Purification and characterization of cytosolic and microsomal cyclophilins from maize (Zea mays).
Methods for the purification and separation of peptidyl prolyl cis-trans isomerase (PPI) from cytosolic and microsomal fractions of etiolated maize are described. On SDS/PAGE, the purified preparations appears as single polypeptides with molecular masses of 17.5 kDa and 17.7 kDa respectively. Instead of using immobilized cyclosporin A derivatives as affinity adsorbents, our methods employ conventional techniques enabling purification of the proteins on a much larger scale than previously described. An antiserum raised against the cytosolic PPI recognizes polypeptides of similar molecular mass from a wide range of plant species on an immunoblot. There is virtually no recognition of the microsomal PPI. The cytosolic and microsomal PPIs are inhibited by cyclosporin A (Ki = 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 mature animal s-cyclophilin (cyclophilin B).
Sheldon
P S
PS
Cell Physiology Department, Horticulture Research International, Wellesbourne, Warwickshire, U.K.
Venis
M A
MA
D000591:Amino Acid Isomerases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification*; D016572:Cyclosporine / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D006801:Humans; D007120:Immunochemistry; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000737:chemistry; D019696:Peptidylprolyl Isomerase; D010658:Phenylglyoxal / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013379:Substrate Specificity; D003313:Zea mays / Q000201:enzymology*
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D009842:Oligopeptides; C043307:succinyl-alanyl-alanyl-prolyl-phenylalanine-4-nitroanilide; D016572:Cyclosporine; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase; D010658:Phenylglyoxal; D005033:Ethylmaleimide