Biochemical properties of the proteasome from Thermoplasma acidophilum.
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 degrees C. The enzyme activity is enhanced severalfold 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-dichloroisocoumarin irreversibly inhibit the enzyme, obviously by modification of both the alpha and beta 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.
Dahlmann
B
B
Diabetes-Forschungsinstitut, Düsseldorf, Federal Republic of Germany.
Kuehn
L
L
Grziwa
A
A
Zwickl
P
P
Baumeister
W
W
D000595:Amino Acid Sequence; D002413:Cations, Divalent / Q000737:chemistry; D002414:Cations, Monovalent / Q000737:chemistry; D003374:Coumarins / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D049934:Isocoumarins; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D015843:Serpins / Q000494:pharmacology; D013696:Temperature; D013822:Thermoplasma / Q000737:chemistry / Q000201:enzymology*
D002413:Cations, Divalent; D002414:Cations, Monovalent; D003374:Coumarins; D049934:Isocoumarins; D009097:Multienzyme Complexes; D010455:Peptides; D015843:Serpins; C046235:3,4-dichloroisocoumarin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex