Purification and properties of urease from bovine rumen.
Urease (urea amidohydrolase, EC 3.5.1.5) was extracted from the mixed rumen bacterial fraction of bovine rumen contents and purified 60-fold by (NH4)2SO4 precipitation, calcium phosphate-gel adsorption and chromatography on hydroxyapatite. The purified enzyme had maximum activity at pH 8.0. The molecular weight was estimated to be 120000-130000. The Km for urea was 8.3 X 10(-4) M+/-1.7 X 10(-4) M. The maximum velocity was 3.2+/-0.25 mmol of urea hydrolysed/h per mg of protein. The enzyme was stabilized by 50 mM-dithiothreitol. The enzyme was not inhibited by high concentrations of EDTA or phosphate but was inhibited by Mn2+, Mg2+, Ba2+, Hg2+, Cu2+, Zn2+, Cd2+, Ni2+ and Co2+. p-Chloromercuribenzenesulfphonate and N-ethylmaleimide inhibited the enzyme almost completely at 0.1 mM. Hydroxyurea and acetohydroxamate reversibly inhibited the enzyme. Polyacrylamide-gel electrophoresis showed that the mixed rumen bacteria produce ureases which have identical molecular weights and electrophoretic mobility. No multiple forms of urease were detected.
D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D002417:Cattle; D004492:Edetic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006877:Hydroxamic Acids / Q000494:pharmacology; D006918:Hydroxyurea / Q000494:pharmacology; D008970:Molecular Weight; D010710:Phosphates / Q000494:pharmacology; D012417:Rumen / Q000201:enzymology*; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014510:Urease / Q000037:antagonists & inhibitors / Q000302:isolation & purification*
D002413:Cations, Divalent; D006877:Hydroxamic Acids; D010710:Phosphates; D013439:Sulfhydryl Reagents; D004492:Edetic Acid; D014510:Urease; D006918:Hydroxyurea