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T Atkinson

Publications and source records attributed to T Atkinson.

At least 37 records · Page 2Linked to original sources
PubMed1995

Hypothalamic NPY and prepro-NPY mRNA in Djungarian hamsters: effects of food deprivation and photoperiod.

Two catabolic states leading to loss of body weight were compared in the Djungarian hamster (Phodopus sungorus campbelli). Hypothalamic neuropeptide Y (NPY) and gene expression for NPY and corticotropin-releasing factor (CRF) were examined after withdrawal of food for 48 h or exposure to short photoperiod for 10 or 20 wk. Food deprivation was accompanied by increases in both NPY and prepro-NPY mRNA in the hypothalamic arcuate nucleus (ARC). Increases in gene expression were limited compared with published data from the rat and were inversely related to predeprivation body weight. Exposure to short photoperiod for 20 wk reduced body weight by 39%, but the activity of the NPY-ergic system was not affected; peptide concentration and gene expression were similar in short photoperiod hamsters and long photoperiod controls. The hypothalamic NPY-ergic system of the Djungarian hamster is sensitive to weight loss due to imposed manipulations of energy balance, but the catabolism observed in short photoperiod gives rise to a body weight that is appropriate to the season encoded by the photoperiod. CRF gene expression was not affected by food deprivation or short photoperiod.

Animals↗
PubMed1995

Cloning, expression and purification of Ppl-1, a kappa-chain binding protein, based upon protein L from Peptostreptococcus magnus.

Protein L is a multi-domain, cell wall constituent of certain strains of Peptostreptococcus magnus, which binds to the variable domain of the L-chains of Ig. A gene fragment which codes for a single Ig-binding domain of protein L (Ppl-1) was cloned into a modified pKK223-3 vector and over-expressed in E. coli JM103. A rapid protein purification protocol is described. In these studies, purified Ppl-1 was immobilised on to an agarose gel and tested against an array of Igs and Ig fragments. It was found that Ppl-1-bound Igs from a number of different sources via interactions with the L kappa-chain. An enzyme linked immunosorbant assay has been developed to assay the binding of Ppl-1 to IgG. The incubation of Ppl-1 with human serum does not produce an immunoprecipitate, thus suggesting one unique interaction per binding domain which has been confirmed by ELISA. These experiments demonstrate the potential value of Ppl-1 as an immunological tool and as an affinity chromatography ligand for the purification of Igs.

Amino Acid Sequence↗
PubMed1994

Erwinia chrysanthemi L-asparaginase: epitope mapping and production of antigenically modified enzymes.

This study shows that the antigenicity of Erwinia chrysanthemi L-asparaginase can be reduced by site-directed mutagenesis. Ten B-cell epitopes of the enzyme were identified using synthetic hexapeptides and polyclonal antisera from rabbits and mice. The region 282GIVPPDEELP292 near the C-terminus was an immunodominant epitope. Binding of two hexapeptides (283IVPPDE288 and 287DEELPG292) to the antibodies was dependent on Pro285, and Pro286, since their replacement by almost any other amino acid resulted in reduced binding. The other residues were less important for binding the antibodies, as binding was relatively unaffected by amino acid substitutions. Three site-directed mutant enzymes, P285T (proline-285-->threonine etc.), P286Q and E288A, were expressed in Escherichia coli. The purified enzymes had subunit M(r) values of 35,000. The pI values of P285T, P286Q and the wild-type enzymes were 8.6, and that for the mutant E288A was 9.2. The kcat. and Km values for the mutants P286Q and E288A with L-asparagine and L-glutamine were comparable with those of the wild-type enzyme. The Km values for the mutant P285T with both substrates was similar to that of the wild-type enzyme, whereas the kcat. was reduced by 2-fold with L-asparagine and by 4-fold with L-glutamine. The change proline-->threonine reduced the antigenicity of the enzyme by 8-fold, as shown in sandwich e.l.i.s.a.s. using monoclonal antibodies raised against the wild-type enzyme.

Amino Acid Sequence↗
PubMed1994

Affinity of chaperonin-60 for a protein substrate and its modulation by nucleotides and chaperonin-10.

The refolding of lactate dehydrogenase fully unfolded in 4 M guanidinium chloride was initiated by dilution into assay buffer, and the emergence of active enzyme was recorded. This was performed in the presence of the following chaperonin complexes in the refolding medium: chaperonin-60 (cpn60), cpn60-MgATP, cpn60-Mgp[NH]ppA, cpn60-MgADP in both the presence and absence of chaperonin-10 (cpn10). For each nucleotide-chaperonin complex studied, the effect of nucleotide concentration was measured. Dissociation constants (Kd) for unfolded LDH bound to the various chaperonin complexes were derived directly from the ability of the complexes to retard the folding of the enzyme. Dissociation constants for the different complexes were found to be in the order: cpn60 < cpn60-MgADP-cpn10 (formed at low [MgADP]) < cpn60-MgADP < cpn60-MgADP-cpn10 < cpn60-Mgp[NH]ppA < cpn60-Mgp[NH]ppA-cpn10 < cpn60-MgATP < cpn60-MgATP-cpn10; i.e. the tightest complex is with cpn60 and the weakest with cpn60-MgATP-cpn10. Only when MgATP is the nucleotide do we see the yield of native enzyme increased on the time scale of 1 h. The results provide estimates of the change in binding energy between the chaperonin and a substrate protein through the cycle of MgATP binding, hydrolysis and dissociation.

Adenosine Triphosphate↗
PubMed1994

Contribution of a buried aspartate residue towards the catalytic efficiency and structural stability of Bacillus stearothermophilus lactate dehydrogenase.

The X-ray structure of lactate dehydrogenase (LDH) shows the side-chain carboxylate group of Asp-143 to be buried in the hydrophobic interior of the enzyme, where it makes hydrogen-bonding interactions with both the side-chain hydroxyl group of Ser-273 and the main-chain amide group of His-195. This is an unusual environment for a carboxylate side-chain as hydrogen bonding normally occurs with water molecules at the surface of the protein. A charged hydrogen-bonding interaction in the interior of a protein would be expected to be much stronger than a similar interaction on the solvent-exposed exterior. In this respect the side-chain carboxylate group of Asp-143 appears to be important for maintaining tertiary structure by providing a common linkage point between three discontinuous elements of the secondary structure, alpha 1F, beta K and the beta-turn joining beta G and beta H. The contribution of the Asp-143 side-chain to the structure and function of Bacillus stearothermophilus LDH was assessed by creating a mutant enzyme containing Asn-143. The decreased thermal stability of both unactivated and fructose-1,6-diphosphate (Fru-1,6-P2)-activated forms of the mutant enzyme support a structural role for Asp-143. Furthermore, the difference in stability of the wild-type and mutant enzymes in guanidinium chloride suggested that the carboxylate group of Asp-143 contributes at least 22 kJ/mol to the conformational stability of the wild-type enzyme. However, there was no alteration in the amount of accessible tryptophan fluorescence in the mutant enzyme, indicating that the mutation caused a structural weakness rather than a gross conformational change. Comparison of the wild-type and mutant enzyme steady-state parameters for various 2-keto acid substrates showed the mutation to have a general effect on catalysis, with an average difference in binding energy of 11 kJ/mol for the transition-state complexes. The different effects of pH and Fru-1,6-P2 on the wild-type and mutant enzymes also confirmed a perturbation of the catalytic centre in the mutant enzyme. As the side-chain of Asp-143 is not sufficiently close to the active site to be directly involved in catalysis or substrate binding it is proposed that the effects on catalysis shown by the mutant enzyme are induced either by a structural change or by charge imbalance at the active site.(ABSTRACT TRUNCATED AT 400 WORDS)

Aspartic Acid↗
PubMed1994

The stability and hydrophobicity of cytosolic and mitochondrial malate dehydrogenases and their relation to chaperonin-assisted folding.

mMDH and cMDH are structurally homologous enzymes which show very different responses to chaperonins during folding. The hydrophilic and stable cMDH is bound by cpn60 but released by Mg-ATP alone, while the hydrophobic and unstable mMDH requires both Mg-ATP and cpn10. Citrate equalises the stability of the native state of the two proteins but has no effect on the co-chaperonin requirement, implying that hydrophobicity, and not stability, is the determining factor. The yield and rate of folding of cMDH is unaffected while that of mMDH is markedly increased by the presence of cpn60, cpn10 and Mg-ATP. In 200 mM orthophosphate, chaperonins do not enhance the rate of folding of mMDH, but in low phosphate concentrations chaperonin-assisted folding is 3-4-times faster.

Adenosine Triphosphate↗
PubMed1994

Substitution of the amino acid at position 102 with polar and aromatic residues influences substrate specificity of lactate dehydrogenase.

The Gln residue at amino acid position 102 of Bacillus stearothermophilus lactate dehydrogenase was replaced with Ser, Thr, Tyr, or Phe to investigate the effect on substrate recognition. The Q102S and Q102T mutant enzymes were found to have a broader range of substrate specificity (measured by kcat/Km) than the wild-type enzyme. However, it is evident that either Ser or Thr at position 102 are of a size able to accommodate a wide variety of substrates in the active site and substrate specificity appears to rely largely on size discrimination in these mutants. The Q102F and Q102Y mutant enzymes have low catalytic efficiency and do not show this relaxed substrate specificity. However, their activities are restored by the presence of an aromatic substrate. All of the enzymes have a very low catalytic efficiency with branched chain aliphatic substrates.

Amino Acid Sequence↗
PubMed1994

An investigation of the thermal stabilities of two malate dehydrogenases by comparison of their three-dimensional structures.

The tertiary structure of Thermus aquaticus malate dehydrogenase (MDH) was predicted based on the known crystal structure of pig heart cytosolic MDH. Guanidinium chloride (GdmCl) unfolding experiments showed that there is only about a 4.2-kjoule/mol difference in delta G 0 between the pig and Thermus MDH. However, the two enzymes varied greatly in their [GdmCl]1/2, with Thermus MDH showing the expected increased stability (3.20 M against 0.58 M for pig MDH). The half-lives were determined for both Thermus MDH (34 min at 90 degrees C) and pig MDH (1.8 min at 60 degrees C). The Thermus MDH model was then examined to see what effect the substituted residues and changes may have on the enzyme, particularly in relation to its high thermal stability.

Amino Acid Sequence↗
PubMed1994

Localization of antigenic domains on the major subunits of Bordetella pertussis serotype 2 and 3 fimbriae.

Antibody-binding domains on the major subunits of Bordetella pertussis serotype 2 (Fim2) and 3 fimbriae (Fim3) have been identified using synthetic peptides which were screened for recognition by anti-protein monoclonal antibodies (mAbs). The presence of non-contiguous fimbrial epitopes was demonstrated by both anti-Fim2 and anti-Fim3 mAbs, several of which recognized at least two peptides that were discontinuous in the amino acid sequence of the corresponding subunits. The specificity of one mAb, 51/24, directed against Fim2, was investigated by replacement-set analysis of a 10-residue peptide, and revealed that antibody binding to the peptide was dependent on the sequence N94PQ96 which is non-conserved in Fim3. Furthermore, proline at residue 95 was found to be essential for mAb 51/24 binding. The specific anti-Fim3 mAb, AG3A, was found to recognize the 10-residue carboxy-terminal peptide from both Fim3 and, unexpectedly, from Fim2. This result suggests that mAb AG3A serospecificity at the protein level is determined by a conformational constraint which prevents mAb AG3A binding to the Fim2 C-terminal domain. Several free peptides containing amino acid residues which comprise part of the Fim2 and Fim3 epitopic domains were prepared as immunogens. One of these peptides was immunogenic in the mouse, indicating the location of a T-helper cell epitope within the peptide sequence, and induced a strong anti-peptide antibody response. The other peptides each required immunization as a conjugate with a carrier protein for anti-peptide antibody stimulation. All four anti-peptide antibody preparations only weakly recognized fimbriae-coated ELISA plates.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

The functional units of a peptostreptococcal protein L.

Protein L is a cell-surface protein from Peptostreptococcus which interacts with immunoglobulin kappa light chains. A gene from Peptostreptococcus strain 3316 coding for protein L and fragments thereof were expressed in Escherichia coli. The peptides were examined for binding to immunoglobulin and serum albumin. The four C units were shown to be responsible for binding to immunoglobulin and the four D units for binding to albumin. This protein L molecule therefore binds to albumin at a site separate from that involved in binding to immunoglobulin. The albumin-binding units have high amino acid sequence identity with the albumin-binding units of streptococcal cell-surface proteins. The gene contains three sites available for internal initiation of translation resulting in three active proteins. The protein L molecule presented in this report was compared with a previously reported protein from Peptostreptococcus strain 312. The two proteins differ in several respects, including size and the number and types of repeat units.

Amino Acid Sequence↗
PubMed1993

Rational design and PCR-based synthesis of an artificial Schizophyllum commune xylanase gene.

A synthetic gene encoding the Schizophyllum commune xylanase XynA was constructed by a novel PCR-based procedure. Three long oligonucleotides were synthesized and used in combination with flanking PCR primers to generate a 607 base pair gene which contained 31 unique locations for restriction enzyme cleavage. The amino acid sequence was tailored for expression in Escherichia coli by using only those codons found in highly expressed E. coli genes. The availability of the gene will facilitate analysis of the structure and function of this and other beta-(1,4) xylanases.

Base Sequence↗
PubMed1993

The identification of a lysine residue reactive to pyridoxal-5-phosphate in the glycerol dehydrogenase from the thermophile Bacillus stearothermophilus.

The glycerol dehydrogenase (GDH) from Bacillus stearothermophilus is inactivated by incubation with pyridoxal-5-phosphate (PALP). The complex formed between the two can be trapped by reduction with sodium borohydride to yield a protein with an absorbance band at 325 nm and a fluorescence emission band at 430 nm, typical of trapped pyridoxal-5-phosphate moieties. Total loss of catalytic activity of the enzyme is associated with the modification of approximately one equivalent of the reagent; the incorporation of the reagent and the loss of activity can be prevented by the additional presence of the oxidised or reduced coenzyme. Peptides derived from the labelled protein have been sequenced and have identified Lys-97 as the reactive residue. Site-directed mutagenesis had been used to replace Lys-97 by a His residue. This mutated enzyme has no catalytic activity and fluorescence spectroscopy studies suggest that it is unable to bind NADH.

Amino Acid Sequence↗
PubMed1993

The energetics and cooperativity of protein folding: a simple experimental analysis based upon the solvation of internal residues.

The reversible unfolding of two dissimilar proteins, phosphoglycerate kinase from Bacillus stearothermophilus (PGK) and Staphylococcus aureus nuclease (SAN), was induced with two denaturants, urea and guanidinium chloride (GuHCl). For each protein, structural transitions were monitored by intrinsic fluorescence intensity changes arising from a unique tryptophan residue. In the case of SAN the single, native tryptophan residue was used, whereas for PGK two versions, one with a tryptophan at position 315 and one at 379, were constructed genetically. The resultant folding curves were analyzed by considering the change in the solvation free energy of internal amino acid residues as the denaturant concentration was varied. We derive the following simple relationship: -RT ln K = delta Gw + n delta Gs,m[D]/Kden. + [D]) where K is the equilibrium constant describing the distribution of folded and unfolded forms at a given denaturant concentration [D], delta Gw is the free energy change for the transition in the absence of denaturant, and n is the number of internal side chains becoming exposed. delta Gs,m and Kden. are constants derived empirically from the solvation energies of model compounds and represent the behavior of an average internal side chain between 0 and 6 M GuHCl and 0 and 8 M urea. For proteins of known structure these values can easily be derived, and for others, average values in guanidinium chloride (delta Gs,m = 0.775 kcal/mol and Kden. = 5.4 M) or urea (delta Gs,m = 1.198 kcal/mol and Kden. = 25.25 M) can be used in the analysis. Results show that the parameters n and delta Gw are independent of the denaturant used for all 12 transitions studied. This supports the hypothesis that the unfolding activity of urea and GuHCl can be accounted for by their effect on the solvation energy of amino acid side chains which are buried in the folded but exposed in the unfolded protein. This simple analytical treatment allows the "cooperativity" of protein folding to be interpreted in terms of the number of side chains becoming exposed to the solvent in a given step and allows accurate estimation of the free energy irrespective of the denaturant concentration needed to induce the transition.

Algorithms↗
PubMed1993

Binding and hydrolysis of nucleotides in the chaperonin catalytic cycle: implications for the mechanism of assisted protein folding.

Cpn60 was labeled with pyrene maleimide in order to follow structural rearrangements in the protein triggered by the binding of nucleotides and cpn10. The conjugate binds ATP, AMP-PNP, and ADP(P(i)) with pyrene fluorescence enhancements of 60%, 60%, and 15%, respectively. In each case, binding is cooperative with half-saturation (K1/2) occurring at 10 microM, 290 microM, and 2500 microM and Hill constants (nH) of 4, 3, and 3, respectively. Inclusion of the co-protein, cpn10, tightens the binding of ATP, AMP-PNP, and ADP(P(i)) to give K1/2 values of 6 microM, 100 microM, and < 0.07 microM, respectively, and cooperativity is increased. Titration of the cpn60/ADP (14-mer) complex with cpn10 (7-mer) gives a stoichiometry of 14:7 with respect to subunits, confirming the molecular asymmetry shown by electron microscopy. Transient kinetics demonstrate that ATP initially forms a weak collision complex with cpn60 (Kd = 4 mM) which isomerizes to the strongly binding state at a rate of 180 s-1. We suggest that the slow structural rearrangement driven by ATP binding is the same event which lowers the affinity of the chaperonin for protein substrates; a suggestion reinforced by the loss of AMP-PNP binding affinity in the presence of an unstructured polypeptide. As such, this rearrangement of cpn60 is analogous to a force-generating step in energy transduction. Measurements of ATP hydrolysis (pH 7.5, 25 degrees C) show that it is slow (0.04 s-1) compared both with the structural rearrangement and with the dissociation of products. This defines the steady-state complex as cpn60/ATP, a form of the chaperonin which binds substrate proteins weakly. The rate of hydrolysis of ATP is stimulated 20-fold upon binding unfolded lactate dehydrogenase, and the yield of folded enzyme is increased even in the absence of cpn10. Addition of this co-protein inhibits hydrolysis on only half of the sites in cpn60 and leads to a faster release of folded LDH. A mechanism for the action of chaperonins is proposed which depends upon cpn60 being cycled between states which have, alternately, low and high affinity for unfolded proteins. This cycle is driven by the binding and hydrolysis of ATP.

Adenine Nucleotides↗
PubMed1993

Dissecting the contributions of a specific side-chain interaction to folding and catalysis of Bacillus stearothermophilus lactate dehydrogenase.

X-ray crystallography predicts hydrogen-bonding interactions between the side chains of Thr198 and two other amino acid residues, Glu194 (adjacent to the catalytic His195) and Ser318 (on the alpha-H helix which rearranges on substrate binding). In order to investigate the contribution of this conserved amino acid residue, Thr198, two mutants of Bacillus stearothermophilus lactate dehydrogenase were created (Val198 and Ile198). The steady-state kinetic parameters for both mutant enzymes were very similar with increased substrate Km and reduced kcat when compared with the wild-type enzyme. The mutation Val198 allowed non-productive binding of pyruvate to the unprotonated form of His195. Steady-state kinetic parameters determined for the Val198 mutant enzyme in high solvent viscosity suggested both an altered rate-limiting step in catalysis and implicated Thr198 in allosteric activation by the effector fructose 1,6-bisphosphate (Fru1,6P2). A shift in the Fru1,6P2 activation constant for the Val198 mutant enzyme suggested that Thr198 stabilises the catalytically competent (Fru1,6P2-activated) form of the enzyme by 6.6 kJ/mol. However, Thr198 was not important for maintaining the thermal stability of the Fru1,6P2-activated form. Equilibrium unfolding in guanidinium chloride indicated that Thr198 contributes 17.2 kJ/mol subunits towards the tertiary structural stability. The results emphasise the importance of the side chain-hydroxyl group of Thr198 which is required for (a) productive substrate binding, (b) allosteric activation and (c) protein conformational stability. The characteristics of the B. stearothermophilus lactate dehydrogenase mutations reported here were significantly different from those of the same mutations made in the corresponding position of the analogous enzyme Thermus flavus malate dehydrogenase [Nishiyama, M., Shimada, K., Horinouchi, S., & Beppu, T. (1991) J. Biol. Chem. 266, 14294-14299].

Base Sequence↗
PubMed1993

Localisation of a receptor-recognition domain on the S3 subunit of pertussis toxin by peptide mapping.

Overlapping 10-amino-acid peptides, which consecutively span the amino acid sequence of the S3 subunit of pertussis toxin, were synthesised on polyethylene pins and screened for their ability to bind the glycoprotein fetuin. Fetuin binding was localised to a single peptide comprising amino acids 46-55. A free peptide, (E)S3c, of longer sequence (S3 amino acids 44-58) was also found to bind alpha-1-acid glycoprotein, mixed brain gangliosides and fetuin. (E)S3c also recognised asialofetuin but with a lower apparent affinity relative to fetuin. The single tryptophan residue of the peptide yielded a fluorescence-emission maximum of 355 nm. In the presence of either ganglioside or the phospholipid L-alpha-lysolecithin, but not N-acetylneuramin-lactose or lactosylceramide, the emission intensity of (E)S3c was enhanced and the emission maximum blue-shifted to 340 nm by ganglioside, or to 345 nm by L-alpha-lysolecithin. Monosialogangliosides, disialogangliosides, and trisialogangliosides, when fluorescence-titrated, were each found to bind the peptide with a similar dissociation constant of 4.4 +/- 2.8 microM. These findings demonstrate that region 44-58 of the pertussis-toxin S3 subunit is likely to be involved in the recognition of both glycosylated and phospholipid constituents of target-cell membranes.

Amino Acid Sequence↗
PubMed1993

The complete amino acid sequence of a hirudin variant from the leech Hirudinaria manillensis.

Unlike the European leech Hirudo medicinalis, the Asian jawed leech Hirudinaria manillensis is specialized for feeding on mammalian blood. In the salivary glands of both these leeches, there is a potent inhibitor of thrombin, called hirudin, which acts as an anticoagulant. We have reported previously the isolation and purification of a variant of hirudin, called bufrudin, from the head portions of Hirudinaria. In the present study, the complete amino acid sequence of bufrudin was determined by automated Edman degradation of peptide fragments generated after cleavage of protein with trypsin or thermolysin. Comparison of the primary structure of bufrudin, with hirudin HV1, show about 70% sequence identity with deletion of two amino acids, but the key amino acids at the C-terminus, involved in the inhibition of thrombin, are conserved. However, similar sequence comparison of bufrudin with hirullin P18, a hirudin variant isolated from the same leech species but from whole leech, instead of heads, reveals even less sequence identity of about 60%. From the amino acid sequence, it is suggested that the conformation of the C-terminal portion of bufrudin may be significantly different from hirullin P18, but similar to hirudin HV1, upon its interaction with thrombin. These results indicate that, as with Hirudo leech, various isoforms of hirudin also exist in Hirudinaria leech, with a significant change occurring in the structure of the molecule during the evolution of leeches.

Amino Acid Sequence↗