Purification and characterization of a mesohalic catalase from the halophilic bacterium Halobacterium halobium.
When subjected to the stress of growth in a relatively low-salt environment (1.25 M NaCl), the halophilic bacterium Halobacterium halobium induces a catalase. The protein has been purified to electrophoretic homogeneity and has an M(r) of 240,000 and a subunit size of approximately 62,000. The enzyme is active over a broad pH range of 6.5 to 10.0, with a peak in activity at pH 7.0. It has an isoelectric point of 4.0. This catalse, which is not readily reduced by dithionite, shows a Soret peak at 406 nm. Cyanide and azide inhibit the enzyme at micromolar concentrations, whereas maleimide is without effect. The addition of 20 mM 3-amino-1,2,4-triazole results in a 33% inhibition in enzymatic activity. The tetrameric protein binds NADP in a 1:1 ratio but does not peroxidize NADPH, NADH, or ascorbate. Although the enzymatic activity is maximal when assayed in a 50 mM potassium phosphate buffer with no NaCl, prolonged incubation in a buffer lacking NaCl results in inactive enzyme. Moreover, purification must be performed in the presence of 2 M NaCl. Equally as effective in retaining enzymatic function are NaCl, LiCl, KCl, CsCl, and NH4Cl, whereas divalent salts such as MgCl2 and CaCl2 result in the immediate loss of activity. The catalase is stained by pararosaniline, which is indicative of a glycosidic linkage. The Km for H2O2 is 60 mM, with inhibition observed at concentrations in excess of 90 mM. Thus, the mesohalic catalase purified from H. halobium seems to be similar to other catalases, except for the salt requirements, but differs markedly from the constitutive halobacterial hydroperoxidase.
Brown-Peterson
N J
NJ
Department of Biochemistry and Molecular Biology, Mississippi State University, Mississippi 39762.
Salin
M L
ML
D000595:Amino Acid Sequence; D002374:Catalase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D002384:Catalysis; D004790:Enzyme Induction; D004795:Enzyme Stability; D019617:Halobacterium salinarum / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012492:Salts; D012965:Sodium Chloride; D013053:Spectrophotometry; D013696:Temperature
D012492:Salts; D012965:Sodium Chloride; D002374:Catalase