Purification and properties of multiple molecular forms of yeast peptidyl prolyl cis-trans isomerase.
By hydrophobic chromatography on a butyl-Toyopearl 650 M column, yeast peptidyl prolyl cis-trans isomerase (PPIase) was separated into at least three molecular components (PPI-I, PPI-II and PPI-III) in their native forms. On the basis of the result of SDS-PAGE, PPI-II and PPI-III were highly purified and their molecular masses were estimated to be 16.5 and 17.2 kDa, respectively. However, PPI-I was still a mixture of two components with molecular masses of 23.3 and 24.1 kDa. The UV absorption spectrum of PPI-II was slightly different from that of PPI-III. In contrast, the CD spectra of the two proteins were essentially identical in the far-UV region. Upon addition of an immunosuppressant, cyclosporine A (CsA), the absorption spectra of the two highly purified proteins were subtly changed, which was indicative of some alterations in the microenvironments of the aromatic amino-acid residues. The two proteins exhibited subtle but clear differences in the kinetic parameters (kc/Km) for the PPIase-catalyzed cis-trans isomerization and in the inhibition constants of CsA for the PPIase activity. These results lead to the conclusions that (1), a family of PPIases exists in one organism and that (2), one member of the family has multiple molecular forms with different substrate specificities and different affinities for the drugs (inhibitors).
Hasumi
H
H
Department of Biophysical Chemistry, Kitasato University School of Medicine, Kanagawa, Japan.
Nishikawa
T
T
D000591:Amino Acid Isomerases / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D016572:Cyclosporine / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008969:Molecular Sequence Data; D019696:Peptidylprolyl Isomerase; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013056:Spectrophotometry, Ultraviolet
D002352:Carrier Proteins; D016572:Cyclosporine; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase