Purification and characterization of a CMP-sialic:LcOse4Cer sialyltransferase from human colorectal carcinoma cell membranes
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.
- Research Organization:
- ER 277 CNRS, IRSC, Villejuif (France)
- OSTI ID:
- 6470336
- Journal Information:
- Biochemistry; (United States), Vol. 27:23
- Country of Publication:
- United States
- Language:
- English
Similar Records
Polysialic acid in human neuroblastoma cells
Biosynthesis of the polysialic acid capsule of Escherichia coli K1: factors influencing cessation of capsule expression at 15/sup 0/C
Related Subjects
LARGE INTESTINE
CARCINOMAS
RECTUM
TRANSFERASES
CHEMICAL COMPOSITION
CARBON 14 COMPOUNDS
CELL MEMBRANES
CHROMATOGRAPHY
ENZYME ACTIVITY
FRACTIONATION
MAN
MEMBRANE PROTEINS
SIALIC ACID
TRACER TECHNIQUES
TUMOR CELLS
ANIMAL CELLS
ANIMALS
BODY
CARBOHYDRATES
CELL CONSTITUENTS
DIGESTIVE SYSTEM
DISEASES
ENZYMES
GASTROINTESTINAL TRACT
INTESTINES
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
MAMMALS
MEMBRANES
MONOSACCHARIDES
NEOPLASMS
ORGANIC COMPOUNDS
ORGANS
PRIMATES
PROTEINS
SACCHARIDES
SEPARATION PROCESSES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques
550901 - Pathology- Tracer Techniques