共查询到20条相似文献,搜索用时 15 毫秒
1.
S J Edelstein W B Beyer A T Elderfrawi M E Elderfrawi 《The Journal of biological chemistry》1975,250(15):6101-6106
Studies are reported on the influence of Triton X-100 on the molecular weight and functional properties of the acetylcholine receptor. Results are presented principally for receptors purified from Torpedo californica and Torpedo marmorata with a limited number of observations on the receptor from Electrophorus electricus. In equilibrium dialysis measurements Trito, X-100 greatly reduced acetylcholine binding to the high affinity sites of the receptor from T. californica, but had only a small effect on the sites of lower affinity. Sedimentation equilibrium experiments on receptor in the absence of added Triton X-100 revealed average apparent molecular weight values of 510,000 for receptor from T. californica and 665,000 for T. marmorata. Under those conditions 0.113 mg of residual Triton X-100 were found per mg of protein as determined by using 3H-labeled Triton X-100. The sedimentation data indicated the presence of more than one molecular species, involving a unit with an apparent molecular weight of 330,000 and higher aggregates. Upon addition of Triton X-100, the higher aggregates were reduced, and above 0.1 percent Triton X-100 the 330,000 unit was the principal component present for receptor from all three species examined. Various structural models are considered in the light of this value, the polypeptide size from Na dodecyl sulfate-gel electrophoresis, and the protomer size determined by the molecular weight of an acetylcholine binding site. 相似文献
2.
The physical properties of the cardiac muscarinic acetylcholine receptor (mAcChR) purified from porcine atria as recently described [Peterson, G.L., Herron, G.S., Yamaki, M., Fullerton, D.S., & Schimerlik, M.I. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 4993-4997] have been examined by D2O/H2O sucrose gradient sedimentation and Sephacryl S-300 gel filtration in Triton X-405 and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). From the sedimentation experiments the partial specific volume and sedimentation constant for the mAcChR-Triton X-405 complex were determined to be 0.813 cm3/g and 5.30 S, respectively, which lead to an estimate of the molecular weight of the complex of 143 000. Gel filtration in Triton X-405 gave an estimate of the Stokes radius (4.29 nm) and an apparent molecular weight of 116 000. Combination of sedimentation and gel filtration gave an apparent molecular weight of 137 000 and a frictional ratio (f/f0) of 1.21 for the complex. The partial specific volume of the receptor calculated from composition was 0.717 cm3/g assuming 26.5% by weight carbohydrate. The amount of bound Triton X-405 was estimated at 1.011 g/g of mAcChR, which gave an apparent molecular weight of 70 900 (sedimentation) or 68 200 (sedimentation plus gel filtration) for the uncomplexed receptor. SDS-PAGE experiments at acrylamide concentrations ranging from 6% T [monomer plus bis(acrylamide)] to 17% T gave a linear range of apparent molecular weight from 67 600 (6% T) to 98 600 (17% T), and calibration against the retardation coefficient, Kr, determined from Ferguson plots gave an apparent molecular weight of 89 100 +/- 6700. From a newly developed, novel evaluation scheme the anomalous migration of the mAcChR in SDS-PAGE was found to be due to both an excess charge density and an abnormally large shape parameter (Kr), and the true molecular weight of the protein portion of the mAcChR ligand binding polypeptide was estimated to be between 50 000 and 60 000. 相似文献
3.
The asymmetric forms of acetylcholinesterase were purified from the electric organs of the electric rays Narke japonica and Torpedo californica, and their properties were compared. Asymmetric acetylcholinesterase was purified by immunoaffinity chromatography with a monoclonal antibody (Nj-601) to acetylcholinesterase. The MgCl2 extracts of these electric organs were applied to a column of Nj-601-Sepharose, and the bound acetylcholinesterase was eluted by lowering the pH of the eluent to 2.8. The purified asymmetric acetylcholinesterases gave peaks of 17 S (A12) and 13 S (A8) on sucrose density gradients. The enzyme from N. japonica contained more A8 than A12, while that of T. californica contained more A12. After treatment with collagenase, the enzymes gave three peaks on sedimentation; 20 S, 16 S and 11 S for N. japonica, and 19 S, 15 S and 11 S for T. californica, indicating the presence of collagen-like tails. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the asymmetric acetylcholinesterase from N. japonica gave bands of Mr 140 000, 100 000, 70 000 and 60 000, while that from T. californica gave bands of Mr 140 000, 100 000, 70 000 and 55 000. The bands of Mr 70 000 and 140 000 were monomers and non-reducible dimers, respectively, of the catalytic subunits. The bands of Mr 60 000 and 55 000 were the tail subunits, since collagenase treatment of the purified enzymes markedly decreased the amounts of these components. The Mr 100 000 subunit constituted less than 3% of the total asymmetric acetylcholinesterase from N. japonica but 18% of that from T. californica. The tail subunits constituted 6-8% of the two preparations. The catalytic subunits and the Mr 100 000 subunits bound concanavalin A, indicating that they are glycoproteins. The amino acid compositions of the enzymes from N. japonica and T. californica were very similar. Both contained hydroxyproline and hydroxylysine, characteristic of the collagen-like tails. The enzyme required divalent metal ions for activity, but only Mn2+, Mg2+ and Ca2+ were effective. Mn2+ was effective at the lowest concentrations, while Mg2+ gave the highest activity. 相似文献
4.
Structural characterization of insulin receptors. I. Hydrodynamic properties of receptors from turkey erythrocytes 总被引:3,自引:0,他引:3
R A Aiyer 《The Journal of biological chemistry》1983,258(24):14992-14999
Insulin receptors from turkey erythrocyte plasma membranes were solubilized in nondenaturing detergents (Triton X-100 and sodium deoxycholate). Their hydrodynamic properties were determined by sedimentation analyses in H2O and D2O, and gel filtration on Sepharose 4B. Two specific insulin-binding species are observed after velocity sedimentation in linear sucrose density gradients: peaks I and II. In Triton X-100, the sedimentation coefficient (s20,w), partial specific volume (Vc), and Stokes radius (a) for peaks I and II are, respectively, 10.2 +/- 0.5 S and 6.6 +/- 0.5 S, 0.75 +/- 0.02 ml/g, and 0.76 +/- 0.02 ml/g, and 89 +/- 3 A and 76 +/- 3 A, to yield Mr = 410,000 +/- 75,000 and 235,000 +/- 55,000, respectively, for the protein-Triton X-100 complex. The corresponding values in deoxycholate solution are: 10.7 +/- 0.5 S and 6.9 +/- 0.5 S, 0.71 +/- 0.03 ml/g and 0.70 +/- 0.04 ml/g, and 86 +/- 3 A and 69 +/- 3 A for peaks I and II, respectively, to yield 360,000 +/- 65,000 and 180,000 +/- 45,000, respectively, for the molecular weight of the protein-deoxycholate complex. These data are consistent with a model whereby each receptor species binds to one micelle of the appropriate detergent. In agreement with this model, it was also found that, in both Triton X-100 and deoxycholate, concentrations higher than the critical micellar concentration are required in order to maintain discrete receptor species in solution. At concentrations below the critical micellar concentration, the receptors aggregate to a broad band that sediments faster than 11.3 S. This is typical of membrane proteins that are stabilized in solution by insertion into detergent micelles. Based on these results, the protein molecular weights of peaks I and II are estimated to be 355,000 +/- 65,000 and 180,000 +/- 45,000, respectively. When membranes are treated with the reducing agent dithiothreitol, peak I is converted to peak II. This fact, together with the estimates obtained for the protein molecular weights of the two receptor species, suggests that peak I is a disulfide-linked dimer of peak II. The sedimentation characteristics of insulin receptors in many different cell types appear to be similar. As with turkey erythrocytes, detergent extracts of membranes from rat liver contained two native receptor species whose sedimentation coefficients were similar to peaks I and II. However, in all the other cell types examined, including rat adipocytes, rat heart muscle, 3T3-L1 adipocytes, 3T3-C2 fibroblasts, and FAO hepatoma cells, peak I (the native dimer) was the predominant species observed. 相似文献
5.
The interaction of a cholinergic depolarizing agent, bromoacetylcholine, with acetylcholine receptor (AcChR) enriched membrane fragments and Triton-solubilized, purified AcChR from Torpedo californica has been studied. The reagent bound to membrane-bound AcChR reversibly with an apparent dissociation constant of 16 +/- 1 nM at equilibrium. This 600-fold higher affinity for the receptor than found from physiological studies [Kact congruent to 10 micrometers; Karlin, A. (1973) Fed. Proc. Fed. Am. Soc. Exp. Biol. 32, 1847--1853] can be attributed to a ligand-induced affinity change of the membrane-bound receptor upon preincubation with bromoacetylcholine. At equilibrium [3H]bromoacetylcholine, like acetylcholine, bound to half the number of alpha-bungarotoxin sites present in the preparation without apparent positive cooperativity, and this binding was competitively inhibited by acetylcholine. In the presence of dithiothreitol, [3H]bromoacetylcholine irreversibly alkylated both membrane-bound and solubilized, purified acetylcholine receptor, with a stoichiometry identical with that for reversible binding. NaDodSO4-polyacrylamide gel electrophoresis of the labeled acetylcholine receptor showed that only the 40 000-dalton subunit contained the label. From these results it is concluded that the 40 000-dalton subunit represents a major component of the agonist binding site of the receptor. 相似文献
6.
The photoreaction center from Rhodospirillum rubrum strain G9 binds about 6 times as much sodium dodecyl sulfate as certain proteins commonly used as molecular weight markers for sodium dodecyl sulfate--polyacrylamide gel electrophoresis. This presumably explains the apparent discrepancy between the molecular weight of the photoreaction center determined by electrophoresis (76 000) and its minimal molecular weight (87 000). The molecular weight of the photoreaction center solubilized with Triton X-100 was determined by three different methods: conventional sedimentation equilibrium, a combination of sedimentation velocity and gel filtration measurements, and sedimentation equilibrium in H2O and in D2O. Each technique required a determination of the amount of bound detergent. All three methods gave molecular weight values close to 60 000. A similar molecular weight was found for the photoactive beta gamma dimer obtained from the photoreaction center of Rhodopseudomonas sphaeroides strain 2.4.1 which, as a whole, had a molecular weight of 87 000. These results indicate that the photoreaction center from Rp. sphaeroides is an oligomer of the type alpha 1 beta 1 gamma 1. In contrast, the photoreaction center from Rs. rubrum appears to be dissociated, in solution, into a photoactive beta gamma dimer and a free polypeptide alpha. 相似文献
7.
The structural basis for the heterogeneity of the two agonist binding sites of the Torpedo californica acetylcholine receptor with respect to antagonist binding and reactivity toward affinity alkylating reagents was investigated. There is one agonist binding site on each of the two alpha subunits in a receptor monomer. One of these sites is easily affinity labeled with bromoacetylcholine, while more extreme conditions are required to label the other. Evidence is presented that the site which is easily labeled with bromoacetylcholine is the site with higher affinity for the antagonist d-tubocurarine. Digestion of purified alpha subunits with staphylococcal V8 protease gave two limit fragments with apparent molecular weights of 17K and 19K. Both of these fragments began at residue 46 of the alpha sequence, and both reacted with monoclonal antibodies specific for the sequence alpha 152-159 but not with antibodies specific for alpha 235-242. Their tryptic peptide maps and reactivity with a number of monoclonal antibodies were virtually identical. Only the 17-kilodalton (17-kDa) fragments stained heavily for sugars with Schiff's reagent. However, both fragments bound 125I-labeled concanavalin A. Complete removal of carbohydrate detectable with concanavalin A from V8 protease digests of alpha subunits resulted in two fragments of lower apparent molecular weights, indicating that these fragments differed not only in carbohydrate content but also in their C-termini or by another covalent modification. Covalent labeling of one of the two agonist sites of the intact receptor with bromo[3H]acetylcholine followed by digestion with V8 protease resulted in labeling of only the 19-kDa fragment.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
8.
We have purified the junctional acetylcholine receptor from normal rat skeletal muscle and compared its structure with that of the extrajunctional receptor from denervated muscle. The two receptors from leg muscle were distinguished by isoelectric focusing and by reaction with sera from patients with myasthenia gravis. The junctional form of the acetylcholine receptor was purified from normal leg muscle by affinity chromatography on concanavalin A/Sepharose and cobrotoxin/Sepharose followed by sucrose gradient centrifugation. Analysis of radioiodinated receptor by polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated that the subunit structure of the junctional receptor was similar to that previously determined for the extra-junctional form (Froehner, S. C., et al. (1977) J. Biol. Chem. 252, 8589-8596), with major polypeptides, whose apparent molecular weights in 9% polyacrylamide gels were 45 000 and 51 000. In addition, several minor polypeptides were found. When the two receptors were labeled with different isotopes of iodine and run together on a sodium dodecyl sulfate gel, the subunits of one receptor could not be resolved from those of the other. As seen earlier with the extrajunctional form, the affinity alkylating reagent [3H]MBTA labeled the 45 000- and 49 000-dalton polypeptides of the junctional receptor. Peptide mapping showed that the two MBTA binding subunits are structurally related, although they are unrelated to the other polypeptides, and that the 45 000- and 51 000-dalton polypeptides of the junctional receptor were indistinguishable from those of the extrajunctional receptor. In addition, peptide mapping of the four subunits of acetylcholine receptor isolated from Torpedo californica electric organ showed that these four polypeptides appear to be structurally unrelated. 相似文献
9.
Xu Y 《Biophysical chemistry》2004,108(1-3):141-163
New graphical procedures have been developed to investigate the heterogeneity of protein preparations using sedimentation equilibrium. The heterogeneous systems that can be studied include self-associating systems contaminated by incompetent monomer, self-associating systems contaminated by non-dissociating oligomer and simple non-interacting monomer-oligmer disperse systems. The new procedures are based on the concentration dependence of the apparent association constants estimated by a non-linear least square fitting program (NONLIN), on the assumption of conservation of mass during sedimentation and on the applications of several standard techniques for statistical inferences of NONLIN estimations. The procedures outlined here can detect various types of heterogeneity, discriminate amongst different types of heterogeneity, estimate the amount of contaminant causing heterogeneity and determine the true equilibrium constant of the self-associating components. The procedures appear to be sensitive, accurate and easily applicable when tested using both protein samples and computer simulated data. 相似文献
10.
J Markl R Schmid S Czichos-Tiedt B Linzen 《Hoppe-Seyler's Zeitschrift für physiologische Chemie》1976,357(12):1713-1725
The haemolymph of the tarantulas, Dugesiella (Eurypelma) californica and Dugesiella (Eurypelma) helluo contains high molecular weight haemocyanin (80-82% of total blood proteins) and a second protein not related to haemocyanin (18-20%). In the Lycosid spider, Cupiennius salei, haemocyanin (75% of total blood protein) occurs in two states of association. The haemocyanins were isolated by ultracentrifugation, gel filtration, isoelectric focusing, or preparative gel electrophoresis. Their sedimentation constants are 36.7 S (both tarantulas), 23.4 S and 15.9 S (Cupiennius). After alkaline dissociation, polypeptides sedimenting at 5.8 S (D. californica) and 4.7 S (Cupiennius) were obtained. The molecular weight of the intact functional subunit is (by sedimentation equilibrium) 70 300 (D. californica) and 69 900 (Cupiennius). Copper analysis results in closely similar values. By sodium dodecylsulphate gel electrophoresis, molecular weights of 71 000 (D. californica), 72 000 (Cupiennius) and 74 000 (D. helluo) were obtained. Denaturation with various agents did not lead to smaller polypeptides. The amino acid composition of the haemocyanins was determined (Table 1). The amino end group is blocked. The haemocyanins contain 1.2-1.5% of neutral carbohydrates and 0.3-0.5% of glucosamine (possibly acetylated). The neutral carbohydrates were identified with glucose, mannose, fucose, and arabinose, glucose being the dominant species. Neuraminic acid was not detected. The haemocyanins of the three species cannot be distinguished by their carbohydrate moieties, while there is a significant difference in amino acid composition between tarantula and Cupiennius haemocyanins. The second, non-respiratory protein isolated from spider blood sediments with 16.1 S (Dugesiella) or 15.9 S (Cupiennius). Its isoelectric point is at pH 5.5 It is stable in weakly alkaline solutions but can be denatured to yield polypeptide chains with molecular weights of 95 000 and 110 000. The amino acid composition is reported. As in the haemocyanins, the N-terminus is blocked. The carbohydrate content is 0.9%, glucose being the only sugar identified. 相似文献
11.
A common molecular weight of the androgen receptor monomer in different target tissues 总被引:1,自引:0,他引:1
Previously reported molecular weights for the monomeric steroid binding subunit of the androgen receptor protein have ranged from 25,000 to 167,000. The molecular weight appeared to vary among different species and target organs, as well as between different investigators. This study has examined androgen receptors from a diverse group of organs and species to determine whether these tissues share a common monomeric form. Gel filtration revealed peaks of specific [3H]dihydrotestosterone binding activity corresponding to Stokes radii of 54, 33, and 20 A in cytosols from several tissues. Phosphocellulose chromatography diminished the appearance of the smaller androgen receptor forms and facilitated the appearance of the larger 54-A form. Mixing experiments suggested that phosphocellulose was stabilizing the 54-A form by binding putative proteases which cleave this larger form. Methods were developed to generate homogenous preparations of a given androgen receptor size for comparative study. Sucrose density gradient analysis showed sedimentation coefficients of 4.5-5.0, 3.5-4.0, and 2.5-3.0 S, respectively. The corresponding calculated molecular weights were 109,000-121,000, 52,000-59,000, and 22,000-27,000. Scatchard analysis of each of these androgen receptor forms demonstrated very similar affinity for [3H]dihydrotestosterone (Kd approximately 1 nM), and each form possessed the ability to bind to DNA-cellulose. Extensively purified preparations of androgen receptor from R3327 tumor contained varying amounts of the three receptor forms even though molybdate and phosphocellulose were used to stabilize the androgen receptor protein during purification.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
12.
Muscarinic acetylcholine receptor structure in acinar cells of mammalian exocrine glands 总被引:5,自引:0,他引:5
S R Hootman T M Picado-Leonard D B Burnham 《The Journal of biological chemistry》1985,260(7):4186-4194
Characterization of muscarinic acetylcholine receptors in acinar cells from rat pancreas and lacrimal and parotid glands was achieved by binding of the reversible muscarinic antagonist [3H]quinuclidinyl benzilate (QNB) and the specific alkylating reagent [3H]propylbenzilylcholine mustard (PrBCM) to intact acini or dispersed acinar cells. Binding studies with [3H]QNB showed that acinar cells from pancreas contain 26,400, from parotid 21,400, and from lacrimal gland 25,700 binding sites/cell. To assess molecular size of the receptor in each gland, acini were prepared by digestion with purified collagenase and singly dispersed acinar cells were prepared by a combination of digestion with crude collagenase, hyaluronidase, and alpha-chymotrypsin and divalent cation chelation using EDTA. Muscarinic receptors on acini or dispersed cells were covalently labeled with 5 nM [3H]PrBCM, solubilized directly in hot sodium dodecyl sulfate buffer, and resolved by polyacrylamide gel electrophoresis. When solubilized acini were electrophoresed, a major labeled peak was observed on gels along with a smaller peak of lower apparent molecular weight. For pancreatic acini, the apparent molecular weights of these peaks were 117,600 and 85,700; for parotid acini, 104,800 and 74,500; and for lacrimal acini, 87,200 and 63,100. Addition of muscarinic antagonists to the labeling medium abolished both peaks. When dispersed acinar cells were labeled, the larger peak was eliminated, and all radioactivity was concentrated in a single peak: 87,600 for pancreas, 78,000 for parotid gland, and 62,800 for lacrimal gland. Digestion of prelabeled acini with the mixture of enzymes used to produce dispersed acinar cells similarly shifted all radioactivity into this second peak. Limited digestion of acini or dispersed cells with 1 mg/ml of papain resulted in the disappearance of these higher molecular weight peaks and the appearance of a broad peak at Mr = 40,000. Cells of nonepithelial origin, IM-9 lymphocytes and NG108 neuroblastoma X glioma hybrids, also were labeled with [3H]PrBCM and electrophoresed.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
13.
The ion-gating ability and the protein electrophoretic band patterns of the acetylcholine receptor from Torpedo californica electroplax were examined after receptor-enriched membrane vesicles were progressively heated. The ion translocation function was lost over a temperature range of 40-55 degrees C. Previous results have shown that the stoichiometry of alpha-bungarotoxin binding is not affected by these temperatures, although bound toxin reversibly dissociates within this temperature range, and that toxin binding is irreversibly lost at somewhat higher temperatures [Soler, G., Farach, M.C., Farach, H. A., Jr., Mattingly, J.R., Jr., & Martinez-Carrion, M. (1983) Arch. Biochem. Biophys. 225, 872]. Thermal gel analysis [Lysko, K. A., Carlson, R., Taverna, R., Snow, J., & Brandts, J.F. (1981) Biochemistry 20, 5570], a sodium dodecyl sulfate-polyacrylamide gel electrophoretic procedure which detects thermally induced aggregation of the components of multimeric systems, was applied to heated acetylcholine receptor enriched membranes. This technique suggests two structural domains susceptible to thermal perturbation within the receptor molecule, one consisting of the Mr 50 000 and the two Mr 40 000 subunits and the other consisting of the Mr 60 000 and 65 000 subunits. Heat disrupts molecular events linking agonist binding with ion-channel opening in the acetylcholine receptor molecule. 相似文献
14.
Viral DNA was isolated from human and murine cytomegalovirus by equilibrium centrifugation in cesium chloride gradients. The size of the DNA was measured relative to T4 DNA by velocity sedimentation in neutral glycerol gradients, and fractions were assayed for infectious DNA. Infectious murine cytomegalovirus DNA sedimented as a single peak with an estimated molecular weight of 136 X 10(6). Infectious human cytomegalovirus DNA was detected in two peaks with molecular weights of 130 X 10(6) and 150 X 10(6). 相似文献
15.
J Imanishi 《Japanese journal of microbiology》1975,19(5):337-342
It was found that a preparation of mouse L cell interferon induced by Newcastle disease virus (NDV) possessed not only interferon activity but also inhibitory activity upon migration of guinea pig peritoneal macrophages (MIF activity). These activities were also observed in a preparation of human leukocyte interferon induced by NDV. The interferon and MIF activities shared common characteristics in the dose response, time course of in vitro production, thermal stability, sensitivity to trypsin and periodate, and elution pattern in CM-Sephadex column chromatography. However, gel filtration pattern with Sephadex G-100 showed two separate peaks. Fractions collected from the first peak, corresponding to a molecular weight of about 45 000, had only the MIF activity, while those collected from the second peak, corresponding to a molecular weight of about 30 000, had both the interferon and MIF activities. A preparation of mouse brain interferon induced by Japanese encephalitis virus had a much weaker MIF activity than the L cell interferon, although these preparations were equal in interferon activity (5000 units/ml). 相似文献
16.
Tryptic digestion of acetylcholine receptor (AChR) from Torpedo californica did not change the pharmacological specificity and the pathological myasthenic acitivity of the receptor molecule. The product obtained after tryptic digestion was repurified by affinity chromatography on a toxin-Sepharose resin and was designated T-AChR. T-AChR has a sedimentation coefficient of 8.0S and in SDS acrylamide gel electrophoresis shows one major band with a molecular weight of 27,000. Immunological studies reveal that T-AChR binds to anti-AChR antibodies directed only against conformational antigenic determinants. 相似文献
17.
Transmembrane topology of acetylcholine receptor subunits probed with photoreactive phospholipids 总被引:3,自引:0,他引:3
The domains of the acetylcholine receptor subunits that contact the lipid phase were investigated by hydrophobic photolabeling of receptor-rich membrane fragments prepared from Torpedo marmorata and Torpedo californica electric organs. The radioactive arylazido phospholipids used carry a photoreactive group, either at the level of the lipid polar head group (PCI) or at the tip of the aliphatic chain (PCII), and thus probe respectively the "superficial" and "deep" regions of the lipid bilayer. The four subunits of T. marmorata and T. californica acetylcholine receptor reacted with both the PCI and PCII probes and thus are all exposed to the lipid phase. Ligands known to stabilize different conformations of the acetylcholine receptor (nicotinic agonists, snake alpha-toxin, and noncompetitive blockers) did not cause any significant change in the labeling pattern. The acetylcholine receptor associated 43 000-dalton v1 protein did not react with any of the probes. A striking difference in labeling between T. marmorata and T. californica acetylcholine receptors occurred at the level of the alpha-subunit when the superficial PCI probe was used. An approximately 5-fold higher labeling of the alpha-subunit as compared to the beta-, gamma-, and delta-subunits was observed by using receptor-rich membranes from T. marmorata but not from T. californica. The same difference persisted after purification of the labeled receptors from the two species and was restricted to an 8000-dalton C-terminal tryptic peptide. The only mutation observed in this region of the complete alpha-subunit sequence of the two species is the substitution of cysteine-424 in T. marmorata by serine-424 in T. californica.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
18.
K U Jansen W G Conroy T Claudio T D Fox N Fujita O Hamill J M Lindstrom M Luther N Nelson K A Ryan 《The Journal of biological chemistry》1989,264(25):15022-15027
Yeast expression vectors were constructed containing complementary DNA encoding the alpha-, beta-, gamma-, and delta-subunits of the Torpedo californica nicotinic acetylcholine receptor under the control of the Saccharomyces cerevisiae alcohol dehydrogenase promoter. All four plasmids were integrated into the yeast genome of a single yeast cell. The resulting yeast strain synthesized polypeptides novel to yeast that had the molecular weights and antigenic properties similar to the authentic T. californica receptor alpha-, gamma, and delta-subunits. The beta-subunit polypeptide could not be detected in this yeast strain, even though the poly(A)+ RNA from this strain contained all the information necessary for the expression of functional acetylcholine receptors in Xenopus laevis oocytes. The replacement of the beta-subunit mRNA 5'-untranslated leader and its N-terminal signal sequence by the corresponding alpha-subunit sequences, however, resulted in the expression of the beta-subunit polypeptide in yeast grown at 5 degrees C. 相似文献
19.
Jiro Imanishi 《Microbiology and immunology》1975,19(5):337-342
It was found that a preparation of mouse L cell interferon induced by Newcastle disease virus (NDV) possessed not only interferon activity but also inhibitory activity upon migration of guinea pig peritoneal macrophages (MIF activity). These activities were also observed in a preparation of human leukocyte interferon induced by NDV. The interferon and MIF activities shared common characteristics in the dose response, time course of in vitro production, thermal stability, sensitivity to trypsin and periodate, and elution pattern in CM-Sephadex column chromatography. However, gel filtration pattern with Sephadex G-100 showed two separate peaks. Fractions collected from the first peak, corresponding to a molecular weight of about 45 000, had only the MIF activity, while those collected from the second peak, corresponding to a molecular weight of about 30 000, had both the interferon and MIF activities. A preparation of mouse brain interferon induced by Japanese encephalitis virus had a much weaker MIF activity than the L cell interferon, although these preparations were equal in interferon activity (5000 units/ml). 相似文献
20.
Several methods were used to analyze for tryptophan in the acetylcholine (ACh) receptors purified from the electric organs of the electric rays, Torpedo californica and Torpedo marmorata. The best value of tryptophan was 2.4 mol %. When excited at 290 nm, both receptors fluoresced with a maximum at 336, but there was no change in the fluorescence emission spectra upon binding of carbamylcholine, d-tubocurarine, ACh, or decamethonium. The free SH content of the Torpedo receptors varied in different preparations, and was highest in that purified from fresh T. californica using deaerated solutions and dialysis under nitrogen, and lowest in that prepared from the aged lyophilized membranes of T. marmorata. The maximum free SH content was 20 nmol/mg of protein or 0.22 mol %, equal to at most 18% of the total cysteic acid residues. Reaction of either 33% or of all the SH residues with p-chloromercuribenzoate reduced maximum ACh binding to the pure receptor prepared from fresh T. californica by only 23%. 相似文献