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81.
[14C]Cholesteryl ester was directly incorporated into human plasma low-density lipoproteins (LDL) for the purpose of preparing a tracer substrate for investigation of the cholesteryl ester transfer reaction between plasma lipoproteins. The radiolabeled cholesteryl oleate was sonicated with egg phosphatidylcholine to form cholesteryl ester-containing liposomes. The liposomes were incubated with plasma fraction of density greater than 1.006 at 37 degrees C in the presence of dithionitrobenzoic acid. When the distribution of the radiolabeled cholesteryl ester was equilibrated among liposomes and lipoprotein fractions, the mixture was applied to an affinity chromatography column of dextran sulfate-cellulose (LA01) (Arteriosclerosis 4, 276-282). LDL was eluted by increasing the NaCl concentration and was finally isolated as a floating fraction by ultracentrifugation at a solvent density of 1.063 (adjusted with NaCl). The chemical composition, electrophoretic mobility and density of the labeled LDL were consistent with those of the native LDL. Radioactivity in this preparation was present exclusively in cholesteryl ester. Apolipoprotein B100 was preserved intact throughout the procedure. When the rate of cholesteryl ester transfer was measured between LDL and high-density lipoproteins by using this labeled LDL, the kinetics was consistent with the equilibrium transfer model, but the apparent rate measured was slightly higher than that measured with the labeled LDL prepared by the method using the intrinsic cholesterol esterification reaction of plasma. 相似文献
82.
Purification of natural human interferon-gamma by antibody affinity chromatography: analysis of constituent protein species in the dimers 总被引:1,自引:0,他引:1
K Miyata Y Yamamoto M Ueda Y Kawade K Matsumoto I Kubota 《Journal of biochemistry》1986,99(6):1681-1688
A simple procedure for purifying human interferon-gamma from leukocytes was established, based on monoclonal antibody affinity chromatography. The recovery of interferon activity was essentially quantitative, and the specific activity of the product was (4-12) x 10(7) international units/mg protein. SDS-polyacrylamide gel electrophoresis reproducibly revealed four components associated with interferon activity (and no other proteins): two major ones with molecular weights (MW) of 24,000-25,000 (25K) and 19,000-20,000 (20K), a minor one with MW 14,000-15,000 (15K) (these three bands were doublets), and a still less prominent one(s) with MV 40,000-48,000. Gel filtration in neutral solution indicated that all the 25K, 20K, and 15K species exist as oligomers, probably dimers. By means of experiments using a cleavable crosslinking reagent, the dimers were shown to comprise both homo-and heterodimers. Gel filtration in alkali (the condition used during purification) indicated that the molecules are largely in a monomeric state. Thus, the molecules once dissociated in alkali appear to reassociate at random upon neutralization; this process takes place without being accompanied by inactivation. 相似文献
83.
84.
85.
Interactions of permeant cations with sodium channels of squid axon membranes. 总被引:6,自引:1,他引:5
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To determine how the permeant cations interact with the sodium channel, the instantaneous current-voltage (I-V) relationship, conductance-ion concentration relationship, and cation selectivity of sodium channels were studied with internally perfused, voltage clamped squid giant axons in the presence of different permeant cations in the external solution. In Na-containing media, the instantaneous I-V curve was almost linear between +60 and -20 mV, but deviated from the linearity in the direction to decrease the current at more negative potentials. The linearity of instantaneous I-V curve extended to more negative potentials with lowering the external Ca concentration. The I-V curve in Li solution was almost the same as that in Na solution. The linearity of the I-V curve improved in NH4 solution exhibiting only saturation at -100 mV with no sign of further decrease in current at more negative potentials. Guanidine and formamidine further linearized the instantaneous I-V curve. The conductance of the sodium channels as measured from the tail current saturated at high concentrations of permeant cations. The apparent dissociation constants determined from the conductance-ion concentration curve at -60 mV were as follows: Na, 378 mM; Li, 247 mM; NH4, 174 mM; guanidine, 111 mM; formamidine, 103 mM. The ratio of the test cation permeability to the sodium permeability as measured from the reversal potentials of tail currents varied with the test cation concentration and/or the membrane potential. These observations are incompatible with the independence principle, and can be explained on the basis of the Eyring's rate theory. It is suggested that the slope of the instantaneous I-V curve is determined by the relative affinity of permeant cations and blocking ions (Ca) for the binding site in the sodium channel. The ionic selectivity of the channel depends on the energy barrier profile of the channel. 相似文献
86.
Location of the SH group of the alkali light chain on the myosin head as revealed by electron microscopy 总被引:1,自引:0,他引:1
K Yamamoto M Tokunaga K Sutoh T Wakabayashi T Sekine 《Journal of molecular biology》1985,183(2):287-290
The location of the single cysteinyl residue of the alkali light chain on the myosin head was determined by electron microscopy. The cysteinyl residue of isolated alkali light chain 2 was biotinylated and the light chain was exchanged with that of heavy meromyosin in 4.7 M-NH4Cl. Avidin was attached to the biotin in the heavy meromyosin and the complex was rotary shadowed and observed in the electron microscope. The distance from the head-rod junction to the centre of avidin was 8(+/- 3) nm (mean value +/- standard deviation: n = 105). 相似文献
87.
Spinach nitrate reductase: purification, molecular weight, and subunit composition 总被引:10,自引:7,他引:3
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Nitrate reductase was purified about 3,000-fold from spinach leaves by chromatography on butyl Toyopearl 650-M, hydroxyapatite-brushite, and blue Sepharose CL-6B columns. The purified enzyme yielded a single protein band upon polyacrylamide gel electrophoresis under nondenaturing conditions. This band also gave a positive stain for reduced methylviologen-nitrate reductase activity. The specific NADH-nitrate reductase activities of the purified preparations varied from 80 to 130 units per milligram protein. Sucrose density gradient centrifugation and gel filtration experiments gave a sedimentation coefficient of 10.5 S and a Stokes radius of 6.3 nanometers, respectively. From these values, a molecular weight of 270,000 ± 40,000 was estimated for the native reductase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the denatured enzyme yielded a subunit band having a molecular weight of 114,000 together with a very faint band possessing a somewhat smaller molecular weight. It is concluded that spinach nitrate reductase is composed of two identical subunits possessing a molecular weight of 110,000 to 120,000. 相似文献
88.
Tajima Shigeyuki; Kanazawa Takeshi; Takeuchi Eiko; Yamamoto Yukio 《Plant & cell physiology》1985,26(5):787-795
The cadaverine content of soybean radicles showed a maximumpeak 34 days after planting. The variation coincidedwith radicle uricase activity during seed germination. The uricase activity could not be fractionate when the bufferpH for the extraction was at 6.0. The addition of 1 M KCl orNaCl to the buffer allowed the extraction of the uricase activity,but an addition of 1 M MgCl2 or BaCl2 inhibited this enzyme'sactivity. The urate-degrading enzyme system was purified 248-fold permilligram of protein from soybean radicles. The respective Kmvalues of the diamine oxidase activity for cadaverine and ofthe urate-degrading activity for hydrogen peroxide and uratewere 1.25, 2.93 and 50.3 µM. Analysis by gel electrophoresisof the partially purified enzyme fraction revealed that theurate-degrading enzyme system consisted of a peroxidase thatdegrades urate with hydrogen peroxide and a diamine oxidasethat releases hydrogen peroxide. These data are evidence that a urate-degrading diamine oxidaseand peroxidase system exists in soybean radicles and that thereaction rate of urate-degradation is controlled by the concentrationof cadaverine. (Received November 28, 1984; Accepted April 8, 1985) 相似文献
89.
A simple and rapid preparation method for apoaspartate aminotransferase from Escherichia coli B was developed. A crude extract of the bacterial cells was treated batchwise with DEAE-cellulose. The enzyme fraction obtained was then applied to a pyridoxamine-Sepharose column. Apoaspartate aminotransferase was eluted with 50 mM potassium phosphate buffer (pH 7.0), and found to be electrophoretically homogeneous. The apoenzyme preparation thus obtained showed very low holoenzyme activity (only 0.4% of the activity seen in the fully saturated condition with pyridoxal 5'-phosphate) and was successfully used for assaying pyridoxal and pyridoxamine 5'-phosphate. 相似文献
90.
Lambda DNA (125 micrograms/ml in Tris buffer, pH 7.4) was irradiated with 60Co gamma-rays and 3H beta-rays, respectively, and the number of strand breaks was determined by electrophoresis. Number of single-strand breaks increased linearly with radiation dose in both gamma- and beta-radiations and the relative effectiveness (beta/gamma) was found to be 1.82 in N2 and 1.16 in O2. Number of double-strand breaks increased with the square of the radiation dose in gamma-irradiation, but it increased linearly with radiation dose in beta-irradiation. Therefore, the relative effectiveness (beta/gamma) is higher at lower doses. O2 effects was observed by gamma-irradiation but was minimal after beta-irradiation. 相似文献