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J E Walker

Publications and source records attributed to J E Walker.

At least 19 recordsLinked to original sources
PubMed1992

Sheep 6-phosphogluconate dehydrogenase. Revised protein sequence based upon the sequences of cDNA clones obtained with the polymerase chain reaction.

Sheep liver 6-phosphogluconate dehydrogenase (6-PGDH) is an enzyme of the pentose phosphate pathway. Evidence has appeared which suggests that the 6-PGDH protein sequence determined previously by direct analysis of the protein isolated from ovine liver is incorrect. Determining the enzyme's DNA sequence was considered to be the best way of solving the problem. In the first instance, a degenerate forward and a degenerate reverse primer were designed on the basis of the known protein sequence, and a partial-length cDNA clone was isolated from total sheep liver cDNA using the polymerase chain reaction. The clone encoded the expected part of the protein sequence. The clone was unsuccessfully used as a prime-cut probe to screen a sheep liver library and a bovine heart library. As a result, the polymerase chain reaction was utilized again to successfully generate a family of overlapping cDNA clones encoding a mature protein of 482 amino acids. The mature protein sequence encoded by the cDNA differs significantly from the sequence derived by direct analysis of the protein, but on closer examination the fundamental difference is caused by the incorrect placement of three enzyme fragments obtained by cyanogen bromide cleavage during the direct sequence analysis of the protein. Placing the fragments in the correct order results in the two sequences being virtually identical except for some minor amino acid changes between the amide and acid forms, and a small number of deletions and insertions.

Amino Acid Sequence↗
PubMed1992

Resolution of NADH:ubiquinone oxidoreductase from bovine heart mitochondria into two subcomplexes, one of which contains the redox centers of the enzyme.

NADH:ubiquinone oxidoreductase (complex I) was purified from bovine heart mitochondria by solubilization with n-dodecyl beta-D-maltoside (lauryl maltoside), ammonium sulfate fractionation, and chromatography on Mono Q in the presence of the detergent. Its subunit composition was very similar to complex I purified by conventional means. Complex I was dissociated in the presence of N,N-dimethyldodecylamine N-oxide and beta-mercaptoethanol, and two subcomplexes, I alpha and I beta, were isolated by chromatography. Subcomplex I alpha catalyzes electron transfer from NADH to ubiquinone-1. It is composed of about 22 different and mostly hydrophilic subunits and contains 2.0 nmol of FMN/mg of protein. Among its subunits is the 51-kDa subunit, which binds FMN and NADH and probably contains a [4Fe-4S] cluster also. Three other potential Fe-S proteins, the 75- and 24-kDa subunits and a 23-kDa subunit (N-terminal sequence TYKY), are also present. All of the Fe-S clusters detectable by EPR in complex I, including cluster 2, are found in subcomplex I alpha. The line shapes of the EPR spectra of the Fe-S clusters are slightly broadened relative to spectra measured on complex I purified by conventional means, and the quinone reductase activity is insensitive to rotenone. Similar changes were found in samples of the intact chromatographically purified complex I, or in complex I prepared by the conventional method and then subjected to chromatography in the presence of lauryl maltoside. Subcomplex I beta contains about 15 different subunits. The sequences of many of them contain hydrophobic segments that could be membrane spanning, including at least two mitochondrial gene products, ND4 and ND5. The role of subcomplex I beta in the intact complex remains to be elucidated.

Animals↗
PubMed1992

Complementary DNA sequences of two 14.5 kDa subunits of NADH:ubiquinone oxidoreductase from bovine heart mitochondria. Completion of the primary structure of the complex?

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.

Amino Acid Sequence↗
PubMed1992

Sequences of 20 subunits of NADH:ubiquinone oxidoreductase from bovine heart mitochondria. Application of a novel strategy for sequencing proteins using the polymerase chain reaction.

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)

Amino Acid Sequence↗
PubMed1992

NADH: ubiquinone oxidoreductase from bovine heart mitochondria. A fourth nuclear encoded subunit with a homologue encoded in chloroplast genomes.

The amino acid sequence has been determined of the precursor of a nuclear encoded 20 kDa subunit of complex I from bovine heart mitochondria. The sequence of the mature protein is related to a protein of uncertain function, hitherto known as psbG, encoded in the chloroplast genomes of higher plants. Open reading frames encoding homologues of psbG have also been detected in bacteria and in the mitochondrial genome of Paramecium tetraurelia. The chloroplast psbG gene is found between ndhC and ndhJ, which encode homologues of ND3, a hydrophobic subunit of complex I encoded in the bovine mitochondrial genome, and of the nuclear encoded 30 kDa subunit of complex I. This 20 kDa protein is the eleventh out of the forty or more subunits of bovine complex I with a chloroplast encoded homologue, and its sequence provides further support for the presence in chloroplasts of a multisubunit enzyme related to complex I that could be involved in chlororespiration. The strict conservation of three cysteines suggests that the subunit might be an iron-sulphur protein.

Amino Acid Sequence↗
PubMed1992

Mitochondrial ATP synthase subunit c storage in the ceroid-lipofuscinoses (Batten disease).

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.

Amino Acid Sequence↗
PubMed1992

Sequence and pattern of expression of a bovine homologue of a human mitochondrial transport protein associated with Grave's disease.

A human cDNA has been isolated previously from a thyroid library with the aid of serum from a patient with Grave's disease. It encodes a protein belonging to the mitochondrial metabolite carrier family, referred to as the Grave's disease carrier protein (GDC). Using primers based on this sequence, overlapping cDNAs encoding the bovine homologue of the GDC have been isolated from total bovine heart poly(A)+ cDNA. The bovine protein is 18 amino acids shorter than the published human sequence, but if a frame shift requiring the removal of one nucleotide is introduced into the human cDNA sequence, the human and bovine proteins become identical in their C-terminal regions, and 308 out of 330 amino acids are conserved over their entire sequences. The bovine cDNA has been used to investigate the expression of the GDC in various bovine tissues. In the tissues that were examined, the GDC is most strongly expressed in the thyroid, but substantial amounts of its mRNA were also detected in liver, lung and kidney, and lesser amounts in heart and skeletal muscle.

Amino Acid Sequence↗
PubMed1992

Sequences of the human and bovine genes for the mitochondrial 2-oxoglutarate carrier.

The oxoglutarate carrier transports 2-oxoglutarate across the inner membranes of mitochondria in an electroneutral exchange for malate or other dicarboxylic acids. The sequences of its human and bovine genes have been determined from overlapping genomic clones generated by polymerase chain reactions by use of primers and probes based on the bovine cDNA sequence. The bovine and human genes are split into 6 and 8 exons, respectively, and five introns are found in the same positions in both genes. The coding and protein sequences are 93% and 96.6% identical, respectively. The human oxoglutarate carrier protein is 314 amino acids in length and, in common with the bovine protein, does not appear to have a processed presequence to help to target it into mitochondria.

Amino Acid Sequence↗
PubMed1991

NADH:ubiquinone oxidoreductase from bovine heart mitochondria. cDNA sequences of the import precursors of the nuclear-encoded 39 kDa and 42 kDa subunits.

The 39 kDa and 42 kDa subunits of NADH:ubiquinone oxidoreductase from bovine heart mitochondria are nuclear-coded components of the hydrophobic protein fraction of the enzyme. Their amino acid sequences have been deduced from the sequences of overlapping cDNA clones. These clones were amplified from total bovine heart cDNA by means of the polymerase chain reaction, with the use of complex mixtures of oligonucleotide primers based upon fragments of protein sequence determined at the N-terminals of the proteins and at internal sites. The protein sequences of the 39 kDa and 42 kDa subunits are 345 and 320 amino acid residues long respectively, and their calculated molecular masses are 39,115 Da and 36,693 Da. Both proteins are predominantly hydrophilic, but each contains one or two hydrophobic segments that could possibly be folded into transmembrane alpha-helices. The bovine 39 kDa protein sequence is related to that of a 40 kDa subunit from complex I from Neurospora crassa mitochondria; otherwise, it is not related significantly to any known sequence, including redox proteins and two polypeptides involved in import of proteins into mitochondria, known as the mitochondrial processing peptidase and the processing-enhancing protein. Therefore the functions of the 39 kDa and 42 kDa subunits of complex I are unknown. The mitochondrial gene product, ND4, a hydrophobic component of complex I with an apparent molecular mass of about 39 kDa, has been identified in preparations of the enzyme. This subunit stains faintly with Coomassie Blue dye, and in many gel systems it is not resolved from the nuclearcoded 36 kDa subunit.

Amino Acid Sequence↗
PubMed1991

Presence of an acyl carrier protein in NADH:ubiquinone oxidoreductase from bovine heart mitochondria.

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.

Acyl Carrier Protein↗
PubMed1991

NADH:ubiquinone oxidoreductase from bovine mitochondria. cDNA sequence of a 19 kDa cysteine-rich subunit.

The sequence of a 19 kDa subunit of NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria has been determined by a new strategy based on the polymerase chain reaction. The subunits of the enzyme were resolved in a polyacrylamide gel by two-dimensional isoelectric focusing and electrophoresis under denaturing conditions, transferred to a poly(vinylidene difluoride) membrane, and the N-terminal sequence was determined on the stained 19 kDa protein up to residue 27. This information was used to design two mixed oligonucleotide primers and a mixed oligonucleotide probe. With total bovine heart cDNA as template, overlapping cDNAs extending to sequences corresponding to both the 5' and 3' extremities of the mRNA coding for the 19 kDa subunit were synthesized in three polymerase chain reactions. These cDNAs were cloned and sequenced and encode a 171-amino-acid mature protein preceded by a methionine residue. The mature protein contains eight cysteine residues spaced at regular intervals through the protein, but the cysteine-rich motifs that are often associated with tetranuclear or binuclear centres in other proteins are not present. However, all eight cysteine residues are strictly conserved in a related protein from Neurospora crassa, suggesting that they have structural and/or functional significance in complex I.

Amino Acid Sequence↗
PubMed1991

Identification of the subunits of F1F0-ATPase from bovine heart mitochondria.

An oligomycin-sensitive F1F0-ATPase isolated from bovine heart mitochondria has been reconstituted into phospholipid vesicles and pumps protons. this preparation of F1F0-ATPase contains 14 different polypeptides that are resolved by polyacrylamide gel electrophoresis under denaturing conditions, and so it is more complex than bacterial and chloroplast enzymes, which have eight or nine different subunits. The 14 bovine subunits have been characterized by protein sequence analysis. They have been fractionated on polyacrylamide gels and transferred to poly(vinylidene difluoride) membranes, and N-terminal sequences have been determined in nine of them. By comparison with known sequences, eight of these have been identified as subunits beta, gamma, delta, and epsilon, which together with the alpha subunit form the F1 domain, as the b and c (or DCCD-reactive) subunits, both components of the membrane sector of the enzyme, and as the oligomycin sensitivity conferral protein (OSCP) and factor 6 (F6), both of which are required for attachment of F1 to the membrane sector. The sequence of the ninth, named subunit e, has been determined and is not related to any reported protein sequence. The N-terminal sequence of a tenth subunit, the membrane component A6L, could be determined after a mild acid treatment to remove an alpha-N-formyl group. Similar experiments with another membrane component, the a or ATPase-6 subunit, caused the protein to degrade, but the protein has been isolated from the enzyme complex and its position on gels has been unambiguously assigned. No N-terminal sequence could be derived from three other proteins. The largest of these is the alpha subunit, which previously has been shown to have pyrrolidonecarboxylic acid at the N terminus of the majority of its chains. The other two have been isolated from the enzyme complex; one of them is the membrane-associated protein, subunit d, which has an alpha-N-acetyl group, and the second, surprisingly, is the ATPase inhibitor protein. When it is isolated directly from mitochondrial membranes, the inhibitor protein has a frayed N terminus, with chains starting at residues 1, 2, and 3, but when it is isolated from the purified enzyme complex, its chains are not frayed and the N terminus is modified. Previously, the sequences at the N terminals of the alpha, beta, and delta subunits isolated from F1-ATPase had been shown to be frayed also, but in the F1F0 complex they each have unique N-terminal sequences.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1991

NADH:ubiquinone oxidoreductase from bovine heart mitochondria. Complementary DNA sequence of the import precursor of the 10 kDa subunit of the flavoprotein fragment.

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.

Amino Acid Sequence↗
PubMed1991

A homologue of a nuclear-coded iron-sulfur protein subunit of bovine mitochondrial complex I is encoded in chloroplast genomes.

The chloroplast genomes of Marchantia polymorpha, Nicotiana tabacum, and Oryza sativa contain open reading frames (ORFs or potential genes) encoding homologues of some of the subunits of mitochondrial NADH:ubiquinone oxidoreductase (complex I). Seven of these subunits (ND1-ND4, ND4L, ND5, and ND6) are products of the mitochondrial genome, and two others (the 49- and 30-kDa components of the iron-sulfur protein fraction) are nuclear gene products. These findings have been taken to indicate the presence in chloroplasts of an enzyme related to complex I, possibly an NAD(P)H:plastoquinone oxidoreductase, participating in chlororespiration. This view is reinforced by the present work in which we have shown that chloroplast genomes encode a homologue of the 23-kDa subunit, another nuclear-encoded component of bovine complex I. The 23-kDa subunit is in the hydrophobic protein fraction of the enzyme, the residuum after removal of the flavoprotein and iron-sulfur protein fractions. The sequence motif CysXXCysXXCysXXXCysPro, which provides ligands for tetranuclear iron-sulfur centers in ferredoxins, occurs twice in its polypeptide chain and is evidence of two associated 4Fe-4S clusters. This is the only iron-sulfur protein identified so far in the hydrophobic protein fraction of complex I, and so it is possible that one of these centers is that known as N-2, the donor of electrons to ubiquinone. The sequence of the 23-kDa subunit is closely related to potential proteins, which also contain the cysteine-rich sequence motifs, encoded in the frxB ORFs in chloroplast genomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Relationship between mitochondrial NADH-ubiquinone reductase and a bacterial NAD-reducing hydrogenase.

Bovine mitochondrial NADH-ubiquinone reductase (complex I), the first enzyme in the electron-transport chain, is a membrane-bound assembly of more than 30 different proteins, and the flavoprotein (FP) fraction, a water-soluble assembly of the 51-, 24-, and 10-kDa subunits, retains some of the catalytic properties of the enzyme. The 51-kDa subunit binds the substrate NAD(H) and probably contains both the cofactor, FMN, and also a tetranuclear iron-sulfur center, while a binuclear iron-sulfur center is located in the 24- or 10-kDa proteins. The 75-kDa subunit is the largest of the six proteins in the iron-sulfur protein (IP) fraction, and its sequence indicates that it too contains iron-sulfur clusters. Partial protein sequences have been determined at the N-terminus and at internal sites in the 51-kDa subunit, and the corresponding cDNA encoding a precursor of the protein has been isolated by using a novel strategy based on the polymerase chain reaction. The mature protein is 444 amino acids long. Its sequence, and those of the 24- and 75-kDa subunits, shows that mitochondrial complex I is related to a soluble NAD-reducing hydrogenase from the facultative chemolithotroph Alcaligenes eutrophus H16. This enzyme has four subunits, alpha, beta, gamma, and delta, and the alpha gamma dimer is an NADH oxidoreductase that contains FMN. The gamma-subunit is related to residues 1-240 of the 75-kDa subunit of complex I, and the alpha-subunit sequence is a fusion of homologues of the 24- and 51-kDa subunits, in the order N- to C-terminal. The most highly conserved regions are in the 51-kDa subunit and probably form parts of nucleotide binding sites for NAD(H) and FMN. Another conserved region surrounds the sequence motif CysXXCysXXCys, which is likely to provide three of the four ligands of a 4Fe-4S center, possibly that known as N-3. Characteristic ligands for a second 4Fe-4S center are conserved in the 75-kDa and gamma-subunits. This relationship with the bacterial enzyme implies that the 24- and 51-kDa subunits, together with part of the 75-kDa subunit, constitute a structural unit in mitochondrial complex I that is concerned with the first steps of electron transport.

Amino Acid Sequence↗
PubMed1991

The 30-kilodalton subunit of bovine mitochondrial complex I is homologous to a protein coded in chloroplast DNA.

In cattle, 7 of the 30 or more subunits of the respiratory enzyme NADH:ubiquinone reductase (complex I) are encoded in mitochondrial DNA, and potential genes (open reading frames, orfs) for related proteins are found in the chloroplast genomes of Marchantia polymorpha and Nicotiana tabacum. Homologues of the nuclear-coded 49- and 23-kDa subunits are also coded in chloroplast DNA, and these orfs are clustered with four of the homologues of the mammalian mitochondrial genes. These findings have been taken to indicate that chloroplasts contain a relative of complex I. The present work provides further support. The 30-kDa subunit of the bovine enzyme is a component of the iron-sulfur protein fraction. Partial protein sequences have been determined, and synthetic oligonucleotide mixtures based on them have been employed as hybridization probes to identify cognate cDNA clones from a bovine library. Their sequences encode the mitochondrial import precursor of the 30-kDa subunit. The mature protein of 228 amino acids contains a segment of 57 amino acids which is closely related to parts of proteins encoded in orfs 169 and 158 in the chloroplast genomes of M. polymorpha and N. tabacum. Moreover, the chloroplast orfs are found near homologues of the mammalian mitochondrial genes for subunit ND3. Therefore, the plant chloroplast genomes have at least two separate clusters of potential genes encoding homologues of subunits of mitochondrial complex I. The bovine 30-kDa subunit has no extensive sequences of hydrophobic amino acids that could be folded into membrane-spanning alpha-helices, and although it contains two cysteine residues, there is no clear evidence in the sequence that it is an iron-sulfur protein.

Amino Acid Sequence↗