Author
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Tabatabai, Louisa |
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Submitted to: Protein Society Symposium
Publication Type: Proceedings Publication Acceptance Date: 4/15/1995 Publication Date: N/A Citation: N/A Interpretive Summary: Technical Abstract: Pasteurella haemolytica O-sialoglycoprotease (E.C.3.4.24.93A) is a neutral metallo-proteinase that is specific for cleaving peptide bonds of glycoproteins that contain sialylated O-linked but not N- linked oligosaccharides. Human erythrocyte glycophorin A is normally used as a substrate for this enzyme. In order to detect the cleavage products, the glycophorin must be tritiated in situ, or iodinated after purification. Radiolabeling procedures are not necessary when bovine fetuin, a glycoprotein of known amino acid sequence, is used as a substrate. The assay consists of incubating glycoprotease- containing fractions with fetuin at neural pH. An aliquot of the incubation mixture is denatured and separated on a gradient gel. The stained gel is examined for the disappearance of the fetuin band and appearance of fetuin cleavage products. N-terminal sequencing of blotted cleavage products established three cleavage sites and four fragments. Based on the amino acid sequence of fetuin these are the N-terminal site at (Lys-49-Val-50) and two C-terminal sites at (Pro- 272-Val-273) and at (Leu-286-His-287). |
