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1.
A R Merrill  W A Cramer 《Biochemistry》1990,29(37):8529-8534
The voltage dependence of channel activity of the bactericidal protein colicin E1 was found to be correlated with insertion into the membrane bilayer of a specific segment of the 178-residue COOH-terminal thermolytic colicin channel peptide. The insertion into the bilayer was detected by an increase in labeling by one of two different lipophilic photoaffinity probes or by a decrease in iodination of peptide tyrosines from the external solution. Imposition of a potassium diffusion potential of -100 mV resulted in an increase of 35-60% in the labeling of the peptide by the lipophilic probe in the bilayer and a concomitant decrease in labeling of Tyr residues in the peptide by the iodination reagent in the external solution. The change in labeling decreased upon dissipation of the membrane potential with a half-time of about 1 min. The labeling change was localized to a 36-residue peptide segment bounded by alanine-425 and by tryptophan-460. This segment containing seven positively charged residues at low pH is a voltage-sensitive region that inserts into the membrane bilayer when the channel is turned on by the potential and is extruded from it when the voltage is removed and the channel is turned off.  相似文献   

2.
S Ganguly 《FEBS letters》1987,224(1):198-200
Insulin receptors from human placenta have been labeled by using an oxidative iodination procedure (iodogen-mediated or chloramine-T-mediated), Bolton-Hunter reagent or [3H]acetic anhydride. The oxidative iodination procedure reduces the affinity for 131I-insulin and the receptor protein becomes fragmented into smaller pieces with an s20,w value of 5-6. However, treatment with Bolton-Hunter reagent or [3H]acetic anhydride does not alter the Kd of 131I-insulin binding and the s20,w value remains unchanged with respect to the native receptor. It is proposed that for labeling multisubunit sulfhydryl-linked protein drastic oxidative iodination procedures should be avoided.  相似文献   

3.
For tracer or analytical studies it is often useful to label proteins by direct iodination or by reacting them with an iodinated reagent. A simple iodination technique with hydrogen peroxide is described for use with either carrier-free or low-specific-activity iodine. The method introduces less oxidative damage to proteins than any other procedure tested, yet the efficiency of labeling approaches that offered by the chloramine T or Iodogen methods. The method has been applied to the facile and inexpensive preparation of the iodinated Bolton-Hunter reagent. This peroxide iodination procedure should be particularly useful for labeling proteins or peptides for structural investigations or for immunoassays.  相似文献   

4.
For tracer or analytical studies it is often useful to label proteins by direct iodination or by reacting them with an iodinated reagent. A simple iodination technique with hydrogen peroxide is described for use with either carrier-free or low-specific-activity iodine. The method introduces less oxidative damage to proteins than any other procedure tested, yet the efficiency of labeling approaches that offered by the chloramine T or Iodogen methods. The method has been applied to the facile and inexpensive preparation of the iodinated Bolton-Hunter reagent. This peroxide iodination procedure should be particularly useful for labeling proteins or peptides for structural investigations or for immunoassays.  相似文献   

5.
M A Markwell  C F Fox 《Biochemistry》1978,17(22):4807-4817
The use of the iodinating reagent 1,3,4,6-tetrachloro-3alpha,6alpha-diphenylglycouril (chloroglycoluril) to selectively label membrane surface proteins was investigated with the following systems: enveloped viruses (Sendai and Newcastle disease viruses), human erythrocytes, and nucleated cells propagated both in suspension (EL-4) and in monolayer culture (BHK-21). Conditions are described for specifically iodinating surface proteins while maintaining full virus integrity or cell viability. Comparison of the chloroglycoluril method with the lactoperoxidase and chloramine-T methods for labeling surface membrane proteins shows that the chloroglycoluril method has a number of advantages: It routinely produces a 3- to 17-fold greater specific radioactivity without sacrificing viral or cellular integrity, it is technically simpler to use, it does not require the addition of extraneous protein to initiate the reaction nor a strong reducing reagent to terminate it. Chloroglycoluril also proved to be an effective substitute for chloramine-T in the nonvectorial labeling of viral and cellular proteins. Membrane protein samples were solubilized with the detergent sodium dodecyl sulfate before iodination or labeled in the presence of high iodide concentrations without prior solubilization. The resulting specific radioactivities generated by the use of chloroglycoluril were equal to or greater than those generated by the chloramine-T method. The effectiveness, simplicity of use, and versatility of chloroglycoluril recommend it as an iodinating reagent for both surface-specific and nonvectorial labeling of membrane systems.  相似文献   

6.
Iodinated derivatives of the hormone avian pancreatic polypeptide   总被引:1,自引:0,他引:1  
Reaction of avian pancreatic polypeptide with an iodine monochloride reagent at both pH 4 and pH 7.5 results in the differential modification of the four tyrosine residues in this peptide hormone. A total of 19 distinct iodinated derivatives were isolated by reverse-phase high-performance liquid chromatography, and their sites of iodination were characterized by both tryptic mapping and leucine aminopeptidase techniques coupled with HPLC. The pH 4 reaction produced 16 derivatives which, overall, represented substantial iodination at each tyrosine residue, whereas the pH 7.5 reaction was more directed, producing only 7 derivatives. Iodination at the C-terminal tyrosineamide 36 predominated at both pH values, and diiodo-Tyr 36 was found in the majority of the pH 7.5 derivatives. The relative of the four tyrosine residues with ICl were as follows: at pH 7.5, Tyr 36 much greater than Tyr 21 much greater than Tyr 27 greater than Tyr 7; at pH 4, Tyr 36 greater than Tyr 27 greater than Tyr 7 greater than Tyr 21.  相似文献   

7.
Studies on the mechanism of the iodination of tyrosine by lactoperoxidase   总被引:1,自引:0,他引:1  
Studies with lactoperoxidase showed that a highly reactive intermediate is produced (on the enzyme) from I- and H2O2 which then diffuses from the enzyme and very rapidly and indiscriminately iodinates any Tyr or peptides containing Tyr which are in the same solution. The evidence supporting these conclusions follows. 1) The rate followed the Michaelis-Menten pattern with I- and H2O2 while the concentration of Tyr peptides had no measurable effect on the rate; 2) the rates of reaction were independent of the type of peptide in which Tyr was located; 3) the amount of iodination which had occurred after the reaction had gone to completion and the amounts of monoiodination and diiodination after completion of the reaction were independent of the peptide type, the pH, the solvent polarity, or the ionic strength; 4) competition for reaction by two very different Tyr peptides depended only on their initial concentrations; and 5) iodination of a large protein occurred through a dialysis membrane. Free Tyr was iodinated at the same rate as Tyr peptides by lactoperoxidase, but monoiodotyrosine and m-fluorotyrosine were iodinated at one-half that rate. The results also showed that one can choose ratios of [peptide] to [H2O2] such that monoiodination is maximized relative to diiodination. It was also found that the iodination capacity of a mixture of I- and H2O2 with lactoperoxidase (when Tyr was absent) was only slowly dissipated. Finally, the results showed that lactoperoxidase can be used to brominate and chlorinate Tyr peptides at a slow rate.  相似文献   

8.
Iodination of the C-terminal half-molecule domain of ovotransferrin (OTF) causes a significant reduction in binding to transferrin receptors on chick reticulocytes when compared to the binding observed with holo-OTF or the N-terminal half-molecule domain. (In such studies binding of iodinated half-molecule is measured in the presence of equimolar unlabelled complementary half-molecule). In particular iodination of the C-terminal half-molecule domain by the solid-phase reagent Iodogen resulted in half the binding found when ICl was used. The iodinated N-terminal half-molecule domain labelled by either Iodogen or ICl showed consistently higher binding than was observed with the C-terminal half-molecule or Fe2OTF. Although the molecular basis for the reduced binding of these proteins relative to the N-terminal half-molecule has not been definitively established, the implication is that there is a Tyr in the C-terminal domain which is involved in receptor recognition and binding. Addition of one or more bulky iodine atoms to the Tyr interferes with the interaction. Tryptic peptide maps of unlabelled holo-OTF and half-molecule domains and of the half-molecule domains labelled by both ICl and Iodogen are presented. The maps indicate limited access of the tyrosine residues to iodination especially in the C-terminal half-molecule domain. Equilibrium binding experiments have been carried out to compare the Kd (the apparent dissociation constant for the interaction between OTF and the transferrin receptors on chick-embryo red blood cells) with the Bmax, (binding at infinite free-ligand concentration) for Fe2OTF labelled using ICl, Iodogen, Enzymobeads and Chloramine-T. The effect of labelling Fe2OTF by Bolton-Hunter reagent has also been assessed. These studies show that ICl appears to be the reagent of choice for labelling Fe2OTF and its half-molecule domains.  相似文献   

9.
J T Dunn  A D Dunn 《Biochimie》1999,81(5):505-509
Thyroglobulin (Tg) is the most important protein in the thyroid because it provides the matrix for thyroid hormone biosynthesis. Here we review experimental work, principally from our laboratory, on the relationship between Tg structure and hormonogenesis. Early work showed that Tg's most important hormonogenic site was located in a fragment of approximately 26 kDa obtained on chemical reduction. With the establishment of the cDNA sequence of Tg, this and other major sites could be localized within Tg's polypeptide chain. The four major hormonogenic sites, designated A, B, C, and D, are located respectively at tyrosyls 5, 2553, 2746, and 1290. In most species, site A accounts for about 40% of Tg's hormone, and site B for about 25%. Site C is associated with increased T3, at least in some species. Site D is prominent in guinea pigs and rabbits, and TSH favors hormonogenesis at it in these species. Sequential iodination of low iodine human Tg shows three consensus sequences associated with early iodination and with T4 formation. Recent work has identified Tyr130 in beef Tg as donor of an outer iodothyronine ring, most likely to Tyr5, the most important hormonogenic site. In addition to its biochemical importance, Tg has clinical interest in familial goiter and autoimmune thyroid disease. Further elucidation of Tg structure and its relation to thyroid hormone synthesis will contribute to thyroid physiology and to its clinical application.  相似文献   

10.
It was established previously that the 15-kDa protein phosphorylated in 3T3-L1 adipocytes treated with insulin and phenylarsine oxide is O-phospho-Tyr19 422(aP2) protein, a fatty acid-binding protein. To assess its capacity to serve as substrate of the insulin receptor tyrosine kinase in vitro, native 422(aP2) protein was isolated from 3T3-L1 adipocytes and purified to homogeneity. Receptor-catalyzed phosphorylation of 422(aP2) protein on Tyr19 was markedly activated when long-chain fatty acid, e.g. oleic acid, is bound to the protein. Fatty acid had no effect on autophosphorylation of the insulin receptor by its intrinsic tyrosine kinase. Both saturated (C14, C16, and C18) and unsaturated (all cis-delta 9 C16, -delta 9 C18, and -delta 9,12 C18, -delta 9,12,15 C18, and -delta 5,8,11,14 C20) fatty acids caused substrate activation. The Km for 422(aP2) protein was greatly reduced (from 170 to 3 microM) by oleic acid with little or no effect on Vmax. Upon binding fatty acid to 422(aP2) protein the susceptibility of Tyr19 and Tyr128 to iodination by the lactoperoxidase method increased greatly. These results indicate that upon binding fatty acid, 422(aP2) protein undergoes a conformational change whereby Tyr19, which lies within a consensus-type sequence for tyrosine kinase substrates, becomes accessible for phosphorylation by the insulin receptor tyrosine kinase and to iodination by lactoperoxidase.  相似文献   

11.
A rapid procedure for the separation of Escherichia coli heat-stable enterotoxin iodination products is described. A two-step procedure using reverse-phase chromatography results in the separation of native, noniodinated heat-stable enterotoxins from several distinctly iodinated species which differ in their ability to bind to brush border receptors. Sequence analysis of the radioligands demonstrated mutually exclusive labeling at the 4Tyr and 18Tyr residues. These tracers have been immunologically and biologically characterized and have been shown to be antigenically similar but to differ in their toxicity for suckling mice. The radioligands separated by this procedure demonstrate stable behavior for a period of months.  相似文献   

12.
Radiolabeling of native proteins conventionally has required iodination using 125Iodine (125I). Although radioiodination can result in high specific activity, there are several drawbacks in the use of 125I (e.g., radiological hazards and short half-life). 14C-Methylamine-glutaraldehyde conjugation to proteins offers an alternative for radiolabeling of proteins that is safer and longer-lived alpha-2-Macroglobulin was radiolabeled by conjugation to a 14C-methylamine-glutaraldehyde conjugate. Analysis of the labeling procedure was performed using scintillation counting, gel filtration chromatography, and protein assays. The radiolabeled alpha-2-macroglobulin was activated using established protocols and tested for functional integrity using competitive binding assays in the presence of recombinant receptor associated protein, an alternative ligand for the alpha-2-macroglobulin cellular receptor. The function of alpha-2-macroglobulin was unaffected by the labeling procedure. Comparison of 14C-methylamine-labeling and iodination by Scatchard analysis yielded nonlinear plots that suggested the presence of two sets of receptors with different binding affinities but that do not show cooperativity. This technique offers an alternative to radioiodination for the sensitive labeling of proteins.  相似文献   

13.
From the cyanogen bromide (CNBr) treatment of porcine thyroglobulin a peptide of mol. wt. 15 000, CNBr-b1, was purified by gel filtration and ion-exchange chromatography. CNBr-b1 contained 50% of the thyroxine (T4) content of the protein. After digestion with trypsin and protease from Staphylococcus aureus V-8, thyroxine-containing peptides were purified and analyzed by microsequence analysis using the colored Edman's reagent dimethylaminoazobenzeneisothiocyanate . Two different sequences harboring T4 were identified: sequence 1, His-Asp-Asp-Asp-T4-Ala-Thr-(Glx,Gly)-Leu-Tyr-Phe-Ser-Ser-Arg, which contains 1 mol T4/mol peptide and sequence 2, Asp-(Tyr/MIT/DIT/T4)-Phe-Ile-Leu-X-Pro-Val-, which is a mixture of the same peptide at different levels of iodination and coupling. These sequences are likely to be representative of distinct hormonogenic sites, the former giving evidence of early iodinated tyrosine residues where preferential coupling into hormonal residues occurs especially at low iodine levels and the latter representing less reactive site(s) operative at higher iodine levels.  相似文献   

14.
A new method has been developed for the chemical modification and labeling of carboxyl groups in proteins. Carboxyl groups are activated with Woodward's reagent K (N-ethyl-5-phenylisoxazolium 3'-sulfonate), and the adducts are reduced with [3H]BH4. The method has been applied to the anion transport protein of the human red blood cell (band 3). Woodward's reagent K is a reasonably potent inhibitor of band 3-mediated anion transport; a 5-min exposure of intact cells to 2 mM reagent at pH 6.5 produces 80% inhibition of transport. The inhibition is a consequence of modification of residues that can be protected by 4,4'-dinitrostilbene-2,2'-disulfonate. Treatment of intact cells with Woodward's reagent K followed by B3H4 causes extensive labeling of band 3, with minimal labeling of intracellular proteins such as spectrin. Proteolytic digestion of the labeled protein reveals that both the 60- and the 35-kDa chymotryptic fragments are labeled and that the labeling of each is inhibitable by stilbenedisulfonate. If the reduction is performed at neutral pH the major labeled product is the primary alcohol corresponding to the original carboxylic acid. Liquid chromatography of acid hydrolysates of labeled affinity-purified band 3 shows that glutamate but not aspartate residues have been converted into the hydroxyl derivative. This is the first demonstration of the conversion of a glutamate carboxyl group to an alcohol in a protein. The labeling experiments reveal that there are two glutamate residues that are sufficiently close to the stilbenedisulfonate site for their labeling to be blocked by 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate and 4,4'-dinitrostilbene-2,2'-disulfonate.  相似文献   

15.
A system for radioactive labeling of compounds of biological interest that, due to their low electronic density, cannot be labeled by the standard iodination techniques is described. Using p-nitroanisole as a model, we have prepared 2-[125I]iodo-4-nitroanisole by treatment with thallium trifluoroacetate, with later displacement of the thallium by iodide according to A. McKillop et al. (J. Amer. Chem. Soc.93, 4841–4844 (1970)). The labeled iodonitroanisole has been used as a photoactive reagent to label a protein (bovine serum albumin), showing that under the irradiation conditions used, the label is incorporated into the polypeptide mainly through modification of ?-amino groups of the lysine residues.  相似文献   

16.
The iodination of vasoactive intestinal peptide (VIP) was studied, using a variety of enzymatic and chemical iodination methods. Reversed phase high performance liquid chromatography (HPLC) was used to purify the reaction products. The lactoperoxidase-glucose oxidase method gave excellent results in terms of reproducibility, iodine incorporation, and yield of the non-oxidized products [Tyr(I)10]VIP and [Tyr(I)22]VIP, and was used to prepare both 125I and 127I labelled derivatives. In both cases, direct application to HPLC and a single column system were used. Although the oxidized peptides [Tyr(I)10,Met(O)17]VIP and [Tyr(I)22,Met(O)17]VIP could be generated to varying degrees directly by iodination of VIP, these were most conveniently prepared by iodination of [Met(O)17]VIP. Iodinated derivatives of the homologous peptides PHI and PHM were likewise prepared by rapid, one-step HPLC procedures. The site and degree of iodination were determined by HPLC peptide mapping of tryptic digests and amino acid analyses, and in the case of [Tyr(I)10]VIP also by sequencing. The vasorelaxant activities of the iodinated peptides in bovine cerebral artery preparations did not differ significantly from those of the corresponding noniodinated peptides, with the exception of [Tyr(I)10,Met(O)17]VIP and [Tyr(I)22,Met(O)17]VIP which, unlike [Met(O)17]VIP itself, had slightly lower potency than VIP.  相似文献   

17.
Recombinant hirudin variant rHV2-Lys47 was radioiodinated using the chloramine-T method. Depending on the reaction pH, the two tyrosine residues, Tyr3 and Tyr63, responded differently to iodination but without change in total iodination yield. Of the incorporated -125 iodine 80% was located on Tyr3 at pH 7.4, but 65% was found on Tyr63 at pH 4. These distinct iodination patterns suggest the existence of a pH-dependent multimerization and/or important conformational changes in the tertiary structure with pH. Each radiotracer was purified to high specific activity by simple low-pressure chromatography including gel filtration and reverse-phase separation, both on short cartridges. The method was validated by reverse-phase and anion-exchange HPLC with on-line radioactivity detection. The iodination sites were characterized following carboxypeptidase Y cleavage coupled with radio-HPLC.  相似文献   

18.
To identify and characterize oxytocin receptors, a 125I-labeled photoreactive oxytocin antagonist was synthesized. The specific oxytocin antagonist [1-(beta-mercapto-beta,beta- cyclopentamethylenepropionic acid), 2-O-methyltyrosine,4-threonine,8- ornithine,9-tyrosylamide]oxytocin ([Mca,Tyr(O-Me)2,Thr4,Orn8,Tyr9-NH2]oxytocin) described by Elands et al. (Elands, J., Barberis, C., Jard, S., Tribollet, E., Dreifuss, J.-J., Bankowski, K., Manning, M., and Sawyer, W. H. (1987) Eur. J. Pharmacol. 147, 192-207) bound to the guinea pig uterine oxytocin receptor with high affinity (apparent Kd = 0.74 nM). The introduction of a 4-azidophenylamidino group at Orn8 resulted in the photoreactive ligand [Mca1,Tyr(O-Me)2,Thr4,Orn(4-azidophenylamidino)8,Tyr9- NH2]oxytocin, which retained the high binding affinity (Kd = 0.69 nM) of the parent compound. The photoreactive antagonist monoiodinated at Tyr9 had approximately double (Kd = 0.39 nM) the affinity of the photoreactive antagonist and several times that of oxytocin (Kd = 2.6 nM) for the guinea pig uterine oxytocin receptor. In photo-affinity labeling experiments using myometrial membranes obtained from guinea pigs during late pregnancy, the 125I-labeled photoreactive antagonist specifically labeled a protein with an apparent molecular mass of between 68 and 80 kDa: the labeling of this protein was completely suppressed by a 100-fold molar excess of oxytocin and oxytocin receptor-specific agonists, but not by vasopressin analogues specific for V1 or V2 receptors or by other peptide hormones. The ability of oxytocin to suppress labeling was decreased in the presence of guanosine 5'-O-(thiotriphosphate) or in the absence of Mn2+. Digestion of the photolabeled oxytocin receptor with endoglycosidase F gave rise to a protein with an apparent molecular mass of 38 +/- 2 kDa. The endoglycosidase F effect and the lack of endoglycosidase H action show that the myometrial oxytocin receptor is highly glycosylated with asparagine-linked complex oligosaccharide chains. Our results suggest that the radioiodinated photoreactive oxytocin antagonist could be a helpful tool in the isolation and further characterization of the oxytocin receptor.  相似文献   

19.
The amino reagent 2,4,6-trinitrobenzenesulfonate (TNBS) was found to inactivate mitochondrial F1-ATPase through covalent labeling, which was not reversed by dithiothreitol. The observed rate of inactivation was retarded by inorganic phosphate, but enhanced by prior labeling of F1 with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-C1). These observations are consistent with the presence of an essential amino group near the bound inorganic phosphate at the catalytic site of F1. A comparison of the observed protection of F1 from NBD-C1 and 5′-p-fluorosulfonyl-benzoyladenosine (FSBA) respectively by inorganic phosphate and by 2′,3′-O-(2,4,6-trinitrophenyl)adenosine 5′-monophosphate (TNP-AMP) suggests that NBD-C1 labels an essential Tyr residue in the positively charged locus for binding the polyphosphate end of ATP, and that FSBA labels an essential Tyr residue in the more hydrophobic locus for binding the adenosine moiety of ATP at the catalytic site of F1.  相似文献   

20.
Chloramine T iodination of intact avian myeloblastosis virus revealed, that the four major group-specific polypeptides were labeled unequally, with the largest polypeptide 4 (m.w. 23,000) being preferentially iodinated, when the labeling time was limited. By contrast, in enzymatic iodination of intact virus only major virion glycoprotein and glycolipids were extensively iodinated. These observations suggest that the protein 4 surrounds other group-specific polypeptides and is itself enveloped by the bilayer of glycolipids.  相似文献   

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