Glycosyltransferase and sialic acid levels of normal and transformed cells

Grimes, William J.

doi:10.1021/bi00729a031

Cited by 73 publications

(14 citation statements)

References 22 publications

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“…Of course the fact that sialyltransferase, as well as other glycosyltransferases, is linked to intracellular structures, namely the Golgi apparatus, is definite. However, increasing evidence is being reported on the presence of plasma membrane-bound sialyltransferase activity in a number of cells and tissues (Grimes, 1973;Datta, 1974;Shur and Roth, 1975;Porter and Bernacki, 1975;Colombino et al, 1978;Painter and White, 1976;Verbert et al, 1977). Thus the possibility that the Golgi apparatus is not the only site of sialyltransferase activity, and that the plasma membranes (namely the synaptosomal membranes) may contain this enzyme, cannot be excluded and is surely worth checking.…”

Section: Discussionmentioning

confidence: 99%

Occurrence of Sialyltransferase Activity in the Synaptosomal Membranes Prepared from Calf Brain Cortex

Preti

Fiorilli

Lombardo

et al. 1980

Journal of Neurochemistry

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The possible occurrence of sialyltransferase activity in the plasma membranes surrounding nerve endings (synaptosomal membranes) was studied, using calf brain cortex. The synaptosomal membranes were prepared by an improved procedure which provided: (a) a „nerve ending fraction” consisting of at least 85% well‐preserved nerve endings and containing only small quantities of membranes of intracellular origin; (b) a „synaptosomal membrane fraction” carrying high amounts of authentic plasma membrane markers (Na+‐K+ ATPase, 5′‐nucleotidase, sialidase, gangliosides) with values of specific activity four to fivefold higher than those in the „nerve ending fraction” and very small amounts of cerebroside sulphotransferase, marker of the Golgi apparatus, and of other markers of intracellular membranes (rotenone‐insensitive NADH and NADPH: cytochrome c reductases), the specific activities of which were, respectively, 0.5‐ and 0.7‐fold that in the „nerve ending fraction”. Thus the preparation of synaptosomal membranes used had the characteristics of plasma membranes and carried a negligible contamination of membranes of intracellular origin. The distribution of sialyltransferase activity in the main brain subcellular fractions (microsomes; P2 fraction; nerve ending fraction; mitochondria) resembled most closely that of thiamine pyrophosphatase, the enzyme known to be linked to the Golgi apparatus and the plasma membranes and of acetylcholine esterase, the enzyme known to be linked to either intracellular or plasma membranes. The enrichment of sialyltransferase activity in the „synaptosomal membrane fraction”, referred to the „nerve ending fraction”, was practically the same as that exhibited by authentic plasma membrane markers. All this is consistent with the hypothesis that in calf brain cortex sialyltransferase has two different subcellular locations: one at the level of intracellular structures, most likely the Golgi apparatus (as described by other authors), the other in the synaptosomal plasma membranes. The basic properties (pH optimum, V/S, V/t and V/protein relationships) and detergent requirements of the synaptosomal membrane‐bound sialyltransferase were established. The highest enzyme activities were recorded on exogenous acceptors, lactosylceramide and ds‐fetuin. The Km values for CMP‐NeuNAc were different using lactosylceramide and ds‐fetuin as acceptor substrates (0.57 and 0.135 mm, respectively); the thermal stability of the enzyme acting on glycolipid acceptor was higher than that on the glycoprotein acceptor; the effect of detergents was different when using glycoprotein from glycolipid acceptors; no competition was observed between lactosylceramide and ds‐fetuin. Thus the synaptosomal membranes carry at least two different sialyltransferase activities: one acting on lactosylceramide (and glycolipid acceptors), the other working on ds‐fetuin (and glycoprotein acceptors). Ganglioside GM3 was recognized as the product of synaptosomal membrane‐bound sialyltransferase activity working on lactosylceramide ...

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Section: Discussionmentioning

confidence: 99%

Occurrence of Sialyltransferase Activity in the Synaptosomal Membranes Prepared from Calf Brain Cortex

Preti

Fiorilli

Lombardo

et al. 1980

Journal of Neurochemistry

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“…Changes in lectin agglutinability, adenylate cyclase activity, glycosyl transferases, and quantities of various cell surface glycolipids and glycoproteins have been demonstrated (1)(2)(3)(4)(5)(6)(7)(8).…”

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confidence: 99%

Isolation of a Major Cell Surface Glycoprotein from Fibroblasts

Yamada

Weston

1974

Proc. Natl. Acad. Sci. U.S.A.

251

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A cell surface component has been isolated in partially purified form from cultured chick embryo and chick heart fibroblasts. This glycoproteiri is similar to a protein recently reported to be present at the surface of normal cells, and missing after neoplastic transformation. It is a major cell surface glycoprotein that is synthesized by cultured fibroblasts, but is not collagen. It is shown to be markedly trypsin-sensitive, and its recovery from the cell surface is dependent on cell density. It is excluded from Sephadex G-200, but is not rapidly sedimented by ultracentrifugation, and has an apparent molecular weight of 220,000. Recently, there have been several reports that a cell surface glycoprotein of 200,000-250,000 daltons is present on several "normal" cell types, but is markedly diminished or absent from their transformed variants (9-13). We report the extraction and preliminary characterization of such a protein from chick embryo and chick heart fibroblasts. The conditions of isolation should be gentle enough to permit future evaluation of its biological function. MATERIALS AND METHODSCulture Methods. Secondary cultures of 10-day chick embryo fibroblasts were prepared by a modification of the methods of Rein and Rubin (14). After 3-4 days of primary culture, cells were harvested with 0.125% (w/v) trypsin (Difco) plus 10 ug/ml of DNase (Schwarz/Mann), passed to 100-mm plastic dishes at 5 X 101 per 12 ml of medium, and cultured to a necessary final cell density of about 2.5 X 105/cm2. Primary cultures of minced 7-day chick embryo heart ventricles were established in 25-cm2 plastic culture flasks (Falcon). Secondary cultures were prepared at densities of 6 to 8 X 100 per 100-mm dish from the outgrowths after 2 days in culture.Abbreviations: HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; NaDodSO4, sodium dodecyl sulfate; CSP, cell surface protein; MW, molecular weight; PAS, periodic acidSchiff. * Isolation of Surface Protein. The following extraction procedure maximized purity and yield of the cell surface protein, and minimized retraction of the cell monolayers. Cultures were rinsed three times with Hanks' balanced salts solution buffered with 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), pH 7.4 (Calbiochem). Cells on 100-mm dishes were then extracted 2. hr at 370 in 5 ml of serum-free Dulbecco's. medium with glutamine on a gyratory shaker (model G-2, New Brunswick Scientific) at 30 rpm.This first extract in serum-free medium was discarded, and the monolayers were washed once with HEPES-buffered Hanks' solution. The cells were then further extracted for 2 hr in serum-free medium to which urea (Schwarz/Mann, ultra-pure) was -added to a final concentration of 0.2 M immediately before use. This second extract was removed, chilled, and centrifuged at 25,000 X g for 15 min.Cell loss after this extraction protocol was minimal: <0.8% of the total was found in the pellets, and no loss was detected when parallel dishes before and after extraction were compared (1.4 X 107...

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“…In addition to differences in the agglutinability of cells by plant lectins (24)(25)(26), transformed cells exhibit incomplete synthesis of carbohydrate chains in membrane glycoproteins (27)(28)(29) and glycolipids (30)(31)(32)(33), and increased synthesis of a sialofucopeptide (34,35).…”

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confidence: 99%

Cholera Toxin and Cell Growth: Role of Membrane Gangliosides

Hollenberg

Fishman

Bennett

et al. 1974

Proc. Natl. Acad. Sci. U.S.A.

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The bindikg of cholera toxin to three transformed mouse cell lines derived from the same parent strain, and the effects of the toxin on DNA synthesis and adenylate cyclase activity, vary in parallel with the ganglioside composition of the cells. TAL/N cells of early passage, which contain large quantities of gangliosides Gm3, GM2, G11, and GDWa, as well as the glycosyltransferases necessary for the synthesis of these gangliosides, bind the most cholera toxin and are the most sensitive to its action.TAL/N cells of later passage, which lack chemically detectable GM, and GD1a and which have no UDP-Gal:GM2 Cholera toxin (choleragen), the principal exoenterotoxin of Vibrio cholerae, is an oligomeric protein (molecular weight, approx. 100,000) (2, 7) which acts at the plasma membrane to exert its biological effect (3-7). The diverse actions of the toxin (2, 8-13) can be attributed to its ability to stimulate specifically the membrane-localized enzyme, adenylate cyclase (EC 4.6.1.1), thereby raising intracellular levels of cAMP. It is presumably by this mechanism that choleragen acts as a potent inhibitor of mitogen-induced DNA synthesis in human fibroblasts (14). Also, stimulation of adenylate cyclase probably explains the toxin's ability to inhibit mitogenic transformation of lymphocytes (15), to stimulate steroidogenesis in adrenal cells (16,17), and to induce differentiation in melanoma cells (18). In all cells so far studied, choleragen is nontoxic by several criteria (14-17) and the action of choleragen is observed to persist for at least two weeks in culture (18).It has been observed that gangliosides can interact specifically with choleragen so as to block the binding of the toxin to cell membranes (3)(4)(5)(6)(7)(19)(20)(21)(22)(23). In particular, GM1 is a highly potent inhibitor of choleragen binding; GD1a and GM2 are at least 50-fold, and GM3 at least 250-fold less effective than

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Glycosyltransferase and sialic acid levels of normal and transformed cells

Cited by 73 publications

References 22 publications

Occurrence of Sialyltransferase Activity in the Synaptosomal Membranes Prepared from Calf Brain Cortex

Occurrence of Sialyltransferase Activity in the Synaptosomal Membranes Prepared from Calf Brain Cortex

Isolation of a Major Cell Surface Glycoprotein from Fibroblasts

Cholera Toxin and Cell Growth: Role of Membrane Gangliosides

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