Purification and characterization of fermodulin, an Fe2+-dependent inhibitor protein of 3-hydroxy-3-methylglutaryl-CoA reductase |
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| Authors: | A S Menon S U Devi T Ramasarma |
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| Affiliation: | 1. School of Agriculture and Wine Sciences, Charles Sturt University, PO Box 883, Orange, New South Wales 2800, Australia;2. Quantitative Consulting Unit, Charles Sturt University, Boorooma Street, Wagga Wagga, New South Wales 2678, Australia;3. Graham Centre (an Alliance Between NSW Department of Primary Industries and Charles Sturt University), Wagga Wagga 2650, New South Wales, Australia;4. State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Institute of Applied Ecology, Fujian Agriculture and Forestry University, Fuzhou 350002, China;5. Joint International Research Laboratory of Ecological Pest Control, Ministry of Education, Fuzhou 350002, China;6. Fujian-Taiwan Joint Innovation Centre for Ecological Control of Crop Pests, Fujian Agriculture and Forestry University, Fuzhou 350002, China;1. Department of Molecular Virology, Institute of Experimental Biology, Faculty of Biology, Adam Mickiewicz University in Poznań, Umultowska 89, PL-61-614 Poznań, Poland;2. Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 3, D-06466 Gatersleben, Germany;1. Department of Physics, Jamia Millia Islamia, Jamia Nagar, New Delhi, 110025, India;2. Inter-University Accelerator Centre, Aruna Asaf Ali Marg, New Delhi, 110067, India |
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| Abstract: | The activity of 3-hydroxy-3-methylglutaryl-CoA (HMGCoA) reductase of rat liver microsomes was inhibited by the addition of FeSO4 and the cytosolic protein, fermodulin. Modulation of the activity was obtained only in the combined presence of Fe2+ and fermodulin. Using ammonium sulfate fractionation, heat treatment, and chromatography on CM-Sephadex and then on an Fe2+-Blue Sepharose affinity matrix, fermodulin was purified to homogeneity. The molecular weight of the purified protein, as determined by filtration through a Sephacryl S-200 column, was 58,000. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the protein resolved into two subunits of Mr 43,000 and 28,000. Fermodulin showed ultraviolet absorption and fluorescence spectra typical of tryptophan-containing proteins, and addition of FeSO4 quenched the fluorescence. Using the Millipore filter assay the binding of 1.6 mol 55FeCl2/mol fermodulin was observed in the presence of Tris-HCl buffer. The inhibitory effect of fermodulin at nonsaturating concentrations was potentiated by bicarbonate, ATP.Mg, and ADP.Mg. |
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