PURIFICATION OF HEPARAN-SULFATE D-GLUCOSAMINYL 3-O-SULFOTRANSFERASE

The cellular generation of proteoglycans with anticoagulant heparan su lfate (HSPG(act)) is determined by microsomal ''HSact conversion activ ity'' that functions in concert with the sulfate doctor 3'-phosphoaden osine 5'phosphosulfate (PAPS) to convert nonanticoagulant heparan sulf ate (HSinact) to anticoagulant heparan sulfate (HSact) (Shworak, N. W. , Fritze, L. M., S., Liu, J., Butler, L. D., and Rosenberg, R. D. (199 6) J. Biol. Chem. 271, 27063-27071), Suspension cultures of L-33(+) ce lls in serum-free medium produce HSPG(act) and secrete HSact conversio n activity, The secreted protein exhibiting HSact conversion activity was isolated by subjecting large volumes of conditioned suspension cul ture medium to heparin-AF Toyopearl affinity chromatography, Mono Q-FP LC, TSK SW3000-HPLC, and 3',5'-ADP-agarose affinity chromatography. Th e final product was purified similar to 700,000-fold relative to cellu lar material with a 5% overall recovery of HSact conversion activity, The isolated protein migrated. on SDS-polyacrylamide gel electrophores is as a broad band of M(r) = 46,000 and co-migrated on nondenaturing a cidic pH gel electrophoresis with HSact conversion activity, The purif ied component was identified as heparan sulfate D-glucosaminyl 3-O-sul fotransferase because it transferred sulfate from [S-35]PAPS to the 3- O-position of D-glucosamine and D-glucosamine 6-O-sulfate of HSact pre cursor and HSinact precursor to produce nearly equivalent amounts of l abeled HSact and HSinact. The exhaustive modification of wild-type LTA cell [S-35]HS with either microsomal HSact conversion activity or pur ified enzyme increased HSact content from 9 to similar to 36%, which i ndicates that microsomal HSact conversion activity predominantly refle cts the level of a 3-O-sulfotransferase that converts HSact precursor into HSact, The kinetic parameters of purified 3-O-sulfotransferase we re determined for modification of HSact precursor and HSinact precurso r, The apparent K(M)--- and V*(max)---, with respect to PAPS concentr ation for sulfation of HSact precursor and HSinact precursor were 2.4 mu M and 23 fmol of sulfate/min/ng of enzyme and 2.1 mu M and 38 fmol of sulfate/min/ng of enzyme, respectively, There was substrate inhibit ion of the sulfation reaction at elevated HS concentration, The appare nt K(M)--- and V*(max)--- with respect to GAG concentration or sulfat ion of HSact precursor and HSinact precursor were 16 nM and 120 fmol o f sulfate/min/ng of enzyme and 17 nM and 240 fmol of sulfate/min/ng of enzyme, respectively The observation that purified 3-O-sulfotransfera se catalyzes sulfation of HSact precursor and HSinact precursor in con junction with a documented discordant regulation of 3-O-sulfate conten t in HSinact and HSact suggests that two discrete forms of the enzyme may exist.