Biomedical subjects

C Ho

Publications and source records attributed to C Ho.

At least 217 records · Page 12Linked to original sources

PubMed1986

Molecular basis for the anti-sickling activity of aromatic amino acids and related compounds: a proton nuclear magnetic resonance investigation.

High-resolution proton nuclear magnetic resonance spectroscopy and relaxation techniques have been used to investigate the interactions of sickle cell hemoglobin (Hb S) and human normal adult hemoglobin (Hb A) with p-bromobenzyl alcohol, L-phenylalanine, L-tryptophan, and L-valine. With the exception of valine, all these compounds inhibit the polymerization of deoxy-Hb S [Noguchi, C. T., & Schechter, A. N. (1978) Biochemistry 17, 5455)). Using transferred nuclear Overhauser effects among the proton resonances of the compound of interest and the corresponding longitudinal relaxation rates (T1(-1], we have shown that the binding of each of the compounds investigated to deoxy-Hb S is comparable to that to deoxy-Hb A. Intermolecular transferred nuclear Overhauser effects have been observed between proton resonances of the anti-sickling compounds and specific protons situated in the heme pockets of Hb. On the basis of these results, we suggest that one binding site, common to all compounds with anti-sickling activity, is at or near the heme pockets in the alpha and beta chains of both deoxy-HB S and deoxy-Hb A. The proton T1(-1) values of the histidyl residues situated over the surface of the hemoglobin molecule indicate that a second binding site is located at or near the beta 6 position, containing the mutation in Hb S (beta 6Glu----Val). The binding of the compounds investigated to the latter site induces conformational changes in the amino-terminal domains of the beta chains.(ABSTRACT TRUNCATED AT 250 WORDS)

Antisickling Agents↗

PubMed1985

19F NMR investigation of molecular motion and packing in sonicated phospholipid vesicles.

Dimyristoylphosphatidylcholine (DMPC) labeled with a C19F2 group in the 4-, 8-, or 12-position of the 2-acyl chain has been investigated in sonicated unilamellar vesicles (SUV) by fluorine-19 nuclear magnetic resonance (NMR) at 282.4 MHz from 26 to 42 degrees C. The 19F NMR spectra exhibit two overlapping resonances with different line widths. Spin-lattice relaxation time measurements have been performed in both the laboratory frame (T1) and the rotating frame (T1 rho) in order to investigate the packing and dynamics of phospholipids in lipid bilayers. Quantitative line-shape and relaxation analyses are possible by using the experimental chemical shift anisotropy (delta nu CSA) and the internuclear F-F vector order parameter (SFF) values obtained from the 19F powder spectra of multilamellar liposomes. The following conclusions can be made: The 19F chemical shift difference between the inside and outside leaflets of SUV can be used to monitor the lateral packing of the phospholipid in the two SUV monolayers. The hydrocarbon chains in the outer layer are found to be more tightly packed than those of the inner one, and the differences between them become smaller near the chain terminals. The effective correlation time [(1-4) x 10(-7) s] obtained from either the motional narrowing of the line widths or off-resonance T1 rho measurements is shorter than that estimated from the Stokes-Einstein diffusion model (10(-6) s), on the basis of a hydrodynamic radius of 110 A for SUV.(ABSTRACT TRUNCATED AT 250 WORDS)

Dimyristoylphosphatidylcholine↗

PubMed1985

Amino acid replacements that compensate for a large polypeptide deletion in an enzyme.

Deletion of more than 400 amino acids from the carboxyl terminus of an enzyme causes a severe reduction in catalytic activity. Selected point mutations within the residual protein partially reverse the effects of the missing segment. The selection can yield mutants with activities at least ten times as high as those of the starting polypeptides. One well-characterized mutation, a single amino acid replacement in the residual polypeptide, increases the catalytic activity of the polypeptide by a factor of 5. The results suggest substantial potential for design of protein elements to compensate for missing polypeptide sequences. They also may reflect that progenitors of large aminoacyl-tRNA (transfer RNA) synthetases--one of which was used in these studies--were themselves much smaller.

Alanine-tRNA Ligase↗

PubMed1985

Proton nuclear Overhauser effect investigation of the heme pockets in ligated hemoglobin: conformational differences between oxy and carbonmonoxy forms.

Proton nuclear Overhauser effect (NOE) measurements have been used extensively to investigate the detailed conformations of peptides, proteins, and nucleic acids in the solution state. However, much of the published work has dealth with molecules of molecular weight less than 15 000. It is generally thought that specific NOEs cannot be observed in larger molecules (due to spin diffusion), so that NOE is of little use in conformational studies of such systems. By use of truncated-driven NOE with an irradiation time of 100 ms, specific NOEs are observed in a protein of the size of human normal adult hemoglobin (Hb A, 65 000 daltons). This technique has permitted us to assign several proton proton resonances arising from heme groups and from amino acid residues situated in the vicinity of the ligand binding site (such as E7 histidine and E11 valine) of the alpha and beta chains of Hb A. In addition, two-dimensional 1H[1H] J-correlated spectroscopy (COSY) experiments as well as theoretical ring-current calculations have confirmed the spectral assignments obtained by the one-dimensional NOE experiments. These new results not only have permitted us to map the heme pockets and to investigate the conformational differences in the heme pockets between oxy and carbonmonoxy forms of Hb A but also have demonstrated that the technique of truncated-driven NOE can be used to investigate the detailed conformations of selected regions in larger macromolecules in a way heretofore thought not to be feasible.

Carboxyhemoglobin↗

PubMed1985

Overproduction and nucleotide sequence of the respiratory D-lactate dehydrogenase of Escherichia coli.

Recombinant DNA plasmids containing the gene for the membrane-bound D-lactate dehydrogenase (D-LDH) of Escherichia coli linked to the promoter PL from lambda were constructed. After induction, the levels of D-LDH were elevated 300-fold over that of the wild type and amounted to 35% of the total cellular protein. The nucleotide sequence of the D-LDH gene was determined and shown to agree with the amino acid composition and the amino-terminal sequence of the purified enzyme. Removal of the amino-terminal formyl-Met from D-LDH was not inhibited in cells which contained these high levels of D-LDH.

Amino Acid Sequence↗

PubMed1984

Fluorine-19 nuclear magnetic resonance investigation of fluorine-19-labeled phospholipids. 1. A multiple-pulse study.

A multiple-pulse nuclear magnetic resonance technique has been used to measure the order parameter, SFF, at 40 MHz for dimyristoylphosphatidylcholine labeled with a difluoromethylene group at the 4-, 8-, or 12-position of the sn-2-acyl chain dispersed in water in the liquid-crystalline phase. The Carr-Purcell-Meiboom-Gill multiple-pulse sequence can resolve the homonuclear dipolar coupling between the two fluorine nuclei, thus making a direct determination of the order parameter, SFF, for the F-F internuclear vector possible. Other interactions, such as the 19F chemical shift anisotropy, heteronuclear dipolar couplings, and field inhomogeneity, which normally obscure the dipolar splitting, are effectively canceled. The order parameters obtained in this work compare well with those obtained by 19F nuclear magnetic resonance line-shape analysis of the 19F-labeled phospholipids reported in the following paper [Dowd, S. R., Simplaceanu, V., & Ho, C. (1984) Biochemistry (following paper in this issue)] as well as comparable SCD order parameters, determined for the deuterium-carbon internuclear vector of deuterium-labeled phospholipids [Oldfield, E., Meadows, M., Rice, D., & Jacobs, R. (1978) Biochemistry 17, 2727-2740]. The present results clearly show the usefulness of using nuclear magnetic resonance spectroscopy to investigate lipid-lipid and protein-lipid interactions, especially for those systems containing a difluoromethylene group in the acyl chain of a phospholipid molecule.

Chemical Phenomena↗

PubMed1984

Fluorine-19 nuclear magnetic resonance investigation of fluorine-19-labeled phospholipids. 2. A line-shape analysis.

Fluorine-19 nuclear magnetic resonance spectra at 282.4 MHz of dimyristoylphosphatidylcholine specifically labeled with a difluoromethylene group at the 4-, 8-, or 12-position of the sn-2-acyl chain and dispersed in excess water show the characteristic powder-pattern line shapes associated with an anisotropic axially symmetric chemical shift tensor, altered by the presence of the homonuclear dipolar interaction of the fluorine nuclei and of heteronuclear dipolar interactions between fluorine and nearby protons. Values for the anisotropy of the fluorine-19 chemical shift and for the fluorine-fluorine internuclear vector order parameter, SFF, as a function of temperature have been determined for the phospholipid dispersions with and without cholesterol. An increased mobility is evidenced in both cases as the temperature is raised. For the phospholipid dispersions in water, the values of SFF parallel quite well the behavior of the carbon-deuterium internuclear vector order parameter, SCD, as determined by deuterium nuclear magnetic resonance spectroscopy for the same labeled position. The effect of adding cholesterol is seen as a restriction of the chain mobility and the eventual disappearance of the phase transition. These new experiments provide a value of 166 ppm for the anisotropy of the axially symmetric chemical shift tensor of a difluoromethylene group in a phospholipid acyl chain. They also demonstrate the feasibility as well as the advantages of using a difluoromethylene group as a probe for molecular motions in the phospholipid bilayers.

Chemical Phenomena↗

PubMed1984

Fluorine-19 nuclear magnetic resonance study of 5-fluorotryptophan-labeled histidine-binding protein J of Salmonella typhimurium.

Fluorine-19 nuclear magnetic resonance has been used to investigate the histidine-binding protein J from Salmonella typhimurium. The protein has been labeled with fluorine-19 by growing the bacterial cells of a tryptophan auxotroph in the presence of 5-fluorotryptophan. Incorporation of up to 70% was achieved. The binding of L-histidine to the 19F-labeled protein is not affected by the isotopic labeling. The protein contains one tryptophan residue, giving rise to a single 19F resonance. Upon binding L-histidine to 19F-labeled histidine-binding protein J, the observed 19F resonance is shifted downfield by about 0.6 parts per million, indicating a conformational change of the protein molecule and a more hydrophobic environment for the 19F nucleus. Additional fluorescence experiments confirm that the tryptophan residue is located inside the hydrophobic core of the protein. 19F spin-lattice relaxation times of the 19F-labeled protein as a function of temperature show no difference between the free protein and the protein-histidine complex. However, the linewidth for the free protein is much larger than that of the protein-substrate complex. This can be explained by slow fluctuations between different conformations of the free protein molecule having slightly different 19F chemical shifts. Both with and without the substrate, the tryptophan residue is immobile inside the protein molecule as shown by the total disappearance of the 19F signal upon broadband irradiation at the 1H frequency. Also, the 19F spin-lattice relaxation times indicate that the protein is a rather rigid structure, in which rapid motions of the tryptophan residue on the time scale of 10(-8) second are not prominent.

Carrier Proteins↗

PubMed1984

A high-resolution proton nuclear-magnetic-resonance investigation of carp hemoglobin. Conformational differences between carp and human normal adult hemoglobins in solution.

The high-resolution proton nuclear magnetic resonance spectra of carp hemoglobin have been compared to those of human normal adult hemoglobin. Carp deoxy and carbonmonoxy hemoglobins in the deoxy-type quaternary state exhibit two downfield exchangeable proton resonances as compared to four seen in human normal adult deoxyhemoglobin. This suggests that two of the hydrogen bonds present in human normal adult deoxyhemoglobin are absent or occur in very different environments in carp hemoglobin. One of the exchangeable proton resonances of carp hemoglobin, while present in the deoxy-type quaternary state of the carbonmonoxy and deoxy derivatives, is absent in the oxy-type quaternary state of both, in agreement with the assignments of these quaternary structures by other methods. The ring-current-shifted proton resonances (sensitive tertiary structural markers) of carp carbonmonoxyhemoglobin are substantially different from those of human normal adult hemoglobin. The aromatic proton resonance region of carp hemoglobin has fewer resonances than that of human normal adult hemoglobin, consistent with its much reduced histidine content. The hyperfine-shifted proximal histidyl NH-exchangeable proton resonances of carp hemoglobin suggest that during the transition from the oxy to the deoxy quaternary structure, there is a greater alteration in the heme pocket of one type of subunits (presumably the beta chain) than that in the other subunit. The present results suggest that there are differences in both tertiary and quaternary structures between carp and human normal adult hemoglobins which could contribute to the great differences in the functional properties between these two proteins.

Adult↗

PubMed1984

Proton nuclear magnetic resonance investigation of cross-linked asymmetrically modified hemoglobins: influence of the salt bridges on tertiary and quaternary structures of hemoglobin.

Asymmetrically modified hemoglobins, [alpha(des-Arg)beta]A[alpha beta]cXL, [alpha(des-Arg-Tyr)beta]A[alpha beta]cXL, [alpha(des-Arg)beta(NES)]A[alpha beta]cXL, and [alpha(des-Arg)beta]A[alpha beta(NES)]cXL, have been prepared from chemically modified human normal adult hemoglobin (Hb A) and mutant hemoglobin C (beta 6Glu----Lys), where the subscript A or C denotes that the alpha beta dimer is from either Hb A or Hb C, respectively, and XL symbolizes a cross-linked hemoglobin prepared by reaction with a bifunctional cross-linking reagent, bis(3,5-dibromosalicyl) fumarate. It has been shown by X-ray crystallography that this bifunctional reagent cross-links the epsilon-amino group of the lysyl residue at position 82 of the two beta chains [Walder, J. A., Walder, R. Y., & Arnone, A. (1980) J. Mol. Biol. 141, 195]. Proton nuclear magnetic resonance spectra of these asymmetrically modified hemoglobins together with their parent hemoglobins, des-Arg(alpha 141) Hb A, des-Arg(alpha 141)-Tyr(alpha 140) Hb A, NES-Hb A and NES-des-Arg(alpha 141) Hb A, have been obtained over the spectral region 5-10 ppm downfield from H2O for the exchangeable proton resonances and 50-80 ppm downfield from H2O for the hyperfine-shifted proximal histidyl N delta H exchangeable proton resonances. The experimental results indicate that the effects on the hyperfine-shifted proximal histidyl N delta H exchangeable proton resonances at pH 6.0 of removing Arg(alpha 141) or Arg(alpha 141)-Tyr(alpha 140) from one of the two alpha subunits are limited to within the alpha subunit from which the carboxyl-terminal amino acids are specifically removed.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspirin↗

PubMed1984

A proton nuclear magnetic resonance investigation of human hemoglobin A2. Implications on the intermolecular contacts in sickle hemoglobin fibers and on the Bohr effect of human normal adult hemoglobin.

High-resolution proton nuclear magnetic resonance spectroscopy at 300 and 600 MHz has been used to investigate the conformation of a minor hemoglobin component of human blood, hemoglobin A2 (alpha 2 delta 2), in solution. We have found that (i) the replacement of the beta chains by the delta chains in hemoglobin A2 conserves the alpha 1 delta 2 interface but slightly perturbs the alpha 1 delta 1 interface, and (ii) one surface histidine residue in the deoxy form and one in the carbonmonoxy form of hemoglobin A2 have local conformations and/or electrostatic environments which are different from the corresponding ones in human normal adult hemoglobin. By comparing the proton nuclear magnetic resonance titration of individual histidine residues in hemoglobin A2 and in human normal adult hemoglobin, we can conclude that in human normal adult hemoglobin, both beta 116 and beta 117 histidine residues are titratable in both the deoxy and the carbonmonoxy forms. Thus, these two histidine residues can contribute to the Bohr effect of human normal adult hemoglobin. The present nuclear magnetic resonance data on hemoglobin A2 and those previously obtained in our laboratory on sickle hemoglobin suggest that the antisickling property of hemoglobin A2 does not originate from an alteration of the intermolecular contact site at the beta 6 position, but involves additional amino-acid residues which are different in the beta and delta chains. We have found that the replacement of the beta 116 and beta 117 histidine residues in the delta chains does not play a significant role in the antisickling effect of hemoglobin A2 and, thus, these amino-acid residues do not participate in the intermolecular interactions responsible for the polymerization of sickle hemoglobin.

Carboxyhemoglobin↗

PubMed1984

A high-resolution 1H-NMR investigation of the histidine-binding protein J of Salmonella typhimurium. Substrate-induced conformational changes.

High-resolution 1H-NMR spectroscopy at 600 MHz has been used to investigate the conformational transitions of the histidine-binding protein J of Salmonella typhimurium in solution as a function of pH and of L-histidine concentration. The dissociation constant for the binding of L-histidine to histidine-binding protein J increases from 6.0 X 10(-8) to 5.1 X 10(-7) M in going from pH 5.57 to 8.00. The conformation of this protein as observed by 1H-NMR also changes over this range of pH. However, when L-histidine is bound, the changes in conformation with pH are much smaller. Also, the pK for the single histidyl residue in histidine-binding protein J changes from 6.75 in the absence of L-histidine to 6.52 when L-histidine is bound. Earlier work in this laboratory resulted in the identification of several proton resonances believed to be at or near the L-histidine-binding site. Two of these resonances have been assigned to a tyrosine and the single histidyl residue in the histidine-binding protein J molecule.

Carrier Proteins↗

PubMed1983

Phosphorus-31 nuclear magnetic resonance investigation of membrane vesicles from Escherichia coli.

Phosphorus-31 nuclear magnetic resonance studies of isolated membrane vesicles prepared from Escherichia coli PSM116 as described by Hunt and Hong [Hunt, A. G., & Hong, J.-S. (1981) J. Biol. Chem. 256, 11988-11991; Hunt, A. G., & Hong, J.-S. (1983) Biochemistry 22, 844-850] are detailed here. This strain harbored a recombinant plasmid containing the phosphoglycerate transport system from Salmonella typhimurium (pJH7). Evidence indicating a surprising metabolic diversity, such as the presence of the enzymes enolase and phosphoglycerate mutase, is presented. The nature of the energization of these membrane vesicles for transport as described by Hugenholtz et al. [Hugenholtz, J., Hong, J.-S., & Kaback, H. R. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 3446-3449] is also discussed. Membrane vesicles prepared from the PSM116 strain do not form a transmembrane pH gradient when phosphoenolpyruvate is added. The present results show that phosphorus-31 nuclear magnetic resonance spectroscopy is an excellent tool to investigate the metabolism of membrane vesicles.

Cell Membrane↗

PubMed1983

Benzoquinolinediones: activity as insect teratogens.

Morphological abnormalities including extra compound eyes, extra heads, and distally duplicated legs were generated in cricket embryos by treating eggs with single doses of either benz[g]isoquinoline-5,10-dione or benzo[h]quinoline-5,6-dione. Slight structural modifications of the molecules resulted in a loss of teratogenic activity, although embryotoxicity occurred. These potent insect teratogens can be used for analysis of developmental events during embryogenesis.

Animals↗

PubMed1983

Proton nuclear magnetic resonance studies of isonitrile-heme protein complexes.

Steric interactions between bound ligand molecules and the valine E11 methyl groups of human hemoglobin and sperm whale myoglobin have been examined directly by high resolution NMR techniques. The methyl proton resonances of this amino acid are shifted markedly upfield and away from the bulk of the protein resonances by the shielding effect of circulating pi electrons in the porphyrin ring. We have monitored the valine resonance in the presence of CO and a series of isonitriles and found considerable shifts in its position, both between the various protein complexes and among the different liganded states. The ring current shifts of the gamma 1-methyl group of Val E11 in the CO forms of isolated alpha and beta chains and myoglobin are -2.70, -2.91, and -3.30 ppm, respectively. In all the proteins, these positions show little change in going from bound CO to bound methyl and ethyl isocyanide. In alpha subunits and myoglobin, n-propyl and n-butyl isocyanide binding produces marked decreases in the magnitude of these shifts, indicating that the valine residue has been forced away from the center of ring by the presence of these large ligand molecules. In the case of beta subunits, however, only tert-butyl isocyanide produces a marked decrease (from -2.91 to -1.99 ppm) in the ring current shift of the valine methyl protons. New peaks were observed in the isonitrile-protein spectra and identified as ligand proton resonances by comparing the spectra of normal and totally deuterated isonitrile complexes. The magnitudes of the ring current shifts for the terminal methyl protons of ethyl isocyanide suggest a linear geometry for the Fe equal to C equal to N - C bonds in beta chains and a bent geometry for alpha chains. The bent geometry in alpha subunits appears to be dictated by the position of the Val E11 methyl group which is located further up from the heme plane but closer to the heme center than the corresponding position of the beta subunit residue. The free energy changes for ethyl isocyanide binding to the two chains are nearly identical, suggesting that the linear and bent geometries are energetically equivalent. Myoglobin ethyl isocyanide complexes exhibit ligand ring current shifts intermediate to those observed for the hemoglobin subunits. Assignment of resonances and positions to the alkyl protons of the longer isonitriles is more difficult.

Animals↗

PubMed1983

Magnetic and spectral properties of carp carbonmonoxyhemoglobin. Competitive effects of chloride ions and inositol hexakisphosphate.

We have extended our studies on the magnetic properties of carp carbonmonoxyhemoglobin and the dependence of these properties upon solution variables. Using an improved version of the superconducting magnetometer, we have found that the magnetic susceptibility of carp carbonmonoxyhemoglobin is sensitive to both inositol hexakisphosphate and chloride ion. The dependence upon chloride ion concentration is complex. At relatively low concentrations this anion reverses the effect of inositol hexakisphosphate, restoring paramagnetism. At higher chloride concentrations the protein is converted to a roughly diamagnetic state in the absence of inositol hexakisphosphate. Along with these susceptibility studies, we have examined the effects of these anions on other properties of carp carbonmonoxyhemoglobin. The positions of the Soret bands of human and carp methemoglobin derivatives are correlated with spin state; changes in the magnetic susceptibility of carbonmonoxyhemoglobin are similarly associated with alterations in this spectral band. We have also examined the effects of these anions on the proton nuclear magnetic resonance spectrum of carp carbonmonoxyhemoglobin. Both chloride and inositol hexakisphosphate alter the position of the proton resonances in the ring-current-shifted region of the spectrum.

Animals↗

PubMed1983

A biochemical study of the reconstitution of D-lactate dehydrogenase-deficient membrane vesicles using fluorine-labeled components.

Fluorine-19 labeled compounds have been incorporated into lipids and proteins of Escherichia coli. 19F-Labeled membrane vesicles, prepared by growing a fatty acid auxotroph of a D-lactate dehydrogenase-deficient strain on 8,8-difluoromyristic acid, can be reconstituted for oxidase and transport activities by binding exogenous D-lactate dehydrogenase. 19F-Labeled D-lactate dehydrogenases prepared by addition of fluorotryptophans to a tryptophan-requiring strain are able to reconstitute D-lactate dehydrogenase-deficient membrane vesicles. Thus, lipid and protein can be labeled independently and used to investigate protein-lipid interactions in membranes.

Cell Membrane↗

PubMed1983

Preparation of oils for bacterial mutagenicity testing.

4 procedures used to prepare fossil-derived oils for bacterial mutagenicity testing have been examined. These are, (a) dewaxing by partitioning the oil between dimethyl sulfoxide (DMSO) and cyclohexane, (b) incorporating a surfactant to increase compatibility of the oil with the bioassay media, (c) directly slurrying the oil in DMSO, and (d) computing the mutagenicity of the oil by summing the contributions of individual chemical class fractions. DMSO slurries generally exhibit higher mutagenicities than computed by summing the contributions of chemical class fractions. Results of testing DMSO-slurries correlate (r = 0.87) well, however, with those obtained by summation. Mutagenicity results agree within a factor of two for the samples tested by 4 sample preparation procedures.

Animals↗