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1.
In order to evaluate the effectiveness and selectivity of various reagents in the solubilization of bacterial membranes, membranes of Micrococcus lysodeikticus were treated with detergents and chaotropic agents. The composition of the extracts so obtained was analyzed by rocket and two-dimensional immunoelectrophoretic techniques. Recovery of succinate-, malate-, and reduced nicotinamide adenine dinucleotide- (NADH) dehydrogenases, ATPase, succinylated lipomannan and cytochromes in the extracts was measured. Treatment with a variety of non-denaturing detergents produced extracts that were generally qualitatively uniform although quantitative differences were observed. The degree of extraction of various components was correlated with the hydrophile-lipophile balance. Several chaotropic agents were also evaluated as reagents for membrane solubilization. These agents were less effective in extraction of bulk protein, but produced extracts enriched in some membrane components. 相似文献
2.
3.
Modification of the salt concentration, composition and/or buffer type in the assay of plasma membrane ATPase activity caused substantial changes in the Km and slight changes in the temperature dependence of this enzyme. The Km and temperature dependence were also affected by detergent solubilization of the ATPase and its subsequent reconstitution into liposomes. Modulation of kinetic properties by assay composition and hydrophobic state reflect the sensitivity of the plasma membrane H+-ATPase to its immediate environment. This may indicate a possible regulatory mechanism for this important plant enzyme. 相似文献
4.
Characterization of Neurotensin Binding Sites in Intact and Solubilized Bovine Brain Membranes 总被引:1,自引:0,他引:1
Analysis of the equilibrium binding of [3H]-neurotensin(1-13) at 25 degrees C to its receptor sites in bovine cortex membranes indicated a single population of sites with an apparent equilibrium dissociation constant (KD) of 3.3 nM and a density (Bmax) of 350 fmol/mg protein (Hill coefficient nH = 0.97). Kinetic dissociation studies revealed the presence of a second class of sites comprising less than 10% of the total. KD values of 0.3 and 2.0 nM were obtained for the higher and lower affinity classes of sites, respectively, from association-dissociation kinetic studies. The binding of [3H]neurotensin was decreased by cations (monovalent and divalent) and by a nonhydrolysable guanine nucleotide analogue. Competition studies gave a potency ranking of [Gln4]neurotensin greater than neurotensin(8-13) greater than neurotensin(1-13). Smaller neurotensin analogues and neurotensin-like peptides were unable to compete with [3H]neurotensin. Stable binding activity for [3H]neurotensin in detergent solution (Kd = 5.5 nM, Bmax = 250 fmol/mg protein, nH = 1.0) was obtained in 2% digitonin/1 mM Mg2+ extracts of membranes which had been preincubated (25 degrees C, 1 h) with 1 mM Mg2+ prior to solubilization. Association-dissociation kinetic studies then revealed the presence of two classes of sites (KD1 = 0.5 nM, KD2 = 3.6 nM) in a similar proportion to that found in the membranes. The solubilized [3H]-neurotensin activity retained its sensitivity to cations and guanine nucleotide. 相似文献
5.
酰尿输出型蚕豆有一定程度合成和同化酰脲的能力(刘承宪和黄维南1987a),蚕豆叶片尿囊素酶(B-ALNase)和酰脲输出型大豆叶片尿囊素酶(S-ALNase)(Thomas等1983)不同,是热敏感的(刘承宪和黄维南 1987b)。我们进一步分离和纯化B-ALNase,并作了初步的鉴 相似文献
6.
A. Touati C. Creuzenet J. M. Chobert E. Dufour T. Haertlé 《Journal of Protein Chemistry》1992,11(6):613-621
The study of the solubility of unstructured proteins (S1-, -, and -casein) and well-structured globulin (-lactoglobulin) in low water binary solvent systems demonstrated the crucial importance of solvent polarity and neutralization of protein polar functions on the final outcome of solubility experiments. The solubilities up to 38, 56, and 96% in CHCl3/CH3OH (1/1, v/v) acidified with HCl and up to 5, 10, and 25% in CHCl3/CH3OH (1/1, v/v) in the presence of triethylamine (TEA) were obtained for -, S1-, and -casein, respectively. The importance of protein charge neutralization was apparent when the solubilization was performed in basified CHCl3/CH3OH media, giving the optimal results when the studied proteins were brought before to their isoionic point. The maximum solubility of -casein at its pI in 30–70% methanol in CHCl3 was reaching 50–60% with triethylamine (TEA) added. -lactoglobulin could be solubilized up to 70% in CHCl3/CH3OH (7/3, v/v) acidified with HCl and up to 40% in CHCl3/CH3OH (3/7, v/v) in the presence of TEA. The observed yield of reductive alkylation of -lactoglobulin was much higher (98%) when performed in studied solvent system than in aqueous conditions (75%). Apparently, steric hindrance of the well-folded -barrel (in aqueous conditions) structure masks the portion of -NH2 groups. In the case of unstructured aqueous media -casein, 90% alkylation yields were obained in organic and aqueous conditions. 相似文献
7.
Summary Unproteolyzed gap junctions isolated from rat heart and liver were analyzed for the presence of inter-subunit disulfide bonds by sodium dodecylsulfate polyacrylamide gel electrophoresis. Rat cardiac junctions contained multiple disulfide bonds connecting theM
r 47,000 subunits of the same connexon and of different connexons. Inter-subunit disulfide bonds were absent in liver junctions. Unproteolyzed rat heart gap junctions were resistant to deoxycholate in their oxidized state, but dissolved readily in the detergent when the disulfide bonds were cleaved with -mercaptoethanol. Disulfide bonding in proteolyzed cardiac junctions was limited to pairs ofM
r 29,500 subunits. These junctions were not soluble in deoxycholate even in the presence of -mercaptoethanol. These results show that heart and liver junctions differ in their quarternary organization. 相似文献
8.
A comparison of techniques for isolation of the outer membrane proteins of Haemophilus influenzae type b 总被引:7,自引:0,他引:7
Terrence L. Stull Karl Mack Joel E. Haas John Smit Arnold L. Smith 《Analytical biochemistry》1985,150(2):471-480
We compared several rapid techniques used for extraction of outer membrane proteins from gram-negative enteric bacteria to Haemophilus influenzae type b. After lysis of cells with a French press, the inner and outer membranes were separated by isopycnic centrifugation. Each membrane was identified by density, morphology, enzymatic activity, and susceptibility to solid-phase iodination of intact cells. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we identified 10 polypeptides which were enriched in the outer membrane band compared to the inner membrane band. Using these proteins, we compared the polypeptide pattern of outer membranes with that obtained by (1) selective solubilization with sodium dodecyl-beta-D-maltoside, octyl-beta-D-glucopyranoside, Triton X-100, sodium, or cholamidopropyl dimethylaminopropanesulfonate; (2) extraction with chaotropic agents and heat; and (3) differential centrifugation of vesicles shed during transition from log growth phase to stationary growth phase. There were definable differences between the polypeptide pattern of membranes obtained with each rapid technique compared to the polypeptide pattern of isolated outer membranes. The polypeptide pattern of lithium extracts and the Triton X-100 insoluble fractions of total membranes most closely approximated the polypeptide pattern of isopycnically isolated outer membranes. Depending on the outer membrane protein sought, one of these rapid techniques can be utilized when a rapid method of outer membrane protein isolation is required. 相似文献
9.
Francisco J. Campoy Maria Dolores Cánovas Encarnación Muñoz-Delgado Cecilio J. Vidal 《Neurochemical research》1989,14(2):197-204
Incubation of membranes derived from sarcotubular system of rabbit skeletal muscle with increasing concentrations of Triton X-100 produced both stimulation of the AChE activity and solubilization of this enzyme. Mild proteolytic treatment of microsomal membranes produced a several fold activation of the still membrane-bound acetylcholinesterase (AChE) activity. Attempts were made to solubilize AChE from microsomal membranes by proteolytic treatment. About 30–40% of the total enzyme activity could be solubilized by means of trypsin or papain. Short trypsin treatment of the microsomal membranes produced first an activation of the membrane-bound enzyme followed by solubilization. Incubation of muscle microsomes for a short time with papain yielded a significant portion of soluble enzyme. Membrane-bound enzyme activation was measured after a prolonged incubation period. These results are compared with those of solubilization obtained by treatment of membranes with progressive concentrations of Triton X-100. The occurrence of molecular forms in protease-solubilized AChE was investigated by means of centrifugation analysis and slab gel electrophoresis. Centrifugation on sucrose gradients revealed two main components of 4.4S and 10–11S in either trypsin or papain-solubilized AChE. These components behaved as hydrophilic species whereas the Triton solubilized AChE showed an amphipatic character. Application of slab gel electrophoresis showed the occurrence of forms with molecular weights of 350,000; 175,000; 165,000; 85,000 and 76,000. The stimulation of membrane-bound AChE by detergents or proteases would indicate that most of the enzyme molecules or their active sites are sequestered into the lipid bilayer through lipid-protein or protein-protein interactions and these are broken by proteolytic digestion of the muscle microsomes. 相似文献
10.
[3H]Spiperone ([3H]SPI) binding sites in rat or bovine striata have been solubilized using CHAPS or digitonin detergents. Solubilized sites retained the binding characteristics of those in native membrane preparations. The same solubilized material, however, did not bind [3H]tyramine ([3H]PTA), thus indicating that [3H]PTA binding sites and DA receptors are different chemico-physical entities. In membrane preparations or crude synaptosomes obtained from the c.striatum of neonatally-rendered hypothyroid rats, when central DA-pathways are impaired, both [3H]PTA binding and [3H]DA uptake processes were markedly decreased, with no effect on [3H]mazindol ([3H]MAZ) binding, compared to euthyroids. Reserpine, a well-known inhibitor of DA-uptake into a variety of secretory vesicles, and a potent in vivo andin vitro inhibitor of [3H]PTA binding, did not affect the [3H]MAZ binding process. This further supported the suggestion that while [3H]PTA binding sites are almost totally associated with the vesicular transporter for DA, [3H]MAZ does label a site involved in the DA-translocation across the neuronal membrane. The latter process seems to be rather insensitive to thyroid hypofunction, when however the intracellular storage of DA might be consistently impaired. In conclusion, PTA might be well exploited as a marker of the DA vesicular transporter through its molecular characterization, whenever possible.Special issue dedicated to Dr. Paola S. Timiras 相似文献