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Biomedical subjects

N C Price

Publications and source records attributed to N C Price.

At least 19 recordsLinked to original sources
PubMed1992

The secondary structure of influenza A M2 transmembrane domain. A circular dichroism study.

Using circular dichroism, this study investigated the secondary structure of the influenza A M2 transmembrane domain. When reconstituted into 1,2-dioleoyl-sn-glycero-3-phosphocholine liposomes, the M2 transmembrane domain was found to adopt a predominantly alpha-helical secondary structure which was unaffected by both temperature and the addition of 1-aminoadamantane hydrochloride. Reconstitution into 1,2-dioleoyl-sn-glycero-3-phosphoglycerol liposomes resulted in a marked decrease in helical content.

Amantadine↗
PubMed1992

Transglutaminase catalyses the modification of glutamine side chains in the C-terminal region of bovine beta-lactoglobulin.

The transglutaminase-catalysed incorporation of primary amines (putrescine and monodansylcadaverine) into bovine beta-lactoglobulin has been studied. In the presence of 1 mM-dithiothreitol between 1 and 2 mol of amine can be incorporated per mol of beta-lactoglobulin subunit. There is very little incorporation of amines in the absence of reducing agent. By isolating and sequencing the modified peptides, the sites of modification have been identified as Gln-159 (preferred) and Gln-155. C.d. has been used to study the structure of beta-lactoglobulin over a range of pH values and in the presence or absence of dithiothreitol. The results are discussed in terms of the X-ray-crystallographically determined structure of beta-lactoglobulin.

Amino Acid Sequence↗
PubMed1992

A comparison of the enzymological and biophysical properties of two distinct classes of dehydroquinase enzymes.

This paper compares the biophysical and mechanistic properties of a typical type I dehydroquinase (DHQase), from the biosynthetic shikimate pathway of Escherichia coli, and a typical type II DHQase, from the quinate pathway of Aspergillus nidulans. C.d. shows that the two proteins have different secondary-structure compositions; the type I enzyme contains approx. 50% alpha-helix while the type II enzyme contains approx. 75% alpha-helix. The stability of the two types of DHQase was compared by denaturant-induced unfolding, as monitored by c.d., and by differential scanning calorimetry. The type II enzyme unfolds at concentrations of denaturant 4-fold greater than the type I and through a series of discrete transitions, while the type I enzyme unfolds in a single transition. These differences in conformational stability were also evident from the calorimetric experiments which show that type I DHQase unfolds as a single co-operative dimer at 57 degrees C whereas the type II enzyme unfolds above 82 degrees C and through a series of transitions suggesting higher orders of structure than that seen for the type I enzyme. Sedimentation and Mr analysis of both proteins by analytical ultracentrifugation is consistent with the unfolding data. The type I DHQase exists predominantly as a dimer with Mr = 46,000 +/- 2000 (a weighted average affected by the presence of monomer) and has a sedimentation coefficient s0(20,w) = 4.12 (+/- 0.08) S whereas the type II enzyme is a dodecamer, weight-average Mr = 190,000 +/- 10,000 and has a sedimentation coefficient, s0(20,w) = 9.96 (+/- 0.21) S. Although both enzymes have reactive histidine residues in the active site and can be inactivated by diethyl pyrocarbonate, the possibility that these structurally dissimilar enzymes catalyse the same dehydration reaction by the same catalytic mechanism is deemed unlikely by three criteria: (1) they have very different pH/log kcat. profiles and pH optima; (2) imine intermediates, which are known to play a central role in the mechanism of type I enzymes, could not be detected (by borohydride reduction) in the type II enzyme; (3) unlike Schiff's base-forming type I enzymes, there are no conserved lysine residues in type II amino acid sequences.

Bacterial Proteins↗
PubMed1992

Reactivation of denatured citrate synthase.

1. The imported mitochondrial enzyme citrate synthase can be partially (less than or equal to 45%) reactivated after denaturation in guanidinium chloride, if the concentration of the denaturing agent is lowered by dialysis, rather than by dilution, when essentially no reactivation is observed. 2. The presence of a reducing agent (dithiothreitol) is necessary for regain of activity. 3. Optimum regain of activity occurs at enzyme concentrations of about 10-20 micrograms/ml; at higher concentrations there is significant formation of aggregates.

Animals↗
PubMed1992

Unfolding and refolding of hen egg-white riboflavin binding protein.

The unfolding and refolding of riboflavin-binding protein (RfBP) from hen egg-white induced by addition of guanidinium chloride (GdnHCl), and its subsequent removal by dialysis have been studied by c.d. and fluorescence for both the native and reduced protein. The reduction of its nine disulphide bonds causes a reduction in the secondary structure (alpha-helix plus beta-sheet) from 63% to 33% of the amino acid residues. Unfolding of the native protein occurred in two phases; the first involving a substantial loss of tertiary structure, followed by a second phase involving loss of secondary structure at higher GdnHCl concentrations. By contrast this biphasic behaviour was not discernible in the reduced protein. The loss of ability to bind riboflavin occurred after the first phase of unfolding. Comparison of unfolding of the holoprotein and apoprotein suggested that riboflavin has only a small stabilizing effect on the unfolding process. After removal of GdnHCl, the holoprotein, apoprotein and reduced protein assumed their original conformation. The significance of the results in relation to various models for protein folding is discussed.

Animals↗
PubMed1992

Phosphoglycerate mutase from Streptomyces coelicolor A3(2): purification and characterization of the enzyme and cloning and sequence analysis of the gene.

The enzyme 3-phosphoglycerate mutase was purified 192-fold from Streptomyces coelicolor, and its N-terminal sequence was determined. The enzyme is tetrameric with a subunit Mr of 29,000. It is 2,3-bisphosphoglycerate dependent and inhibited by vanadate. The gene encoding the enzyme was cloned by using a synthetic oligonucleotide probe designed from the N-terminal peptide sequence, and the complete coding sequence was determined. The deduced amino acid sequence is 64% identical to that of the phosphoglycerate mutase of Saccharomyces cerevisiae and has substantial identity to those of other phosphoglycerate mutases.

2,3-Diphosphoglycerate↗
PubMed1991

The aromatic amino acid content of the bacterial chaperone protein groEL (cpn60). Evidence for the presence of a single tryptophan.

Studies of the absorption and fluorescence properties of the chaperone protein groEL (cpn60) from Escherichia coli show that tryptophan is present, in contrast to the proposed amino acid sequence of the protein (Hemmingsen, S.M. et al. (1988) Nature 333, 330-334). By determining a suitable value for the specific absorption coefficient of the protein at 280 nm, it has been shown that the content of the aromatic amino acids corresponds to a single tryptophan and (most probably) seven tyrosines per subunit (Mr 57,200).

Amino Acids↗
PubMed1991

Stabilization of the shikimate pathway enzyme dehydroquinase by covalently bound ligand.

Reversible binding of a ligand to an enzyme active site can elicit a variety of changes in the protein, such as conformational changes (close to the site of binding or communicated over long distances), changes in the ionization state of surrounding amino acid side chains, changes in the interaction of the target protein with other subunits (or other proteins), or even changes in the thermodynamic stability of the protein. Relatively little attention has been given to studying these effects in proteins to which the ligand has been irreversibly bound, yet this can be a convenient way of studying the effects of ligand binding in the absence of association/dissociation equilibria. We report the dramatic changes which occur to the shikimate pathway enzyme dehydroquinase when ligand is attached to its active site after borohydride reduction of the mechanistically important Schiff's base intermediates. The effects of this modification have been characterized by limited proteolysis, circular dichroism, guanidine hydrochloride denaturation, and differential scanning calorimetry. The conclusions from these studies are that although anchoring the ligand at the active site does not cause a gross change in conformation, it does increase markedly the conformational stability of the protein. This is conclusively established by three separate experiments: 1) the modified protein is completely resistant to proteases, whereas the unmodified protein is very susceptible to proteolysis; 2) the concentration of guanidine hydrochloride required to unfold the ligand-linked dehydroquinase is 3-4-fold greater than that of the unmodified protein; 3) the melting temperature (Tm) of the modified protein is 40 degrees C higher than that of the unmodified protein. These results are a very clear example of the thermodynamic link between ligand binding, conformational stability, and proteolytic susceptibility in vitro and will be a useful system for dissecting the contributions of individual protein-ligand interactions to these parameters.

Calorimetry, Differential Scanning↗
PubMed1991

Structural properties of long- and short-chain alcohol dehydrogenases. Contribution of NAD+ to stability.

Structural studies were undertaken on long-chain and short-chain alcohol dehydrogenases (from horse liver and Drosophila respectively). Far-u.v. c.d. measurements were used to estimate the secondary structure contents of the enzymes. For the horse liver enzyme, the results agree well with the X-ray data; for the Drosophila enzyme (for which a crystal structure is not yet available), the results are in good agreement with those obtained by applying a range of structure-prediction procedures to the amino acid sequence of this enzyme. The conformational stabilities of the two enzymes were investigated by studying the unfolding brought about by guanidinium chloride (GdnHCl) by using activity and c.d. measurements. The unfolding of the Drosophila enzyme was analysed in terms of a two-state model; the presence of the substrate NAD+ leads to considerable protection against unfolding. By contrast, the unfolding of the horse liver enzyme shows a plateau effect at intermediate concentrations of GdnHCl, indicating that a two-state model is not appropriate in this case. NAD+ affords little, if any, protection against unfolding for the horse liver enzyme.

Alcohol Dehydrogenase↗
PubMed1991

The unfolding and refolding of pig heart fumarase.

The unfolding of pig heart fumarase in solutions of guanidinium chloride (GdnHCl) has been examined. Loss of activity occurs at lower concentrations of GdnHCl than the structural changes detected by fluorescence or c.d. After denaturation, regain of activity can be observed provided that a reducing agent (dithiothreitol) is present and that the concentration of GdnHCl is lowered by dialysis rather than by dilution. The regain of secondary structure occurs with high efficiency even when little or no activity is recovered.

Animals↗
PubMed1991

Solution structures of calcitonin-gene-related-peptide analogues of calcitonin-gene-related peptide and amylin.

Near-u.v. and far-u.v. c.d. spectra of human alpha-calcitonin-gene-related peptide (h alpha CGRP), analogues and fragments of CGRP and amylin were recorded in aqueous solution and in trifluoroethanol (TFE)/water mixtures. All peptides contained significant amounts of alpha-helix in aqueous solution, and this amount increased on adding TFE. The helical content was unaffected by pH and salt. However, amylin contained much less helix than CGRP and the c.d. spectrum was more temperature-sensitive. A band in the near-u.v. c.d. spectrum of CGRP (but not present in the spectrum of amylin) was attributed to the disulphide bond in CGRP. The intensity of this band was pH-dependent and titrated with a pKa of 6.5, suggesting the involvement of histidine ionization.

Amino Acid Sequence↗
PubMed1991

Selective modification by transglutaminase of a glutamine side chain in the hinge region of the histidine-388----glutamine mutant of yeast phosphoglycerate kinase.

The transglutaminase-catalysed incorporation of putrescine and monodansylcadaverine into yeast phosphoglycerate kinase has been studied. There is little incorporation of the amines into wild-type enzyme, but nearly stoichiometric incorporation into the histidine-388----glutamine mutant enzyme. C.d. studies show that the overall structure of the mutant enzyme is very similar to that of the wild-type enzyme. Incorporation of the amines into the mutant enzyme causes no significant change in its activity. Glutamine-388 was shown, by isolation and sequencing of the modified peptide, to be the site of incorporation of monodansylcadaverine into the mutant enzyme. The specificity of the transglutaminase reaction is discussed in the light of available data.

Amino Acid Sequence↗
PubMed1991

A comparative study of the structure of egg-white riboflavin binding protein from the domestic fowl and Japanese quail.

1. The riboflavin binding proteins from domestic fowl and Japanese quail have been isolated and their structures compared by circular dichroism, fluorescence and peptide mapping. 2. The two proteins have similar secondary structures, but differ in their tertiary structures as reflected in the environments of aromatic amino acid side chains. 3. Differences in amino acid sequence between the proteins are indicated by the digestion patterns obtained with thermolysin, chymotrypsin and V8 proteinase from Staphylococcus aureus. Both proteins are resistant to digestion by trypsin.

Animals↗
PubMed1991

Secondary structures of narcissus mosaic virus coat protein.

Circular dichroism (CD) measurements on the coat protein of narcissus mosaic virus particles show that the dominant secondary structure is the alpha-helix, with a 45 (+/- 2)% content. The beta-sheet content is much lower at 5 (+/- 3)%. Both values are essentially the same as those found in potato virus X coat protein. The CD results are used to assess the results of secondary structure prediction methods.

Capsid↗
PubMed1990

The unfolding and attempted refolding of citrate synthase from pig heart.

The unfolding of the dimeric enzyme citrate synthase from pig heart in solutions of guanidinium chloride (GdnHCl) was studied. Data from fluorescence, circular dichroism (CD) and thiol group reactivity studies indicated that the enzyme was almost completely unfolded at GdnHCl concentrations greater than or equal to 4 M. On dilution of GdnHCl, essentially no reactivation of the enzyme occurred. The implications of this finding for the process of folding and assembly in vivo of this and other mitochondrial enzymes are discussed. Exposure of the enzyme to high pH (9-10) led to only a small loss of secondary structure and partial reactivation could be observed on readjustment of the pH to 8.0.

Animals↗