title: Preliminary characterization of a cloned neutral isoelectric form of the human peptidyl prolyl isomerase cyclophilin.
abstract: We report the cloning of a neutral isoelectric form of the human peptidyl prolyl isomerase, cyclophilin, its expression in Escherichia coli, and its purification and comparison to bovine thymus cyclophilin. The cloned protein exhibited a pI of approximately 7.8 and formed a simple 1:1 complex with cyclosporin A. This cloned form had a pI similar to that observed for the neutral isoform (pI approximately 7.4) of human splenocyte cyclophilin. The bovine thymus proteins exhibited anomalous behavior on CM-cellulose chromatography but were resolved into alkaline (pI approximately 9.3) isoforms and a new neutral (pI approximately 7.8) isoform by isoelectric focusing gel electrophoresis and ultimately into at least four discrete isoforms by capillary electrophoresis. For cyclosporin A binding we observe a Kd of approximately 160 nM for an electrophoretically heterogeneous preparation of the natural bovine protein and approximately 360 nM for the more homogeneous preparation of the cloned human neutral isoform. Stopped-flow measurements of the activation energies for peptidyl-prolyl isomerase activity indicate the recombinant human protein has an activation enthalpy of 3.67 kcal/mol and an activation entropy of -47.3 cal/K-mol for cis----trans isomerization.
authors.affiliation: Department of Molecular Biology, Abbott Laboratories, Abbott Park, Illinois 60064.
authors.forename: T F
authors.initials: TF
authors.lastname: Holzman
authors.forename: D A
authors.initials: DA
authors.lastname: Egan
authors.forename: R
authors.initials: R
authors.lastname: Edalji
authors.forename: R L
authors.initials: RL
authors.lastname: Simmer
authors.forename: R
authors.initials: R
authors.lastname: Helfrich
authors.forename: A
authors.initials: A
authors.lastname: Taylor
authors.forename: N S
authors.initials: NS
authors.lastname: Burres
mesh_terms: D000591:Amino Acid Isomerases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003524:Cyclosporins / Q000378:metabolism*; D004789:Enzyme Activation; D015870:Gene Expression; D006801:Humans; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D019696:Peptidylprolyl Isomerase; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D003524:Cyclosporins; D011994:Recombinant Proteins; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase