Partial sequence of human platelet heparitinase and evidence of its ability to polymerize

被引:12
作者
Gonzalez-Stawinski, GV
Parker, W
Holzknecht, ZE
Huber, NS
Platt, JL
机构
[1] Duke Univ, Med Ctr, Dept Surg, Durham, NC 27710 USA
[2] Duke Univ, Ctr Med, Dept Pediat, Rochester, MN 55905 USA
[3] Duke Univ, Ctr Med, Dept Immunol, Rochester, MN 55905 USA
来源
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY | 1999年 / 1429卷 / 02期
关键词
heparan sulfate; heparan sulfate proteoglycan; heparitinase; glycosaminoglycan; platelet;
D O I
10.1016/S0167-4838(98)00254-4
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Heparitinase cleaves heparan sulfate, a glycosaminoglycan associated with all nucleated mammalian cells and extracellular matrices. Despite the important physiologic role heparitinase is postulated to play in such processes as tumor metastasis and inflammation, the identity of the enzyme remains a matter of controversy and there is a question of whether heparitinase is CTAP III. We report a 900000-fold purification of heparitinase from human platelets. A multi-step procedure utilizing chromatography on heparin, DEAE, hydroxyapatite and size exclusion matrices was employed and yielded a single protein as judged by Coomassie staining of protein separated by SDS-PAGE. The purified protein had an apparent molecular mass of 35 kDa by size exclusion chromatography and 55 kDa by SDS-PAGE. During purification, heparitinase activity co-eluted from the hydroxyapatite and size exclusion columns with the 35-55 kDa protein, confirming that the purified protein was indeed heparitinase. The 35-55 kDa protein reacted strongly with concanavalin A, a lectin known to bind to heparitinase, further confirming that the protein was heparitinase. Platelet heparitinase formed dimers and tetramers upon storage in a purified form, possibly accounting for the various molecular weights previously reported for the enzyme. A partial amino acid sequence of the protein revealed that heparitinase has not been previously sequenced. (C) 1999 Elsevier Science B.V. All rights reserved.
引用
收藏
页码:431 / 438
页数:8
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