Search PubMed⌕ Search

Biomedical subjects

A B Mason

Publications and source records attributed to A B Mason.

At least 55 records · Page 3Linked to original sources
PubMed1995

Targeting the fungal plasma membrane proton pump.

The need for new mechanistic classes of broad spectrum antifungal agents has prompted development of the membrane sector and ectodomain of the plasma membrane proton pumping ATPase as an antifungal target. The fungal proton pump is a highly abundant, essential enzyme in Saccharomyces cerevisiae. It belongs to the family of P-type ATPases, a class of enzymes that includes the Na+,K(+)-ATPase and the gastric H+,K(+)-ATPase. These enzymes are cell surface therapeutic targets for the cardiac glycosides and several anti-ulcer drugs, respectively. The effects of acid-activated omeprazole show that extensive inhibition of the S. cerevisiae ATPase is fungicidal. Fungal proton pumps possess elements within their transmembrane loops that distinguish them from other P-type ATPases. These loops, such as the conformationally sensitive transmembrane loop 1+2, can attenuate the activity of the enzyme. Expression in S. cerevisiae of fully functional chimeric ATPases that contain a foreign target comprising transmembrane loops 1+2 and/or 3+4 from the fungal pathogen Candida albicans suggests that these loops operate as a domain. The chimera containing C. albicans transmembrane loops 1+2 and 3+4 provides a prototype for mutational analysis of the target region and the screening of inhibitors directed against opportunistic fungal pathogens. Panels of mutants with modified ATPase regulation or with altered cell surface cysteine residues are also described. Information about the ATPase membrane sector and ectodomain has been integrated into a model of this region.

Amino Acid Sequence↗
PubMed1994

Calorimetric studies of serum transferrin and ovotransferrin. Estimates of domain interactions, and study of the kinetic complexities of ferric ion binding.

Human serum transferrin and hen ovotransferrin have been studied by differential scanning calorimetry (DSC), in an effort to quantitatively estimate the free energy of interaction of the N- and C-domains in each protein and to further understand their interaction with chelated ferric ions. In the case of serum transferrin, separate DSC transitions are observed for the two domains while only a single, coupled transition is seen for ovotransferrin. Although domain interactions are somewhat larger for ovotransferrin (-4100 cal/mol) than for serum transferrin (-3100 cal/mol), the major cause of separated transitions for serum transferrin is that the difference in intrinsic folding stability of the N- and C-domains is about 4-fold larger than for ovotransferrin. Chelated ferric ions bind strongly to each site in both proteins and produce changes in Tm by as much as 30 degrees C. When apparent binding constants are estimated from DSC results, these appear to be substantially larger than those estimated previously from equilibrium methods at low temperatures, where very long equilibrium times must be used because of slow ligand release. Although second DSC upscans on each protein show good "reversibility", downscans on serum transferrin revealed that liganded forms of the protein are in fact not in true equilibrium during upscanning, which causes Tm values during upscans to be higher than the true reversible Tm values. The likely reason for this kinetic control over unfolding is the slow release of bound ferric ions and those effects, for technical reasons, cannot be totally eliminated by lowering the scan rate.

Animals↗
PubMed1994

Salt effects on the physical properties of the transferrins.

Salts are known to have a pronounced effect on the spectroscopic, thermodynamic and kinetic properties of human serum transferrin. The present study was undertaken to examine the effect of NaCl on the related proteins ovotransferrin and lactoferrin. EPR difference spectroscopy was used to probe changes in the metal site of these proteins. Sodium chloride was found to perturb the g' = 4.3 EPR spectra of both ovotransferrin and lactoferrin but in different ways. The spectrum of ovotransferrin is reduced in amplitude with a broad feature appearing at g' = 4.8 whereas there is a loss of resolution of the doublet feature at the peak of the EPR derivative spectrum for lactoferrin. The increase in the amplitude of the ovotransferrin EPR difference spectrum (spectrum without NaCl minus spectrum with NaCl) as a function of NaCl concentration is suggestive of saturation binding. A Hill plot binding isotherm gave n = 1.87 +/- 0.32 and log K = 1.49 +/- 0.03 for ovotransferrin, where n is the number of C1- ions binding to either one or both iron containing lobes of the protein and K is the overall association constant. Preliminary measurements with lactoferrin gave n = 1.95 +/- 0.34 and log K = 1.41 +/- 0.06. These results are similar to those previously reported for serum transferrin and suggest that Cl- binds to all the transferrins with strong pairwise cooperativity. This binding may reflect a functional role for chloride and other physiological anions in the uptake and release of iron by the transferrins.

Chlorides↗
PubMed1993

Calorimetric studies of the binding of ferric ions to human serum transferrin.

The binding of ferric ions, chelated with nitrilotriacetate, to human serum transferrin (hTF) has been studied using ultrasensitive titration calorimetry. Studies were done in both the presence and the absence of the synergistic bicarbonate anion. It was found that the C-site of hTF is capable of weakly binding bicarbonate (K of 250 M-1, delta H of -8 kcal) at the binding site even before ferric ion is added, although this does not happen to the same extent at the N-site. When preinsertion of the bicarbonate ion occurs, then ferric ion can subsequently bind very quickly to the C-site. Although the chelated ferric ion can bind weakly to the N-site in a fast reaction, the insertion of the bicarbonate ion occurs subsequently in a slow endothermic reaction. Binding of ferric ion to both sites is quickly reversible in the absence of bicarbonate but becomes kinetically controlled for long periods of time once bicarbonate has inserted into the metal-binding site due to the long time required for release of ferric ion. Estimates of the heats of binding to each site, apparent binding constants, and heat capacities of binding are made for different sets of solution conditions. Results from this study are compared to earlier results with ovotransferrin (Lin, L.-N., Mason, A. B., Woodworth, R. C., & Brandts, J. F. (1991) Biochemistry 30, 11660-11669), with major differences and some similarities noted.

Bicarbonates↗
PubMed1993

Calorimetric studies of the N-terminal half-molecule of transferrin and mutant forms modified near the Fe(3+)-binding site.

The effects of single amino acid substitution on the thermal stability of the N-terminal half-molecule of human transferrin and its iron-binding affinity have been studied by high-sensitivity scanning calorimetry. All site-directed mutations are located on the surface of the binding cleft, and they are D63-->S, D63-->C, G65-->R, H207-->E and K206-->Q. Differential scanning calorimetry results show that the mutations do not significantly alter the conformational stability of the apo-forms of the proteins. The changes in free energy of unfolding relative to the wild-type protein range from 0.83 to -2.4 kJ/mol. The D63-->S, G54-->R and H207-->E mutations slightly destabilize the apo-protein, while the D63-->C and K206-->Q mutations increase its stability by a small amount. However, there are large compensating enthalpy-entropy changes caused by all mutations. All mutants bind ferric ion, but with different affinities. Replacement of Asp-63 by either Ser or Cys decreases the apparent binding constant by 5-6 orders of magnitude. The G65-->R mutation also decreases the apparent binding constant by 5 orders of magnitude. The K206-->Q mutation increases the apparent binding constant by 20-fold, while the H207-->E mutation does not significantly change the apparent iron-binding affinity of the half-molecule.

Binding Sites↗
PubMed1993

Asp ligand provides the trigger for closure of transferrin molecules. Direct evidence from X-ray scattering studies of site-specific mutants of the N-terminal half-molecule of human transferrin.

Recent X-ray crystallographic and solution X-ray scattering studies have shown that transferrins (serum transferrin, lactoferrin and ovotransferrin) undergo a major conformational change when iron is incorporated into the molecule. Apo-proteins show a structure with open interdomain clefts which close when iron is bound. The closed conformation has been suggested as an important step in the receptor recognition. Here, we report X-ray solution scattering experiments of the mutated N-terminal fragment of human serum transferrin with Asp63-->Ser (Cys). The data provide the first direct experimental evidence for the existence of a trigger mechanism for the closure of the interdomain cleft and that this trigger mechanism is disrupted by mutation of Asp63, the only ligand of iron from domain I.

Aspartic Acid↗
PubMed1993

Expression of glycosylated and nonglycosylated human transferrin in mammalian cells. Characterization of the recombinant proteins with comparison to three commercially available transferrins.

The coding sequence for human serum transferrin was assembled from restriction fragments derived from a full-length cDNA clone isolated from a human liver cDNA library. The assembled clone was inserted into the expression vector pNUT and stably transfected into transformed baby hamster kidney (BHK) cells, leading to secretion of up to 125 mg/L recombinant protein into the tissue culture medium. As judged by mobility on NaDodSO4-PAGE, immunoreactivity, spectral properties (indicative of correct folding and iron binding), and the ability to bind to receptors on a human cell line, initial studies showed that the recombinant transferrin, is identical to three commercial human serum transferrin samples. Electrospray mass spectrometry (ESMS), anion-exchange chromatography, and urea gel analysis showed that the recombinant protein has an extremely complex carbohydrate pattern with 16 separate masses ranging from 78,833 to 80,802 daltons. Mutation of the two asparagine carbohydrate linkage sites to aspartic acid residues led to the expression and secretion of up to 25 mg/L nonglycosylated transferrin. ESMS, anion-exchange chromatography, and urea gel analysis showed a single molecular species that was consistent with the expected theoretical mass of 75,143 daltons. In equilibrium binding experiments, the nonglycosylated mutant bound to HeLa S3 cells with the same avidity and to the same extent as the glycosylated protein and the three commercial samples. These studies demonstrate conclusively that carbohydrate has no role in this function.

Animals↗
PubMed1993

Oxalate- and Ga(3+)-induced structural changes in human serum transferrin and its recombinant N-lobe. 1H NMR detection of preferential C-lobe Ga3+ binding.

(1) The binding of the synergistic anion oxalate and Ga3+ to human serum transferrin (HTF, 80 kDa) and its recombinant N-lobe (HTF/2N, 40 kDa) has been studied by one- and two-dimensional 1H NMR spectroscopy, at 310 K, pH*7.25. (2) Specific protein resonances are sensitive to oxalate binding (fast exchange on the NMR time scale) and allowed determination of the apparent binding constant for oxalate binding to the N-lobe (log K 4.04). (3) Slow exchange between apo-HTF and Ga-loaded HTF or HTF/2N was observed. Binding of Ga3+ appeared to be accompanied by small changes in the orientations of residues in hydrophobic pockets in the interdomain hinge region close to the metal binding site. (4) Under the conditions used, preferential binding of Ga3+ (added as Ga(NTA)2) to the C-lobe of HTF was observed. Binding to the C-lobe markedly perturbed resonances in the glycan N-acetyl region of the spectrum, suggesting that metal binding is communicated to the surface of the protein. This could be important in receptor recognition of metallotransferrins. (5) The displacement of Ga3+ from Ga-ox-HTF with Fe3+ was studied, and the paramagnetic broadening effects allowed identification of resonances from groups close to Fe3+. The passage of Fe3+ from the exterior to the interior of the protein was followed by 1H NMR spectroscopy, and the half-life for Ga(3+)-Fe3+ exchange was determined to be 4.3 h (310 K).

Gallium↗
PubMed1993

Molecular cloning and characterization of the Candida albicans enolase gene.

A DNA clone containing the putative Candida albicans enolase gene (ENO1) was isolated from a genomic DNA library. The sequenced insert contained a continuous open reading frame of 1,320 bp. The predicted 440-amino-acid protein is 78 and 76% identical, respectively, to Saccharomyces cerevisiae enolase proteins 1 and 2. Only one enolase gene could be detected in C. albicans genomic DNA by Southern analysis with a homologous probe. Northern (RNA) analysis detected a single, abundant C. albicans ENO1 transcript of approximately 1,600 nucleotides. When cells were grown on glucose, levels of ENO1 mRNA were markedly increased by comparison with ENO1 mRNA levels in cells grown on ethanol, a gluconeogenic carbon source. In contrast to this glucose-mediated transcriptional induction, the carbon source had no dramatic effect on the levels of enolase protein or enzyme activity in the C. albicans strains tested. These results suggest that posttranscriptional mechanisms are responsible for modulating expression of the C. albicans enolase gene.

Amino Acid Sequence↗
PubMed1992

Preliminary crystallographic analyses of the N-terminal lobe of recombinant human serum transferrin.

The N-terminal lobe of recombinant human serum transferrin (residues 1 to 337) has been crystallized in a form suitable for high-resolution three-dimensional X-ray crystallographic analyses. Crystals are of the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions of a = 44.9 A, b = 57.0 A and c = 135.9 A, and diffract to beyond 2 A resolution. Further studies show that isomorphous crystals of specifically designed mutants of this protein can also be grown. Structural studies of both recombinant and mutant protein forms will provide a basis for understanding the mechanism by which human serum transferrin functions.

Crystallization↗
PubMed1992

Uptake of Al3+ into the N-lobe of human serum transferrin.

We have studied the binding of Al3+ to human serum apotransferrin (80 kDa) and recombinant N-lobe human apotransferrin (40 kDa) in 0.1 M-sodium bicarbonate solution at a pH meter reading in 2H2O (pH*) of 8.8 using 1H n.m.r. spectroscopy. The results show that for the intact protein, preferential binding of Al3+ to the N-lobe occurs. Molecular modelling combined with an analysis of ring-current-induced shifts suggest that n.m.r. spectroscopy can be used to probe hinge bending processes which accompany metal uptake in solution.

Aluminum↗
PubMed1991

Calorimetric studies of the binding of ferric ions to ovotransferrin and interactions between binding sites.

Transferrins are two-domain proteins with a very strong site for iron binding located in each domain. Using ultrasensitive titration calorimetry, the binding of ferric ion (chelated with a 2-fold molar excess of nitrilotriacetate) to the two sites of ovotransferrin was studied in detail as well as the binding to the single site in the N- and C-terminal half-molecules. In the presence of excess bicarbonate ion, the binding occurs in two kinetic steps. The fast process of contact binding is instantaneous with respect to instrument response time, is strongly exothermic for the N site and less so for the C site, and corresponds to binding of the chelated ferric ion. The slower process of bicarbonate insertion with concomitant release of nitrilotriacetate occurs on a time scale of 2-20 min over the temperature range 7-37 degrees C and is endothermic for the N site and exothermic for the C site, with rates being significantly slower for insertion at the C site. The delta H of binding is strongly temperature-dependent for both sites, arising from a large negative delta Cp of binding which probably indicates removal of hydrophobic groups from contact with water. When bicarbonate ion is absent, only the fast process of contact binding is seen. Each site within a half-molecule is qualitatively similar to the same site in intact ovotransferrin, although quantitative differences were detected. It was shown that contact binding to ovotransferrin occurs reversibly with free exchange of Fe+3 between N and C sites, while the attachment to either site becomes essentially irreversible after bicarbonate insertion. The strong preference for the first ferric ion to bind to the N site is shown to be due to its larger contact binding constant and the faster rate of bicarbonate insertion, relative to the C site, and is not due to stronger thermodynamic binding after bicarbonate insertion. True equilibrium is achieved only over much longer periods of time. In another series of experiments, direct binding studies were carried out between the two half-molecules under different states of ligation with Fe+3 in the presence of bicarbonate. The results indicate that the two binding sites in ovotransferrin, separated by ca. 40 A, are not independent of one another but communicate as a result of ligand-dependent changes in the heats and free energies of domain-domain interactions.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Expression and initial characterization of five site-directed mutants of the N-terminal half-molecule of human transferrin.

Five site-directed mutants of the N-terminal half-molecule of human serum transferrin have been expressed in baby hamster kidney cells and purified to homogeneity. Expression levels and overall yields varied considerably from the wild-type protein, depending on the mutant in question. The mutants are D63S, D63C, G65R, K206Q, and H207E and are based on mutations observed in a variety of transferrins of known sequence. Their molecular masses, determined by electrospray mass spectrometry, agree with theory, except for the D63C mutant, which appears to be cysteinylated. All mutants bind iron but with varying affinities; qualitatively, in increasing order D63S approximately D63C approximately G65R much less than wild type less than or equal to H207E much less than K206Q. In general, reduction of formal negative charge within the binding cleft shifts the visible spectral maximum of the iron complex toward the blue and reduces the affinity for iron, and increasing the formal negative charge shifts the visible maximum toward the red and increases the affinity for iron. The K206Q mutant is exceptional inasmuch as its visible maximum shows a blue shift, but its affinity for iron is the greatest of all of the mutants studied. All mutants reported, in addition to the wild-type protein, exhibit very similar visible molar extinction coefficients for the iron complex and very similar changes in extinction coefficients at 240 nm on binding Fe(III) or Ga(III). These results suggest that in all cases the bound metal ion is coordinated by two tyrosyl side chains.

Amino Acid Sequence↗
PubMed1991

Exosome formation during maturation of mammalian and avian reticulocytes: evidence that exosome release is a major route for externalization of obsolete membrane proteins.

We have assessed whether exosome formation is a significant route for loss of plasma membrane functions during sheep reticulocyte maturation in vitro. Although the recovery of transferrin binding activity in exosomes is at best approximately 25-30% of the lost activity, recoveries of over 50% of the lost receptor can be obtained if 125I-labelled transferrin receptor is measured using an that receptor instability may contribute to the less than quantitative recovery of the transferrin receptor. Significantly higher (75-80%) levels of the nucleoside transporter can be recovered in exosomes during red cell maturation using 3H-nitrobenzylthioinosine binding to measure the nucleoside transporter. These data suggest that exosome formation is a major route for removal of plasma membrane proteins during reticulocyte maturation and plasma membrane remodelling. We have also shown that both in vivo and in vitro, embryonic chicken reticulocytes form exosomes which contain the transferrin receptor. Thus, exosome formation is not restricted to mammalian red cells, but also occurs in red cells, which retain organelles, such as nuclei and mitochondria, into the mature red cell stage.

Animals↗
PubMed1991

Electrophoretic characterization of human, equine and bovine transferrins.

1. Human, bovine and equine transferrins have been characterized with respect to mol. wt, and behavior on urea-polyacrylamide gels, and isoelectric focussing gels. 2. As shown by SDS-polyacrylamide gel electrophoresis human transferrin has one major polypeptide whereas both bovine and equine transferrins have two polypeptides. 3. The transferrins show multiple banded patterns on urea-polyacrylamide and isoelectric focussing gels, particularly when iron saturated. The various forms are not resolved by neuraminidase treatment.

Animals↗
PubMed1991

Monoclonal antibodies to the amino- and carboxyl-terminal domains of human transferrin.

Seven high affinity antibodies to human serum transferrin which recognize at least four different epitopes are described. Apparent dissociation constants (Kd's) have been determined for the binding of the antibodies to human transferrin in the presence and absence of iron. Small differences in reactivity were found. Five of the antibodies bind to the isolated amino-terminal half-molecule of human transferrin. Two of the antibodies appear to be to the C-terminal lobe since they bind to holo-transferrin but do not recognize the N-terminal half-molecule. Immunoblotting shows that six of the antibodies recognize both reduced and nonreduced transferrin. In addition, all of the antibodies bind with sufficiently high avidity to transferrin to make them useful as probes in studies in which binding of transferrin to the specific transferrin receptor is examined.

Animals↗