HYDROLYSIS OF GLUCURONIDE-BASED SUBSTRATES MEDIATED BY TUNGSTEN, CU2+, FE2+, AND ZN2+

A variety of metal microprojectiles are currently used for carrying fo reign DNA into living cells via particle-acceleration techniques. Whil e developing a microprojectile-mediated protocol for transforming cell s of sugarbeet (Beta vulgaris L.), formation of a blue precipitate was observed with the indigoqenic substrate 5-bromo-4-chloro-3-indolyl-be ta-D-glucuronic acid (X-gluc) in the absence of gusA DNA encoding beta -D-glucuronidase (GUS). Tungsten microcarriers, but not gold or silico n carbide, proved capable of catalyzing the cleavage of the glucuronid e residue from three histochemical substrates evaluated: X-gluc, salmo n X-gluc and magenta X-gluc. Indigo-stained sugarbeet cells were obser ved following bombardment with tungsten in the absence of DNA. Additio n of oxidative catalysts to tungsten microcarriers during substrate in cubation had no apparent effect on the metal-mediated catalysis. Treat ment of microcarriers with Proteinase K and heat ruled out the presenc e of enzymes. Microbiological evaluation indicated the absence of cont aminating microbes. Similarly, metal-catalyzed hydrolysis of the fluor ogenic substrate 4-methylumbelliferyl-beta-D-glucuronic acid (4-MUG) w as observed in the presence of tungsten spheres but not with gold or s ilicon carbide particles. With this substrate. hydrolysis also occurre d with millimolar concentrations of Cu2+, Fe2+ and Zn2+ ions. Conseque ntly, careful monitoring of DNA-minus controls and avoidance of millim olar concentrations of Cu2+, Fe2+ and Zn2+ ions are recommended in mic roprojectile bombardment experiments where transient assays for gusA e xpression are performed.