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J W Thomas

Publications and source records attributed to J W Thomas.

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

Oxygen reaction and proton uptake in helix VIII mutants of cytochrome bo3.

The oxygen reaction of wild-type and helix VIII mutants of cytochrome bo3 from Escherichia coli, and the associated proton uptake during this reaction, has been studied using flash photolysis of the CO complex of the reduced protein after rapid mixing with oxygen. We have focused on mutations in the transmembrane helix VIII where protonatable residues have been exchanged, and mainly on the inactive mutants (i.e., T352A, T359A, and K362L, -M, and -Q). The kinetics for electron transfer during oxidation for the mutants are similar to the wild-type; two rate constants of 3.2 x 10(4) and 3.4 x 10(3) s-1 (at 1 mM oxygen) are detected. Proton uptake is observed for wild-type as well as for the mutant enzymes, but the mutations within helix VIII have affected the rate of proton uptake; it is significantly accelerated in the mutants. These results show that none of the protonatable residues in helix VIII are required in the reaction between the fully reduced cytochrome bo3 and oxygen. We have also studied electron redistribution after photolysis of CO from the mixed-valence compound; we found three kinetic components for wild-type and the mutants T352A and T359A, but for K362M only the first and third components are observed, with amplitudes that are lower than those for the corresponding components in the wild-type enzyme, suggesting that the characteristics of internal electron transfer in the K362M mutant are different from those of the wild-type enzyme.

Carbon Monoxide↗
PubMed1995

Possible proton relay pathways in cytochrome c oxidase.

As the final electron acceptor in the respiratory chain of eukaryotic and many prokaryotic organisms, cytochrome c oxidase (EC 1.9.3.1) catalyzes the reduction of oxygen to water and generates a proton gradient. To test for proton pathways through the oxidase, site-directed mutagenesis was applied to subunit I of the Rhodobacter sphaeroides enzyme. Mutants were characterized in three highly conserved regions of the peptide, comprising possible proton loading, unloading, and transfer sites: an interior loop between helices II and III (Asp132Asn/Ala), an exterior loop between helices IX and X (His411Ala, Asp412Asn, Thr413Asn, Tyr414Phe), and the predicted transmembrane helix VIII (Thr352Ala, Pro358Ala, Thr359Ala, Lys362Met). Most of the mutants had lower activity than wild type, but only mutants at residue 132 lost proton pumping while retaining electron transfer activity. Although electron transfer was substantially inhibited, no major structural alteration appears to have occurred in D132 mutants, since resonance Raman and visible absorbance spectra were normal. However, lower CO binding (70-85% of wild type) suggests some minor change to the binuclear center. In addition, the activity of the reconstituted Asp132 mutants was inhibited rather than stimulated by ionophores or uncoupler. The inhibition was not observed with the purified enzyme and a direct pH effect was ruled out, suggesting an altered response to the electrical or pH gradient. The results support an important role for the conserved II-III loop in the proton pumping process and are consistent with the possibility of involvement of residues in helix VIII and the IX-X loop.

Amino Acid Sequence↗
PubMed1995

VH gene structure predicts a large potential anti-insulin repertoire.

The majority of insulin antibodies derived from immunization are IgG antibodies that cross-react extensively with the autologous hormone. To examine the relationship between VH genes expressed by such self-reactive antibodies and their germline (non-rearranged) counterparts, we used the polymerase chain reaction (PCR) to amplify and isolate the germline progenitors of anti-insulin VH genes derived from BALB/c mice immunized with beef or human insulin. Results indicate that two anti-insulin mAbs (123 and 124) express VH genes which arise from a small subset of the J558 gene family and are highly homologous to the VH gene used by the murine CD5 + B-cell tumor, BCL1. The anti-insulin IgG mAb 127 belongs to the VH-VIII (Vgam 3.2) family and the amplification and isolation of germline VH genes from this small family precisely identified only two somatic mutation events in the CDRH2 of mAb 127. Another anti-insulin mAb, 133, also shows two replacement substitutions in the CDRHs when compared to the germline encoded anti-dextran antibody 19.1.2. These findings indicate that the IgG response to this small self-protein uses multiple VH genes which are largely germline encoded with only a low level of somatic mutation in their CDRHs. Additionally, analysis of N-segment additions in CDRH3s indicates anti-insulin B cells may originate from both early (fetal) and adult repertoires. These data are consistent with the concept that the mechanisms of clonal anergy or deletion do not regulate anti-insulin B cells and indicate that there is a large potential VH gene repertoire for insulin.

Amino Acid Sequence↗
PubMed1995

Current management of proliferative vitreoretinopathy.

The recognition of APVR and its dissection, along with the use of perfluorocarbon liquids, has greatly improved the success rate in surgery for severe PVR. In reviewing our first 71 cases using these techniques, 75% were attached with one operation and 90% were attached with one or more operations. These techniques have greatly improved our operative results and have obviated the use of retinal tacks and suturing, retinotomy (in most cases), and the need for very high scleral buckles. (We rarely alter an in-place scleral buckle.)

Fluorocarbons↗
PubMed1994

Site-directed mutagenesis of residues within helix VI in subunit I of the cytochrome bo3 ubiquinol oxidase from Escherichia coli suggests that tyrosine 288 may be a CuB ligand.

The heme-copper oxidase superfamily contains all of the mammalian mitochondrial cytochrome c oxidases, as well as most prokaryotic respiratory oxidases. All members of the superfamily have a subunit homologous to subunit I of the mammalian cytochrome c oxidases. This subunit provides the amino acid ligands to a low-spin heme component as well as to a heme-copper binuclear center, which is the site where dioxygen is reduced to water. The amino acid sequence of transmembrane helix VI of subunit I is the most highly conserved within the superfamily. Previous efforts have demonstrated that one of the residues in this region, H284, is critical for oxidase activity and for the assembly of CuB. This paper presents the analysis of additional site-directed mutants in which other highly conserved residues in helix VI (P285, E286, Y288, and P293) have been substituted. Most of the mutants are enzymatically inactive. Structural perturbations reported by Fourier transform infrared absorption difference spectroscopy of CO adducts of the mutant oxidases confirm the previous suggestion that this region is adjactent to CuB. Furthermore, the analysis of five different substitutions for Y288 indicates that all lack CuB. On the basis of these data, it is proposed that Y288 may be a CuB ligand along with H333, H334, and H284, and a plausible molecular model of the CuB site is presented.

Amino Acid Sequence↗
PubMed1994

A loop between transmembrane helices IX and X of subunit I of cytochrome c oxidase caps the heme a-heme a3-CuB center.

Site-directed mutants were prepared of four consecutive and highly conserved residues (His-411, Asp-412, Thr-413, Tyr-414) of an extramembrane loop that connects putative transmembrane helices IX and X of subunit I of Rhodobacter sphaeroides cytochrome c oxidase. The modified enzymes were purified and analyzed by optical, resonance Raman, FTIR, and EPR spectroscopies. Consistent with our recent model in which both hemes are ligated to histidines of helix X [Hosler, J. P., et al. (1993) J. Bioenerg. Biomembr. 25, 121-136], substitutions for three of these four residues cause perturbations of either heme a or heme a3. Resonance Raman spectra of the mutant Y414F demonstrate that Tyr-414 does not participate in a hydrogen bond with the heme a formyl group, but its alteration does result in a 5-nm red-shift of the alpha-band of the visible spectrum, indicating proximity to heme a. The mutant D412N shows changes in resonance Raman and FTIR difference spectra indicative of an effect on the proximal ligation of heme a3. Changing His-411 to alanine has relatively minor effects on the spectral and functional properties of the oxidase; however, FTIR spectra reveal alterations in the environment of CuB. Conversion of this residue to asparagine strongly disrupts the environment of heme a3 and CuB and inactivates the enzyme. These results suggest that His-411 is very near the heme a3-CuB pocket. We propose that these residues form part of a cap over the heme a-heme a3-CuB center and thus are important in the structure of the active site.

Amino Acid Sequence↗
PubMed1994

Flash photolysis of the carbon monoxide compounds of wild-type and mutant variants of cytochrome bo from Escherichia coli.

The carbon monoxide compounds of the fully reduced and mixed valence forms of cytochrome bo from Escherichia coli were laser photolysed under anaerobic conditions at room temperature. The carbon monoxide recombined with characteristic rate constants of 50 s-1 or 35 s-1 in the fully reduced and mixed valence forms, respectively. Rates of CO recombination with the fully reduced enzyme were examined in a variety of mutant forms of cytochrome bo, produced by site-directed mutagenesis. A method was developed to deconvolute cytochromes bo and bd, leading to some reassessment of histidine ligands to the metals. Significant changes in the rate constant of recombination of carbon monoxide occurred in many of these mutants and these results could be rationalised generally in terms of our current working model of the folding structure of subunit I. In the mixed valence form of the enzyme the transient photolysis spectra in the visible region are consistent with a rapid electron redistribution from the binuclear centre to the low-spin haem. This electron transfer is biphasic, with rate constants of around 10(5) and 8000 s-1. The process was also examined in the His-333-Leu mutant, in which a putative histidine ligand to CuB is replaced by leucine, and which results in the loss of the CuB. It appeared that rapid haem-haem electron transfer could still occur. The observation that CuB is apparently not required for rapid haem-haem electron transfer is consistent with the recently proposed model in which the two haems are positioned on opposite sides of transmembrane helix X in subunit I of the oxidase.

Amino Acid Sequence↗
PubMed1994

Detection of T cells responsive to a vascular growth factor in rheumatoid arthritis.

The primary lesion in rheumatoid arthritis (RA) is a destructive synovitis characterized by proliferation of endothelial cells, fibroblasts, and vascular smooth muscle cells, and with perivascular lymphocyte aggregates. A nonhematopoietic growth factor, acidic fibroblast growth factor (aFGF), may induce many of the biological features found in rheumatoid synovium, including T cell activation. To determine if aFGF-responsive T cells are increased in RA, we developed an assay to measure the frequency of peripheral blood T cells that are costimulated by aFGF. The data indicate that the frequency of aFGF-responsive T cells is increased in RA and may change with disease activity and treatment.

Arthritis, Rheumatoid↗
PubMed1994

The cytochrome oxidase superfamily of redox-driven proton pumps.

Most respiratory oxidases of eukaryotic and prokaryotic organisms are members of a superfamily of enzymes that couple the redox energy available from the reduction of molecular oxygen to the mechanism of pumping protons across the membrane. The recent applications of site-directed mutagenesis and of a variety of spectroscopic techniques have allowed major advances in our understanding of the structure and function of these proteins.

Amino Acid Sequence↗
PubMed1993

Spectroscopic characterization of mutants supports the assignment of histidine-419 as the axial ligand of heme o in the binuclear center of the cytochrome bo ubiquinol oxidase from Escherichia coli.

The bo-type ubiquinol oxidase of Escherichia coli is a member of the superfamily of heme-copper oxidases which also includes the aa3-type cytochrome c oxidases. The oxygen-binding binuclear center of cytochrome bo is located in subunit I and consists of a heme (heme o; heme a3 in the aa3-type oxidases) and a copper (Cu(B)). Previous spectroscopic studies have shown that heme o is bound to the protein via a single histidine residue. Site-directed mutagenesis of conserved histidine residues in subunit I has identified two residues (H284 and H419) which are candidates for the ligand of heme o, while spectroscopic studies of mutants at H284 definitively demonstrated that this residue cannot be the axial ligand. Consequently, the single remaining conserved histidine in subunit I (H419) was assigned as the ligand for the heme of the binuclear center. In this paper, this assignment is tested by characterization of additional mutants in which the putative heme o axial ligand, H419, is replaced by other amino acids. All mutations at H419 result in the loss of enzyme activity. Analyses via UV-visible and Fourier transform infrared spectroscopies reveal that substantial perturbation has occurred at the binuclear center as a result of the amino acid substitutions. In contrast with the wild-type enzyme, the mutant enzymes bind very little carbon monoxide. Three other amino acid residues which are potential ligands for heme o are shown tob e nonessential for enzyme activity. Mutations in these residues do not perturb the UV-visible or FTIR spectroscopic characteristics of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Site-directed mutagenesis of highly conserved residues in helix VIII of subunit I of the cytochrome bo ubiquinol oxidase from Escherichia coli: an amphipathic transmembrane helix that may be important in conveying protons to the binuclear center.

Cytochrome bo from Escherichia coli is a ubiquinol oxidase which is a member of the superfamily of heme-copper respiratory oxidases. This superfamily, which includes the eukaryotic cytochrome c oxidases, has in common a bimetallic center consisting of a high-spin heme component and a copper atom (CuB) which is the site where molecular oxygen is reduced to water. Subunit I, which contains all the amino acid ligands to the metal components of the binuclear center, has 15 putative transmembrane spanning helices, of which 12 are common to the entire superfamily. Transmembrane helix VIII has been noted to contain highly conserved polar residues that fall along one face of the helix. These residues could, in principle, be important components of a pathway providing a conduit for protons from the cytoplasm to gain access to the binuclear center. These conserved residues include Thr352, Thr359, and Lys362. In addition, Pro358, in the middle of this transmembrane helix, is totally conserved in the superfamily. Some substitutions for Thr352 (Ala, Asn) result in major perturbations at the binuclear center as judged by the low-temperature Fourier transform infrared (FTIR) absorbance difference spectroscopy of the CO adducts. Whereas Thr352Ala is inactive enzymatically, both Thr352Asn and Thr352Ser have substantial activity. Substitutions for Thr359 (Ala or Ser) also do not perturb the spectroscopic properties of the binuclear metal center, but the Thr359Ala mutant is devoid of enzyme activity. Changing the neighboring Pro358 to Ala has no detectable effect on the properties of the oxidase. However, all substitutions for Lys362 (Leu, Met, Gln, or Arg) are inactive.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Identity of the axial ligand of the high-spin heme in cytochrome oxidase: spectroscopic characterization of mutants in the bo-type oxidase of Escherichia coli and the aa3-type oxidase of Rhodobacter sphaeroides.

Prokaryotic and eukaryotic cytochrome c oxidases and several bacterial ubiquinol oxidases compose a superfamily of heme-copper oxidases. These enzymes are terminal components of aerobic respiratory chains, the principal energy-generating systems of aerobic organisms. Two such heme-copper oxidases are the aa3-type cytochrome c oxidase of Rhodobacter sphaeroides and the bo-type ubiquinol oxidase of Escherichia coli. These enzymes catalyze the reduction of oxygen to water at a heme-copper binuclear center. Energy conservation is accomplished by coupling electron transfer through the metals of the oxidases to proton translocation across the cellular membrane. The Rb. sphaeroides and E. coli enzymes have previously been utilized in site-directed mutagenesis studies which identified two histidines which bind the low-spin heme (heme a), as well as additional histidine residues which are probable ligands for copper (CuB). However, the histidine that binds the heme of the binuclear center (heme a3) could not be unequivocally identified between two residues (His284 and His419). Additional characterization by Fourier transform infrared spectroscopy of the CO-bound forms of the E. coli enzyme in which His284 is replaced by glycine or leucine demonstrates that these mutations cause only subtle changes to CO bound to the heme of the binuclear center. Resonance Raman spectroscopy of the Rb. sphaeroides enzyme in which His284 is replaced by alanine shows that the iron-histidine stretching mode of heme a3 is maintained, in contrast with the loss of this mode in mutants at His419. These results demonstrate that His284 is not the heme a3 ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Substitution of asparagine for aspartate-135 in subunit I of the cytochrome bo ubiquinol oxidase of Escherichia coli eliminates proton-pumping activity.

The terminal quinol oxidase, cytochrome bo, of Escherichia coli is a member of the large terminal oxidase family, which includes cytochrome aa3-type terminal oxidases from bacteria, plants, and animals. These enzymes conserve energy by linking electron transfer to vectorial proton translocation across mitochondrial or bacterial cell membranes. Site-directed mutagenesis of the five most highly conserved acidic amino acids in subunit I of cytochrome bo was performed to study their role in proton transfer. Mutation of only one of these sites, Asp135, to the corresponding amide, results in a dramatic decrease in proton pumping but with little change in electron-transfer activity. However, the conservative mutation Asp135Glu is active in proton translocation. It is proposed that an acidic residue at position 135 in subunit I may be important to form a functional proton input channel of the proton pump.

Amino Acid Sequence↗
PubMed1993

Monoclonal antibody to rubella virus capsid protein recognizes a beta-cell antigen.

The high frequency of insulin-dependent diabetes (IDDM) in children with congenital rubella suggests that the infectious agent may trigger the autoimmune process. To explore the immunologic relationship between rubella virus and IDDM, we examined a panel of mAb that recognizes rubella virus capsid and envelope glycoproteins for reactivity with islet cell Ag. One mAb, C9, which recognizes a defined domain within the capsid protein of rubella virus, was found to react with extracts from a rat beta-cell tumor and normal rat and human islets. Using one and two dimensional immunoblot analysis of rat beta-cell tumor extracts, the C9-like epitope was found to reside on a 52 kDa protein that is also the target of autoantibodies from human IDDM and nonobese diabetic mice. To confirm this cross-reactivity, antibodies in diabetic sera were absorbed to the recombinant rubella virus capsid protein, eluted, and then shown to react with the 52 kDa insulinoma protein. These data show that an immunogenic epitope on the rubella virus capsid protein is mimicked by a similar structure on a beta-cell protein. These findings suggest that rubella virus has the potential to sensitize susceptible individuals for an autoimmune response to beta-cell Ag and identify one mechanism that may contribute to beta destruction in IDDM.

Amino Acid Sequence↗
PubMed1993

V region diversity in human anti-insulin antibodies. Preferential use of a VHIII gene subset.

Data on structures used by human antibody repertoires are derived principally from lymphoid malignancies and from autoantibodies that often express VH genes from the developmentally regulated fetal repertoire. To determine whether human immune responses generated by exogenous Ag use a pool of VH genes distinct from the fetal repertoire, nucleotide and predicted amino acid sequences were determined for five anti-insulin B cell clones from a type I diabetic patient treated with human insulin. The data show that a set of VHIII genes is preferentially used by human anti-insulin B cells. Structural features indicate that these expressed VH are derived from germ-line genes that are not frequent in fetal repertoires and these genes have undergone Ag-driven somatic mutation. The preferential use of related VH segments contrasts with the BALB/c anti-insulin response, which uses multiple V genes elements largely unmutated from germ-line sequences. In addition, long CDRH3 structures in human anti-insulin mAb are generated by complex gene interaction mechanisms that are not seen in murine anti-insulin mAb. Interestingly, similar potential insulin-binding structures are used by antibodies from both species. These findings suggest that human responses to exogenous insulin may express a limited number of VH genes and depend upon somatic mutation and complex D gene interactions in CDRH3 to expand the repertoire. Although these antibodies bind autologous insulin, VH gene usage and structural features that predominate in the response are not characteristic of the fetal repertoire.

Amino Acid Sequence↗
PubMed1993

Insight into the active-site structure and function of cytochrome oxidase by analysis of site-directed mutants of bacterial cytochrome aa3 and cytochrome bo.

Cytochrome aa3 of Rhodobacter sphaeroides and cytochrome bo of E. coli are useful models of the more complex cytochrome c oxidase of eukaryotes, as demonstrated by the genetic, spectroscopic, and functional studies reviewed here. A summary of site-directed mutants of conserved residues in these two enzymes is presented and discussed in terms of a current model of the structure of the metal centers and evidence for regions of the protein likely to be involved in proton transfer. The model of ligation of the heme a3 (or o)-CuB center, in which both hemes are bound to helix X of subunit I, has important implications for the pathways and control of electron transfer.

Amino Acid Sequence↗