Partial sequence of human platelet heparitinase and evidence of its ability to polymerize.
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 900,000-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.
Gonzalez-Stawinski
G V
GV
Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA.
Parker
W
W
Holzknecht
Z E
ZE
Huber
N S
NS
Platt
J L
JL
D000180:Acrylic Resins; D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000201:enzymology*; D002845:Chromatography / Q000379:methods; D003636:DEAE-Cellulose; D017886:Durapatite; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011133:Polysaccharide-Lyases / Q000737:chemistry* / Q000302:isolation & purification; D012685:Sepharose