Quantitative continuous assay for hyaluronan synthase.

Joanne C Krupa; David Shaya; Lianli Chi; Robert J Linhardt; Miroslaw Cygler; Stephen G Withers; John S Mort

doi:10.1016/j.ab.2006.11.011

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PubMed · 17173853

Quantitative continuous assay for hyaluronan synthase.

Joanne C Krupa·David Shaya·Lianli Chi·Robert J Linhardt·Miroslaw Cygler·Stephen G Withers·John S Mort

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Abstract

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.

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Fluorescent Dyes Glucuronosyltransferase Hyaluronan Synthases Hyaluronic Acid Kinetics Mutagenesis, Site-Directed N-Acetylglucosaminyltransferases NAD Pasteurella multocida Spectrophotometry, Ultraviolet Uridine Diphosphate Uridine Diphosphate Sugars Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

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Joanne C Krupa, David Shaya, Lianli Chi, Robert J Linhardt, Miroslaw Cygler, Stephen G Withers, John S Mort. 2006-11-27. Quantitative continuous assay for hyaluronan synthase.. https://doi.org/10.1016/j.ab.2006.11.011

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