Purification and characterization of proteasome from ostrich liver.
The proteasome (EC 3.4.99.46) is a high molecular mass (approximately 700 kDa) multisubunit enzyme complex which is the focus of worldwide research in order to identify the structure, mechanism of action and specificity of the complex. The purpose of the present study was to investigate the tryptic, chymotryptic and peptidylglutamyl-peptide hydrolysing (PGPH) activities of ostrich liver proteasome. The proteasome was purified from ostrich liver by employing ammonium sulphate fractionation, followed by three sequential chromatographic steps on Toyopearl Super Q-650 S, Sephadex G-150 and phenyl-Toyopearl columns. Temperature and pH optima were examined and the effect of inhibitors, detergents, fatty acids and cations on the peptidase activities was determined. Ostrich proteasome exhibited a relative M(r) of approximately 665,000 using non-denaturing gradient PAGE and dissociated into the characteristic "ladder" associated with the proteasome subunits during SDS-PAGE. The pH optima for the peptidase activities were found to be slightly alkaline (tryptic activity) and neutral (chymotryptic-like and PGPH activities). Ostrich liver proteasome was found to be activated in terms of the PGPH activity by fatty acids and SDS, whereas the chymotryptic and tryptic-like activities were differentially inhibited. Ostrich proteasome, in its inhibition by monovalent cations, was similar to the proteasomes extracted from other sources. The specificity of the proteasome appears to be very broad, although it lacks aminopeptidase activity. The yield compared favourably with similar extraction procedures which have been reported. On the basis of the physicochemical and kinetic properties which ostrich liver proteasome exhibited, it can be safely concluded that it corresponds well with the proteasomes isolated from many other sources.
Klinkradt
S
S
Department of Biochemistry and Microbiology, University of Port Elizabeth, South Africa.
Naudé
R J
RJ
Muramoto
K
K
Oelofsen
W
W
D000595:Amino Acid Sequence; D000818:Animals; D001717:Birds; D002845:Chromatography; D003546:Cysteine Endopeptidases / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D016415:Sequence Alignment
D009097:Multienzyme Complexes; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex