Chemical studies on hamycin. I. Purification, counter-current distribution and chemical degradation.
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Rat liver plasma membranes, enriched in blood-sinusoidal or bile-canalicular regions by differential and sucrose-gradient centrifugation, were further purified by partitioning in an aqueous polymer two-phase system. This method separates membranes according to differences in surface properties rather than size and density. A several-fold increase in the ratio of leucine aminopeptidase (a bile-canalicular marker) and 5'-nucleotidase to asialo-orosomucoid binding (a blood-sinusoidal marker) was obtained in one fraction, whereas another fraction gave a 2-3-fold increase in ratio of blood-sinusoidal to bile-canalicular markers. Furthermore, the markers for both regions of the plasma membrane, as well as markers for Golgi membranes and lysosomes, showed a heterogeneous behaviour on counter-current distribution.
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Levorin and mycoheptin, polyenic antibiotics were separated into 3 and 7 heptaenic components respectively by the method of disc electrophoresis in 7.5 per cent polyacryl amide gel prepared with tris-HCl buffer, pH 8.9. It was shown that nystatin had different composition as dependent on the organism producing it. The results of electrophoresis confirmed the data of the counter-current distribution.
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The formation constants of the 1:1 and 1:2 complexes of Hg(II) with glutathione and their protonated species have been determined by using a competitive potentiometric titration with the competing ligand diethylenetriaminepentaacetic acid (DTPA). The formation constants of the 1:1 complex and its protonated species have not been reported previously. The formation constant of the 1:2 complex of Hg(II) and glutathione is substantially smaller than the accepted values that has been reported in the literature. These results have important implications in the models that have been employed to explain the mobilization and distribution of Hg(II) in biological systems.
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We describe the influence of sample dilution on the measurement of free or non-protein-bound hormones in serum or plasma. The concentration of free hormone in the sample will alter after dilution or addition of binding reagents. The extent of this equilibrium perturbation depends on the binding properties of the endogenous carrier proteins, which vary from sample to sample and from sample to reference standard. As examples, we demonstrate the different changes in concentrations of free thyroxin and free cortisol after dilution, both experimentally and by theoretical modeling of the hormone-protein distribution. We also show how this systematic error can be minimized in the design of free hormone assays.
Rat sperm isolated from the caput and caudal epididymis and the vas deferens were subjected to multiple partition in aqueous two-phase systems. The technique was used to reveal heterogeneity of a sperm population with respect to particular surface properties. Sperm from all three regions gave broad distributions indicative of heterogeneous cell populations. Greatest heterogeneity was observed for cauda sperm with caput and vas sperm producing similar distributions. Following multiple partition sperm from different regions of the distribution profiles were immunostained with three antibodies known to recognise maturation antigens. The results show that some antigens are acquired during epididymal transit whilst others are present throughout. The partition (surface heterogeneity) seen cannot therefore be explained solely by the distribution of the antigens recognized by 2D6, 6B2 and 3D5.
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In conventional chromatography, a solute is usually viewed to be longitudinally transported only in the mobile phase, remaining longitudinally motionless in the stationary phase. In counter-current chromatography, both phases undergo intense mixing in the variable force field of a coil planet centrifuge and longitudinal dispersion of matter in the stationary phase is not to be excluded. To take into account longitudinal mixing in both phases, a cell model of chromatographic process is proposed in which the number of perfectly mixed cells n is determined by the rates of mixing in stationary (Ds) and mobile (Dm) phases by the equation n = LF/(2ADc)/(1 + Sf(lambda - 1)) with A = K(D)D(S)/Dm (F, L, Ac and KD are the mobile phase flow-rate, column length, column cross-section and distribution ratio, respectively). This equation has been derived by comparing the discontinuous cell model with continuous diffusion assuming equilibrium conditions. Parameter determination and their relationships are discussed.
Ligands with an apparent affinity for various structural elements on the surface of synaptic membrane fragments have been bound to the polymers poly(ethylene glycol) and dextran. The ligand-polymer derivatives have been included in aqueous two-phase systems composed of water, poly(ethylene glycol) and dextran. The uneven distribution of the polymers resulted in the concentration of the polymer-bound ligand in one of the two phases. The effect of the ligand-polymer on the partition of membranes was studied by using synaptic membranes from calf brain, obtained by standard centrifugation methods. By using ligand-containing two-phase systems for nine-step counter-current distribution of membranes, it was shown that the distribution behaviour of various parts of the membrane preparation could be affected. The distribution was followed by determination of opiate binding, acetylcholinesterase, and total membrane (using protein and light-scattering measurements).