期刊
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
卷 290, 期 4, 页码 1206-1213出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1006/bbrc.2001.6268
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资金
- NCI NIH HHS [CA 090940] Funding Source: Medline
- NIGMS NIH HHS [GM 057073, 5T32GM08334] Funding Source: Medline
Heparan sulfate (HS) glycosaminoglycans are a structurally diverse class of complex biomolecules that modulate many important events at the cell surface and within the extracellular matrix and whose structural heterogeneity derives largely from the sequence-specific N- and O-sulfations catalyzed by an extensive repertoire of sulfating enzymes. We have expressed the human heparan sulfate 3-OST-1 isoform in Escherichia coli and subsequently purified a soluble, active enzyme. To assess its functionality, we determined the kinetic parameters for the recombinant 3-O-sulfotransferase-1 using a radiochemical assay that directly measures the 3-O-sulfation of unlabeled bovine kidney heparan sulfate in vitro using [S-35]PAPS as the sulfate donor. The apparent Km values measured were in the low micromolar range (K-m(HS) = 4.3 muM; K-m(PAPS) = 38.6 muM); V-max values of 18 and 21 pmol sulfate/min/pmol of enzyme for HS and PAPS, respectively. These values were compared with kinetic parameters likewise measured for recombinant 3-OST-1 purified from baculovirus-infected sf9 cells. The two enzymes appear to modify heparan sulfate in vitro to roughly the same extent and with comparable specificities. The expression of 3-OST-1 in E. coli represents an important step in subsequent structure-function studies. (C) 2002 Elsevier Science (USA).
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