Diffusion of macromolecules through compartments containing polysaccharides.
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Biomedical subjects
Publications and source records attributed to H Pertoft.
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Heparan sulfate from human aorta has been subjected to a physico-chemical analysis in buffers of physiological ionic strength containing either sodium chloride or calcium chloride. A molecular weight of 50,000 was obtained both in sodium and calcium solutions by sedimentation equilibrium and from sedimentation and diffusion coefficients. The values obtained for So20,w in sodium and calcium chloride solutions were 2.28 X 10(-13) and 2.70 X 10(-13) sec, respectively, and the corresponding values for Dl20,w were 2.7 X 10(-7) and 3.1 X 10(-7) cm2/sec, respectively. Diffusion coefficients calculated from data obtained by gel chromatography were in excellent agreement with those determined by conventional techniques. The results indicated that the molecule contracts in the presence of calcium, presumably due to an increased binding of counter-ions with a concomitant decrease in charge density. Circular dichroism spectra above 200 nm, where the substituted amino group contributes to the absorption, gave no indication of a conformational change in the polysaccharide upon transformation from the sodium to the calcium salt. When the polysaccharide was dissolved in a salt solution, physiological both in ionic strength and in sodium to calcium ratio, it sedimented as the sodium salt. The sedimentation coefficient, the diffusion coefficient and the apparent molecular weight all displayed a concentration dependence. This dependence was much less in calcium chloride than in sodium chloride in determinations of molecular weight. Therefore there are advantages in performing molecular weight determinations of glycosaminoglycans in calcium solutions.
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The glycosaminoglycan synthesis in Furth solid mastocytoma tissue has been studied. Approx. 10% of the polysaccharide isolated after incubation in vitro with [(14)C]-glucosamine was digestible with chondroitinase ABC and the product of digestion was identified as 2-acetamido-2-deoxy-3-O-(beta-d-gluco-4-enepyranosyluronic acid)-4-O-sulpho-d-galactose. Similarly, labelling of polysaccharide in vivo with (35)SO(4) (2-) followed by isolation of mast-cell fractions by density-gradient centrifugation on colloidal silica revealed the presence of a polysaccharide which migrated as did chondroitin sulphate on electrophoresis in barium acetate. Chondroitinase ABC produced the same digestion product as before. Finally, the presence of the UDP-N-acetylgalactosamine-chondroitin 6-sulphate hexasaccharide N-acetylgalactosaminyltransferase previously implicated in chondroitin sulphate biosynthesis was demonstrated in microsomal particles from fractions of purified mast cells.
Using a colloidal silica density gradient, HeLa cells in mitosis were found to have a density of 1.040-1.046 g/cc, lighter than the remaining interphase cells. The mitotic cells could be harvested and cultured after centrifugation, showing growth synchrony by measurement of a peak in mitotic index 21 hr after establishing the culture. By using Colcemid or vinblastine sulfate, HeLa cells were arrested in metaphase and centrifuged on the colloidal silica density gradient. The blocked metaphase cells were lighter in density than the interphase cells but somewhat more dense than untreated cells selected by the density gradient centrifugation. Near-equilibrium conditions were established during the centrifugation of cells so that cell density measurements could be made, and the gradient medium employed was not measurably toxic to those cells tested.
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