B5 Molecule Research Articles

The adsorption of cytochromes on a modified surface of gold electrodes

The adsorption of cytochromes b 5 and c on the surface of gold electrodes, including the surface modified with cysteine, was studied. The quartz crystal microbalance method with parallel dissipation energy measurements, microcontact printing, and atomic-force microscopy were used to show that the special features of the structure and morphology of two-component cytochrome b 5 and c films were determined by the nature of the proteins themselves and the influence of the modifying “sublayer.” The largest changes in the weight of films and dissipation energy were observed in the adsorption of cytochrome b 5 on a cytochrome c film deposited on a cysteine sublayer. Atomic-force microscopy measurements showed that strong interaction between cytochrome c and b 5 molecules on the surface of gold modified with cysteine could be related to the formation of the corresponding protein complex.

Chemical modification of Tyr34 and Tyr129 in rabbit liver microsomal cytochrome b5 affects interaction with cytochrome P-450 2B4.

Rabbit liver microsomal cytochrome b5 was allowed to react with tetranitromethane. Up to three tyrosine residues in each cytochrome b5 molecule were found to be accessible to the nitrating agent. Co-modification of tryptophan and histidine residues could be disregarded. CD-spectral measurements disproved gross changes in cytochrome b5 structure as a consequence of derivatization. Introduction of 1.6 nitro groups/polypeptide chain resulted in a fivefold increase in binding affinity for cytochrome P-450 2B4 (P-450 2B4), whereas spectral interaction with cytochrome c remained unaffected. Furthermore, the capacity of nitrated cytochrome b5 to shift the spin equilibrium to the high-spin conformer of P-4502B4 was diminished by 44% compared with the control. This corresponded with the partial disruption of NADH-dependent electron flow to ferric P-450 2B4. Changes in the redox potential of cytochrome b5 could be discounted as being responsible for this effect. The overall oxidative turnover of 4-nitroanisole did not respond to cytochrome b5 modification. MS analysis and sequencing of peptide fragments produced by tryptic digestion of modified cytochrome b5 permitted the detection of three nitrated tyrosine residues located at positions 11, 34 and 129. Derivatization of cytochrome b5 in the presence of a protective amount of P-450 2B4 provided evidence of the involvement of Tyr34 and Tyr129 in complexation of the two hemoproteins. It is proposed that Tyr129 might control docking of cytochrome b5 to P-450 2B4, whereas Tyr34 could be of functional importance in electron transfer.

Fluorescence study of a temperature-induced conversion from the "loose" to the "tight" binding form of membrane-bound cytochrome b5.

Cytochrome b5 is a liver integral membrane protein that has now been expressed in, and isolated from, Escherichia coli. The structure-function relationships of the 43 amino acid membrane-binding domain (nonpolar peptide) have been examined in both native and mutant forms of the protein; in the latter, tryptophan residues at positions 108 and 112 were replaced by leucine. The temperature dependence of the fluorescence quantum yield of the Trp residues in the isolated membrane-binding domain was examined while the domain was bound to lipid vesicles. Both the lipid-bound mutant domain and lipid-bound native domain showed an irreversible increase in fluorescence above 50 degrees C. When the whole cytochrome b5 molecule, bound to lipid vesicles, was heated to this temperature, there was a conversion of the metastable, intermembrane-exchangeable ("loosely" bound), conformation to a final, virtually unexchangeable ("tightly" bound), conformation. It has been suggested previously that the protein exists in a "looped back" conformation and a "bilayer penetrating" conformation. Although the present studies are not designed to determine the absolute conformations of the loose and tight forms, the changes observed in steady-state and frequency-modulated fluorescence and the lack of change in depth of Trp 109 in the bilayer are consistent with a movement of the C-terminal segment from a looped back to a bilayer penetrating conformation as the tight form is generated.

Expression of cytochrome b5 in yeast and characterization of mutants of the membrane-anchoring domain.

The soluble and membrane-bound forms of the synthetic rat cytochrome b5 gene have been expressed in Saccharomyces cerevisiae. In order to examine the topology and function of the COOH-terminal membrane binding domain of cytochrome b5, mutants have been constructed, expressed, purified, and partially characterized. Pro-115 is located in the middle of the putative alpha-helical membrane-anchoring domain of cytochrome b5 and has been hypothesized to give rise to either a hairpin-like loop or approximately equal to 26 degrees kink in the helix, depending on whether it exists, respectively, in the cis or trans configuration. The Pro-115----Ala mutant, which is expected to have a straight transmembrane helix, inserted normally into the endoplasmic reticulum and exhibited wild type levels of activity in yeast microsomes and in vitro in the cytochrome P-450 mixed function oxidation system. Since a hairpin structure does not appear to be essential, it is likely that the membrane binding domain of cytochrome b5 spans the membrane. Characterization of the truncated cytochrome b5 molecule, Pro-115----Stop, lacking 19 amino acids at the COOH terminus indicates that the distal part of the membrane binding domain of cytochrome b5 is necessary for in vivo binding to the endoplasmic reticulum and for functioning with its membrane-associated electron transfer partners. Replacement of Ser-104 to Met-125, the putative membrane-anchoring domain of cytochrome b5, with 22 leucine residues results in a protein which targets to the endoplasmic reticulum but the extent of its reduction is only 50% of that of the wild type in yeast microsomes. In vitro, the polyleucine mutant is unable to support substrate oxidation by cytochrome P-450. The mutation of Ala-131 and Glu-132, amino acids flanking the transmembrane domain, to lysines resulted in a protein with normal membrane topology and function.

Restriction of Epstein-Barr virus-specific cytotoxic T cells by HLA-A, -B, and -C molecules

HLA-loss variants of an Epstein-Barr virus-transformed B-lymphoblastoid cell line (EBV-LCL) 721 were used as target cells to identify HLA molecules utilized by EBV-LCL-specific cytotoxic T cells. Split culture analysis of cytotoxic T cells plated at limiting dilution showed killing of HLA-loss variants bearing either HLA-A2 or -B5 molecules, with 10 times higher frequency of cytotoxic T cells restricted by the HLA-B5 molecule. Clonal analysis confirmed the restriction by HLA-A2 or -B5 of some cytotoxic T-cell clones and identified cytotoxic T-cell clones cytolytic for target cells which do not express HLA-A or -B but do express the HLA-C determinant. Thus, our results show immunodominance of the HLA-B5 restriction determinant for EBV-induced antigens in the donor of the HLA-loss variants and provide evidence that the HLA-C molecule can also serve as restriction determinant for EBV-LCL-specific cytotoxic T cells.

The identification of the heat-stable microsomal protein required for methoxyflurane metabolism as cytochrome b5.

Methoxyflurane is an anesthetic whose metabolism by cytochrome P-450LM2 has been shown to be dependent upon a heat-stable microsomal protein (Canova-Davis, E., and Waskell, L. A. (1982) Biochem. Biophys. Res. Commun. 108, 1264-1270). Treatment of this protein with diethylpyrocarbonate, which modifies selected amino acids, caused a dose-dependent loss in its ability to effect the metabolism of methoxyflurane by purified cytochrome P-450LM2. This protein factor has been identified as cytochrome b5 by demonstrating that cytochrome b5 and the heat-stable factor coelute during cytochrome b5 purification. Neither ferriheme nor apocytochrome b5 was able to substitute for the activating factor, while cytochrome b5 reconstituted from apocytochrome b5 and heme exhibited an activity similar to that of native b5. Examination of the cytochrome b5 molecule by computer graphics suggested that diethylpyrocarbonate did not inactivate b5 by reacting with the anionic surface of the cytochrome b5 molecule. Maximal rates of methoxyflurane metabolism were obtained at a ratio of 1:1:1 of the three proteins, cytochrome P-450LM2:reductase:cytochrome b5. In summary, it has been demonstrated that the heat-stable protein, cytochrome b5, is obligatory for the metabolism of methoxyflurane by cytochrome P-450LM2. These data also suggest that cytochrome b5 may be acting as an electron donor to P-450LM2 in the O-demethylation of methoxyflurane.

Calorimetric and fluorescence characterization of interactions between cytochrome b5 and phosphatidylcholine bilayers.

The interactions of cytochrome b5 with dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine lipid bilayers have been studied with high-sensitivity differential scanning calorimetry and fluorescence spectroscopy. The incorporation of cytochrome b5 into large single lamellar vesicles causes a reduction in the enthalpy change associated with the lipid phase transition. Analysis of the dependence of this enthalpy change on the protein/lipid molar ratio indicates that each cytochrome b5 molecule prevents 14 +/- 1 lipid molecules from participating in the gel to liquid-crystalline transition and that this number is independent of the phospholipid acyl chain length. Resonance energy transfer between the intrinsic tryptophan fluorescence of cytochrome b5 and pyrenedecanoic acid indicates that, in the liquid-crystalline phase, protein and lipid molecules are uniformly distributed within the bilayer plane. In the gel phase, pyrenedecanoic acid partitions into the boundary layer lipid causing a dramatic decrease in the fluorescence intensity of cytochrome b5. The excimer/monomer ratios of pyrenedecanoic acid decrease upon increasing the protein/lipid molar ratio, indicating that the presence of protein molecules within the bilayer slows down the lateral mobility of the lipid probes. The picture that emerges from this set of experiments is that cytochrome b5 perturbs one layer of lipid around the hydrophobic segment of the protein and that this layer is unable to undergo the gel-liquid-crystalline transition, remaining instead in a relatively disordered configuration above and below the transition temperature of the bulk lipid.

The nonpolar peptide segment of cytochrome b5. Binding to phospholipid vesicles and identification of the fluorescent tryptophanyl residue.

The nonpolar peptide segment of cytochrome b5 consisting of the COOH-terminal 43 amino acid residues binds rapidly to dimyristyl lecithin vesicles above the transition temperature of the phospholipid. The binding is complete as indicated by density gradient centrifugation and is accompanied by approximately a 2-fold increase in the fluorescence emission of the protein, and insertion in the bilayer is apparently in an orientation indistinguishable from that of the whole cytochrome b5 molecule. Quantitative reaction of the protein with N-bromosuccinimide destroys the fluorescence of the protein with a stoichiometry which indicates that a single tryptophanyl residue is responsbile for the fluorescence. The fluorescent tryptophanyl residue is one of 2 adjacent residues, Trp-108 or Trp-109, as shown by carboxypeptidase digestion of N-bromosuccinimide-reacted nonpolar peptide.

Endogenous Synthesis of Membrane b5 by Lymphocytes from Rabbits Recovering from b5 Allotype Suppression

Abstract In the rabbit, production of b-locus allotypic determinants present on kappa-type light chains can be suppressed by perinatal exposure to anti-b5 antisera (allotype suppression) (1, 2). We have previously demonstrated that b5-bearing lymphocytes are deleted in totally suppressed rabbits with no circulating b5, and that in spontaneous escape from suppression the appearance of b5-bearing lymphocytes immediately precedes the appearance of detectable serum b5 (3). However, during recovery from allotype suppression, the proportion of peripheral blood lymphocytes with membrane b5 approaches normal levels, while circulating b5 levels remain chronically and disproportionately depressed (3). Because there is this unusual discrepancy between the number of b5-bearing lymphocytes and the amount of circulating b5 in rabbits recovering from allotype suppression, and because passive adsorption of circulating b5 molecules to lymphocytes not themselves capable of synthesizing b5 could account for the inappropriately large number of b5-bearing cells, we have investigated these lymphocytes for their ability to synthesize actively their own membrane b5 Ig after enzymatic stripping of the cell surface.

Selective simulation of allelic expression: effectf antibodies to allotypic markers on lymphoid cells.

Peripheral blood leukocytes from rabbits which were heterozygous (b(5)/b(9)) for markers on their immunoglobulin light chains were maintained in vitro for up to 24 hours in the presence or absence of antibody to b9. After culture they were transferred into lethally irradiated b(4)/b(4)hosts. Recipients of cells exposed to antibodies to allotype markers showed a striking increase in concentration of circulating b9 molecules and number of b9 plasma cells in their spleens compared pared to control animals receiving untreated cells from the same donor. There was no appreciable difyerence between the two groups of recipients with respect to their content of b5 molecules and immunocytes.

Distribution of Allelic Allotypes among γG-Immunoglobulins and Purified Anti-Hapten Antibodies from the Same Rabbit

Summary Antibodies were prepared to the two haptenic groups 2,4-dinitrophenyl (DNP) and p-azobenzenearsonate (R-azo) in 17 rabbits heterozygous at either or both the a or b allotypic specificity loci. By qualitative Ouchterlony test, every purified antibody preparation contained the same allotypic specificities as that of the immune serum from which it had been isolated. By quantitative Oudin analysis in agar gel tubes, the quantitative variation of allotypes among the antibody preparations of individual rabbits was considerable. Among five b4/b5 heterozygous rabbits, the concentration of b4 and b5 molecules in the purified anti-DNP population was not consistently different from that in the bulk γG-immunoglobulins from which they were isolated (P > 0.20). On the other hand, among the same five b4:b5 heterozygotes as well as two more, the concentration of b4 molecules was consistently greater (P < 0.05) and of b5 molecules consistently less (P < 0.01) in the purified anti-R-azo population than in the bulk γG-immunoglobulins from which they were isolated. Furthermore, among four a1/a3 heterozygous rabbits, the concentration of a1 molecules was consistently less (P < 0.01) and of a3 molecules consistently more (P < 0.09) in the anti-DNP population than in the bulk γG-immunoglobulins from which they were isolated. While these results suggest that antibodies of a given specificity are selectively formed from certain allotypes, confidence in the biologic significance of the present results must be restrained until additional experimental conditions outlined in this article are satisfied.