title: Purification and characterization of aspartate aminotransferase from the halophile archaebacterium Haloferax mediterranei.
abstract: Aspartate aminotransferase from the archaebacterium Haloferax mediterranei was purified and found to be homogeneous. An average Mr of 66,000 was estimated. The native halophilic transaminase exhibited no maximum absorption at 410 nm, which indicates that the apo form is obtained by our purification procedure, and the molar absorption coefficient at 275 nm in 3.5 M-KCl (pH 7.8) was found to be 78.34 mM-1.cm-1. Plots of titration data show that 1 mol of halophilic aspartate aminotransferase binds 2 mol of pyridoxal 5'-phosphate. The halophilic transaminase behaved as a dimer with two similar subunits and had a maximum activity in the pH range 7.6-7.9 and at 65 degrees C in 3.5 M-KCl. By differential scanning calorimetry, the denaturation temperature of the halophilic holo- and apo-transaminase was determined to be 78.5 and 68.0 degrees C respectively at 3.3 M-KCl (pH 7.8). At low salt concentration the halophilic transaminase was inactivated, following first-order kinetics. The Km values for 2-oxoglutarate and L-aspartate, in 3 M-KCl (pH 7.8), were 0.75 mM and 12.6 mM respectively.
authors.affiliation: Departamento de Bioquímica, Facultad de Farmacia, Universidad de Sevilla, Spain.
authors.forename: F J
authors.initials: FJ
authors.lastname: Muriana
authors.forename: M C
authors.initials: MC
authors.lastname: Alvarez-Ossorio
authors.forename: A M
authors.initials: AM
authors.lastname: Relimpio
mesh_terms: D000596:Amino Acids / Q000032:analysis; D001105:Archaea / Q000201:enzymology*; D001219:Aspartate Aminotransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D008970:Molecular Weight; D011489:Protein Denaturation; D011732:Pyridoxal Phosphate / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
chemical_list: D000596:Amino Acids; D046911:Macromolecular Substances; D011732:Pyridoxal Phosphate; D001219:Aspartate Aminotransferases