Search PubMed⌕ Search

Biomedical subjects

A J Wilkinson

Publications and source records attributed to A J Wilkinson.

At least 73 records · Page 4Linked to original sources
PubMed1993

The mechanism of autooxidation of myoglobin.

Time courses for the autooxidation of native and mutant sperm whale and pig myoglobins were measured at 37 degrees C in the presence of catalase and superoxide dismutase. In sperm whale myoglobin, His64(E7) was replaced with Gln, Gly, Ala, Val, Thr, Leu, and Phe; Val68(E11) was replaced with Ala, Ile, Leu, and Phe; Leu29(B10) was replaced with Ala, Val, and Phe. In pig myoglobin, His64(E7) was replaced with Val; Val68(E11) was replaced with Thr and Ser; Thr67(E10) was replaced with Ala, Val, Glu, and Arg; Lys45(CD3) was replaced with Ser, Glu, His, and Arg. The observed pseudo-first order rate constants varied over 4 orders of magnitude, from 58 h-1 (H64A) to 0.055 h-1 (native) to 0.005 h-1 (L29F) at 37 degrees C, pH 7, in air. The dependences of the observed autooxidation rate constant on oxygen concentration and pH were measured for native and selected mutant myoglobins. In the native proteins and in most mutants still possessing the distal histidine, autooxidation occurs through a combination of two mechanisms. At high [O2], direct dissociation of the neutral superoxide radical (HO2) from oxymyoglobin dominates, and this process is accelerated by decreasing pH. At low [O2], autooxidation occurs by a bimolecular reaction between molecular oxygen and deoxymyoglobin containing a weakly coordinated water molecule. The neutral side chain of the distal histidine (His64) inhibits autooxidation by hydrogen bonding to bound oxygen, preventing both HO2 dissociation and the oxidative bimolecular reaction with deoxymyoglobin. Replacement of His64 by amino acids incapable of hydrogen bonding to the bound ligand markedly increases the rate of autooxidation and causes the superoxide mechanism to predominate. Increasing the polarity of the distal pocket by substitution of Val68 with Ser and Thr accelerates autooxidation, presumably by facilitating protonation of the Fe(II).O2 complex. Increasing the net anionic charge at the protein surface in the vicinity of the heme group also enhances the rate of autooxidation. Decreasing the volume of the distal pocket by replacing small amino acids with larger aliphatic or aromatic residues at positions 68 (E11) and 29 (B10) inhibits autooxidation markedly by decreasing the accessibility of the iron atom to solvent water molecules.

Animals↗
PubMed1992

Role of B13 Glu in insulin assembly. The hexamer structure of recombinant mutant (B13 Glu-->Gln) insulin.

The assembly of the insulin hexamer brings the six B13 glutamate side-chains at the centre into close proximity. Their mutual repulsion is unfavourable and zinc co-ordination to B10 histidine is necessary to stabilize the well known zinc-containing hexamers. Since B13 is always a carboxylic acid in all known sequences of hexamer forming insulins, it is likely to be important in the hormone's biology. The mutation of B13 Glu-->Gln leads to a stable zinc-free hexamer with somewhat reduced potency. The structures of the zinc-free B13 Gln hexamer and the 2Zn B13 insulin hexamer have been determined by X-ray analysis and refined with 2.5 A and 2.0 A diffraction data, respectively. Comparisons show that in 2Zn B13 Gln insulin, the hexamer structure (T6) is very like that of the native hormone. On the other hand, the zinc-free hexamer assumes a quaternary structure (T3/R3) seen in the native 4Zn insulin hexamer, and normally associated only with high chloride ion concentrations in the medium. The crystal structures show the B13 Gln side-chains only contact water in contrast to the B13 glutamate in 2Zn insulin. The solvation of the B13 Gln may be associated with this residue favouring helix at B1 to B8. The low potency of the B13 Gln insulin also suggests the residue influences the hormone's conformation.

Animals↗
PubMed1992

Oxidation of residue 45 mutant forms of pig deoxymyoglobin with [Fe(CN)6]3-.

The effect of replacement of the highly conserved Lys45 residue in pig myoglobin (Mb) with His, Ser, Glu, and Arg has been investigated. Rate constants/M-1 s-1 at 25 degrees C and pH 8.0, I = 0.100 M (NaCl), for the oxidation of deoxyMb with [Fe(CN)6]3- have been determined, and are for wild-type Lys45 (2.83 x 10(6)), His45 (1.02 x 10(6)), Ser45 (1.12 x 10(6)), Glu45 (0.87 x 10(6)), and Arg45 (3.06 x 10(6)). It is concluded that charge on the residue at position 45 has only a mild effect on reactivity, and that this is unlikely to be the site for electron transfer.

Animals↗
PubMed1992

High-resolution X-ray structures of pig metmyoglobin and two CD3 mutants: Mb(Lys45----Arg) and Mb(Lys45----Ser).

The structure of pig aquometmyoglobin has been refined to a crystallographic R-factor of 19.8% against X-ray diffraction data between 10- and 1.75-A spacing. The final structural model comprises two molecules of pig myoglobin, 233 water molecules, and two sulfate ions. A water molecule is coordinated to each of the heme iron atoms with an average Fe-OH2 bond distance of 2.19 A, and the mean Fe-N epsilon (proximal histidine-93) distance is 2.20 A. In contrast to the structure of sperm whale metmyoglobin, the iron is not significantly displaced from the plane of the heme. At the entrance to the heme pocket, the side-chain amino group of lysine-45 (CD3) is well-defined in the electron density map and forms salt-bridging interactions with the heme 6-propionate and with a sulfate ion. Serine and arginine replacements have been made previously at position 45 to examine the proposal that the CD3 side chain acts as a barrier to ligand entry into the protein. Crystal structures of the arginine-45 and serine-45 mutant metmyoglobins have been solved to 1.9 and 2.0 A resolution, respectively. In both cases the structural changes are confined to the site of mutation. Arginine-45 takes up a conformation closely similar to that observed for this residue in wild-type sperm whale myoglobin, in which it makes more extensive charge-charge and charge-dipole interactions and appears to restrict the movement of the distal histidine away from the ligand. The hydroxyl group of serine-45 is disordered, but it is clear that the effect of the mutation is to open up the solvent-exposed face of the heme pocket.

Animals↗
PubMed1991

Structure and molecular model refinement of Aspergillus oryzae (TAKA) alpha-amylase: an application of the simulated-annealing method.

Monoclinic crystals of a neutral alpha-amylase from Aspergillus oryzae, containing three molecules in the asymmetric unit, have been reported previously and studied at 3 A resolution [Matsuura, Kunusoki, Harada & Kakudo (1984). J. Biochem. 95, 697-702]. Here we report the solution of the structure of this enzyme in a different crystal form (space group P2(1)2(1)2(1), a = 50.9, b = 67.2, c = 132.7 A), with only one molecule in the asymmetric unit. The structure was solved by the molecular replacement method, using a model of acid alpha-amylase from a related fungus A. niger [Brady, Brzozowski, Derewenda, Dodson & Dodson (1991). Acta Cryst. B47, 527-535]. Conventional least-squares crystallographic refinement failed to converge in a satisfactory manner, and the technique of molecular dynamics in the form of the XPLOR package [Brunger (1988). XPLOR Manual. Yale Univ., USA] was used to overcome the problem. A large rigid-body type movement of the C-terminal domain was identified and accounted for. The final round of restrained least-squares refinement (at 2.1 A resolution) including 3675 protein atoms and 247 water molecules resulted in a conventional crystallographic R factor of 0.183 and an atomic model which conforms well to standard stereochemical parameters (standard deviation of bond lengths from their expected values is 0.028 A, while that for planar groups is 0.029 A).

Aspergillus oryzae↗
PubMed1991

Distal pocket polarity in ligand binding to myoglobin: structural and functional characterization of a threonine68(E11) mutant.

Site-directed mutagenesis studies have confirmed that the distal histidine in myoglobin stabilizes bound O2 by hydrogen bonding and have suggested that it is the polar character of the imidazole side chain rather than its size that limits the rate of ligand entry into the protein. We constructed an isosteric Val68 to Thr replacement in pig myoglobin (i) to investigate whether the O2 affinity could be increased by the introduction of a second hydrogen-bonding group into the distal heme pocket and (ii) to examine the influence of polarity on the ligand binding rates more rigorously. The 1.9-A crystal structure of Thr68 aquometmyoglobin confirms that the mutant and wild-type proteins are essentially isostructural and reveals that the beta-OH group of Thr68 is in a position to form hydrogen-bonding interactions both with the coordinated water molecule and with the main chain greater than C=O of residue 64. The rate of azide binding to the ferric form of the Thr68 mutant was 60-fold lower than that for the wild-type protein, consistent with the proposed stabilization of the coordinated water molecule. However, bound O2 is destabilized in the ferrous form of the mutant protein. The observed 17-fold lowering of the O2 affinity may be a consequence of the hydrogen-bonding interaction made between the Thr68 beta-OH group and the carbonyl oxygen of residue 64. Overall association rate constants for O2, NO, and alkyl isocyanide binding to ferrous pig myoglobin were 3-10-fold lower for the mutant compared to the wild-type protein, whereas that for CO binding was little affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1991

The solution structure of human transforming growth factor alpha.

The solution structure of transforming growth factor alpha has been determined by a combination of high-resolution 1H-nuclear magnetic resonance and distance geometry and restrained molecular dynamics. The 382 restraints derived from the NMR experiments were used to calculate many distance geometry structures, which were then refined by restrained molecular mechanics. Five of these structures were further refined using a variety of methods. Comparison of independently measured parameters, such as calculated hydrogen bonding patterns and experimental amide exchange rates, have been used to evaluate the accuracy of the structures. Also, possible mechanisms to explain the pH-dependent conformational interconversion observed are suggested. Finally comparisons between this work and others on this topic have been made.

Amino Acid Sequence↗
PubMed1991

Contributions of residue 45(CD3) and heme-6-propionate to the biomolecular and geminate recombination reactions of myoglobin.

Overall association and dissociation rate constants were measured at 20 degrees C for O2, CO, and alkyl isocyanide binding to position 45 (CD3) mutants of pig and sperm whale myoglobins and to sperm whale myoglobin reconstituted with protoheme IX dimethyl ester. In pig myoglobin, Lys45(CD3) was replaced with Arg, His, Ser, and Glu; in sperm whale myoglobin, Arg45(CD3) was replaced with Ser and Gly. Intramolecular rebinding of NO, O2, and methyl isocyanide to Arg45, Ser45, Glu45, and Lys45(native) pig myoglobins was measured following 35-ps and 17-ns excitation pulses. The shorter, picosecond laser flash was used to examine ligand recombination from photochemically produced contact pairs, and the longer, nanosecond flash was used to measure the rebinding of ligands farther removed from the iron atom. Mutations at position 45 or esterification of the heme did not change significantly (less than or equal to 2-fold) the overall association rate constants for NO, CO, and O2 binding at room temperature. These data demonstrate unequivocally that Lys(Arg)45 makes little contribution to the outer kinetic barrier for the entry of diatomic gases into the distal pocket of myoglobin, a result that contradicts a variety of previous structural and theoretical interpretations. However, the rates of geminate recombination of NO and O2 and the affinity of myoglobin for O2 were dependent upon the basicity of residue 45. The series of substitutions Arg45, Lys45, Ser45, and Glu45 in pig myoglobin led to a 3-fold decrease in the initial rate for the intramolecular, picosecond rebinding of NO and 4-fold decrease in the geminate rate constant for the nanosecond rebinding of O2. (ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Proximal gastric vagotomy: follow-up at 10-20 years.

From August 1969 to December 1989, 600 patients had elective proximal gastric vagotomy for duodenal ulceration with an operative mortality of 0.2 per cent. Of these, 372 patients had surgery over 10 years ago. Three hundred and forty-two patients survived for more than 10 years and, in a prospective study, 305 were reviewed, forming the basis of this 10-20-year follow-up report. Forty-six (15 per cent) have had recurrent ulceration; 80 per cent of these developed symptoms within 5 years and no patient has had recurrence after 13 years. Although 29 patients required reoperation for recurrent ulceration, the current patient satisfaction rate for Visick grades I and II is 92 per cent. Only two patients required reoperation because of gastric stasis. It is concluded that proximal gastric vagotomy is a safe and satisfactory first choice operation for duodenal ulceration.

Adolescent↗
PubMed1991

Reduction in CD4 binding affinity associated with removal of a single glycosylation site in the external glycoprotein of HIV-2.

The role of selected glycosylation sites in the ability of the envelope glycoprotein of HIV-2 (gp 105) to bind to CD4 has been investigated. Loss of glycosylation sites at amino acids 410 and 447 did not affect the CD4 binding ability of gp 105 even when removed in pair combination. Loss of a single glycosylation site at amino acid 400, however, was sufficient to cause a reduction of at least 50-fold in the efficiency of receptor binding. These data support the hypothesis that some of the carbohydrate side chains on gp 105 have a profound effect on biological activity.

Base Sequence↗
PubMed1990

Determination of the crystal structure of recombinant pig myoglobin by molecular replacement and its refinement.

As part of a protein engineering study, the X-ray crystal structure of recombinant pig myoglobin, prepared and crystallized from E. coli, has been determined. Diffraction data were collected to 2.5 A spacing using a synchrotron X-ray source. The structure was solved using the molecular-replacement method and refined using least-squares minimization procedures to a crystallographic R factor of 18.5% using 14,481 reflections between 10 and 2.5 A. A preliminary comparison of the structure of pig myoglobin with other myoglobin structures is presented.

Animals↗
PubMed1990

Expression of HIV-1 gp120 and human soluble CD4 by recombinant baculoviruses and their interaction in vitro.

The soluble domains of the envelope glycoprotein of HIV-1 (gp120) and human CD4 (sCD4) have been individually expressed in insect cells using recombinant baculoviruses. Each product is secreted from infected cells and accumulates in the surrounding media to levels of 1-2 mg/liter of 2 x 10(9) cells. Both molecules have full biological activity, and conditioned media from infected cells have been used to establish a simple assay for gp120-sCD4 interaction that is highly specific and amenable to mass screening. The crystallization of sCD4 purified from this source is reported.

Animals↗
PubMed1990

Early gastric cancer: a report of 30 cases.

Thirty patients with early gastric cancer have been reviewed. Endoscopy demonstrated gastric abnormality in 93% of patients examined and biopsy was accurate in 96% of cases. Twenty-nine patients were treated by partial gastrectomy, although three required further procedures. The commonest morphological type was type III (45%). Five patients with early gastric cancer had lymph node spread at the time of presentation. The relative 5-year survival rate was 68%. Four patients eventually died of gastric cancer. Three patients (10%) developed a second primary carcinoma. The significance of this diagnosis is emphasized and selective screening is considered.

Adult↗
PubMed1988

Apomyoglobin as a molecular recognition surface: expression, reconstitution and crystallization of recombinant porcine myoglobin in Escherichia coli.

Recombinant porcine myoglobin has been produced in Escherichia coli using the lambda cII fusion expression system of Nagai and Thøgersen [Nature, 309, 810-812 (1984)]. After processing and reconstitution with haem, the protein is gel-electrophoretically and spectrophotometrically indistinguishable from native pig myoglobin. Large crystals of both native and recombinant porcine myoglobin were grown from 50 mM sodium phosphate, pH 7.1, 80% ammonium sulphate. The crystals belong to space group C2 (a = 156.9 A, b = 42.0 A, c = 92.2 A, beta = 127.9 degrees) and diffract to a nominal 2.5 A resolution. We plan to explore apomyoglobin as a binding surface in studies combining site-directed mutagenesis and X-ray analysis. These experiments will be extended by studying the binding of haem analogues to the mutant apoproteins.

Animals↗
PubMed1987

Impedance plethysmography. A screening procedure to detect deep-vein thrombosis.

Impedance plethysmography has great potential in the non-invasive detection of dangerous iliofemoral thrombosis. It was used to examine 198 patients undergoing total hip replacement for evidence of proximal venous segment thrombosis. There were 13 abnormal results, and subsequent venography in 12 of these revealed a false-positive rate of 4.0%; there was one false-negative result giving a sensitivity of 85.7%. A venographic study of 46 other patients clinically suspected of having a proximal deep-vein thrombosis confirmed this sensitivity. The iliofemoral thrombosis rate was 3.9% after total hip replacement and this rate increased significantly in the group of patients over 70 kg in weight. Impedance plethysmography was found to be a useful non-invasive screening procedure for potentially fatal proximal venous thrombosis.

Adult↗