Abstract
A microsomal cytochrome b5 cDNA from the house fly, Musca domestica, was cloned and sequenced. The deduced amino acid sequence of the full-length house fly cytochrome b5 (134 residues) is 48% identical to that of rat microsomal cytochrome b5. The house fly cytochrome b5 protein was overexpressed in Escherichia coli, purified, and characterized. Absorption and EPR spectroscopy reveal properties very similar to cytochromes b5 from vertebrates. NMR spectra indicate that the orientation of the heme in the protein relative to its alpha,gamma meso axis is about 1:1. A redox potential of -26 mV versus standard hydrogen electrode was measured by cyclic voltammetry on a modified gold electrode in the presence of hexamminechromium(III) chloride. The cytochrome b5 is reduced by house fly cytochrome P450 reductase in a reconstituted system at a high rate (5.5 s-1), and it stimulates heptachlor epoxidation when reconstituted with house fly cytochrome P450 reductase, cytochrome P450 6A1, phospholipid, and detergent. Cytochrome b5 decreases the apparent Km for P450 reductase and increases the Vmax for heptachlor epoxidation at constant cytochrome P450 6A1 concentrations. The results indicate that cytochrome b5 stimulates a step following the first electron transfer during cytochrome P450 6A1 turnover.
Highlights
Cyt b51 was first discovered in Cecropia silkworm larvae [1], but the functions of this membrane-bound heme protein have been most extensively studied in mammals [2, 3]
Metabolism of insecticides by P450s is a major mechanism of insecticide resistance in insects [22, 23], and detoxification of plant toxins by P450s is thought to be an adaptation to the hazards of herbivory [24]
Both CYP6A1, an insect P450, which is overproduced in insecticide-resistant strains of the house fly, and NADPH-dependent cytochrome P450 reductase, which provides electrons to P450s from NADPH, have been cloned from the house fly, Musca domestica [25, 26] and expressed in E. coli [27]
Summary
Amplification of a Partial cDNA Encoding House Fly Cytochrome b5 by RT-PCR—Four-day-old larvae of the diazinon-resistant strain “Rutgers” of the house fly, M. domestica, were used as the source of poly(A)ϩ RNA. University of Oregon, Eugene, OR) was used for the expression of the house fly cyt b5 This plasmid contains an NdeI site adjacent to the ATG codon. 1H NMR spectra were recorded at 25 °C on a Varian Unity 300 spectrometer operating in the quadrature mode with a proton frequency of 299.997 MHz. Recombinant house fly cyt b5 solutions (1 mM) were obtained by repeated exchanges of a concentrated aqueous solution of the protein with 30 mM phosphate buffer in D2O at pH* ϭ 7.0. CYP6A1 was incubated for 15 min on ice with P450 reductase and, where indicated, cyt b5 in the presence of 1 mg/ml L-␣-dilauroyl-sn-glycero-3-phosphocholine and 0.2% CHAPS in a 0.1 M potassium phosphate buffer, pH 7.6 (enzyme mixture). P450 reductase was calculated based on protein content [36]
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