Purification and characterization of yeast protein disulfide isomerase.
Protein disulfide-isomerase (PDI), which reactivates inactive scrambled RNase, was purified from Saccharomyces cerevisiae. The enzyme was purified 1,850-fold to apparent homogeneity by five purification steps: 30-70% ammonium sulfate fractionation, DEAE Toyopearl-650S and Butyl Toyopearl-650S chromatographies, and differential Phenyl-5PW HPLC with or without cysteine. The native enzyme had an apparent Mr of 140,000 on gel filtration chromatography, and its NH2-terminal was blocked. The Mr of its subunits were estimated to be 70,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that the enzyme is probably composed of two identical subunits. The Mr of the subunits changed to 60,000 on endoglucosaminidase H treatment, indicating that the enzyme is transported into the endoplasmic reticulum. The enzyme has a pH optimum of 8.5, and pI of 4.02. Its enzymic properties were compared with those of purified bovine liver PDI. The Km values of yeast and bovine PDIs for scrambled RNase were 1 x 10(-5) and 2 x 10(-5) M, and their Vmax values were 6 and 7 units/mg protein, respectively. The two enzymes showed no significant differences in Km or Vmax values with respect to thiol compounds. Bacitracin inhibited both PDIs in the same fashion. These results indicate that this yeast PDI corresponds to mammalian PDI.
Mizunaga
T
T
Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo.
Katakura
Y
Y
Miura
T
T
Maruyama
Y
Y
D000596:Amino Acids / Q000032:analysis; D001414:Bacitracin / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006596:Hexosaminidases / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007535:Isomerases / Q000737:chemistry*; D007700:Kinetics; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008970:Molecular Weight; D019704:Protein Disulfide-Isomerases; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology*; D013438:Sulfhydryl Compounds / Q000494:pharmacology
D000596:Amino Acids; D013438:Sulfhydryl Compounds; D001414:Bacitracin; D006596:Hexosaminidases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D007535:Isomerases; D019704:Protein Disulfide-Isomerases