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S J Leach

Publications and source records attributed to S J Leach.

At least 19 recordsLinked to original sources
PubMed1990

Location of the dihydroorotase domain within trifunctional hamster dihydroorotate synthetase.

Mammalian dihydroorotase (DHOase, EC 3.5.2.3) is part of a trifunctional protein, dihydroorotate synthetase which catalyzes the first three reactions of de novo pyrimidine biosynthesis. We have subcloned a portion of the cDNA from the plasmid pCAD142 and obtained a nucleotide sequence which extends 2.1 kb in the 5' direction from the sequence encoding the aspartate transcarbamoylase (ATCase) domain at the 3'-end of the cDNA. The DHOase and ATCase domains have been purified from an elastase digest of the trifunctional protein and subjected to amino acid (aa) sequencing from their N termini. The sequence of the N-terminal 24 aa of the DHOase domain has been obtained and aligned with the cDNA sequence. The C-terminal residues of the DHOase domain have been identified as Leu followed by Val which, when taken with partial sequences of the CNBr fragments of this domain, defines the coding sequence of the active, globular DHOase domain released by proteolysis. Prediction of protein secondary structure from the deduced aa sequence showed that the DHOase domain (Mr 37,751) is separated from the C-terminal ATCase domain (Mr 34,323) by a bridging sequence (Mr 12,532) consisting of multiple beta-turns.

Amino Acid Sequence↗
PubMed1988

Synthesis, antiviral activity, and conformational characterization of mouse-human alpha-interferon hybrids.

Reciprocal hybrids were constructed between human and mouse interferons (IFNs), and their antiviral activity was examined on different target cells and compared to the activity of the parental molecules. In addition, we used a number of predictive algorithms on a data base of the available alpha-interferon sequences to propose a working model for the overall conformation of the alpha-interferon molecule that is consistent with the structural predictions. Remarkable conservation within the predicted alpha-helical segments of the interferon molecule was observed. We propose that the observed changes in the activity and specificity of the hybrids obtained are largely due to the sequences present in the loops at the ends of the major helical structures; these are less conserved, contain beta-bends, and are generally hydrophilic and flexible. The data on the constructed mouse-human hybrids have shown that the activity on human cells is contributed by determinants present in the N-terminal 122 amino acids of human IFN, thus implicating one or more loops within this region (e.g. loops 1-12, 25-38, 70-74, and 103-113). The activity on bovine cells appears to be localized mainly in sequence 60-121, implicating the role of loops 70-74 and/or 103-113 of the human IFN molecule. The specificity of mouse IFN for mouse cells is in some or all of the loops (70-74, 103-113, 134-139, and 163-166) in the C-terminal sequence. The proposed working model should provide guidelines for the study of the specificity of action in molecular terms.

Amino Acid Sequence↗
PubMed1988

Conformational homologies among cytokines: interleukins and colony stimulating factors.

Some 30 cytokine amino acid sequences (mainly interleukins, colony stimulating factors and tumor necrosis factors) have been examined for evidence of secondary structure as well as longer-range interactions of a type likely to lead to stable alpha-helical bundles. Most, though not all, of the cytokines examined have a high predicted alpha-helical content (40-60%) and quasi-repeating heptads containing i/i + 3 apolar periodicities. This major subset of the cytokines is predicted to be characterized by molecules in which 4-alpha-helical bundles with an average length of 25A are the most marked conformational features. Based on these conclusions, we suggest structures for huG-CSF, huGM-CSF and muIL-5 in which defined loop segments at the ends of helical bundles are the most likely sites for binding and recognition by specific cell receptors. As such, they provide a means for testing or refining the three working models we have defined, using currently available methods of site-directed substitution and deletion mutagenesis, as well as synthetic peptides corresponding to the proposed loop sequences and the use of monoclonal antibodies of defined epitopic specificity. The structure arrived at for huGM-CSF is consistent with the limited data currently available concerning the residues which are important for binding and activity.

Algorithms↗
PubMed1986

Antigenic repeat structures in proteins of Plasmodium falciparum.

The majority of malaria antigens that have been cloned contain short sequence repeats which encode antigenic epitopes that are naturally immunogenic. Synthetic peptides have been used to show that natural antibody responses to a strain-specific Plasmodium falciparum S antigen are largely directed against epitopes encoded in an 11-amino acid sequence that is repeated approximately 100 times in the molecule. A 16-amino acid peptide conjugated to bovine serum albumin induced antibodies specific for the S antigen of the homologous isolate. Synthetic peptides have also been used to confirm the natural immunogenicity of epitopes encoded within two blocks of related repeats in the Ring-infected Erythrocyte Surface Antigen (RESA). A 16-amino acid peptide, comprising four repeats of the tetrameric sequence EENV, induced antibodies reactive with the native molecule. Detailed analyses of these anti-peptide antisera indicate that short sequence repeats express more than one epitope, some of which may cross-react with other repeat structures.

Amino Acid Sequence↗
PubMed1986

Antibody response to the C-terminal peptide sequence in beef myoglobin.

The nature of the surface determinants on the beef myoglobin molecule which direct the distinctive antibody response of sheep, have been further defined. Antisera raised to beef myoglobin in sheep, rabbits and mice have been compared for their ability to recognize the synthetic C-terminal beef myoglobin peptide (140-153), which contains four of the six amino acid substitutions between sheep and beef myoglobins. The antibodies raised in rabbits, directed to the entire surface of beef myoglobin, contain only a minor population directed to the C-terminal sequence: in mice, even fewer antibodies are specific for this sequence. In sheep, however, antibodies to beef myoglobin appear to be directed almost exclusively to topographic domains which include the (140-153) sequence. This specificity is most apparent in "early" antisera in which all antibodies display equal avidity for "native" beef myoglobin and the peptide; further immunization produces antibodies which recognize a larger overlapping set of domains and only 20% of these antibodies have effective avidity for the (140-153) peptide. The antibodies to beef myoglobin raised in sheep comprise two discrete populations. One ("common") population is directed to regions of similarity between the beef and sheep myoglobin molecules, in which the region represented by the C-terminal peptide of beef myoglobin is less important in defining the antibody-binding site and/or affinity, while still being directly involved in the topographic determinant. The other ("non-common") appears to be directed almost exclusively to the C-terminal sequence (140-153) of beef myoglobin. The findings are discussed in relation to our previous findings on the effect of the host species on the nature of the antibody response and in relation to views on the possibility of direct vs indirect effects of evolutionary amino acid substitution on immuno-cross-reactivity among homologous proteins.

Animals↗
PubMed1985

Two large immunogenic and antigenic myoglobin peptides and the effects of cyclisation.

Peptides corresponding to sequences (72-88) and (26-54) of beef myoglobin have been synthesised in their open-chain and cyclised forms (using a disulphide bridge) and tested for their antigenicity and immunogenicity. Antibodies raised to beef myoglobin bound to both peptides but more strongly to the 29-residue than to the 17-residue peptide. Cyclisation increased the antigenicity of the larger peptide. In this form the peptide competed much more strongly than in the uncyclised form for specific antibodies to beef myoglobin. The peptides are immunogenic in mice without being coupled to a protein carrier and produce antibodies which bind to beef myoglobin. Peptide (26-54) is the more immunogenic in producing a larger antibody titre to the parent myoglobin and cyclisation again enhances this property. The findings lend weight to the view that longer peptide sequences might be expected to favour the folded state, therefore binding more strongly to antibodies raised to the native protein and eliciting a population of antibodies which contain a larger proportion specific for that conformation. Cyclisation enhances antigenicity and immunogenicity presumably by decreasing the number of degrees of conformational freedom of a peptide without excluding native-like conformations.

Amino Acid Sequence↗
PubMed1985

Original antigenic sin and the production of autoantibodies.

Autoantibodies to sheep myoglobin have been raised by priming sheep with beef myoglobin and boosting with sheep myoglobin. The autoantibodies appear to be a subset of those produced when beef myoglobin is used for both priming and boosting. This subset of antibodies is presumably directed to the surface regions which are common to both myoglobins. The antibodies which bind to sheep myoglobin in the 2 types of antisera differ. Those elicited by boosting with beef myoglobin bind better to beef myoglobin than to sheep myoglobin, while those obtained by boosting with sheep myoglobin bind with equal avidity to the 2 myoglobins. It would seem therefore that the boosting immunogen determines which fraction of antibodies is selected from the antibody repertoire established by the priming immunogen. Our results also show that tolerance at the T-cell level can be circumvented by exposing the immune system to a protein closely related to a homologous self protein.

Animals↗
PubMed1985

Conformational analysis of cyclized dipeptide models for specific types of beta-bends by two-dimensional nuclear Overhauser spectroscopy.

The solution conformations in DMSO-d6 of the two cyclized dipeptides, cyclo(L-alanyl-L-alanyl-epsilon-aminocaproyl) and cyclo(L-alanyl-D-alanyl-epsilon-aminocaproyl), have been analyzed by means of the two-dimensional nuclear Overhauser effect (2D-NOE). The preferred conformations for the two compounds have been deduced by comparing proton-proton distances, derived from the 2D-NOE data and relaxation-time measurements, with the corresponding distances in several possible computed low-energy conformations. The predominant conformations are a type III bend and a type II bend, respectively, for the two compounds. These conclusions agree with those deduced earlier on the basis of infrared and Raman spectra and circular dichroism measurements.

Magnetic Resonance Spectroscopy↗
PubMed1979

Leghaemoglobin from Trifolium subterraneum. Purification and characterization.

Leghaemoglobin from the subclover, Trifolium subterraneum cultivar Woogenellup, has been fractionated into at least four electrophoretically distinct components using the ion-exchange chromatographic procedure described by Appleby et al. (Appebly, C.A., Nicola, N.A., Hurrell, J.G.R. and Leach, S.J. (1975) Biochemistry 14, 4444--4450) for soybean leghaemoglobins. Unlike those of soybean, the subclover leghaemoglobins showed no evidence of autoxidation under identical isolation procedures, implying that these proteins have an unusually stable ferrous oxidation state. Circular dichroism in the far-ultraviolet (200--240 nm) indicated a high helicity (approx. 70%) as has been reported for other species of leghaemoglobins. However, circular dichroism in the near-ultraviolet region (240--300 nm) indicated that the haem-protein interactions may be considerably different in the subclover leghaemoglobins and this may explain their atypical resistance to autoxidation and the absence of nicotinate binding in these proteins.

Binding Sites↗
PubMed1979

Cyclized dipeptide model for a beta-bend.

A cyclic dipeptide in which L-Ala-Gly was cyclized with epsilon-aminocaproic acid has been synthesized as a model for a beta-bend. Its conformational properties have been examined by means of conformational energy calculations and nuclear magnetic resonance, infrared, Raman, and circular dichroism spectroscopy in various solvents. These calculations and experiments suggest that a type II beta-bend exists in the Ala-Glymoiety, with an NH...O = C hydrogen bond in the epsilon-aminocaproic acid portion of the molecule, and that the molecule adopts a unique conformation in solution. In contrast, an open-chain analog of this compound exists in solution as an ensemble of conformations but with a significant amount of a type II beta-bend structure in the ensemble.

Circular Dichroism↗
PubMed1977

Structural rearrangements due to ligand binding and haem replacement in myoglobin and leghaemoglobins.

Structural rearrangements in sperm whale myoglobin and leghaemoglobins caused by changes in the spin or oxidation state of the iron as a consequence of ligand binding have been measured by difference spectroscopy in the ultraviolet. When compared with the high-spin acetate complex, ligands which cause a transition to the low-spin state also cause large perturbations of tyrosine(s) remote from the haem pocket in myoglobin but only minor perturbations of tryptophan (s) in leghaem calobin. This may indicate a weaker coupling between events at the haem site and conformational changes in the protein in leghaemoglobins. The absorption spectra of various haem-liganded forms of the two proteins as well as the binding of the dye rose Bengal to the two apoproteins are consistent with weaker interactions between the haem ano apoprotein and a more solvent-exposed haem pocket in leghaemoglobin compared with myoglobin.

Animals↗