Purification and characterization of a CMP-sialic:LcOse4Cer sialyltransferase from human colorectal carcinoma cell membranes

Abstract

Purified lactotetraosylceramide (Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc1-Cer) was tested for its ability to accept [14C]sialic acid from CMP-[14C]sialic into monosialoganglioside fractions in the presence of membrane fractions purified from human colorectal carcinoma cells (SW1116). Membrane fractions were isolated by three different methods: sucrose density centrifugation, CMP-agarose gel column chromatography, and LcOse4 gel chromatography. We optimized the incubation conditions for detergent dependency (taurocholate), pH (6.3), and acceptor concentration. The sialyltransferase activity was dependent on membrane protein and linear for time up to at least 4 h. The LcOse4 affinity chromatography of the crude microsomal membrane pellet from these cells yielded a membrane fraction that was 136-fold enriched in LcOse4 acceptor specific activity compared to cell homogenates. The apparent Km for the sialyltransferase activity with LcOse4Cer acceptor in the presence of affinity-purified membranes was 20 microM and the Vmax was 7 pmol h-1 (100 micrograms of protein)-1. Acceptor capabilities of other core structures were 5-20-fold lower: LcOse4Cer much greater than GgOse4Cer greater than nLcOse4Cer much greater than GbOse4Cer. The enzymatic activity was purified further (900-fold) by a combination of LcOse4 and CMP affinity gels. SDS-PAGE electrophoresis of this material showed a major set of closely migrating bands of Mr 58,000-54,000 compared to authentic proteins, as well as a minor band at 27,000. We analyzed picomole quantities of the radioactive product by convenient controlled short-term hydrolyses with an endoglycoceramidase and sialidases (from four different sources) in comparison to sialylated tetrasaccharides of known structure.(ABSTRACT TRUNCATED AT 250 WORDS)

An optimised assay for quantitative, high-throughput analysis of polysialyltransferase activity

“…Several methods have been reported for the assessment of the activity of different mono-ST enzymes, each with significant shortcomings when applied more specifically to polyST activity measurement. These methods can be broadly divided into four groups: radioactive assays, [37][38][39][40] non-radioactive assays, 41,42 phosphatase-coupled assays 43 and chemical conjugation methods. 44 In summary, none of these approaches is satisfactory for polyST analysis, requiring huge quantities of enzyme and acceptor proteins, at considerable cost.…”

Apical release of base‐labile fatty acyl groups commensurate with stimulation of glycoprotein sialosyl Lewisa secretion in colorectal carcinoma cells

“…The main band incorporating radioactivity from (3H) palmitate of membrane antigens appeared to be non-saponifiable complex lipid which had an almost 3-fold greater radioactivity from RA-treated sources than the corresponding band in control cells. This band co-migrated with sialosyl Lewis" ganglioside in the solvent system CHC13/CH30H/0.02% CaC12, 60/40/9 (Fredman and Svennerholm, 1980;Liepkalns et al, 1985Liepkalns et al, , 1988Jolif and Liepkalns, 1988).…”

Monosialoganglioside biosynthesis by subcellular membranes of rhabdomyosarcoma cell lines differing in metastatic potential

“…Sialyltransferase and fucosyltransferase assays to LcOse,Cer acceptors (purified from human meconium) have been published (Liepkans and Larson, 1987;Liepkans et al, 1988;Jolif and Liepkans, 1988) and were modified for these experiments. Briefly, each incubation contained 1.5 m~ taurocholate, 60 p~ glycolipid, 0.6 mM ATP, 0.05 M MES, 0.01 M dithiothreitol, 70 p~ radioactive nucleotide CMP-(I4C) sialic acid 288 pci/pM (NEN, Boston, MA) or GDP (14C) fucose (283 p C i / p~) (NEN), and purified membranes from RMS cell variants harvested at specified cell densities (10 to 15 pg protein), all in a total volume of 45 pl, PH 6.1.…”

“…LcOse,Cer was purified from human meconium (Karlsson and Larson, 1979;Liepkans et al, 1988). GgOse,Cer was obtained from Supelco (Belle Fonte, PA).…”

“…We have reported the characterization of sialyltransferases and fucosyltransferases to a purified glycolipid acceptor, LcOse,Cer, a core structure of many "tumor-associated'' and peripheral-tissue antigens (Liepkans and Larson, 1987;Jolif and Liepkans, 1988;Liepkans et al, 1988). In view of the above reports on differences in the sialylation of specific cell surface molecules in cell variants of metastasis, we tested membrane fractions of metastatic and non-metastatic strains of cultured rat rhabdomyosarcoma (RMS) cells for the presence of sialyltransferase activities to LcOse,Cer and GgOse,Cer glycolipid acceptors.…”