CELL-FREE SYNTHESIS OF ANTICOAGULANT HEPARAN-SULFATE REVEALS A LIMITING CONVERTING ACTIVITY THAT MODIFIES AN EXCESS PRECURSOR POOL

LTA cells synthesize a minor population of heparan sulfate proteoglyca ns (HSPG(act)) bearing anticoagulant heparan sulfate (HSact) with a sp ecific monosaccharide sequence that accelerates the action of antithro mbin (AT), LTA cells also synthesize a major population of heparan sul fate proteoglycans endowed with nonanticoagulant heparan sulfate (HSin act) lacking the AT-binding site. To investigate the pathway specific features of HSPG(act) generation, we established a novel detergent-con taining cell-free system with unlabeled and labeled microsomes from wi ld-type and variant LTA cells, respectively, The unlabeled microsomes provide ''HSact conversion activity'' that requires 3'-phosphoadenosin e 5'-phosphosulfate to convert [S-35]HSPG(inact) into [S-35] HSPG(act) , presumably by sulfation, The reaction kinetics demonstrate that the rate of HSact synthesis is constant over the first 4 h of incubation, During this time, the rate of HSact production is linearly dependent o n the amount of unlabeled LTA microsomal protein over a range of 10 to 50 mu g as well as on the level of [S-35]HS substrate over a range of 0.4 to 4.0 mu g, microsomal protein, Compared with labeled microsomes , equivalent or slightly greater levels of HSact were generated from S -35-labeled HSPG, microsomal HS, or cell surface HS, which demonstrate s that HSinact is the minimal substrate and that large amounts of HSac t precursor exit the Golgi apparatus, Indeed, extensive modification o f wild-type LTA cell surface [S-35]HS elevated HSact content from 9 to 35%, The hypothesis that microsomal HSact conversion activity predict s the cellular rate of HSact generation was tested with wild-type or v ariant LTA cells in which production of HSact has been significantly a ltered by mutagenesis or overexpression of core protein or growth cond itions, The data demonstrate that microsomal HSact conversion activity accurately reflects the cellular rate of HSact synthesis over a very wide range of conditions, The possibility that the reduced HSact gener ation is due to an inhibitor was excluded by mixing experiments, The p ossibility that reduced HSact generation is caused by decreased levels of HSact precursor was excluded as equivalent levels of HSact were fo rmed from wild-type and variant [S-35]HS, Based upon the above data, t he LTA cell microsomal HSact conversion activity contains one or more limiting components that kinetically regulate the rate of cellular HSa ct generation and the levels of HSact precursor in HS greatly exceed H Sact production.