title: Biochemical characterisation of urease from urease-positive thermophilic campylobacter (UPTC).
abstract: This study aims to characterise biochemically urease from an atypical Campylobacter lari, namely urease-positive thermophilic Campylobacter (UPTC). Urease was purified from cells of a Japanese UPTC isolate (CF89-12) using phenyl-Sepharose chromatography. Two protein components (estimates molecular masses 24 kDa and 61 kDa) were obtained that appeared to be structural proteins of urease (subunits A and B), and these were fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (PAGE). The native molecular weight for the final purified UPTC urease was estimated to be approximately 186,000 Da which is close to the calculated molecular weight (182,738 Da) based on all six open reading frames of UPTC CF89-12 urease genes (ureA, B, E, F, G and H), as described previously. Moreover, an active band was observed on phenol red staining after a nondenaturing native PAGE of the crude extract from the UPTC cells. In addition, the purified urease of UPTC CF8912 showed enzyme activity over a broad pH range (pH 6-10), with maximal activity at pH 8.0. The urease was also stable against heat treatment, with almost no loss of enzyme activity seen following 60-min incubation at temperatures of 20-60 degrees C. Urease subunits A and B were identified immunologically by Western blot analysis with rabbit anti-urease alpha (A) and beta (B) raised against Helicobacter pylori.
authors.affiliation: Laboratory of Molecular Biology, School of Environmental Health Sciences, Azabu University, Sagamihara, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Tazumi
authors.forename: T
authors.initials: T
authors.lastname: Nakajima
authors.forename: A
authors.initials: A
authors.lastname: Sekizuka
authors.forename: K
authors.initials: K
authors.lastname: Arikawa
authors.forename: S
authors.initials: S
authors.lastname: Nakanishi
authors.forename: K
authors.initials: K
authors.lastname: Hayashi
authors.forename: E
authors.initials: E
authors.lastname: Tasaki
authors.forename: J E
authors.initials: JE
authors.lastname: Moore
authors.forename: B C
authors.initials: BC
authors.lastname: Millar
authors.forename: M
authors.initials: M
authors.lastname: Matsuda
mesh_terms: D015153:Blotting, Western; D044863:Campylobacter lari / Q000201:enzymology* / Q000378:metabolism; D002384:Catalysis / Q000187:drug effects; D002847:Chromatography, Agarose; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004983:Ethanolamines / Q000737:chemistry / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D006918:Hydroxyurea / Q000494:pharmacology; D012685:Sepharose / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D013696:Temperature; D013890:Thiourea / Q000494:pharmacology; D014510:Urease / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D004983:Ethanolamines; C020927:phenyl-sepharose; D012685:Sepharose; D014510:Urease; D013890:Thiourea; D005033:Ethylmaleimide; C007369:2-diethylaminoethanol; D006918:Hydroxyurea