Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 46
Filtrar
1.
J Membr Biol ; 248(3): 431-42, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25837994

RESUMO

Staphylococcus epidermidis has emerged as one of the major nosocomial pathogens associated with infections of implanted medical devices. The most important factor in the pathogenesis of these infections is the formation of bacterial biofilms. Bacteria grown in biofilms are more resistant to antibiotics and to the immune defence system than planktonic bacteria. In these infections, the antimicrobial therapy usually fails and the removal of the biofilm-coated implanted device is the only effective solution. In this study, three proteomic approaches were performed to investigate membrane proteins associated to biofilm formation: (i) sample fractionation by gel electrophoresis, followed by isotopic labelling and LC-MS/MS analysis, (ii) in-solution sample preparation, followed by isotopic labelling and LC-MS/MS analysis and (iii) in-solution sample preparation and label-free LC-MS/MS analysis. We found that the commensal strain S. epidermidis CECT 231 grown in biofilms expressed higher levels of five membrane and membrane-associated proteins involved in pathogenesis: accumulation-associated protein, staphylococcal secretory antigen, signal transduction protein TRAP, ribonuclease Y and phenol soluble modulin beta 1 when compared with bacteria grown under planktonic conditions. These results indicate that a commensal strain can acquire a pathogenic phenotype depending on the mode of growth.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Biofilmes , Staphylococcus epidermidis/fisiologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/metabolismo , Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Espectrometria de Massas em Tandem , Regulação para Cima , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
2.
Nucleic Acids Res ; 40(13): 6109-21, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22453275

RESUMO

Mitochondrial ribosomes and translation factors co-purify with mitochondrial nucleoids of human cells, based on affinity protein purification of tagged mitochondrial DNA binding proteins. Among the most frequently identified proteins were ATAD3 and prohibitin, which have been identified previously as nucleoid components, using a variety of methods. Both proteins are demonstrated to be required for mitochondrial protein synthesis in human cultured cells, and the major binding partner of ATAD3 is the mitochondrial ribosome. Altered ATAD3 expression also perturbs mtDNA maintenance and replication. These findings suggest an intimate association between nucleoids and the machinery of protein synthesis in mitochondria. ATAD3 and prohibitin are tightly associated with the mitochondrial membranes and so we propose that they support nucleic acid complexes at the inner membrane of the mitochondrion.


Assuntos
Mitocôndrias/genética , Proteínas Mitocondriais/biossíntese , Nucleoproteínas/fisiologia , Biossíntese de Proteínas , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/fisiologia , Linhagem Celular Tumoral , DNA Mitocondrial/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/fisiologia , Proteínas Nucleares/fisiologia , Proibitinas , RNA/análise , RNA/isolamento & purificação , RNA Mensageiro/análise , RNA Mitocondrial , Proteínas Repressoras/fisiologia , Ribossomos/metabolismo
3.
Nucleic Acids Res ; 40(13): 6097-108, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22447445

RESUMO

The bacterial homologue of C4orf14, YqeH, has been linked to assembly of the small ribosomal subunit. Here, recombinant C4orf14 isolated from human cells, co-purified with the small, 28S subunit of the mitochondrial ribosome and the endogenous protein co-fractionated with the 28S subunit in sucrose gradients. Gene silencing of C4orf14 specifically affected components of the small subunit, leading to decreased protein synthesis in the organelle. The GTPase of C4orf14 was critical to its interaction with the 28S subunit, as was GTP. Therefore, we propose that C4orf14, with bound GTP, binds to components of the 28S subunit facilitating its assembly, and GTP hydrolysis acts as the release mechanism. C4orf14 was also found to be associated with human mitochondrial nucleoids, and C4orf14 gene silencing caused mitochondrial DNA depletion. In vitro C4orf14 is capable of binding to DNA. The association of C4orf14 with mitochondrial translation factors and the mitochondrial nucleoid suggests that the 28S subunit is assembled at the mitochondrial nucleoid, enabling the direct transfer of messenger RNA from the nucleoid to the ribosome in the organelle.


Assuntos
DNA Mitocondrial/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Mitocôndrias/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Linhagem Celular Tumoral , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/isolamento & purificação , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos
4.
Nucleic Acids Res ; 39(12): 5098-108, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21398640

RESUMO

Mitochondrial DNA maintenance and segregation are dependent on the actin cytoskeleton in budding yeast. We found two cytoskeletal proteins among six proteins tightly associated with rat liver mitochondrial DNA: non-muscle myosin heavy chain IIA and ß-actin. In human cells, transient gene silencing of MYH9 (encoding non-muscle myosin heavy chain IIA), or the closely related MYH10 gene (encoding non-muscle myosin heavy chain IIB), altered the topology and increased the copy number of mitochondrial DNA; and the latter effect was enhanced when both genes were targeted simultaneously. In contrast, genetic ablation of non-muscle myosin IIB was associated with a 60% decrease in mitochondrial DNA copy number in mouse embryonic fibroblasts, compared to control cells. Gene silencing of ß-actin also affected mitochondrial DNA copy number and organization. Protease-protection experiments and iodixanol gradient analysis suggest some ß-actin and non-muscle myosin heavy chain IIA reside within human mitochondria and confirm that they are associated with mitochondrial DNA. Collectively, these results strongly implicate the actomyosin cytoskeleton in mammalian mitochondrial DNA maintenance.


Assuntos
Actinas/fisiologia , DNA Mitocondrial/metabolismo , Cadeias Pesadas de Miosina/fisiologia , Miosina não Muscular Tipo IIA/fisiologia , Miosina não Muscular Tipo IIB/fisiologia , Actinas/análise , Actinas/antagonistas & inibidores , Animais , Células Cultivadas , DNA Mitocondrial/química , DNA Mitocondrial/isolamento & purificação , Inativação Gênica , Humanos , Camundongos , Mitocôndrias/química , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/isolamento & purificação , Cadeias Pesadas de Miosina/antagonistas & inibidores , Miosina não Muscular Tipo IIA/análise , Miosina não Muscular Tipo IIA/antagonistas & inibidores , Miosina não Muscular Tipo IIB/antagonistas & inibidores , Ratos
5.
J Mol Biol ; 242(4): 408-21, 1994 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-7932700

RESUMO

Four subunits of the F1F0-ATPase from bovine heart mitochondria have been produced by heterologous over-expression in Escherichia coli. They are the oligomycin sensitivity conferral protein (OSCP), coupling factor 6 (F6) and subunits b and d. Likewise, fragments b', bI, bC, and bM (amino acid residues 79 to 214, 121 to 214, 165 to 214 and 79 to 164, respectively, of subunit b), and fragment d' (subunit d lacking residue 1 to 14) have been produced in abundant quantities by bacterial expression. These subunits, and the fragments of subunits b and d, have been assayed singly and in various combinations by gel-filtration chromatography for their abilities to bind to bovine heart F1-ATPase. Only the OSCP was found to be capable of forming a stable binary complex with F1-ATPase. When fragments b', bI or bC were added to F1-ATPase together with the OSCP, the ternary complexes F1.OSCP.b', F1.OSCP.bI or F1.OSCP.bC were formed, but b', bI and bC appeared to be present in sub-stoichiometric amounts. When F6 was added also, then the stoichiometric quaternary complexes F1.OSCP.b'.F6 and F1.OSCP.bI.F6 were obtained, as was a fourth quaternary complex containing approximately equivalent amounts of F1 and OSCP, and sub-stoichiometric quantities of bC and F6. Finally, three pentameric complexes F1.OSCP.b'.F6.d, F1.OSCP.b'.F6.d' and F1.OSCP.b.F6.d were isolated. In a further series of reconstitution experiments, the binary complexes b'.OSCP and b'.d, the ternary complex b'.d'.F6, and the quaternary complex OSCP.b'.F6.d were obtained. The pre-formed quaternary complex produced a stoichiometric pentameric complex with F1-ATPase. It was shown by S-carboxymethylation of cysteine residues with iodo-[2-14C]acetic acid that bovine F1F0-ATPase and the reconstituted F1.stalk complex, F1.OSCP.b'.d.F6, each contained one copy per complex of subunits b (or b'), OSCP and d, and that the separate stalk complex contained the same three subunits in the approximate molar ratio 1:1:1. The ratio of b to d in purified F0 was 1:1. Finally, it was demonstrated that the binding of the various subunits to F1-ATPase increases the ATP hydrolase activity and diminishes its inactivation by exposure to cold. These assembly experiments help to define some of the inter-subunit interactions in the stalk region of the F1F0-ATPase complex, and they are an essential step forward towards the goal of extending the high-resolution structure of bovine F1-ATPase into the stalk.


Assuntos
Mitocôndrias Cardíacas/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Clonagem Molecular , Primers do DNA , Escherichia coli/genética , Dados de Sequência Molecular , ATPases Translocadoras de Prótons/genética
6.
J Mol Biol ; 226(4): 1051-72, 1992 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-1518044

RESUMO

NADH:ubiquinone oxidoreductase, the first enzyme in the respiratory electron transport chain of mitochondria, is a membrane-bound multi-subunit assembly, and the bovine heart enzyme is now known to contain about 40 different polypeptides. Seven of them are encoded in the mitochondrial DNA; the remainder are the products of nuclear genes and are imported into the organelle. The primary structures of 12 of the nuclear coded subunits have been described and those of a further 20 are described here. The subunits have been sequenced by following a strategy based on the polymerase chain reaction. This strategy has been tailored from existing methods with the twofold aim of avoiding the use of cDNA libraries, and of obtaining a cDNA sequence rapidly with minimal knowledge of protein sequence, such as can be determined in a single N-terminal sequence experiment on a polypeptide spot on a two-dimensional gel. The utility and speed of this strategy have been demonstrated by sequencing cDNAs encoding 32 nuclear-coded-membrane associated proteins found in bovine heart mitochondria, and the procedures employed are illustrated with reference to the cDNA sequence of the 20 subunits of NADH:ubiquinone oxidoreductase that are presented. Extensive use has also been made of electrospray mass spectrometry to measure molecular masses of the purified subunits. This has corroborated the protein sequences of subunits with unmodified N terminals, and their measured molecular masses agree closely with those calculated from the protein sequences. Nine of the subunits, B8, B9, B12, B13, B14, B15, B17, B18 and B22 have modified alpha-amino groups. The measured molecular masses of subunits B8, B13, B14 and B17 are consistent with the post-translational removal of the initiator methionine and N-acetylation of the adjacent amino acid. The initiator methionine of subunit B18 has been removed and the N-terminal glycine modified by myristoylation. Subunits B9 and B12 appear to have N-terminal and other modifications of a hitherto unknown nature. The sequences of the subunits of bovine complex I provide important clues about the location of iron-sulphur clusters and substrate and cofactor binding sites, and give valuable information about the topology of the complex. No function has been ascribed to many of the subunits, but some of the sequences indicate the presence of hitherto unsuspected biochemical functions. Most notably the identification of an acyl carrier protein in both the bovine and Neurospora crassa complexes provides evidence that part of the complex may play a role in fatty acid biosynthesis in the organelle, possibly in the formation of cardiolipin.(ABSTRACT TRUNCATED AT 400 WORDS)


Assuntos
Sequência de Aminoácidos , Mitocôndrias Cardíacas/enzimologia , NAD(P)H Desidrogenase (Quinona)/química , Reação em Cadeia da Polimerase/métodos , Animais , Sequência de Bases , Transporte Biológico , Bovinos , Núcleo Celular , Clonagem Molecular/métodos , DNA de Cadeia Simples/genética , Transporte de Elétrons/fisiologia , Espectrometria de Massas/métodos , Proteínas de Membrana/genética , Dados de Sequência Molecular , NAD(P)H Desidrogenase (Quinona)/genética , Conformação Proteica , Homologia de Sequência do Ácido Nucleico
7.
J Mol Biol ; 260(2): 251-60, 1996 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-8764404

RESUMO

The major sperm protein (MSP) of Ascaris suum mediates amoeboid motility by forming an extensive intermeshed system of cytoskeletal filaments analogous to that formed by actin in many amoeboid cells. We have used a combination of biochemical and NMR methods to show that, in contrast to actin, MSP exist in solution as a symmetrical dimer. This result has important implications for the mechanism of both MSP filament assembly and the recognition of different MSP isoforms in vivo.


Assuntos
Ascaris suum/química , Proteínas de Helminto/química , Conformação Proteica , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Animais , Cromatografia em Gel , Escherichia coli/genética , Espectroscopia de Ressonância Magnética , Masculino , Espectrometria de Massas , Peso Molecular , Dobramento de Proteína , Proteínas Recombinantes/química , Ultracentrifugação
8.
J Mol Biol ; 184(4): 677-701, 1985 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-2864455

RESUMO

The enzyme complex F1-ATPase has been isolated from bovine heart mitochondria by gel filtration of the enzyme released by chloroform from sub-mitochondrial particles. The five individual subunits alpha, beta, gamma, delta and epsilon that comprise the complex have been purified from it, and their amino acid sequences determined almost entirely by direct protein sequence analysis. A single overlap in the gamma-subunit was obtained by DNA sequence analysis of a complementary DNA clone isolated from a bovine cDNA library using a mixture of 32 oligonucleotides as the hybridization probe. The alpha, beta, gamma, delta and epsilon subunits contain 509, 480, 272, 146 and 50 amino acids, respectively. Two half cystine residues are present in the alpha-subunit and one in each of the gamma- and epsilon-chains; they are absent from the beta- and delta-subunits. The stoichiometry of subunits in the complex is estimated to be alpha 3 beta 3 gamma 1 delta 1 epsilon 1 and the molecular weight of the complex is 371,135. Mild trypsinolysis of the F1-ATPase complex, which has little effect on the hydrolytic activity of the enzyme, releases peptides from the N-terminal regions of the alpha- and beta-chains only; the C-terminal regions are unaffected. Sequence analysis of the released peptides demonstrates that the N terminals of the alpha- and beta-chains are ragged. In 65% of alpha-chains, the terminus is pyrrolidone carboxylic acid; in the remainder this residue is absent and the chains commence at residue 2, i.e. lysine. In the beta-subunit a minority of chains (16%) have N-terminal glutamine, or its deamidation product, glutamic acid (6%), or the cyclized derivative, pyrrolidone carboxylic acid (5%). A further 28% commence at residue 2, alanine, and 45% at residue 3, serine. The delta-chains also are heterogeneous; in 50% of chains the N-terminal alanine residue is absent. The sequences of the alpha- and beta-chains show that they are weakly homologous, as they are in bacterial F1-ATPases. The sequence of the bovine delta-subunit of F1-ATPase shows that it is the counterpart of the bacterial epsilon-subunit. The bovine epsilon-subunit is not related to any known bacterial or chloroplast H+-ATPase subunit, nor to any other known sequence. The counterpart of the bacterial delta-subunit is bovine oligomycin sensitivity conferral protein, which helps to bind F1 to the inner mitochondrial membrane.(ABSTRACT TRUNCATED AT 400 WORDS)


Assuntos
Mitocôndrias/enzimologia , ATPases Translocadoras de Prótons , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Bovinos , Eletroforese em Gel de Poliacrilamida , Escherichia coli/enzimologia , Focalização Isoelétrica , Substâncias Macromoleculares , Peso Molecular , ATPases Translocadoras de Prótons/isolamento & purificação , ATPases Translocadoras de Prótons/metabolismo , Compostos de Sulfidrila/análise
9.
Mech Dev ; 43(1): 21-36, 1993 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-8240969

RESUMO

The Drosophila position-specific integrins (PS integrins or PS antigens) comprise two heterodimeric complexes, alpha PS1 beta PS and alpha PS2 beta PS. With the cloning of alpha PS1 described here, we complete the characterization of the primary structure of the three PS integrin subunits. We have purified the alpha PS1 subunit, obtained peptide sequence and isolated genomic and cDNA clones. The encoded alpha PS1 protein contains the cysteine pattern of the cleaved alpha integrins, three putative metal binding domains and shows the other characteristic features of alpha integrins. Regions of sequence variation indicate that alpha PS1 is distinct from all other alpha chains. The transcript analysis shows that the patterns of both alpha PS1 mRNA and protein expression are the same, suggesting that the gene is controlled transcriptionally. We compare the gene structures of the Drosophila alpha PS1, alpha PS2, the human alpha IIb and alpha X (p150,95) and the C. elegans F54G8.3 integrins. We find several positions and phases of introns conserved which, supported by conservation also in the amino acid sequence, indicates that they all derive from a common ancestral gene.


Assuntos
Proteínas de Drosophila , Drosophila melanogaster/metabolismo , Genes de Insetos , Integrinas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Caenorhabditis elegans/genética , Clonagem Molecular , DNA Complementar/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Embrião não Mamífero/metabolismo , Embrião não Mamífero/ultraestrutura , Proteínas de Helminto/química , Humanos , Cadeias alfa de Integrinas , Integrinas/isolamento & purificação , Dados de Sequência Molecular , Morfogênese/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie
10.
FEBS Lett ; 438(3): 301-5, 1998 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-9827566

RESUMO

The sequences of 41 subunits of complex I (NADH:ubiquinone oxidoreductase) from bovine heart mitochondria have been described previously. Seven of them are encoded in mitochondrial DNA, and the remainder are nuclear gene products that are imported into the organelle from the cytoplasm. By electrospray mass spectrometry experiments conducted on complex I and on two related subcomplexes, an additional protein has been identified with a mass not corresponding to any of the known subunits of the enzyme. This protein has also been found in samples of the enzyme fractionated on two dimensional polyacrylamide gels. Material from these gels has been digested with trypsin and peptide sequences have been determined, confirming that the protein did not correspond to any of the known subunits of complex I. The cDNA sequence of this protein, determined with the aid of the peptide sequences, demonstrates that it is a novel subunit of complex I, and that it is related to a 13-kDa human protein associated with differentiation.


Assuntos
Mitocôndrias Cardíacas/enzimologia , NADH NADPH Oxirredutases/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , DNA Mitocondrial/genética , Complexo I de Transporte de Elétrons , Humanos , Substâncias Macromoleculares , Dados de Sequência Molecular , NADH NADPH Oxirredutases/genética , Reação em Cadeia da Polimerase , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
11.
FEBS Lett ; 286(1-2): 121-4, 1991 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-1907568

RESUMO

The amino-acid sequence of a subunit of NADH:ubiquinone oxidoreductase from bovine heart mitochondria has been determined and is closely related to those of acyl carrier proteins that are involved in fatty acid biosynthesis in Escherichia coli and plants. Evidence for the presence of covalently attached pantetheine-4'-phosphate in the bovine protein has been obtained by determination of the molecular mass of the isolated subunit by electrospray mass spectrometry, before and after incubation of the protein at alkaline pH under reducing conditions. This decreased the molecular mass from 10,751.6 to 10,449.4, a difference of 302.2 mass units; the value calculated from the protein sequence with one covalently attached pantetheine-4'-phosphate is 10,449.8. The acyl group which is removed by alkaline reduction, appears to be attached via a thioester linkage. By analogy with the bacterial protein it is likely that the attachment site of the pantetheine-4-phosphate is serine-44, which is found in a highly conserved region of the sequence. At present the function of the acyl carrier protein in mitochondrial complex I is not understood.


Assuntos
Proteína de Transporte de Acila/genética , Mitocôndrias Cardíacas/enzimologia , Quinona Redutases/genética , Proteína de Transporte de Acila/química , Proteína de Transporte de Acila/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , DNA , Dados de Sequência Molecular , NAD(P)H Desidrogenase (Quinona) , Panteteína/análogos & derivados , Panteteína/metabolismo , Quinona Redutases/química , Quinona Redutases/metabolismo , Alinhamento de Sequência
12.
FEBS Lett ; 282(1): 135-8, 1991 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-1902801

RESUMO

The amino acid sequence of the 10 kDa subunit of the flavoprotein (FP) fragment of complex I from bovine heart mitochondria has been determined by protein sequence analysis, thereby completing the sequence of the FP fragment. The calculated molecular weight of the 10 kDa subunit agrees exactly with the value of 8438 determined by electrospray mass spectrometry, and further confirmation of the sequence has been obtained by sequencing cDNAs amplified from total bovine heart cDNA by the polymerase chain reaction, using mixed oligonucleotides based upon the protein sequence as primers and hybridization probes. The sequence of the 10 kDa subunit is not related to that of any known protein. Being devoid of cysteine residues, it has none of the characteristic features of known iron-sulfur proteins and it is improbable that it is involved in liganding Fe-S centers in the FP fragment.


Assuntos
Flavoproteínas/genética , Mitocôndrias Cardíacas/enzimologia , Precursores de Proteínas/genética , Quinona Redutases/genética , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Sequência de Bases , Bovinos , Cisteína/química , DNA , Flavoproteínas/química , Proteínas Ferro-Enxofre/química , Espectrometria de Massas , Dados de Sequência Molecular , Peso Molecular , NAD(P)H Desidrogenase (Quinona) , Fragmentos de Peptídeos/genética , Reação em Cadeia da Polimerase , Precursores de Proteínas/química , Quinona Redutases/metabolismo
13.
FEBS Lett ; 313(1): 80-4, 1992 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-1426273

RESUMO

The amino acid sequences of two nuclear-encoded subunits of complex I from bovine heart mitochondria have been determined. Both proteins have an apparent molecular weight of 14.5 kDa and their N-alpha-amino groups are acetylated. They are known as subunits B14.5a and B14.5b. Neither protein is evidently related to any known protein and their functions are obscure. A total of 34 nuclear-encoded subunits of bovine complex I have now been sequenced and it is thought that the primary structure of the complex is now complete, although with such a complicated structure it is difficult to be certain that there are no other subunits remaining to be sequenced. Seven additional hydrophobic subunits of the enzyme are encoded in mitochondrial DNA, and therefore bovine heart complex I is an assembly of about 41 different proteins. If it is assumed that there is one copy of each protein in the assembly, these polypeptides contain 7,955 amino acids in their sequences, more than are found in the Escherichia coli ribosome, which contains 7,336 amino acids in its 32 polypeptides.


Assuntos
DNA , Mitocôndrias Cardíacas/enzimologia , NAD(P)H Desidrogenase (Quinona)/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Peso Molecular , NAD(P)H Desidrogenase (Quinona)/química
14.
Am J Med Genet ; 42(4): 561-7, 1992 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-1535179

RESUMO

The ceroid-lipofuscinoses (Batten disease) are neurodegenerative inherited lysosomal storage diseases of children and animals. A common finding is the occurrence of fluorescent storage bodies (lipopigment) in cells. These have been isolated from tissues of affected sheep. Direct protein sequencing established that the major component is identical to the dicyclohexylcarbodiimide (DCCD) reactive proteolipid, subunit c, of mitochondrial ATP synthase and that this protein accounts for at least 50% of the storage body mass. No other mitochondrial components are stored. Direct sequencing of storage bodies isolated from tissues of children with juvenile and late infantile ceroid-lipofuscinosis established that they also contain large amounts of complete and normal subunit c. It is also stored in the disease in cattle and dogs but is not present in storage bodies from the human infantile form. Subunit c is normally found as part of the mitochondrial ATP synthase complex and accounts for 2-4% of the inner mitochondrial membrane protein. Mitochondria from affected sheep contain normal amounts of this protein. The P1 and P2 genes that code for it are normal as are mRNA levels. Oxidative phosphorylation is also normal. These findings suggest that ovine ceroid-lipofuscinosis is caused by a specific failure in the degradation of subunit c after its normal inclusion into mitochondria, and its consequent abnormal accumulation in lysosomes. This implies a unique pathway for subunit c degradation. It is probable that the human late infantile and juvenile diseases and the disease in cattle and dogs involve lesions in the same pathway.


Assuntos
Proteínas de Transporte/metabolismo , Dicicloexilcarbodi-Imida/metabolismo , Lipídeos , Mitocôndrias/enzimologia , Lipofuscinoses Ceroides Neuronais/enzimologia , Proteolipídeos/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Cães , Humanos , Lisossomos/enzimologia , Dados de Sequência Molecular , Pigmentos Biológicos/metabolismo , Ovinos
15.
Adv Exp Med Biol ; 266: 211-22; discussion 223, 1989.
Artigo em Inglês | MEDLINE | ID: mdl-2535017

RESUMO

The ceroid lipofuscinoses (Batten's disease) are a group of neuro-degenerative lysosomal storage diseases of children and animals that are recessively inherited. In the diseased individuals fluorescent storage bodies accumulate in a wide variety of cells, including neurons. The material stored in the cells of sheep affected with ceroid lipofuscinosis is two-thirds protein. The stored material does not arise from lipid peroxidation or a defect in lipid metabolism, and the lipid content is consistent with a lysosomal origin for the storage bodies. The major protein stains poorly with Coomassie blue dye and is soluble in organic solvents. It has an apparent molecular weight of 3,500 and its amino acids sequence is identical to that of the dicyclohexylcarbodiimide (DCCD) reactive proteolipid, subunit c, of mammalian mitochondrial ATP synthases. Apart from removal of mitochondrial import sequences, it has not been modified post-translationally. At least 50% of the mass of the storage bodies is composed of this protein. A minor protein sequence related to the 17-kDa subunit of vacuolar H(+)-ATPase is also found in storage bodies isolated from pancreas. As in humans and cattle, the ovine protein is the product of two expressed genes named P1 and P2. In normal and diseased animals there are no differences in sequences between P1 cDNAs or P2 cDNAs, nor do levels of mRNAs in liver for P1 or P2 differ substantially between normal and diseased animals. Both normal and diseased sheep also express a spliced pseudogene encoding amino acids 1 to 31 of the mitochondrial import presequence. The peptides they encode differ by one amino acid; arginine-23 is changed to glutamine in the diseased sheep. Storage bodies isolated from brains and pancreas of children affected with the juvenile and late infantile forms of ceroid lipofuscinosis also contain large amounts of material that is identical to subunit c of ATP synthase. However, the protein is not present in storage bodies isolated from brains of patients affected with the infantile form of the disease, and these storage bodies contain other unidentified proteins. It is possible that the cause of ovine, juvenile and late infantile ceroid lipofuscinoses is related to a defect in degradation of the subunit c of mitochondrial ATP synthase.


Assuntos
Lisossomos/enzimologia , Mitocôndrias/enzimologia , Lipofuscinoses Ceroides Neuronais/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Sequência de Aminoácidos , Animais , Dicicloexilcarbodi-Imida , Humanos , Dados de Sequência Molecular , Lipofuscinoses Ceroides Neuronais/patologia , Lipofuscinoses Ceroides Neuronais/veterinária , Proteínas/análise , ATPases Translocadoras de Prótons/genética , Ovinos , Doenças dos Ovinos/enzimologia
16.
Can J Vet Res ; 54(1): 15-21, 1990 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-2306665

RESUMO

The pathogenesis of the ceroid-lipofuscinoses, inherited storage diseases of children, was studied in an ovine model. This was shown to have clinical and pathological features most in common with the late infantile and juvenile human forms of the disease. The ability to study sequential changes allowed the retinal lesions to be described as a dystrophy of photoreceptor outer segments which preceded loss of the photoreceptor cells. An early decrease in amplitude of the c-wave electroretinograph was attributed to a decrease in the transpigment epithelial component. The decreased a- and b-wave amplitudes were attributed to the changes in and loss of, photoreceptor cells. The chemical components of isolated storage cytosomes were analyzed and shown to consist mostly of protein. Sequence analysis of the dominantly stored protein showed that it was identical to the DCCD reactive proteolipid or subunit c of mitochondrial adenosine triphosphate synthase and that it comprised approximately 50% of storage material. Based on the adage that the dominantly stored species should reflect the underlying biochemical anomaly, it was concluded that it was of pathogenic significance. This highly hydrophobic protein tends to extract with lipids in chloroform/methanol and is thus known as a proteolipid. Some of the remainder of the stored proteins also had this characteristic. It was concluded that ovine ceroid-lipofuscinosis was a proteinosis, more specifically a proteolipid proteinosis and as such it forms the prototype of a new class of storage diseases. Recognition of the nature of the dominantly stored chemical species has helped understanding of a variety of chemical and physical characteristics attributed to the whole pigment.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Lipofuscinoses Ceroides Neuronais/veterinária , Proteolipídeos/análise , Doenças dos Ovinos/patologia , Sequência de Aminoácidos , Animais , Modelos Animais de Doenças , Humanos , Dados de Sequência Molecular , Lipofuscinoses Ceroides Neuronais/patologia , Ovinos
18.
EMBO J ; 5(8): 2003-8, 1986 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-2875870

RESUMO

Two hydrophobic proteins have been purified to homogeneity from a mixture of about 13 proteins that are extracted from bovine mitochondria with a chloroform:methanol mixture. Sequence analysis shows that the smaller is a protein of 66 amino acids and is the product of a mitochondrial gene, A6L. The larger, a protein of 226 amino acids, is ATPase-6, a membrane component of ATP synthase, also encoded in mitochondrial DNA. The protein sequences determined establish that the genes for the two proteins overlap by 40 bases and indicate that translation of the second gene, ATPase-6, is initiated within the coding region of A6L. The A6L and the ATPase-6 proteins have also been isolated from the ATP synthase complex and so appear to be bona fide components of the enzyme. The function of A6L is unknown. However, weak structural homology suggests a functional similarity to the yeast mitochondrial protein, aapI, which is required for assembly of the fungal ATP synthase complex. Homologies between ATPase-6 and subunit a of the Escherichia coli ATP synthase complex indicate that the ATPase-6 protein has a similar role in the mitochondrial complex to its bacterial counterpart, being essential for the formation of an active proton channel.


Assuntos
DNA Mitocondrial/genética , Genes , Mitocôndrias Cardíacas/enzimologia , ATPases Translocadoras de Prótons/genética , Adenosina Trifosfatases/isolamento & purificação , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Brometo de Cianogênio , Membranas Intracelulares/enzimologia , Fragmentos de Peptídeos/análise , Proteolipídeos/isolamento & purificação , ATPases Translocadoras de Prótons/isolamento & purificação , Partículas Submitocôndricas/enzimologia
19.
Biochemistry ; 26(25): 8247-51, 1987 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-2964865

RESUMO

The bovine mitochondrial gene products ND2 and ND4, components of NADH dehydrogenase, have been purified from a chloroform/methanol extract of mitochondrial membranes, and the human mitochondrial gene products ND2 and cytochrome b have been obtained by similar procedures. They have been identified by comparison of their amino-terminal protein sequences with those predicted from DNA sequences of bovine and human mitochondrial DNA. All of the proteins have methionine as their amino-terminal residue. In bovine ND2, this residue is encoded by the "universal" isoleucine codon AUA, and the sequences of human cytochrome b and bovine ND2 demonstrate that AUA also encodes methionine in the elongation step of mitochondrial protein synthesis. In human ND2, the amino-terminal methionine is encoded by AUU, which, as in the "universal" genetic code, is also used as an isoleucine codon in elongation. Thus, AUU has a dual coding function which is dependent upon its context.


Assuntos
Adenosina Trifosfatases/genética , Códon , Redutases do Citocromo/genética , Grupo dos Citocromos b/genética , DNA Mitocondrial/genética , Genes , Mitocôndrias Cardíacas/enzimologia , Mitocôndrias/enzimologia , NADH Desidrogenase/genética , RNA Mensageiro , Adenosina Trifosfatases/isolamento & purificação , Aminoácidos/análise , Animais , Bovinos , Grupo dos Citocromos b/isolamento & purificação , Feminino , Humanos , Substâncias Macromoleculares , Peso Molecular , NADH Desidrogenase/isolamento & purificação , Placenta/enzimologia , Partículas Submitocôndricas/enzimologia
20.
Biochem J ; 237(1): 73-84, 1986 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-3800890

RESUMO

A solid-phase protein microsequencer is described that has been designed to determine protein sequences with subnanomolar quantities of protein. Its utility has been demonstrated by the determination of many sequences in subunits of mitochondrial F1-ATPase, in a protein isolated from mouse gap junctions and in the mitochondrial phosphate-transporter protein. It has a number of advantages over liquid- and gas-phase sequencers. Firstly, the degradation cycle takes 24 min, more than twice as fast as any other sequencer. This helps to reduce exposure of proteins to inimical reagents and increases throughput of samples. Secondly, polar amino acids such as phosphoserine, and polar derivatives formed by active-site photoaffinity labelling with 8-azido-ATP, are recovered quantitatively from the reaction column and can be positively identified. In other types of sequencer these polar derivatives, being somewhat insoluble in butyl chloride, tend to remain in the reaction chamber of the instrument and so are more difficult to identify. The solid-phase protein sequencer is also more suited than the liquid-phase instrument for analysis of proteolipids from membranes. These hydrophobic proteins tend to dissolve in organic solvents during washing steps in the liquid-phase instrument and are lost. Covalent attachment as used in the solid-phase instrument solves this problem.


Assuntos
Sequência de Aminoácidos , Proteínas , Autoanálise/instrumentação , Desenho de Equipamento , Microquímica/instrumentação , Microcomputadores , Feniltioidantoína , Fatores de Tempo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA