Mucopolysaccharides associated with nuclei of cultured mammalian cells.

Abstract

Mucopolysaccharides have been isolated, fractionated, and characterized from the nuclei of cultured B16 mouse melanoma cells grown in the presence of Digestion of the nuclei with DNase followed by Pronase gave a mixture of complex carbohydrates from which the mucopolysaccharides were isolated by precipitation with cetylpyridinium chloride. After fractionation by differential salt extraction and chromatography on controlled pore glass bead columns, the components were identified by chemical and enzymatic methods. The major polysaccharide components were a family of high-molecular-weight chondroitin sulfates with different degrees of sulfation; a minor component has been characterized as heparan sulfate.The production of mucopolysaccharides by cultured cells and the effect of virus transformation on this has been demonstrated for several cell lines (1-5). A major portion of the mucopolysaccharide synthesized by the cells can be detected in the culture media and as intercellular material, not a surprising result, since mucopolysaccharides are considered to be extracellular components. In addition, mucopolysaccharide is detectable in cell pellets, although the nature of the association of this material with the cell has not been fully established. The presence of heparan sulfate as a surface component of several cell lines has been reported (3) but it is not clear whether the heparan sulfate-protein complex is an integral part of the plasma membrane of cells or is present as a cell coat (fuzz) immobilized by ionic interaction with cell membrane proteins.In this paper we present evidence for the presence of mucopolysaccharides associated with cell nuclei. The presence of anionic saccharides in the nucleus of mammalian cells may have considerable significance. While cell surface complex carbohydrates can be involved in determining antigenicity, intercellular recognition and adhesion, contact inhibition, and other surface-related phenomena, anionic saccharides of the nuclei are more likely to be involved in controlling nuclear and cytoplasmic events. Alternatively these components may have a common function in all membranes as structural elements maintaining organellar integrity or regulating membrane properties, such as transport of nutrients. MATERIALS AND METHODSB16 mouse melanoma cell line 3 and an amelanotic clone were propagated as described previously (5). Cells utilized for the isolation of nuclei were harvested in the late-log phase of growth at a density of about 15 X 106 cells per 16 oz (0.5 liter) bottle. In experiments requiring labeling of complex carbohydrates, the cells were cultured for 48 hr prior to harvest in sulfate-free medium containing per milliliter 10 MACi of [3H]glucosamine.HCl (New England Nuclear, 7.3 Ci/-mmol) and 50 MuCi of Na2aSO4 (New England Nuclear, 738 mCi/mmol).Cells were harvested by pouring off the media, washing the cell layer three times with serum-free medium, and treating with 0.01 M EDTA in calcium, magnesium-free phosphatebuffered saline (10 ml) at 370 for 5 min. Se...

Histochemical demonstration of acidic glycosaminoglycans in the cell nuclei of the iris and other tissues

“…The main components were shown to be chondroitin sul-fates of high molecular weight. Small quantities of heparan suifate were also found by Bhavanandan and Davidson [1975]. Frommeet at.…”

“…DNA, RNA, histones, nonbasic proteins and lipids arc recognized as being generally traceable components of the cell nucleus [Rose andFrenster, 1965: Ris, 1969;Buddecke, 1971;Marquardt, 1971], PAS-positive material was found by Stick (1954], Onishiet al [1973], Bhavanandan and Davidson [1975] and Stein el al. [1975].…”

“…Khachaturov et al [1975] also recognized a specific stainability of aGAG in the nuclei of human cells which they could block by prior mcthylation. Using biochemical meth ods, Bhavanandan and Davidson [1975] and Stein et al [1975] analyzed aGAG in the nuclei of mammalian cells. The GAG were proteolytically isolated and were then characterized by means of chromatography, electrophoresis, enzymatic digestion, incorporation of isotopes and analysis of the monosaccharides.…”

Proteoglycans in normal physiology and carcinogenesis

“…Первые сообщения, демонстрирующие локализа-цию ПГ в ядре клеток, относятся к середине 1970-х го-дов [104,105], когда в клеточных ядрах были иденти-фицированы различные классы ГАГ -ГС, ДС и/или ХС [106,107]. Концентрация, состав и степень сульфати-рования ГАГ изучались в очищенных ядрах, выделен-ных из мозга крыс, где были обнаружены ГС, ХС и ги-алуроновая кислота [105], а в ядре и цитозоле сосудистых гладкомышечных клеток аорты крысы -внутриклеточный ХСПГ версикан [108].…”

“…Концентрация, состав и степень сульфати-рования ГАГ изучались в очищенных ядрах, выделен-ных из мозга крыс, где были обнаружены ГС, ХС и ги-алуроновая кислота [105], а в ядре и цитозоле сосудистых гладкомышечных клеток аорты крысы -внутриклеточный ХСПГ версикан [108]. Основными полисахаридными компонентами ядер клеток мела-номы мыши B16 являлись ХС с разной степенью суль-фатирования, в то время как ГС был представлен в не-значительном коли честве [104]. В ядрах нормальной печени мышей были обнаружены ГСПГ и ДСПГ [109], а ядра клеток мыши ной гепатомы содержали ГСПГ, ДСПГ и ХСПГ [110].…”