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S H Koenig

Publications and source records attributed to S H Koenig.

At least 91 records · Page 5Linked to original sources
PubMed1984

Prazosin as initial antihypertensive therapy: correlates of sympathetic function.

Abnormal sympathetic function has been proposed as a factor in the development of essential hypertension. If this is the case, prazosin hydrochloride, which works by a selective, peripheral, antisympathetic effect--postsynaptic alpha blockade--may have an advantage over other antihypertensive agents. In this study, blood pressure response and measures of sympathetic and baroreflex function were followed in 13 hypertensive patients. Prazosin alone significantly reduced standing and sitting diastolic blood pressures without affecting pulse rates, plasma catecholamines or baroreflex slopes in all patients. The addition of a thiazide diuretic in persons who did not achieve goal blood pressure on prazosin alone was generally successful in reducing blood pressure to desired levels, and increased both plasma renin activity and aldosterone concentrations. No significant relation was apparent between specific characteristics of sympathetic function and response to prazosin as initial therapy, although patients responding tended to have initially higher plasma norepinephrine concentrations.

Adult↗
PubMed1984

Determinants of proton relaxation rates in tissue.

It is well established that the longitudinal magnetic relaxation rate of solvent water protons, 1/T1, increases markedly in homogeneous protein solutions as the magnetic field is reduced well below the traditional NMR range. For a 5% solution of protein of 10(5) Da, for example, 1/T1 increases from about 50% above the pure water rate of 20 MHz to five times the water rate at 0.01 MHz. For tissue, including blood, the behavior is similar. Data for blood show that extracellular water has ready access to the hemoglobin inside red blood cells, which causes the enhanced relaxation. The extent to which cell water can sample the spatial structure of soft tissue, and how this structure influences relaxation rates, is as yet unknown. Nonetheless, the relaxation data for tissue can be accommodated within the conceptual framework developed earlier for analyzing homogeneous solutions of diamagnetic proteins. The variation of 1/T1 with field differs among tissues, and its magnitude at a given field can vary by more than a factor of three, far more than does the water content of the tissues. Solute complexes of paramagnetic ions with macromolecules, which increase the relaxation rates of solvent protons, can be introduced intravenously in tissue. They are known to accumulate in specific organs, and therefore have potential utility as contrast-enhancing agents in NMR imaging. Mn2+ and Gd3+, for example, produce characteristic dependences of 1/T1 on magnetic field that vary with the chemical state of the agent. The possibility exists, therefore, for monitoring the in vivo biochemistry of these agents.

Animals↗
PubMed1984

Relaxation of solvent protons by paramagnetic ions and its dependence on magnetic field and chemical environment: implications for NMR imaging.

Paramagnetic ions have been used to alter the magnetic relaxation rates 1/T1 and 1/T2 of solvent water protons since the first observations of a proton resonance signal almost four decades ago. The earliest theories of relaxation indicated that the influence of solute paramagnetic ions on relaxation rates of solvent protons should depend both on the chemical environment of the solute ions and on the magnetic field strength. Much knowledge, both experimental and theoretical, has since been amassed regarding relaxation effects in solutions of hydrated ions (aquoions) and of complexes of these ions with macromolecules, mainly proteins. The phenomena are well understood at this point, though the understanding is more retrospective than predictive for ion-protein complexes. Nonetheless, from what is now known about homogeneous solutions, and from current reports on the introduction of paramagnetic ions into tissue to alter contrast in NMR images by affecting relaxation rates, it is clear that the solution results are particularly germane, and transferable, to tissue investigations. The main features of relaxation of solvent protons in the presence of solute paramagnetic ions, as hydrated aquoions and complexed with protein, are presented here, with attention to those ions most relevant to in vivo studies, and with emphasis on the influence of the magnetic field and the chemical environment of these ions on solvent proton relaxation rates.

Cobalt↗
PubMed1984

Magnetic field dependence of solvent proton relaxation induced by Gd3+ and Mn2+ complexes.

The effect of solute paramagnetic ions on the longitudinal magnetic relaxation rate 1/T1 of solvent water protons depends on magnetic field strength and on the chemical environment of the ions. The variation of 1/T1 with field has been measured for solutions of Gd3+ and Mn2+ ions in three grossly different environments near physiological pH: the hydrated aquoion; chelated by EDTA (ethylenediaminetetraacetic acid) and DTPA (diethylenetriaminepentaacetic acid); and bound to the protein concanavalin A. It is demonstrated that over the field range at which NMR imaging is currently being done, the chemical environment can alter 1/T1 of solvent protons by more than an order of magnitude. The relevance of these results to the potential utility of these ions as agents for enhancement of contrast in NMR images is discussed.

Concanavalin A↗
PubMed1984

Ligand exchange at the active site of the carbonic anhydrases: a "ping-ping-pong" view.

We emphasize that the interconversion of CO2 and HCO-3 catalyzed by carbonic anhydrase must be regarded as two-substrate, two-product reactions, involving CO2, H2O and HCO-3, H+. Questions then arise regarding the interactions of substrates and products with enzyme during catalysis. Hydration of CO2 has been described by others as a pair of half reactions in which HCO-3 product is released from enzyme, followed by (usually buffer-catalyzed) release of H+. This "ping-pong" view is extended here to a "ping-ping-pong" sequence; based on analyses of specific models for the several intermediate complexes, we conclude that the second "ping" relates to the rate of ligand exchange of H2O from a pentacoordinate enzyme-H2O-HCO-3 complex, about which little is known at present and that is difficult to investigate. We discuss the types of experiments used to measure enzymatic activity and note that (1) they generally measure the rate of the combined "ping-ping" sequence rather than either independently, and (2) the pathway for exchange of substrate H2O is unrelated to the exchange of H2O measured by proton magnetic relaxation and by loss of isotopically labeled oxygen from the CO2-HCO-3 system. The "ping-ping-pong" view, while giving new insights and raising new questions, is consistent with OH- as the ligand of the high-pH form of isozymes I and II, as well as with the results of magnetic relaxation, isotope mixing and loss, 13C NMR linewidths of CO2 and HCO-3 at equilibrium, and stopped-flow kinetic measurements.

Animals↗
PubMed1983

Stoichiometry of manganese and calcium ion binding to concanavalin A.

Using measurements of solvent nuclear (proton) magnetic relaxation dispersion (NMRD), we have previously shown that concanavalin A (Con A) can exist in two conformational forms and that, in the absence of Ca2+, Mn2+ can bind to both the S1 and S2 sites of each monomer of Con A of at least one conformer [Brown, R.D., III, Brewer, C.F., & Koenig, S.H. (1977) Biochemistry 16, 3883-3896]. Recently other investigators have claimed that the stoichiometry of Mn2+ binding to Con A is only 1:1 for this conformational state, both in the absence and presence of saccharide; the same was claimed for Ca2+ under similar conditions. We now present titration and equilibrium dialysis experiments, both in the absence and presence of saccharide, using NMRD and atomic absorption spectroscopy, to investigate the stoichiometry of Mn2+ and Ca2+ binding to Con A. We have extended the NMRD method to include the determination of the total concentration of Mn2+ in samples of Con A. This, coupled with our previous use of NMRD to measure the concentration of free Mn2+ in protein solutions as well as the distribution of bound Mn2+ among different sites, allows us to measure the stoichiometry of binding with precision. We reconfirm that, at equilibrium in the presence of excess Mn2+, the binding stoichiometry of Mn2+ to Con A is 2:1, both in the absence and presence of saccharide. Addition of Ca2+ to a solution of Mn2+-Con A results in stoichiometric displacement of Mn2+ from the S2 site under the conditions investigated. Under nonequilibrium conditions, Mn2+ forms a metastable binary complex with the protein that persists for days at 5 degrees C. We also report, for the first time, values for all of the dissociation constants of binary and ternary complexes of Mn2+ with both conformations of Con A in solution. Atomic absorption measurements also indicate that Ca2+, in the absence of Mn2+, binds to both S1 and S2 sites in the absence and presence of saccharides.

Calcium↗
PubMed1983

Kinetics of conformational transitions of demetalized Concanavalin A.

Demetalized Concanavalin A exists in two conformational states, known as locked (PL) and unlocked (P) [Brown et al., Biochemistry 16, 3883 (1977)]. The equilibrium ratio [PL]/[P] is 0.14 +/- 0.01 at 25 degrees C, pH 6.4 [Brown et al., Biochemistry 21, 465 (1982)]. We now report values of the rate constants for the P in equilibrium; k1 = (33 +/- 4 h)-1 and k-1 = (4.6 +/- 0.6 h)-1 for the P leads to PL and PL leads to P transitions, respectively, at 25 degrees C, pH 6.4. The experiments utilize the fact that saccharide binds to PL [Koenig et al., Biochemistry 17, 4251 (1978)], producing a time-dependent increase in the total concentration of locked forms at equilibrium, and use a new technique for measuring this concentration.

Chemical Phenomena↗
PubMed1983

Water exchange at the active site of carbonic anhydrase. A synthesis of the OH- and H2O-models.

We have measured the paramagnetic contribution to the magnetic relaxation rate of solvent protons in highly purified, buffer- and salt-free solutions of Co(2+)-substituted human carbonic anhydrase B (HCAB), as a function of pH in the range 5.5-10 and as a function of magnetic field. We have also measured the optical absorption at 640 nm to characterize the enzyme. The relaxation rates vary with pH much as does the CO(2) hydration activity, increasing with increasing pH. We find that the relaxation rates at all intermediate values of pH can be described as linear combinations of the rates obtained at the extremes of pH used, indicating the existence of low- and high-pH forms of the enzyme with pH-dependent concentrations. The optical data can be similarly represented. The fraction of high-pH form present, determined from either the relaxation or optical data, has a pK(a) of approximately 7.6 when approximated by a single ionization. The data are very similar to that for HCAB in the presence of buffer, in contrast to the bovine enzyme for which the pK(a) is affected substantially by the presence of sulfate. Previous analysis of the high relaxation rates at high pH indicated rapid exchange of Co(2+)-liganded protons, possible only if these exchanging protons were conveyed by water molecules. On the other hand, the present demonstration of the existence of two forms of HCAB in highly purified solutions, coupled with other data, argues strongly for ionization of a water molecule ligand of the metal ion at the active site, with OH(-) as the solvent-donated ligand at high pH. We propose a mechanism of ligand exchange at high pH that reconciles these ostensibly conflicting requirements by invoking a pentacoordinate intermediate having both OH(-) and H(2)O as ligands. Proton exchange can be rapid between these ligands because charge transfer without net ionization can occur, so that the leaving water can carry away the initial OH(-). The low-pH form is a thermal mixture of tetra- and pentacoordinate species, the latter having low relaxation rates by analogy with inhibitor derivatives of the enzyme and model systems. The proposed associative ligand-exchange mechanism reconciles the distinctions between the OH- and H(2)O-models of carbonic anhydrase by merging them, providing the first model is consistent with the observed pH dependence of hydration activity, optical absorption, and solvent magnetic relaxation.

Binding Sites↗
PubMed1983

Clonidine in patients with diabetes and mild hypertension.

Clonidine has been reported to adversely affect glucose tolerance in experimental animals and normal man. We assessed its short- and long-term effects in 10 patients with both mild hypertension and diabetes mellitus. Patients were studied before and 10 wk after treatment with 0.1 mg clonidine twice daily, which induced reductions in blood pressure (from 148 +/- 5/93 +/- 2 mm Hg sitting, to 125 +/- 4/80 +/- 2) and control of hypertension in all patients. Clonidine increased the glycemic response to intravenous glucose (incremental glucose AUC from 161 +/- 13 to 184 +/- 14) but did not significantly change long-term diabetic control as assessed by weekly fasting serum glucose, glycosylated hemoglobin, and 24-hr urinary glucose excretions before and after treatment. We conclude that low-dose clonidine controlled blood pressure and impaired the response to an acute glucose challenge in midly hypertensive, type II diabetic patients but did not adversely affect diabetic control over 10 wk.

Adult↗
PubMed1983

Metal ion binding and conformational transitions in concanavalin A: a structure-function study.

The affinity of the lectin Concanavalin A (Con A) for saccharides, and its requirement for metal ions such as Mn2+ and Ca2+, have been known for about 50 years. However the relationship between metal ion binding and the saccharide binding activity of Con A has only recently been examined in detail. Brown et al. (Biochemistry 16, 3883 (1977)) showed that Con A exists as a mixture of two conformational states: a "locked" form and an "unlocked" form. The unlocked form of the protein weakly binds metal ions and saccharide, and is the predominate conformation of demetallized Con A (apo-Con A) at equilibrium. The locked form binds two metal ions per monomer with the resulting complex(es) possessing full saccharide binding activity. Brown and coworkers measured the kinetics of the transition of the unlocked form to the fully metallized locked conformation containing Mn2+ and Ca2+. They also demonstrated that Mn2+ alone could form a locked ternary complex with Con A, and that rapid removal of the ions resulted in a metastable form of apo-Con A in the locked conformation which slowly (hours at 25 degrees C) reverted back to (predominantly) the unlocked conformation. The ability to form either conformation in the absence or presence of metal ions has thus allowed us to explore the relationship between metal ion binding and conformational transitions in Con A as determinants of the saccharide binding activity of the lectin. Based on the kinetics of the transition of unlocked apo-Con A to fully metallized locked Con A, and X-ray crystallographic data, it appears that the transition between the two conformations of Con A involves a cis-trans isomerization of an Ala-Asp peptide bond in the backbone of the protein, near one of the two metal ion binding sites. The relatively large activation energy for the transition (approximately 22 kcal M-1) results in relatively slow interconversions between the conformations (from minutes to days), whereas the equilibria with metal ions and saccharide are rapid. Thus, many metastable complexes can be formed and a variety of transition pathways between the two conformations studied. We have identified and characterized binary, ternary, and quaternary complexes of both conformations of Con A containing Mn2+ and saccharide, and have determined both metal ion and saccharide dissociation constants for all of them, as well as equilibrium and kinetic values for the conformational transitions between them.(ABSTRACT TRUNCATED AT 400 WORDS)

Carbohydrates↗
PubMed1982

Conformational equilibrium of demetalized concanavalin A.

Concanavalin A (Con A) is known to exist in two conformations [Brown, R. D., III, Brewer, C. F., & Koenig, S. H. (1977) Biochemistry 16, 3883-3896] that differ in their metal ion and saccharide binding properties. The conformation that binds metal ions tightly, and which is associated with saccharide binding, has been designated as "locked" and that which binds metal ions only weakly as "unlocked". In the presence of excess metal ions, such as Mn2+ and Ca2+, essentially 100% of the protein is in the locked conformation. The scheme proposed to explain these effects [Koenig, S. H., Brewer, C. F., & Brown, R. D., III (1978) Biochemistry 17, 4251-4260] predicts an equilibrium between these conformations for the apoprotein. By monitoring the solvent proton relaxation dispersion as equimolar concentrations of Mn2+ and Ca2+ are titrated, at 5 degrees C, into an apo-Con A solution that had been equilibrated at 25 degrees C, we find that 12.5% of the apoprotein is in the locked conformation, corresponding to an energy separation of 1.2 kcal mol-1. We also show that these conformations can be separated by column chromatography at 5 degrees C and that the 100% unlocked form prepared in this way returns to the expected equilibrium mixture when kept at 25 degrees C.

Apoproteins↗
PubMed1982

Nuclear magnetic relaxation dispersion in monoclinic lysozyme crystals.

Nuclear magnetic relaxation measurements are reported as a function of field strength corresponding to the frequency range from 0.01 to 20 MHz for water protons in monoclinic lysozyme crystals at 278 and 298 K. Though the instrumentation used selects only a portion of the total magnetization to sample, the data clearly indicate a field dependence of the relaxation rate that signals the presence of slow motions characterized by time constants in the range of tenths of microseconds and slower. The data support, but do not uniquely prove, the hypothesis that this time scale is that appropriate to the isotropic averaging of locally anisotropic water molecule motion at the protein surface.

Animals↗
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

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↗
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↗