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Volume 271, Number 43, Issue of October 25, 1996 pp. 27063-27071
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Cell-free Synthesis of Anticoagulant Heparan Sulfate Reveals a Limiting Converting Activity That Modifies an Excess Precursor Pool

(Received for publication, March 29, 1996, and in revised form, July 3, 1996)

Nicholas W. Shworak ^§ , Linda M. S. Fritze ^§ , Jian Liu , Lynne D. Butler and Robert D. Rosenberg ^§

From the  Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 and the ^§ Department of Medicine, Harvard Medical School, Beth Israel Hospital, Boston, Massachusetts 02215

LTA cells synthesize a minor population of heparan sulfate proteoglycans (HSPG^act) bearing anticoagulant heparan sulfate (HS^act) with a specific monosaccharide sequence that accelerates the action of antithrombin (AT). LTA cells also synthesize a major population of heparan sulfate proteoglycans endowed with nonanticoagulant heparan sulfate (HS^inact) lacking the AT-binding site. To investigate the pathway-specific features of HSPG^act generation, we established a novel detergent-containing cell-free system with unlabeled and labeled microsomes from wild-type and variant LTA cells, respectively. The unlabeled microsomes provide ``HS^act conversion activity'' that requires 3-phosphoadenosine 5-phosphosulfate to convert [³⁵S]HSPG^inact into [³⁵S] HSPG^act, presumably by sulfation. The reaction kinetics demonstrate that the rate of HS^act synthesis is constant over the first 4 h of incubation. During this time, the rate of HS^act production is linearly dependent on the amount of unlabeled LTA microsomal protein over a range of 10 to 50 µg as well as on the level of [³⁵S]HS substrate over a range of 0.4 to 4.0 µg, microsomal protein. Compared with labeled microsomes, equivalent or slightly greater levels of HS^act were generated from ³⁵S-labeled HSPG, microsomal HS, or cell surface HS, which demonstrates that HS^inact is the minimal substrate and that large amounts of HS^act precursor exit the Golgi apparatus. Indeed, extensive modification of wild-type LTA cell surface [³⁵S]HS elevated HS^act content from 9 to 35%. The hypothesis that microsomal HS^act conversion activity predicts the cellular rate of HS^act generation was tested with wild-type or variant LTA cells in which production of HS^act has been significantly altered by mutagenesis or overexpression of core protein or growth conditions. The data demonstrate that microsomal HS^act conversion activity accurately reflects the cellular rate of HS^act synthesis over a very wide range of conditions. The possibility that the reduced HS^act generation is due to an inhibitor was excluded by mixing experiments. The possibility that reduced HS^act generation is caused by decreased levels of HS^act precursor was excluded as equivalent levels of HS^act were formed from wild-type and variant [³⁵S]HS. Based upon the above data, the LTA cell microsomal HS^act conversion activity contains one or more limiting components that kinetically regulate the rate of cellular HS^act generation and the levels of HS^act precursor in HS greatly exceed HS^act production.

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