Mapping of resistance to the potato cyst nematode Globodera rostochiensis from the wild potato species Solanum vernei

A population of diploid potato (Solanum tuberosum) was used for the genetic analysis and mapping of a locus for resistance to the potato cyst nematode Globodera rostochiensis, introgressed from the wild potato species Solanum vernei. Resistance tests of 108 genotypes of a F₁ population revealed the presence of a single locus with a dominant allele for resistance to G. rostochiensis pathotype Ro1. This locus, designated GroV1, was located on chromosome 5 with RFLP markers. Fine-mapping was performed with RAPD and SCAR markers. The GroV1 locus was found in the same region of the potato genome as the S. tuberosum ssp. andigena H1 nematode resistance locus. Both resistance loci could not excluded to be allelic. The identification of markers flanking the GroV1 locus offers a valuable strategy for marker-assisted selection for introgression of this nematode resistance.Abbreviations BSA bulked segregant analysis - RAPD random-amplified polymorphic DNA - RFLP restriction fragment length polymorphism - SCAR sequence-characterized amplified region     

Cytochrome b5 reductase–cytochrome b5 as an active P450 redox enzyme system in Phanerochaete chrysosporium: Atypical properties and in vivo evidence of electron transfer capability to CYP63A2

Two central redox enzyme systems exist to reduce eukaryotic P450 enzymes, the P450 oxidoreductase (POR) and the cyt b₅ reductase–cyt b₅. In fungi, limited information is available for the cyt b₅ reductase–cyt b₅ system. Here we characterized the kinetic mechanism of (cyt b₅r)–cyt b₅ redox system from the model white-rot fungus Phanerochaete chrysosporium (Pc) and made a quantitative comparison to the POR system. We determined that Pc-cyt b₅r followed a “ping-pong” mechanism and could directly reduce cytochrome c. However, unlike other cyt b₅ reductases, Pc-cyt b₅r lacked the typical ferricyanide reduction activity, a standard for cyt b₅ reductases. Through co-expression in yeast, we demonstrated that the Pc-cyt b₅r–cyt b₅ complex is capable of transferring electrons to Pc-P450 CYP63A2 for its benzo(a)pyrene monooxygenation activity and that the efficiency was comparable to POR. In fact, both redox systems supported oxidation of an estimated one-third of the added benzo(a)pyrene amount. To our knowledge, this is the first report to indicate that the cyt b₅r–cyt b₅ complex of fungi is capable of transferring electrons to a P450 monooxygenase. Furthermore, this is the first eukaryotic quantitative comparison of the two P450 redox enzyme systems (POR and cyt b₅r–cyt b₅) in terms of supporting a P450 monooxygenase activity.     

Evaluation of soybean RFLP marker diversity in adapted germ plasm

Summary Soybean RFLP markers have been primarily developed and genetically mapped using wide crosses between exotic and adapted genotypes. We have screened 38 soybean lines at 128 RFLP marker loci primarily to characterize germ plasm structure but also to evaluate the utility of RFLP markers identified in unadapted populations. Of these DNA probes 70% detected RFLPs in this set of soybean lines with an average polymorphism index of 0.30. This means that only 1 out of 5 marker loci was informative between any particular pair of adapted soybean lines. The variance associated with the estimation of RFLP genetic distance (GD_R) was determined, and the value obtained suggested that the use of more than 65–90 marker loci for germ plasm surveys will add little precision. Cluster analysis and principal coordinate analysis of the GD_R matrix revealed the relative lack of diversity in adapted germ plasm. Within the cultivated lines, several lines adapted to Southern US maturity zones also appeared as a separate group. GD_R data was compared to the genetic distance estimates obtained from pedigree analysis (GD_P). These two measures were correlated with r = 0.54 for all 38 lines, but the correlation increased to r = 0.73 when only adapted lines were analyzed.     

The effect of the r a and r b loci on the lipid content of the seed of Pisum sativum

Summary The lipid content of seed from a set of isogenic lines for the R _a : r _a locus has been determined; the results show that this locus as well as affecting the starch, sugar and storage protein content and composition, also has a marked effect on the lipid content of the seed. Genotypes having different combinations of alleles at the r _a and r _b loci have also been examined; an r _a r _a r _b r _b , genotype had 5.57% purified total lipid in its seed — a more than 2-fold increase over that found in the round-seeded varieties (R _a R _a R _b R _b ) usually grown for the dry sed crop.     

Ascorbate-independent electron transfer between cytochromeb 561 and a 27 kDa ascorbate peroxidase of bean hypocotyls

Summary Cytochromeb ₅₆₁ (cytb ₅₆₁) is a trans-membrane cytochrome probably ubiquitous in plant cells. In vitro, it is readily reduced by ascorbate or by juglonol, which in plasma membrane (PM) preparations from plant tissues is efficiently produced by a PM-associated NAD(P)Hquinone reductase activity. In bean hypocotyl PM, juglonol-reduced cytb ₅₆₁ was not oxidized by hydrogen peroxide alone, but hydrogen peroxide led to complete oxidation of the cytochrome in the presence of a peroxidase found in apoplastic extracts of bean hypocotyls. This peroxidase active on cytb ₅₆₁ was purified from the apoplastic extract and identified as an ascorbate peroxidase of the cytosolic type. The identification was based on several grounds, including the ascorbate peroxidase activity (albeit labile), the apparent molecular mass of the subunit of 27 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the dimeric native structure, the typical spectral properties of a heme-containing peroxidase, and an N-terminal sequence strongly conserved with cytosolic ascorbate peroxidases of plants. Cytb ₅₆₁ used in the experiments was purified from bean hypocotyl PM and juglonol was enzymatically produced by recombinant NAD(P)H:quinone reductase. It is shown that NADPH, NAD(P)H:quinone reductase, juglone, cytb ₅₆₁, the peroxidase interacting with cytb ₅₆₁, and H₂O₂, in this order, constitute an artificial electron transfer chain in which cytb ₅₆₁ is indirectly reduced by NADPH and indirectly oxidized by H₂O₂.Abbreviations APX ascorbate peroxidase - b ₅₆₁PX cytochrome 6561 peroxidase - CPX coniferol peroxidase - cyt cytochrome - GPX guaia-col peroxidase - IWF intercellular washing fluid - MDHA monodehydroascorbate - PM plasma membrane     

Modulation of the reductive metabolism of halothane by microsomal cytochrome b5 in rat liver

To study the modulation of the reductive metabolism of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) by microsomal cytochrome b₅, formation of 2-chloro-1,1,1-trifluoroethane (CTE) and 2-chloro-1,1-difluoroethylene (CDE), major reduced metabolites of halothane, was analyzed in vivo and in vitro. Rats were pretreated with both malotilate (diisopropyl-1,3-dithiol-2-ylidenemalonate) and sodium phenobarbital (malotilate-treated rats) or only with sodium phenobarbital (control rats). The microsomes of malotilate-treated rats had significantly more cytochrome b₅ than the controls, whereas the cytochrome P-450 content was not different between the two groups. At the end of 2-h exposure to 1% halothane in 14% oxygen, the ratio of CDE to CTE in arterial blood was significantly higher in malotilate-treated rats than in the controls. Under anaerobic conditions, the formation of CDE and the ratio of CDE to CTE were significantly greater in microsomal preparations of malotilate-treated rats than those of the controls. In a reconstituted system containing cytochrome P-450_PB purified from rabbit liver, addition of cytochrome b₅ to the system enhanced the formation of CDE and increased the ratio of CDE to CTE. These results suggested that cytochrome b₅ enhances the formation ratio of CDE to CTE by stimulating the supply of a second electron to cytochrome P-450, which might reduce radical reactions in the reductive metabolism of halothane.     

The role of the membrane bound cytochromes of b- and c-type in the electron transport chain of Rhodobacter capsulatus

(1) The electron transport system of heterotrophically dark-grown Rhodobacter capsulatus was investigated using the wild-type strain MT1131 and the phototrophic non-competent (Ps^-) mutant MT-GS18 carrying deletions of the genes for cytochrome c ₁ and b of the bc ₁ complex and for cytochrome c ₂. (2) Spectroscopic and thermodynamic data demonstrate that deletion of both bc ₁ complex and cyt. c ₂ still leaves several haems of c- and b-type with Em_7.0 of +265 mV and +354 mV at 551–542 nm, and +415 mV and +275 mV at 561–575 nm, respectively. (3) Analysis of the oxidoreduction kinetic patterns of cytochromes indicated that cyt. b ₄₁₅ and cyt. b ₂₇₅ are reduced by either ascorbate-diaminodurene or NADH, respectively. (4) Growth on different carbon and nitrogen sources revealed that the membrane-bound electron transport chain of both MT1131 and MT-GS18 strains undergoes functional modifications in response to the composition of the growth medium used. (5) Excitation of membrane fragments from cells grown in malate minimal medium by a train of single turnover flashes of light led to a rapid oxidation of 32% of the membrane-bound c-type haem complement. Conversely, membranes prepared from peptone/yeast extract grown cells did not show cyt. c photooxidation. These results are discussed within the framework of an electron transport chain in which alternative pathways bypassing both the cyt. c ₂ and bc ₁ complex might involve high-potential membrane bound haems of b- and c-type.Abbreviations AA antimycin A - CCCP carbonylcyanide m-chlorophenyl hydrazone - CN^- cyanide - DAD diaminodurene - Q₂H₂ ubiquinol-2 - Q-pool ubiquinone-10 pool - RC photochemical reaction center     

Initial purification study of the cytochromeb 561 of bean hypocotyl plasma membrane

Summary The plasma membrane (PM) of higher plants contains a major ascorbate-reducible, high-potentialb-type cytochrome, named cytochromeb ₅₆₁ (cytb ₅₆₁). In this paper a rapid purification protocol for the cytb ₅₆₁ of bean hypocotyls PM is described. An almost 200-fold increase of cytb ₅₆₁ specific concentration was achieved with respect to the PM fraction, which contained about 0.2 nmol of ascorbate-reducible heme per mg protein. The procedure can be performed in one day starting from purified PMs obtained by the phase-partitioning procedure. However, cytb ₅₆₁ proved to be unstable during chromatographic purification and the amount of protein finally recovered was low. Purified cytb ₅₆₁ eluted as a 130,000 Da protein-detergent complex from gel-filtration columns. It was completely reduced by ascorbate and reduced-minus-oxidized spectra showed -, - and -bands at 561, 530, and 429 nm respectively, not unlike the spectra of whole PMs. This work represents an initial approach to the biochemical characterization of the cytb ₅₆₁ of higher plants, formerly suggested to be related to cytb ₅₆₁ of animal chromaffin granules.Abbreviations cytb ₅₆₁ cytochromeb ₅₆₁ - PM plasma membrane - UPV upper-phase vesicles - GSII glucan synthase II - CCR NADH-dependent cytochromec reductase - CCO cytochromec oxidase - TX-100R reduced Triton X-100     

Physical linkage of the SLG and SRK genes at the self-incompatibility locus of Brassica oleracea

Summary In Brassica oleracea, the pollen-stigma interaction of self-incompatibility is controlled by a single genetically defined locus designated S. Molecular studies have identified two genes that are tightly linked to the classically defined S locus: The S-Locus Glycoprotein (SLG) gene and the S-Receptor Kinase (SRK) gene. In previous RFLP linkage analyses with probes specific for SLG and SRK, we were unable to identify any recombination events between SLG, SRK, and self-incompatibility phenotype. In this paper, we use pulsed-field gel electrophoresis (PFGE) in conjunction with DNA blot analysis to characterize the S-locus region from two highly divergent self-incompatibility genotypes, S ₂ and S ₆. We establish the physical linkage of SLG and SRK in each genotype, and demonstrate that the two genes are separated by a maximum distance of 220 kb in the S ₆ genotype and 350 kb in the S ₂ genotype. Furthermore, a comparison of the data from the two genotypes reveals that a high level of polymorphism exists across the entire S-locus region.     

Regulation of biosynthesis ofN-glycolylneuraminic acid-containing glycoconjugates: characterization of factors required for NADH-dependent cytidine 5′monophosphate-N-acetylneuraminic acid hydroxylation

The hydroxylation of CMP-NeuAc has been demonstrated to be carried out by several factors including the soluble form of cytochromeb ₅. In the present study, mouse liver cytosol was subjected to ammonium sulfate fractionation and cellulose phosphate column chromatography for the separation of two other essential fractions participating in the hydroxylation. One of the fractions, which bound to a cellulose phosphate column, was able to reduce the soluble cytochromeb ₅, using NADH as an electron donor. The other fraction, which flowed through the column, was assumed to contain the terminal enzyme which accepts electrons from cytochromeb ₅, activates oxygen, and catalyses the hydroxylation of CMP-NeuAc. Assay conditions for the quantitative determination of the terminal enzyme were established, and the activity of the enzyme in several tissues of mouse and rat was measured. The level of the terminal enzyme activity is associated with the expression ofN-glycolylneuraminic acid in these tissues, indicating that the expression of the terminal enzyme possibly regulates the overall velocity of CMP-NeuAc hydroxylation.Abbreviations CMP cytidine 5-monophosphate - NeuAc N-acetylneuraminic acid - NeuGc N-glycolylneuraminic acid - NADH reduced nicotinamide adenine dinucleotide - NADPH reduced nicotinamide adenine dinucleotide phosphate - DTT dithiothreitol     

Identification of RAPD markers linked to a major blast resistance gene in rice

Rice blast, caused byPyricularia grisea, is a major production constraint in many parts of the world. The Korean rice variety Tongil showed high levels of resistance for about six years when widely planted under highly disease-conducive conditions, before becoming susceptible. Tongil was found to carry a single dominant gene, designatedPi-10t, conferring resistance to isolate 106 of the blast pathogen from the Philippines. We report here the use of bulked segregant RAPD analysis for rapid identification of DNA markers linked toPi-10t. Pooled DNA extracts from five homozygous blast-resistant (RR) and five susceptible (rr) BC₃F₂ plants, derived from a CO39 × Tongil cross, were analyzed by RFLP using 83 polymorphic probes and by RAPD using 468 random oligomers. We identified two RAPD markers linked to thePi-10t locus: RRF6 (3.8 ± 1.2 cM) and RRH18 (2.9 ± 0.9 cM). Linkage of these markers withPi-10t was verified using an F2 population segregating forPi-10t. The two linked RAPD markers mapped 7 cM apart on chromosome 5. Chromosomal regions surrounding thePi-10t gene were examined with additional RFLP markers to define the segment introgressed from the donor genome.Pi-10t is likely to be a new blast-resistance locus, because no other known resistance gene has been mapped on chromosome 5. These tightly linked RAPD markers could facilitate early selection of thePi-10t locus in rice breeding programmes.     

Ascaris suum NADH-methemo(myo)globin reductase systems recovering differential functions of hemoglobin and myoglobin, adapting to environmental hypoxia

We reported previously that Ascaris suum cytochrome b₅, specifically expressed in this nematode at the adult stage and dually localized in extracellular perienteric fluid and hypodermis, is involved in both perienteric NADH-methemoglobin and cytosolic NADH-metmyoglobin reduction, where cytochrome b₅ functions as an electron carrier between NADH-mediated cytochrome b₅ reductase and substrates, methemo(myo)globins to reduce the nonfunctional globins back to functional ferrous hemo(myo)globins. To further characterize NADH-methemo(myo)globin reductase systems, the midpoint potentials of A. suum perienteric hemoglobin and body wall myoglobin, as well as the affinities of Ascaris methemoglobin and metmyoglobin toward cytochrome b₅, were evaluated using potentiometric titration and surface plasmon resonance techniques, respectively. Midpoint potentials of + 7.2 mV and + 19.5 mV were obtained for Ascaris perienteric hemoglobin and body wall myoglobin, respectively. The affinities of Ascaris perienteric methemoglobin and body wall metmyoglobin toward the nematode cytochrome b₅ were comparable to that for mammalian hemoglobin and cytochrome b₅; association constants were 0.585 × 10³ M^− 1 and 2.32 × 10³ M^− 1, respectively, with rapid equilibration kinetics. These observations highlight the physiological importance of A. suum perienteric NADH-methemoglobin and cytosolic metmyoglobin reductase systems. Differential roles of A. suum perienteric hemoglobin and body wall myoglobin are also discussed from the viewpoint of oxygen homeostasis under hypoxic conditions.     

Purification of NADH-cytochromeb 5 reductase from rat liver plasma membranes

Summary An NADH-cytochromeb ₅ reductase was purified from rat liver plasma membranes. Rat liver plasma membranes were prepared by aqueous two-phase partition. Peripheral proteins were removed by EDTA extraction and integral membrane proteins were solubilized with Triton X-100. The NADH-cytochromeb ₅ reductase was purified by hydroxyapatite, anion exchange, and gel filtration chromatographies. The purified preparation was homogeneous and estimated to have an apparent molecular weight of 32 kDa on SDS-polyacrylamide gel electrophoresis. Two tryptic peptides of the purified enzyme had sequence homologies with rat, human, and bovine NADH-cytochromeb ₅ reductases.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate - BCA bicinchonicic acid - EDTA ethylenediamine tetraacetate acid disodium salt - FeCN ferricyanide - HPLC high-performance liquid chromatography - NADH nicotinamide adenine dinucleotide reduced form - PMSF -phenylmethylsulfonyl fluoride     

Recombination rates of soybean varieties from different periods of introduction and release

Theory predicts that selection for adaptability during the short term also favors selection for a reduced recombination rate in the population. The objective of this study was to test whether the cyclic short-term selection which has taken place in soybean breeding programs in the USA since the introduction of the crop has measurably reduced recombination frequencies. Thirteen soybean varieties separated into four different release periods (prior to 1940, 1940–1954, 1955–1969, after 1970) were evaluated for their recombination frequencies within three locus pairs. Recombination frequencies among the individual varieties ranged from 7.6 to 24.1 % at thep ₁ r locus pair, from 20.9 to 30.1 % at thelnp ₂ locus pair, and from 28.7 to 41.6% at thedt ₁ l ₁ locus pair. Recombination frequencies were significantly different among varieties within a release period for thep ₁ r andlnp ₂ locus pairs, but recombination frequencies did not differ among release periods for any locus pair. Thus, apparently, plant breeders have developed soybean varieties with improved adaptation without influencing recombination rates.     

Restriction fragment length polymorphisms of the phytohemagglutinin genes inPhaseolus andVigna (Leguminosae)

Restriction fragment length polymorphisms at the phytohemagglutinin (PHA) locus were determined among 21 genotypes ofPhaseolus vulgaris, P. coccineus, P. acutifolius, P. lunatus, and threeVigna species, using five restriction enzymes and one double digestion, in order to provide molecular evidence for their genetic relatedness. The dissimilarity between genotypes was estimated from binary RFLP data. The dissimilarity was high among species (from 0.75 to 0.95), and of variable extent among genotypes of the same species (0.33–0.89). InP. vulgaris, two different DNA hybridization patterns were found, giving further evidence for two major gene pools in that species. The restriction patterns ofP. vulgaris var.aborigineus, the putative ancestral form ofP. vulgaris, exhibit clear homology toP. vulgaris genotypes. An undefined landrace from Taiwan could be identified as aP. vulgaris genotype. RFLP-based trees for the phytohemagglutinin genes of the species studied were computed with several distance matrix and parsimony methods.     

The interaction of microsomal cytochrome P450 2B4 with its redox partners, cytochrome P450 reductase and cytochrome b(5)

Cytochrome P450 2B4 is a microsomal protein with a multi-step reaction cycle similar to that observed in the majority of other cytochromes P450. The cytochrome P450 2B4-substrate complex is reduced from the ferric to the ferrous form by cytochrome P450 reductase. After binding oxygen, the oxyferrous protein accepts a second electron which is provided by either cytochrome P450 reductase or cytochrome b₅. In both instances, product formation occurs. When the second electron is donated by cytochrome b₅, catalysis (product formation) is ∼10- to 100-fold faster than in the presence of cytochrome P450 reductase. This allows less time for side product formation (hydrogen peroxide and superoxide) and improves by ∼15% the coupling of NADPH consumption to product formation. Cytochrome b₅ has also been shown to compete with cytochrome P450 reductase for a binding site on the proximal surface of cytochrome P450 2B4. These two different effects of cytochrome b₅ on cytochrome P450 2B4 reactivity can explain how cytochrome b₅ is able to stimulate, inhibit, or have no effect on cytochrome P450 2B4 activity. At low molar ratios (<1) of cytochrome b₅ to cytochrome P450 reductase, the more rapid catalysis results in enhanced substrate metabolism. In contrast, at high molar ratios (>1) of cytochrome b₅ to cytochrome P450 reductase, cytochrome b₅ inhibits activity by binding to the proximal surface of cytochrome P450 and preventing the reductase from reducing ferric cytochrome P450 to the ferrous protein, thereby aborting the catalytic reaction cycle. When the stimulatory and inhibitory effects of cytochrome b₅ are equal, it will appear to have no effect on the enzymatic activity. It is hypothesized that cytochrome b₅ stimulates catalysis by causing a conformational change in the active site, which allows the active oxidizing oxyferryl species of cytochrome P450 to be formed more rapidly than in the presence of reductase.     

The Nir1 locus in barley is tightly linked to the nitrite reductase apoprotein gene Nii

pBNiR1, a cDNA clone encoding part of the barley nitrite reductase apoprotein, was isolated from a barley (cv. Maris Mink) leaf cDNA library using the 1.85 kb insert of the maize nitrite reductase cDNA clone pCIB808 as a heterologous probe. The cDNA insert of pBNiR1 is 503 by in length. The nucleotide coding sequence could be aligned with the 3 end of other higher plant nitrite reductase apoprotein cDNA sequences but diverges in the 3 untranslated region. The whole-plant barley mutant STA3999, previously isolated from the cultivar Tweed, accumulates nitrite after nitrate treatment in the light, has very much lowered levels of nitrite reductase activity and lacks detectable nitrite reductase cross-reacting material due to a recessive mutation in a single nuclear gene which we have designated Nir1. STA3999 has the characteristics expected of a nitrite reductase apoprotein gene mutant. Here we have used pB-NiR1 in RFLP analysis to determine whether the mutation carried by STA3999 is linked to the nitrite reductase apoprotein gene locus Nii. An RFLP was identified between the wild-type barley cultivars Tweed (major hybridising band of 11.5 kb) and Golden Promise (major hybridising band of 7.5 kb) when DraI-digested DNA was probed with the insert from the partial barley nitrite reductase cDNA clone, pBNiR1. DraI-digested DNA from the mutant STA3999 also exhibited a major hybridising band of 11.5 kb after hybridisation with the insert from pBNiR1. F₁ progeny derived from the cross between the cultivar Golden Promise and the homozygous nir1 mutant STA3999 were heterozygous for these bands as anticipated. Co-segregation of the Tweed RFLP band of 11.5 kb and the mutant phenotype (leaf nitrite accumulation after nitrate treatment/loss of detectable nitrite reductase cross-reacting material at M_r 63000) was scored in an F₂ population of 312 plants derived from the cross between the cultivar Golden Promise and the homozygous mutant STA3999. The Tweed RFLP band of 11.5 kb and the mutant phenotype showed strict co-segregation (in approximately one quarter (84) of the 312 F₂ plants examined). Only those F₂ individuals heterozygous for the RFLP pattern gave rise to F₃ progeny which segregated for the mutant phenotype. We conclude that the nir1locus and the nitrite reductase apoprotein gene Nii are very tightly linked.     

Preparation of a biologically active apo-cytochrome b5 via heterologous expression in Escherichia coli

Cytochrome b₅ (b₅) has been shown to modulate many cytochrome P450 (CYP)-dependent reactions. In order to elucidate the mechanism of such modulations, it is necessary to evaluate not only the effect of native b₅ on CYP-catalyzed reactions, but also that of the apo-cytochrome b₅ (apo-b₅). Therefore, the apo-b₅ protein was prepared using a heterologous expression in Escherichia coli. The gene for rabbit b₅ was constructed from synthetic oligonucleotides using polymerase chain reaction (PCR), cloned into pUC19 plasmid and amplified in DH5α cells. The gene sequence was verified by DNA sequencing. The sequence coding b₅ was cleaved from pUC19 by NdeI and XhoI restriction endonucleases and subcloned to the expression vector pET22b. This vector was used to transform E. coli BL-21 (DE3) Gold cells by heat shock. Expression of b₅ was induced with isopropyl β-d-1-thiogalactopyranoside (IPTG). The b₅ protein, produced predominantly in its apo-form, was purified from isolated membranes of E. coli cells by chromatography on a column of DEAE–Sepharose. Using such procedures, the homogenous preparation of apo-b₅ protein was obtained. Oxidized and reduced forms of the apo-b₅ reconstituted with heme exhibit the same absorbance spectra as native b₅. The prepared recombinant apo-b₅ reconstituted with heme can be reduced by NADPH:CYP reductase. The reconstituted apo-b₅ is also fully biologically active, exhibiting the comparable stimulation effect on the CYP3A4 enzymatic activity towards oxidation of 1-phenylazo-2-hydroxynaphthalene (Sudan I) as native rabbit and human b₅.     

b-Type cytochromes in plasma membranes ofPhaseolus vulgaris hypocotyls,Arabidopsis thaliana leaves, andZea mays roots

Summary The plasma membrane of higher plants contains more than one kind ofb-type cytochromes. One of these has a high redox potential and can be fully reduced by ascorbate. This component, the cytochromeb ₅₆₁ (cytb ₅₆₁), has its characteristic -band absorbance close to 561 nm wavelength at room temperature. Cytb ₅₆₁ was first isolated from etiolated bean hook plasma membranes by two consecutive anion exchange chromatography steps. During the first step performed at pH 8, cytb ₅₆₁ did not bind to the anion exchange column, but otherb-type cytochromes did. In the second step performed at pH 9.9, cytb ₅₆₁ was bound to the column and was eluted from the column at an ionic strength of about 100 mM KCl. However, when the same protocol was applied to the solubilized plasma membrane proteins fromArabidopsis thaliana leaves and maize roots, the ascorbate-reducible cytb ₅₆₁ bound already to the first anion exchange column at pH 8 and was eluted also at an ionic strength of about 100 mM KCl. Otherb-type cytochromes than the ascorbate-reducible cytb ₅₆₁ from the plasma membranes of Arabidopsis leaves and maize roots showed similar Chromatographic characteristics to that of bean hypocotyls. These results demonstrate particular differences in the Chromatographic behavior of cytb ₅₆₁ from different sources.Abbreviations cyt b ₅₆₁ cytochromeb ₅₆₁ - PM plasma membrane - PAGE polyacrylamide gel electrophoresis