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231.
232.
Karin Dahlgren Caldwell Rolf Axn Margareta Bergwall Ingmar Olsson Jerker Porath 《Biotechnology and bioengineering》1976,18(11):1605-1614
Hexyl-groups have been introduced into crosslinked Sepharose 6B, yielding gels with degrees of substitution which range from 0.02 to 0.70 mol hexyl-side chain per mole galactose residue. The gels were exposed to β-amylase in solution, and the resulting adsorbates indicated a monotonic increase in adsorption capacity with an increasing hexyl-content. Adsorbate activity, by contrast, displayed a maximum for a carrier gel with a hexyl–galactose ratio of 0.51. Adsorbates based on gels with different hexyl-content were used in column reactors for continuous maltose production from a soluble starch substrate. 相似文献
233.
Site-site interactions among insulin receptors. Characterization of the negative cooperativity. 总被引:38,自引:0,他引:38
By studying the dissociation of 125I-instulin from its receptors in the absence and phe negatively cooperative type for the insulin receptors. In the present study we extend oy purified mouse and rat liver membranes as well as in human circulating monocytes and human cultured lymphocytes demonstrated negative cooperativity that was extraordinarily simn membranes more slowly than it does from its receptors on whole cells. The dissociaty a small percentage of the receptor sites (1 to 5%), are sufficient to accelerate dissociation of hormone from receptor. At these insulin concentrations insulin is entirely monomeric, and in fact at higher concentrations of insulin (greater than 10(-7) M) where insulin dimers predominate, the cooperativity effect is progressively lost. The dissociation rate of 125I-insulin alone (that is at very low fractional saturation of receptors) was markedly accelerated by dripping the pH from 8.0 to 5.0, whereas the dissociation of 125I-insulin at high receptor occupancy was only slightly accelerated by the fall in pH. The dissociation rate was directly related to temperature, but the dissociation rate of 125I-insulin at low receptor occupancy was much more affected by reduction in temperature and showed a sharp transition at 21 degrees. Urea at concentrations as low as 1 M produced a marked acceleration of 125I-insulin dissociation. Divalent cations (calcium and magnesium) appear to stabilize the insulin-receptor interaction, since higher degrees of receptor occupancy were required to achieve a given rate of dissociation of 125I-insulin. These data make it likely that the insulin receptors exist as oligomeric structures or clusters in the plasma membrane. Insulin receptor sites appear to switch from a "slow dissociating" state to a "fast dissociating" state when their occupancy increases; the proportion of sites in each state is a function of occupancy of the receptor sites by the insulin monomer as well as of the physiochemical environment. Other models which could explain apparent negative cooperativity besides site-site interactions, i.e. polymerization of the hormone, steric or electrostatic hindrance due to ligand-ligand interactions, or unstirred (Noyes-Whitney) layers are considered unlikely in the case of insulin receptors on both experimental and theoretical grounds. 相似文献
234.
Cooperativity in ligand binding: a new graphic analysis. 总被引:16,自引:0,他引:16
When analyzing binding of ligands to macromolecules, the existence of site-site interactions complicates a straightforward interpretation of the binding parameters obtained through classical analytical methods, such as the Scatchard plot. For describing site-site interactions, we propose a new parameter, the of the receptor sites, , calculated as . Plotting as a function of fractional occupancy (), reveals that: (1) at very low occupancy a limiting high is obtained (e) (“empty sites” conformation); (2) when the fraction of sites filled increases above a certain threshold, begins to fall due to increasing site-site interactions until (3) a limiting low (f) is obtained (“filled sites” conformation). This method has been successfully applied to the negative cooperativity of insulin receptors. 相似文献
235.
V H Morgenroth L R Hegstrand R H Roth P Greengard 《The Journal of biological chemistry》1975,250(5):1946-1948
Addition of adenosine 3':5'-monophosphate (cAMP) to high speed supernatant preparations obtained from rat brain caused a 3- to 4-fold increase in tyrosine 3-monooxygenase (tyrosine hydroxylase) activity. The tyrosine 3-monooxygenase remained in an activated state upon removal of the cAMP by passing the enzyme through a Sephadex G-25 column. Substances which inhibit cAMP-dependent protein kinase, namely, EDTA, ADP, and adenosine, and protein kinase modulator, each antagonized the activation of tyrosine 3-monooxygenase produced by cAMP. Furthermore, addition of partially purified brain cAMP-dependent protein kinase caused a several-fold increase in tyrosin 3-monooxygenase activity. The activation of tyrosine 3-monooxygenase by added cAMP and protein kinase required the presence of ATP and Mg-2+. These data suggests that the cAMP activation of tyrosine 3-monooxygenase may be mediated by a cAMP-dependent protein kinase. 相似文献
236.
G H Wada J H Fellman T S Fujita E S Roth 《The Journal of biological chemistry》1975,250(17):6720-6726
Avian liver p-hydroxyphenylpyruvate hydroxylase (EC 1.13.11.27) was purified to a 1000-fold increase in specific activity over crude supernatant, utilizing a substrate analogue, o-hydroxyphenylpyruvate, to stabilize the enzyme. The preparation was homogeneous with respect to sedimentation with a sedimentation velocity (s20,w) of 5.3 S. The molecular weight of the enzyme was determined to be 97,000 +/- 5,000 by sedimentation equilibrium, and the molecular weight of the subunits was determined to be 49,000 +/- 3,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis revealed heterogeneity of the purified enzyme. The multiple molecular forms were separable by isoelectric focusing, and their isoelectric points ranged from pH 6.8 to 6.0. The amino acid compositions and tryptic peptide maps of the three forms isolated by isoelectric focusing were very similar. The forms of the enzyme had the same relative activity toward p-hydroxyphenylpyruvate and phenylpyruvate. Conditions which are known to accelerate nonenzymic deamidation of proteins caused interconversion of the multiple molecular forms. Iron was the only transition metal found to be associated with the purified enzyme at significant levels. The amount of enzyme-bound iron present in equilibrium-dialyzed samples was equivalent to 1 atom of iron per enzyme subunit. Purification of the enzyme activity correlated with the purification of the enzyme-bound iron. An EPR scan of the purified enzyme gave a signal at g equal 4.33, which is characteristic of ferric iron in a rhombic ligand field. 相似文献
237.
Scanning electron microscopy of surface and internal features of developing perithecia of Neurospora crassa. 下载免费PDF全文
Stages in the development of perithecia of Neurospora crassa, designated by the time elapsed after crossing, were investigated with the scanning electron microscope, from protoperithecia through perithecia. The usual examination of external features of whole specimens with this instrument was augmented by a freeze-fracture technique which allowed the viewing of development internally as well. Rapid increases in perithecial size soon after crossing were followed by the appearance, in section, of a centrum, at first undifferentiated but subsequently developing ascogenous hyphae. The perithecial beak appeared as a compact mass easily distinguishable in whole specimens from the surrounding hyphae by means of texture as well as shape. Two ascospores were photographed during emergence from an ostiole, but ostioles were found more frequently closed than open. 相似文献
238.
Summary The preparation of a mannan-iron complex is described. The mannan-iron complex can be used for electron microscopic demonstration of membrane bound Concanavalin A or Lens culinaris lectin because the high reactivity of these lectins toward the mannan. 相似文献
239.
M McMillan B Chernow B L Roth 《Biochemical and biophysical research communications》1986,134(2):970-974
We investigated the actions of two biologically active phorbol esters, phorbol dibutyrate (PDB) and phorbol myristate acetate (PMA), on receptor-stimulated phosphoinositide hydrolysis in rat aorta. We found both PDB and PMA potently inhibited norepinephrine (NE) stimulated PI hydrolysis in rat aortic rings. The biologically inactive phorbol, 4-alpha-phorbol was ineffective. In the presence of the calcium channel antagonist nitrendipine, PDB potently inhibited both the phasic and tonic components of NE-induced contraction. These results suggest a functional coupling between receptor-stimulated PI turnover and vascular contraction. They also suggest a mode of feed-back regulation in vascular tissue involving phorbol esters in receptor-stimulated PI hydrolysis. 相似文献
240.
Microtiter assay for acetylcholinesterase 总被引:4,自引:0,他引:4
A microtiter plate adaptation of the classical Ellman colorimetric procedure for measurement of acetylcholinesterase activity is described. This method permits use of an enzyme-linked immunosorbent assay plate reader for rapid analysis of multiple samples and is particularly suitable for analysis of acetylcholinesterase activity on sucrose gradients. The novel procedure is rapid and sensitive and does not require use of radioactive material. 相似文献