Purification and characterization of protein disulfide isomerase from soybean.
Protein disulfide isomerase (PDI), which catalyses the folding of newly synthesized or denatured proteins through correct disulfide formation, was purified from soybean (Glycine max). The enzyme was purified 12,000-fold over crude extracts to apparent homogeneity in six purification steps: 60-70% ammonium sulfate fractionation, and chromatography on DEAE Toyopearl 650M, Q-Sepharose Fast Flow, Hiload Superdex 200 pg, Phenyl Sepharose HP, and TSK G-3000 SW. The native enzyme had a molecular weight of 120 kDa on gel filtration. Subunit molecular weight was estimated as 63 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, thus indicating the enzyme to be comprised of two identical subunits. The enzyme pH optimum was 8.0 with reactivation of scrambled RNase, and the pI 7.65. The N-terminal amino acid sequence of soybean PDI was homologous to that of mature alfalfa as deduced from the cDNA sequence. Two identical active site sequences, APWCGHCK, were obtained from different proteolytic peptide fragments of soybean PDI. Soybean PDI facilitated reactivation not only of scrambled RNase, but denatured and reduced lysozyme and the Bowman Birk soybean trypsin inhibitor as well. This is the first report to appear on the the purification, characterization and amino acid sequence analysis of the active site of a plant PDI.
Kainuma
K
K
Protein Science Laboratory, National Food Research Institute, Ibaraki.
Ookura
T
T
Kawamura
Y
Y
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D004789:Enzyme Activation; D007535:Isomerases / Q000302:isolation & purification*; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D009113:Muramidase / Q000378:metabolism; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D019704:Protein Disulfide-Isomerases; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D013025:Glycine max / Q000201:enzymology*; D014358:Trypsin Inhibitor, Bowman-Birk Soybean / Q000737:chemistry
D008670:Metals; D014358:Trypsin Inhibitor, Bowman-Birk Soybean; D012259:Ribonuclease, Pancreatic; D009113:Muramidase; D007535:Isomerases; D019704:Protein Disulfide-Isomerases