Biochemical properties of the proteasome from Thermoplasma acidophilum

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Abstract
We have purified proteasomes to apparent homogeneity from the archaebacterium Thermoplasma acidophilum. This proteinase has a molecular mass of about 650 kDa and an isoelectric point of 5.6. The proteasome hydrolyses peptide substrates containing an aromatic residue adjacent to the reporter group, as well as [14C]methylated casein optimally at pH 8.5 and 90°C. The enzyme activity is enhanced severalford by Mg2+ and Ca2+ at 25–500 mM. This increase in activity results primarily from a change in Km. The serine‐proteinase inhibitors diisopropylfluorophosphate and 3,4‐dichlorosiocoumarin irreversibly inhibit the enzyme, obviously by modification of both the α and β subunits in the proteasome. The inhibition of proteasomal activity by the peptidylchloromethanes, Cbz‐Leu‐Leu‐CH2Cl and Cbz‐Ala‐Ala‐Phe‐Ch2Cl (Cbz, benzyloxycarbonyl), is reversible and predominantly of a competitive type. The enzyme is not activated by any of the compounds that typically stimulate the activities of the eukaryotic proteasome.