Abstract
Effective resolution of detergent-solubilized Tetrahymena microsomal cytochrome b560ms, which is similar to mammalian cytochrome b5, was obtained by chromatography on DEAE-cellulose and gel filtration. On the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight of Tetrahymena cytochrome b560ms is 22,000. The absorption peaks are 414 nm in the oxidized form and 560, 528, and 425 nm in the dithionite-reduced form. The alpha-peak of the reduced form, which is situated at 560 nm and asymmetric with a shoulder at 556 nm, is different from that of mammalian liver microsomal cytochrome b5. The spectra of the reduced and the oxidized cytochrome b560ms are not altered by the presence of cyanide, azide, and carbon monoxide. The alpha-peak is resolved into two distinct peaks at 551 and 558 nm at low temperature. The absorption peaks at 557, 527, and 418 nm in the pyridine ferrohemochrome spectrum indicate that protoheme is the prosthetic group of the purified b-type cytochrome. The oxidation-reduction potential (E'0) is -42 mV. It is reducible by NADH in the presence of an NADH-cytochrome b5 reductase purified from rat liver microsomes. The present results suggest that the pathways of electrons to microsomal cytochrome b560ms-linked oxidative desaturation of fatty acyl-CoA in protozoan Tetrahymena pyriformis are via either of the two microsomal flavoprotein reductases, NADH-ferricyanide reductase or NADPH-cytochrome c reductase.
Highlights
On the basisof sodium dodecyl sulfate-polyacrylamide in microsomal desaturation systems,alsovariedwith ingel electrophoresis, the molecular weight of Tetrahymena cytochrome bS6Oms is22,000
The electron transport in Tetrahymena mitochondria has been described in a number of studies [20, 21], less is known about microsomal electron transport and functions of b-type hemoprotein in a ciliated protozoan Tetrahymena microsomes [22]
Cytochrome b, is widely accepted as a well characterized membrane protein involved in the microsomal electron transport systemof various organisms
Summary
Diluted cytochrome b5wmsfraction was applied to asmallDEAEcellulose column (2 X 5 cm) that had been pre-equilibrated with 20 mM Tris-HC1 (pH 8.0) containing 0.2 mM EDTA plus 0.1% Triton X-100. The concentrated solution (6 ml) was applied to a Sephadex G-100 column (2.6 X 90 cm) that had been previously equilibrated with 20 mM Tris-HC1 buffer (pH 8.0) containing 0.2 mM EDTA plus 1.0% sodium cholate. The concentrated cytochrome bes fraction was subsequently applied to a Sephadex G-100 column (2.6 X 90 cm) that had been previously equilibrated with 20 mM Tris-HC1 buffer (pH 8.0)containing 0.2 mM EDTAplus 1.0% sodium cholate. For removal of residual Triton X-100, salt, and sodium cholate,cytochrome bWmsfractions were applied to a small DEAE-cellulose column (1.5 X 3 cm) that had been previously equilibrated with 20 mM Tris-HC1 buffer (pH 8.0)containing 0.2 mM EDTA.
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