A Microsomal Ecdysone-Binding Cytochrome P450 from the Insect Locusta migratoria Purified by Sequential Use of Type-II and Type-I Ligands
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Jochen Winter
Abstract
A dualaffinity method was established to purify, for the first time, a microsomal ecdysonebinding cytochrome P450 protein from locust Malpighian tubules. This method involved, after prepurification on ωoctylaminoagarose and hydroxylapatite, binding of cytochrome P450 to an immobilized triazolebased general P450 inhibitor (typeII ligand) followed by elution with the substrate ecdysone (typeI ligand) of the bound cytochrome. The isolated material showed a typical cytochrome P450 spectrum, a specific heme content of 13 nmol/mg protein, and a prominent protein of about 60 kDa on SDSPAGE. Based on a tryptic undecapeptide sequence the isolated protein may be identical to CYP6H1, a putative ecdysone 20-monooxygenase recently cloned from the same tissue. Ecdysone 20-monooxygenase activity could be partially reconstituted from microsomal detergent extracts, when supplemented with purified bovine cytochrome P450 reductase and detergentextracted microsomes; reconstitution was not successful with any chromatographic fraction, however. Therefore, purification of the locust cytochrome P450 was monitored by ecdysoneinduced typeI difference spectra, whenever applicable, in addition to carbon monoxide spectra. Affinity columns with matrixbound diethylstilbestrol and testosterone 3-thiosemicarbazone, but not with the 17β hemisuccinate, yielded elution profiles with ecdysone that were comparable to those of the triazole matrix. The concept of dualaffinity chromatography described here may be generally applicable to the isolation of cytochromes P450.
Copyright © 2001 by Walter de Gruyter GmbH & Co. KG
Articles in the same Issue
- An Overview of Endosymbiotic Models for the Origins of Eukaryotes, Their ATP-Producing Organelles (Mitochondria and Hydrogenosomes), and Their Heterotrophic Lifestyle
- A Microsomal Ecdysone-Binding Cytochrome P450 from the Insect Locusta migratoria Purified by Sequential Use of Type-II and Type-I Ligands
- The Large Cytoplasmic Loop of the Glucose Transporter GLUT1 Is an Essential Structural Element for Function
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- Elimination of Hydrocortisone from the Medium Enables Tissue Plasminogen Activator Gene Expression by Normal and Immortalized Nonmalignant Human Epithelial Cells
- Molecular Cloning and Biochemical Characterisation of Proteases from Staphylococcus epidermidis
- Splice Variants of Human Cathepsin L mRNA Show Different Expression Rates
- Osmolytes as Modulators of Conformational Changes in Serpins
- Protective Activity of Aromatic Amines and Imines against Oxidative Nerve Cell Death
- Nitric Oxide Generation in Aqueous Solutions of Cigarette Smoke and Approaches to Its Origin
- A Recombinant scFv/Streptavidin-Binding Peptide Fusion Protein for the Quantitative Determination of the Scorpion Venom Neurotoxin AahI
Articles in the same Issue
- An Overview of Endosymbiotic Models for the Origins of Eukaryotes, Their ATP-Producing Organelles (Mitochondria and Hydrogenosomes), and Their Heterotrophic Lifestyle
- A Microsomal Ecdysone-Binding Cytochrome P450 from the Insect Locusta migratoria Purified by Sequential Use of Type-II and Type-I Ligands
- The Large Cytoplasmic Loop of the Glucose Transporter GLUT1 Is an Essential Structural Element for Function
- Visual Representation by Atomic Force Microscopy (AFM) of Tomato Spotted Wilt Virus Ribonucleoproteins
- Elimination of Hydrocortisone from the Medium Enables Tissue Plasminogen Activator Gene Expression by Normal and Immortalized Nonmalignant Human Epithelial Cells
- Molecular Cloning and Biochemical Characterisation of Proteases from Staphylococcus epidermidis
- Splice Variants of Human Cathepsin L mRNA Show Different Expression Rates
- Osmolytes as Modulators of Conformational Changes in Serpins
- Protective Activity of Aromatic Amines and Imines against Oxidative Nerve Cell Death
- Nitric Oxide Generation in Aqueous Solutions of Cigarette Smoke and Approaches to Its Origin
- A Recombinant scFv/Streptavidin-Binding Peptide Fusion Protein for the Quantitative Determination of the Scorpion Venom Neurotoxin AahI
