Simultaneous purification and characterization of cytochrome b5 reductase and cytochrome b5 from sheep liver

https://doi.org/10.1016/S1357-2725(98)00099-5Get rights and content

Abstract

Cytochrome b5 was purified from detergent solubilized sheep liver microsomes by using three successive DEAE-cellulose, and Sephadex G-100 column chromatographies. It was purified 54-fold and the yield was 23.5% with respect to microsomes. The apparent Mr of cytochrome b5 was estimated to be 16,200±500 by SDS-PAGE. Absolute absorption spectrum of the purified cytochrome b5 showed maximal absorption at 412 nm and dithionite-reduced cytochrome b5 gave peaks at 557, 526.5 and 423 nm. The ability of the purified sheep liver cytochrome b5 to transfer electrons from NADH-cytochrome b5 reductase to cytochrome c was investigated. The Km and Vmax values were calculated to be 0.088 μM cytochrome b5 and 315.8 μM cytochrome c reduced/min/mg enzyme, respectively. Also the reduction of cytochrome b5 by reductase was studied and Km and Vmax values were determined to be 5 μM cytochrome b5 and 5200 nmol cytochrome b5 reduced/min/mg enzyme, respectively. The Km and Vmax values for the cofactor NADH in the presence of saturating concentration of cytochrome b5 were found to be 0.0017 mM NADH and 6944 nmol cytochrome b5 reduced/min/mg enzyme, respectively.

NADH-cytochrome b5 reductase was also partially purified from the same source, detergent solubilized sheep liver microsomes, by using two successive DEAE-cellulose, and 5′-ADP-agarose affinity column chromatographies. It was purified 144-fold and the yield was 7% with respect to microsomes. The apparent monomer Mr of reductase was estimated to be 34,000 by SDS-PAGE.

When ferricyanide was used as an electron acceptor, reductase showed maximum activity between 6.8 and 7.5. The Km and Vmax values of the enzyme for ferricyanide were calculated as 0.024 mM ferricyanide and 673 μmol ferricyanide reduced/min/mg enzyme, respectively. The Km and Vmax values for the cofactor NADH in the presence of saturating amounts of ferricyanide were found to be 0.020 mM NADH and 699 μmol ferricyanide reduced/min/mg enzyme, respectively.

Introduction

Cytochrome b5 and cytochrome b5 reductase are amphipathic membrane-bound proteins which exist in relatively high concentration in endoplasmic reticulum of liver tissues. These proteins are involved in NADH-dependent electron transport in endoplasmic reticulum. Two electrons are transferred from NADH to two molecules of cytochrome b5 through a FAD-containing cytochrome b5 reductase. Cytochrome b5 plays a central role in diverse metabolic reactions such as in lipid metabolism including Δ9-, Δ6-, and Δ5- fatty acyl desaturation1, 2, 3, 4, in elongation of fatty acids[5], in biosynthesis of cholestrol[6], in plasmalogen synthesis[7], in prostaglandin synthesis[8]and in drug metabolism9, 10, 11. Physiological functions of this system are depicted in Fig. 1.

Cytochrome b5 reductase-cytochrome b5 system is also involved in the synthesis of biologically active highly polyunsaturated fatty acids called eicososanoids which are prostaglandins, prostacylins, thromboxanes and leukotriens. These compounds are important mediators and regulators of various cell functions12, 13and the synthesis of these compounds mostly takes place in the liver. Except for the structural studies carried out with bovine liver (including calf liver)14, 15, 16, most of the studies related to the biochemical and physiological functions of cytochrome b5 reductase-cytochrome b5 system have been carried out by using small laboratory animals such as rat2, 3, 4, 5, 6, hen1, 17, rabbit18, 19, 20and mice[21]and recently house fly, Musca domestica22, 23.

In recent years, there has been a great deal of interest in the study of metabolism of endogenous and exogenous compounds, drug-metabolizing enzymes, biochemical mechanisms in veterinary animals especially in sheep. However, very little information is available on these systems in sheep, although the sheep liver has a very high nutritional value and is extensively consumed by man.

This study is undertaken to purify both cytochrome b5 and b5 reductase from sheep liver in biocatalytically active form and to characterize some structural and kinetic properties.

Section snippets

Chemicals

Glycerol, sodium dithionite, phenylmethylsulfonyl fluoride (PMSF) were the products of Merck, Darmstadt, cholic acid sodium salt, d,l-dithiothreitol (DTT), bovine serum albumin (BSA), l-glutamate dehydrogenase type IV, horse cytochrome c (type VI), ε-amino caproic acid (ε-ACA), sodium dodecyl sulfate (SDS), 5′-ADP-agarose were purchased from Sigma Chemical Company, Saint Louis, MI. Preswollen diethylaminoethyl cellulose (DEAE-cellulose, DE-52) was obtained from Whatman Biochemical Ltd., Kent.

Purification of cytochrome b5 from sheep liver microsomes

The results of a typical purification of cytochrome b5 from sheep liver microsomes are presented in Table 1. It was found that cytochrome b5 was purified 516-fold and the recovery of cytochrome b5 in the final purified fraction was about 23.5% with respect to microsomal cytochrome b5 content. The overall procedure used to purify cytochrome b5 from sheep liver microsomes is outlined in Fig. 2.

As shown in Fig. 3, cytochrome b5 was eluted from first DEAE-cellulose column as a sharp peak with 0.1 M

Discussion

Many important metabolic reactions are carried out in the liver and NADH dependent cytochrome b5 reductase-cytochrome b5 electron transport system plays a pivotal role in some of these reactions. Both proteins are amphipathic integral membrane proteins, with two domains. In cytochrome b5, hydrophillic heme-containing peptide fragment, Mr of 11,000, occupies N-terminal of this protein and hydrophobic nonpolar peptide, Mr 5000, present in C-terminus, anchors cytochrome b5 to the membranes.

Acknowledgements

A financial support provided by a grant from Turkish AFP-01-08-DPT-96K121570 is gratefully acknowledged.

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