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Biomedical subjects

J E Walker

Publications and source records attributed to J E Walker.

At least 91 records · Page 5Linked to original sources
PubMed1993

F1F0-ATP synthase from bovine heart mitochondria: development of the purification of a monodisperse oligomycin-sensitive ATPase.

A new procedure for the isolation of ATP synthase from bovine mitochondria has been developed, with the primary objective of producing enzyme suitable for crystallization trials. Proteins were extracted from mitochondrial membranes with dodecyl-beta-D-maltoside, and the ATP synthase was purified from the extract in the presence of the same detergent by a combination of ion-exchange and gel-filtration chromatography and ammonium sulphate precipitation. This simple and rapid procedure yields 20-30 mg of highly pure and monodisperse enzyme, evidently consisting of 14 different subunits, amongst them, in apparently stoichiometric amounts with the established subunits, subunit e, a recently discovered subunit of unknown function. The enzyme preparation has an oligomycin-sensitive ATP hydrolysis activity, and so the F1 domain is functionally associated with the membrane domain, F0. In contrast with the N-termini of some of the subunits of bovine mitochondrial F1-ATPase, those of the F1F0-ATP synthase are not degraded by proteolysis during the isolation procedure. This preparation therefore satisfies prerequisites for crystallization trials.

Adenosine Triphosphate↗
PubMed1993

Polyclonal antibody generation in rabbit by administration of an organophosphorus acid anhydrolase (OPAA) from squid.

When a nerve gas hydrolyzing enzyme [organophosphorus acid anhydrolase (OPAA), formerly DFPase] purified from squid hepatopancreas was injected into rabbits, the resulting sera (RAS) inhibited OPAA purified from either squid hepatopancreas or squid optic ganglia. The inhibition was non-competitive, with 50% inhibition at a 1:1,000 serum dilution, and with the limit of inhibition (in effect, a "titer") at approximately 1:10,000. This RAS did not inhibit the distinctly different OPAAs from a mammalian and two bacterial sources. The hepatopancreas-generated RAS also reacted positively to the appropriate enzyme-linked immunosorbent assay (ELISA) at a titer of 1:100,000. In marked contrast, when OPAA purified from squid optic ganglion was injected into rabbits, the resulting sera did not inhibit squid OPAA, and did not give a positive ELISA. Control sera taken from the same rabbits prior to any injection (RS) did not inhibit the OPAAs. These results show another major difference between squid type OPAAs and the OPAAs from other sources, sometimes termed "Mazur type" OPAAs.

Animals↗
PubMed1993

Organization and sequences of genes for the subunits of ATP synthase in the thermophilic cyanobacterium Synechococcus 6716.

The sequences of the genes for the nine subunits of ATP synthase in the thermophilic cyanobacterium Synechococcus 6716 have been determined. The genes were identified by comparison of the encoded proteins with sequences of ATP synthase subunits in other species, and confirmed for subunits alpha, beta, delta and epsilon, by determining their N-terminal sequences. They are arranged at three separate loci. Six of them are in one cluster in the order a: c: b': b: delta: alpha, and those for the beta and epsilon subunits form a second and separate cluster. The gene for the gamma-subunit is at a third site. As in other bacteria, the gene for subunit a is immediately preceded by a gene coding for a small hydrophobic protein of unknown function, known as uncI in Escherichia coli. The gene orders in Synechococcus 6716 are related to the orders of ATP synthase genes in the plastid genomes of higher plants, and particularly of a red alga and a diatom. The sequences of the subunits are similar to those of chloroplast ATP synthase, the alpha, beta and c subunits being particularly well conserved. Differences in the primary structures of the Synechococcus 6716 and chloroplast gamma subunits probably underlie different mechanisms of activation of ATP synthase. The nucleotide sequences that are presented also contain 12 other open reading frames. One of them encodes a protein sequence related to the E. coli DNA repair enzyme, photolyase, and another codes for a protein that contains internal repeats related to sequences in the myosin heavy chain.

Amino Acid Sequence↗
PubMed1993

Abundant bacterial expression and reconstitution of an intrinsic membrane-transport protein from bovine mitochondria.

The oxoglutarate carrier, an intrinsic membrane-transport protein of the inner membranes of bovine-heart mitochondria, has been expressed at an abundant level in Escherichia coli. It accumulates in the bacterium as inclusion bodies, and none of the protein was detected in the bacterial inner membrane. The mitochondrial ADP/ATP carrier, a member of the same super-family of transport proteins as the oxoglutarate carrier, has also been expressed in E. coli. However, the expression of the ADP/ATP carrier in bacteria retards their growth, and so the levels of expression that were attained were lower than those of the oxoglutarate carrier. The oxoglutarate carrier inclusion bodies have been disaggregated with the detergent N-dodecanoyl-sarcosine, and the protein has been incorporated into liposomes. In its ability to transport oxoglutarate and malate and other known substrates of the carrier in mitochondria, and in its inhibition characteristics by a wide range of non-competitive and competitive inhibitors, this reconstituted oxoglutarate carrier is similar to the natural protein in the inner membranes of mitochondria, and to the carrier that has been purified from mitochondria and reconstituted in liposomes. These experiments remove significant obstacles to crystallization trials and to site-directed mutagenesis of the oxoglutarate carrier.

Amino Acid Sequence↗
PubMed1993

Sequences of members of the human gene family for the c subunit of mitochondrial ATP synthase.

Subunit c is an intrinsic membrane component of ATP synthase, and in mammals it is encoded by two expressed nuclear genes, P1 and P2. Both genes encode the same mature c subunit, but the mitochondrial import pre-sequences in the precursors of subunit c are different. The DNA sequences of the human P1 and P2 genes are described. They occupy about 3.0 and 10.9 kb respectively of the human genome, and both genes are split into five exons. The human genome also contains about 14 related spliced pseudogenes, and the sequence of one such pseudogene related to P2 is described. Sequences flanking the 5' ends of the human P1 and P2 coding sequences each contain a CpG-rich island. Potential promoter elements (TATA and CCAAT boxes) are present in the 5' sequences of the P1 gene, but not that of P2, although there is no direct experimental evidence to show the involvement of these sequences in transcription of the genes.

Animals↗
PubMed1993

Characterization of the expressed genes for subunit c of mitochondrial ATP synthase in sheep with ceroid lipofuscinosis.

The human and bovine genomes each contain two expressed nuclear genes, called P1 and P2, for subunit c, a hydrophobic subunit of the membrane sector, Fo, of mitochondrial ATP synthase. Both P1 and P2 encode the same mature protein, but the associated mitochondrial import sequences are different. In sheep with the neurodegenerative disease ceroid lipofuscinosis, and also in humans with Batten's disease, unmodified subunit c accumulates in lysosome-derived organelles in a variety of tissues. However, the sequences of cDNAs for P1 and P2 from sheep with ceroid lipofuscinosis were identical to those in healthy control animals. Therefore, since there was no mutation in either of the mitochondrial import sequences of subunit c in the diseased animals, ceroid lipofuscinosis does not arise from changes in an import sequence causing mis-targeting of the c subunit to lysosomes. The levels of expression of P1 and P2 genes were approximately the same in diseased and healthy animals, and so the protein is unlikely to accumulate because of excessive transcription of either gene. Transcription of a spliced pseudogene related to P2 was detected in both a control animal and a sheep with ceroid lipofuscinosis. The transcripts encode amino acids 1-31 of the P2 mitochondrial targeting sequence. In the diseased animal, an arginine replaced a glutamine in the control sequence. However, restriction fragment analysis of genomic DNA from a further 12 sheep established that the sequence differences were not linked to ceroid lipofuscinosis.

ATP Synthetase Complexes↗
PubMed1993

Crystallization of F1-ATPase from bovine heart mitochondria.

Crystals of the F1-ATPase sector of the ATP synthase complex from bovine heart mitochondria have been grown from solutions containing polyethylene glycol 6000. The crystals diffract to 2.9 A resolution on a laboratory X-ray source. They are orthorhombic and belong to the space group P2(1)2(1)2(1). The unit cell axes are a = 285 A, b = 108 A, c = 140 A. There is one molecule of F1-ATPase in the asymmetric unit.

Animals↗
PubMed1993

Inherent asymmetry of the structure of F1-ATPase from bovine heart mitochondria at 6.5 A resolution.

ATP synthase, the assembly which makes ATP in mitochondria, chloroplasts and bacteria, uses transmembrane proton gradients generated by respiration or photosynthesis to drive the phosphorylation of ADP. Its membrane domain is joined by a slender stalk to a peripheral catalytic domain, F1-ATPase. This domain is made of five subunits with stoichiometries of 3 alpha: 3 beta: 1 gamma: 1 delta: 1 epsilon, and in bovine mitochondria has a molecular mass of 371,000. We have determined the 3-dimensional structure of bovine mitochondrial F1-ATPase to 6.5 A resolution by X-ray crystallography. It is an approximately spherical globule 110 A in diameter, on a 40 A stem which contains two alpha-helices in a coiled-coil. This stem is presumed to be part of the stalk that connects F1 with the membrane domain in the intact ATP synthase. A pit next to the stem penetrates approximately 35 A into the F1 particle. The stem and the pit are two examples of the many asymmetric features of the structure. The central element in the asymmetry is the longer of the two alpha-helices in the stem, which extends for 90 A through the centre of the assembly and emerges on top into a dimple 15 A deep. Features with threefold and sixfold symmetry, presumed to be parts of homologous alpha and beta subunits, are arranged around the central rod and pit, but the overall structure is asymmetric. The central helix provides a possible mechanism for transmission of conformational changes induced by the proton gradient from the stalk to the catalytic sites of the enzyme.

Animals↗
PubMed1993

The mitochondrial transport protein superfamily.

The ADP/ATP, phosphate, and oxoglutarate/malate carrier proteins found in the inner membranes of mitochondria, and the uncoupling protein from mitochondria in mammalian brown adipose tissue, belong to the same protein superfamily. Established members of this superfamily have polypeptide chains approximately 300 amino acids long that consist of three tandem related sequences of about 100 amino acids. The tandem repeats from the different proteins are interrelated, and probably have similar secondary structures. The common features of this superfamily are also present in nine proteins of unknown functions characterized by DNA sequencing in various species, most notably in Caenorhabditis elegans and Saccharomyces cerevisiae. The high level expression in Escherichia coli of the bovine oxoglutarate/malate carrier, and the reconstitution of active carrier from the expressed protein, offers encouragement that the identity of superfamily members of known sequence but unknown function may be uncovered by a similar route.

Amino Acid Sequence↗
PubMed1993

Biopolymer metal catalysts for the hydrolysis of nerve gases.

Glucosamine oligomers--monomer through tetramer--form complexes with Cu2+ that catalyse the hydrolysis of the 'nerve gas' 1,2,2-trimethylpropyl- methylphosphonofluroidate (soman) by cleaving the P-F bond. A 1/1 glucosamine/Cu2+ ratio whether as glucosamine or glucosamine units, gives the highest hydrolytic rate over the 11.5/1 to 1/1 range. This trend also appears to hold for a glucosamine polymer, chitosan, which, when complexed with Cu2+ also hydrolyzes soman. The relatively low rate of hydrolysis by this polymer-Cu2+ complex, while not yet explainable, is consistent with an extrapolation of the monomer-through-tetramer series. The question may be raised as to whether these biopolymer metal complexes provide any clues to the involvement of Mn2+ in the functioning of one class of P-F cleaving enzymes.

Catalysis↗
PubMed1993

Stereoselectivity of soman detoxication by organophosphorus acid anhydrases from Escherichia coli.

Three organophosphorus acid anhydrases have been isolated from E. coli by gel filtration and ion exchange column procedures, and further identified by gel electrophoresis. All three have molecular weights in the 120,000-140,000 range. Two of them hydrolyze racemic 1,2,2-trimethylpropylmethylphosphonofluoridate (soman) to completion at a single rate and, in parallel with this, detoxify soman at a comparable rate. The third enzyme appears to show stereoselectivity with respect to the two pairs of isomers of soman in that it hydrolyzes the racemic mixture at a fast and a slow rate, the latter approaching the non-enzymatic rate, and detoxifies soman only at the slower rate. In the past, organophosphorus acid anhydrases from bacterial and mammalian sources have been assayed either as crude sonicates or homogenates, or as cold ethanol precipitated fractions. Major discrepancies among laboratories have probably been due either to the assay of mixtures of varying proportions of these three enzymes depending on the various organs or organisms used as the source, or to the purification of one of the enzymes at the expense of the others. For E. coli, a fourth organophosphorus acid anhydrase is also present but at a considerably lower activity.

Aryldialkylphosphatase↗
PubMed1993

Partial purification and identification of an immunosuppressive factor in AIDS sera.

The cause of the profound immunodeficiency state induced by the human immunodeficiency virus (HIV) is not yet fully understood. Although direct cytopathic effects of the virus leading to the loss of CD4+ lymphocytes are believed to play a major role, soluble factors that cause lymphocyte dysfunction have also been identified. Here, we demonstrate that sera of AIDS patients contain a viral component capable of profoundly inhibiting normal T lymphocyte and natural killer cell functions. This factor (M(r) 30,000-50,000) is relatively resistant to proteolytic degradation, heat denaturation, extreme pH, and chemical modifications. It has been determined by Western blotting to share epitopes with the gp41 viral transmembrane component of HIV-1. This immunosuppressive factor has the characteristic property of inhibiting lymphocyte activation in the early stage (within 3 hr) of mitogenic stimulation. Concomitant with the inhibition of lymphocyte activation and interleukin 2 (IL-2) production, transcription of the IL-2 message is also reduced in a time-dependent manner. The unique time dependency of this immunosuppressive factor in lymphocyte activation could serve as a valuable tool to study the early steps of signal transduction in lymphocyte activation beyond membrane interaction.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Complementary DNA sequence of bovine cpn10 (Hsp10), a chaperone protein from mitochondria.

A cDNA sequence encoding a chaperone protein from bovine heart mitochondria is described. It is deduced from overlapping partial cDNAs amplified in polymerase chain reactions from bovine heart cDNA, using in the first instance mixtures of synthetic oligonucleotide primers based on the incomplete sequence of the cpn10 (Hsp10) chaperone protein from rat liver mitochondria. The encoded bovine protein sequence is 101 amino acids in length. By analogy with the rat homologue it is likely that the initiator methionine is removed after translation and that the mature N-terminal is modified. The mitochondrial proteins are members of the chaperonin 10 family which also includes the bacterial GroES chaperones. These proteins act in conjunction with members of the chaperonin-60 protein family, such as bacterial GroEL, to facilitate the folding of newly synthesised or translocated proteins.

Amino Acid Sequence↗
PubMed1993

Congestive heart failure in the elderly.

Changes occurring in the cardiovascular system as healthy subjects age, specifically vascular rigidity and decreased ventricular compliance, increase risks for the development of systolic hypertension, left ventricular hypertrophy, and diastolic dysfunction. These changes of "usual aging" act as independent risk factors which increase the frequency of morbid events accompanying hypertension and coronary artery disease as these diseases increase with age. The rate of congestive heart failure, for example, increases exponentially, developing a three-year mortality of 80% in patients older than 65. Advances in prevention, diagnosis, and management over the past decade have delayed the onset of cardiovascular events, extending healthy life span remarkably. These advances are reviewed, placing special attention on the changes of aging that effect the clinical management of older patients.

Aged↗
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↗