Biomedical subjects

J M Brewer

Publications and source records attributed to J M Brewer.

At least 55 records · Page 3Linked to original sources

PubMed1993

Fluoride inhibition of yeast enolase: crystal structure of the enolase-Mg(2+)-F(-)-Pi complex at 2.6 A resolution.

Enolase in the presence of its physiological cofactor Mg2+ is inhibited by fluoride and phosphate ions in a strongly cooperative manner (Nowak, T, Maurer, P. Biochemistry 20:6901, 1981). The structure of the quaternary complex yeast enolase-Mg(2+)-F(-)-Pi has been determined by X-ray diffraction and refined to an R = 16.9% for those data with F/sigma (F) > or = 3 to 2.6 A resolution with a good geometry of the model. The movable loops of Pro-35-Ala-45, Val-153-Phe-169, and Asp-255-Asn-266 are in the closed conformation found previously in the precatalytic substrate-enzyme complex. Calculations of molecular electrostatic potential show that this conformation stabilizes binding of negatively charged ligands at the Mg2+ ion more strongly than the open conformation observed in the native enolase. This closed conformation is complementary to the transition state, which also has a negatively charged ion, hydroxide, at Mg2+. The synergism of inhibition by F- and Pi most probably is due to the requirement of Pi for the closed conformation. It is possible that other Mg(2+)-dependent enzymes that have OH- ions bound to the metal ion in the transition state also will be inhibited by fluoride ions.

Fluorides↗

PubMed1993

Preparation and characterization of the E168Q site-directed mutant of yeast enolase 1.

Yeast has two enolase isozymes (called 1 and 2), either of which suffices for growth. We cloned DNA encoding the enolase 1 protein coding and promoter regions flanked by BamHI termini using the PCR. The DNA, which contained no nucleotide base changes altering the protein sequence, was cloned into the multicopy shuttle vector pRS314 and transformed into a yeast strain with a deletion in its enolase 1 gene. The resulting plasmid-containing strain makes enolase 1 in quantities which depend on cell growth. A "charge shuttle" mechanism of action of enolase based on X-ray crystallographic evidence (Lebioda and Stec, Biochemistry 30:2817, 1991) involves Glu-168 accepting a proton from a water molecule that in turn accepts a proton from carbon-2 of the substrate. We prepared the E168Q mutant of enolase 1 by oligonucleotide-directed site-directed mutagenesis. Its identity was confirmed by N-terminal sequence analysis, HPLC on Superose 12, SDS-gel electrophoresis, and the sequence of the mutated DNA protein-coding region. The E168Q mutant has approximately 0.01% of the activity of native enolase. It binds substrate/product, AEP (3-aminoenolpyruvate-2-phosphate, the 3-amino analogue of the product phosphoenolpyruvate) and TSP (D-tartronate semialdehyde-2-phosphate, the aldehyde analogue of the substrate 2-phosphoglycerate), the latter two at least with affinities similar to those of the native enzyme. The E168Q enolase also produces absorbance changes in the analogues. The reaction with AEP is consistent with the "charge shuttle" mechanism; the reaction with TSP, which presumably requires proton removal from carbon-2, is complex but shows a very slow phase consistent with expectations.

Base Sequence↗

PubMed1992

Characterization of the interaction of yeast enolase with polynucleotides.

Yeast enolase is inhibited under certain conditions by DNA. The enzyme binds to single-stranded DNA-cellulose. Inhibition was used for routine characterization of the interaction. The presence of the substrate 2-phospho-D-glycerate reduces inhibition and binding. Both yeast enolase isozymes behave similarly. Impure yeast enolase was purified by adsorption onto a single-stranded DNA-cellulose column followed by elution with substrate. Interaction with RNA, double-stranded DNA, or degraded DNA results in less inhibition, suggesting that yeast enolase preferentially binds single-stranded DNA. However, yeast enolase is not a DNA-unwinding protein. The enzyme is inhibited by the short synthetic oligodeoxynucleotides G6, G8 and G10 but not T8 or T6, suggesting some base specificity in the interaction. The interaction is stronger at more acid pH values, with an apparent pK of 5.6. The interaction is prevented by 0.3 M KCl, suggesting that electrostatic factors are important. Histidine or lysine reverse the inhibition at lower concentrations, while phosphate is still more effective. Binding of single-stranded DNA to enolase reduces the reaction of protein histidyl residues with diethylpyrocarbonate. The inhibition of yeast enolase by single-stranded DNA is not total, and suggests the active site is not directly involved in the interaction. Binding of substrate may induce a conformational change in the enzyme that interferes with DNA binding and vice versa.

Animals↗

PubMed1992

The adjuvant activity of non-ionic surfactant vesicles (niosomes) on the BALB/c humoral response to bovine serum albumin.

The ability of non-ionic surfactant vesicles (NISV) to enhance antibody production against bovine serum albumin (BSA) was compared with Freund's complete adjuvant (FCA), in the BALB/c mouse. Two subcutaneous inoculations with NISV entrapped BSA induced antibody levels comparable to, and persisting as long as those produced by FCA by either the subcutaneous or intraperitoneal route of inoculation. Intraperitoneal inoculation of NISV did not generate as strong an antibody response. The adjuvant activity of NISV was wholly dependent on the BSA being entrapped within preformed vesicles; mixing free BSA with vesicles was not effective. Analysis of the anti-BSA IgG subclasses induced by NISV and FCA showed that NISV were generally better stimulators of IgG2a than was FCA, but poorer stimulators of IgG1. From this, we deduce that NISV are potentially better stimulators of the Th1 lymphocyte subset than is FCA and by inference, potent stimulators of cellular immunity. We believe that NISV may offer many advantages over other adjuvants in terms of immunological selectivity, low toxicity and stability.

Adjuvants, Immunologic↗

PubMed1991

Comparison of the amino acid sequences of tissue-specific parvalbumins from chicken muscle and thymus and possible evolutionary significance.

Chicken leg muscle parvalbumin was digested with cyanogen bromide or trypsin or trypsin after citraconylation. Peptides isolated by reverse phase HPLC at pH 7.0 were subjected to acid hydrolysis and amino acid analysis and, in some cases, sequencing. The chicken muscle parvalbumin amino acid sequence has ca. 80% sequence identity with alpha-type parvalbumins from mammalian (rabbit, human and rat) muscle. By contrast, the chicken thymus parvalbumin ("avian thymic hormone") sequence is very similar to reptile (turtle, salamander and frog) muscle beta-type parvalbumins. We hypothesize that the evolutionary appearance of the warm-blooded reptiles was accompanied by recruitment of the beta parvalbumin isozyme for promotion of lymphocyte maturation.

Amino Acid Sequence↗

PubMed1991

Subunit structure of deglycosylated human and swine trachea and Cowper's gland mucin glycoproteins.

The oligosaccharide chains in human and swine trachea and Cowper's gland mucin glycoproteins were completely removed in order to examine the subunit structure and properties of the polypeptide chains of these glycoproteins. The carbohydrate, which constitutes more than 70% of these glycoproteins, was removed by two treatments with trifluoromethanesulfonic acid for 3 h at 3 degrees and periodate oxidation by a modified Smith degradation. All of the sialic acid, fucose, galactose, N-acetylglucosamine and N-acetylgalactosamine present in these glycoproteins was removed by these procedures. The deglycosylated polypeptide chains were purified and characterized. The size of the monomeric forms of all three polypeptide chains were very similar. Data obtained by gel filtration, release of amino acids during hydrolysis with carboxypeptidase B and gel electrophoresis in the presence of 0.1% dodecyl sulfate showed that a major fraction from each of the three mucin glycoproteins had a molecular size of about 67 kDa. All of the deglycosylated chains had a tendency to aggregate. Digestion with carboxypeptidases showed that human and swine trachea mucin glycoproteins had identical carboxyl terminal sequences, -Val-Ala-Phe-Tyr-Leu-Lys-Arg-COOH. Cowper's gland mucin glycoprotein had a similar carboxyl terminal sequence, -Val-Ala-Tyr-Leu-Phe-Arg-Arg-COOH. The yield of amino acids after long periods of hydrolysis with carboxypeptidases showed that at least 85% of the polypeptide chains in each of the deglycosylated preparations have these sequences. These results suggested that the polypeptide chains in these deglycosylated mucin glycoprotein preparations were relatively homogeneous. The deglycosylated polypeptide chains as well as the intact mucin glycoproteins had blocked amino terminii. The purified polypeptide chains were digested with trypsin-TCPK, and S. aureus V8 protease and the resulting peptides were isolated by gel electrophoresis in the presence of 0.1% dodecyl sulfate and by HPLC. Two partial amino acid sequences from swine trachea mucin glycoprotein, two partial sequences from human trachea mucin glycoprotein and three partial sequences from Cowper's gland mucin glycoprotein were determined. The partial amino acid sequences of the peptides isolated from swine trachea mucin glycoprotein showed more than 70% sequence homology to a repeating sequence present in porcine submaxillary mucin glycoprotein. Five to eight immunoprecipitable bands with sizes ranging from about 40 kDa to 46 kDa were seen when the polypeptide chains were digested with S. aureus V8 protease. All of the bands had blocked amino terminii and differed by a constant molecular weight of about 1.5 kDa.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1991

Inhibition of enolase: the crystal structures of enolase-Ca2(+)- 2-phosphoglycerate and enolase-Zn2(+)-phosphoglycolate complexes at 2.2-A resolution.

Enolase is a metalloenzyme which catalyzes the elimination of H2O from 2-phosphoglyceric acid (PGA) to form phosphoenolpyruvate (PEP). Mg2+ and Zn2+ are cofactors which strongly bind and activate the enzyme. Ca2+ also binds strongly but does not produce activity. Phosphoglycolate (PG) is a competitive inhibitor of enolase. The structures of two inhibitory ternary complexes: yeast enolase-Ca2(+)-PGA and yeast enolase-Zn2(+)-PG, were determined by X-ray diffraction to 2.2-A resolution and were refined by crystallographic least-squares to R = 14.8% and 15.7%, respectively, with good geometries of the models. These structures are compared with the structure of the precatalytic ternary complex enolase-Mg2(+)-PGA/PEP (Lebioda & Stec, 1991). In the complex enolase-Ca2(+)-PGA, the PGA molecule coordinates to the Ca2+ ion with the hydroxyl group, as in the precatalytic complex. The conformation of the PGA molecule is however different. In the active complex, the organic part of the PGA molecule is planar, similar to the product. In the inhibitory complex, the carboxylic group is in an orthonormal conformation. In the inhibitory complex enolase-Zn2(+)-PG, the PG molecule coordinates with the carboxylic group in a monodentate mode. In both inhibitory complexes, the conformational changes in flexible loops, which were observed in the precatalytic complex, do not take place. The lack of catalytic metal ion binding suggests that these conformational changes are necessary for the formation of the catalytic metal ion binding site.

Amino Acid Sequence↗

PubMed1991

Immunological characterization of deglycosylated human and swine trachea and Cowper's gland mucin glycoproteins.

Antibodies were raised in rabbits against purified swine and human trachea and Cowper's gland mucin glycoproteins and their deglycosylated polypeptide chains. Three monospecific antibody fractions that recognize the carbohydrate, the deglycosylated or unglycosylated regions of the polypeptide chains in these glycoproteins, were isolated by immunoaffinity chromatography. The human and swine trachea mucin glycoproteins showed extensive immunological homology in both their carbohydrate and polypeptide chains. The carbohydrate chains and unglycosylated region of the polypeptide chain in Cowper's gland mucin glycoprotein showed little or no cross-reaction with comparable regions in respiratory mucin glycoproteins. However, the polypeptide chains in the deglycosylated regions of all three mucin glycoproteins showed extensive homology. Five bands with molecular masses ranging from 40 to 46 kDa that differed by a constant molecular mass of approximately 1.5 kDa were detected in hydrolysates of all of the polypeptide chains after treatment with S. aureus V8 protease. Monospecific antibodies to the deglycosylated region of these chains reacted with the peptides, whereas those directed against the unglycosylated region did not. The results suggest that these chains contain tandem repeating sequences of amino acids.

Amino Acids↗

PubMed1990

The amino acid sequence of avian thymic hormone, a parvalbumin.

The complete amino acid sequence of 'avian thymic hormone' (ATH), a protein from thymus tissue that appears to promote immune maturation in chicken bone marrow cells in culture, is presented. The sequence was obtained from sequences of ATH peptides isolated by HPLC after tryptic, chymotryptic, peptic or S aureus V8 protease digestions. The protein is a parvalbumin consisting of 108 residues with a blocked amino terminus, a single cysteine, tyrosine, proline and arginine and no histidine, methionine or tryptophan. This is the first amino acid sequence of a parvalbumin which is not derived from muscle tissue.

Amino Acid Sequence↗

PubMed1990

Ocular cicatricial pemphigoid.

A series of 17 patients with ocular cicatricial pemphigoid (OCP) is described retrospectively. The importance of early recognition, especially of disease involving the medial canthus and caruncular region, diagnosis by biopsy, and adequate immunosuppressive and surgical therapy, are emphasised. A detailed grading scheme has been developed and this has enabled the authors to determine the success or failure of the therapy during the active treatment period. A combined ophthalmological and immunological approach to treatment can result in a successful visual outcome of this potentially blinding disease.

Aged↗

PubMed1989

Avian thymic hormone (ATH) is a parvalbumin.

Amino acid sequence analysis of a protein from chicken thymus tissue which promotes immunological maturity in chicken bone marrow cells in culture has established sequences of a 45-residue fragment, a 24-residue fragment and a 9-residue and an 8-residue peptide. Independent comparison of the 45- and 24-residue fragments with known amino acid sequences by computer search has unequivocally identified avian thymic hormone as a parvalbumin. This is the first demonstration that a protein previously identified by a biological function is a parvalbumin.

Amino Acid Sequence↗

PubMed1989

The structure of yeast enolase at 2.25-A resolution. An 8-fold beta + alpha-barrel with a novel beta beta alpha alpha (beta alpha)6 topology.

The three-dimensional structure of yeast enolase has been determined by the multiple isomorphous replacement method followed by the solvent flattening technique. A polypeptide model, corresponding with the known amino acid sequence, has been fitted to the electron density map. Crystallographic restrained least-squares refinement of the model without solvent gave R = 20.0% for 6-2.25-A resolution with good geometry. A model with 182 water molecules and 1 sulfate which is still being refined has presently R = 17.0%. The molecule is a dimer with subunits related by 2-fold crystallographic symmetry. The subunit has dimensions 60 X 55 X 45 A and is built from two domains. The smaller N-terminal domain has an alpha + beta structure based on a three-stranded antiparallel meander and four helices. The main domain is an 8-fold beta + alpha-barrel. The enolase barrel is, however, different from the triose phosphate isomerase barrel; its topology is beta beta alpha alpha (beta alpha)6 rather than (beta alpha)8 as found in triose phosphate isomerase. The inner beta-barrel is not entirely parallel, the second strand is antiparallel to the other strands, and the direction of the first helix is also reversed with respect to the other helices. This supports the hypothesis that some enzymes evolved independently producing the stable structure of beta alpha barrels with either enolase or triose phosphate isomerase topology. The active site of enolase is located at the carboxylic end of the barrel. A fragment of the N-terminal domain and two long loops protruding from the barrel domain form a wide crevice leading to the active site region. Asp246, Glu295, and Asp320 are the ligands of the conformational cation. Other residues in the active site region are Glu168, Asp321, Lys345, and Lys396.

Binding Sites↗

PubMed1987

Investigation of conformational changes in yeast enolase using dynamic fluorescence and steady-state quenching measurements.

Conformational changes in yeast enolase were investigated using steady state quenching and dynamic (fluorescence decay and fluorescence anisotropy decay) measurements. The tryptophan fluorescence rotational correlation time increases from 24 to 38 ns on subunit association. The acrylamide quenching constant decreases two-fold when the subunits associate. The conformational metal ion effect suggests a more compact molecule. Under conditions of catalysis, the correlation time decreases 25%, though the sedimentation constant does not change (Holleman, 1973). The enzyme may undergo a hinge-bending motion during catalysis.

Catalysis↗

PubMed1987

Fluorescence lifetime and spectral study of the acid expansion of bovine serum albumin.

The fluorescence lifetimes of the tryptophan residues of bovine serum albumin were measured in the native and acid-expanded conformation. A three-exponential process is required to fit the fluorescence decay data. The results are interpreted empirically in terms of two emitting species. The emission at longer wavelength (360 nm) has slower rates of decay than that at shorter wavelength (325 nm). For both emitting species the average lifetime decreases when the N-F transition occurs and shortens further when the protein expands. Rotational correlation times, derived from the decay of the fluorescence anisotropy of the tryptophan residues, suggest that longer emission wavelengths are associated with somewhat shorter correlation times. There is no certain indication of any independent motion of the tryptophans in any conformation, although some very fast process, perhaps Raman scattering, appears to occur. On acid expansion the long correlation times decrease to around 10 ns in the fully expanded form. Static quenching experiments using I- or acrylamide suggest a greater average exposure of the tryptophans when the protein is most greatly expanded. This is despite the fact that the fluorescence emission maximum shifts to shorter wavelength under these conditions. Also, there is no difference in accessibility to quenching between the longer and shorter wavelength emissions.

Fluorescence Polarization↗

PubMed1986

Arginyl residues and thermal stability in proteins.

Guanidination and amidination of bovine serum albumin, yeast enolase and yeast alcohol dehydrogenase were accompanied by increases in thermal stability at lower extents of modification. Decreases in thermal stability result from greater modification. These results support suggestions that surface guanidino groups (arginyl groups) are an important factor in thermal stability of proteins.

Alcohol Dehydrogenase↗

PubMed1986

Yeast enolase carboxyl modification using Woodward's reagent K.

Yeast enolase is inactivated by Woodward's reagent K. Substantial protection is afforded by binding of 1 mol of "conformational" metal ion/subunit. Inactivation is correlated with modification of 13 carboxyl groups/subunit in the absence of conformational metal ion and 17 in its presence. Ten tryptic peptides labeled by Woodward's reagent K can be isolated, mostly from the C-terminal half of the protein. The changes in reactivity of these peptides produced by conformational metal ion suggest direct coordination to Glu-181 together with a contraction of the protein.

Amino Acids↗

PubMed1985

A spectrophotometric method for quantitation of carboxyl group modification of proteins using Woodward's Reagent K.

Reaction of proteins with Woodward's Reagent K in 0.05 ionic strength Tris-HCl, pH 7.8, followed by removal of excess reagent by chromatography on Sephadex G-25 in the same buffer, results in covalently attached chromophores with an absorption maximum at 340 nm and an extinction coefficient of 7000 M-1 cm-1. This absorbance can be used to quantitate the reaction of Woodward's Reagent K with carboxyl groups in proteins, provided sulfhydryl groups do not react. The chromophore also enables specific detection and identification of carboxyl-modified peptides upon separation by chromatography or electrophoresis.

Indicators and Reagents↗

PubMed1985

Cadmium(II)-113 NMR studies of the mechanism of metal ion activation of yeast enolase.

Yeast enolase binds one mole of 113Cd2+ per subunit at a site that consists of all oxyligands in a distorted octahedral environment. This "conformational" metal ion's environment undergoes further distortion on addition of substrate/product or analogs. At pH's below the optimum value the shifted resonance tends to break up into several, suggesting the existence of several slowly exchanging intermediate forms. At acid pH's, on addition of one additional mole/subunit of 113Cd2+, which greatly increases catalysis, "conformational" resonance(s) further broadens, suggesting that the second, "catalytic" metal ion increases the rates of interconversion between "conformational" species. At more alkaline pH's, near the optimum pH, the "conformational" peak is sharpened, which suggests that very fast interconversion is occurring. The position of the "catalytic" metal ion resonance also suggests all oxyligands in a distorted octahedral geometry. The "catalytic" resonance is often broadened to the point where it cannot be seen, suggesting rapid changes in its geometry due to interconversion of substrate and product.

Binding Sites↗