. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 224 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS 3-fucose. A nonreactive compound was identified as a sulfated jjaUctose-linked tetrasaccharide (5). On the basis of these structures, we predict that sponge tissue contains specific sul- fotransferases (sulfo-T) as the biosynthetic enzymes involved in the sulfation process. To explore this possibility, we performed assays for sulfo-T using Triton-extracted sponge microsomes ( mg/ml) in the presence of chemically defined monosaccharide acceptor substrates at
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 224 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS 3-fucose. A nonreactive compound was identified as a sulfated jjaUctose-linked tetrasaccharide (5). On the basis of these structures, we predict that sponge tissue contains specific sul- fotransferases (sulfo-T) as the biosynthetic enzymes involved in the sulfation process. To explore this possibility, we performed assays for sulfo-T using Triton-extracted sponge microsomes ( mg/ml) in the presence of chemically defined monosaccharide acceptor substrates at concentrations of 20-320 nM. The co-substrate and active sulfate donor was radiolabeled 3'-phosphoadenosine 5'- phosphosulfate ((35S)PAPS) 7 uM (2145 dpm/pmol) in the presence of pyrophosphatase and sulfatase inhibitors and 10 mAf Mg2+ buffered at pH The total volume of the reaction mix- ture was 40 fi\ (6). After incubation for 1 h at 37°, the reaction was terminated by adding 10/jl of 2% sodium borate/20 mM EDTA. Separation of unreacted PAPS from sulfated product was accomplished by thin-layer chromatography using a solvent system containing acetonitrile. water and methanol (4:1). or high-voltage electrophoresis using 1% sodium borate buffer at pH The figures for product yield, expressed as picomoles. were the average of duplicate values from which had been sub- tracted picomoles present in endogenous assays in which acceptor had been omitted. The biosynthesized product was expressed as picomoles per hour per milligram of protein (pmol/h/mg). Acceptor substrates were monosaccharides or derivatized monosaccharides representing sugars known to occur in Micro- dona AP. They included 0-D-fucose, a-L-fucose, j3-r>glucuronide phenyl, /J-phenyl-A'-acetyl-D-glucosamine(GlcNAc), phenyl-/V acetyl-«-r>galactosamine(GalNAc), phenyl-«-D-mannoside and phenyl-^-D-galactoside(Gal). Of these. phenyl-GlcNAc and phenyl-Gal gave measurable product yields, but assays using
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Keywords: ., bookauthorlilliefrankrat, booksubjectbiology, booksubjectzoology
