Abstract
An endoglycosidase which cleaves heparin and heparan sulfate was isolated from outdated human platelets by freeze-thaw solubilization, heparin-Sepharose chromatography, DEAE-cellulose chromatography, hydroxylapatite chromatography, octyl-agarose chromatography, concanavalin A-Sepharose chromatography, and Sephacryl S-200 gel filtration. The overall extent of purification of the platelet heparitinase is about 240,000-fold and the overall yield of the enzyme is about 5.6% as compared to the initial freeze-thaw solubilization preparation. The final product is physically homogeneous as judged by disc gel electrophoresis at acidic pH as well as gel filtration chromatography and exhibits an apparent molecular weight of approximately 134,000. Furthermore, our results indicate that the above enzyme is present within platelet lysosomes. The biologic potency of the endoglycosidase was examined as a function of pH. The data show that the platelet heparitinase is maximally active from pH 5.5 to pH 7.5. However, the enzyme possesses minimal ability to cleave heparin at pH less than 4.0 or greater than 9.0. The substrate specificity of the platelet endoglycosidase was determined by identifying susceptible linkages within the heparin molecule that can be cleaved by the above component. Our studies indicate that this enzyme is only able to hydrolyze glucuronsylglucosamine linkages. Furthermore, investigation of the structure of the disaccharide which lies on the nonreducing end of the cleaved glucuronic acid residue suggests that N-sulfation of the glucosamine moiety or ester sulfation of the adjacent iduronic acid groups are not essential for bond scission.
Highlights
The apparentmolecular weightof platelet heparitinasewas established by gel fitration chromatography
The 125I-labeled enzyme and proteinosf known molecular weight were filtered at 2 ml/hon a Sephacryl S-200 column (0.6 X 128 cm) equilibrated with 0.5 M NaCl in 0.01
H with the platelet enzyme faint al concentrations of 1000 and our studies demonstrate that the platelet heparitinase
Summary
Purification of Platelet Heparitinase individually filtered a t 10 ml/hthrough a hydroxylapatite column (2.5 X 28 cm) that had been equilibrated at4 "C with 0.15 M NaCl in 0.01 M sodium phosphate, pH6.0. Step I: Solubilization of Platelet Heparitinase-Approxi- ylapatite productswere pooled, concentrated by ultrafiltration mately 150units of outdated human platelets stored-20at "C from about 600 to about 50 ml utilizing PM-30 membranes were rapidly thawed at 37 "C, acidified to pH 5.0 with glacial and stored at -80 "C prior to use. Under these conditions, acetic acid, and centrifuged at 8000 X g for 30 min a t 4 "C. preparations of platelet endoglycosidase were stable for at Directmeasurement of the resultant supernatant solution least 12 weeks. These data represent average results obtained employing 900 units of outdated human Platelets
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