Format

Send to

Choose Destination
J Biol Chem. 1996 Oct 25;271(43):27072-82.

Purification of heparan sulfate D-glucosaminyl 3-O-sulfotransferase.

Author information

1
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

Abstract

The cellular generation of proteoglycans with anticoagulant heparan sulfate (HSPGact) is determined by microsomal "HSact conversion activity" that functions in concert with the sulfate donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to convert nonanticoagulant heparan sulfate (HSinact) to anticoagulant heparan sulfate (HSact) (Shworak, N. W., Fritze, L. M. S., Liu, J., Butler, L. D., and Rosenberg, R. D. (1996) J. Biol. Chem. 271, 27063-27071). Suspension cultures of L-33(+) cells in serum-free medium produce HSPGact and secrete HSact conversion activity. The secreted protein exhibiting HSact conversion activity was isolated by subjecting large volumes of conditioned suspension culture medium to heparin-AF Toyopearl affinity chromatography, Mono Q-FPLC, TSK SW3000-HPLC, and 3',5'-ADP-agarose affinity chromatography. The final product was purified approximately 700,000-fold relative to cellular material with a 5% overall recovery of HSact conversion activity. The isolated protein migrated on SDS-polyacrylamide gel electrophoresis as a broad band of Mr = 46,000 and co-migrated on nondenaturing acidic pH gel electrophoresis with HSact conversion activity. The purified component was identified as heparan sulfate D-glucosaminyl 3-O-sulfotransferase because it transferred sulfate from [35S]PAPS to the 3-O-position of D-glucosamine and D-glucosamine 6-O-sulfate of HSact precursor and HSinact precursor to produce nearly equivalent amounts of labeled HSact and HSinact. The exhaustive modification of wild-type LTA cell [35S]HS with either microsomal HSact conversion activity or purified enzyme increased HSact content from 9 to approximately 36%, which indicates that microsomal HSact conversion activity predominantly reflects the level of a 3-O-sulfotransferase that converts HSact precursor into HSact. The kinetic parameters of purified 3-O-sulfotransferase were determined for modification of HSact precursor and HSinact precursor. The apparent KM* and Vmax* with respect to PAPS concentration for sulfation of HSact precursor and HSinact precursor were 2.4 microM and 23 fmol of sulfate/min/ng of enzyme and 2.1 microM and 38 fmol of sulfate/min/ng of enzyme, respectively. There was substrate inhibition of the sulfation reaction at elevated HS concentration. The apparent KM* and Vmax* with respect to GAG concentration for sulfation of HSact precursor and HSinact precursor were 16 nM and 120 fmol of sulfate/min/ng of enzyme and 17 nM and 240 fmol of sulfate/min/ng of enzyme, respectively. The observation that purified 3-O-sulfotransferase catalyzes sulfation of HSact precursor and HSinact precursor in conjunction with a documented discordant regulation of 3-O-sulfate content in HSinact and HSact suggests that two discrete forms of the enzyme may exist.

PMID:
8900198
DOI:
10.1074/jbc.271.43.27072
[Indexed for MEDLINE]
Free full text

Supplemental Content

Full text links

Icon for HighWire
Loading ...
Support Center