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91.
Characterization of epitopes on the cationic peanut peroxidase by four monoclonal antibodies 总被引:1,自引:0,他引:1
The epitope sites on the cationic peanut peroxidase were characterized by four monoclonal antibodies raised against this isozyme. Evidence is presented showing that the epitope for monoclonal antibody 1B is located on the polypeptide. Sensitivity of the epitopes recognized by 1M and 2F to 0.1M HCl, boiling, 10 mM periodate and trifluoromethane sulfonic acid treatment indicate that they occur at regions where oligosaccharides are linked to the polypeptide backbone. The antigenic specificity of 2A is, in addition, dependent on the conformation of the epitope site which is destroyed after partial proteolysis of the peroxidase. 相似文献
92.
93.
Regulation of differentiation of the BC3H1 muscle cell line through cAMP-dependent and -independent pathways 总被引:7,自引:0,他引:7
Serum mitogens, fibroblast growth factor (FGF), and type beta transforming growth factor (TGF-beta) suppress differentiation of the mouse muscle cell line BC3H1; however, the signal transduction pathways whereby these growth factors exert their effects on this system are unknown. The goal of this study was to determine whether the program for differentiation of BC3H1 cells was susceptible to negative regulation by signaling pathways involving cAMP or protein kinase C and whether these intracellular effectors participate in the mechanism by which growth factors prevent establishment of the myogenic phenotype. Exposure of BC3H1 cells to dibutyryl cAMP, 8-bromo-cAMP, or compounds that stimulate adenylate cyclase, i.e. forskolin, prostaglandin E1, and cholera toxin, prevented up-regulation of muscle-specific gene products following growth arrest in mitogen-deficient medium. Conversely, addition of cAMP to differentiated BC3H1 myocytes caused down-regulation of muscle-specific mRNAs. In contrast to the ability of cAMP to block differentiation, chronic exposure to O-tetradecanoylphorbol-13-acetate, the potent activator of protein kinase C, exhibited no apparent effects on expression of muscle-specific gene products. The proto-oncogenes c-myc and c-fos were up-regulated rapidly by cAMP in a manner similar to that observed previously by serum, FGF, and TGF-beta. However, these growth factors failed to increase intracellular cAMP levels, and they did not induce ornithine decarboxylase, which was subject to positive regulation by cAMP and O-tetradecanoyl-13-acetate. Together, these data indicate that differentiation of BC3H1 cells is subject to negative regulation through a cAMP-dependent pathway and that serum mitogens, FGF, and TGF-beta inhibit differentiation through a mechanism independent of cAMP or protein kinase C. 相似文献
94.
M?ssbauer spectra of 57Fe-enriched NADH-reduced yeast cytochrome c oxidase reveal two quadrupole doublets of unequal intensity; one (approximately 33%) is typical of high-spin ferrous heme with histidine coordination and is assigned to heme a3, while the other (approximately 67%) is typical of low-spin heme with two nitrogeneous axial ligands as expected from heme a. The excess intensity (approximately 17%) of the low-spin doublet must therefore be assigned to heme a3 in a modified environment. The M?ssbauer spectra of the same sample exposed to CO show that 50% of the heme iron forms a CO adduct, consistent with heme a3 being inhibited by CO. While low-spin hem a has the same M?ssbauer parameters as in the reduced sample, its intensity has dropped to 35%. A distinctly new high-spin species (approximately 15%) is observed and assigned to heme a in a modified environment. The comparable size of the unexpected high-spin heme a fraction in the CO adduct and the low-spin heme a3 fraction in the reduced enzyme suggest that they arise from the same material. This material is likely to be the inactive fraction that has been found in all preparations of resting yeast cytochrome c oxidase (Siedow, J.N., Miller, S., and Palmer, G. (1981) J. Bioenerg. Biomembr. 14, 171-179). The kinetics of CO recombination following photolysis of the CO complex further confirms the coexistence of two distinct fractions associated with active and inactive protein. The majority (approximately 74%), presumably active protein, recombines exponentially from 160 to 270 K following an Arrhenius law. The large activation enthalpy, delta H approximately 35 kJ/mol, is comparable to that found in the beef heart enzyme, suggesting that the flashed-off CO is bound by the nearby CuB as in the mammalian system (Fiamingo, F.G., Altschuld, R.A., Moh, P.P., and Alben, J.O. (1982) J. Biol. Chem. 250, 1639-1650). In the minority, presumably inactive, fraction the CO recombination has fast nonexponential kinetics with a distribution of activation enthalpies peaking near delta Hp = 13 kJ/mol reminiscent of CO binding to myoglobin. In this inactive fraction CuB is apparently not accessible to the flashed-off CO. 相似文献
95.
The proton-pumping site of cytochrome c oxidase: a model of its structure and mechanism 总被引:8,自引:0,他引:8
Cytochrome c oxidase is an electron-transfer driven proton pump. In this paper, we propose a complete chemical mechanism for the enzyme's proton-pumping site. The mechanism achieves pumping with chemical reaction steps localized at a redox center within the enzyme; no indirect coupling through protein conformational changes is required. The proposed mechanism is based on a novel redox-linked transition metal ligand substitution reaction. The use of this reaction leads in a straightforward manner to explicit mechanisms for achieving all of the processes previously determined (Blair, D.F., Gelles, J. and Chan, S.I. (1986) Biophys. J. 50, 713-733) to be needed to accomplish redox-linked proton pumping. These processes include: (1) modulation of the energetics of protonation/deprotonation reactions and modulation of the energetics of redox reactions by the structural state of the pumping site; (2) control of the rates of the pump's redox reactions with its electron-transfer partners during the turnover cycle (gating of electrons); and (3) regulation of the rates of the protonation/deprotonation reactions between the pumping site and the aqueous phases on the two sides of the membrane during the reaction cycle (gating of protons). The model is the first proposed for the cytochrome oxidase proton pump which is mechanistically complete and sufficiently specific that a realistic assessment can be made of how well the model pump would function as a redox-linked free-energy transducer. This assessment is accomplished via analyses of the thermodynamic properties and steady-state kinetics expected of the model. These analyses demonstrate that the model would function as an efficient pump and that its behavior would be very similar to that observed of cytochrome oxidase both in the mitochondrion and in purified preparations. The analysis presented here leads to the following important general conclusions regarding the mechanistic features of the oxidase proton pump. (1) A workable proton-pump mechanism does not require large protein conformational changes. (2) A redox-linked proton pump need not display a pH-dependent midpoint potential, as has frequently been assumed. (3) Mechanisms for redox-linked proton pumps that involve transition metal ligand exchange reactions are quite attractive because such reactions readily lend themselves to the linked gating processes necessary for proton pumping. 相似文献
96.
By the use of a highly specific monoclonal antibody (designated MC), we were able to detect three radiolabeled bands with molecular weights of 60,000, 63,000, and 66,000 daltons in the ts-110 Moloney murine sarcoma virus mutant-transformed rat kidney cells known as 6M2. Expression of transformation properties as well as these three bands in 6M2 cells was found to be temperature sensitive. Therefore, MC detected factors that are apparently associated with the transformation of 6M2 cells. These factors are tentatively referred to as transformation associated proteins. These transformation proteins were found in two other Moloney murine sarcoma virus-transformed rat cell lines. These proteins were found to differ from known gene products of the ts-110 Moloney murine sarcoma virus mutant and do not have kinase activity. The transformation associated proteins may represent rat cellular factors activated during the transformation of rat cells by Moloney murine sarcoma virus. 相似文献
97.
Temperature dependence of the reduction potential of CuA in carbon monoxide inhibited cytochrome c oxidase 总被引:2,自引:0,他引:2
The temperature dependence of the reduction potential of the CuA site in carbon monoxide inhibited cytochrome c oxidase has been measured with a spectroelectrochemical method adapted to the relatively weak near-infrared absorption of this copper ion. These measurements, together with parallel measurements on the 604-nm absorption due to Fea, indicate that an interaction between CuA and Fea causes the reduction potential for one of these sites to be decreased by approximately 40 mV upon reduction of the other. The temperature dependence of the CuA reduction potential indicates a relatively large and negative standard entropy of reduction of CuA (delta So' = -48.7 +/- 2.3 eu). Possible implications of the intersite redox interaction and the large standard entropy of reduction of the CuA site are discussed. 相似文献
98.
Pigment patterns in mutants affecting the biosynthesis of pteridines and xanthommatin in Drosophila melanogaster 总被引:3,自引:0,他引:3
Juan Ferré Francisco J. Silva M. Dolores Real José L. Ménsua 《Biochemical genetics》1986,24(7-8):545-569
Eye-color mutants of Drosophila melanogaster have been analyzed for their pigment content and related metabolites. Xanthommatin and dihydroxanthommatin (pigments causing brown eye color) were measured after selective extraction in acidified butanol. Pteridines (pigments causing red eye color) were quantitated after separation of 28 spots by thin-layer chromatography, most of which are pteridines and a few of which are fluorescent metabolites from the xanthommatin pathway. Pigment patterns have been studied in 45 loci. The pteridine pathway ramifies into two double branches giving rise to isoxanthopterin, drosopterins, and biopterin as final products. The regulatory relationship among the branches and the metabolic blockage of the mutants are discussed. The Hn locus is proposed to regulate pteridine synthesis in a step between pyruvoyltetrahydropterin and dihydropterin. The results also indicate that the synthesis and accumulation of xanthommatin in the eyes might be related to the synthesis of pteridines.Support for this work was provided to J.F. in part by a grant from the Ministerio de Universidades e Investigación (Spain) and to F.J.S. by a grant from the Ministerio de Educación y Ciencia (Spain). 相似文献
99.
Epitopes, structural domains, and asymmetry of amino acid residues in SS-B/La nuclear protein 总被引:21,自引:0,他引:21
E K Chan A M Francoeur E M Tan 《Journal of immunology (Baltimore, Md. : 1950)》1986,136(10):3744-3749
SS-B/La is a conserved cellular phosphoprotein of 46 to 48 KD that is the target antigen of autoantibodies in sera of patients with Sjogren's syndrome and systemic lupus erythematosus. SS-B/La is also known to be associated with certain small cellular and viral RNA, including adenovirus VAI and VAII RNA. Two relatively protease-resistant domains (X and Y) were defined in SS-B from HeLa cells by using human autoantibodies as reagents. Domain X, a methionine-containing nonphosphorylated 28 KD polypeptide, was found to be resistant to partial digestion with six different proteases. Similar domains were also found in calf and rabbit SS-B. Domain Y, a 23 KD polypeptide, was detected after limited digestion with S. aureus V8 and trypsin. This domain contained little if any methionine, but all the detectable phosphorylated amino acids. Among 16 anti-SS-B sera tested by immunoblotting, 11 (69%) were reactive with both domains, three (19%) only with domain X, and two (13%) only with domain Y. These results showed that there are at least two distinct antigenic epitopes on the 46 to 48 KD SS-B/La protein, each located on a separate structural domain. The asymmetric distribution of methionine and phosphorylated amino acid residues in SS-B/La show striking similarity to the two reported domains of the adenovirus 72 KD DNA-binding protein, and raises questions concerning functional similarities that await investigation. 相似文献
100.
C W Hu T A Griffin K S Lau R P Cox D T Chuang 《The Journal of biological chemistry》1986,261(1):343-349
To characterize the lipoyl-bearing domain of the dihydrolipoyl transacylase (E2) component, purified branched-chain alpha-keto acid dehydrogenase complex from bovine liver was reductively acylated with [U-14C] alpha-ketoisovalerate in the presence of thiamin pyrophosphate and N-ethylmaleimide. Digestion of the modified complex with increasing concentrations of trypsin sequentially cleaved the E2 polypeptide chain (Mr = 52,000) into five radiolabeled lipoyl-containing fragments in the order of L1 (Mr = 28,000), L2 (Mr = 24,500), L3 (Mr = 21,000), L4 (Mr = 15,000) to L5 (Mr = 14,000) as determined by the autoradiography of sodium dodecyl sulfate-polyacrylamide gel. In addition, a lipoate-free inner E2 core consisting of fragment A (Mr = 26,000) and fragment B (Mr = 22,000) was produced. Fragment A contains the active site for transacylation reaction and fragment B is the subunit-binding domain. Fragment L5 and fragment B were stable and resistant to further tryptic digestion. Mouse antiserum against E2 reacted only with fragments L1, L2, and L3, and did not bind fragments L4, L5, A, and B as judged by immunoblotting analysis. The anti-E2 serum strongly inhibited the overall reaction catalyzed by the complex, but was without effect on the transacylation activity of E2. Measurement of incorporation of [1-14C]isobutyryl groups into the E2 subunit indicated the presence of 1 lipoyl residue/E2 chain. Based on the above data, a model is proposed in which the lipoyl-bearing domain is connected to the inner E2 core via a trypsin-sensitive hinge. The lipoyl-bearing domain contains five consecutive tryptic sites (L1 to L5), with the L1 site in the hinge region, and the L5 site next to the terminal lipoyl-binding sequence. An exposed and antigenic region is located between L1 and L4 tryptic sites of the lipoyl-bearing domain. The region accounts for about 24% of the E2 chain length. Binding of antibodies to this region probably impairs the mobility of the lipoyl-containing polypeptide, resulting in an interruption of the active-site interactions that are necessary for the overall reaction. The lack of antigenicity and resistance to tryptic digestion indicate a highly folded conformation for fragment L5, the limit polypeptide carrying the single lipoyl residue. 相似文献