Abstract
Crystals of the hydrophilic, catalytic domain (30 kDa) of pig liver NADH-cytochrome b5 reductase solubilized by the protease (cathepsin D) have been grown in a solution of polyethylene glycol by the vapor-diffusion procedure. The crystals belong to the orthorhombic system, space group P2(1)2(1)2(1) with unit-cell dimensions of a = 87.1, b = 73.2, and c = 49.0 A. The asymmetric unit contains one molecule of the enzyme. The x-ray diffraction patterns extend to 2.0-A resolution. On the other hand, the intact enzyme (35 kDa) containing the hydrophobic membrane-binding domain solubilized by the detergent (Triton N-101) has been crystallized also from the polyethylene glycol solution. The crystals are needle-shaped and still too small for x-ray diffraction study.
Highlights
KDa) of pig liver NADH-cytochrome b5 reductase sol- cytochrome b, whose three-dimensional structure has already ubilized by the protease have been grown been elucidated by x-ray diffraction study [6]. in a solution of polyethylene glycol by the vapor-diffusion procedure
In thecase of the detergentsolubilized enzyme, 0.1% Triton N-101 and 10% glycerol were contained in the solution
NADH-cytochrome bs reductase, which accepts two electrons from NADH to transfer to one electronacceptorcytochrome b5, catalyzesto reduce cytochrome bs in an electron-transport chain from NADH to a terminal oxidase desaturase in theendoplasmic reticulum [1]
Summary
KDa) of pig liver NADH-cytochrome b5 reductase sol- cytochrome b, whose three-dimensional structure has already ubilized by the protease (cathepsinD) have been grown been elucidated by x-ray diffraction study [6]. in a solution of polyethylene glycol by the vapor-diffusion procedure. Purification-The detergent-and lysosome-solubilizedenzymes were prepared according to the methodpreviously described [7].The purity of the sample was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which showed a singbleanding pattern corresponding to 35 and 30 kDa for detergent- and lysosome-solubilized enzymes, respectively [7]. Crystallization-Protein solutions a t a concentration of 5-40 mg/ ml were preparedinthe presence of 3-20% PEG 40001 (w/v)as lution.
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