Isolation, characterization, and esterase and CO2 hydration activities of ubiquitin from bovine erythrocytes.
Ubiquitin was isolated from bovine erythrocytes by a relatively simple procedure involving extraction with chloroform and ethanol, chromatography on DEAE-cellulose, and gel filtration. Amino acid and partial sequence analyses showed it to be identical to previously isolated material. Ubiquitin released p-nitrophenolate from p-nitrophenyl acetate, but did not cleave other esterase substrates that were tested. It had a turnover number of 116 mmol for p-nitrophenyl acetate at pH 7.7 and 30 degrees C, and this activity was relatively stable to heat treatment. Electrophoretic studies indicated that the ubiquitin was sequentially acetylated by p-nitrophenyl acetate, as judged by the appearance of more anodically migrating components. The reactions of ubiquitin with p-nitrophenyl acetate at pH 7.0 were biphasic and consisted of (a) an initial phase, during which the release of p-nitrophenol resulted from monoacetylation of the ubiquitin and from ubiquitin-catalyzed hydrolysis of the ester; and (b) a second phase, during which the release of p-nitrophenol resulted only from the breakdown and reformation of the acetyl-enzyme complex. Ubiquitin also showed CO2 hydration activity and could be localized following gel electrophoresis by the CO2-bromthymol blue staining method. The strong inhibitor of carbonic anhydrase, acetazolamide, also inhibited the CO2 hydration activity and p-nitrophenyl acetate activity of ubiquitin. An antibody against this protein did not precipitate bovine carbonic anhydrase II. The esterase activity of ubiquitin was much higher than those previously reported for the carbonic anhydrases.
D000107:Acetylation; D000818:Animals; D002245:Carbon Dioxide / Q000378:metabolism*; D002417:Cattle; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004912:Erythrocytes / Q000032:analysis*; D004950:Esterases / Q000097:blood*; D006609:High Mobility Group Proteins / Q000097:blood*; D006358:Hot Temperature; D006868:Hydrolysis; D005779:Immunodiffusion; D009596:Nitrophenols / Q000378:metabolism; D010446:Peptide Fragments; D014357:Trypsin; D014452:Ubiquitins / Q000097:blood*
D006609:High Mobility Group Proteins; D009596:Nitrophenols; D010446:Peptide Fragments; D014452:Ubiquitins; D002245:Carbon Dioxide; C008642:4-nitrophenyl acetate; D004950:Esterases; D014357:Trypsin