Localization of cytochrome b566 at the outer surface of the inner mitochondrial membrane]

The membrane-impermeable protein reagent p-diazonium benzene sulfonate (DABS) inhibits the mitochondrial respiratory chain in the cytochromes b-c1 region. It has been shown that DABS-treatment brings about a dramatic change in a low-temperature absorption spectrum of rat liver mictochondria, so that the long-wavelength alpha-band of ferrocytochrome b566 is sharply decreased, which is indicative of this hemoprotein modification. This observation suggest that cytochrome b566 is accessible to DABS at the outer surface of the coupling mitochondrial membrane.     

Mitochondrial localization of cytochrome c1 with specific antibodies]

A specific antibody against cytochrome c1 (pig heart mitochondria) has been obtained. It inhibits the electron transport of the respiratory chain in the intact mitochondria at the cytochrome c1 site of inner mitochondrial membrane ; but it has no effect on the isolated submitochondrial particles (inside-out inner mitochondrial membrane vesicles free of any outer membrane or outside-out inner membrane). Thus the topologic position of cytochrome c1 in the inner mitochondrial membrane is asymetrically lcoated on the outer side of the inner mitochondrial membrane. These results agree with our previous researches on ATP-ase and cytochromes b, c and a, indicating the location on the inner side for the first one, transmembranous for the last one, on the outer side for the others respiratory chain components. Thus the electron transport from cytochrome b to a takes place in the outer region of inner mitochondrial membrane and the transmembranous location of cytochrome-oxidase facilitates the transfer of the electrons to oxygen.     

Identification of mosquito bloodmeals using mitochondrial cytochrome oxidase subunit I and cytochrome b gene sequences

Abstract.  Primer pairs were designed and protocols developed to selectively amplify segments of vertebrate mitochondrial cytochrome oxidase subunit 1 (COI) and cytochrome b (Cyt b ) mtDNA from the bloodmeals of mosquitoes (Diptera: Culicidae). The protocols use two pairs of nested COI primers and one pair of Cyt b primers to amplify short segments of DNA. Resultant sequences are then compared with sequences in GenBank, using the BLAST function, for putative host identification. Vertebrate DNA was amplified from 88% of our sample of 162 wild-caught, blood-fed mosquitoes from Oregon, U.S.A. and GenBank BLAST searches putatively identified 98% of the amplified sequences, including one amphibian, seven mammalian and 14 avian species. Criteria and caveats for putative identification of bloodmeals are discussed.     

Localization of lipophosphonoglycan in membranes of Acanthamoeba by using specific antibodies.

Antibodies against two electrophoretically distinct forms of lipophosphonoglycan (LPG) were produced in rabbits. Antibody specificity was demonstrated by the coupled antibody 125I-protein A assay (Adair et al., J. Cell Biol. 79:281-285, 1978). Indirect immunofluorescent labeling of intact Acanthamoeba showed that antibodies to both LPG components had the same uniform distribution on the cell surface. Both antibodies also bound to the cytoplasmic surface of isolated phagosomes. The location of LPG in other membranes of the amoeba was demonstrated on sections by the unlabeled antibody method. Although LPG was absent from the nuclear membrane, virtually all of the internal vacuole membranes were labeled, including the contractile vacuole. Antibodies directed against LPG were utilized to label lipophosphonoglycan in the plasma membrane of living amoebae. Labeled membrane was internalized and then localized by immunofluorescence in cytoplasmic vacuoles within 10 min of incubation. Although these results are evidence for exchange between plasma and cytoplasmic vacuolar membranes, the contractile vacuole remained unlabeled and can be considered, therefore, a separate membrane compartment. Concanavalin A also was bound and internalized by the amoeba, but electron microscopy showed that this label caused pronounced membrane perturbation, limiting its usefulness as a membrane marker in this system.     

Detection and quantification of Pfiesteria piscicida by using the mitochondrial cytochrome b gene

Mitochondrial cytochrome b was isolated from the dinoflagellate Pfiesteria piscicida, and the utility of the gene for species identification was examined. One of the primer sets designed was shown to be highly specific for P. piscicida. A time step PCR protocol was used to demonstrate the potential of this primer set for quantification of this species.     

Properties of bovine heart mitochondrial cytochrome b560

A large-scale preparation of the two-subunit protein complex (QPs) that converts succinate dehydrogenase into succinate-ubiquinone reductase from cytochrome b-c1 particles is achieved by a procedure involving Triton X-100 solubilization and calcium phosphate column chromatography at different pH values. The isolated two-subunit QPs contains 25 nmol of cytochrome b560/mg of protein and is able to reconstitute with soluble succinate dehydrogenase to form a TTFA-sensitive succinate-ubiquinone reductase. The maximum reconstitutive activity is 100 mumol of succinate oxidized per min per mg of QPs protein at 23 degrees C. Although cytochrome b560 in isolated QPs is not succinate reducible and its dithionite reduced form is reactive to carbon monoxide, cytochrome b560 is shown to be physically associated with succinate dehydrogenase by the following observations. The dithionite reduced form of cytochrome b560 in isolated QPs has a symmetrical alpha-absorption peak, which upon reconstitution with succinate dehydrogenase becomes slightly broadened and shows a shoulder at around 553 nm, identical to that of cytochrome b560 in succinate-ubiquinone reductase. Upon addition of succinate dehydrogenase to QPs, about 50% of the reduced form of cytochrome b560 in the QPs becomes insensitive to carbon monoxide treatment. The redox potential of cytochrome b560 in QPs is -144 mV which is higher than that of cytochrome b560 in succinate-ubiquinone reductase (-185 mV). Upon addition of succinate dehydrogenase, the redox potential of about 46% of the cytochrome b560 in QPs preparation becomes identical to that of cytochrome b560 in succinate-ubiquinone reductase. Cytochrome b560 in the QPs preparation shows two epr signals, g = 3.07 and g = 2.92, whereas cytochrome b560 in succinate-ubiquinone reductase exhibits only one epr signal at g = 3.46. When QPs is reconstituted with succinate dehydrogenase to form succinate-ubiquinone reductase, the g = 3.46 epr signal reappears at the expense of the g = 3.07 signal. Based on epr measurement at liquid helium temperature, about 18% of the total cytochrome b in the isolated active succinate-cytochrome c reductase is cytochrome b560, indicating that cytochrome b560 is indeed a unique cytochrome b and not a denatured product of cytochrome b562 or b565.     

Localization of binding sites for carboxyl terminal specific anti-rhodopsin monoclonal antibodies using synthetic peptides

The binding sites for four monoclonal antibodies, rho 1D4, rho 3C2, rho 3A6, and rho 1C5, have been localized within the C-terminal region of bovine rhodopsin: Asp18'-Glu-Ala16'-Ser-Thr-Thr-Val12'-Ser-Lys-Thr-Gl u8'-Thr-Ser-Gln-Val4'-Ala-Pr o -Ala1'. Antibody binding sites were localized by using synthetic C-terminal peptides in conjunction with solid-phase competitive inhibition assays and limited proteolytic digestion of rhodopsin in conjunction with electrophoretic immunoblotting techniques. Binding of the rho 1D4 and rho 3C2 antibodies to immobilized rhodopsin was inhibited with peptides of length 1'-8' and longer. Antibody rho 1D4 binding was not inhibited by peptides 2'-13' or 3'-18', indicating that the C-terminal alanine residue of rhodopsin was required. Similar competitive inhibition studies indicated that the antibody rho 3A6 required peptides of length 1'-12' and longer whereas rho 1C5 required peptide 1'-18'. Peptide 3'-18' was as effective as 1'-18' in inhibiting rho 3A6 binding to rhodopsin, but replacement of glutamic acid in position 8' with glutamine abolished competition. This substitution had little effect on the binding of antibody rho 1C5. Thus, Glu8' was essential for rho 3A6 binding but not for the binding of the rho 1C5 antibody. Cleavage of the seven amino acid C-terminus from rhodopsin and further cleavage to F1 (Mr 25 000) and F2 (Mr 12 000) fragments with Staphylococcus aureus V8 protease abolished binding of rho 1D4 antibody to the membrane-bound rhodopsin fragments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutational analysis of the mouse mitochondrial cytochrome b gene

The protonmotive cytochrome b protein of the mitochondrial bc1 respiratory chain complex contains two reactions centers, designated Qo and Qi, which can be distinguished by the effects of different inhibitors. The nucleotide sequences have been determined of the mitochondrial cytochrome b genes from a series of mouse cell mutants selected for increased inhibitor resistance. Each mutant contains a single nucleotide change which results in an amino acid substitution. When the proximity of the altered amino acid residues to the histidines involved in heme ligation is considered, the results support a model for cytochrome b folding in which there are eight transmembrane domains rather than the nine of the Widger-Saraste model. Replacement of the Gly38 residue by valine results in resistance to the Qi inhibitors antimycin A and funiculosin but not 2-n-heptyl-hydroxyquinoline-N-oxide. Based upon sequence comparisons of mitochondrial and bacterial cytochrome b and chloroplast b6 proteins, the region of the molecule involved in antimycin binding is as highly conserved as those domains involved in heme ligation. It is suggested that the antimycin binding domain of cytochrome b is involved in forming the Qi reaction center. Alterations of the Gly142 and Thr147 residues result in resistance to myxothiazol and stimatellin, respectively. While both inhibitors block the Qo reaction center, the two mutations do not confer cross-resistance to each other. This region of cytochrome b is the most highly conserved during evolution and these inhibitor binding sites probably occur within the protein domain constituting the Qo reaction center. In addition, there is a less conserved region of the protein, defined by the Leu294 residue, which may function in binding the hydrophobic portions of Qo inhibitors.     

Localization of the redox-center of cytochrome b562 on the internal (M-) side of the mitochondrial membrane

In the inside-out submitochondrial particles, cytochrome b-562 is readily reduced by a hydrophilic redox mediator Ru(NH3)6(2)+; this reaction is not inhibited by antimycin and myxothiazol. In mitochondria, cytochromes b do not virtually interact with Ru(NH3)6(2)+. The accessibility of cytochrome b-562 to Ru(NH3)6(2)+ in submitochondrial particles and its inaccessibility in mitochondria suggest the localization of the hemoprotein redox center on the inner surface of the mitochondrial membrane.     

Location of haem-binding sites in the mitochondrial cytochrome b

Amino acid sequences of 6 mitochondrial cytochromes b are analysed to locate the binding sites of the two protohaems. Four invariant histidines are found in two protein segments which span the membrane. In each, two histidines are separated by 13 residues. This would place them on the same sides of alpha-helices, and the protohaems could be sandwiched as bis-imidazole complexes between the two transmembrane segments. In this model the haems are located in different halves of the bilayer; the Fe-Fe distance is about 20 A.     

Targeting of cytochrome b2 into the mitochondrial intermembrane space: specific recognition of the sorting signal.

Cytochrome b2 contains 2-fold targeting information: an amino-terminal signal for targeting to the mitochondrial matrix, followed by a second cleavable sorting signal that functions in directing the precursor into the mitochondrial intermembrane space. The role of the second sorting sequence was analyzed by replacing one, two or all of the three positively charged amino acid residues which are present at the amino-terminal side of the hydrophobic core by uncharged residues or an acidic residue. With a number of these mutant precursor proteins, processing to the mature form was reduced or completely abolished and at the same time targeting to the matrix space occurred. The accumulation in the matrix depended on a high level of intramitochondrial ATP. At low levels of matrix ATP, the mutant proteins were sorted into the intermembrane space like the wild-type precursors. The results: (i) suggest the existence of one or more matrix components that specifically recognize the second sorting signal and thereby trigger the translocation into the intermembrane space; (ii) indicate that the mutant signals have reduced ability to interact with the recognition component(s) and then embark on the default pathway into the matrix by interacting with mitochondrial hsp70 in conjunction with matrix ATP; (iii) strongly argue against a mechanism by which the hydrophobic segment of the sorting sequence stops translocation in the hydrophobic phase of the inner membrane.     

Radiometric assay for direct quantitation of rat liver cytochrome P-450b using monoclonal antibodies

A simple and sensitive assay has been developed that is capable of detecting as little as 0.2 ng of the major isozyme of cytochrome P-450 (P-450b) isolated from the livers of phenobarbital-induced rats. This assay employs monoclonal antibodies generated against cytochrome P-450b to directly quantify the levels of this enzyme in various tissues. Separation of bound from free labeled antibody is achieved by using 6,9-diaminoacridine lactate (Rivanol). The useful range of the assay is between 1 and 100 ng of P-450b.     

Genetic diversity of mitochondrial cytochrome b gene in swamp buffalo

The swamp buffalo (Bubalus carabanensis) is mainly bred for meat, transport and rice cultivation in China and Southeast Asian countries. In the current study, we investigated the genetic diversity, maternal origin and phylogenetic relationship of swamp buffalo by analyzing 1,786 mitochondrial cytochrome b (cytb) sequences from China, Vietnam, Laos, Thailand, India and Bangladesh. Our results indicated that the swamp buffalo can be divided into two major lineages (SA and SB with the sublineages) and three rare lineages (SC, SD and SE), which showed strong geographic differentiation. The SA1 lineage represented a major domestication event, which involved population expansion. Regions III and V showed higher haplotype diversity than the other regions, indicating that the regions of Southwest China and IndoChina are potential domestication centers for the swamp buffalo. In addition, the swamp buffalo showed a closer phylogenetic relationship to tamaraw. In conclusion, our findings revealed a high level of genetic diversity and the phylogenetic pattern of the swamp buffalo.     

从细胞色素b基因序列探讨笛鲷属的分子系统发生关系

测定了9种中国南海的笛鲷属鱼类的细胞色素b基因的部分序列,结合来自GenBank中1种分布于菲律宾和9种分布于美国大西洋的笛鲷属鱼类的相应同源序列,用邻接法和最大简约法构建分子系统树。结果显示:红鳍笛鲷(Lutjanuserythropterus)与红笛鲷(L.sanguineus)之间的同源序列碱基差异百分率只有0.32%,支持二者是同种异名的观点;中国南海的笛鲷属鱼类间的平均碱基差异要高于美国大西洋笛鲷属鱼类。在MP和NJ树中,美国大西洋笛鲷表现为亲缘关系较近,来源于中国南海的笛鲷鱼类相对集中在树的基部,分歧较大。这与所研究的笛鲷地理分布和地理隔离基本相一致,同时也说明中国南海笛鲷分化较早并且分歧较大。     

Mutations in putative intervening sequences of the mitochondrial cytochrome b gene of yeast produce abnormal cytochrome b polypeptides.

The apoprotein of yeast cytochrome b is translated on mitochondrial ribosomes and coded for by a split gene which is located in the COB-BOX region on mitochondrial DNA. With the aid of an antibody against cytochrome b, we identified the cytochrome b-cross-reacting polypeptides of respiration-deficient mutants mapping either in coding or intervening sequences of the cytochrome b gene. Most mutations in the coding regions caused the accumulation of a single apocytochrome b fragment whose apparent molecular weight (12,000 to 26,600) depended on the map position of the mutation. In contrast, mutations in putative intervening sequences often led to multiple new polypeptides immunologically related to apocytochrome b. Some of these abnormal polypeptides were considerably larger than wild type apocytochrome b. This suggests that mutations in intervening sequences can thus generate aberrant polypeptide products.     

Localization of an ascorbate-reducible cytochrome b561 in the plant tonoplast

As a free radical scavenger, and cofactor, ascorbate (ASC) is a key player in the regulation of cellular redox processes. It is involved in responses to biotic and abiotic stresses and in the control of enzyme activities and metabolic reactions. Cytochromes (Cyts) b561 catalyze ASC-driven trans-membrane electron transport and contribute to ASC-mediated redox reactions in subcellular compartments. Putative Cyts b561 have been identified in Arabidopsis (ecotype Columbia) on the basis of sequence similarity to their mammalian counterparts. However, little is known about the function or subcellular localization of this unique class of membrane proteins. We have expressed one of the putative Arabidopsis Cyt b561 genes (CYBASC1) in yeast and we demonstrate that this protein encodes an ASC-reducible b-type Cyt with absorbance characteristics similar to that of other members of this family. Several lines of independent evidence demonstrate that CYBASC1 is localized at the plant tonoplast (TO). Isoform-specific antibodies against CYBASC1 indicate that this protein cosediments with the TO marker on sucrose gradients. Moreover, CYBASC1 is strongly enriched in TO-enriched membrane fractions, and TO fractions contain an ASC-reducible b-type Cyt with alpha-band absorbance maximum near 561 nm. The TO ASC-reducible Cyt has a high specific activity, suggesting that it is a major constituent of this membrane. These results provide evidence for the presence of trans-membrane redox components in this membrane type, and they suggest the coupling of cytoplasmic and vacuolar metabolic reactions through ASC-mediated redox activity.