Insulin receptors on Xenopus laevis oocytes: effects of injection of ob/ob mouse liver mRNA

1991 ◽  
Vol 100 (1) ◽  
pp. 167-171
Author(s):  
D.A. Diss ◽  
B.D. Greenstein
Keyword(s):  
Xenopus Laevis ◽  
Binding Sites ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Cell Types ◽  
Follicle Cells ◽  
Vitelline Membrane ◽  
Xenopus Laevis Oocytes ◽  
Endogenous Insulin ◽  
Order Of Magnitude

We describe here conditions for the detection of insulin binding sites on Xenopus laevis oocytes. The binding of 125I-labelled insulin displayed sigmoidal behaviour, which is characteristic of the binding relationship between insulin and its receptor. Resolution of the resulting curvilinear Scatchard plot into two components revealed KD values of 8.86 × 10(−10) +/− 1.9 × 10(−10) and 5.32 × 10(−9) +/− 2.4 × 10(−9) M and n values of 9.7 × 10(7) +/− 0.4 × 10(7) and 3.3 × 10(8) +/− 0.5 × 10(8) binding sites per oocyte, respectively. The possibility cannot be excluded, however, that receptors for IGF-1 were also being detected. Also described are conditions for the rapid and efficient removal of all tissues surrounding the oocyte, including the vitelline membrane. We could not detect any specific 125I-labelled insulin binding to oocytes that had their follicle cells or vitelline membrane removed and this was not due to the enzymic treatment used in the process. Microinjection of oocytes without follicular layers did not result in the appearance of any detectable insulin binding sites, which were, however, observed if oocytes were first stripped of the vitelline membrane. We suggest that oocytes may possess endogenous insulin receptors on their surface in numbers of the same order of magnitude as those present on somatic cells. The removal of tissues surrounding the oocyte should facilitate studies aimed at determining functional interactions of the various cell types during oocyte development and for studying insulin receptors on the oocyte-follicular cell complex.

Insulin Binding Sites Induced in the Tetrahymena by Rat Liver Receptor Antibody

1984 ◽  
Vol 39 (1-2) ◽  
pp. 183-185 ◽  
Author(s):  
G. Csaba ◽  
P. Kovács ◽  
Ágnes Inczefi-Gonda
Keyword(s):  
Rat Liver ◽  
Concanavalin A ◽  
Binding Sites ◽  
Binding Capacity ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Receptor Antibody ◽  
Receptor Antibodies

Abstract Tetrahvmena cells treated with purified rabbit anti­ bodies to rat hepatocellular membrane exhibited a consider­ able increase in binding capacity on reexposure to the antibody 24 h later. Insulin binding was similarly enhanced by preexposure to the antibody, and vice versa, preex­ posure to insulin enhanced the later binding of rat liver receptor antibodies. This suggests that (1) the Tetrahymena and the rat possess similar insulin receptors, and (2) the receptor antibody is also able to induce imprinting for itself as well as for insulin. Concanavalin-A, noted for binding overlap with insulin, failed to induce imprinting either for insulin or for antibodies to receptors, whereas the latter did induce imprinting for Concanavalin-A.

scholarly journals Identification of Host Insulin Binding Sites on Schistosoma japonicum Insulin Receptors

PLoS ONE ◽  
2016 ◽  
Vol 11 (7) ◽  
pp. e0159704 ◽  
Author(s):  
Rachel J. Stephenson ◽  
Istvan Toth ◽  
Jiening Liang ◽  
Amanjot Mangat ◽  
Donald P. McManus ◽  
...  
Keyword(s):  
Schistosoma Japonicum ◽  
Binding Sites ◽  
Insulin Receptors ◽  
Insulin Binding

Distribution of 125I-labelled insulin-binding sites in peripheral tissues of the normal and diabetic rat: an autoradiographic study

1991 ◽  
Vol 128 (1) ◽  
pp. 85-NP ◽  
Author(s):  
C. S. Thompson ◽  
R. M. Sykes ◽  
J. Muddle ◽  
M. R. Dashwood
Keyword(s):  
Binding Sites ◽  
Regional Distribution ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Autoradiographic Study ◽  
Diabetic Rat ◽  
Peripheral Tissues ◽  
Liver And Kidney ◽  
Impaired Insulin Action

ABSTRACT In-vitro autoradiography was used to demonstrate the regional distribution of 125I-labelled insulin-binding sites in the liver, kidney and heart of normal rats and rats made diabetic with streptozotocin. The distribution of insulin-binding sites in the liver of control rats was uniformly high, while in the kidney of control rats there was weak 125I-labelled insulin binding in the medulla and dense binding in the cortex. In the hearts of control rats a high density of 125I-labelled insulin-binding sites was evident both in the atrial and ventricular muscle. Non-ketotic diabetes mellitus caused a marked increase in 125I-labelled insulin-binding sites in both the liver and kidney with the former tissue exhibiting a time-dependent (7 to 62 days) increase. There was no apparent effect of diabetes on insulin-binding sites in the heart. Since experimental diabetes causes (1) a decrease in circulating insulin concentration and (2) impaired insulin action at many target tissues, the increase in 125I-labelled insulin-binding sites observed in the present study may represent a compensatory 'up regulation' of insulin receptors. Journal of Endocrinology (1991) 128, 85–89

Kinetics of Accumulation and Processing of Simian Virus 40 RNA in Xenopus laevis Oocytes Injected with Simian Virus 40 DNA

1982 ◽  
Vol 2 (12) ◽  
pp. 1581-1594
Author(s):  
Timothy J. Miller ◽  
Donald L. Stephens ◽  
Janet E. Mertz
Keyword(s):  
Xenopus Laevis ◽  
Transcription Termination ◽  
Simian Virus 40 ◽  
Cell Types ◽  
Simian Virus ◽  
Xenopus Laevis Oocytes ◽  
Sequence Specificity ◽  
The Stability ◽  
Sv40 Dna ◽  
Kinetics Of

We examined the kinetics of accumulation and processing of simian virus 40 (SV40) RNA in stage 6 oocytes of Xenopus laevis microinjected intranuclearly with SV40 DNA. The rates of synthesis and degradation, cellular distribution, size, and sequence specificity of radiolabeled SV40-specific and endogenous oocyte RNA were determined. The kinetics of accumulation of SV40 RNA were biphasic, with greater than 90% of the viral RNA turning over in the nucleus with a half-life of 20 to 40 min. Although most of the primary transcription products were multigenomic in length, some stable polyadenylated SV40-specific RNA similar in size and sequence to late 19S mRNA accumulated in the cytoplasm with time. Differences in strand preference, efficiencies of transcription termination and polyadenylation, and the splice sites used in the synthesis and processing of SV40 RNA in Xenopus oocytes and monkey cells were noted. However, these differences were quantitative, rather than qualitative, in nature. Consequently, they are probably due to regulatory rather than mechanistic differences between the two cell types. We therefore conclude that Xenopus oocytes may be a useful system for studying both mechanistic and cell type-specific regulatory aspects of mRNA biogenesis from cloned DNAs. However, since only a small percentage of the initially synthesized RNA ends up in stable mRNA, it will be important to determine whether mutants of cloned DNAs that produce abnormal amounts of stable mRNAs are altered in promotion and initiation of RNA synthesis, transcription termination, RNA processing, or the stability of the resultant mRNAs.

Elaboration of a novel technique for purification of plasma membranes from Xenopus laevis oocytes

2007 ◽  
Vol 292 (3) ◽  
pp. C1132-C1136 ◽  
Author(s):  
Alexandre Leduc-Nadeau ◽  
Karim Lahjouji ◽  
Pierre Bissonnette ◽  
Jean-Yves Lapointe ◽  
Daniel G. Bichet
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Xenopus Laevis ◽  
Western Blot ◽  
Plasma Membranes ◽  
Transient Receptor Potential ◽  
Expression System ◽  
Vitelline Membrane ◽  
Transient Receptor Potential Vanilloid ◽  
Xenopus Laevis Oocytes

Over the past two decades, Xenopus laevis oocytes have been widely used as an expression system to investigate both physiological and pathological properties of membrane proteins such as channels and transporters. Past studies have clearly shown the key implications of mistargeting in relation to the pathogenesis of these proteins. To unambiguously determine the plasma membrane targeting of a protein, a thorough purification technique becomes essential. Unfortunately, available techniques are either too cumbersome, technically demanding, or require large amounts of material, all of which are not adequate when using oocytes individually injected with cRNA or DNA. In this article, we present a new technique that permits excellent purification of plasma membranes from X. laevis oocytes. This technique is fast, does not require particular skills such as peeling of vitelline membrane, and permits purification of multiple samples from as few as 10 and up to >100 oocytes. The procedure combines partial digestion of the vitelline membrane, polymerization of the plasma membrane, and low-speed centrifugations. We have validated this technique essentially with Western blot assays on three plasma membrane proteins [aquaporin (AQP)2, Na+-glucose cotransporter (SGLT)1, and transient receptor potential vanilloid (TRPV)5], using both wild-type and mistargeted forms of the proteins. Purified plasma membrane fractions were easily collected, and samples were found to be adequate for Western blot identification.

scholarly journals Insulin and insulin-like-growth-factor-I (IGF-I) receptors in Xenopus laevis oocytes. Comparison with insulin receptors from liver and muscle

1991 ◽  
Vol 273 (3) ◽  
pp. 673-678 ◽  
Author(s):  
P Hainaut ◽  
A Kowalski ◽  
S Giorgetti ◽  
V Baron ◽  
E Van Obberghen
Keyword(s):  
Growth Factor ◽  
Xenopus Laevis ◽  
Insulin Receptors ◽  
Beta Subunit ◽  
Beta Subunits ◽  
Xenopus Laevis Oocytes ◽  
Insulin Like Growth Factor ◽  
Factor I ◽  
Alpha Subunits ◽  
Igf I

Insulin and insulin-like-growth-factor-I (IGF-I) receptors were partially purified from full-grown (stages V-VI) Xenopus laevis oocytes by affinity chromatography on wheat-germ agglutinin-agarose. Competitive-binding assays revealed high-affinity binding sites for both insulin and IGF-I (Kd = 2.5 x 10(-10) M and 8 x 10(-10) M respectively). However, IGF-I receptors were about 15 times more abundant than insulin receptors (22.5 x 10(11) versus 1.5 x 10(11)/mg of protein). Moreover, comparison of intact and collagenase-treated oocytes showed that most of the insulin receptors were in the oocyte envelopes, whereas IGF-I receptors were essentially at the oocyte surface. Oocyte receptors were composed of alpha-subunits of approximately 130 kDa and a doublet of beta-subunits of 95 and 105 kDa, which both had ligand-induced phosphorylation patterns compatible with IGF-I receptor beta-subunits. Accordingly, the receptor tyrosine kinase was stimulated at low IGF-I concentrations [half-maximally effective concentration (EC50) approximately 0.5-1 nM], and at higher insulin concentrations (EC50 approximately 20-50 nM). Partially purified glycoproteins from Xenopus liver and muscle contained mainly receptors of the insulin-receptor type, with alpha-subunits of 140 kDa in liver and 125 kDa in muscle, and doublets of beta-subunits of 92-98 kDa in liver and 85-94 kDa in muscle. Immunoprecipitation of receptors from oocytes, liver and muscle by receptor-specific anti-peptide antibodies suggested that the beta-subunit heterogeneity resulted from the existence of two distinct IGF-I receptors in oocytes and of two distinct insulin receptors in both liver and muscle. In the different tissues, the two receptor subtypes differed at least by their beta-subunit C-terminal region.

scholarly journals Guanosine nucleotides regulate hormone binding of insulin receptors

1992 ◽  
Vol 281 (3) ◽  
pp. 735-743 ◽  
Author(s):  
E R Mortensen ◽  
J Drachman ◽  
G Guidotti
Keyword(s):  
Binding Sites ◽  
Plasma Membranes ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Scatchard Analysis ◽  
Hormone Binding ◽  
Temperature Dependent ◽  
High Affinity ◽  
Mild Reduction ◽  
Adipocyte Plasma Membranes

Insulin receptors in turkey erythrocyte and rat adipocyte plasma membranes display non-linear hormone binding by Scatchard analysis. This result is consistent with evidence that the insulin-binding sites are heterogeneous and have at least two affinities for the hormone. Mild reduction of plasma membranes with dithiothreitol, before insulin binding, increased the fraction of hormone binding with high affinity without significantly changing the total number of receptor-binding sites. In the presence of guanosine 5′-[gamma-thio]triphosphate, the amount of receptor with high affinity for insulin in the reduced membranes decreased to that present in the absence of reduction; the effect of the nucleotide was concentration- and temperature-dependent. This decrease in insulin binding was specific for guanine nucleotides.

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