共查询到20条相似文献,搜索用时 15 毫秒
1.
The disulphide bridges of a mouse immunoglobulin G1 protein 总被引:7,自引:3,他引:4
[(35)S]Cystine-labelled immunoglobulin MOPC21 (IgG1) was prepared from myeloma cells in tissue culture. Carrier myeloma protein was added and the protein was digested with pepsin. The digest was fractionated on Sephadex G-50 into two fractions, further digested with trypsin and again fractionated on Sephadex. Disulphide-bridge peptides were purified by electrophoresis and chromatography and identified by radioautography. A peptide of 96 residues was isolated, which contains both the heavy-light interchain disulphide bridge and all the inter-heavy-chain disulphide bridges. Other peptides were isolated, accounting for all the intrachain disulphide bridges (which could be placed by homology with proteins of other species), except for the variable section of the light chain. Sequences describing this missing disulphide bridge were obtained from totally reduced and alkylated light chains. Peptides related to the interchain disulphide-bridge peptide were isolated from partially reduced and alkylated myeloma protein and from totally reduced heavy chain. The interchain disulphide-bridge peptide was placed at the C-terminal position of the F(ab')(2) fragment, prepared by digestion of the protein with pepsin at pH4.0. Sequences from the heavy-chain intrachain disulphide bridges of MOPC 21 immunoglobulin are compared with homologous sequences from mouse myeloma proteins of other subclasses and proteins of other species. 相似文献
2.
Mouse IgM (immunoglobulin M) was selectively and partially reduced and treated with iodo[2-14C]acetate to label the interchain disulphide bridges. The carboxymethylation was studied in some detail. The labelled peptides were purified, sequenced and positioned by homology with human IgM. Only peptides originating from three interchain disulphide bridges were labelled, in contrast with the four labelled bridges obtained in human IgM under the same conditions. These peptides are homologous to human bridge peptides forming the heavy-light bridge and two inter-heavy bridges, one present in the CMU2 region and the other in the C-terminal region. The inter-heavy bridge in the Cmu2 region was alone cleaved and radioactively labelled in selectively reduced IgM held together as a pentamer by non-covalen interactions. The same bridge was the only one to be totally cleaved in subunits released after more extensive, though still selective, reduction. In the light of these results a possible arrangement of the disulphide bridges of the mouse IgM. 相似文献
3.
4.
5.
F Pochon V Favaudon J Bieth 《Biochemical and biophysical research communications》1983,111(3):964-969
Free thiol groups released on proteolytic attack of alpha 2-macroglobulin by trypsin or chymotrypsin bind covalently to thiopropyl-Sepharose, indicating that they are located at the surface of the complexes. These cysteine sulfhydryl groups appear to be in contact with the alpha 2M-bound proteases from singlet-singlet energy transfer measurements between fluorescein isothiocyanate-labeled proteinases and N-(iodoacetylaminoethyl)-5-naphtylamine-1-sulfonic acid-labeled thiols in alpha 2-macroglobulin. 相似文献
6.
7.
We have shown previously that immunoglobulin M (IgM) is present within IgM-forming cells mainly in its 7S subunit form (IgMs), whereas only fully assembled IgM pentamers are secreted. There is no spontaneous polymerization of intracellular IgMs in cell lysates, suggesting that the 7S subunits had blocked cysteine residues. This suggestion was explored and confirmed in the present paper. Radioactive IgM (secreted) and IgMs (intracellular) were prepared by sucrose-density-gradient centrifugation after incubation of cells of the IgM-producing mouse myeloma MOPC 104E with [(3)H]leucine. We investigated the susceptibility to reduction of fully assembled mouse IgM and its reconstitution from subunits by analysis by polyacrylamide-gel electrophoresis under dissociating conditions. With increasing concentrations of dithioerythritol, interchain disulphide bonds were cleaved in the following order: inter-IgMs subunit, intra-IgMs subunit H-H, intra-IgMs subunit H-L. Removal of the reducing agent from IgM-reduction mixtures by filtration through Sephadex G-25 caused partial reconstitution of IgM at low protein concentrations (5-100mug/ml) and total reconstitution at higher protein concentrations (300mug/ml or more). Isolated radioactive intracellular IgMs showed no tendency to polymerize unless first treated with a reducing agent; under optimum conditions removal of the reducing agent caused 70% of the subunits to be assembled into IgM. Similar assembly occurred when IgMs was isolated from cells that had been lysed in the presence of an irreversible alkylating reagent (iodoacetamide). The intracellular IgMs cysteine residues responsible for inter-IgMs linkage therefore appear to be reversibly blocked within the cells. Assembly into IgM is thus controlled by removal of this block during secretion. 相似文献
8.
9.
Ochoa WF Torrecillas A Fita I Verdaguer N Corbalán-García S Gomez-Fernandez JC 《Biochemistry》2003,42(29):8774-8779
Protein kinase C(alpha) (PKC(alpha)) is a key enzyme regulating the physiology of cells and their growth, differentiation, and apoptosis. PKC activity is known to be modulated by all-trans retinoic acid (atRA), although neither the action mechanism nor even the possible binding to PKCs has been established. Crystals of the C2-domain of PKC(alpha), a regulatory module in the protein that binds Ca(2+) and acidic phospholipids, have now been obtained by cocrystallization with atRA. The crystal structure, refined at 2.0 A resolution, shows that RA binds to the C2-domain in two locations coincident with the two binding sites previously reported for acidic phospholipids. The first binding site corresponds to the Ca(2+)-binding pocket, where Ca(2+) ions mediate the interactions of atRA with the protein, as they do with acidic phospholipids. The second binding site corresponds to the conserved lysine-rich cluster localized in beta-strands three and four. These observations are strongly supported by [(3)H]-atRA-binding experiments combined with site-directed mutagenesis. Wild-type C2-domain binds 2 mol of atRA per mol of protein, while the rate reduces to one in the case of C2-domain variants, in which mutations affect either Ca(2+) coordination or the integrity of the lysine-rich cluster site. Competition between atRA and acidic phospholipids to bind to PKC is a possible mechanism for modulating PKC(alpha) activity. 相似文献
10.
Disulfide bond formation between the active-site thiol and one of the several free thiol groups of chymopapain 总被引:2,自引:0,他引:2
Chymopapain, a cysteine protease of papaya latex, has been purified with the use of fast protein liquid chromatography. Two homogeneous fractions were analyzed for thiol content and thiol reactivity. It was found that peak 1 and peak 2 contained two and three thiol groups, respectively, per mole of enzyme. This result is inconsistent with the general belief that chymopapain contains one essential and one nonessential thiol group and suggests that a significant portion of the thiol groups was oxidized in the previous preparations. Such an oxidation can account for some of the inconsistent results reported in the literature. An irreversibly oxidized nonessential thiol group may modify the catalytic function of chymopapain especially if it is close to the active site. That one thiol group resides indeed in the vicinity of the essential thiol group is clearly demonstrated by the biphasic reactions of chymopapain with disulfide compounds such as 2,2'-dipyridyl disulfide and 5,5'-dithiobis(2-nitrobenzoate). In the first step of these reactions a mixed disulfide is formed between the enzyme and the reactant, which is followed by a first-order, intramolecular reaction leading to the liberation of the second half of the disulfide compound. Furthermore, on addition of one Hg2+ ion, 2 mol of thiol group, one essential and one nonessential, disappears concomitantly. Formation of a disulfide bond between the catalytically competent thiol group and another free thiol group of chymopapain under physiological conditions may be of regulatory importance. 相似文献
11.
12.
13.
Inactivation of human alpha 1-proteinase inhibitor by thiol proteinases. 总被引:10,自引:0,他引:10
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Human plasma alpha1 proteinase inhibitor is the body's principal modulator of serine proteinases (such as those released from phagocytic cells). Cysteine-active-site proteinases, which are not inhibited, have now been found to inactivate this important inhibitor by proteolytic cleavage of a scissile peptide bond. Papain carries out this inactivation catalytically, whereas cathepsin B1 acts stoicheiometrically. Thus thiol proteinases could easily disrupt the delicately regulated balance between serine proteinases and alpha1 proteinase inhibitor. 相似文献
14.
Six peptides containing eight half-cystine residues were isolated in good yield, after either oxidation or reduction and carboxymethylation of fragment C-1, which contains the N-terminal half of the heavy chain of rabbit immunoglobulin G. The sequences of five of these peptides had been reported previously (Cebra, Steiner & Porter, 1968b; Wilkinson, 1969) and that of the sixth was established. Other peptides containing half-cystine residues were isolated in much lower yield and are presumed to be derived from minor sequence variants. The cystine-containing peptides from enzymic digests of whole immunoglobulin G and Fc fraction were studied by several techniques and the results obtained enable us to put forward a scheme of the arrangement of the inter- and intra-chain disulphide bonds. 相似文献
15.
16.
S Y Venyaminov E Rajnav?lgyi G A Medgyesi J Gergely P Závodszky 《European journal of biochemistry》1976,67(1):81-86
The hydrogen-deuterium exchange data of human immunoglobulin G1 (IgG1) are interpreted by assuming fast fluctuations of the protein conformation, through which the peptide groups become exposed to the solvent. The probability of solvent exposure of peptide hydrogens reflects a rather loose conformation for native IgG in comparison with other globular proteins. The probability of solvent exposure is greater than 10(-3) for half of the peptide groups, which shows that the conformational transitions by which these groups are exposed to the solvent are accompanied by changes in standard free energy less than 17 kJ/mol (4 kcal/mol). In the range of pH 6.2-8.45, at 25 degrees C no gross conformational changes are reflected in the hydrogen-deuterium exchange behaviour of the native, the reduced-nonalkylated-reassociated and the reduced-S-alkylated-reassociated IgG1. No difference could be detected in the conformational stability of the native and reoxidised reassociated IgG1 proteins. The lack of inter-subunit disulphide bridges in S-alkylated-reassociated molecules results in an increased conformational motility. This destabilization of protein conformation affects about 90% of the peptide groups covered by the measurements, and corresponds to changes in standard free energy of 8 kJ/mol on the average. 相似文献
17.
Participation of cellular thiol/disulphide groups in the uptake, degradation and bioactivity of insulin in primary cultures of rat hepatocytes. 总被引:1,自引:1,他引:1
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The effects on the uptake (cell-associated 125I) and degradation (125I-labelled products released into the medium) of 125I-insulin and bioactivity (protein, glycogen and lipid synthesis) of insulin caused by altering the cellular thiol/disulphide status in primary cultures of rat hepatocytes were studied. Incubation of hepatocyte cultures with various exogenous thiol compounds (reduced glutathione, 2-mercaptoethanol, cysteamine, dithiothreitol) resulted in increased insulin binding, but markedly decreased degradation and bioactivity. These effects could be reversed by washing or by the addition of oxidized glutathione, which alone had no effect. When cultures were exposed to certain thiol-modifying reagents (N-ethylmaleimide, p-chloromercuribenzoate, p-chloromercuribenzenesulphonate, iodoacetamide, iodoacetate), some decreases in bioactivity were evident, but the pronounced decrease in insulin degradation observed with the thiol-containing compounds was not observed with this class of compounds. None of the thiol-containing or -modifying agents tested had any significant effect on cellular ATP concentrations, indicating that the effects observed were due to perturbation of the thiol/disulphide status. Depletion of intracellular glutathione by DL-buthionine SR-sulphoximine (a specific inhibitor of glutathionine biosynthesis) decreased the syntheses of glycogen and lipid by about one-half, while having essentially no effect on protein synthesis, ATP concentrations or on the binding and degradation of insulin. The data presented here indicate that although intracellular thiol (glutathione) concentrations may be important for the maintenance of full expression of certain biological activities (glycogen and lipid synthesis), the thiol/disulphide groups on the cell surface and those immediately inside the cell membrane may be more critical in the mediation of insulin action, including the degradation and bioactivity of insulin in primary cultures of rat hepatocytes. 相似文献
18.
19.
Summary
Bennett and Yphantis (1948) introduced into histochemistry the use of organic mercurials for the detection of sulphydryl groups. The new fluorescent method which is now reported is similarly based upon the reaction of mercaptans with an organic mercurial, fluorescein mercuric acetate (FMA). This method is sensitive; moreover it is not as elaborate as the dihydroxy-dinaphthyl-disulphide (DDD) technique of Barrnett and Seligman (1952). 相似文献
20.
A convenient method of preparation of high-activity urease from Canavalia ensiformis by covalent chromatography and an investigation of its thiol groups with 2,2''-dipyridyl disulphide as a thiol titrant and reactivity probe.
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
1. A convenient method of preparation of jack-bean urease (EC3.5.1.5) involving covalent chromatography by thiol-disulphide interchange is described. 2. Urease thus prepared has specific activity comparable with the highest value yet reported (44.5 +/- 1.47 kat/kg, Km = 3.32 +/- 0.05 mM; kcat. = 2.15 X 10(4) +/- 0.05 X 10(4)s-1 at pH7.0 and 38 degrees C). 3. Titration of the urease thiol groups with 2,2'-dipyridyl disulphide (2-Py-S-S-2-Py) and application of the method of Tsou Chen-Lu [(1962) Sci. Sin. 11, 1535-1558] suggests that the urease molecule (assumed to have mol.wt. 483000 and epsilon280 = 2.84 X 10(5) litre-mol-1-cm-1) contains 24 inessential thiol groups of relatively high reactivity (class-I), six 'essential' thiol groups of low reactivity (class-II) and 54 buried thiol groups (class-III) which are exposed in 6M-guanidinium chloride. 4. The reaction of the class-I thiol groups with 2-Py-S-S-2-Py was studied in the pH range 6-11 at 25 degrees C(I = 0.1 mol/l) by stopped-flow spectrophotometry, and the analogous reaction of the class-II thiol groups by conventional spectrophotometry. 5. The class-I thiol groups consist of at least two sub-classes whose reactions with 2-Py-S-S-2-Py are characterized by (a) pKa = 9.1, k = 1.56 X 10(4)M-1-s-1 and (b) pKa = 8.1, k = 8.05 X 10(2)M-1-s-1 respectively. The reaction of the class-II thiol groups is characterized by pKa = 9.15 and k = 1.60 X 10(2)M-1-s-1. 6. At pH values 7-8 the class-I thiol groups consist of approx. 50% class-Ia groups and 50% class-Ib groups. The ratio class Ia/class Ib decreases an or equal to approx. 9.5, and at high pH the class-I thiol groups consist of at most 25% class-Ia groups and at least 75% class-Ib groups. 7. The reactivity of the class-II thiol groups towards 2-Py-S-S-2-Py is insensitive to the nature of the group used to block the class-I thiols. 8. All the 'essential' thiol groups in urease appear to be eeactive only as uncomplicated thiolate ions. The implications of this for the active-centre chemistry of urease relative to that of the thiol proteinases are discussed. 相似文献