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1.
Abstract

A 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   

2.
Abstract: Properties so far studied of the 125-kDa 14C-arginylated protein from rat brain show remarkable similarities with those of the STOP (stable tubule only polypeptide) protein. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the 125-kDa 14C-arginylated protein moves to the same position as the STOP protein. The 125-kDa 14C-arginylated protein was immunoprecipitated by the monoclonal Mab 296 antibody specific for neuronal STOP protein. The 125-kDa 14C-arginylated protein was retained by a calmodulin column like STOP protein. As occurs with the STOP protein, the 125-kDa 14C-arginylated protein is found in higher proportion in cold-stable than in cold-labile microtubules. However, the modified protein associates with microtubules in a lower proportion than the STOP protein. We conclude that the STOP protein incorporates arginine by a posttranslational reaction but that only a small fraction of the STOP protein shows acceptor capacity in vitro.  相似文献   

3.
Transformation of Pseudomonas putida with chromosomal DNA   总被引:3,自引:0,他引:3  
The in vitro deiodination of [125I]-4-iodobiphenyl, [125I]-4-iodonitrobenzene and [125I]-4-iodoaniline was investigated. No deiodination was detected in rat thyroid homogenates. However, at least three biodeiodination mechanisms were indicated for substrates in rat liver subcellar fractions. Microsomal dehalogenation occurred to a minor extent with increased dehalogenation taking place in the cytosol fraction. The cytosol deiodination was extensive for 4-iodonitrobenzene and was mediated by glutathione. A second cytosol deiodination mechanism, not mediated by glutathione, was evident when 4-iodobiphenyl was the substrate. This soluble enzyme system could be enhanced by Arochlor 1254 or 4-iodobiphenyl pretreatment.  相似文献   

4.
Binding sites for calcitonin (CT), as assayed by the displacable binding of [125-I] iodo salmon CT ([125-I]sCT), were found on a membrane fraction prepared from rat brain. The half times of association varied between 23 and 7 min as a function of the temperatures used in the incubation medium, ranging from 6° to 37°C. Salmon CT in amounts as low as 10?10 M inhibited the binding of [125-I]sCT to the membranes, whereas the virtually biologically inactive free acid of human CT and human CT sulfone did not affect the binding. The specific binding of [125-I]sCT to the membranes was directed to structural and/or conformational features in the COOH-terminal half of salmon CT. 133 to 8,900 times higher amounts of porcine CT and human CT and analogues thereof were required to achieve an inhibition of binding equal to that produced by salmon CT. Sixty-seven percent of specific binding of labeled hormone was not dissociable, even after 6 h of incubation with an excess of unlabeled hormone. [125-I]sCT extracted from the membranes was not degraded, as judged by gel permeation chromatography, and retained binding activity. Specific binding was highest in the hypothalamus, followed by the brainstem. It was intermediate in the midbrain-thalamus and the striatum, lower in the cortex and negligible in the hippocampus, and cerebellum and the spinal cord.  相似文献   

5.
Neonatal brain development in the rat is adversely affected by malnutrition. Alterations in tissue binding of IGF-I in the malnourished brain were tested in rat pups from mothers who were fed a 20% protein diet (C) or a 4% protein diet (M) starting from day 21 of gestation and continued throughout suckling. IGF-I binding in both cortex and cerebellum decreased progressively in C and M groups from day 6 to day 13. At day 9, 11, and 13, the binding was significantly greater (p < 0.02) in M compared to C groups. To investigate whether these changes might be related to the alteration in receptor activity, membranes were incubated with 125I-IGF in the presence of excess insulin with or without unlabeled IGF-I. In the absence of insulin, specific IGF-I binding in the M group was increased by 41.8 ± 13.8% (mean ± SEM p < 0.05) relative to C group. Insulin produced a consistent but incomplete inhibition of binding in both C and M, of 75% and 67% respectively. In addition, the specific IGF-I binding in the presence of insulin was increased in M group by 70.2 ± 9.4% relative to C, p < 0.05. To characterize the nature of this binding, cerebral cortical membranes, from both groups, incubated with 125I-IGF-I were cross-linked, and electro-phoresed on 6% and 10% SDS-PAGE gels under reducing conditions. Autoradiography of the 6% gel showed two specific bands at 115 kD and 240 kD, consistent with monomeric and dimeric forms of the IGF-I receptor, which were inhibited by excess insulin. In contrast, a 10% gel showed an additional band at 35 kD (IGF-binding protein) that was not inhibited by insulin. In both gels, membrane preparations from the M group showed a heightened intensity of the bands relative to C. The increase in binding protein relative to the receptor suggests a disequilibrium that may limit the availability of exogenous IGF-I to the tissues.  相似文献   

6.
—A separation of soluble brain proteins and Con A-binding glycoproteins by chromatography on calcium hydroxylapatite in the presence of SDS is described. Seventeen Coomassie Blue-stained bands were detected by polyacrylamide gel electrophoresis in SDS of Con A-binding glycoproteins of the soluble fraction of rat brain: 16 of these were found by in vivo uptake of [3H]fucose to be fucosylglycoproteins. Hydroxylapatite chromatography yielded several glycoprotein pools, each of which was shown by gel electrophoresis to contain between 4 and 8 individual glycoproteins. Such pools were enriched in [36H]fucose relative to the brain soluble fraction by factors of between 6 and 21. Preliminary experiments demonstrate that this method is also applicable to the fractionation of membrane-bound glycoproteins.  相似文献   

7.
Many steroid and thyroid hormones and some drugs are bound by circulating red cells. Red cell-bound ligand may not be physiologically inert, as recent studies show that red cell-bound drug is available for uptake by brain. To investigate whether triiodothyronine (T3) is available for uptake by brain in vivo from the circulating red cell pool, the present investigations measure the effects of human erythrocytes on rat brain uptake of [125I]T3 in vivo. The fraction of circulating T3 available for uptake in vivo in the presence of 0, 2, 5, 10, 22, or 44% red cells was essentially identical to the fraction of [125I]T3 unbound in vitro. Therefore, [125I]T3 bound to red cells obtained from normal volunteers is not available for uptake by brain in vivo.  相似文献   

8.
In this study we have examined whether rat brain nuclear thyroid hormone receptors bind T4 or metabolites of T4 and whether there is a developmental change in brain T4 metabolism and binding. Developing animals were injected with trace [125I]3,5-tetraiodothyronine ([125I]T4) and after sacrifice brain nuclear and cytoplasmic fractions were examined to determine whether their radioactivity was represented by the injected [125I]T4 or any of its metabolites. Of the radiothyronines specifically bound to the nucleus, 90% was found to be triiodothyronine ([125I] T3) and 10% was [125I]T4. Of the cytoplasmic, protamine sulfate-precipitable fraction, 40% was [125I]T4 and 60% [125I]T3. Inasmuch as the percentage of [125I] T3 found in plasma during the same postinjection interval was similar to that present as contaminant of the injected material, it was concluded that brain [125I] T3 derives from local monodeiodination of T4 to T3. The main developmental change observed was a marked decline in the total cytoplasmic and nuclear [125I] T4 uptake. However, with development, the T3/T4 ratio remained constant in the nuclear fraction while it decreased in the cytoplasmic fraction. It is concluded that although T3, deriving from monodeiodianation of T4, is the main form of thyroid hormone that regulates brain development by its binding to brain nuclear receptors, the fact that T4 is the most available from during the critical period makes it, indirectly, very important to brain development. Further, the decline observed with development in T4 uptake and monodeiodination to T3, may contribute to the concomitantly declining role of thyroid hormones on brain tissue.  相似文献   

9.
Abstract: Specific binding of glucagon-like peptide (GLP)-1(7–36)amide was detected in several rat brain areas, with the highest values being found in hypothalamic nuclei and the nucleus of the solitary tract. In hypothalamus and brainstem homogenate binding of 125I-GLP-1(7–36)amide was time, temperature, and protein content dependent and was inhibited by unlabeled proglucagon-derived peptides. The rank order of potency was GLP-1(7–36)amide ? GLP-1(1–36)amide > GLP-1(1–37) ? GLP-2 > glucagon. Scatchard analysis of the steady-state binding data was consistent with the presence of both high- and low-affinity binding sites in hypothalamus and brainstem. Brain 125I-GLP-1(7–36)amide-binding protein complexes were covalently cross-linked using disuccinimidyl suberate and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single radiolabeled band of Mr 56,000 identified in both hypothalamus and brainstem homogenates was unaffected by reducing agents. An excess of unlabeled GLP-1(7–36)amide abolished the band labeling, whereas glucagon had no effect. Other unlabeled GLPs inhibited Mr 56,000 complex labeling with the following order of potency: GLP-1(1–36)amide > GLP-1(1–37) > GLP-2. The binding of 125I-GLP-1(7–36)amide and the intensity of the cross-linked band were similarly inhibited in a dose-response manner by increasing concentrations of unlabeled GLP-1(7–36)amide. Covalent Mr 56,000 125I-GLP-1(7–36)amide-binding protein complexes solubilized by Triton X-100 were adsorbed onto wheat germ agglutinin. Our results suggest that the GLP-1(7–36)amide receptor in rat brain is a glycoprotein with a single binding subunit that has a greater molecular weight but binding features and ligand specificity similar to those of its peripheral tissue counterparts.  相似文献   

10.
In this study we examined whether adult rat brain tissue (cerebral hemispheres) would under cold exposure respond with changes in the local metabolism and nuclear binding of thyroid hormones (T3, T4). Adult, control rats kept at 22°C and cold exposed (4°C, 20 h) rats were injected with trace of 125I-T4 or 125I-T3 returned to their respective environment and sacrificed four hours later. The radioactive hormonal forms were identified and quantified in the cytoplasmic and nuclear fractions. It was found that in cold exposed rats injected with 125I-T4, the total cytoplasmic radioactivity was higher than that of controls. This increase was not associated with 125I-T4 but it reflected an increase (88 %) in its deiodination product 125I-T3 (125I-T3 (T4)). Although total cytoplasmic 125I-T4 did not change, there was a decrease (28%) in its protein free cytoplasmic fraction. 125I-T3 (T4) and 125I-T4 bound to the nuclear fraction were found to decrease by 58 and 46% respectively. Cold exposed animals injected with 125I-T3 also showed an increase in cytoplasmic 125I-T3 (81%) and a decrease in 125I (40%) whereas 125I-T3 bound to the nuclear fraction decreased by 64%. These results indicate that cold exposure of rats decreases brain local T3 metabolism and nuclear binding while it does not effect local T4 metabolism.  相似文献   

11.
The effect of elevated heliox pressure (101 ATA) on activity of Na+,K+-ATP-ase and some characteristics of fatty acid composition was studied in membrane phospholipids of trout and rat brain synaptosomal and mitochondrial fractions. The Na+,K+-ATP-ase activity was shown to decrease by 25% in both fractions of the rat brain; in mitochondrial fraction of the trout brain it decreased by 47%, while in synaptosomal fraction, only by 11%. It has also been established that under experimental conditions, the unsaturation index of fatty acids of phosphatidylcholine and phosphatidylethanolamine of trout synaptosomes decreased, with no changes in these lipids in mitochondrial fraction. The phosphatidylcholine unsaturation index in rats did not practically change in both fractions, while in rat phosphatidylethanolamine it increased in mitochondrial fraction and slightly decreased in synaptosomal fraction. Thus, under conditions of high pressure the reduction of the enzyme activity is also determined, specifically, by peculiarities of the phospholipid fatty acid composition in the subcellular fractions studied. A possibility of changes of the enzyme activity as a result of transition of its lipid component from the liquid crystalline to the gel state under effect of an enhancement of lipid peroxidation under conditions of elevated pressure is discussed.  相似文献   

12.
Synaptic vesicles from rat brain were labeled with125I, and the association of the vesicles with other subcellular components of brain was examined using a centrifugation assay. Copper at micromolar concentrations enhances the binding of the vesicles to the synaptic membrane as well as other fractions. Magnesium, Ca2+, and calmodulin with Ca2+ are ineffective. There is virtually no binding of synaptic vesicles to the microtuble fraction and only a slight enhancement with Cu2+. These findings support the hypothesis that Cu may serve as a bridge between synaptic vesicles and the plasma membrane.  相似文献   

13.
Calmodulin binding proteins in bovine thyroid plasma membranes were investigated using the 125I-labeled calmodulin gel overlay technique. The purified thyroid plasma membranes contained two calmodulin binding proteins with molecular weights of approx. 220 000 and 150 000 respectively. The binding of 125I-labeled calmodulin to the calmodulin binding proteins was inhibited by excess unlabeled calmodulin, 100 μM trifluoperazine or 1 mM EGTA, indicating that the binding was calmodulin-specific and calcium-dependent. The calmodulin binding proteins appear to be components of the cytoskeleton since they remained in the pellet after treatment of the thyroid plasma membranes with 1% Triton X-100. Similar calmodulin binding proteins were present in rat liver plasma membranes, but not in human red blood cell plasma membranes. These two calmodulin binding proteins may interact with other components of the cytoskeleton and regulate endocytosis, exocytosis and hormone secretion in thyroid cells.  相似文献   

14.
Cedel TE 《Plant physiology》1980,66(4):696-703
This study employs 125I-labeled phytochrome (125I-P) from oats to quantitate the binding of phytochrome to a membrane fraction from oats that is highly enriched for mitochondria, and it examines several parameters that influence this attachment. The binding of 125I-Pfr to the mitochondrial fraction of unirradiated oat seedlings is significantly higher than that of 125I-Pr. However, 125I-Pfr and 125I-Pr bind in equal quantities to mitochondrial preparations isolated from light-exposed seedlings. Maximum 125I-Pfr binding to membranes from light-exposed plants occurs within 30 seconds and is optimized in a reaction buffer containing 5 millimolar MgCl2 at pH 6.8. Scatchard plots of the binding data for Pfr indicate a single high-affinity site with an affinity constant of 1.79 × 1011 per molar. When optimal binding conditions are used, over 20% of the 125I-P added is bound and a stoichiometry of about 100 molecules per mitochondrion is attained. When the specificity of binding is tested using competition experiments with a 15-fold excess of unlabeled phytochrome, 125I-Pfr shows no specific binding to rat liver mitochondria.  相似文献   

15.
To cross-link the 3′-terminus of 5 S RNA to its neighbouring proteins, ribosomal 60 S subunits of rat liver were oxidized with sodium periodate and reduced with sodium borohydride. 5 S RNP was then isolated by EDTA treatment followed by sucrose density-gradient centrifugation and subjected to SDS-polyacrylamide gel electrophoresis. The protein with a slower mobility than the L5 protein, which was thought to be cross-linked 5 S RNP, was labeled with 125I, treated with RNAase, and analyzed by two-dimensional polyacrylamide gel electrophoresis, followed by radioautography. A radioactive spot located anodically from L5 protein was observed, suggesting that it is the L5 protein-oligonucleotide complex. When analyzed by SDS slab polyacrylamide gel electrophoresis followed by radioautography, the peptide pattern of the α-chymotrypsin digest of this 125I-labeled protein-oligonucleotide complex was similar to that of the digest of 125I-labeled L5 protein. The results indicate that L5 protein binds to the 3′-terminal region of 5 S RNA in rat liver 60 S subunits.  相似文献   

16.
Low temperature and the lysosomotropic agent, chloroquine, were used to study the degradation of 125I-insulin in a perfused rat liver. Insulin (1.5 × 10?9m) was removed from the perfusate at 35 °C with a T12 of 12 min, and this process was slowed to 35 min at a temperature of 17 °C. Essentially no degradation of 125I-insulin took place in the liver at 17 °C. After 90 min at that temperature 64% of the liver radioactivity had accumulated in the microsomal fraction of the tissue homogenate, while at 35 °C 60% of the radioactive material was in the supernatant fraction. Greater than 80% of the supernatant radioactivity was acid soluble. Rapid warming of a 17 °C-treated liver to 35 °C allowed the accumulated 125I-insulin in the microsomal fraction to be degraded to acid-soluble products in the normal manner. Chloroquine (0.2 mm) also caused the liver to degrade insulin more slowly. At 60 min after adding 125I-insulin to the chloroquine-treated liver, 50% of the radioactivity in the tissue was still present in the lysosome-rich fraction of the homogenate, while less than 10% was in this fraction in a control liver. The effects of low temperature show transfer of insulin to its degradative site is rate limiting for hormone catabolism and the inhibition by chloroquine suggests lysosomes have a role in insulin degradation by the liver.  相似文献   

17.
The effect of platelet-activating factor (PAF) on protein tyrosine phosphorylation was studied in rat hippocampal slices. PAF caused an increase in the tyrosine phosphorylation of two phosphoproteins, which we identified by immunoprecipitation assays as the focal adhesion kinase p125FAK and crk-associated substrate p130Cas. The PAF effect was time- and dose-dependent. In addition, the involvement of PAF receptor was demonstrated by using PCA-4248, a specific receptor antagonist. When NO synthase was inhibited by NG-monomethyl-L-arginine (L-NMA), PAF-stimulated protein tyrosine phosphorylation was inhibited. In conclusion, our results indicate that PAF increased the tyrosine phosphorylation of both p125FAK and p130Cas proteins by the production of NO in hippocampus, suggesting that PAF may play a role in the functioning of this cerebral area.  相似文献   

18.
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   

19.
Histamine H3 receptor therapeutics have been proposed for several diseases such as schizophrenia, attention deficit hyperactivity disorder, Alzheimer''s disease and obesity. We set out to evaluate the novel compound, [125I]WYE-230949, as a potential radionuclide imaging agent for the histamine H3 receptor in brain. [125I]WYE-230949 had a high in vitro affinity for the rat histamine H3 receptor (Kd of 6.9 nM). The regional distribution of [125I]WYE-230949 binding sites in rat brain, demonstrated by in vitro autoradiography, was consistent with the known distribution of the histamine H3 receptor. Rat brain uptake of intravenously injected [125I]WYE-230949 was low (0.11 %ID/g) and the ratio of specific: non-specific binding was less than 1.4, as determined by ex vivo autoradiography. In plasma, metabolism of [125I]WYE-230949 into a less lipophilic species occurred, such that less than 38% of the parent compound remained 30 minutes after injection. Brain uptake and metabolism of [125I]WYE-230949 were increased and specific binding was reduced in anaesthetised compared to conscious rats. [125I]WYE230949 is not a potential radiotracer for imaging rat histamine H3 receptors in vivo due to low brain uptake, in vivo metabolism of the parent compound and low specific binding.  相似文献   

20.
Changes in the activity of a collagen peptidase, PZ-peptidase, acting on a synthetic substrate [4-Phenylazobenzyloxycarbonyl(PZ)-l-Pro-l-Leu-Gly-l-Pro-l-Arg] for bacterial collagenase were examined in developing rat brain regions. The hypothalamus, pons-medulla, colliculi, cerebellum, ceerbrum, midbrain and pituitary gland were studied in rats ranging in age from 1 week to adult; PZ-peptidase activity continuously decreased with maturation in all of the brain regions examined except the hypothalamus. The pituitary gland showed the highest activity in all of the brain regions. PZ-peptidase activity in crude mitochondrial and supernatant fractions from rat whole brain had an optimum pH between 7.5–8.0. It was strongly inhibited by p-chloromercuribenzoic acid, N-ethylmaleimide or EDTA. whereas iodoacetic acid did not affect the enzyme activity. Among various metal ions, the enzyme activity was inhibited by Zn+2 or Cu+2 but not by Mn+2, Ca+2, Mg+2 or Na+. There is no inhibition of the activity by serine protease inhibitors, including diisopropylfluorophosphate and phenylmethylsulphonyl fluoride. An approximate molecular weight of this enzyme was estimated to be 68,000 by gel filtration. Since these properties of rat brain PZ-peptidase were similar to those of other peripheral PZ-peptidases, we suppose that PZ-peptidase in the brain may be the same molecule as the enzyme which hydrolyses collagen peptides in peripheral tissues, but it may have some different physiological roles.  相似文献   

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