Preliminary characterization of a cloned neutral isoelectric form of the human peptidyl prolyl isomerase cyclophilin.
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.
Holzman
T F
TF
Department of Molecular Biology, Abbott Laboratories, Abbott Park, Illinois 60064.
Egan
D A
DA
Edalji
R
R
Simmer
R L
RL
Helfrich
R
R
Taylor
A
A
Burres
N S
NS
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
D002352:Carrier Proteins; D003524:Cyclosporins; D011994:Recombinant Proteins; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase