Preliminary characterization of a cloned neutral isoelectric form of the human peptidyl prolyl isomerase cyclophilin
| Autor: | T F, Holzman, D A, Egan, R, Edalji, R L, Simmer, R, Helfrich, A, Taylor, N S, Burres |
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| Rok vydání: | 1991 |
| Předmět: |
Base Sequence
Molecular Sequence Data Gene Expression Cyclosporins Peptidylprolyl Isomerase Recombinant Proteins Enzyme Activation Animals Humans Cattle Amino Acid Sequence Isoelectric Point Cloning Molecular Isoelectric Focusing Carrier Proteins Chromatography High Pressure Liquid Amino Acid Isomerases |
| Zdroj: | The Journal of biological chemistry. 266(4) |
| ISSN: | 0021-9258 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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