共查询到20条相似文献,搜索用时 15 毫秒
1.
Effect of insulin on glucagon binding to rat epididymal adipocytes was studied in vitro. [125I]iodoglucagon binding to isolated adipocytes was increased by preincubation of the cells with insulin. Maximal increase was observed with 7 X 10(-10) M insulin. In Scatchard analysis, [125I]iodoglucagon competition data generated one binding site with a single affinity for glucagon binding in the cells pretreated with buffer alone. Pretreatment of the cells with insulin increased the affinity without changes in the number of binding sites. [125I]iodoglucagon binding to isolated adipocytes was not affected by pretreatment of the cells with luteinizing hormone, follicle-stimulating hormone, growth hormone, or with prolactin. These results suggest that insulin stimulates glucagon binding to adipocytes. 相似文献
2.
Comparison of Insulin Binding Proteins In Plasma and Nuclear Membranes of Obese and Lean Mouse Liver
《Journal of receptor and signal transduction research》2013,33(5-6):419-433
AbstractPlasma membranes obtained from obese (ob/ob) and lean (+/+ or +/ob) mouse livers were chemically crosslinked to [125I] -insulin and examined by electrophoresis and autoradiography. The pattern of crosslinked hormone was qualitatively similar in obese and lean plasma membranes. A major insulin binding protein of approximately M 120,000 was observed. Two additional bands were apparent, one which remained near the top of the gel and one about M 90,000. A minor band at approximately M 50,000 was also detected. For each of the insulin binding proteins a reduction in the amount of [125I]-insulin bound was observed with obese plasma membranes as compared with lean. For all proteins the insulin binding was specific as determined by competition with unlabeled hormone. In addition to plasma membrane receptors, insulin has also been reported to bind to nuclear membranes. The autoradiographic patterns of gels of [125]-insulin bound and crosslinked to nuclear membranes from obese and lean mouse livers indicated the presence of proteins of the same M as those described for plasma membranes. Nuclear membrane proteins bound less insulin than plasma membranes and, again, the obese was decreased relative to the lean. Contamination of the nuclear membrane fraction by plasma membranes was ruled out. Scatchard analyses of [125]-insul in bound to plasma and nuclear membranes indicated that the decrease in hormone binding in the obese mouse is a result of a reduction in the absolute number of receptors. The findings presented in this study provide additional support for this conclusion by demonstrating that membranes from obese mice are comprised of the same set of apparently unaltered insulin binding proteins. Further, the presence of similar insulin binding proteins in both nuclear and plasma membranes suggests a physiological relationship between these structures with respect to hormone binding and/or in the mechanism of action of insulin. 相似文献
3.
Unmasking of insulin receptors in rat submaxillary gland microsomes: effect of high ionic strength, phospholipase C and S-adenosyl-L-methionine 总被引:1,自引:0,他引:1
The specific binding of [125I]insulin to submaxillary gland microsomes was significantly enhanced by increasing the ionic strength of the incubation medium. This effect was neither related to changes in receptor or hormone degradation nor in the polymerization of the tracer. When equilibrium binding data from competition curves of unlabelled insulin versus [125I]insulin were analyzed, a marked increase in total binding capacity in high ionic strength was observed (from 890 to 2440 fmol/mg protein), with no change in binding affinity. Phospholipase C digestion was also able to increase specific [125I]insulin binding to microsomes. These results suggest the presence of masked receptors in submaxillary gland microsomes. Methylation of rat submaxillary gland microsomes by using S-adenosyl-L-methionine as the methyl donor significantly increased [125I]insulin binding. Scatchard analysis of the equilibrium binding data showed that addition of S-adenosyl-L-methionine (0.46 mM) to microsomes resulted in an enhancement of the total binding capacity (from 990 to 1520 fmol/mg protein) with no change in the affinity constants, which suggests the exposure of masked insulin receptors under such conditions. Both the methyl group incorporation into membrane phospholipids and the effect on insulin binding were dependent on the S-adenosyl-L-methionine concentration used and were partially suppressed in the presence of S-adenosyl-L-homocysteine, a specific competitive inhibitor of the methyltransferases activity. When microsomes were treated with S-adenosyl-L-[methyl-3H]methionine, the 3H-labelled methyl groups incorporated were found mainly in the lipid fraction associated to phosphatidylcholine, suggesting in this case that the unmasking of insulin receptors could be a consequence of alterations produced in membrane composition. The effects of phospholipase C, S-adenosyl-L-methionine and high ionic strength on insulin binding were not additive, suggesting that these procedures unmask receptors from the same pool. 相似文献
4.
E M Rutanen T K?rkk?inen C Lundqvist F Pekonen O Ritvos P Tanner M Welin T Weber 《Biochemical and biophysical research communications》1988,152(1):208-215
Monoclonal antibodies were prepared against the 27-34K insulin-like growth factor (IGF)-binding protein purified from human placenta/decidua and designated placental protein 12 (PP12). Four different antibodies were characterized. Each recognized the major band at 32K on immunoblots of the purified PP12 preparation and amniotic fluid. In liquid phase RIA, IGF-I did not affect the binding of [125I] PP12 to one antibody (Mab 6303), it slightly increased the binding to two antibodies (Mab 6301 and 6304), and it slightly decreased the binding to one antibody (Mab 6302). All antibodies immunoprecipitated the cross-linked PP12-[125I] IGF-I complex, but Mab 6302 considerably less effectively than the others. Preincubation of PP12 with Mab 6302 completely inhibited the binding of [125I] IGF-I to PP12, whereas preincubation with Mab 6303 had no effect, and Mab 6301 as well as Mab 6304 increased it. These results suggest that Mab 6302 binds to an epitope at or near to the IGF-binding site, whereas the other antibodies react at other sites of the PP12 molecule. Conformational changes in PP12 probably account for the IGF-I-induced increase in the binding of Mabs 6301 and 6304 to [125I] PP12, and vice versa, for Mabs 6301- and 6304-induced increase in the binding of [125I] IGF-I to PP12. 相似文献
5.
Distribution of receptors for insulin and insulin-like growth factor I (somatomedin C) in the adrenal gland 总被引:2,自引:0,他引:2
D J Pillion M Yang W E Grizzle 《Biochemical and biophysical research communications》1988,154(1):138-145
Rat adrenal glands contain cell surface high-affinity receptors for several peptide hormones. Receptors for IGF-I were abundant in this tissue, but receptors for insulin were relatively scarce. The behavior of adrenal membrane IGF-I receptors in radioligand binding assays was similar to the behavior of IGF-I receptors from other tissues, with a KD congruent to 6.2 x 10(-9) M. Covalent cross-linking studies with [125I]IGF-I revealed an IGF-I receptor alpha-subunit with Mr congruent to 135,000 on dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions, as well as a smaller radiolabeled peptide, Mr = 116,000. In contrast, little binding of [125I]insulin to adrenal membranes was observed and no labeling occurred in cross-linking studies using [125I]insulin. These results contrast with the findings of whole-body autoradiographic studies that indicated substantial binding of [125I]insulin to adrenal glands and suggest that IGF-I, rather than insulin, may play a critical role in the growth and development of the adrenal gland. 相似文献
6.
Hydrocortisone and dexamethasone produced a time-dependent increase [125I]epidermal growth factor [( 125I]EGF) binding in primary cultures of isolated rat hepatocytes. Maximally effective doses of glucocorticoids resulted in a 70-100% increase in binding. The effect was similar when hepatocytes were maintained on collagen-coated plates or directly on culture dishes. The glucocorticoid-mediated increase in [125I]EGF binding could be detected after 4 h exposure to glucocorticoid and was substantial by 8 h. The major effect of glucocorticoid appeared to be to increase the number of EGF receptors. While insulin (100 nM) had no effect on basal [125I]EGF binding, it significantly inhibited the increase produced by the glucocorticoid. Since the inhibitory effect of insulin was only observed when insulin was added with the inducing glucocorticoid, insulin appears to inhibit an early hydrocortisone-mediated event. 相似文献
7.
Regulation of high density lipoprotein receptors in cultured macrophages: role of acyl-CoA:cholesterol acyltransferase 总被引:4,自引:1,他引:3
下载免费PDF全文
![点击此处可从《The EMBO journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The interaction of human serum high density lipoproteins (HDL) with mouse peritoneal macrophages and human blood monocytes was studied. Saturation curves for binding of apolipoprotein E-free [125I]HDL3 showed at least two components: non-specific binding and specific binding that saturated at approximately 40 micrograms HDL protein/ml. Scatchard analysis of specific binding of apo E-free [125I]-HDL3 to cultured macrophages yielded linear plots indicative of a single class of specific binding sites. Pretreatment of [125I]HDL3 with various apolipoprotein antibodies (anti apo A-I, anti apo A-II, anti apo C-II, anti apo C-III and anti apo E) and preincubation of the cells with anti-idiotype antibodies against apo A-I and apo A-II prior to the HDL binding studies revealed apolipoprotein A-I as the ligand involved in specific binding of HDL. Cellular cholesterol accumulation via incubation with acetylated LDL led to an increase in HDL binding sites as well as an increase in the activity of the cytoplasmic cholesterol esterifying enzyme acyl-CoA:cholesterol acyltransferase (ACAT). Incubation of the cholesterol-loaded cells in the presence of various ACAT inhibitors (Sandoz 58.035, Octimibate-Nattermann, progesterone) revealed a time- and dose-dependent amplification in HDL binding and HDL-mediated cholesterol efflux. It is concluded that the homeostasis of cellular cholesterol in macrophages is regulated in part by the number of HDL binding sites and that ACAT inhibitors enhance HDL-mediated cholesterol efflux from peripheral cells. 相似文献
8.
1. 125I-labelled secretin bound rapidly and specifically to membranes from cat pancreas. Binding of labelled hormone was competitively inhibited by unlabelled secretin in the same range of concentrations that stimulated pancreatic adenylate cyclase in these membranes. The dissociation constant of the membrane binding sites for unlabelled secretin as evaluated by these displacement experiments was 4.1-10(-9) M and the number of binding sites 1.0 pmol per mg of membrane protein. 2. Studies using different concentrations of [125I]secretin (at a constant ratio of labelled to unlabelled hormone) revealed a similar value of 4-4-10(-9) M for the dissociation constant. 3. Both the association and dissociation rate constants of [125I]secretin binding were temperature sensitive; the dissociation rate constant increased more rapidly with increase in temperature. The ratio k-1/k+1 (at 22 degrees C) gave a dissociation constant of 3.7-10(-9)M which agrees closely with the figure obtained from equilibrium data. These data indicate that 125I-labelled secretin and unlabelled secretin bind to the same binding site on pancreatic membranes, with high affinity. 4. Unlabelled secretin stimulated pancreatic adenylate cyclase with an apparent Km of 8.4-10(-9) M, while [125I]secretin apparently did not stimulate the adenylate cyclase. Together with the binding data this might suggest that different portions of the secretin molecule are responsible for binding and adenylate cyclase activation. 5. Studies on the specificity of [125I]secretin binding carried out with various peptide hormones (glucagon, human gastrin, pancreozymin and caerulein) which are all inefficient in stimulating pancreatic fluid secretin, showed that these hormones have no influence on the binding of [125I]secretin. In contrast, vasoactive intestinal polypeptide, which stimulates pancreatic fluid and bicarbonate secretion, showed a competitive inhibition of secretin binding to the plasma membrane preparation. 相似文献
9.
N M Gulaia Iu V Bezdrobny? E S Gavriliuk M D Lutsik O F Peredere? 《Ukrainski? biokhimicheski? zhurnal》1988,60(3):3-10
The presence of insulin receptors in neuroblastoma C 1300 N18 cells was shown by the method of the [125I]insulin binding with cells as well as by the electron-cytochemical methods based on the analysis of the binding of colloid gold-labelled insulin and of agglutinin of wheat embryos with the surface of plasma membrane. It is established that the number and distribution of insulin receptors depend on the functional state of cells. Expression of receptors on the membrane increases after 5'-deoxyuridine-induced differentiation of cells. Due to changes in the lipid composition of cells caused by the incubation with lecithin-cholesterol liposomes (an increase in the content of cholesterol and its esters, as well as of unsaturated fatty acids and appearance of lysolecithin) the quantity of insulin receptors decreases and cell membranes are damaged. The lowering insulin regulation is not observed for insulin receptors of the neuroblastoma C 1300 N18 cells. 相似文献
10.
A method is described in which high-speed centrifugation of membranes through an oil phase is used to separate membrane-bound and detergent-solubilized polypeptide receptor-iodinated ligand complexes from unbound ligands. Three centrifuges, the Brinkmann Eppendorf (5412), the Beckman Microfuge B and the Beckman Airfuge were evaluated for this capability. Under the conditions described, the Beckman Airfuge surpassed the others in recovering previously 125I- and 32P-labelled cell membranes. The Airfuge method was compared with the more classically employed membrane filtration method to measure specific [125I]insulin and [125I]thrombin binding to human placental membranes and an enriched plasma membrane fraction from mouse embryo fibroblasts, respectively, are found to be 4 to 6 times more sensitive. For example, specific binding of ligand to its receptor was demonstrated with 5 micrograms of protein. With slight modifications, the polyethyleneglycol 6000 method of precipitating 125I-labelled ligand-soluble receptor complexes can be adapted to the Airfuge sedimentation through oil procedure. 相似文献
11.
[125I]VIP (vasoactive intestinal peptide) bound to apical membranes isolated from the bovine tracheal epithelium with a half maximal inhibition by unlabeled VIP (IC50) of 0.6 x 10(-9)M and binding was reversible. Glucagon did not affect [125I]VIP binding to the membranes. [125I]VIP was covalently cross-linked to tracheal membrane proteins using disuccinimidyl suberate. SDS-polyacrylamide gel electrophoresis of labeled tracheal membranes revealed one major [125I]-receptor complex of Mr = 71,000 to which binding of [125I]VIP was inhibited by 10 microM unlabeled VIP. These results are consistent with the presence of a specific, high-affinity receptor for VIP, with a Mr = 71,000, in apical membrane vesicles isolated from the bovine tracheal epithelium. 相似文献
12.
125I-Iodohydroxybenzylpindolol ([125I] IHYP), a potent beta-adrenergic receptor antagonist, has been used to study beta-adrenergic receptors in rat brain. Binding of [125I] IHYP (30 pM) to a membrane fraction min and dissociation took place with a half time of about 16 min. Phentolamine (10(-4) M) decreased non-receptor binding but it had no effect on the binding of [125I] IHYP to beta-adrenergic receptors in cortex, cerebellum or caudate. In the presence of phentolamine specific binding (defined as binding which was blocked by 0.3 muM dl-propranolol) represented 70-85% of total binding. The binding of [125I] IHYP was inhibited by beta-adrenergic agonists and antagonists. d-Stereoisomers were 2-3 orders of magnitude less potent than the corresponding 1-isomers. The denstiy of [125I] IHYP binding sites was studied in membrane fractions from cerebral cortex, cerebellum, and caudate nucleus by means of Scatchard analysis. The K(D) of [125I] IHYP was similar in the three regions studied, and the density of [125I] IHYP binding sites was approximately 50% greater in the cortex and caudate than in the cerebellum. The Hill coefficient for the binding of [125I] IHYP to membranes from cerebral cortex was 1.02. The properties of the binding of [125I] IHYP are similar to those which would be expected of binding to beta-adrenergic receptors in vitro. 相似文献
13.
In the radioreceptor assay for growth hormone (RRA-GH) using [125I]iodo-hGH, hGH and human liver membrane particulate fractions as tracer, hormone standard and receptors, respectively, ovine placental lactogen (oPL) is capable of inhibiting the binding of [125I]iodo-hGH in a parallel manner with hGH and in equipotency. Similarly, in the RRA-GH by employing [125I]iodo-oPL, oPL and human liver membrane particulate fractions as tracer, hormone standard and receptors, respectively, hGH is also equipotent as oPL in inhibiting the binding of [125I]iodo-oPL in a parallel fashion. The addition of monoclonal antibodies against oPL in the assay was effective in inhibiting the binding of [125I] iodo-oPL to human liver, but could not, however, inhibit the binding of [125I]iodo-hGH to human liver. Furthermore, the addition of the monoclonal antibodies in the RRA-GH did not affect the parallelism of the oPL standard but lowered the total binding of oPL. Our studies indicate that the structure of the binding sequence in oPL which binds to the GH receptor of human liver is not identical to the equivalent sequence of hGH and that the monoclonal antibodies compete with GH receptors in human liver for the binding of oPL. 相似文献
14.
Insulin-like growth factors in sheep thyroid cells: action, receptors and production 总被引:2,自引:0,他引:2
L K Bachrach M C Eggo R L Hintz G N Burrow 《Biochemical and biophysical research communications》1988,154(3):861-867
Sheep thyroid cells cultured in serum-free medium were used to study the biologic activity, binding, and production of the insulin-like growth factors (IGFs). IGF-I, IGF-II, and insulin stimulated thyroid cell division. Abundant, specific IGF receptors on sheep thyroid cell membranes were identified by binding displacement studies. Maximal specific binding of [125I]-labeled IGF-I and IGF-II to 25 micrograms of membrane protein averaged 21% and 27% respectively. The presence of type I and type II IGF receptors was confirmed by polyacrylamide gel electrophoresis of [125I]IGFs covalently cross-linked to cell membranes. Under reducing conditions, [125I]IGF-I bound to a moiety of approximate Mr = 135,000 and [125I]IGF-II to a moiety of approximate Mr = 260,000. Cross-linking of [125I]IGF-I to medium conditioned by thyroid cells indicated the presence of four IGF binding proteins with apparent Mr = 34,000, 26,000, 19,000 and 14,000. Thyroid cells also secreted IGF-I and II into the medium. IGF synthesis was enhanced consistently by recombinant growth hormone. These data indicate that sheep thyroid cells are a site for IGF action, binding, and production and provide further evidence that IGFs may modulate thyroid gland growth in an autocrine or paracrine manner. 相似文献
15.
S Hasumura B Rossi R Alderson I Pastan S Cheng 《Biochemical and biophysical research communications》1984,124(3):956-962
To develop antibodies against the plasma membrane 3,3',5-triiodo-L-thyronine (T3) binding protein (M.W. 55,000), rabbits were immunized with formalin-fixed GH3 cells or highly purified plasma membranes from these cells. Antibodies were screened by immunoprecipitation using detergent solubilized N-bromoacetyl-[125I]T3-labeled 55K protein. Among the nine detergents tested, 0.18% CHAPS was found to be the best in its solubilization efficiency and its ability to maintain the integrity of the antigenicity of the 55K protein. The N-bromoacetyl-[125I]T3-labeled 55K protein was also immunoprecipitated by anti-T3 antibodies. The anti-55K protein antibodies cross-reacted with plasma membrane T3 binding proteins from cultured cells and tissues of human and rodent origin. These results indicate that structural similarities exist in human and rodent plasma membrane T3 binding proteins. These antibodies should provide a powerful tool in the characterization and in probing the function(s) of the plasma membrane T3 binding protein in cells. 相似文献
16.
Nancy R. Zahniser M. Beth Goens Patrick J. Hanaway John V. Vinych 《Journal of neurochemistry》1984,42(5):1354-1362
An in vitro receptor binding assay, using filtration to separate bound from free [125I]insulin, was developed and used to characterize insulin receptors on membranes isolated from specific areas of rat brain. The kinetic and equilibrium binding properties of central receptors were similar to those of hepatic receptors. The binding profiles in all tissues were complex and were consistent with binding in multiple steps or to multiple sites. Similar binding properties were found among receptors in olfactory tubercle/bulb, cerebral cortex, hippocampus, striatum, hypothalamus, and cerebellum. High affinity [125I]insulin binding sites (KD = 3-11 nM) were distributed evenly between membranes isolated from P1 and P2 fractions of these brain areas, with the exception of the olfactory tubercle in which binding to P2 membranes was four-fold greater (Bmax = 150 fmol/mg protein). One difference between insulin receptors in brain and peripheral target tissues, however, was observed. Following exposure to 0.17 microM insulin for 3 h at 37 degrees C, the number of specific [125I]insulin binding sites on adipocytes decreased by 40%, while the number of binding sites on minces of cerebral cortex/olfactory tubercle remained constant. The results suggest that although the binding characteristics of central and peripheral insulin receptors are similar, these receptors do not appear to be regulated in the same manner. 相似文献
17.
The specific binding of 125I-labelled low density lipoprotein ([125I]LDL to human adipocyte plasma membranes was higher at 37 than at 0 degree C. Prior treatment of membranes with pronase had no effect on LDL binding measured at 0 degree C but consistently stimulated binding at 37 degrees C. Plasmin was similar to pronase in enhancing LDL-specific binding, but thrombin was not as effective. 125I-labelled high density lipoprotein ([125I]HDL2) specific binding to human adipocyte plasma membranes was similarly sensitive to temperature and pronase treatment. Addition of the protease inhibitor aprotinin in the adipocyte membrane binding assay significantly reduced [125I]LDL binding at 37 degrees C (p less than 0.05), suggesting the involvement of a protease activity intrinsic to the lipoproteins and (or) membranes. These data demonstrate that both LDL and HDL binding in human adipocyte plasma membranes can be "up-regulated" by specific proteolytic perturbations in a temperature-dependent manner. 相似文献
18.
G Loo E Berlin P C Allen 《Comparative biochemistry and physiology. B, Comparative biochemistry》1990,96(2):361-366
1. Male chicks were deprived of feed for 48 hr to study the effect of metabolic stress on hepatic membrane and lipoprotein fluidity and binding of radioiodinated lipoproteins to hepatic membranes. 2. Plasma levels of low density lipoprotein (LDL) and high density lipoprotein (HDL) were markedly and slightly elevated, respectively. 3. There was a reduction in lipoprotein and hepatic membrane fluidity. 4. Binding of [125I]LDL, but not [125I]HDL, to hepatic membranes was decreased. 5. It is suggested that a reduction in the fluidity of LDL and/or hepatic membranes impedes LDL catabolism in vivo. 相似文献
19.
Binding of epidermal growth factor by human colon carcinoma cell (Caco-2) monolayers 总被引:4,自引:0,他引:4
I J Hidalgo A Kato R T Borchardt 《Biochemical and biophysical research communications》1989,160(1):317-324
The objective of this study was to investigate whether Caco-2 cells bind and internalize epidermal growth factor (EGF). [125I]EGF was presented to the apical (AP) or basolateral (BL) side of Caco-2 monolayers, grown on microporous membranes, at different times in culture. At day 10, [125I]EGF binding (at 37 degrees C) to the BL membrane was 2-3 times greater than binding to the AP membrane. Of that [125I]EGF bound to the AP membrane 76% was internalized within 3 h while internalization from the BL membrane was 90%. At lower temperatures membrane-bound [125I]EGF increased while internalization decreased. At day 16, AP and BL binding decreased and then remained constant through day 25. [125I]EGF was bound to the BL membrane of 10 days old monolayers with a Kd of 0.67 nM. There was a single binding site whose numbers in the BL membrane was about 5500/cell. 相似文献
20.
The glycoproteinic nature of the insulin receptor was indicated using two different approaches: 1. [125I]insulin binding to soluble receptors from mouse liver was inhibited by digestion with β-galactosidase or pretreatment with Ricinus communis I or concanavalin A. An other enzyme (neuraminidase) and lectins (wheat germ agglutinin, Dolichos biflorus) did not affect the binding reaction. These data confirmed that insulin directly interacts with the galactoglycoproteins of liver membranes. 2. The galactose oxidase-sodium boro[3H]hydride technique, previously used for labeling accessible membrane galactoglycoproteins, was again utilized to discern the components that interact with insulin. When liver membranes were equilibrated with 10?7 M insulin prior to labeling, the SDS gel radioactive profiles were specifically modified within two galactoglycoproteins of apparent molecular sizes 195 000 and 145 000, compatible with their participation in the insulin binding interaction. Membrane pretreatment with β-galactosidase or Sophora japonica lectin reduced the labeling in most peaks, thus supporting the argument for labeling sensitivity. Preincubation of membranes with 10?7 M proinsulin slightly hindered labeling while pretreatment with 10?7 M glucagon was ineffective, suggesting a specificity of the insulin effect. These data indicate the glycoprotein nature of the insulin receptor for two reasons: alteration of insulin binding after modification of the galactoglycoproteins, and alteration of galactoglycoprotein labeling after insulin binding. Two galactoglycoproteins, with apparent molecular weights 145 000 and 195 000, respectively, were identified and they are suggested to have insulin binding properties. 相似文献