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Biomedical subjects

N C Price

Publications and source records attributed to N C Price.

At least 37 records · Page 2Linked to original sources
PubMed1999

The role of the C-terminal region in phosphoglycerate mutase.

Removal of the C-terminal seven residues from phosphoglycerate mutase from Saccharomyces cerevisiae by limited proteolysis is associated with loss of mutase activity, but no change in phosphatase activity. The presence of the cofactor 2, 3-bisphosphoglycerate, or of the cofactor and substrate 3-phosphoglycerate together, confers protection against proteolysis. The substrate alone offers no protection. Replacement of either or both of the two lysines at the C-terminus by glycines has only limited effects on the kinetic properties of phosphoglycerate mutase, indicating that these residues are unlikely to be involved in crucial electrostatic interactions with the substrate, intermediate or product in the reaction. However, the double-mutant form of the enzyme is more sensitive to proteolysis and is no longer protected against proteolysis by the presence of cofactor. The proteolysed wild-type and two of the mutated forms of the enzyme show a reduced response to 2-phosphoglycollate, which enhances the instability of the phospho form of the native enzyme. The phosphoglycerate mutase from Schizosaccharomyces pombe, which lacks the analogous C-terminal tail, has an inherently lower mutase activity and is also less responsive to stimulation by 2-phosphoglycollate. It is proposed that the C-terminal region of phosphoglycerate mutase helps to maintain the enzyme in its active phosphorylated form and assists in the retention of the bisphosphoglycerate intermediate at the active site. However, its role seems not to be to contribute directly to ligand binding, but rather to exert indirect effects on the transfer of the phospho group between substrate, enzyme, intermediate and product.

Amino Acid Sequence↗
PubMed1999

Site-directed mutagenesis of proline 204 in the 'hinge' region of yeast phosphoglycerate kinase.

Site-specific mutants have been produced in order to investigate the role of proline 204 in the 'hinge' region of yeast phosphoglycerate kinase (PGK). This totally conserved proline has been shown to be the only cis-proline in the high resolution crystal structures of yeast, B. stearothermophilus, T. brucei and T. maritima PGK, and may therefore have a role in the independent folding of the two domains or in the 'hinge' bending of the molecule during catalysis. The residue was replaced by a histidine (Pro204His) and a phenylalanine (Pro204Phe), and the resulting proteins characterised by differential scanning calorimetry (DSC), circular dichroism (CD), tryptophan fluorescence emission and kinetic analysis. Although the secondary and tertiary structure of the Pro204His protein is generally similar to that of the wild-type enzyme as assessed by CD, the enzyme is less stable to heat and guanidinium chloride denaturation than the wild-type. In the denaturation experiments two transitions were observed for both the wild-type and the Pro204His mutant, as have been previously reported for yeast PGK [Missiakas, D., Betton, J.M., Minard, P. & Yon, J.M. (1990) Biochemistry 29, 8683-8689]. The first transition is accompanied by an increase in fluorescence intensity leading to a hyperfluorescent state, followed by the second, corresponding to a decrease in fluorescence intensity. However, for the Pro204His mutant, the first transition proceeded at lower concentrations of guanidinium chloride and the second transition proceeded to the same extent as for the wild-type protein, suggesting that sequence-distant interactions are more rapidly disrupted in this mutant enzyme than in the wild-type enzyme, while sequence-local interactions are disrupted in a similar way. The Michaelis constants (K(m)) for both 3-phospho-D-glycerate and ATP are increased only by three or fourfold, which confirms that, as expected, the substrate binding sites are largely unaffected by the mutation. However, the turnover and efficiency of the Pro204His mutant is severely impaired, indicating that the mechanism of 'hinge' bending is hindered. The Pro204Phe enzyme was shown to be significantly less well folded than the wild-type and Pro204His enzymes, with considerable loss of both secondary and tertiary structure. It is proposed that the proline residue at 204 in the 'hinge' region of PGK plays a role in the stability and catalytic mechanism of the enzyme.

Adenosine Triphosphate↗
PubMed1999

Activation of the acidic isoform of phospholipase A2 from Naja mossambica mossambica venom by oleoyl imidazolide requires the cooperative action of two ionizing groups.

The acidic phospholipase A2 isoform from the spitting cobra Naja mossambica mossambica is activated irreversibly by treatment with a molar equivalent of oleoyl imidazolide. The kinetics of the chemical modification of the enzyme can also be monitored by measuring the large reduction of tryptophan fluorescence, which is accompanied by a distinct red shift. The addition of a single molar equivalent of oleic acid to the enzyme produces an instantaneous reduction in fluorescence but with a barely detectable red shift, confirming that the response to oleoyl imidazolide results from covalent modification of the protein rather than hydrolysis of the reagent. The pH dependence of both activation and fluorescence reduction by oleoyl imidazolide has an optimum rate near pH 8.0. We propose that long-chain fatty acids and long-chain acyl imidazolides bind at a single activation site and that the reaction of the imidazolides involves two protein residues, one of which is a nonessential histidine residue and the other a primary amino group.

Animals↗
PubMed1999

Colorimetric protein assay techniques.

There has been an increase in the number of colorimetric assay techniques for the determination of protein concentration over the past 20 years. This has resulted in a perceived increase in sensitivity and accuracy with the advent of new techniques. The present review considers these advances with emphasis on the potential use of such technologies in the assay of biopharmaceuticals. The techniques reviewed include Coomassie Blue G-250 dye binding (the Bradford assay), the Lowry assay, the bicinchoninic acid assay and the biuret assay. It is shown that each assay has advantages and disadvantages relative to sensitivity, ease of performance, acceptance in the literature, accuracy and reproducibility/coefficient of variation/laboratory-to-laboratory variation. A comparison of the use of several assays with the same sample population is presented. It is suggested that the most critical issue in the use of a chromogenic protein assay for the characterization of a biopharmaceutical is the selection of a standard for the calibration of the assay; it is crucial that the standard be representative of the sample. If it is not possible to match the standard with the sample from the perspective of protein composition, then it is preferable to use an assay that is not sensitive to the composition of the protein such as a micro-Kjeldahl technique, quantitative amino acid analysis or the biuret assay. In a complex mixture it might be inappropriate to focus on a general method of protein determination and much more informative to use specific methods relating to the protein(s) of particular interest, using either specific assays or antibody-based methods. The key point is that whatever method is adopted as the 'gold standard' for a given protein, this method needs to be used routinely for calibration.

Colorimetry↗
PubMed1998

Structural plasticity of the feline leukaemia virus fusion peptide: a circular dichroism study.

The secondary structure of the feline leukaemia virus (FeLV) fusion peptide was investigated using circular dichroism (CD). Our results show that this peptide can readily flip between random, alpha-helical and beta-sheet conformations, depending upon its environment. The CD spectrum changes from one characteristic of random coil to predominantly beta-sheet type, and finally to that showing the characteristics of alpha-helical structure on moving from an aqueous solvent, through several increasingly hydrophobic systems, to a highly hydrophobic solvent. Electron microscopy confirmed the presence of beta structure. We propose that the structural plasticity demonstrated here is crucial to the ability of the fusion peptide to perturb lipid bilayers, and thus promote membrane fusion.

Animals↗
PubMed1998

Stopped-flow analysis of the refolding of hen egg white riboflavin binding protein in its native and dephosphorylated forms.

In earlier work (D.A. McClelland, S.H. McLaughlin, R.B. Freedman, and N.C. Price, Biochem. J. 311, 133-137 (1995)), we had shown that during the refolding of hen egg white riboflavin binding protein (RfBP) after denaturation in 6 M guanidinium chloride, most of the native properties of the protein are regained within 10-15 s of the dilution of the denaturing agent. We have now employed stopped-flow measurements of CD, protein fluorescence and regain of riboflavin binding ability to examine the rapid phases of the refolding process. Essentially, all of the native secondary structure as judged by the CD signal at 230 nm was regained within the dead-time of the instrument in CD mode (approximately 8 ms). 80% of the native protein fluorescence was regained within the dead-time of the instrument in fluorescence mode (1.7 ms). A further 10% was regained with a half time of 30 ms in the case of the apo-protein, though the half time was approximately doubled in the presence of riboflavin. This second phase corresponded with the regain of riboflavin binding ability. Two slow phases, with half-times of 46 s and 1 h involved the regain of the final 10% of fluorescence signal. Binding of the fluorescent probe, 1-anilino-8-naphthalenesulphonate (ANS) preceded the formation of the riboflavin binding site. Dephosphorylation of RfBP by treatment with acid phosphatase did not affect the binding of riboflavin, nor did it alter the kinetics of the refolding process. This is consistent with the proposal that in vivo phosphorylation occurs on a surface-exposed portion of the protein after the major portion of the folding process is complete.

Animals↗
PubMed1998

Purification and characterization of pyruvate kinase from Schizosaccharomyces pombe: evidence for an unusual quaternary structure.

Earlier attempts to purify and characterize nonrecombinant pyruvate kinase from Schizosaccharomyces pombe proved difficult due to problems associated with the instability of the protein. The enzyme has been overexpressed in Saccharomyces cerevisiae strain AH22, permitting studies to determine the conditions required to stabilize the enzyme during purification. Recombinant S. pombe pyruvate kinase was purified by a combination of ion-exchange chromatography and gel filtration. The purified enzyme showed sigmoidal kinetics with respect to PEP; in the presence of FBP, the kinetics were restored to Michaelis-Menten behavior. With respect to ADP, the Hill coefficient was not affected by FBP. Determination of the molecular mass of the purified enzyme by ultracentrifugation showed that it behaved as a dimer-tetramer system with a Kd of approximately 1 microM.

Adenosine Diphosphate↗
PubMed1998

Comparison of the cardiovascular effects of 2.5% phenylephrine and 10% phenylephrine during ophthalmic surgery.

PURPOSE: The recommended concentration of topical phenylephrine for mydriasis is still a matter of debate. Our purpose was to compare the cardiovascular effects of 10% and 2.5% topical aqueous phenylephrine. METHODS: We carried out a double-masked randomised study on 54 consecutive patients undergoing routine local anaesthetic cataract extraction, comparing the effects on blood pressure and heart rate of either 2.5% or 10% topical aqueous phenylephrine in combination with 1% topical aqueous tropicamide in those with no history of cardiovascular disease. RESULTS: No difference was found in the rise in blood pressure produced by 2.5% and 10% topical aqueous phenylephrine. We also found no sustained changes in blood pressure or heart rate after instillation of either 2.5% or 10% topical aqueous phenylephrine. CONCLUSION: We recommend the routine use of 2.5% topical aqueous phenylephrine as a mydriatic agent during cataract surgery and acknowledge the role of 10% topical aqueous phenylephrine as an effective mydriatic agent in cases where 2.5% phenylephrine may not be so effective, such as in subjects with darkly pigmented irides.

Aged↗
PubMed1998

The herpes simplex virus triplex protein, VP23, exists as a molten globule.

Two proteins, VP19C (50,260 Da) and VP23 (34,268 Da), make up the triplexes which connect adjacent hexons and pentons in the herpes simplex virus type 1 capsid. VP23 was expressed in Escherichia coli and purified to homogeneity by Ni-agarose affinity chromatography. In vitro capsid assembly experiments demonstrated that the purified protein was functionally active. Its physical status was examined by differential scanning calorimetry, ultracentrifugation, size exclusion chromatography, circular dichroism, fluorescence spectroscopy, and 8-anilino-1-naphthalene sulfonate binding studies. These studies established that the bacterially expressed VP23 exhibits properties consistent with its being in a partially folded, molten globule state. We propose that the molten globule represents a functionally relevant intermediate which is necessary to allow VP23 to undergo interaction with VP19C in the process of capsid assembly.

Base Sequence↗
PubMed1997

The Ov20 protein of the parasitic nematode Onchocerca volvulus. A structurally novel class of small helix-rich retinol-binding proteins.

Ov20 is a major antigen of the parasitic nematode Onchocerca volvulus, the causative agent of river blindness in humans, and the protein is secreted into the tissue occupied by the parasite. DNA encoding Ov20 was isolated, and the protein was expressed in Escherichia coli. Fluorescence-based ligand binding assays show that the protein contains a high affinity binding site for retinol, fluorescent fatty acids (11-((5-dimethylaminonaphthalene-1-sulfonyl)amino)undecanoic acid, dansyl-DL-alpha-aminocaprylic acid, and parinaric acid) and, by competition, oleic and arachidonic acids, but not cholesterol. The fluorescence emission of dansylated fatty acids is significantly blue-shifted upon binding in comparison to similarly sized beta-sheet-rich mammalian retinol- and fatty acid-binding proteins. Secondary structure prediction algorithms indicate that a alpha-helix predominates in Ov20, possibly in a coiled coil motif, with no evidence of beta structures, and this was confirmed by circular dichroism. The protein is highly stable in solution, requiring temperatures in excess of 90 degrees C or high denaturant concentrations for unfolding. Ov20 therefore represents a novel class of small retinol-binding protein, which appears to be confined to nematodes. The retinol binding activity of Ov20 could possibly contribute to the eye defects associated with onchocerciasis and, because there is no counterpart in mammals, represents a strategic target for chemotherapy.

Amino Acid Sequence↗
PubMed1997

Characterisation of the molybdenum-responsive ModE regulatory protein and its binding to the promoter region of the modABCD (molybdenum transport) operon of Escherichia coli.

Molybdenum-dependent repression of transcription of the Escherichia coli modABCD operon, which encodes the high-affinity molybdate transporter, is mediated by the ModE protein. This regulatory protein was purified as an N-terminal His6-tagged derivative and characterised both with and without the N-terminal oligohistidine extension. Equilibrium centrifugation showed that ModE is at least a 57-kDa homodimer. Circular dichroism spectroscopy indicated that when molybdate or tungstate bind to ModE there is little change in its alpha-helical content, but a major change in the environment of tryptophan and tyrosine residues occurs. Addition of molybdate or tungstate to the protein results in almost 50% quenching of the fluorescence attributed to tryptophan. Titration of fluorescence quenching showed that two molecules of molybdenum bind to each dimer of ModE with a Kd of 0.8 microM. DNA mobility-shift assays showed that ModE requires molybdenum, or tungstate, to bind with high affinity (approximate Kd of 30 nM ModE) to the modABCD promoter region. In accord with ModE's role as a molybdenum-dependent transcriptional repressor, DNase I footprinting experiments showed that the ModE-molybdenum complex binds to a single 31-bp region around the transcription start of the modABCD promoter. This region contains a 6-base palindromic sequence CGTTAT-N12-ATAACG.

Bacterial Proteins↗
PubMed1997

Synthetic, structural and biological studies of the ubiquitin system: synthesis and crystal structure of an analogue containing unnatural amino acids.

Ubiquitin is a 76-amino acid protein involved in the targeting for destruction of proteins in the cell. The protein can readily be synthesized chemically affording an extra dimension to studies of protein stability. Ubiquitin with various modifications to the hydrophobic core has been synthesized. In particular, two core amino acids have been replaced by aminobutyric acid (Val-26) and norvaline (for Ile-30) and the product crystallized. The refined crystal structure shows an overall contraction of the molecule and the side chain of Nva-30 rotates relative to Ile-30. However, the side chain rotation is not sufficient to compensate for the effect of the loss of the methyl group and hence a small cavity is introduced into the structure, which decreases the stability of the protein. The biological behaviour of the modified protein is unaltered. The observed changes in stability are of the magnitude expected for the removal of methyl groups from the hydrophobic core of a protein. Interestingly, the effect appears to be independent of the position of the removed methyl group. The intact structure, but not its stability, is important for recognition by the biological conjugating system.

Aminobutyrates↗
PubMed1997

Surface activity properties of cysteine-substituted C-terminal melittin analogues.

In order to extend our knowledge of factors important in the surface activity of melittin, cysteine was substituted for lysine-21 and lysine-21/glutamine-25 in a pair of synthetic peptide analogues. The first of these changes resulted in only modest effects on secondary structure (determined in 50% trifluoroethanol), emulsification and surface tension properties. Introduction of a second cysteine greatly reduced both the rate of surface tension decay and the equilibrium surface tension attained, although secondary structure (determined in 50% trifluoroethanol) was only slightly affected by this modification. This latter peptide completely lacked emulsification and haemolytic properties and was found to oligomerise readily due to the formation of intermolecular, disulphide bridges. These results indicate that oligomerisation abolishes surface activity in melittin.

Animals↗
PubMed1997

Glucose modification of human serum albumin: a structural study.

Structural changes associated with the exposure of human serum albumin (HSA) to glucose with or without the presence of Cu (II) have been characterized using a bank of methods for structural analysis including circular dichroism (CD), amino acid analysis (AAA), fluorescence measurements, SDS-PAGE, and boronate binding (which is a measure of Amadori product formation). We show that in the short-term (10 d) incubation mixtures, HSA is resistant to Cu (II)-mediated oxidative damage and that the early products of glycation of HSA had minimal effects on the folded structure. Amino acid analysis showed that there was no formation of advanced glycation endproducts (AGE), which can be measured by loss of lysine. This remained the case in longer term incubation of HSA (56 d) in the hyperglycemic concentration range (5-25 mM glucose) despite increased levels of Amadori product (60% boronate binding) and the formation of glycophore (Excitation 350, Emission 425). At high, nonphysiological concentrations (100 mM and 500 mM) of glucose, glycophore formation increased and 3 and 11 mol Lysine-glucose adduct/mol HSA were converted to AGE, respectively. This was accompanied by increased damage to tryptophan and protein-protein crosslinking but only minor tertiary structural change. In the presence of Cu (II), however, AGE formation was accompanied by extensive damage to histidine and tryptophan side chains, main chain fragmentation, and loss of both secondary and tertiary structure. Thus, changes in structure appear to be the result of oxidation as opposed to glycation, per se.

Amino Acids↗