PURIFICATION AND CHARACTERIZATION OF CYTOSOLIC AND MICROSOMAL CYCLOPHILINS FROM MAIZE (ZEA-MAYS)

Methods for the purification and separation of peptidyl prolyl cis-tra ns isomerase (PPI) from cytosolic and microsomal fractions of etiolate d maize are described. On SDS/PAGE, the purified preparations appear a s single polypeptides with molecular masses of 17.5 kDa and 17.7 kDa r espectively. Instead of using immobilized cyclosporin A derivatives as affinity adsorbents, our methods employ conventional techniques enabl ing purification of the proteins on a much larger scale than previousl y described. An antiserum raised against the cytosolic PPI recognizes polypeptides of similar molecular mass from a wide range of plant spec ies on an immunoblot. There is virtually no recognition of the microso mal PPI. The cytosolic and microsomal PPIs are inhibited by cyclospori n A (K-i = 6 nM in both cases), indicating that they are cyclophilins. The cytosolic enzyme is inactivated by 5 mM N-ethylmaleimide and 2 mM phenylglyoxal. N-terminal sequencing of the microsomal PPI indicates a high level of sequence similarity with the N-terminal sequence of ma ture animal s-cyclophilin (cyclophilin B).