Purification and enzymic characterization of the cytoplasmic pyrophosphatase from the thermoacidophilic archaebacterium Thermoplasma acidophilum.
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.
Richter
O M
OM
Institut für Biochemie, Medizinische Universität zu Lübeck, Federal Republic of Germany.
Schäfer
G
G
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*
D002413:Cations, Divalent; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D008274:Magnesium; D010658:Phenylglyoxal