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Biomedical subjects

R D Brown

Publications and source records attributed to R D Brown.

At least 361 records · Page 20Linked to original sources
PubMed1981

Metal ion substitution at the catalytic site of horse-liver alcohol dehydrogenase: results from solvent magnetic relaxation studies. 2. Binding of manganese(II) and competition with zinc(II) and cadmium(II) ions.

The interaction of Mn2+ aquo ions with native horse-liver alcohol dehydrogenase demetalized specifically at the catalytic sites has been investigated by studying the magnetic field dependence and time dependence of the magnetic spin-lattice relaxation rate of solvent water protons. We find no detectable binding of Mn2+ ions to the catalytic sites in times on the order of hours; however, we do find that these ions bind to the enzyme at two previously unreported types of sites: one, characterized by a low dissociation constant (0.01 mM at pH 7.7, 5 degrees C), low relaxivity, and a stoichiometry of one per two catalytic sites, and a second, with a high dissociation constant (1.5 mM at pH 7.7, 5 degrees C) and high relaxivity. The stoichiometry of the second type of site could not be determined because of the relatively weak bindng of Mn2+ ions to these sites. Both Zn2+ and Cd2+ ions bind to the newly found tight-binding sites, displacing Mn2+ ions and thereby altering the relaxation rates of solvent protons. By monitoring the return to equilibrium of these altered rates, we find that Zn2+ ions enter the catalytic sites from the new tight-binding sites with an on-rate of approximately 0.1 M-1 s-1. It is not clear whether binding to these new sites is an obligatory intermediate for reintroduction of Zn2+ ions into the catalytic sites, but a small excess of Zn2+ ions beyond one per monomer causes the protein to precipitate. Cd2+ ions, by contrast, enter the catalytic sites at least 1 order of magnitude more rapidly than do Zn2+ ions, a rate too rapid to observe by our techniques. However, once the catalytic sites are filled, Cd2+ ions displace Mn2+ ions at the new sites as do Zn2+ ions.

Alcohol Dehydrogenase↗
PubMed1981

Identification and properties of terminal deoxynucleotidyltransferase from Xenopus embryos prior to the development of the thymus.

The appearance of terminal deoxynucleotidyltransferase (TdT), a marker for early lymphoid cells, has been measured during the development of the South African clawed frog, Xenopus laevis. The enzyme can first be detected in 2-day-old embryos, at approximately the same time as the first detectable embryonic immunoglobulin and well before the appearance of the thymus. Xenopus TdT has been partially purified and some of its properties have been investigated. The partially purified enzyme is 68,000 daltons in size, as measured by gel filtration, and cross-reacts immunologically with anti-calf thymus TdT.

Animals↗
PubMed1981

Ototoxic drugs and noise.

Drugs that produce tinnitus can be subdivided into those which produce temporary or permanent hearing loss and those which apparently do not cause any hearing loss. The tinnitus occurring with drugs of the first group is probably secondary to the hearing loss. However, most of the drugs that produce tinnitus without an accompanying hearing loss probably do so because of their effect on biogenic amines in the central nervous system and/or as an extension of their proconvulsant side-effects. A pre-existing cochlear impairment is the underlying factor in most patients who experience tinnitus. Not only can ototoxic drugs or high levels of noise produce cochlear impairment but the interaction of the two can place humans in more jeopardy than when exposed to either agent alone. Chloramphenicol has little ototoxic potential when administered systemically in humans. However, our studies show that when chloramphenicol is combined with noise exposure in rats, considerably more cochlear damage results than from the noise alone (chloramphenicol alone does no produce any cochlear damage). We are presently conducting more detailed studies of this ototoxic interaction to determine whether it occurs with other antibiotics (such as erythromycin) which are also commonly considered to have minimal ototoxicity.

Acoustic Stimulation↗
PubMed1981

Exchange of labeled nuclei in the CO2--HCO3--solvent system catalyzed by carbonic anhydrase.

Silverman et al. (1979. J. Am. Chem. Soc. 101:6734-6740) have reported measurements of the loss of 18O to solvent from the isotopically labeled CO2--HCO3-system and of the mixing of 18O and 13C labels within the system, as catalyzed by human carbonic anhydrase C in the pH range 6-8. This work is an extension of earlier work (Silverman and Tu. 1976. J. Am. Chem. Soc. 98:978-984) on the very similar bovine enzyme. The more recent work is analyzed by its authors in terms of the "hydroxide" model for the apparent pH-dependence of enzymatic activity, a model in which the pH-dependence is associated with the presumed ionization of an H2O ligand of the active-site metal ion to OH-. From a comparison of their data with a solution of the coupled differential equations that describe the kinetics of isotope exchange in terms of the model, Silverman et al. derived a pH-dependent rate of exchange for the water molecule which is formed at the active site of the enzyme during dehydration. By contrast, using the same data and a model in which active enzyme has a water molecule on the metal ion at the active site, and similar differential equations, we derive a value for the rate of exchange of water that is pH-independent. This model has the attraction that it explains the magnetic relaxation rate of solvent water protons in the Co2+-substituted enzyme, whereas the hydroxide mechanism cannot explain these data without the introduction of unfounded ad hoc assumptions; further, the presence of an OH- ligand of the metal has never been demonstrated. We also include an analysis of analogous data for the bovine enzyme. One result of our analysis is that the pKa for activity of the enzyme samples used is near 6.0, implying that the bulk of the data were taken when the enzyme was essentially all active. It is straightforward to account for the pH-dependence of the data near and below the pKa by using an empirically-derived value for the pKa. However, we have recently developed a model for the low pH (inactive) enzyme that has been successful in interpreting a wide range of data, and we show that this new view can explain the few points at low pH quite adequately. Additionally, we consider the recent kinetic results for the human C enzyme, obtained at chemical equilibrium by studies of the linewidths of nuclear magnetic resonances of 13C in labeled substrate (Simonsson et al. 1979. Eur. J. Biochem. 93:409-417) and show that these experiments and those of Silverman et al. are all consistent with kinetic data from nonequilibrium stopped-flow experiments, viewed in terms of our model, in the limit of low substrate concentration. Results at higher concentrations indicate that the Michaelis constants and equilibrium constants differ somewhat.

Animals↗
PubMed1981

Interactions of solvent with the heme region of methemoglobin and fluoro-methemoglobin.

It is now more than 20 years since Davidson and collaborators (1957, Biochim. Biophys, Acta. 26:370-373; J. Mol. Biol. 1:190-191) applied the theoretical ideas of Bloembergen et al. (1948. Phys. Rev. 73:679-712) on outer sphere magnetic relaxation of solvent protons to studies of solutions of methemoglobin. From then on, there has been debate regarding the relative contributions to paramagnetic solvent proton relaxation by inner sphere (ligand-exchange) effects and by outer sphere (diffusional) effects in methemoglobin solutions. Gupta and Mildvan (1975. J. Biol. Chem 250:146-253) extended the early measurements, attributed the relatively small paramagnetic effects to exchange with solvent of the water ligand of the heme-Fe3+ ion, and interpreted their data to indicate cooperativity and an alkaline Bohr effect in the presence of inositol hexaphosphate. They neglected the earlier discussions entirely, and made no reference to outer sphere effects. We have measured the relaxation rate of solvent protons as a function of magnetic field for solutions of methemoglobin, under a variety of conditions of pH and temperature, and have given careful consideration to the relatively large diamagnetic corrections that are necessary by making analogous measurements on oxyhemoglobin, carbonmonoxyhemoglobin, and cyano- and azide-methemoglobin. (The latter two, because of their short electronic relaxation times, behave as though diamagnetic). We show that the paramagnetic contribution to solvent relaxation can be dominated by outer sphere effects, a result implying that many conclusions, including those of Gupta and Mildvan, require reexamination. Finally, we present data for fluoro-methemoglobin, which relaxes solvent protons an order of magnitude better than does methemoglobin. Here one has a startling breakdown of the dogma that has been the basis for interpreting many ligand-replacement studies; in contrast to the prevailing view that replacement of a water ligand of a protein-bound paramagnetic ion by another ligand should decrease relaxation rates, replacement of H2O by F- increases the relaxation rate drastically. The data can all be reconciled, however, with what is anticipated from knowledge of ligand interactions in the heme region.

Electron Spin Resonance Spectroscopy↗
PubMed1981

Construction of recombinant plasmids containing Xenopus immunoglobulin heavy chain DNA sequences.

A recombinant cDNA plasmid containing Xenopus immunoglobulin heavy chain sequence has been constructed from Xenopus spleen poly(A)-containing RNA. The plasmid was identified by colony hybridization and a hybridization-translation assay and its identity was confirmed by DNA sequence analysis. The portion of the heavy chain sequence contained in the plasmid is 35% homologous to mammalian mu and gamma sequences. The mRNA corresponding to this plasmid is 2.5 kilobases, in close agreement with the size of mouse mu mRNA. RNA sequences complementary to the cloned sequence appear in embryos about 24 hr after fertilization, which corresponds to 24 hr before the first detectable immunoglobulin.

Animals↗
PubMed1981

Molecular cloning of genes for cellobiose utilization and their expression in Escherichia coli.

The genes for cellobiose utilization in Escherichia adecarboxylata were cloned by using recombinant deoxyribonucleic acid techniques and transferred to Escherichia coli. Preliminary analysis of the beta-glucosidase activity expressed in these host cells indicated that the enzyme is membrane bound and required magnesium ions, phosphate ions, and heat-stable, non-dialyzable factors from the bacterial cytoplasm.

Cell Membrane↗
PubMed1981

The plasma aldosterone response to angiotensin II infusion in aldosterone-producing adenoma and idiopathic hyperaldosteronism.

To determine if the adrenal sensitivity to angiotensin II in patients with an aldosterone-producing adenoma differs from that in patients with idiopathic hyperaldosteronism, we infused graded doses of angiotensin II into 17 patients with primary aldosteronism and measured their plasma aldosterone concentrations after each dose. At a rate of 0.5 ng angiotensin II/kg.min, the mean increase in the plasma aldosterone concentration in the 8 patients from whom an aldosterone-producing adenoma was subsequently removed was 4 +/ 2.4 ng/dl (mean +/- SE), which was significantly less (P less than 0.01) than the mean increase (23 +/- 4.8 ng/dl) in the 9 patients with idiopathic hyperaldosteronism. The threshold dose of angiotensin II in the patients with aldosterone-producing adenoma was 1.0 +/- 0.24 ng/kg.min, significantly greater (P less than 0.05) than the threshold dose (0.3 +/- 0.07 ng/kg.min) in the patients with idiopathic hyperaldosteronism. We conclude that the sensitivity of aldosterone-producing adenomas to angiotensin II is significantly less than that of the hypersecreting adrenal tissue in patients with idiopathic hyperaldosteronism. This difference in adrenal sensitivity might in part explain the difference in the response of plasma aldosterone concentrations to upright posture in these two subsets of aldosteronism with low renin activity.

Adenoma↗
PubMed1981

Effect of metoclopramide on the secretion and metabolism of aldosterone in man.

We performed this study to determine whether metoclopramide increases the concentration of plasma aldosterone in normal man by increasing the secretion rate of aldosterone or by decreasing aldosterone metabolic clearance. On the first day that metoclopramide was administered orally to seven normal subjects, the secretion rate of aldosterone increased significantly (P less than 0.05) from the rate during the preceding placebo period. By the fourth day of treatment, the secretion rate had returned to control values and remained there during an ensuing placebo period. The excretion rate of aldosterone followed a similar pattern. The increase in aldosterone secretion was accompanied by a transient but significant decrease in urinary sodium excretion. Metoclopramide administered iv had no effect on the metabolic clearance of aldosterone. Metoclopramide stimulated aldosterone-producing adenomas and nodular hyperplastic adrenal tissue resected from patients with primary aldosteronism to produce aldosterone in vitro. We conclude that metoclopramide increases the concentration of aldosterone in plasma by stimulating the secretion of aldosterone rather than by decreasing aldosterone metabolic clearance, and that metoclopramide probably stimulates aldosterone secretion by acting directly on adrenal tissue.

Adult↗
PubMed1981

Genetic and pathophysiologic studies of a new kindred with glucocorticoid-suppressible hyperaldosteronism manifest in three generations.

This report describes investigations in a new kindred with dexamethasone-suppressible hyperaldosteronism affecting three successive generations. The presumptive diagnosis was first made in a 7-yr-old boy and led to the identification of the disorder in his mother and grandmother. Several other members of the family were investigated. Genotyping and HLA typing were also performed. To further explore the nature of this unusual disorder, urine from the three patients documented to have the syndrome was assayed for an aldosterone-stimulating factor recently reported to be found in patients with idiopathic aldosteronism. None of these patients showed measurable activity of such a urinary factor. The identification of members in three generations strongly supports the heritable nature of the disorder and probable autosomal dominant type of transmission. The absence of urinary aldosterone-stimulating factor in these patients further supports the tenet that the disorder is pathogenetically distinct from idiopathic aldosteronism, since both disorders are usually associated with bilateral adrenal hyperplasia.

Adult↗
PubMed1980

Interaction of bovine carbonic anhydrase with (neutral) aniline, phenol, and methanol.

We have investigated the interaction of bovine carbonic anhydrase with neutral aniline, phenol, and methanol molecules. The measurements are of optical spectra and solvent water and methanol proton magnetic relaxation rates of solutions of Co2+-substituted enzyme. We recently proposed a model [Koenig, S. H., Brown, R. D., & Jacob, G. S. (1980) Proceedings of the Symposium on Biophysics and Physiology of Carbon Dioxide, Springer-Verlag, West Berlin and Heidelberg], based on the interaction of enzyme with monovalent anions, that accounts for the pH dependences observed for a wide variety of phenomena, including the apparent pKa for enzymatic activity. We now extend the model to include the observed effects of neutral molecules. Aniline and phenol, though isoelectronic, shift the observed pKa values in opposite directions, and both appear to bind at the aromatic binding site to which sulfonamide inhibitors and aromatic esters are known to bind. The resulting binary complexes behave as altered enzymes, with different values of the pKa for activity, but otherwise are similar to the native enzyme. In terms of our model, aniline and phenol alter the relative affinities of water and anions for the same coordination position of the metal ion at the active site. The effect is opposite in sign for the two nolecules becuase of the differing proton affinities of the NH2 and OH moieties of the phenol ring in each case. By extension, our results indicate that data from experiments using aromatic buffers such as imidazole and lutidine should be analyzed with some care; effects previously attributed to buffer molecules to the aromatic binding site in the active region of the enzyme. The interaction of methanol with carbonic anhydrase is quite different, and very weak. Methanol does displace water at the metal, but to first order there is little, if any, preferential binding of methanol compared to water. Observations by others that alcohols inhibit esterase activity with inhibition constants on the order of 1 M are not attributable to binding of alcohol to enzyme but rather, in our view, result from the increased solubility of aromatic ester substrates in the alcohol-modified solvent.

Aniline Compounds↗
PubMed1980

The identification of messenger RNA coding for immunoglobulin heavy and light chains of Xenopus laevis.

Total poly(A)-containing mRNA isolated from Xenopus spleens was translated in a rabbit reticulocyte lysate in vitro protein-synthesizing system. Approx. 1% of the radioactivity incorporated into the protein was precipitated by an antibody directed against adult Xenopus IgM. The immunoprecipitated proteins were characterized as IgM heavy and light chains by their molecular weight as determined by polyacrylamide-sodium dodecyl sulfate gel electrophoresis The sequence variability of the synthesized light c hain proteins was analyzed by isoelectric focusing and shown to be indistinguishable from authentic Xenopus immunoglobulin light chain proteins derived from IgM. The data presented here identify Xenopus spleen mRNA as a potential source of a natural immunoglobulin mRNA population with which the development of the immune system can be studied.

Amino Acid Sequence↗