The X-linked chronic granulomatous disease gene codes for the beta-chain of cytochrome b-245

Chronic granulomatous disease (CGD) is a rare inherited disorder associated with a profound predisposition to infection due to the lack of a microbicidal oxidase system in the phagocytes of these patients. This syndrome is most commonly inherited through a defect on the X chromosome and the only clearly defined component of the oxidase system, the very unusual cytochrome b (b-245), has been shown to be missing from the cells of these patients. This cytochrome is a heterodimer composed of an alpha-chain of relative molecular mass (Mr) 23,000 (23K) and a 76-92K beta-chain; neither are detectable in neutrophils from X-linked CGD subjects. The defective X-CGD gene has recently been cloned by 'reverse genetics' but the protein predicted from the proposed complementary DNA sequence was not identified. We have purified the beta-chain of the cytochrome and sequenced 43 amino acids from the N terminus. Almost complete homology was obtained between this sequence and that of the complementary nucleotides 19-147 of the sequence of the X-CGD gene, originally designated as a non-coding region.     

Association of a Ras-related protein with cytochrome b of human neutrophils

Activation of the superoxide generating system in human neutrophils is thought to involve the interaction or assembly of cytochrome b with other cytosolic and membrane proteins. We have now co-isolated by conventional purification procedures a protein of relative molecular mass 22,000 with cytochrome b. This Ras-related protein is not a fragment of either of the subunits of cytochrome b, and its primary structure, as determined by the sequencing of its complementary DNA, is identical to that predicted from a recently cloned ras-related gene, rap1 (also termed Krev-1). Immunoaffinity purification on anti-cytochrome and anti-Ras immunoaffinity matrices indicates an association between cytochrome b and the Ras-related protein. The association of a Ras-related GTP-binding protein with cytochrome b of human neutrophils could indicate a role for such a protein in the transduction, regulation or structure of the superoxide generating system.     

Identification of the BAL-labile factor

One of us has previously reported that treatment of the Keilin and Hartree heart-muscle preparation with 2,3-dimercaptopropanol (BAL), in the presence of air, leads to the complete inactivation of the succinate oxidase system with little if any effect on the activities of succinate dehydrogenase (until more than half the BAL was oxidized) or cytochrome c oxidase. The inactivation of the complete succinate oxidase system requires the oxidation of BAL by air in the presence of the enzyme. It is not caused by H2O2 or BAL disulphides produced during the oxidation of BAL. Spectroscopic studies identified the block as lying between cytochromes b and c. It was suggested that a BAL-labile factor is present which transfers electrons from cytochrome b to cytochrome c and which is destroyed by coupled oxidation with BAL. The factor is also required for NADH oxidation. Subsequent work showed it is not identical with cytochrome c1 (ref. 4), myoglobin present in the preparation or the antimycin-binding site. We report here that this factor is identical to the iron-sulphur protein in the central portion of the respiratory chain first identified by Rieske.     

Stimulated neutrophils from patients with autosomal recessive chronic granulomatous disease fail to phosphorylate a Mr-44,000 protein

Phagocytosing neutrophils, monocytes, macrophages and eosinophils produce a burst of non-mitochondrial respiration that is important for the killing and digestion of microbes. Much of the information about the oxidase system involved comes from studies on patients with chronic granulomatous disease (CGD), a syndrome in which an undue predisposition to infection results from complete absence of this burst of stimulated respiratory activity. The basis of the oxidase activity is an electron transport chain, the only established component of which is a very unusual b-type cytochrome (b-245) (ref. 2). The molecular defect in the X-linked subgroup of CGD is the absence of this cytochrome b-245, which, however, appears to be normal in those subjects with the autosomal recessive mode of inheritance. In an attempt to identify an abnormality of activation, or an absence or malfunction of a proximal component of the electron transport chain in this latter group, we examined protein phosphorylation in neutrophils after activation of the oxidase with phorbol myristate acetate. All four of the patients studied demonstrated a selective lack of the enhanced phosphorylation of a protein of relative molecular mass (Mr) 44,000 (44K) that was observed in normal subjects and in two CGD patients with an X-linked inheritance. This molecule, therefore, could be an important functional component of the oxidase.     

Influence of site-directed mutation of cytochrome b5 Phe35 on the protein’s structure and properties

Kinetic studies of the heme dissociation from the wild type and Phe35Tyr, Phe35Leu mutants of bovine liver microsomal ferricytochrome b 5 indicate that the oxidized Phe35Tyr mutant is more stable towards denaturant than wild type but Phe35Leu mutant proceeded with a different mechanism compared with wild type cytochrome b 5 and Phe35Tyr mutant protein. Because of the decrease of side chain volume in Phe35Leu mutant, a cavity produced in the interior of the protein may offer a channel for urea molecule to enter the hydrophobic pocket. When urea concentration is larger than 5 mol/L, the urea molecule may compete to coordinate the iron of heme with His39, that results in sharp increase of the rate of heme dissociation. The interaction between cytochrome b 5 and cytochrom c demonstrated that a 1:1 protein complex was formed between the two proteins. The binding constants of cytochrome b 5 with cytochrome c are: wild type K A=4.2(±0.01)×10 6(mol/L) -1 , Phe35Tyr K A=3.7(±0.01)×10 6(mol/L) -1 and Phe35Leu K A=4.7(±0.01)×10 6(mol/L) -1 respectively ( I =1 m mol/L, pH 7.0 soldium phosphate buffer, 25℃). These results clearly show that the mutation at Phe35 has no influence on the binding of cytochrome b 5 with cytochrome c and that the hydrophilic patch residues are not involved in the binding of cytochrome b 5 and cytochrome c.     

Requirement of hormone for thermal conversion of the glucocorticoid receptor to a DNA-binding state

A central question arising from the model of eukaryotic gene regulation by steroid hormone receptors is whether or not proteins represent pre-existing gene regulatory proteins that are activated on exposure to the extracellular signal. It has been generally believed that the ligand-binding of steroid hormone receptors triggers an allosteric change in receptor structure, manifested by an increased affinity of the receptor for DNA in vitro and nuclear target elements in vivo, as monitored by nuclear translocation. But this model has been challenged by recent reports indicating that glucocorticoid and progesterone receptors bind specifically in vitro to target DNA sequences even in the absence of hormone. On the other hand, it appears that the hormone induces protection in vivo of the glucocorticoid response element of the tyrosine amino transferase gene. Here we show that under conditions permitting minimal in vitro manipulation, the steroid-free glucocorticoid receptor in crude cytosol associates with the hsp90 heat shock protein (relative molecular mass Mr approximately equal to 90,000) to form a large 300K complex, rather than the 94K liganded receptor monomer. More importantly, we have developed an assay to demonstrate the requirement of hormone to dissociate the 300K complex by heat treatment. Specific DNA-binding activity of the receptor becomes apparent in this process, showing that DNA binding occurs but is inhibited in the large heteromeric complex. We propose a model in which receptor function is repressed by association of the receptor with hsp90. Dissociation of this complex is induced by the binding of steroid and is apparently an irreversible process.     

A different genetic code in human mitochondria.

Comparison of the human mitochrondial DNA sequence of the cytochrome oxidase subunit II gene and the sequence of the corresponding beef heart protein shows that UGA is used as a tryptophan codon and not as a termination codon and suggests that AUA may be a methionine and not an isoleucine codon. The cytochrome oxidase II gene is contiguous at its 5' end with a tRNAAsp gene and there are only 25 bases at its 3' end before a tRNALys gene. These tRNA'S are different from all other known tRNA sequences.     

Activation of the NADPH oxidase involves the small GTP-binding protein p21rac1

Professional phagocytes, such as neutrophils and monocytes, have an NADPH oxidase that generates superoxide and other reduced oxygen species important in killing microorganisms. Several components of the oxidase complex have been identified as targets of genetic defects causing chronic granulomatous disease. The complex consists of an electron transport chain that has as its substrate cytosolic NADPH and which discharges superoxide into the cavity of the intracellular phagocytic vacuole. The only electron transport component identified so far is a low-potential cytochrome b, apparently the only membrane component required. At least three cytosolic factors are also necessary, two of which, p67phOx and p47phOx, have been identified by their absence in patients with chronic granulomatous disease. A third component, sigma 1, is required for stimulation of oxidase activity in a cell-free system. The active components of purified sigma 1 are two proteins that associate as heterodimers, and here we report that these are the small GTP-binding protein p21rac1 and the GDP-dissociation inhibitor rhoGDI.     

Functional relationship of cytochrome c(6) and plastocyanin in Arabidopsis

Photosynthetic electron carriers are important in converting light energy into chemical energy in green plants. Although protein components in the electron transport chain are largely conserved among plants, algae and prokaryotes, there is thought to be a major difference concerning a soluble protein in the thylakoid lumen. In cyanobacteria and eukaryotic algae, both plastocyanin and cytochrome c(6) mediate electron transfer from cytochrome b(6)f complex to photosystem I. In contrast, only plastocyanin has been found to play the same role in higher plants. It is widely accepted that cytochrome c(6) has been evolutionarily eliminated from higher-plant chloroplasts. Here we report characterization of a cytochrome c(6)-like protein from Arabidopsis (referred to as Atc6). Atc6 is a functional cytochrome c localized in the thylakoid lumen. Electron transport reconstruction assay showed that Atc6 replaced plastocyanin in the photosynthetic electron transport process. Genetic analysis demonstrated that neither plastocyanin nor Atc6 was absolutely essential for Arabidopsis growth and development. However, plants lacking both plastocyanin and Atc6 did not survive.     

暗纹东方鲀线粒体若干tRNA基因的克隆及序列分析

利用分离纯化的暗纹东方鲀肝脏mtDNA为模板，按照红鳍东方纯（Takifugu rubripes）mtDNA序列设计合成特异引物进行PCR扩增，首次克隆并测定了暗纹东方纯mtDNA的Cytb、COⅠ、COⅡ、COⅢ、D-loop等5个重要基因及其侧翼的8个tRNA基因。上述基因均已在GenBank登录。8个tRNA基因含有69-72个碱基。推定了各个tRNA的二级结构并进行了初步的序列分析。结果表明：8个tRNA基因具有较为典型的三叶草型结构，各臂的碱基配对率大多数较高，稳定性较好.     

Characterization of an expressed CD3-associated Ti gamma-chain reveals C gamma domain polymorphism

The majority of human T cells express an antigen receptor consisting of a disulphide-linked heterodimer (Ti) of relative molecular mass 80,000-90,000 (Mr 80-90K) which is noncovalently associated with a set of at least three proteins of Mr 20-28K termed CD3 (Leu4, T3). Whereas both chains of Ti, an acidic alpha-chain of Mr 48-54K and a more basic beta-chain of Mr 40-44K, contain variable and constant region domains, the component peptides of CD3 are invariant. Several laboratories have more recently reported the expression of CD3 in association with a novel protein. On the surface of long-term T-cell lines and one thymocyte clone this novel structure consists of a 40K protein noncovalently linked to a 55 or 62K protein identified as the protein product of the Ti gamma-chain gene, a T-cell specific gene which like the Ti alpha- and Ti beta-chain genes undergoes rearrangement of variable (V) and joining (J) region gene segments. On the human T-cell leukaemic line PEER we have detected only a single 55K glycoprotein associated with CD3. We here demonstrate that an anti-Ti gamma-peptide antiserum reacts with the 55K CD3-associated protein on PEER. Most previously described human Ti gamma-chain complementary DNA clones encode the products of non-functional rearrangements. One of the Ti gamma cDNAs isolated from PEER, however, represents a functional rearrangement reported for the first time in a cell which expresses a Ti gamma-chain protein product on the cell surface. Interestingly, a 48-base-pair (bp) sequence in the constant (C) region domain of this functional Ti gamma-chain cDNA is triplicated in PEER and duplicated in other cDNAs isolated from PEER and other cell lines.     

Structural insights into electron transfer in caa3-type cytochrome oxidase

Cytochrome c oxidase is a member of the haem copper oxidase superfamily (HCO). HCOs function as the terminal enzymes in the respiratory chain of mitochondria and aerobic prokaryotes, coupling molecular oxygen reduction to transmembrane proton pumping. Integral to the enzyme's function is the transfer of electrons from cytochrome c to the oxidase via a transient association of the two proteins. Electron entry and exit are proposed to occur from the same site on cytochrome c. Here we report the crystal structure of the caa3-type cytochrome oxidase from Thermus thermophilus, which has a covalently tethered cytochrome c domain. Crystals were grown in a bicontinuous mesophase using a synthetic short-chain monoacylglycerol as the hosting lipid. From the electron density map, at 2.36?? resolution, a novel integral membrane subunit and a native glycoglycerophospholipid embedded in the complex were identified. Contrary to previous electron transfer mechanisms observed for soluble cytochrome c, the structure reveals the architecture of the electron transfer complex for the fused cupredoxin/cytochrome c domain, which implicates different sites on cytochrome c for electron entry and exit. Support for an alternative to the classical proton gate characteristic of this HCO class is presented.     

Proton-coupled electron transfer drives the proton pump of cytochrome c oxidase

Electron transfer in cell respiration is coupled to proton translocation across mitochondrial and bacterial membranes, which is a primary event of biological energy transduction. The resulting electrochemical proton gradient is used to power energy-requiring reactions, such as ATP synthesis. Cytochrome c oxidase is a key component of the respiratory chain, which harnesses dioxygen as a sink for electrons and links O2 reduction to proton pumping. Electrons from cytochrome c are transferred sequentially to the O2 reduction site of cytochrome c oxidase via two other metal centres, Cu(A) and haem a, and this is coupled to vectorial proton transfer across the membrane by a hitherto unknown mechanism. On the basis of the kinetics of proton uptake and release on the two aqueous sides of the membrane, it was recently suggested that proton pumping by cytochrome c oxidase is not mechanistically coupled to internal electron transfer. Here we have monitored translocation of electrical charge equivalents as well as electron transfer within cytochrome c oxidase in real time. The results show that electron transfer from haem a to the O2 reduction site initiates the proton pump mechanism by being kinetically linked to an internal vectorial proton transfer. This reaction drives the proton pump and occurs before relaxation steps in which protons are taken up from the aqueous space on one side of the membrane and released on the other.     

An embryo protein induced by SV40 virus transformation of mouse cells

A specific protein of molecular weight (MW) approximately 55,000 (55K) was found recently by immunoprecipitation in all SV40 virus-transformed mammalian cells, in addition to the SV40 large T antigen (appoximately 94K) and small antigen (approximately 17K), which are the only proteins coded by the 'early half' of the SV40 genome. The 55K protein is encoded by cellular DNA; its peptide pattern is different from that of the SV40 antigens and it is species specific in mouse, rat, hamster, monkey and human SV40-transformed (or infected) cells. A 55K protein with a similar peptide pattern was found in mouse embryonal carcinoma cells not exposed to SV40. Similar proteins were reported in mouse sarcomas and leukaemias induced by a great variety of aetiological agents and also in a spontaneously transformed mouse fibroblast cell line, and it has been suggested that the protein may be a general correlated of cellular tumorigenicity. We now report that the approximately 55K protein is present in primary cell cultures from 12-14 day old mouse embryos, but not in 16-day old mouse embryos. The embryo protein has a peptide pattern virtually indistinguishable from that of the SV40-induced protein. We also show by comparing closely related cell families that spontaneously transformed highly tumorigenic mouse cells do not possess the 55K protein.     

Mitochondrial cytochrome b sequence variations and phylogeny of the East Asian bagrid catfishes

The mitochondrial DNA cytochrome b gene was sequenced from 8 bagrid catfishes in China. Aligned with cytochrome b sequences from 9 bagrid catfishes in Japan, Korea and Russia retrieved from GenBank, and selected Silurus meridionalis, Liobagrus anguillicauda, Liobagrus reini and Phenacogrammus interruptus as outgroups, we constructed a matrix of 21 DNA sequences. The Kimura's two-parameter distances were calculated and molecular phylogenetic trees were constructed by using the maximum parsimony (MP) and neighbor-joining (NJ) methods. The results show that (i) there exist 3-bp deletions of mitochondrial cytochrome b gene compared with cypriniforms and characiforms; (ii) the molecular phylogenetic tree suggests that bagrid catfishes form a monophyletic group, and the genus Mystus is the earliest divergent in the East Asian bagrid catfishes, as well as the genus Pseudobagrus is a monophyletic group but the genus Pelteobagrus and Leiocassis are complicated; and (iii) the evolution rate of the East Asian bagrids mitochondrial cytochrome b gene is about 0.18%～0.30% sequence divergence per million years.     

Pumping of protons from the mitochondrial matrix by cytochrome oxidase

The stoichiometry and mechanism of redox-linked proton translocation by the mitochondrial respiratory chain is a major issue of debate in membrane bioenergetics. The function of cytochrome oxidase is a focal point of disagreement. In 1977 it was suggested that the terminal component of the respiratory chain, cytochrome oxidase, functions as a redox-linked proton pump. That and subsequent studies were based mainly on measurements of proton ejection from mitochondria or from vesicles reconstituted with isolated cytochrome oxidase, or on measurements of translocation of electrical charge equivalents across mitochondrial and vesicle membranes. This proton-translocating function of cytochrome oxidase is confirmed here by a quantitative determination of proton uptake from the inside (matrix) of intact mitochondria.     

Antiglucocorticosteroid effects suggest why steroid hormone is required for receptors to bind DNA in vivo but not in vitro

Sequence-specific interaction between steroid hormone receptors (R) and DNA hormone-responsive elements (HRE) takes place in vitro irrespective of the presence of hormone and even when R is liganded with an antagonist. In vivo, in contrast, the presence of hormone is mandatory for glucocorticosteroid (G) receptor-HRE interaction to occur and no HRE occupancy is detected in the presence of an antagonist. One possible explanation is that in vivo R is originally complexed with a protein that prevents its binding to target HREs. The hormone would then induce the dissociation of the oligomer, thus unmasking the functional DNA binding domain of the receptor. The unliganded, non DNA-binding 8S-form of the chick GR is a hetero-oligomer including the relative molecular mass (Mr) 94,000 steroid-binding unit (4S-GR), and the non-steroid-binding, non-DNA-binding 90,000 protein common to all classes of 8S-R and identified as heat-shock protein (hsp 90). We report here that triamcinolone acetonide (TA) promotes the transformation of 8S-GR to 4S-GR complexes both in explants and in cell-free conditions and that the high-affinity antiglucocorticosteroid RU 486 stabilizes the 8S-GR, as assessed by gradient sedimentation and HPLC. However, in vitro TA- and RU 486- 4S-GR showed comparable DNA-binding activity. These results suggest that the lack of affinity for DNA of the 8S form of GR may be attributable in vivo to the interaction of the 4S-GR protein with hsp 90, and that hormone binding might trigger a conformational change which results in the release of active 4S-GR.