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N C Price

Publications and source records attributed to N C Price.

At least 55 records · Page 3Linked to original sources
PubMed1997

Use of the Ober2 Model B-1200 system for analysis of eye movements made during reading.

BACKGROUND: The Ober2 Model B-1200 system uses infrared reflections to record and analyze eye movements made during reading. The system's ability to analyze data from normal subjects and the reliability of the data produced by subjects who read standard paragraphs were investigated. METHODS: Forty-two optometry students and 20 junior high school students, all self-assessed normal readers, read five different Taylor test paragraphs during each of two sessions. Ober2 Model B-1200 analysis was attempted for each paragraph; when analysis was successful, grade-equivalent scores based on fixations, span of recognition, regressions, fixation duration, and reading rate were determined. RESULTS: The Ober2 Model B-1200 system was unsuccessful in analyzing data from 30% of the paragraph presentations; manual calibration procedures allowed no additional data analysis. Using mean grade-equivalent data from 16 optometry student subjects for whom all 10 paragraphs could be analyzed, significant differences were found between results for two of the test paragraphs. Split-half inter-session reliability coefficients for grade-equivalent data ranged from 0.84 to 0.95. No intra-session learning or fatigue effects on reading data were found. CONCLUSIONS: Although the Ober2 Model B-1200 potentially can provide valuable information on eye movements made during reading, problems exist with respect to its ability to analyze data. The analysis failures significantly limited the value of this system.

Adolescent↗
PubMed1996

Analysis of the structural stability of the multidomain enzyme flavocytochrome P-450 BM3.

The unfolding and refolding of flavocytochrome P-450 BM3 and its constituent haem and flavin domains have been analysed, using guanidinium chloride (GdnHCl) as a denaturant. Enzyme activities are lost at GdnHCl concentrations too low to cause major changes in secondary structure (0.1-0.5 M). The losses are primarily due to time-dependent FMN removal. Fluorescence and visible CD spectroscopies show that FMN dissociation is complete by 0.7 M GdnHCl, whereas FAD removal is complete by 1.5 M GdnHCl. Limited regain of activity is achieved by dilution of enzyme from solutions of < or = 0.75 M GdnHCl into fresh buffer. Supplementation of GdnHCl-free assay media with flavins (FAD and FMN) causes small additional regains in flavin domain (cytochrome-c reductase) activity lost at low [GdnHCl]. However, flavin addition during the denaturation step affords greater protection against inactivation, suggesting that conformational changes may occur subsequent to flavin loss and that these changes are not readily reversed on dilution of GdnHCl. Loss of catalytically competent haem ligation occurs over the same [GdnHCl] range for P-450 BM3 and its haem domain. In both cases, the 'denatured' P-420 form accumulates in the reduced/carbon monoxide-bound visible spectrum from 0.5 to 2 M GdnHCl. Secondary structure loss also occurs over similar [GdnHCl] ranges for P-450 BM3 and its two domains (80-90% lost from 0.5-3 M GdnHCl), indicating that there is little mutual stabilisation of domains in the holoenzyme. Differential scanning calorimetry measurements support this conclusion, but show that the haem domain is more thermostable than the flavin domain.

Bacillus megaterium↗
PubMed1996

Phosphoglycerate mutase from Schizosaccharomyces pombe: development of an expression system and characterisation of three histidine mutants of the enzyme.

The small, monomeric, phosphoglycerate mutase (PGAM) from Schizosaccharomyces pombe has been overexpressed in a strain of Saccharomyces cerevisiae in which the gene encoding PGAM has been deleted, with a yield of purified enzyme of 10-15 mg per litre cell culture. Three mutants in which histidine residues in S. pombe PGAM have been substituted by glutamine have been purified and characterised. Two mutants (H151Q and H196Q) have kinetic and structural properties very similar to wild-type enzyme, consistent with the proposed location of these (non-conserved) histidines on the surface of the enzyme. The third mutant (H163Q) involving a histidine thought to be part of the active site has greatly reduced mutase and phosphatase activities. Mass spectrometry shows that the phosphorylated form of the H163Q is several 100-times more stable towards hydrolysis than the phosphorylated form of wild-type enzyme. The H163Q mutant appears to be structurally quite distinct from wild-type enzyme. 600 MHz 1D proton NMR spectra of good quality have been obtained for wild-type enzyme and the H151Q and H196Q mutants.

Base Sequence↗
PubMed1996

An analysis of the conformational changes that accompany the activation and inhibition of gelatinase A.

The latent precursors of the matrix metalloproteinases (MMPs) are converted by (4-aminophenylmercuric)acetate to active forms that lose their propeptide as a result of autolysis. C.D. and an active site mutant of progelatinase A (MMP2) were used to demonstrate that, although propeptide removal is accompanied by a decrease in the enzyme's beta-sheet content, the initial activation is achieved with only minor modifications to the conformation. Mixing activated gelatinase A with the natural inhibitor, TIMP-1, resulted in conformational changes that were absent when a synthetic inhibitor was used. The relevance of these results to MMP activation and inhibition is discussed.

Amino Acid Sequence↗
PubMed1996

Cytotoxicity of prion protein peptide (PrP106-126) differs in mechanism from the cytotoxic activity of the Alzheimer's disease amyloid peptide, A beta 25-35.

The abnormal form of the prion protein (PrPSc), a synthetic prion protein peptide fragment (PrP106-126) and fragments of the Alzheimer's protein precursor, APP, have been shown to be cytotoxic in vitro. We have used synchronous, clonal cell models originally developed to study the toxicity of the Alzheimer's disease amyloid peptide, A beta 25-35, to investigate the actions of PrP peptides. We found that the cytotoxicity of the PrP106-126 depends on its state of aggregation and the cellular expression of PrPc, and is independent of a loss of MTT reduction activity in the absence of cell death associated with the cellular effects of A beta 25-35. These factors may play a role in the lesion specificity of different pathological phenotypes of prion-protein related diseases.

Amino Acid Sequence↗
PubMed1996

Conformational changes associated with activation of bee venom phospholipase A2.

Bee venom PLA2 possesses a binding site for long-chain fatty acids that can be acylated by long-chain fatty acid imidazolides [Drainas, D. and Lawrence, A.J. (1978) Eur. J. Biochem. 91, 131-138]. Occupation of the site either by oleic acid or the oleoyl residue enhances the catalytic activity by 45.7-fold in the presence of 20% 1-propanol and occupation of the site by the oleoyl residue increases the lytic activity against rabbit erythrocytes by 60-fold. Treatment of the enzyme with oleic acid and glutaraldehyde is known to produce irreversible activation [Lawrence, A.J. and Moores, G.R. (1975) FEBS Lett. 49, 287-291]. Here we show that reduction of the glutaraldehyde-treated enzyme with borohydride stabilizes the activated state and enables the fatty acid to be removed, revealing that a large proportion of the induced activation does not require the presence of oleic acid and indicating that activation is due to a change in the conformation rather than the hydrophobicity of the protein. A kinetic study of enzyme activated by oleoyl imidazolide showed that this modification stabilizes the protein against reversible inactivation by 1-propanol. Comparison of the CD spectra of native and oleoyl imidazolide-activated enzyme shows a change in secondary structure with apparent increase in both alpha-helix and beta-sheet content. During reaction of the enzyme with oleoyl imidazolide, the protein fluorescence shows a biphasic response with an initial fall attributed to occupation of the binding site followed by a progressive decrease with a shift of the emission maximum from 341 to 348 nm. The rate of the second phase closely matched the rate of increase in catalytic activity of the enzyme. Free oleic acid caused a rapid fall in fluorescence emission without the subsequent slow change. These results support the proposal that oleic acid or the oleoyl residue occupy a very similar site on the protein and that occupation of this site increases the exposure of one or both of the Trp residues to the aqueous environment. Binding studies show that activation by oleoyl imidazolide does not increase the affinity of the enzyme for the erythrocyte membrane. It is proposed that occupation of a long-chain fatty acid binding site on the enzyme enhances catalytic activity by changing the conformation of the protein rather than acting as a hydrophobic anchor to the substrate surface.

1-Propanol↗
PubMed1996

Ligand-induced conformational changes in wild-type and mutant yeast pyruvate kinase.

A mutant form of pyruvate kinase in which serine 384 has been mutated to proline has been engineered in the yeast Saccharomyces cerevisiae. Residue 384 is located in a helix in a subunit interface of the tetrameric enzyme, and the mutation was anticipated to alter the conformation of the helix and hence destabilize the interface. Previous results indicate that the mutant favours the T quaternary conformation over the R conformation, and this is confirmed by the results presented here. Addition of phosphoenol-pyruvate (PEP), ADP and fructose-1, 6-bisphosphate (Fru-1.6-P2) singly to the wild-type and mutant enzymes results in a significant quenching of tryptophan fluorescence (12-44%), and for Fru-1,6-P2, a red shift of 15 nm in the emission maximum. Fluorescence titration experiments showed that PEP, ADP and Fru-1,6-P2 induce conformations which have similar ligand-binding properties in the wild-type and mutant enzymes. However, the Fru-1,6-P2 induced conformation is demonstrably different from those induced by either ADP or PEP. The enzymes differ in their susceptibility to trypsin digestion and N-ethylmaleimide inhibition. The thermal stability of the enzyme is unaltered by the mutation. Far-UV CD spectra show that both enzymes adopt a similar overall secondary structure in solution. Taken together, the results suggest that the Ser384-Pro mutation causes the enzyme to adopt a different tertiary and/or quaternary structure from the wild-type enzyme and affects the type and extent of the conformational changes induced in the enzyme upon ligand binding. A simplified minimal reaction mechanism is proposed in which the R and T states differ in both affinity and kcat. Thus, in terms of the models of cooperativity and allosteric interaction, pyruvate kinase is both a K and a V system.

Adenosine Diphosphate↗
PubMed1995

Kinetics of acid-mediated disassembly of the B subunit pentamer of Escherichia coli heat-labile enterotoxin. Molecular basis of pH stability.

The B-subunit pentamer of Escherichia coli heat-labile enterotoxin (EtxB) is highly stable, maintaining its quaternary structure in a range of conditions that would normally be expected to cause protein denaturation. In this paper the structural stability of EtxB has been studied as a function of pH by electrophoretic, immunochemical, and spectroscopic techniques. Disassembly of the cyclic pentameric structure of human EtxB occurs only below pH 2. As determined by changes in intrinsic fluorescence this process follows first-order kinetics, with the rate constant for disassembly being proportional to the square of the H+ ion concentration, and with an activation energy of 155 kJ mol-1. A C-terminal deletion mutant, hEtxB214, similarly shows first-order kinetics for disassembly but with a higher pH threshold, resulting in disassembly being seen at pH 3.4 and below. These findings are consistent with the rate-limiting step for disassembly of human EtxB being the simultaneous disruption of two interfaces by protonation of two C-terminal carboxylates. By comparison, disassembly of the B-subunit of cholera toxin (CtxB), a protein which shows 80% sequence identity with EtxB, exhibits a much lower stability to acid conditions; with disassembly of CtxB occurring below pH 3.9, with an activation energy of 81 kJ mol-1. Reasons for the observed differences in acid stability are discussed, and the implications of these findings to the development of oral vaccines using EtxB and CtxB are considered.

Bacterial Toxins↗
PubMed1995

Cloning and sequencing of a gene encoding pyruvate kinase from Schizosaccharomyces pombe; implications for quaternary structure and regulation of the enzyme.

A cDNA encoding pyruvate kinase from Schizosaccharomyces pombe has been isolated from a lambda ZAPII library. This cDNA was sequenced and found to contain an open reading frame of 1524 nucleotides, giving a predicted protein subunit M, of 55470. The sequence shows a high degree of identity with other pyruvate kinase sequences, with residues implicated in the binding of substrate and metal ion co-factors conserved. However, there are significant differences in the putative subunit interface and effector binding regions which may account for the unusual quaternary structure and regulatory properties of the S. pombe enzyme.

Amino Acid Sequence↗
PubMed1995

NADPH oxidase activity of cytochrome P-450 BM3 and its constituent reductase domain.

Cytochrome P-450 BM3 from Bacillus megaterium catalyses NADPH oxidation in the absence of added substrate. This activity is also associated with the independently expressed flavin-containing reductase domain of the protein. The rates of these activities are more than two orders of magnitude lower than those in the presence of fatty acid P-450 substrates or artificial electron acceptors. Electrons derived from NADPH in this fashion are transferred onto oxygen, generating superoxide (O2-) anions. The formation of these active oxygen species is detectable by luminometry and the chemiluminescence can be inhibited through the addition of superoxide dismutase (but not catalase). This activity is reminiscent of the microbicidal NADPH oxidase activity associated with neutrophils and other leukocyte blood cell types. Diphenyliodonium, a potent inhibitor of the neutrophil NADPH oxidase, effectively inhibits fatty acid hydroxylase and electron transferase activities catalysed by P-450 BM3 and its reductase domain. CD studies on the native and NADPH-reduced P-450 BM3 and BM3 reductase indicate that no secondary structural alteration is caused by pre-incubation with the reductant. Therefore, the previously recognised reversible time-dependent inactivation of P-450 BM3 by NADPH may be attributed to the NADPH oxidase activity associated with the reductase domain of the enzyme.

Bacillus megaterium↗
PubMed1995

The refolding of hen egg white riboflavin-binding protein: effect of protein disulphide isomerase on the reoxidation of the reduced protein.

Hen egg white riboflavin-binding protein (RfBP) contains nine disulphide bonds. Provided these remain intact, the refolding of RfBP after incubation in 6 M guanidinium chloride is highly efficient with at least 95% of the binding activity regained within 3 min. Kinetic studies indicate that this regain consists of at least two phases. When the disulphide bonds of RfBP are reduced, reoxidation using a mixture of oxidized and reduced glutathione leads to less than 5% recovery of activity. However, if protein disulphide isomerase (PDI; EC 5.3.4.1) is present during the reoxidation nearly 50% activity can be regained, suggesting that PDI may play an important role in the maturation of RfBP in vivo.

Animals↗
PubMed1995

Conformational changes induced by DNA binding of NF-kappa B.

The transcription factor NF-kappa B makes extensive contacts with its recognition site over one complete turn of the double helix. Structural transitions, in both protein and DNA, that accompany formation of the DNA-protein complex were analysed by proteinase sensitivity and circular dichroism (CD) spectroscopy. In the absence of DNA chymotrypsin cleaved p50 after residues Y60 and N78, while proteinase K cleaved p50 after residues S74 and Q180. Previous experiments had indicated that trypsin cleaved p50 after K77. Cleavages after Y60, S74, K77 and N78 were blocked in the presence of bound DNA, whereas cleavage after Q180 was enhanced. Y60, S74, K77 and N78 are all located in the p50 N-terminal domain AB loop, whereas Q180 is located in the mainly alpha-helical region between p50 N-terminal domain beta-strands G' and H. As only Y60 makes direct contact with the DNA it is likely that the AB loop is highly unstructured in the absence of DNA, but is held in a rigid, proteinase-resistant structure by bound DNA. These conclusions were supported by CD spectroscopic studies of recombinant p50 and p65 homodimers, which indicated that both species changed conformation when binding DNA. Examination of the near UV CD spectra revealed that with some DNA sequences the bound and free forms of the DNA assumed different conformations. While this was evident for a fully symmetrical, high affinity recognition site DNA, it was not apparent with less tightly bound DNA.

Base Sequence↗