Form Of Cytochrome B5 Research Articles

Protein-protein interactions in the systems of cytochromes P450 3A4 and 3A5

Molecular interactions between protein partners of the monooxygenase system involved in drug biotransformation (cytochromes P450 3A4, 3A5 and cytochrome b 5) have been investigated. Human cytochromes P450 3A4 and 3A5 (CYP3A4 and CYP3A5) form complexes with various forms of cytochrome b 5, including microsomal (b 5 mc) and mitochondrial (b 5 om) forms of this protein, as well as two chimeric constructs (b 5(om-mc),(b 5(mc-om). Interestingly, significant differences were observed only during interactions with b 5 om. Electroanalytical characteristics of electrodes with immobilized hemoproteins have been determined for CYP3A4, CYP3A5, b 5 mc, b 5 om, b 5 mc(om-mc), and b 5 mc(mc-om). The electrochemical analysis revealed that these proteins are characterized by close reduction potentials ranged from −0.435 V to −0.350 V (vs. Ag/AgCl). Cytochrome b 5 mc stimulated the electrocatalytic activity of CYP3A4.

The role of porcine cytochrome b5A and cytochrome b5B in the regulation of cytochrome P45017A1 activities

Male pigs are routinely castrated to prevent the accumulation of testicular 16-androstene steroids, in particular 5α-androst-16-en-3-one (5α-androstenone), which contribute to an off-odour and off-flavour known as boar taint. Cytochrome P450C17 (CYP17A1) catalyses the key regulatory step in the formation of the 16-androstene steroids from pregnenolone by the andien-β synthase reaction or the synthesis of the glucocorticoid and sex steroids via 17α-hydroxylase and C17,20 lyase pathways respectively. We have expressed CYP17A1, along with cytochrome P450 reductase (POR), cytochrome b5 reductase (CYB5R3) and cytochrome b5 (CYB5) in HEK-293FT cells to investigate the importance of the two forms of porcine CYB5, CYB5A and CYB5B, in both the andien-β synthase as well as the 17α-hydroxylase and C17,20 lyase reactions. Increasing the ratio of CYB5A to CYP17A1 caused a decrease in 17α-hydroxylase ( p < 0.013), a transient increase in C17,20 lyase, and an increase in andien-β synthase activity ( p < 0.0001). Increasing the ratio of CYB5B to CYP17A1 also decreased 17α-hydroxylase, but did not affect the andien-β synthase activity; however, the C17,20 lyase, was significantly increased. These results demonstrate the differential effects of two forms of CYB5 on the three activities of porcine CYP17A1 and show that CYB5B does not stimulate the andien-β synthase activity of CYP17A1.

Two tail-anchored protein variants, differing in transmembrane domain length and intracellular sorting, interact differently with lipids

C-tail-anchored (TA) proteins often require a transmembrane domain of moderate hydrophobicity to maintain their endoplasmic reticulum residence, but the suggested role of protein-lipid interactions in this phenomenon has not been established. Here, we studied the interaction of TA proteins with lipids by differential scanning calorimetry by using a model system consisting of liposomes embedding either of two forms of cytochrome b(5): the endoplasmic reticulum-resident wild-type (b(5)wt) and a mutant thereof (b(5)ext), that contains five extra nonpolar amino acids in its transmembrane domain and, therefore, reaches the plasma membrane. The proteins were incorporated into liposomes of palmitoyl-oleyl-phosphatidylcholine (POPC) or POPC mixed with either distearoyl-phosphatidylserine (DSPS), palmitoyl-oleyl-phosphatidylserine (POPS), distearoyl-phosphatidylcholine (DSPC), or C16-ceramide (CER). POPC liposomes displayed a single thermotropic transition centered at -3.4 degrees C. When present, the second lipid formed a domain within the POPC bilayer, as indicated by the appearance of an additional peak. This peak was centered at temperatures close to 0 degrees C in the case of liposomes containing 10% CER, DSPS, and POPS and at 23 degrees C in the case of DSPC, likely reflecting a higher degree of molecular packing for DSPC domains. In DSPS/POPC, POPS/POPC, or CER/POPC, but not in DSPC/POPC liposomes, the insertion of b(5)wt increased, whereas b(5)ext decreased, the relative contribution to the total enthalpy of the higher temperature, phase-separated component. These results were confirmed with fluorescence measurements by using pyrene-labeled phospholipids. The dissimilar interaction with lipids of these two differently localized TA proteins could have implications for their intracellular sorting.

Site-directed mutagenesis of cytochrome b5 for studies of its interaction with cytochrome P450.

We have shown earlier that microsomal cytochrome b5 can form a specific complex with mitochondrial cytochrome P450 (cytochrome P450scc). The formation of the complex between these two heme proteins was proved spectrophotometrically, by affinity chromatography on immobilized cytochrome b5, and by measuring the cholesterol side-chain cleavage activity of cytochrome P450scc in a reconstituted system in the presence of cytochrome b5. To further study the mechanism of interaction of these heme proteins and evaluate the role of negatively charged amino acid residues Glu42, Glu48, and Asp65 of cytochrome b5, which are located at the site responsible for interaction with electron transfer partners, we used site-directed mutagenesis to replace residues Glu42 and Glu48 with lysine and residue Asp65 with alanine. The resulting mutant forms of cytochrome b5 were expressed in E. coli, and full-length and truncated forms (shortened from the C-terminal sequence due to cleavage of 40 amino acid residues) of these cytochrome b5 mutants were purified. Addition of the truncated forms of cytochrome b5 (which do not contain the hydrophobic C-terminal sequence responsible for interaction with the membrane) to the reconstituted system containing cytochrome P450scc caused practically no stimulation of catalytic activity, indicating an important role of the hydrophobic fragment of cytochrome b5 in its interaction with cytochrome P450scc. However, full-length cytochrome b5 and the full-length Glu48Lys and Asp65Ala mutant forms of cytochrome b5 stimulated the cholesterol side-chain cleavage reaction catalyzed by cytochrome P450scc by 100%, suggesting that residues Glu48 and Asp65 of cytochrome b5 are not directly involved in its interaction with cytochrome P450scc. The replacement of Glu42 for lysine, however, made the Glu42Lys mutant form of cytochrome b5 about 40% less effective in stimulation of the cholesterol side-chain cleavage activity of cytochrome P450scc, indicating that residue Glu42 of cytochrome b5 is involved in electrostatic interactions with cytochrome P450scc. Residues Glu42 and Glu48 of cytochrome b5 appear to participate in electrostatic interaction with microsomal type cytochrome P450.

Purification and characterization of two forms of microsomal cytochrome b5 from sheep lung

Two forms of cytochrome b5 were purified from detergent solubilized sheep lung microsomes by three successive DEAE-cellulose, Sephadex G-100 and Sephadex G-200 column chromatographies. The specific contents of cytochromes b5-I and b5-II were determined to be 45.4 and 43.8 nmol b5/mg protein, which represented up to 567 and 547-fold purification compared with that of the lung microsomes. The most striking difference between b5-I and b5-II was observed in their elution pattern from the third DEAE-cellulose column. Cytochromes gave one protein band with a Mr of 16400±500 on SDS-PAGE. Both forms of reduced b5 showed a major peak at 423 nm while reduced b5-I had two minor peaks at 527 and 556 and reduced b5-II gave two well-defined peaks at 526 and 555 nm. The ability of the purified b5-I and b5-II fractions to transfer the electrons from NADH-cytochrome b5 reductase to cytochrome c was investigated. Apparent K m, 0.055 μM, of b5-II was found to be 38% lower than that of b5-I. In addition, cytochrome b5-I was found to be more sensitive to heat treatment than b5-II when cytochromes were subjected to 62°C for varying periods of time and the coupling of b5 reduction to cytochrome c reduction was determined. These results may indicate that two forms may exist in lung endoplasmic reticulum.

Isolation and characterization of the rabbit cytochrome b5 gene.

In mammals, two forms of cytochrome b5 (b5) are present and they are called according to their subcellar distribution as the microsomal-form and the soluble-form. These two forms of b5 s were encoded by separate mRNAs. In order to elucidate the mechanism of the expression of these two mRNAs, we isolated genomic clones that cover the gene for b5 from the rabbit genomic library. Analysis of these clones revealed that b5 gene spans over 20kbase pairs. A solube-form specific exon was found to be present between the two exons, which are in common to both forms of b5s. These observation demonstrate that the two forms of b5 mRNA are derived from the same gene by the alternative exon skip splicing.

Purification and characterization of two forms of cytochrome b5 from an arachidonic acid-producing fungus, Mortierella hygrophila

Two forms of cytochrome b 5 have been purified from the microsomes of an arachidonic acid-producing fungus, Mortierella hygrophila IFO 5941, after detergent solubilization. They have monometic molecular masses of about 16 kDa and 19 kDa. Their absorption spectra are similar to those of mammalian cytochrome b 5s. Their amino acid compositions show some similarity to those of mammalian cytochrome b 5s, but the contents of some amino acids ( glycine, alanine, aspartic acid + asparagine, glutamic acid + glutamine, arginine, proline, histidine, leucine and lysine) are unique to the cytochrome b 5s of M. hygrophila. Some of their internal peptide sequences also show close homology with those of some mammals (approx. 65 to 67%), while some others show no or little homology. The addition of various acyl-CoAs to NADH-reduced microsomes caused an abrupt shiftdown of the steady state reduction level of cytochrome b 5. This indicates the increased utilization of electrons for the desaturation process and may suggest that the cytochrome b 5s of this fungus actually take part in its microsomel desaturation system for polyunsaturated fatty acid biosynthesis as electron carriers.

CMP‐N‐acetylneuraminic acid hydroxylase from mouse liver and pig submandibular glands

In this report, the nature of the protein components involved in the functioning of cytidine-5'-monophosphate-N-acetylneuraminic acid (CMP-Neu5 Ac) hydroxylase in high-speed supernatants of mouse liver has been investigated. Fractionation and reconstitution experiments showed that this enzyme system consists of NADH-cytochrome b5 reductase, cytochrome b5 and a 56-kDa terminal electron acceptor having the CMP-Neu5 Ac hydroxylase activity. This enzyme system is extracted in a soluble protein fraction; however, the amphipathic, usually membrane-associated, forms of cytochrome b5 and the reductase were found to predominate and are presumably the forms which support the turnover of the hydroxylase in vivo. Although the majority of cellular cytochrome b5 and cytochrome b5 reductase is membrane-bound, the addition of intact microsomes elicited no significant increase in the hydroxylase activity of supernatants. Detergent-solubilised microsomes, however, potently activated the hydroxylase, probably due to the greater accessibility of the cytochrome b5. Accordingly, in reconstitution experiments, pure hydrophilic cytochrome b5 interacts more effectively with the hydroxylase than isolated amphipathic cytochrome b5. Studies on the CMP-Neu5 Ac hydroxylase system in fractionated porcine submandibular glands and bovine liver suggest that the composition of this enzyme system is conserved in all mammals possessing sialoglycoconjugates containing N-glycolylneuraminic acid.

S18.10 Interaction of CMP-Neu5ac hydroxylase with cytosolic and microsomal forms of cytochrome b5 and cytochrome b5 reductase

S18.10 Interaction of CMP-Neu5ac hydroxylase with cytosolic and microsomal forms of cytochrome b5 and cytochrome b5 reductase

The specific subcellular localization of two isoforms of cytochrome b5 suggests novel targeting pathways.

Two forms of cytochrome b5 are present in rat tissues, with a sequence identity of approximately 60% in the cytoplasmically exposed, tryptic fragments (Lederer, F., Ghrir, R., Guiard, B., Cortial, S., and Ito, A. (1983) Eur. J. Biochem. 132, 95-102). It has been suggested that the two isoforms have partially overlapping subcellular distributions, with each form localized to some extent on both endoplasmic reticulum and outer mitochondrial membranes (Ito, A. (1980) J. Biochem. (Tokyo) 87, 73-80). To investigate the degree of specificity of the localization of cytochrome b5 isoforms, we studied their subcellular distributions with antipeptide antibodies, one specific for microsomal cytochrome b5, one specific for outer membrane cytochrome b, and one against a sequence common to the two cytochromes. We first identified outer membrane Cyt b as a tightly bound, Triton X-114-extractable, 23-kDa polypeptide. We then analyzed biochemically characterized rat liver subcellular fractions by Western blotting and found that outer mitochondrial membrane cytochrome b was not present on endoplasmic reticulum membranes. Conversely, microsomal cytochrome b5 was present on outer mitochondrial membranes in extremely low concentration, at a level < 5% of that on endoplasmic reticulum membranes. Thus, the subcellular distribution of microsomal cytochrome b5 is more restricted than previously thought, suggesting that novel posttranslational targeting mechanisms direct it to the endoplasmic reticulum.

Proton linkage of complex formation between cytochrome c and cytochrome b5: electrostatic consequences of protein-protein interactions.

Two potentiometric methods have been used to study the pH-dependent changes in proton binding that accompany complex formation between cytochrome c and cytochrome b5. With one method, the number of protons bound or released upon addition of one cytochrome to the other has been measured as a function of pH. The results from these studies are correlated with the complexation-induced difference titration curve calculated from the titration curves of the preformed complex and of the individual proteins. Both methods demonstrate that complex formation at acid pH is accompanied by proton release, that complex formation at basic pH is accompanied by proton uptake, and that the change in proton binding at neutral pH, where stability of complex formation is maximal, is relatively small. Under all conditions studied, the stoichiometry of cytochrome c-cytochrome b5 complex formation is 1:1 with no evidence of higher order complex formation. Although the dependence of complex formation on pH for interaction between different species of cytochrome c and cytochrome b5 are qualitatively similar, they are quantitatively different. In particular, complex formation between yeast iso-1-cytochrome c and lipase-solubilized bovine cytochrome b5 occurs with a stability constant that is 10-fold greater than observed for the other two pairs of proteins under all conditions studied. Interaction between these two proteins is also significantly less dependent on ionic strength than observed for complexes formed by horse heart cytochrome c with either form of cytochrome b5.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics of flavin semiquinone reduction of the components of the cytochrome c-cytochrome b5 complex.

The kinetics of flavin semiquinone reduction of the components of the 1:1 complex formed by cytochrome c with either cytochrome b5 or a derivative of cytochrome b5 in which the heme propionates are esterified (DME-cytochrome b5) have been studied. The rate constant for the reduction of horse heart cytochrome c by the electrostatically neutral lumiflavin semiquinone (LfH) is unaffected by complexation with native cytochrome b5 at pH 7. However, complex formation with DME-cytochrome b5 (pH 7) decreases by 35% the rate constant for cytochrome c reduction by LfH. At pH 8, complex formation with native cytochrome b5 decreases the rate constant for cytochrome c reduction by LfH markedly, whereas the rate constant for cytochrome c reduction, either unbound or in the complex formed with DME-cytochrome b5, is increased 2-fold relative to pH 7. These results indicate that the accessibility of the cytochrome c heme is not the same in the complexes formed with the two cytochrome b5 derivatives and that the docking geometry of the complex formed by the two native cytochromes is pH dependent. Binding of horse heart and tuna cytochromes c to native and DME-cytochromes b5 decreases the rate constants for reduction of cytochrome c by the negatively charged flavin mononucleotide semiquinone (FMNH) by approximately 30% and approximately 40%, respectively. This finding is attributed to substantial neutralization of the positive electrostatic potential surface of cytochrome c that occurs when it binds to either form of cytochrome b5.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochrome b5 induced flip-flop of phospholipids in sonicated vesicles.

Cytochrome b5 induced flip-flop of phosphatidylethanolamine (PE) in sonicated vesicles prepared from a 9:1 mixture of phosphatidylcholine (PC) to phosphatidylethanolamine was determined as follows. First, vesicles having a nonequilibrium distribution of PE across the bilayer were prepared by amidinating the external amino groups with isethionyl acetimidate. Amidinated cytochrome b5 was then added, and after the protein was completely bound, the rate of appearance of fresh PE on the outer surface was determined by removing aliquots at timed intervals and titrating the external amino groups with trinitrobenzenesulfonic acid. The results show an initial rapid phase of flip-flop (especially in the presence of salt) followed by a very slow phase, at 25 degrees C. Similar results were obtained when cytochrome b5 was introduced into the amidinated vesicles by spontaneous transfer from PC donor vesicles. These results indicate that the accumulation of the transferable ("loose") form of cytochrome b5 on the outer surface of a vesicle causes a transient, global destabilization of the bilayer that is relieved by lipid flip-flop. We speculate that this mechanism may be a significant driving force for the transfer of amphipathic molecules across membranes.