The oligosaccharide-cleaving enzymes, peptide:N-glycosidase F (PNGase F) and endo Beta-N-acetylglucosaminidase F (endo F), have been isolated and partially purified from the cultural filtrates of Flavobacterium meningosepticum. They cleave many of the N-linked glycopeptides and glycoproteins by hydrolysis at the glycosyl-asparagine junction or at the di-N-acetylchitobiosyl core, respectively. These enzymes will be purified to homogeneity and their substrate specificity will be well-defined by our present series of N-linked glycopeptides plus proposed additions of carbohydrate-modified glycopeptides. PNGase and endo F offer potential as the next generation of carbohydrate chain-cleaving enzymes releasing oligosaccharides beyond the range and limits of endo D or H or PNGase A. PNGase F shows particular promise for use in structure/function studies of glycosylated proteins in their native conformation. Several N-linked glycopeptides have been modified into dns- and/or dabsyl-derivatives to increase their sensitivity of detection and resistance to proteolysis. Enzymatic digests of bovine fetuin will be used as a source of O-linked glycopeptides which will be similarly modified. These N- and O-linked derivatives will be used as enzyme substrates suitable for detection of mammalian endo- and PNGase-type enzymes in crude extracts. Detection of these enzyme activities in mammalian tissues will explain the many oligosaccharide fragments released in urine or accumulated in tissues in human disease involving a deficiency of a carbohydrate hydrolase. Futhermore, purification of any of these enzyme activities from mammalian tissues by use of our sensitive assays will allow a better understanding of their function and role in glycoprotein metabolism.
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