Characterization of human platelet UDPglucose-collagen glucosyltransferase using a new rapid assay.
A rapid and specific assay has been developed for UDPglucose-collagen glucosyltransferase (UDPglucose: 5-hydroxylysine-collagen glucosyltransferase, EC 2.4.1.66) using galactosylhydroxylysine (Gal-Hyl) as acceptor. Studies with intact human platelets and isolated plasma membranes indicated that about 5--10% of the total activity was surface bound and the rest was of cytoplasmic origin. The two forms of the enzyme had similar broad pH optima (6.5--8.0), Km values for UDPglucose (5 muM) and Gal-Hyl (approx. 4 mM) and for optimal manganese concentrations (25 mM). The soluble form of the enzyme was purified 80-fold. The reaction mechanism was determined as being rapid equilibrium random BiBi + dead end complex or ordered BiBi with UDPglucose being the first substrate to bind. Using Gal-Hyl bound in purified alpha 1 chain of chick skin collagen, a Km value three orders of magnitude less (2 muM) was found than for free Gal-Hyl and the manganese requirement decreased to 2 mM. These results suggest that the binding to the enzyme of Gal-Hyl in the collagen molecule is enhanced by the presence of the protein portion so that the enzyme may be capable of recognizing not only the carbohydrate side chains but also the primary structure of collagen.
D000818:Animals; D001792:Blood Platelets / Q000201:enzymology*; D002462:Cell Membrane / Q000201:enzymology; D002645:Chickens; D003094:Collagen / Q000378:metabolism; D003593:Cytoplasm / Q000201:enzymology; D005964:Glucosyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D006027:Glycosides / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006901:Hydroxylysine / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D012867:Skin; D014532:Uridine Diphosphate Glucose / Q000378:metabolism
D006027:Glycosides; D006901:Hydroxylysine; C007817:galactosylhydroxylysine; D008345:Manganese; D003094:Collagen; D005964:Glucosyltransferases; C023247:UDP glucose-collagen glucosyltransferase; D014532:Uridine Diphosphate Glucose