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1.
Shuichiro Gohda Akio Shimizu Masamichi Ikeguchi Shintaro Sugai 《The protein journal》1995,14(8):731-737
The disulfide reduction kinetics in equine lysozyme (ELZ), which is a Ca2+-binding lysozyme, and human (HLA) and equineα-lactalbumin (ELA) at pH 8.5 and 25°C by excess dithiothreitol were studied, and it was found that in ELZ there is no superreactive disulfide bond, while one of the disulfides is reduced very quickly by the reducing agent in HLA and ELA, as in bovineα-lactalbumin. The local conformation around the surface disulfide in ELZ seems to be more similar to that in hen egg-white lysozyme than inα-lactalbumin. The four disulfides in ELZ were reduced slowly in an apparently single-exponential form, and the bound Ca2+ lowered the reduction rate. The torsion energy on each of the disulfides in threeα-lactalbumin and eight c-type lysozymes whose native conformations have been experimentally or theoretically analyzed was calculated, and it was found that torsion imposed on the surface disulfide between Cys 6 and Cys 120 inα-lactalbumin is a main cause of the superreactivity and all of lysozymes, including the Ca2+-binding ones, have no such strained surface bond. 相似文献
2.
Summary The fluorescent behaviour and the photodynamic effect was studied in native and structurally modified lysozyme and-lactalbumin.The Tyr residues in lysozyme and-lactalbumin show different sensitivities to the photodynamic effect. The effect is zero in the case of Tyr from native lysozyme. In contrast, the Tyr residues in-lactalbumin are susceptible to photooxidation, which indicates a greater degree of exposure to the solvent. The three His residues of-lactalbumin have different degrees of exposure and show two different kinetics of photooxidation whereas the His residue of lysozyme is photooxidized with a single kinetic.Two photooxidation kinetics were obtained for the Trp residues of both native proteins, an indication that in both cases there are Trp residues that are differently exposed to the solvent. The wavelengths of maximum fluorescent emission of the Trp residues were different for the two proteins, an effect which can also be explained in terms of a difference in the environment of these residues. The modified form of these proteins emit at wavelengths longer than those of the native forms. When modified the proteins photooxidize with noticeably greater quantum yields. 相似文献
3.
《Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology》1989,994(3):195-199
The positions of the inter- and intra-chain disulfide bonds of human plasma α2HS-glycoprotein were determined. α2HS-glycoprotein was digested with acid proteinase and then with thermolysin. The disulfide bonds containing peptides were separated by reversed-phase HPLC and detected by SBD-F (7-fluorobenzo-2-oxa-1,3-diasole-4-sulfonic acid ammonium salt) method. One inter-disulfide bond containing peptide and five intra-disulfide bond containing peptides (A-chain) were purified and identified as Cys-18 (B-chain)-Cys-14 (A-chain), Cys-71-Cys-82, Cys-96-Cys-114, Cys-128-Cys-131, Cys-190-Cys-201 and Cys-212-Cys-229, respectively. The location of the intra-disulfide bonds revealed that the A-chain of α2HS-glycoprotein is composed of three domains. Two domains were shown to possess intramolecular homology judging from the total chain length of the domains, size of the loops formed by the SS bonds, the location of two disulfide loops near the C-terminal end of domains A and B, the distance between two SS bonds of each domain, the amino acid sequence homology between these two domains (22.6%), number of amino acid residues between the second SS loops and the end of domains A and B, and the positions of the ordered structures. 相似文献
4.
Roger T. Dean 《The Biochemical journal》1974,138(3):407-413
1. The subcellular distribution of beta-glucuronidase and other hydrolases in rabbit liver was investigated. beta-Glucuronidase was found in both microsomal and lysosomal fractions. 2. Multiple forms of beta-glucuronidase were present in extracts of microsomal and lysosomal fractions. All forms were common to both fractions. 3. A specific antiserum against beta-glucuronidase was raised, and characterized by immunoprecipitation and affinity-chromatography procedures. 4. The immunological identity of the multiple forms in the pure beta-glucuronidase preparation, and the immunological identity of the beta-glucuronidase complement of lysosomal extracts with that of microsomal extracts, were demonstrated by means of the antiserum. The presence of inactive enzyme in various enzyme preparations was shown. 相似文献
5.
Satya S. Sadhu Jiashu Xie Hongwei Zhang Omathanu Perumal Xiangming Guan 《Biochemistry and Biophysics Reports》2016
Glutathione disulfide (GSSG) is the oxidized form of glutathione (GSH). GSH is a tripeptide present in the biological system in mM concentration and is the major antioxidant in the body. An increase in GSSG reflects an increase in intracellular oxidative stress and is associated with disease sates. The increase has also been demonstrated to lead to an increase in protein S-glutathionylation that can affect the structure and function of proteins. Protein S-glutathionylation serves as a regulatory mechanism during cellular oxidative stress. Though GSSG is commercially available, its roles in various GSSG-associated normal/abnormal physiological functions have not been fully delineated due to the reason that GSSG is not cell membrane permeable and a lack of method to specifically increase GSSG in cells. We have developed cationic liposomes that can effectively deliver GSSG into cells. Various concentrations of GSSG liposomes can be conveniently prepared. At 1 mg/mL, the GSSG liposomes effectively increased intracellular GSSG by 27.1±6.9 folds (n=3) in 4 h and led to a significant increase in protein S-glutathionylation confirming that the increased GSSG is functionally effective. The Trypan blue assay demonstrated that GSSG liposomes were not cytotoxic; the cell viability was greater than 95% after cells were treated with the GSSG liposomes for 4 h. A stability study showed that the dry form of the GSSG liposomes were stable for at least 70 days when stored at ?80 °C. Our data demonstrate that the GSSG liposomes can be a valuable tool in studying GSSG-associated physiological/pathological functions. 相似文献
6.
Human α1-acid glycoprotein (AGP) is an acute phase plasma glycoprotein containing two disulfide bridges. As a member of the lipocalin superfamily, it binds and transports several basic and neutral ligands, but a number of other activities have also been described. Thanks to its binding properties, AGP is also a good candidate for the development of biosensors and affinity chromatography media, and in this context detailed structural information is needed. The structural properties of AGP at different p2Hs and under reducing conditions were analysed by FT-IR spectroscopy. The obtained data indicate that AGP, when denatured, does not aggregate at neutral or basic p2Hs whilst it does at acidic p2Hs. Under reducing conditions the protein is remarkably less thermostable than its oxidized counterpart and presents an enhanced tendency to aggregate, even at neutral p2H. A heat-induced molten globule-like state (MG) was detected at 55 °C at p2H 7.4 and 5.5. At p2H 4.5 the MG occurred at 45 °C with an onset of formation at 40 °C. The MG was not observed under reducing conditions. A lower affinity of chlorpromazine and progesterone for the MG formed at p2H 4.5 and 40 °C was observed, suggesting that ligand(s) may be released near the negative surfaces of biological membranes. Furthermore, the reduced AGP displays an enhanced affinity for progesterone, indicating the importance of disulfide bonds for the binding capacity of AGP. 相似文献
7.
Summary The effect of iodide ion on the tryptophyl fluorescence of the homologous proteins lysozyme and-lactalbumin in their native form, as well as in their modified structures and in fragments from these proteins was studied. By assessing the contribution to the total fluorescence of the exposed and buried Trp residues, and of the respective fluorescence quantum yields, the quantization of the number of Trp exposed to the solvent for all the species studied was possible. Both native proteins show an important increase in the number of Trp residues exposed to the solvent when treated with denaturing agents. The peptides L-II (aa 13-105) and-I (aa 1-90) from lysozyme and-lactalbumin, respectively, showed Trp residues with different degree of exposure, whereas the smaller fragments, L-III (aa 106-129) and-II (aa 91–123), had all their Trp residues exposed to the solvent. 相似文献
8.
Barackov I Mause A Kapoor S Winter R Schembecker G Burghoff B 《Journal of biotechnology》2012,161(2):138-146
Purification and separation of proteins play a major role in biotechnology. Nowadays, alternatives to multistep operations suffering from low product yields and high costs are investigated closely amidst which one of the promising options is foam fractionation. The molecular behavior at the gas-liquid interface plays an important role in the formation and stabilization of enriched foam. This study for the first time correlates the physico-chemical parameters to the molecular structure in view of protein enrichment during foam fractionation of the two relatively different proteins lysozyme and β-casein employing biophysical techniques such as circular dichroism (CD) spectroscopy and infrared reflection absorption spectroscopy (IRRAS). In case of lysozyme, high enrichment was achieved at pH相似文献
9.
10.
High-quality spray-dried latex of Carica papaya L was fractionated by using SP-Sephadex-C50. The four major cysteine-proteinase components—papain(E.C.3.4.22.2), chymopapains A and B(jointly designated currently as E.C.3.4.22.6), and papaya peptidase A—were isolated and characterized by protein chemical methods and by study of their thiol groups using2,2′-dipyridyl disulfide as a two-protonic-state titrant and reactivity probe. Papain and papaya peptidase A each contain one thiol group/molecule, which in each case is part of the catalytic site, as evidenced by high reactivity toward2,2′-dipyridyl disulfide in acidic media. Chymopapains A and B each contain two thiol groups/molecule, only one of which is essential for catalytic activity. The reactivities of the thiol groups of these enzymes toward2,2′-dipyridyl disulfide at pH4 and10 and activity loss analysis by Tsou Chen-Lu plots each provides a ready means of distinguishing among the four cysteine proteinases. The nonessential thiol groups of chymopapains A and B readily undergo irreversible oxidation. The reactivity characteristics of the essential thiol groups of the four enzymes suggest the presence of somewhat similar interactive cysteine-histidine catalytic center systems in papain, papaya peptidase A, and chymopapain B but a different type of catalytic center environment in chymopapain A. 相似文献
11.
《生物化学与生物物理学报:生物膜》2022,1864(1):183776
Alpha-synuclein (α-syn) aggregation and mitochondrial dysfunction are considered as two of the main factors associated with Parkinson's disease (PD). In the present investigation, the effectiveness of the amyloid fibrils obtained from α-syn with those of hen egg white lysozyme (HEWL), as disease-related and-unrelated proteins, to damage rat brain and rat liver mitochondria have been investigated. This was extended by looking at SH-SY5Y human neuroblastoma cells and erythrocytes, thereby investigating the significance of structural characteristics of amyloid fibrils related to their interactions with biomembranes obtained from various sources. Results presented clearly demonstrate substantial differences in the response of tested biomembranes to toxicity induced by α-syn/HEWL amyloid fibrils, highlighting a structure-function relationship. We found that fibrillar aggregates of α-syn, but not HEWL, caused a significant increase in mitochondrial ROS, loss of membrane potential, and mitochondrial swelling, in a dose-dependent manner. Toxicity was found to be more pronounced in brain mitochondria, as compared to liver mitochondria. For SH-SY5Y cells and erythrocytes, however, both α-syn and HEWL amyloid fibrils showed the capacity to induce toxicity. Taken together, these results may suggest selective toxicity of α-syn amyloid fibrils to mitochondria mediated likely by their direct interaction with the outer mitochondrial membrane, indicating a correlation between specific structural characteristics of α-syn fibrils and an organelle strongly implicated in PD pathology. 相似文献
12.
13.
Kunio Takeda Kyoko Ogawa Masami Ohara Satoshi Hamada Yoshiko Moriyama 《The protein journal》1995,14(8):679-684
Four disulfide bridges of bovineα-lactalbumin (α-lact) were selectively reduced to obtain its derivatives with three, two, and zero disulfide bridges (designated as 3SS, 2SS, and OSSα-lact, respectively). The original helicity was almost maintained in 3SSα-lact missing only the Cys6-Cysl20 bridge. Upon the reduction of both Cys28-Cys111 and Cys6-Cys120 bridges, various changes occurred in the protein. In particular, the maximum fluorescence of 1-anilinonaphthalene-8-sulfonic acid was observed in this stage. Upon the reduction of all disulfide bridges, the hydrophobic box of the protein, formed by Trp60, Ile95, Tyr103, and Trp104, was disrupted and an internal helical structure was destroyed. The conformation of each derivative was examined mainly in a solution of sodium dodecyl sulfate. In the surfactant solution, the helicity increased from 33% to 37% in 3SSα-lact, from 26% to 31% in 2SSα-lact, and from 18% to 37% in OSSα-lact, as against from 34% to 44% in intactα-lact. On the other hand, the tryptophan fluorescence of each derivative was affected in very low surfactant concentrations, suggesting that the tertiary structure considerably changed prior to the secondary structural change in the surfactant solution. 相似文献
14.
Meunier-Jamin C Kapp U Leonard GA McSweeney S 《The Journal of biological chemistry》2004,279(24):25830-25837
The three-dimensional structure of the organic hydroperoxide resistance protein (OHRP) from Deinococcus radiodurans as determined using single crystal xray diffraction techniques is reported. Comparison of the structure with that obtained for OHRP from Pseudomonas aeruginosa reveals that the polypeptide chain of OHRPs can adopt two significantly different conformations ("in" and "out") in the region of the active site disulfide moiety. It is postulated that the closed configuration is consistent with efficient catalysis of the reduction of organic hydroperoxides, whereas the open form is required for enzyme recycling. Comparison of the structures of OHRP and that of the osmotically induced protein C (OsmC) from Mycoplasma pneumoniae shows that OHRPs and OsmCs are structurally homologous, perhaps indicating related functions for the two families of proteins. 相似文献
15.
《Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology》1986,869(2):147-157
Porcine pancreatic α-amylase (1,4-α-d-glucan glucanohydrolase EC 3.2.1.1), a single polypeptide chain, contains nine residues of methionine. Eight different fragments resulting from cleavage of this molecule by cyanogen bromide were characterized. The sequences of six of them have previously been reported. Two missing fragments, CN2 (82 residues) and CN3b1 (76 residues) were purified after breaking of the interpeptidic disulfide bridge and their complete sequence as well as that of the previously purified CN1 peptide (102 residues) are reported here. The location of the three disulfide bridges present in these peptides was determined. Ordering of the carboxymethylated cyanogen bromide fragments was carried out by pulse labeling the amylase chain in vivo. The complete sequence of the porcine pancreatic amylase chain (496 residues) and the location of its five disulfide bridges is presented. Comparison with human and mouse pancreatic and salivary α-amylases and with rat pancreatic amylase obtained from the corresponding cDNA nucleotidic sequences shows a high degree of homology between mammalian α-amylases. 相似文献
16.
H. Kobayashi Hiroko Oyamada Naoko Iwadate Hiromi Suzuki Hideko Mitobe Kaori Takahashi Nobuyuki Shibata Shigeo Suzuki Yoshio Okawa 《Archives of microbiology》1998,169(3):188-194
A mannan of Candida glabrata IFO 0622 digested by Arthrobacter exo-α-mannosidase and a β-1,2-linked mannobiose obtained from the parent mannan by acid treatment was analyzed using 13C nuclear magnetic resonance spectroscopy. The results show that the β-1,2-linked mannobiosyl residue is esterified to a phosphate
group through position C-1 in the α-configuration, Manβ1– 2Manα1–HPO3–. The results of immunochemical assays of these mannans using the commercial antigenic factor sera of the genus Candida (Candida Check, Iatron) indicate that the main recognition site of serum no. 6 in this kit is the mannotetraosyl side-chain
Manβ1–2Manα1– 2Manα1–2Man in C. glabrata mannan and also suggest that the phosphate-containing unit (such as Manβ1– 2Manα1–HPO3– in this mannan) behaves as one of the antigenic determinants of serum no. 6, but not of serum no. 5. Therefore, the present
and previous findings indicate that serum no. 5 recognizes relatively longer β-1,2-linked oligomannosyl side-chains, Manβ1–[2Manβ1–]n 2Man (n = 1–6), attached to the phosphate groups previously observed in the cell wall mannans of Candida albicans, Candida stellatoidea, and Candida tropicalis.
Received: 18 March 1997 / Accepted: 16 September 1997 相似文献
17.
The early intermediates in the protein aggregation pathway, the elusive soluble aggregates, play a pivotal role in growth and maturation of ordered aggregates such as amyloid fibrils. Blocking the growth of soluble oligomers is an effective strategy to inhibit aggregation. To decipher the molecular mechanisms and develop better strategies to arrest aggregation, it is imperative to understand how the size, molecular dynamics, activity and growth kinetics of soluble aggregates are affected when aggregation is inhibited. With this objective, in the present study we have investigated the influence of additives such as SDS, CTAB (cetyltrimethylammonium bromide) and DTT (dithiothreitol) on the slow aggregation of HEWL (hen eggwhite lysozyme) at pH 12.2. For this purpose, techniques such as steady-state and time-resolved fluorescence anisotropy of covalently labelled dansyl probe, gel-filtration chromatography, estimation of free thiol groups, thioflavin T and ANS (8-anilinonaphthalene-1-sulfonic acid) fluorescence, CD and atomic-force microscopy were employed to monitor the soluble oligomers over a period spanning 30 days. The results of the present study reveal that: (i) the spontaneous formation of soluble aggregates is irreversible and abolishes activity; (ii) the initial growth of aggregates (0-24 h) is promoted by a gradual increase in the exposure of hydrophobic surfaces; (iii) subsequently intermolecular disulfide bonds are critical for the assembly and stability of aggregates; (iv) the tight molecular packing inside large aggregates which contributed to slow (approximately 5 ns) and restricted segmental motion of dansyl probe was clearly loosened up in the presence of additives, enabling fast (1-2 ns) and free motion (unlike DTT, the size of lysozyme complexes with surfactants, was large, due to a conglomeration of proteins and surfactants); (v) the aggregates show reduced helical content compared with native lysozyme, except in the presence of SDS; and (vi) DTT was more potent than SDS/CTAB in arresting the growth of aggregates. 相似文献
18.
Wadahama H Iwasaki K Matsusaki M Nishizawa K Ishimoto M Arisaka F Takagi K Urade R 《Plant physiology》2012,158(3):1395-1405
β-Conglycinin, one of the major soybean (Glycine max) seed storage proteins, is folded and assembled into trimers in the endoplasmic reticulum and accumulated into protein storage vacuoles. Prior experiments have used soybean β-conglycinin extracted using a reducing buffer containing a sulfhydryl reductant such as 2-mercaptoethanol, which reduces both intermolecular and intramolecular disulfide bonds within the proteins. In this study, soybean proteins were extracted from the cotyledons of immature seeds or dry beans under nonreducing conditions to prevent the oxidation of thiol groups and the reduction or exchange of disulfide bonds. We found that approximately half of the α'- and α-subunits of β-conglycinin were disulfide linked, together or with P34, prior to amino-terminal propeptide processing. Sedimentation velocity experiments, size-exclusion chromatography, and two-dimensional polyacrylamide gel electrophoresis (PAGE) analysis, with blue native PAGE followed by sodium dodecyl sulfate-PAGE, indicated that the β-conglycinin complexes containing the disulfide-linked α'/α-subunits were complexes of more than 720 kD. The α'- and α-subunits, when disulfide linked with P34, were mostly present in approximately 480-kD complexes (hexamers) at low ionic strength. Our results suggest that disulfide bonds are formed between α'/α-subunits residing in different β-conglycinin hexamers, but the binding of P34 to α'- and α-subunits reduces the linkage between β-conglycinin hexamers. Finally, a subset of glycinin was shown to exist as noncovalently associated complexes larger than hexamers when β-conglycinin was expressed under nonreducing conditions. 相似文献
19.
The immunoglobulin G receptor FcRIIIB belongs to the immunoglobulin superfamily as two extracellular domains show homology to the immunoglobulin domains. Since some residues in these domains, such as the two cysteines, are supposed to form an intrachain disulfide bridge are so commonly conserved, they may be of importance for correct folding. Site-directed mutagenesis and expression in BHK21 confirmed this supposition for the FcRIIIB. Replacing both cysteines in the first and/or second domain by serines reduced the surface expression level by 50%, whereas the ligand binding capability was 20–30% of that seen in cells expressing the wild-type receptor. Replacing one of the four cysteines resulted in the loss of surface expression. Exchanging the conserved tryptophan in the first domain by phenylalanine only slightly affected the ligand binding (25%), whereas the surface expression remained unchanged. 相似文献
20.