Quantitative continuous assay for hyaluronan synthase.
A rapid, continuous, and convenient three-enzyme coupled UV absorption assay was developed to quantitate the glucuronic acid and N-acetylglucosamine transferase activities of hyaluronan synthase from Pasteurella multocida (PmHAS). Activity was measured by coupling the UDP produced from the PmHAS-catalyzed transfer of UDP-GlcNAc and UDP-GlcUA to a hyaluronic acid tetrasaccharide primer with the oxidation of NADH. Using a fluorescently labeled primer, the products were characterized by gel electrophoresis. Our results show that a truncated soluble form of recombinant PmHAS (residues 1-703) can catalyze the glycosyl transfers in a time- and concentration-dependent manner. The assay can be used to determine kinetic parameters, inhibition constants, and mechanistic aspects of this enzyme. In addition, it can be used to quantify PmHAS during purification of the enzyme from culture media.
Krupa
Joanne C
JC
Joint Diseases Laboratory, Shriners Hospital for Children, Montreal, Que., Canada H3G 1A6.
Shaya
David
D
Chi
Lianli
L
Linhardt
Robert J
RJ
Cygler
Miroslaw
M
Withers
Stephen G
SG
Mort
John S
JS
D005456:Fluorescent Dyes / Q000737:chemistry; D014453:Glucuronosyltransferase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D000076002:Hyaluronan Synthases; D006820:Hyaluronic Acid / Q000032:analysis*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D017351:N-Acetylglucosaminyltransferases / Q000032:analysis; D009243:NAD / Q000737:chemistry; D016979:Pasteurella multocida / Q000201:enzymology*; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D014530:Uridine Diphosphate / Q000737:chemistry; D014539:Uridine Diphosphate Sugars / Q000737:chemistry
D005456:Fluorescent Dyes; D014539:Uridine Diphosphate Sugars; D009243:NAD; C046669:UDP-N-acetylglucosaminuronic acid; D014530:Uridine Diphosphate; D006820:Hyaluronic Acid; D017351:N-Acetylglucosaminyltransferases; D014453:Glucuronosyltransferase; D000076002:Hyaluronan Synthases