Purification and characterization of urease from schizosaccharomyces pombe.
The urease from the ascomycetous fission yeast Schizosaccharomyces pombe was purified about 4000-fold (34% yield) to homogeneity by acetone precipitation, ammonium sulfate precipitation, DEAE-Sepharose ion-exchange column chromatography, and if required, Mono-Q ion-exchange fast protein liquid chromatography. The enzyme was intracellular and only one species of urease was detected by nondenaturing polyacrylamide gel electrophoresis (PAGE). The native enzyme had a M(r) of 212 kDa (Sepharose CL6B-200 gel filtration) and a single subunit was detected with a M(r) of 102 kDa (PAGE with sodium dodecyl sulfate). The subunit stoichiometry was not specifically determined, but the molecular mass estimations indicate that the undissociated enzyme may be a dimer of identical subunits. The specific activity was 700-800 micromols urea.min-1.mg protein-1, the optimum pH for activity was 8.0, and the Km for urea was 1.03 mM. The sequence of the amino terminus was Met-Gln-Pro-Arg-Glu-Leu-His-Lys-Leu-Thr-Leu-His-Gln-Leu-Gly-Ser-Leu-Ala and the sequence of two tryptic peptides of the enzyme were Phe-Ile-Glu-Thr-Asn-Glu-Lys and Leu-Tyr-Ala-Pro-Glu-Asn-Ser-Pro-Gly-Phe-Val-Glu-Val-Leu-Glu-Gly-Glu-Ile- Glu- Leu-Leu-Pro-Asn-Leu-Pro. The N-terminal sequence and physical and kinetic properties indicated that S. pombe urease was more like the plant enzymes than the bacterial ureases.
Lubbers
M W
MW
Department of Microbiology and Genetics, Massey University, Palmerston North, New Zealand.
Rodriguez
S B
SB
Honey
N K
NK
Thornton
R J
RJ
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009532:Nickel / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000254:growth & development; D017386:Sequence Homology, Amino Acid; D014510:Urease / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D007527:Isoenzymes; D046911:Macromolecular Substances; D010446:Peptide Fragments; D008345:Manganese; D009532:Nickel; D014510:Urease