title: Purification and enzymic characterization of the cytoplasmic pyrophosphatase from the thermoacidophilic archaebacterium Thermoplasma acidophilum.
abstract: Cytoplasmic pyrophosphatase has been isolated from the thermoacidophilic archaebacterium Thermoplasma acidophilum. The enzyme was purified to electrophoretic homogeneity by combining ion-exchange and affinity-chromatographic separations. This soluble pyrophosphatase probably consists of six identical subunits, since SDS/PAGE gave an estimate of about 22 kDa for a single subunit and size-exclusion chromatography under non-denaturing conditions indicates a molecular mass of 110 +/- 5 kDa. The two most prominent catalytic features of this enzyme are the absolute requirement for divalent cations for catalytic action, Mg2+ conferring the highest activity, and the pronounced specificity for PPi. The catalytic behavior apparently follows simple Michaelis-Menten kinetics with a Km of about 7 microM for PPi and a specific activity of about 1200 U/mg at 56 degrees C. Surprisingly, maximum activity could be observed at 85 degrees C which is more than 20 degrees C above the temperature for optimal growth. Several cytoplasmic extracts of eubacteria and archaebacteria have been probed with a polyclonal antiserum raised against the purified archaebacterial protein. The only noticeable cross-reactivity could be detected with an extract from the methanogen Methanosarcina barkeri although this probably does not reflect the inferred phylogenetic relationship between methanogens and Thermoplasma acidophilum.
authors.affiliation: Institut für Biochemie, Medizinische Universität zu Lübeck, Federal Republic of Germany.
authors.forename: O M
authors.initials: OM
authors.lastname: Richter
authors.forename: G
authors.initials: G
authors.lastname: Schäfer
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D002413:Cations, Divalent; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003593:Cytoplasm / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007118:Immunoassay; D043564:Inorganic Pyrophosphatase; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D017020:Methanosarcina / Q000201:enzymology; D008969:Molecular Sequence Data; D010658:Phenylglyoxal / Q000494:pharmacology; D011755:Pyrophosphatases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013379:Substrate Specificity; D013696:Temperature; D013822:Thermoplasma / Q000201:enzymology*
chemical_list: D002413:Cations, Divalent; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D008274:Magnesium; D010658:Phenylglyoxal