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Peptidyl-prolyl cis-trans isomerases (PPIases), enzymes that catalyze the cis-trans isomerization of peptide bonds to which proline contributes the nitrogen, were purified from Escherichia coli. In this organism, at least two PPIases are present. Both the cationic (periplasmic) and anionic (cytoplasmic) PPIases are idubited by cyclosporin A with a Ki of 25 -50 pM, a concentration 1000-fold higher than that required for eukaryotic PPIases. Although isoelectric focusing indicates that the two enzymes differ in isoelectric point by at least 4.0 pH units, the specific activities of the enzymes toward the tetrapeptide substrate succinyl-Ala-Aka-Pro-Phe-methyl-coumarylamide are equivalent. The activity of both enzymes for a series of substituted succinyl-Ala-Xaa-Pro-Phe-para-nitroanilide tetrapeptides suggests that the structure and function of the active site of the prokaryotic proteins is similar to that of eukaryotic cyclophilins. Both enzymes are capable of catalyzing the refolding of thermally denatured type 111 collagen. Antibodies against the periplasmic PPIase do not recognize the cytoplasmic enzyme, indicating significant differences in epitopes between the two forms. Circular dichroism spectroscopy indicates that the secondary structure of the cationic protein consists of 17% a-helix, 34% B-sheet, 17% turns, 33% random coil and is very similar to human cytosolic PPIase.

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We generated anti-peptide antibodies against four highly conserved sequences in the kinase domain and against two nonconserved sequences surrounding autophosphorylation sites in the carboxyl-terminal domain of the epidermal growth factor receptor (EGFR). These antibodies were used to examine topology and function in catalysis of specific sequences. Two of the highly conserved sites, HRD (residues 811-818) and DFG (residues 827-838), appeared to participate in catalysis since alpha HRD and alpha DFG but not the other anti-peptide antibodies inhibited EGFR kinase activity. Examination of the topology of the six sites revealed that epitopes in all except the HRD site appeared to be exposed to antibody binding in the EGFR. The conditions that caused increased exposure of the HRD site to interaction with antibody included autophosphorylation, addition of the ionic detergent sodium dodecyl sulfate (SDS), and elevation in temperature from 4 to 34 degrees C.