hepatic receptor protein and mRNA content in the oldest animals indicate some age-related changes in insulin signaling.

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“…We have synthesized a new biotinylated probe for angiotensin I1 receptors studies: biotinyl-NH(CH,),-SS-(CH2)2-CO-Gly-cAhx-[Ala', Phe(4N3)']angiotensin I1 (5). This molecule can be photoactivated through an arylazido group.…”

Section: Synthesis Of a Biotinylated Probe With An Extended Cleavablementioning

confidence: 99%

Synthèse d'une sonde biotinylée à bras clivable allongé pour l'isolement des récepteurs de l'angiotensine II

Seyer

Aumelas

Marie

et al. 1989

Helvetica Chimica Acta

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We have synthesized a new biotinylated probe for angiotensin I1 receptors studies: biotinyl-NH(CH,),-SS-(CH2)2-CO-Gly-cAhx-[Ala', Phe(4N3)']angiotensin I1 (5). This molecule can be photoactivated through an arylazido group. 'H-NMR studies suggest that it adopts an extended conformation which should allow a simultaneous recognition of both streptavidin and hormone receptor. It has a good affinity for receptors (Kd = 1 nM) and hence is a promising tool in their detection (autoradiography, gold-, ferritin-, enzyme-, or fluorescent streptavidin derivatives) and separation (cell sorting, affinity chromatography). It can be monoiodinated ( 4 ) at its tyrosine residue without a significant loss of affinity. Its extended cleavable arm allows an easy recovery of the 'probe-receptor' complex from streptavidin. An HPLC monitoring of the synthesis is described, particularly of the segment coupling 1 + 2 in presence of (benzotriazol-l-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP). This method can be used as well for synthesis of the D-Phe' derivative that has antagonist properties.

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The rat liver insulin receptor

Cited by 22 publications

References 18 publications

ATP sensitizes the insulin receptor to insulin.

ATP sensitizes the insulin receptor to insulin.

Age-related changes of insulin receptors, plasma insulin and glucose level

Synthèse d'une sonde biotinylée à bras clivable allongé pour l'isolement des récepteurs de l'angiotensine II

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