Purification and characterization of Candida albicans 20S proteasome: identification of four proteasomal subunits.
The 20S proteasome from yeast cells of Candida albicans was purified by successive chromatographic steps to apparent homogeneity, as judged by nondenaturing and denaturing polyacrylamide gel electrophoresis. Its molecular mass was estimated to be 640 kDa by gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate gave at least 10 bands in the range 20-32 kDa. Two-dimensional electrophoresis revealed the presence of at least 14 polypeptides. By electron microscopy after negative staining, the proteasome preparation appeared as typical symmetrical barrel-shaped particles. The enzyme cleaved the peptidyl-arylamide bonds in the model synthetic substrates Cbz-G-G-L-p-nitroanilide, Cbz-G-G-R-beta-naphthylamide, and Cbz-L-L-E-beta-naphthylamide (chymotrypsin-like, trypsin-like, and peptidylglutamyl-peptide-hydrolyzing activities). The differential sensitivity of these activities to aldehyde peptides and sodium dodecyl sulfate supported the multicatalytic nature of this enzyme. Three proteasomal subunits were identified as alpha6/Pre5, alpha3/Y13, and alpha5/Pup2 by internal sequencing of tryptic fragments. Their sequences perfectly matched the corresponding deduced amino acid sequences of the C. albicans genes. A fourth subunit was identified as alpha7/Prs1 by immunorecognition with a monoclonal antibody specific for C8, the human proteasome subunit homologue. Treatment of the intact isolated 20S proteasome with acid phosphatase and Western blot analysis of the separated components indicated that the alpha7/Prs1 subunit is obtained as a multiply phosphorylated protein.
Fernández Murray
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Cátedra de Microbiología, Facultad de Agronomía, Universidad de Buenos Aires, CIBYF-CONICET, Avda. San Martín 4453, Buenos Aires, 1417, Argentina. murray@mail.agro.uba.ar
Biscoglio
M J
MJ
Passeron
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S
D000135:Acid Phosphatase / Q000378:metabolism; D000447:Aldehydes / Q000494:pharmacology; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002176:Candida albicans / Q000166:cytology / Q000201:enzymology*; D002384:Catalysis / Q000187:drug effects; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism / Q000648:ultrastructure; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism / Q000648:ultrastructure; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism
D000447:Aldehydes; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D011480:Protease Inhibitors; D012967:Sodium Dodecyl Sulfate; D000135:Acid Phosphatase; D014357:Trypsin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex