Purification and Characterization of Microsomal Cytochrome b₅ and NADH Cytochrome b₅ Reductase from Pisum sativum

Abstract

In this communication we document the reproducible protocols for the purification of milligram quantities of cytochrome b5 and NADH-cytochrome b5 reductase from the microsomal fraction of Pisum sativum. The cytochrome b5 component of this NADH linked electron transport chain was found to have a molecular mass of 16,400 daltons and the reductase a molecular mass of 34,500 daltons. These components could be reconstituted into a functional NADH oxidase activity active in the reduction of exogenous cytochrome c or ferricyanide. In the latter assay the purified reductase exhibited a turnover number of 22,000 per minute. The aminoterminal amino acid sequence of the cytochrome b5 component was determined by sequential Edmund degredation, thus providing crucial information for the efficient cloning of this central protein of plant microsomal electron transfer.The microsomal electron transport systems of mammalian tissue play important roles in a variety of oxidative reactions including fatty acid desaturation and the support of mixed function oxidative biotransformations catalyzed by the Cyt P-450 monooxygenases. Reductive reactions of the latter class involve NADPH dehydrogenation by a flavoprotein, NADPH-Cyt P-450 reductase, containing both FAD and FMN prosthetic groups, followed by sequential one electron tansfers to the cytochrome P450 heme center. In addition, some P450 biotransformations can be mediated by electron transfer from a short redox transfer chain which links to the dephosphorylated form of pyridine nucleotide, NADH. This system includes NADH-Cyt b5 reductase, a flavoprotein with a single FAD prosthetic group, and the heme protein Cyt b5. Both the NADPH and NADH flavoprotein reductases as well as Cyt b5 have been purified to homogeneity from a variety of mammalian microsomal fractions and have received intensive study during the past decade (20,23,24). Both of these proteins of the NADH linked chain are amphipathic (15) in nature, consisting oftwo definite domains, a water soluble catalytic portion and a hydrophobic fragment that binds the protein to the microsomal membrane. The catalytic domain can be separated from the holenzyme by limited proteolytic digestion ofthe microsomal fraction (9, 21). Alternately, the intact enzyme can be obtained by detergent solubilization (16,20,24 Unfortunately, much less is known about the corresponding electron transport and P-450 monoxygenase systems from plant tissue. Galle et al. (5) have recently purified an NADH-ferricyanide reductase from potato tuber that is apparently involved in the desaturation of oleate to form linoleic acid in higher plants. Two mitochondrial forms of a reductase have also been isolated by Klein and Burke (8), the first of which contained two peptide fragments and was specific for NADH whereas the second was apparently comprised of five polypeptides, displayed a broad pH maxima, and utilized both NADH and NADPH. Efforts at the isolation of a Cyt P-450 reductase from the microsomal fraction of higher plants began with the partial pur...

NADH-Ferricyanide Reductase of Leaf Plasma Membranes

“…4 and 5). Notably, several molecular masses have been reported for membrane-bound (microsomal) NADH-(acceptor) oxidoreductases from different plant sources ( 13,15,17,23).…”

Cytochrome P-450 from the Mesocarp of Avocado (Persea americana)

“…Cyt P-450 has been purified from tulip (Tulipa gesneriana) bulbs (12), and from tubers of Jerusalem artichoke (Helianthus tuberosus) (10). Additionally, an NADPH:Cyt P450 reductase has been purified from Jerusalem artichoke (2), and the related Cyt b5 and NADH:Cyt b5 reductase have been purified from Pisum sativum (16). Unfortunately, the tulip bulb Cyt P450 has not been associated with any enzymic activity, and polyclonal antibodies raised against it were not cross reactive with proteins from other species (1 1).…”

Cytochrome-b5 reductase

“…Monomer (30000-33000, rat [24], rabbit [25], human [27], erythrocytes, soluble form, SOS-PAGE [24,25,27],27000-28000, Saccharomyces cerevisiae, SOS-PAGE [35]) [24,25,27,35] Oligomer (possibly tetramer [27], membrane bound form, x x 27000-45000, values depending on organism and method of solubilization, Phascolopsis gouldii [1], human [6,7,17,20,27], rat [6,24,33,37], Pisum sativum [12], bovine [16,21,39], rabbit [ …”

“…Human [2,3,6,7,10,17,20,23,27,29,32]; Pig [4,11,19,39]; Rat [5,14,18,24,33,37]; Phascolopsis gouldii [1,9]; Pisum sativum [12]; Potato [13]; Maize [13]; Bovine (calf [38,39]) [8,15,16,21,22,28,38,39]; Tetrahymena pyriformis [26]; Rabbit [25,31,34,36,39]; Saccharomyces cerevisiae (grown anaerobically) [35] Source tissue Erythrocytes (soluble form) [1,3,6,9,10,20,24,25,…”

“…Triton X-1 00, activation) [4,23]; Spermine (activation) [3); 9-Amino-1 ,2,3A-tetrahydroacridine (activation) [3] Metal compounds/salts More (rate of reduction dependent on ionic strength) [12,19,25] Turnover number (min-1 ) 2 40600 (ferricyanide, human red cell membrane) [20]; 42000 (ferricyanide, human, cytosol) [20]; 49600 (ferricyanide, human liver, detergent solubilized) [20]; 5900 (ferricyanide, human liver, protease solubilized) [20); 1280 (cytochrome b5) [32); 30000 (NADH) [38]; More [1,12,33] Specific activity (U/mg) 230.5 (high MW aggregate) [1]; 611.1 (low MW aggregate) [1]; 628 [13]; 790 [27]; More [4,6,7,12,18,21,36,37] Km-value (mM) 0.0001 EMl.0009 (NADH) [16,19,20,27,32]; 0.001--0.008 (NADH) [6,21,25]; 0.01--0.06 (NADH) [1,4,18,35]; O.D1--O. 04 (ferricytochrome b s ) [4,6,19,20,34,38]; 0.002--0.009 (ferricytochrome b s ) …”