Characterization of a structural glycoprotein from bovine ligamentum nuchae exhibiting dual amine oxidase activity.
A structural glycoprotein has been extracted from bovine ligamentum nuchae by using 5 M guanidine hydrochloride containing a disulfide bond reducing agent and purified by preparative gel electrophoresis. The isolated material appeared to be monodisperse, with a molecular weight of approximately 34000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by analytical ultracentrifugation. It contains 10% carbohydrate comprising mannose, N-acetylglucosamine, galactose, and sialic acid in a 6:5:3:3 molar ratio. The glycoprotein has been assayed for peptidyl-lysine oxidase activity by using [3H]lysine-aortic elastin, prepared from 15- to 17-day-old chick embryos, as a substrate. In the absence of free lysine, the specific activity of the preparation over a 2-h incubation was approximately 60 X 10(4) dpm/mg of purified protein. Addition of 10 mM lysine resulted in an approximately 50% decrease in the specific activity. Free lysine was shown to act as a substrate for the glycoprotein preparation as indicated by control experiments using [3H]lysine in place of the aortic substrate. These results demonstrate that the glycoprotein exhibits a dual amine oxidase activity. In the presence of 0.27 mM beta-aminopropionitrile fumarate, a concentration which completely inhibits peptidyl-lysine oxidase activity in other lysyl oxidases, the glycoprotein preparation was inhibited by approximately 14%. In the absence of 5 M guanidine hydrochloride and reducing agent, the glycoprotein undergoes aggregation which in the presences of copper ions results in the formation of cylindrical tactoids, the diameter of which (11 nm) corresponds closely to that of the fibrils which in the majority of connective tissue matrices constitute the microfibrillar component mainly associated with elastic fibers.
D000594:Amino Acid Oxidoreductases / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002417:Cattle; D002642:Chick Embryo; D006023:Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D008022:Ligaments / Q000201:enzymology*; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase / Q000302:isolation & purification / Q000378:metabolism*; D008249:Protein-Lysine 6-Oxidase / Q000302:isolation & purification / Q000378:metabolism*
D000596:Amino Acids; D002241:Carbohydrates; D006023:Glycoproteins; D046911:Macromolecular Substances; D006899:Mixed Function Oxygenases; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase; D000594:Amino Acid Oxidoreductases; D008249:Protein-Lysine 6-Oxidase