Biomedical subjects

G L Mendz

Publications and source records attributed to G L Mendz.

At least 55 records · Page 3Linked to original sources

PubMed1991

Physicochemical characterization of dodecylphosphocholine/palmitoyllysophosphatidic acid/myelin basic protein complexes.

The stoichiometry of dodecylphosphocholine/palmitoyllysophosphatidic acid/myelin basic protein complexes and the location of the protein in the micelles have been investigated by electron paramagnetic resonance, ultracentrifugation, small-angle X-ray scattering, 31P, 13C, and 1H nuclear magnetic resonance spectroscopy, and electron microscopy. Ultracentrifugation measurements indicated that well-defined complexes are formed by association of one protein molecule with approximately 133 detergent molecules. The spin-labels 5-, 12-, and 16-doxylstearate have been incorporated into detergent/protein aggregates. Electron paramagnetic resonance spectral parameters and 13C and 1H nuclear magnetic resonance relaxation times showed that the addition of myelin basic protein does not affect the environment and location of the labels or the organization of the micelles. Previous results suggesting that the protein lies primarily near the surface of the micelles have been confirmed by comparing 13C spectra of the detergents with and without protein with spectra of detergent/protein aggregates containing the spin labels. Electron micrographs of the complexes taken by using the freeze-fracture technique revealed the presence of particles with an estimated radius about three times the radius of the micelles measured by small-angle X-ray scattering. The structural integrity of the complexes appears to be based on intramolecular protein interactions as well as protein-detergent interactions.

Animals↗

PubMed1991

Stimulation of mutases and isomerases by vanadium.

The effects of vanadate and vanadate complexes on the rates of exchange of phosphoryl groups in the reactions catalyzed by the enzymes phosphoglucomutase and the coupled system formed by phosphoglycerate mutase and enolase, and the effects of vanadyl complexes on the interconversion of aldehyde and keto groups catalyzed by the enzymes phosphomannose isomerase, phosphoribose isomerase, and phosphoglucose isomerase, were measured using one-dimensional 31P nuclear magnetic resonance spectroscopy. Chemical exchange was investigated by observing the transfer of magnetization achieved by selective irradiation of resonances using the DANTE pulse sequence. The presence of vanadium stimulated the catalytic activity of the enzymes in vitro, with the exception of enolase whose activity was not affected. Addition of vanadate also increased the rate constants of the interconversion of glucose 6-phosphate and fructose 6-phosphate in hemolysates. 51V nuclear magnetic resonance spectroscopy and electron paramagnetic resonance spectroscopy were employed to investigate the interactions between ammonium vanadate and sugar phosphates and the formation of vanadium--sugar phosphate complexes that may be involved in the stimulation of the catalytic activity of the isomerases.

Animals↗

PubMed1990

Interactions of myelin basic protein with mixed dodecylphosphocholine/palmitoyllysophosphatidic acid micelles.

The interactions of myelin basic protein and peptides derived from it with detergent micelles of lysophosphatidylglycerol, lysophosphatidylserine, palmitoyllysophosphatidic acid, and sodium lauryl sulfate, and with mixed micelles of the neutral detergent dodecylphosphocholine and the negatively charged detergent palmitoyllysophosphatidic acid, were investigated by 1H NMR spectroscopy and circular dichroic spectropolarimetry. The results with single detergents suggested that there are discrete interaction sites in the protein molecule for neutral and anionic detergent micelles and that at least some of these sites are different for each type of detergent. The data on the binding of the protein and peptides to mixed detergent micelles suggested that intramolecular interactions in the intact protein and in one of the longer peptides limited the formation of helices and also that a balance between hydrophobic and ionic forces is achieved in the interactions of the peptides with the detergents. At high detergent/protein molar ratios, hydrophobic interactions appeared to be favored.

Animals↗

PubMed1990

Stimulation of human erythrocyte 2,3-bisphosphoglycerate phosphatase by vanadate.

The rates of vanadate-stimulated hydrolysis of 2,3-bisphosphoglycerate in metabolically competent erythrocytes and in hemolysates were determined from data on time courses up to 35 min employing 31P nuclear magnetic resonance spectroscopy. The enhanced rate of hydrolysis of the bisphosphate was attributed principally to the activation of the phosphatase activity of 2,3-bisphosphoglycerate synthase both in cell suspensions and in hemolysates. Information on the concentrations of vanadate and vanadyl present in the preparations was obtained employing 51V nuclear magnetic resonance spectroscopy and electron paramagnetic resonance spectroscopy. Redox reactions involving vanadium ions appeared to be important in establishing the final equilibrium concentrations of the oxy- and oxo-ions (vanadate and vanadyl, respectively), but the data suggested that the activation of the enzyme resulted from direct action of the vanadium ions on the enzyme and not as a consequence of the alteration in the equilibrium of intracellular oxidants and reductants.

Biological Transport↗

PubMed1989

Magnetic potential and field gradients of a model cell.

The magnetic potential within and outside a nucleated cell placed in a uniform magnetic field is described for a model consisting of two concentric diamagnetic spheres. The analytical description of the magnetic potential in and around a system consisting of a finite number of concentric diamagnetic spheres in a uniform magnetic field was derived. The solution was employed to calculate the field gradients outside and inside a model chicken red blood cell. The form and magnitude of the gradients provide a theoretical basis on which to discuss experimental results relating to the attenuation of signals obtained using proton nuclear magnetic resonance spectroscopy with chicken erythrocyte suspensions. The form of the magnetic field, field difference and field gradients in the external medium of a cell suspension was simulated for a distribution of spheres in a uniform magnetic field, such as might occur in an idealised dilute cell suspension.

Animals↗

PubMed1989

Characterization of leukocyte enzymes involved in the release of amino acids in incubated blood cell lysates.

The neutral leukocyte proteases involved in the release of free amino acids in incubation mixtures of blood cell lysates and of human serum albumin with leukocyte lysates were characterized by several techniques including 1H nuclear magnetic resonance spectroscopy, electrophoresis, high performance liquid chromatography, gel filtration, amino acid analysis, and NH2-terminal analysis. The data suggested that the enzymes which contributed significantly to the extensive protein hydrolysis observed were two endopeptidases and three exopeptidases. Identification and analysis of the products obtained in incubation mixtures of human serum albumin with elastase and leucine aminopeptidase were compared with the products obtained in incubation mixtures of the protein with leukocyte lysates.

Amino Acids↗

PubMed1989

Identification of methyl resonances in the 1H NMR spectrum of incubated blood cell lysates.

The origin of low frequency methyl resonances which appear in the spin-echo 1H nuclear magnetic resonance spectra of incubated blood cell lysates was investigated by several techniques including 1H and 13C nuclear magnetic resonance spectroscopy, electrophoresis, high performance liquid chromatography, gel filtration, and amino acid analysis. These resonances were identified as arising from methyl moieties of leucine and valine. Other peaks which also appeared in the spectra of incubated blood cell lysates were assigned to methyl groups of alanine and threonine. The free amino acids are products of neutral proteases located on the leukocyte membrane or able to act on the extracellular medium. Since more than one enzyme appears to be implicated, it is possible that both membrane and granule proteases take part in the hydrolysis. Comparison of rates of product formation in white cell lysates incubated with human serum albumin, and with red cell lysate, suggests that erythrocyte peptidases also contribute to proteolysis in the latter case.

Amino Acids↗

PubMed1988

Conformation of a heptadecapeptide comprising the segment encephalitogenic in rhesus monkey.

The 17-residue peptide FKLGGRDSRSGSPMARR derived from myelin basic protein, containing an epitope encephalitogenic in rhesus monkey, has been studied in aqueous solution by high-resolution one- and two-dimensional carbon and proton nuclear magnetic resonance spectroscopy. The resonances of the spectra from both nuclei were assigned with the aid of two-dimensional correlated spectroscopy, pH and solvent titrations, and one-dimensional spin-decoupling techniques and by comparison of the spectra of the heptadecapeptide with those of a phosphorylated form of the peptide, the pentadecapeptide FKLGGRDSRSGSPMA, and the nonapeptide FKLGGRDSR. Amide proton temperature coefficients, coupling constants, 13C- spin-lattice relaxation times, and nuclear Overhauser effect data suggest the existence of three structured regions comprising residues 3-6, 7-12, and 12-14 in the solution conformations of the encephalitogenic heptadecapeptide.

Amino Acid Sequence↗

PubMed1988

Characterization of dodecylphosphocholine/myelin basic protein complexes.

The stoichiometry of myelin basic protein (MBP)/dodecylphosphocholine (DPC) complexes and the location of protein segments in the micelle have been investigated by electron paramagnetic resonance (EPR), ultracentrifugation, photon correlation light scattering, 31P, 13C, and 1H nuclear magnetic resonance (NMR), and electron microscopy. Ultracentrifugation measurements indicate that MBP forms stoichiometrically well-defined complexes consisting of 1 protein molecule and approximately 140 detergent molecules. The spin-labels 5-, 12-, and 16-doxylstearate have been incorporated into DPC/MBP aggregates. EPR spectral parameters and 13C and 1H NMR relaxation times indicate that the addition of MBP does not affect the environment and location of the labels or the organization of the micelles except for a slight increase in size. Previous results indicating that the protein lies primarily near the surface of the micelle have been confirmed by comparing 13C NMR spectra of the detergent with and without protein with spectra of protein/detergent aggregates containing spin-labels. Electron micrographs of the complexes taken by using the freeze-fracture technique confirm the estimated size obtained by light-scattering measurements. Overall, these results indicate that mixtures of MBP and DPC can form highly porous particles with well-defined protein and lipid stoichiometry. The structural integrity of these particles appears to be based on protein-lipid interactions. In addition, electron micrographs of aqueous DPC/MBP suspensions show the formation of a small amount of material consisting of large arrays of detergent micelles, suggesting that MBP is capable of inducing large changes in the overall organization of the detergent.

Animals↗

PubMed1988

31P magnetization transfer in the phosphoglyceromutase-enolase coupled enzyme system.

The rate of exchange of phosphoryl groups between 2-phosphoglycerate, 3-phosphoglycerate and phosphoenolpyruvate by the coupled phosphoglyceromutase-enolase enzyme system using one- and two-dimensional 31P NMR spectroscopy was measured. Magnetization exchange in one-dimensional experiments was achieved by saturation transfer with selective irradiation at both one and two sites in this three-site exchange system using the DANTE pulse sequence. The two-dimensional magnetization exchange experiment avoids the need to selectively saturate at one or more frequencies which may be difficult in complex exchange systems. Analysis of the two-dimensional exchange experiment by the back transformation method yielded exchange rate constants in good agreement with the saturation transfer method.

Animals↗

PubMed1988

Interactions of Na+ with haemoglobin-organic phosphate complexes.

Longitudinal and transverse relaxation times were measured in aqueous solutions containing haemoglobin and 2,3-bisphosphoglycerate and in dilute lysates of human erythrocytes. Analysis of the data in terms of calculated excess relaxation rates shows that Na+ interacts with the protein-organic phosphate complex. The comparable magnitude of the effect in the model system and in dilute lysate suggests that intracellular Na+ binds to the haemoglobin-bisphosphate complex. Results obtained with adenosine triphosphate and D-glucose indicate that there is also interaction between Na+ and haemoglobin complexes of these molecules.

2,3-Diphosphoglycerate↗

PubMed1986

Determination of erythrocyte pyrimidine 5'-nucleotidase activity by 31P nuclear magnetic resonance: comparison of normal controls and multiple sclerosis patients.

A technique to assay erythrocyte pyrimidine 5'-nucleotidase activity in situ using 31P nuclear magnetic resonance spectroscopy is presented. The assay is chemically specific, simple and applicable to untreated lysates. A comparison of enzyme levels in normal controls and in multiple sclerosis patients employing the assay yielded no significant differences between both groups. Difficulties encountered in the quantitative analysis of the assay using 1H-NMR spectroscopy are briefly discussed.

5'-Nucleotidase↗

PubMed1986

NMR studies of myelin basic protein. XIII. Assignment of histidine residues in rabbit, bovine and porcine proteins.

Myelin basic protein from three species (rabbit, cow and pig) and peptides from enzymatic digests or cleavage of the proteins have been examined in aqueous solutions by proton nuclear magnetic resonance (NMR) at 400 MHz. The epsilon 1-CH and delta 2-CH resonances of all the histidine residues in the three proteins have been assigned and the pK values have been measured. The heterogeneity of chemical shifts among these resonances can be variously ascribed to persistent localized secondary structures and to effects arising from charged side-chains, particularly those of aspartic acid residues, and from side-chains of aromatic moieties.

Animals↗

PubMed1985

Conformation of two antigenic regions in myelin basic protein.

Four different regions of myelin basic protein from various species have been reported to be the antigenic sites (epitopes) for seven monoclonal antibodies evoked in rats or mice by guinea pig or monkey basic protein. The structures of the epitopes located in the amino-terminal region and in the eight-residue sequence including S-133, were examined by proton n. m. r at 400 MHz in aqueous solutions of peptides obtained by enzymatic cleavage of the rabbit protein. The data suggest conformational similarities between the two regions.

Amino Acid Sequence↗

PubMed1985

N.m.r. studies of myelin basic protein. Conformation of a peptide that is an antigenic determinant for B-cell reactivity.

The peptide Gly-Arg-Ala-Ser-Asp-Tyr-Lys-Ser, derived from myelin basic protein (MBP), is part of an epitope to monoclonal antibodies to human MBP. Its conformation has been studied in aqueous solution by high-resolution one- and two-dimensional 1H and 13C n.m.r. Two-dimensional correlated spectroscopy, pH titrations and one-dimensional spin-decoupling techniques were employed to assign the spectra observed from both nuclei. Amide proton temperature coefficients, coupling constants, 13C spin-lattice relaxation times and nuclear-Overhauser-effect data provide evidence that the solution conformations of the octapeptide include a type-II beta-turn with a hydrogen bond between the CO group of Arg2 and the NH group of Asp5. The results are discussed in view of a possible conformation of the antibody receptor site.

Antibodies, Monoclonal↗

PubMed1985

Proton-n.m.r. study of interaction of myelin basic protein with a monoclonal antibody.

Proton n.m.r. at 400 MHz has been applied to study the interactions of bovine or porcine myelin basic protein (b- or p-MBP) with a monoclonal antibody to human (h-) MBP. The antibody, an IgG immunoglobulin that contains a sequential epitopic region, cross-reacts with b-MBP but not with p-MBP, the presumed epitope being identical in h- and b-MBP. N.m.r. spectra were recorded from the Fab fragment of the antibody and for mixtures of Fab and MBP at various molar ratios. The n.m.r. spectrum of MBP in the mixture consists mostly of well resolved peaks against a broad background due to the Fab. With b-MBP, but not p-MBP, specific interactions are observed at the residue tyrosine-135, which is part of the epitopic sequence. Other interactions occur between the Fab and both b- and p-MBP at residues distant from the epitopic region. Standard radioassay techniques were employed to calculate the binding constants of both basic proteins with the immunoglobulin. The binding constant, Kb, for IgG to column-immobilized b-MBP at 298K is (0.95 +/- 0.07) X 10(7) dm3/mol. The value of Kb decreases with the ionic strength of the medium, suggesting a coulombic interaction between antigen and antibody. N.m.r. spectra were also measured for mixtures of the Fab fragment and peptides containing the epitopic site, with results in agreement with those for the whole protein.

Antibodies, Monoclonal↗

PubMed1984

Interaction of myelin basic protein with micelles of dodecylphosphocholine.

Interactions of myelin basic protein (MBP) and peptides derived from it with micelles of dodecylphosphocholine (DPC) and perdeuterated DPC have been studied by proton nuclear magnetic resonance (NMR) at 400 MHz and by circular dichroism (CD). When MBP binds to DPC micelles, it acquires about 18% alpha-helicity. The CD spectra of various peptides derived by cleavage of MBP indicate that a major alpha-helical region occurs in residues 85-99 just before the sequence of three prolyl residues 100-102. From line broadenings by fatty acid spin-labels in the micelles and from changes in chemical shifts, the NMR data identify specific residues in MBP that participate in lipid binding. One such sequence is an alpha-helical region from residues 85 to 95, and others occur around methionine-21 and between residues 117 and 135. The different effects of C5, C12, and C16 spin-labels suggest that some segments of the protein may penetrate beyond the dipolar interfacial region of the micelles into the hydrophobic interior, but no part of the protein is protected by the micelles against rapid exchange of its amide groups with the aqueous environment. Even at a lipid to protein molar ratio of 200/1, most NMR resonances from side chains of amino acid residues are not appreciably broadened, suggesting that much of the polypeptide remains highly mobile.

Animals↗

PubMed1983

NMR studies of myelin basic protein. IX. Complete assignments of the tyrosine residues by proton NMR of proteins from six species.

All the proton resonances from the tyrosine residues are assigned in 400 MHz NMR spectra in aqueous solution of myelin basic proteins from human, cow, pig, rabbit, rat (small protein) and chicken. Assignments are based on species comparisons, spectra of enzymatic cleavage products of the basic protein, pH titrations, broadening effects of Gd(III), and nuclear Overhauser effects. The mobile extended polypeptide chain structure of the protein facilitates the detection of interactions between nearest neighbors. Evidence is found for reverse turns in the structure in regions of encephalitogenic determinants.

Animals↗