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Biomedical subjects

J E Walker

Publications and source records attributed to J E Walker.

At least 73 records · Page 4Linked to original sources
PubMed1995

Determination of the structures of respiratory enzyme complexes from mammalian mitochondria.

Knowledge of the atomic resolution structures of the respiratory enzymes from mammalian mitochondria is likely to be essential for understanding the molecular basis of human diseases involving their dysfunction. Because they are membrane-bound multisubunit assemblies, the determination of their high resolution structures is a rather challenging undertaking. The greatest progress has been made with the ATP synthase. The structure of its catalytic domain, F1-ATPase, has been solved by X-ray crystallography at 2.8 A resolution. It supports a binding change mechanism of catalysis in intact ATP synthase in which the catalytic subunits are in different states of the catalytic cycle at any instant. Interconversion of the states may be achieved by rotation of an alpha-helical domain of the gamma-subunit relative to the alpha 3 beta 3 sub-assembly. The membrane domain of ATP synthase has been purified, and the stalk linking the catalytic and membrane domains has been reassembled in vitro from its constituent subunits. Considerable progress has also been made in analyzing the structure of bovine complex I. It has about 43 different subunits and 42 of them have been sequenced. All of the known prosthetic groups have been localized in its extrinsic membrane arm, which has been split away from the membrane subunits and purified. The next stage is to crystallize the domain and to solve its structure.

Animals↗
PubMed1995

Multiple deficiencies of mitochondrial DNA- and nuclear-encoded subunits of respiratory NADH dehydrogenase detected with peptide- and subunit-specific antibodies in mitochondrial myopathies.

Antibodies have been raised against synthetic peptides corresponding to several computer-predicted epitopes of three mtDNA-encoded subunits, ND4, ND5 and ND6, of the human respiratory chain NADH dehydrogenase (Complex I). Antibodies were characterized by a sensitive immunoblotting assay using proteins from human skeletal muscle mitochondria and by immunoprecipitation of radio-labeled HeLa cell mitochondrial translation products. Only antibodies against two of six selected peptides of the ND4 subunit, i.e., the C-terminal peptide and an internal peptide close to the C-terminus, reacted in both assays with the subunit. Antibodies raised against an internal peptide close to the N-terminus of the ND5 subunit and antibodies raised against an internal epitope of the ND6 subunit also reacted in both the immunoblotting and immunoprecipitation assays. The antibodies described above and other Complex I subunit- or holoenzyme-specific antibodies were used to investigate the subunit deficiencies of the respiratory NADH dehydrogenase in the skeletal muscle of patients affected by mitochondrial myopathies associated with Complex I defects. The reduction in enzyme activity correlated in an immunoblot assay with a decrease of four mtDNA-encoded subunits of the enzyme, as well as with a decrease of other subunits of Complex I encoded in the nDNA. The present work provides the first evidence of a decrease in NADH dehydrogenase subunits encoded in the mitochondrial genome in myopathy patients.

Adolescent↗
PubMed1995

Hydrolysis of tetriso by an enzyme derived from Pseudomonas diminuta as a model for the detoxication of O-ethyl S-(2-diisopropylaminoethyl) methylphosphonothiolate (VX).

An enzyme termed organophosphorus hydrolase (OPH), derived from Pseudomonas diminuta, had been found previously to hydrolyze the powerful acetylcholinesterase (AChE) inhibitor O-ethyl S-(2-diisopropylaminoethyl) methylphosphonothiolate (VX). This enzyme has now been shown to be correlated with the loss of AChE inhibitory potency (detoxication). OPH also hydrolyzed and detoxified the VX analogue, O,O-diisopropyl S-(2-diisopropylaminoethyl) phosphorothiolate (Tetriso), also a potent AChE inhibitor, about five times faster than VX. The Km for the hydrolysis of the P-S bond of Tetriso was 6.7 x 10(-3) M. OPH also hydrolyzed diisopropylphosphorofluoridate (DFP) 50-60 times faster than Tetriso, and 1,2,2-trimethylpropyl methylphosphonofluoridate (Soman) about seven times faster than Tetriso. DFP was a non-competitive inhibitor of Tetriso hydrolysis, Ki = 8.7 x 10(-4) M. The DFP hydrolysis product, diisopropyl phosphate, was a competitive inhibitor, Ki = 2.3 x 10(-4) M. The rate of detoxication of Tetriso compared with the rate of hydrolysis suggests that OPH may not be totally specific for P-S bond cleavage. OPH was inhibited completely by 1.5 x 10(-4) M 8-hydroxyquinoline-5-sulfonate or 1,10-phenanthroline, both transition element chelators, but inhibited only partially by EDTA, a much more potent chelator.

Aryldialkylphosphatase↗
PubMed1994

ATP synthase from bovine heart mitochondria: identification by proteolysis of sites in F0 exposed by removal of F1 and the oligomycin-sensitivity conferral protein.

The exposure to trypsinolysis of subunits of F1F0-ATPase and of its F0 domain have been compared in everted inner membrane vesicles (submitochondrial particles) made from bovine mitochondria. Treatment of submitochondrial particles with guanidine hydrochloride removed the subunits of F1-ATPase and the oligomycin-sensitivity conferral protein (OSCP), and exposed sites that were occluded in the intact F1F0-ATPase complex. These sites were identified by purifying the subunits from the isolated F0 and F1F0-ATPase complexes before and after proteolysis of the vesicles, and by characterizing them by N-terminal sequencing and electrospray-ionization mass spectrometry. In the stripped vesicles, subunit F6 was completely digested away by either trypsin or chymotrypsin. Trypsin also cleaved subunit b, first at the bond arginine-166-glutamine-167, and then at the consecutive linkages, lysine-120-arginine-121 and arginine-121-histidine-122. Chymotrypsin-sensitive sites were observed after the adjacent methionines 164 and 165. Trypsin also removed amino acids 1-3 of subunit d, and minor cleavage sites were observed in subunit d between amino acids 24 and 25, in subunit g between amino acids 5 and 6, and after amino acid 40 in subunit e. The other subunits remained protected from proteolysis. In membrane-bound F1F0-ATPase, the N-terminus of subunit d was also accessible to trypsin, and subunit e was more susceptible to proteolysis than in F0. Otherwise the F0 subunits and the OSCP were protected. Subunits alpha and beta were cleaved by trypsin at the same sites in their N-terminal regions as in purified F1-ATPase. The trypsinized F0 was incapable of binding F1-ATPase in the presence of the OSCP. These experiments and in vitro re-assembly experiments described elsewehere, that were guided by the results of the proteolysis experiments, have helped to establish a central role for subunit b in the formation of the stalk connecting the F1 and F0 domains of the F1F0-ATPase complex.

Adenosine Triphosphatases↗
PubMed1994

ATP synthase from bovine heart mitochondria. In vitro assembly of a stalk complex in the presence of F1-ATPase and in its absence.

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.

Amino Acid Sequence↗
PubMed1994

Structure at 2.8 A resolution of F1-ATPase from bovine heart mitochondria.

In the crystal structure of bovine mitochondrial F1-ATPase determined at 2.8 A resolution, the three catalytic beta-subunits differ in conformation and in the bound nucleotide. The structure supports a catalytic mechanism in intact ATP synthase in which the three catalytic subunits are in different states of the catalytic cycle at any instant. Interconversion of the states may be achieved by rotation of the alpha 3 beta 3 subassembly relative to an alpha-helical domain of the gamma-subunit.

Amino Acid Sequence↗
PubMed1994

Fo membrane domain of ATP synthase from bovine heart mitochondria: purification, subunit composition, and reconstitution with F1-ATPase.

The Fo membrane domain of the F1Fo-ATP synthase complex has been purified from bovine heart mitochondria. The purification procedure involves the removal of peripheral membrane proteins, including F1-ATPase, from submitochondrial particles with guanidine hydrochloride, followed by extraction of Fo and other membrane proteins from the stripped membranes in the presence of the detergent n-dodecyl beta-D-maltoside. Fo was then purified by ion-exchange and dye ligand chromatography in the presence of the same detergent. Approximately 15 mg of pure Fo was recovered from 1.8 g of mitochondrial membrane protein. The purified Fo is a complex of nine different polypeptides. They are subunits a, b, c, d, e, F6, and A6L characterized before in F1Fo-ATPase preparations, and two new hitherto undetected subunits, named f and g. The sequences of subunits f and g have been determined. They are not related significantly to any known protein, but subunit f appears to contain a membrane-spanning alpha-helix. Proteins f and g are also present in approximately stoichiometric amounts in a highly purified preparation of intact F1Fo-ATPase, and so it is concluded that they are authentic subunits of the bovine enzyme with unknown functions. Dibutyltin 3-hydroxyflavone, an inhibitor of F1Fo-ATPase, also binds to the purified Fo in detergent and competes for binding with venturicidin. In the presence of F1 and OSCP, the purified Fo was reassembled into the intact F1Fo-ATPase complex. Therefore, this procedure provides a relatively abundant source of pure and functional Fo that is suitable for structural analysis.

Adenosine Triphosphatases↗
PubMed1994

The role of the stalk in the coupling mechanism of F1F0-ATPases.

The extrinsic and intrinsic membrane sectors of F1F0-ATPases are linked by a slender stalk 40-50 A in length. The stalk transmits the energy produced by oxidative or photosynthetic phosphorylation from the intrinsic sector, F0, to the catalytic sites in the extrinsic F1 sector. How this is achieved is unknown, but long-range conformational changes linked to transmembrane proton transport may be involved. In bacterial and chloroplast F1F0-ATPases, the stalk is probably a composite of subunits delta and epsilon, part of the gamma-subunit, and the extrinsic membrane domains of 2 subunits (identical or non-identical according to the species) that are bound to the membrane by their N-terminal regions. The stalk in the bovine mitochondrial enzyme appears to be more complex, and the gamma, delta, epsilon, OSCP, F6, b and d subunits all contribute to it. A bovine stalk complex has been assembled in vitro from bacterially expressed OSCP, F6, b and d, both in the presence and in the absence of F1-ATPase. One molecule of each of these subunits is present in the assembled complex, as there is also in each native F1F0-ATPase assembly. Providing that suitable crystals can be obtained, the stalk complex and the F1.stalk complex may permit the high resolution structure of bovine F1-ATPase to be extended into the stalk domain.

Animals↗
PubMed1994

The sequences of human and bovine genes of the phosphate carrier from mitochondria contain evidence of alternatively spliced forms.

The sequences of the human and bovine genes for the phosphate carrier from the inner membranes of mitochondria have been determined. The genes have similar structures and each is divided into nine exons. In both genes, two exons, named IIIA and IIIB, are closely related, and they appear to the alternatively spliced. The human exon IIIB sequence is found in a published human heart cDNA sequence, and bovine exon IIIA forms part of a published bovine heart cDNA sequence. By further examination of the human heart cDNA library, sequences arising from both alternatively spliced forms of the phosphate carrier have been characterized. Both forms were also found in several bovine tissues, but the ratios of expression of the two forms varied. The form containing exon IIIA was expressed most highly in bovine heart and liver, less highly in brain and kidney, and only in low amounts in lung. The opposite hierarchy was found for the form containing exon IIIB; it was most highly expressed in lung and least in heart and liver. The alternative splicing mechanism affects amino acids 4-45 of the mature phosphate carrier protein, which is believed to form one of six transmembrane segments of the phosphate carrier and to emerge into a large extramembranous loop. The alternative splicing mechanism changes 13 and 11 amino acids in the human and bovine carrier proteins, respectively. As the function of this region of the phosphate carrier is not known, the effects of the changes on carrier function are not understood at present.

Alternative Splicing↗
PubMed1994

Predicting physical and verbal aggression on a brain trauma unit.

Brain injury often results in cognitive impairments and neurobehavioral deficits that effect recovery, rehabilitation, and general adjustment. Aggressive behaviors and agitation, well-known consequences of cerebral damage, are the most difficult for caregivers to evaluate and manage and the most stressful for patients and families. This study was designed to determine the efficacy of an observational protocol (Cognitive Behavioral Rating Scale, CBRS) in evaluating cognition and its usefulness with demographic, medical, and psychological information in predicting aggressive behaviors of cerebrally damaged patients. Twenty-eight brain-injured patients consecutively admitted to a brain injury unit were evaluated by nurses who used the CBRS after first establishing interrater reliability in using the instrument. Relationships were determined between results of the CBRS, demographic information, medical and psychological information; the variable in each category that was most highly correlated with aggressive behaviors was then used in a multiple linear regression to predict the frequency of aggressive behaviors. Discussion focused on disorientation to place and time as the most significant predictors of aggressive behaviors, as well as the prescriptive use of antiseizure medications and the number of medical comorbidities.

Adult↗
PubMed1994

The regulation of catalysis in ATP synthase.

ATP synthase is regulated so as to prevent futile hydrolysis of ATP when the transmembrane proton electrochemical gradient, delta mu H+, falls. Mitochondria and chloroplasts have different mechanisms for inhibition of ATP synthase: by binding an inhibitor protein, and by stabilization of the ADP-inhibited state by making an intramolecular disulphide bond, respectively. The recently determined structure of bovine F1-ATPase is locked in a conformation that probably represents the ADP-inhibited state of the enzyme.

Amino Acid Sequence↗
PubMed1994

Extension of the mitochondrial transporter super-family: sequences of five members from the nematode worm, Caenorhabditis elegans.

The sequences are presented of cDNAs encoding five related proteins from the nematode worm, Caenorhabditis elegans. Three of them can be recognised as the homologues of the ADP/ATP, phosphate and oxoglutarate/malate carrier proteins that have been found in the inner membranes of mitochondria in other species. These carrier proteins, and the uncoupling protein from the mitochondria in mammalian brown adipose tissue, have common features in their primary and secondary structures, and are members of the same protein super-family. Members of this super-family have polypeptide chains approximately 300 amino acid long that consist of three tandem related sequences of about 100 amino acids. The tandem repeats from the different proteins are inter-related, and each repeat is probably folded into a common secondary structural motif consisting of two hydrophobic stretches of amino acids with the potential to form membrane spanning alpha-helices, linked by an extensive hydrophilic region. The common characteristic features of this family of proteins are also present in sequences of two further proteins, named C1 and C2, encoded in nematode cDNAs, and in four published protein sequences from various sources. Neither the transport properties nor the subcellular locations of any of this latter group of six proteins are known. Therefore, currently the super-family of mitochondrial carrier proteins has at least ten different members.

Amino Acid Sequence↗