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1.
A possible receptor for thrombin on the platelet membrane has been identified. Whole platelets were treated with 125I-labelled thrombin followed by washing of the platelets, solubilization in Triton X-100, crossed immunoelectrophoresis and autoradiography. A heavily labelled antigen which migrated slightly more slowly than albumin was observed. No corresponding arc was seen on the same immunoplate when stained with Coomassie brilliant blue, indicating that the antigen possessed weak antigenic properties and/or was present in very small amounts. When 125I-labelled thrombin that had been inactivated by phenylmethylsulphonyl fluoride was used, no such labelled arc was seen. The radiolabelled immunoprecipitate does not represent any of the antigens identified hitherto in the immunoelectrophoretic patterns obtained with platelets or platelet material. The electrophoretic mobility of the antigen was influenced neither by neuraminidase treatment of the platelets prior to the 125I-labelled thrombin exposure nor by inclusion of concanavalin A, wheat-germ lectin or lentil lectin in the gel during the first-dimension electrophoresis. This suggests that the antigen does not represent a glycoprotein. Upon subcellular fractionation the radioactively labelled arc was observed in the cytosol fraction following crossed immunoelectrophoresis and autoradiography. Analysis of the secreted proteins after induction of the release reaction with 125I-labelled thrombin revealed labelling of immunoprecipitates representing thrombospondin, albumin and the ‘line’ form of platelet factor 4. This confirms that stable complexes of 125I-labelled thrombin and platelet proteins can exist in the presence of Triton X-100 and during electrophoresis. 相似文献
2.
《Journal of receptor and signal transduction research》2013,33(4):529-539
AbstractA photoaffinity derivative of highly purified 125I-labelled epidermal growth factor (125I-EGF) has been synthesized. The heterobifunctional crosslinking reagent p-azidophenylglyoxal (PAPG) was bound to arginine residues in 125I-EGF. PAPG-125I-EGF bound to EGF receptors on rat fibroblasts and human A431 epidermoid carcinoma cells in culture. An apparent decreased affinity of PAPG-125I-EGF for the EGF receptor is in accord with at least one arginine being at or near the EGF receptor binding site. The PAPG-125I-EGF:EGF receptor complexes on rat cells were internalized to the same extent as control EGF:receptor complexes. A431 cells treated with PAPG-125I-EGF were irradiated with ultraviolet light and the labelled proteins were analyzed by SDS-polyacrylamide gel electrophoresis. The 3 major labelled proteins had apparent molecular weights ranging from 75,000 to 200,000. Only the labelling of the 200,000-Mr protein was prevented by the addition of excess unlabelled EGF with the PAPG-125I-EGF. This molecular weight is in agreement with the reported size of the EGF receptor plus EGF. A protein with apparent molecular weight of 100,000 was labelled by 125I-EGF by an unknown mechanism which was dependent on the dose of UV light and blocked by the addition of excess unlabelled EGF. 相似文献
3.
Membrane proteins of the intestinal brush border were labelled in vivo by intraluminal injection of diazotised [125I]iodosulfanilic acid, a highly polar molecule. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of brush border membranes labelled in this manner showed 20 protein bands, 11 of which contained significant radioactivity. The most heavily labelled proteins had molecular weights greater than 150 000, indicating that they were the most exposed to the intestinal lumen. Little radioactivity was detected in proteins with molecular weights of less than 94 000. The majority of these smaller proteins were likely to have been brush border core proteins. The evidence that diazotised [125I]iodosulfanilic acid bound primarily to brush border membrane proteins when administered in this way, was: (a) the specific activity of brush border proteins was up to 3-fold greater than that of total cell particulate proteins (pelleted at 27 000 × from mucosal homogenates); (b) principal peaks in the gel radioactivity profile of total cell particulate proteins corresponded to the most heavily labelled proteins of the isolated brush border membrane; and (c) brush border core proteins showed minimal radioactivity in vivo, but considerably higher radioactivity when brush border membranes were labelled in vitro. A small amount of label was absorbed across the intestinal mucosa. However, secondary labelling of brush border proteins by this absorbed label was minimal, since the specific activity of brush border proteins in jejunum adjacent to the labelled loop was only 0.22% of the level for those proteins in the labelled segment. Since this technique did not affect the cellular morphology, enzyme activity or biochemical integrity of the membrane, it should prove useful as a means of accurately studying in vivo turnover rates of brush border membrane proteins. 相似文献
4.
Intact HeLa cells and isolated HeLa cell plasma membranes were subjected to lactoperoxidase-catalysed iodination. The 125I-labelled proteins were separated by SDS-polyacrylamide gel electrophoresis. Six protein species with apparent molecular weights from 32 000 to 200 000 were accessible to labelling from the outer cell surface, while most of the proteins present in the plasma membrane were labelled when isolated plasma membranes were iodinated. Iodination of synchronized intact cells revealed that the labelling obtained was cell cycle dependent with maximal labelling at mitosis. No changes in the distribution of radioactivity among the labelled proteins were observed when cells from different phases were iodinated. 相似文献
5.
During incubation of 6,7-3H-ethynylestradiol with rat liver microsomes up to 20 % of the radioactivity was bound irreversibly to the microsomal proteins. Incubations in presence of albumin resulted in a further radioactive labelling of the albumin. The irreversible nature of the steroid-protein bond was established by solvent extraction and charcoal treatment. Further evidence was obtained after hydrolyzing the microsomal protein with trypsin and submitting the labelled tryptic peptides to ion exchange chromatography and electrophoresis. The labelled albumin was applied to sephadex gel filtration which showed the association of the ethynylestradiol radioactivity to the albumin peak.The binding reaction required supply of NADPH, could be stimulated by pretreatment of the animals with phenobarbital and was inhibited by CO and SKF 525 A. On these characteristics the concept was based that, in analogy to the well known binding of estradiol and estrone, 2hydroxylation is also an essential prerequisite for the binding of ethynylestradiol. The concept was confirmed by trapping off the 2-hydroxy-ethynylestradiol with glutathione, which led to a decrease of the ethynylestradiol-protein binding.Further evidence resulted from experiments , dosing rats with 6,7-3H-ethynylestradiol and 6,7-3H-estradiol 48 hrs prior to sacrifice and examining the amount of radioactivity irreversibly bound to the liver endoplasmic reticulum. 3H-ethynylestradiol caused a radioactive labelling of microsomes twice as much as that after 3H-estradiol. 相似文献
6.
A mixture of 4 purified proteins at equal concentrations was radiolabelled with [125I] and lactoperoxidase and analysed by SDS-polyacrylamide gel electrophoresis. Bovine serum albumin took up 9 times as much label as ovalbumin or lysozyme and 3.3 times as much as α? chymotrypsinogen A. These results suggest that when applying the [125I]- lactoperoxidase technique to labelling unknown mixtures of proteins, such as may exist on the surfaces of cells, caution should be exercised in interpreting the degree of labelling of particular proteins in terms only of surface abundance or accessibility. 相似文献
7.
Phosvitin (PV), a subunit of a female-specific protein, vitellogenin, binds to oocyte membranes with a KD of 10?6 M. Binding reaches equilibrium within 30 min after incubation at 25°C. Bound 125I-PV dissociates from the membrane with a t1/2 of 13 h when incubated in buffer. However, when 125I-PV-labeled membranes are incubated in buffer containing 10?5 M unlabeled PV, 50% of the initially bound 125I-PV dissociates from the membrane within 10 min. These results support the conclusion that PV binds to a membrane-associated receptor. Solubilization studies show that Triton X-100 solubilizes up to 45% of the total membrane-bound 125I-PV. Gel-exculsion chromatography of the solubilized material yields a 500,000 dalton 125I-PV-containing complex separated from free 125I-PV. The 500,000 dalton complex completely dissociates to yield free 125I-PV when incubated with excess unlabeled PV. However, when incubated with (1) no addition, (2) IgG, or (3) serum albumin, the extent of dissociation is significantly reduced and is consistent with that which would be predicted on the basis of the observed dissociation rate in the absence of unlabeled PV. These results suggest that bound 125I-PV can only be displaced by unlabeled PV. These results also indicate that the 500,000 dalton species is a solubilized PV-receptor complex and that it is possible to solubilize the PV-receptor in an active form. 相似文献
8.
Cell-surface proteins of the embryonal carcinoma line C17-S1 1003 (1003) and of some of its mesenchymal derivatives were studied. The surface proteins were labelled with 125I using the lactoperoxidase-glucose-glucose oxidase system either on the cells attached to the culture dishes or after their dissociation. Iodinated proteins were analyzed by two-dimensional gel electrophoresis. The patterns obtained with embryonal carcinoma cells 1003 and with two mesenchymal cell types derived from them, namely embryonic mesenchymal cells (line 10035) and fibroblastic cells (line 10031), were different one from the other, especially when considering the group of proteins labelled on the attached cells. The pattern of cell-surface proteins of the myoblastic line 1168, also derived from C17-S1, was found to be similar to that of 10031 fibroblastic cells. This result is discussed in the light of the phenotypic transition toward myogenesis, which can be obtained with 10031 fibroblastic cells but not with 10035 embryonic mesenchymal cells. A direct method of detection of lectin-binding proteins permitted us to identify the major concanavalin A-binding proteins. Two of them are common to all cell lines studied. They were labeled with 125I on the attached undifferentiated 1003 cells, while in all differentiated derivatives they became available for labelling after the cell detachment only. 相似文献
9.
Wolfgang Stremmel Barry J. Potter Paul D. Berk 《Biochimica et Biophysica Acta (BBA)/General Subjects》1983,756(1):20-27
To characterize a previously proposed hepatocyte albumin receptor, we examined the binding of native and defatted 125I-labeled rat albumin to rat liver plasma membranes. After incubation for 30 min, binding was determined from the distribution of radioactivity between membrane pellet and supernatant following initial centrifugation (15 000 × g for 15 min), after repeated cycles of washing with buffer and re-centrifugation. 125I-labeled albumin recovered in the initial membrane pellet averaged only 4% of that incubated. Moreover, this albumin was only loosely associated with the membrane, as indicated by recovery in the pellet of under 0.5% of the counts after three washes. Binding of 125I-labeled albumin to the plasma membranes was no greater than to erythrocyte ghosts, was not inhibited by excess unlabeled albumin, and was not decreased by heat denaturation of the membranes, all suggestive of a lack of specific binding. Failure to observe albumin binding to the membranes was not due to a rapid dissociation rate or ‘off-time’, as incubations in the presence of sufficient ultraviolet light to promote covalent binding of ligands to receptors did not increase 125I counts bound to the membrane. Finally, affinity chromatography over albumin/agarose gel of solubilized membrane proteins provided no evidence of a membrane protein with a high affinity for albumin. These studies, therefore, do not support the hypothesis that liver cell plasma membranes contain a specific albumin receptor. 相似文献
10.
The binding of 125I-labelled rat uterine luminal proteins from Day-5 pregnant rats showed higher binding affinity to blastocysts than did the binding of proteins in uterine fluid from pro-oestrous rats (Day 0), rat serum albumin (RSA) or bovine serum albumin (BSA). Apparently little uptake of proteins into cells by phagocytosis or entry into the blastocoelic cavity occurred since similar results were obtained in the presence of sodium azide or cytochalasin B. Autoradiographic studies showed that the proteins were localized on the outer surface of the blastocyst. The binding was Ca2+-dependent. Denaturation of Day-5 uterine proteins at 80 degrees C reduced the counts to the values obtained with undenatured RSA and Day-0 fluids; this residual binding was considered as non-specific. The binding of labelled Day-5 uterine proteins was substantially reduced in the presence of unlabelled Day-5 proteins but to a lesser extent in the presence of RSA or rat serum. The dissociation of the bound labelled Day-5 uterine proteins occurred most rapidly in the presence of unlabelled Day-5 proteins. However, dissociation occurred within 2 h in the presence of other macromolecules, suggesting that the binding was not strong. 相似文献
11.
《International journal of radiation applications and instrumentation. Part B, Nuclear medicine and biology》1992,19(6):685-695
In the present in vivo study the uptake kinetics of radioiodinated albumin were determined in normal organs, and tumours of rats using sequential scintigraphy. Rat serum (RSA) was radioiodinated either directly at a tyrosine residue (d-RSA), or indirectly at a residualizing marker tagged to the albumin (rm-RSA). These labelling procedures did not alter the kinetics of labelled albumin, as shown by blood disappearance curves. Directly labelled albumin was shown to have tumour uptake. Residualizing markers like tyramine-cellobiose (TCB), tyramine-deoxysorbitol (TDS) and aminonaphthaltyrimide-deoxysorbitol (ANTDS) are metabolically inert. After the intracellular degradation of the albumin carrier the TCB-, TDS- and ATNDS-residues accumulate in the lysosomes, particularly those of tumour cells. It was able to be demonstrated that residualizing-marker tagged albumin-bound radioactivity was five times higher after 72 h than the tumour radioactivity after use of directly labelled RSA. These data found support when whole-body retention of directly labelled RSA, and residualizing marker-RSAs, were determined. After 72 h, 60% of 131I bound to RSA directly had been excreted, compared to only 25% of the activity attached indirectly to RSA with a residualizing marker. Whole-body autoradiography of rats injected with directly labelled RSA, or residualizing marker-RSA, support these results. Most of the radioactivity of directly labelled RSA was excreted within 24 h, whereas labelled residualizing marker-RSAs were also stored in tumour and liver tissue. ANTDS bound to RSA allows fluorescence microscopy. Cryosections of tumours from rats preinjected 10 min and 24 h with ANTDS-RSA before dissection, demonstrated that the fluorescence is localized on and in tumour cells. This indicates that cellular uptake of the marker takes place. Fluorescence was not observed in muscle tissue. This appears to suggest that the albumin uptake is greater in tumours than in normal tissue, and that it is metabolized in the tumour cells. 相似文献
12.
Nicole Remy-Heintz Richard Magous Jean-Pierre Bali 《Biochemical and biophysical research communications》1980,93(4):1217-1224
The labelling of α1-acid glycoprotein (AGP) with (3H)-sodium borohydride was compared to the labelling with (125I)-sodium iodide by the chloramine T method in view to its use in a radioimmunoassay. The tritium labelling allowed to reach a high specific radioactivity similar to that obtained with iodide ((3H)-AGP: 29.8 mCi/mg; (125I)-AGP: 30.5 mCi/mg). Each mole of sialic acid residue of AGP contains one atom of tritium. The stability of (3H)-AGP was better than that of (125I)-AGP as indicated by its immunoreactivity as a function of time. Immunoreactivities and standard curves were similar for the two tracers but affinity of antiserum was higher for (125I)-AGP than for (3H)-AGP. Tritium labelling by (3H)-borohydride will be very useful for glycoprotein antigens which cannot be labelled with (125I)-iodide. 相似文献
13.
Uptake and degradation of vitamin D binding protein and vitamin D binding protein-actin complex in vivo in the rat.
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We have labelled the rat vitamin D binding protein (DBP), DBP-actin and rat albumin with 125I-tyramine-cellobiose (125I-TC). In contrast with traditional 125I-labelling techniques where degraded radioactive metabolites are released into plasma, the 125I-TC moiety is trapped intracellularly in the tissues, where the degradation of the labelled proteins takes place. By using this labelling method, the catabolism of proteins can be studied in vivo. In this study we have used this labelling technique to compare the tissue uptake and degradation of DBP, DBP-actin and albumin in the rat. DBP-actin was cleared from plasma at a considerably faster rate than DBP. After intravenous injection of labelled DBP-actin complex, 48% of the radioactive dose was recovered in the liver after 30 min, compared with 14% when labelled DBP was administered. Only small amounts of DBP-actin complex were recovered in the kidneys. In contrast with the results obtained with DBP-actin complex, liver and kidneys contributed about equally in the uptake and degradation of DBP determined 24 h after the injection. When labelled DBP was compared with labelled albumin, the amount of radioactivity taken up by the liver and kidneys by 24 h after the injection was 2 and 5 times higher respectively. In conclusion, liver and kidneys are the major organs for catabolism of DBP in the rat. Furthermore, binding of actin to DBP enhances the clearance of DBP from circulation as well as its uptake by the liver. 相似文献
14.
1. A new method is described for labelling proteins to high specific radioactivities with 125I. The protein is treated with a 125I-labelled acylating agent, iodinated 3-(4-hydroxyphenyl)propionic acid N-hydroxysuccinimide ester, which reacts with free amino groups in the protein molecule to attach the 125I-labelled groups by amide bonds. 2. Three protein hormones have been labelled by this method, human growth hormone, human thyroid-stimulating hormone and human luteinizing hormone. Specific radioactivities of up to 170, 120 and 55μCi/μg respectively have been obtained for these hormones. 3. The immunoreactivity of these labelled hormones has been investigated by using a radioimmunoassay system specific for each hormone. These preparations have also been compared with and found to be equal or superior to labelled hormones prepared by chemical substitution of 125I into tyrosine residues of the proteins by using the chloramine-t-oxidation procedure. 4. With some antisera the immunoreactivity of the antigen was diminished by the introduction of a single I atom into the tyrosyl groups, whereas antigen containing a single 125I-labelled 3-(4-hydroxyphenyl)propionamide group showed the same immunoreactivity as the unmodified antigen. 相似文献
15.
It was found that native, as well as125I labelled fibrinogen, may be coupled by means of transglutaminase to surface proteins of malignant plasma cells of the mouse. Binding of fibrinogen, although greatly affecting the [14C] putrescine labelling of several surface proteins, leaves the cells viable and malignant. 相似文献
16.
A possible receptor for thrombin on the platelet membrane has been identified. Whole platelets were treated with 125I-labelled thrombin followed by washing of the platelets, solubilization in Triton X-100, crossed immunoelectrophoresis and autoradiography. A heavily labelled antigen which migrated slightly more slowly than albumin was observed. No corresponding arc was seen on the same immunoplate when stained with Coomassie brilliant blue, indicating that the antigen possessed weak antigenic properties and/or was present in very small amounts. When 125I-labelled thrombin that had been inactivated by phenylmethylsulphonyl fluoride was used, no such labelled arc was seen. The radiolabelled immunoprecipitate does not represent any of the antigens identified hitherto in the immunoelectrophoretic patterns obtained with platelets or platelet material. The electrophoretic mobility of the antigen was influenced neither by neuraminidase treatment of the platelets prior to the 125I-labelled thrombin exposure nor by inclusion of concanavalin A, wheat-germ lectin or lentil lectin in the gel during the first-dimension electrophoresis. This suggests that the antigen does not represent a glycoprotein. Upon subcellular fractionation the radioactively labelled arc was observed in the cytosol fraction following crossed immunoelectrophoresis and autoradiography. Analysis of the secreted proteins after induction of the release reaction with 125I-labelled thrombin revealed labelling of immunoprecipitates representing thrombospondin, albumin and the 'line' form of platelet factor 4. This confirms that stable complexes of 125I-labelled thrombin and platelet proteins can exist in the presence of Triton X-100 and during electrophoresis. 相似文献
17.
Summary Chinese hamster cells (Cl : 1) were labelled with3H-thymidine or125Iododeoxyuridine for 18 h and after 3 h in non-radioactive medium they were stored at 0° C up to 6 h. The number of DNA strand breaks observed after the labelling period (37° C) or after treatment at 0° C was determined using the DNA-unwinding technique.125I-decays in DNA were significantly more efficient than3H-decays in introducing unrepairable DNA strand breaks during the labelling period. 32% of125I-induced and 3% of3H-induced DNA strand breaks were unrepaired after 21 h at 37° C. Comparison between the effects of125I- or 3H-disintegrations in DNA in three different ways shows 7–12 times more pronounced effects for125I-decays. For125I-labelled cells 3–4 DNA strand breaks were found per decay and the corresponding value for3H- labelled cells was 2. 相似文献
18.
《Journal of receptor and signal transduction research》2013,33(2):233-253
AbstractThis study was undertaken in order to improve photoaffinity labelling efficiency of pancreatic cholecystokinin receptor by the cleavable probe 125I-ASD-(Thr28, Ahx31)-CCK-25-33 and to further characterize the denaturated receptor and is agonist binding domain. Membrane bound pancreatic cholecystokinin receptor was specifically labelled by 125I-ASD-(Thr28, Ahx31)-CCK-25-33 as a component of Mr ≈ 85,000-100,000. The efficiency of the photolabelling was 3–4%. Performing photolysis on [125I-ASD-(Thr28, Ahx31)-CCK-25-33-receptor] complexes solubilized by CHAPS did not affect specificity of the labelling reaction but enhanced its efficiency so that up to 10% of the receptor site population could be cross-linked. Several lectins were tested for their ability to recognize and purify the cholecystokinin receptor denaturated by Nonidet P-40. Wheat germ agglutinin provided the best recovery and purification rate. The receptor was fully adsorbed on immobilized wheat germ agglutinin, while only a fraction was retained on ricin II (28%) and Ulex europaeus (28%), thus suggesting that the receptor is heterogeneously glycosylated. Finally, major labelled receptor fragments were generated by enzymatic digestion. There were: endoproeinase Glu-Mr → C ≈ 34,000; endoproteinase Glu-C/trypsin → Mr ≈ 12,000; chymotrypsinlendoproteinase Glu-C → Mr ≈ 16,000 and 12,000. The fragment of Mr 2 34,000 was deglycosylated to a component of Mr ≈ 22,000 whereas the other fragments were insensitive to deglycosylation Such results strongly suggest that cholecystokinin binding occurs in a non-glycosylated domain of the cholecystokinin receptor protein. 相似文献
19.
Brian R. Booth 《Biochemical and biophysical research communications》1980,94(4):1029-1036
Cell surface proteins of K12 have been labelled with 125I using a lactoperoxidase method. Results suggest that most outer membrane proteins so far characterised appear to have at least part of their polypeptide chain on the cell surface. These include major outer membrane protains I and II1, the maltose and vitamin B12 binding proteins and proteins involved in iron transport. The labelling of an antibiotic sensitive mutant and its parent were compared but their labelling patterns did not appear to differ in any way which would suggest the cause of the permeability difference between these two strains. 相似文献
20.