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I Gibbons

Publications and source records attributed to I Gibbons.

At least 19 recordsLinked to original sources
PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗
PubMed2001

Multiplexed enzyme assays in capillary electrophoretic single-use microfluidic devices.

We describe a method of performing multiple enzyme assays in a single reaction vessel. The resolving power of capillary electrophoresis enables several enzyme assays to be analyzed at high speed in microfluidic arrays. Multiplexed measurement can increase throughput significantly without requiring highly dense microfluidic arrays. Enzyme assays in a multiplexed format for selected kinases in this work show essentially identical performance to assays performed individually. This establishes an approach for screening one compound against multiple enzyme targets simultaneously. Another potential application for performing multiplexed enzyme assay is to study protein-protein (especially enzyme-enzyme) interaction by monitoring the enzymatic activity changes.

Amino Acid Sequence↗
PubMed1998

Discrimination of site-specific alterations in gastrointestinal permeability in the rat.

BACKGROUND & AIMS: Detection of gastrointestinal damage can be accomplished by permeability testing. However, current methodology presents several problems, including lack of site specificity and an inability to easily measure colonic damage. We hypothesized that probes of similar size could be used to simultaneously assess permeability along the length of the gastrointestinal tract. Differentiation of damage sites could be made, provided that probes were selectively destroyed at different levels. Class 1 probes should be destroyed after leaving the stomach, class 2 after leaving the small intestine, and class 3 not at all. In this manner, class 1 probes would report damage to the proximal gut, class 2 to the small intestine, and class 3 to the gut as a whole. METHODS: We defined saccharide probes with these characteristics and observed their permeability patterns in defined models of localized gastrointestinal damage. RESULTS: With gastric damage, permeability of only class 1 probes increased, whereas with colonic damage only permeation rates of class 3 probes was increased. Furthermore, the permeation rates of both class 2 and 3 probes increased in the presence of small intestinal disease. CONCLUSIONS: These techniques allow a single screening test that is sensitive to damage at any level of the gastrointestinal tract and may be used in either animals or humans.

Animals↗
PubMed1994

A family of dynein genes in Drosophila melanogaster.

We report the identification and initial characterization of seven Drosophila dynein heavy chain genes. Each gene is single copy and maps to a unique genomic location. Sequence analysis of partial clones reveals that each encodes a highly conserved portion of the putative dynein hydrolytic ATP-binding site in dyneins that includes a consensus phosphate-binding (P-loop) motif. One of the clones is derived from a Drosophila cytoplasmic dynein heavy chain gene, Dhc64C, that shows extensive amino acid identity to cytoplasmic dynein isoforms from other organisms. Two other Drosophila dynein clones are 85 and 90% identical at the amino acid level to the corresponding region of the beta heavy chain of sea urchin axonemal dynein. Probes for all seven of the dynein-related sequences hybridize to transcripts that are of the appropriate size, approximately 14 kilobases, to encode the characteristic high molecular weight dynein heavy chain polypeptides. The Dhc64C transcript is readily detected in RNA from ovaries, embryos, and testes. Transcripts from five of the six remaining genes are also detected in much lesser amounts in tissues other than testes. All but one of the dynein transcripts are expressed at comparable levels in testes suggesting their participation in flagellar axoneme assembly and motility.

Adenosine Triphosphate↗
PubMed1992

Negative complementation in aspartate transcarbamylase. Analysis of hybrid enzyme molecules containing different arrangements of polypeptide chains from wild-type and inactive mutant catalytic subunits.

A comprehensive set of hybrid molecules of aspartate transcarbamylase (ATCase) from Escherichia coli has been constructed of wild-type and mutationally altered catalytic chains. The mutant enzymes that were virtually devoid of activity contained a replacement of Gly-128 in the catalytic polypeptide chains by either Asp or Arg. The kinetic properties of these hybrid enzyme-like molecules were analyzed to evaluate the basis for the unusual quaternary constraint demonstrated by an intersubunit hybrid containing one wild-type catalytic subunit, one inactive mutant subunit (containing the Gly to Asp replacement), and three wild-type regulatory subunits. A similar intersubunit hybrid was constructed from the wild-type catalytic subunit and the mutant in which Gly-128 was replaced by Arg, and it too demonstrated a pronounced decrease in activity relative to that expected for a hybrid containing three active sites. Moreover, neither of these hybrid holoenzymes exhibited the cooperativity with respect to aspartate that is characteristic of wild-type ATCase. In contrast, hybrid holoenzymes containing at least one wild-type chain in each catalytic subunit showed cooperativity. Also, hybrid enzymes containing different arrangements of five, four, three, or two wild-type catalytic chains with an appropriate complement of mutant chains had specific activities proportional to the number of wild-type chains in the holoenzymes. Exceptions were observed only in hybrids in which one of the two subunits in the holoenzyme was composed completely of mutant catalytic chains. For these hybrids the negative complementation was manifested as a much lower enzyme activity than expected from the number of wild-type chains in the enzyme and the loss of cooperativity. Thus, the activity and allosteric properties of these hybrids is dependent on the arrangement of catalytic chains in the holoenzyme, in contrast to results obtained for hybrids containing native and chemically modified catalytic chains. Intrasubunit hybrid catalytic trimers containing one or two wild-type chains exhibited one-third and two-thirds the activity of the intact wild-type catalytic subunit, respectively, indicating the dominant negative effect that was seen in intersubunit hybrid holoenzymes is absent within trimers.

Aspartate Carbamoyltransferase↗
PubMed1989

Patient-side immunoassay system with a single-use cartridge for measuring analytes in blood.

We describe an immunoassay system suited to patient-side assay of therapeutic drugs and blood proteins. The system consists of an electronic monitor and single-use plastic cartridges containing dry reagents and liquid diluents. The monitor is turned on by insertion of a cartridge. To run the test, the user applies an unmeasured drop of blood to the cartridge when prompted by the monitor. All subsequent steps are performed without further user intervention and results are provided in less than 3 min. The system hemolyzes and precisely dilutes the blood. Hemoglobin concentration is measured, then the diluted blood is precisely diluted further and mixed with two dry reagents. The drug concentration is measured by a turbidimetric latex agglutination inhibition reaction. Theophylline and hemoglobin assay results for clinical samples correlate well with results of widely used comparison methods.

Hemoglobins↗
PubMed1987

Monoclonal antibodies to glucose-6-phosphate dehydrogenase (G6PDH) form cyclic 1:1 complexes with G6PDH and act as regulatory subunits.

A number of IgG monoclonal antibodies against L. mesenteroides glucose-6-phosphate dehydrogenase (G6PDH) have been prepared. Four of the antibodies form 1:1 enzyme-antibody complexes which are stabilized in the presence of glucose-6-phosphate (G6P) and have greatly reduced enzyme activity. In the absence of G6P, the 1:1 complexes convert gradually to a more active multimeric form. Reduction of the IgG inter-heavy chain disulfides partially relieves inhibition and removes the G6P requirement for stability. F(ab')2 fragments of one of the antibodies behave similarly to the intact IgG. Reduction of the disulfides in the G6PDH-F(ab')2 complex leads to complete recovery of activity. The activity of complexes of G6PDH with reduced antibodies or Fab with digoxin bound to the antibody or Fab sulfhydryl groups can be modulated with antibodies to digoxin. The anti-G6PDH antibodies bridge two identical epitopes of this two subunit enzyme and simulate the function of regulatory subunits in which anti-digoxin acts as an activator. The system can be used to provide a sensitive homogeneous immunoassay for digoxin.

Animals↗
PubMed1987

Use of liposome encapsulation in a combined single-liquid reagent for homogeneous enzyme immunoassay.

A technique has been developed to permit mutually reactive macromolecular reagents used in immunoassays to be combined without premature reaction. A conjugate of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and theophylline has been encapsulated in 0.2-micron-diameter bi-lamellar liposomes. Suspensions of these liposomes had excellent stability. Whereas the enzyme activity of the free conjugate is rapidly inhibited by anti-theophylline antibody, a suspension of the encapsulated conjugate in a solution of the antibody and NAD+ (6.0 mmol/L) retained greater than 92% of the initial enzyme activity after standing for one year at 4 degrees C. At higher NAD+ concentrations the liposomes aggregated, and enzyme activity was inhibited by leakage of the NAD+ hydrolysis product, adenosine diphosphoryl 5-ribose (ADP-ribose), into the liposomes. Inhibition by ADP-ribose could be blocked and partly reversed by adding semicarbazide. The liposomes were efficiently lysed by Triton X-100, deoxycholate, or octyl glucoside, the kinetics and extent of lysis being affected by liposome size and correlating with the acid strength of various cholate derivatives. Addition of a serum sample and a solution of buffer, substrate, and detergent to a single reagent containing the liposomes and anti-theophylline antibody provided assay results equivalent to those obtained by conventional two-reagent EMIT homogeneous enzyme immunoassay for theophylline.

Adenosine Diphosphate Ribose↗
PubMed1985

Action of beta-galactosidase on novel synthetic macromolecular substrates. A processive enzymic reaction controlled by coulombic interactions.

Macromolecular beta-galactosidase substrates were prepared by attaching o-nitrophenyl-beta-galactoside to carboxymethyldextran with positively charged linking groups. Almost all of the substituents were susceptible to enzymic hydrolysis by two distinct pathways. Under some conditions, there was random reaction to give a soluble product. In other conditions, in the initial stages of the reaction, most of the substituents of some, but not all, of the substrate polymers were hydrolyzed to give a product which precipitated as a second aqueous phase. Kinetics of hydrolysis were studied with respect to charge and molecular weight of both the enzyme and substrate. Factors that caused a decrease in Km favored formation of the second phase product. The reaction has similarities to the processive catalytic reactions found in naturally occurring enzyme systems with polymeric charged substrates.

Dextrans↗
PubMed1985

Improved sensitivity in homogeneous enzyme immunoassays using a fluorogenic macromolecular substrate: an assay for serum ferritin.

A new highly sensitive nonseparation enzyme immunoassay for human serum ferritin is described. Reagents include a beta-galactosidase-ferritin conjugate, sheep anti-ferritin, anti-sheep IgG, and dextran-linked beta-galactosylumbelliferone as enzyme substrate. The method is based on inhibition of enzyme activity when anti-ferritin binds to the enzyme-ferritin conjugate. Ferritin in the sample and enzyme-labeled ferritin compete for a limited quantity of anti-ferritin. The enzyme activity of the reaction mixture is directly related to the ferritin content of the sample. Some patients' samples caused strong interference in the assay due to the presence of antibody to beta-galactosidase. Several ways of eliminating the interference are presented. When measures were adopted to suppress sample interference, the assay results correlated well with those of other immunoassay methods.

Antibody Affinity↗
PubMed1981

Enzyme-enhancement immunoassay: a homogeneous assay for polyvalent ligands and antibodies.

A homogeneous enzyme immunoassay for proteins has been developed that avoids the need for a labeled antigen. The technique involves antibody labeled with beta-galactosidase (EC 3.2.1.23), succinylated antibody, and a macromolecular o-nitrophenyl-beta-galactoside substrate. The enzyme-labeled antibody and the succinylated antibody form an immune complex in the presence of sample antigen. An enzyme within this negatively charged microenvironment produces a product that forms a second light-scattering phase, whereas the product produced by free enzyme remains soluble. Thus the antigen modulates the rate of increase in light scattering. The technique has been applied to assays for human immunoglobulin G and C-reactive protein as well as for specific antibodies.

Antibodies↗
PubMed1979

Extended amino acid sequences around the active-site lysine residue of class-I fructose 1,6-bisphosphate aldolases from rabbit muscle, sturgeon muscle, trout muscle and ox liver.

1. Amino acid sequences covering the region between residues 173 and 248 [adopting the numbering system proposed by Lai, Nakai & Chang (1974) Science 183, 1204-1206] were derived for trout (Salmo trutta) muscle aldolase and for ox liver aldolase. A comparable sequence was derived for residues 180-248 of sturgeon (Acipenser transmontanus) muscle aldolase. The close homology with the rabbit muscle enzyme was used to align the peptides of the other aldolases from which the sequences were derived. The results also allowed a partial sequence for the N-terminal 39 residues for the ox liver enzyme to be deduced. 2. In the light of the strong homology evinced for these enzymes, a re-investigation of the amino acid sequence of rabbit muscle aldolase between residues 181 and 185 was undertaken. This indicated the presence of a hitherto unsuspected -Ile-Val-sequence between residues 181 and 182 and the need to invert the sequence -Glu-Val- to -Val-Glx- at positions 184 and 185. 3. Comparison of the available amino acid sequences of these enzymes suggested an early evolutionary divergence of the genes for muscle and liver aldolases. It was also consistent with other evidence that the central region of the primary structure of these enzymes (which includes the active-site lysine-227) forms part of a conserved folding domain in the protein subunit. 4. Detailed evidence for the amino acid sequences proposed has been deposited as Suy Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.

Amino Acid Sequence↗
PubMed1977

Ligand-promoted weakening of intersubunit bonding domains in aspartate transcarbamolylase.

THE COOPERATIVITY AND FEEDBACK INHIBITION EXHIBITED BY THE REGULATORY ENZYME, ASPARTATE TRANSCARBAMOYLASE (CARBAMOYLPHOSPHATE: L-aspartate carbamoyltransferase; EC 2.1.3.2), from Escherichia coli are generally attributed to ligand-promoted conformational changes involving alterations in the subunit interactions. However, no quantitative estimates have been made of the effect of ligands on the strength of the intersubunit bonding domains. The native enzyme, composed of two catalytic trimers "crosslinked" by three regulatory dimers, shows little tendency to dissociate in neutral buffers at room temperature. In addition, very little exchange was observed in 2 hr between subunits within the intact enzyme and free subunits. Although exchange was enhanced in solutions of low ionic strength containing the bisubstrate analog, N-(phosphonacetyl)-L-aspartate, the rates of exchange were too small to permit reliable estimates of the weakening of the bonding domains caused by the ligand. Studies were conducted, therefore, on a less stable oligomeric complex which resembles the native enzyme in structure and allosteric behavior but lacks one regulatory subunit. These molecules, containing only four bonding domains between the catalytic and regulatory polypeptide chains (compared to six in the native enzyme), disproportionate to form the more stable native enzyme and free catalytic subunits. An electrophoretic technique is described for measuring the rate of disproportionation which is controlled by the rupture of the intersubunit bonding domains. This rate is enhanced about 300-fold upon the addition of the active-site ligand. Hence the ligand-promoted allosteric conversion of the enzyme-like complex from the constrained to the relaxed conformation involves a substantial weakening of the intersubunit interactions corresponding to about 1.7 kcal/mole (7.1 kJ/mole) per bonding domain between a catalytic and a regulatory chain.

Aspartate Carbamoyltransferase↗
PubMed1976

Concerted allosteric transition in hybrids of aspartate transcarbamoylase containing different arrangements of active and inactive sites.

Various hybrids of aspartate transcarbamoylase of Escherichia coli were constructed from native regulatory subunits and mixtures of active and inactive (pyridoxylated) catalytic chains in specific arrangements within the two catalytic subunits. The kinetic and physical properties of these well-defined hybrids were studied in order to determine the effects of reducing the number of substrate binding sites and distributing the active and inactive chains in different ways. Experiments on enzyme-like molecules containing six, four, three, two, and one active sites showed that the Hill coefficient decreased and the apparent Km increased as the number of active chains in the hybrids was reduced. The maximum inhibition and activation by the nucleotide effectors, CTP and ATP, were independent of the composition of the enzyme-like molecules. Two hybrids were of particular interest since one contained two active sites in one catalytic subunit and none in the other, and the second hybrid had one active site in each catalytic subunit. These two hybrids exhibited identical kinetic behavior despite the markedly different structural arrangements. The ligand-promoted conformational changes of the hybrids monitored both by sedimentation velocity measurements and the reactivity toward p-hydroxymercuribenzoate were similar to those of the native enzyme. These results indicate that there are no discrete "cooperative units" within the enzyme molecules but rather that the allosteric transition promoted by ligands is fully concerted. The various kinetic and physical properties can be accounted for satisfactorily in terms of the two-state model of Monod et al.

Allosteric Regulation↗