Biomedical subjects
Publications and source records attributed to C Ho.
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The thermotropic behavior of three fluorinated phospholipids has been studied by means of high-sensitivity differential scanning calorimetry. The three lipids are 1,2-di(X,X-difluoromyristovyl)phosphatidylcholine with X = 4,8, or 12. The compound with X = 4 has a higher transition temperature than dimyristoylphosphatidylcholine whereas the other two have lower values. All three lipids have transition enthalpies approximately twice that of the unsubstituted lipid. It was found the substituted lipids form strikingly nonideal mixtures with the parent compound. In view of these observations it is evident that results obtained using these or related compounds as probes of bilayer or membrane behavior must be interpreted with due caution. Some of the properties exhibited by geminal fluorine atoms in fluorinated phospholipids appear to be similar to those of cis double bonds in lipids.
The structural changes associated with cooperative oxygenation of human adult hemoglobin as a function of oxygen saturation in aqueous media at neutral pH and at 25-27 degrees C have been investigated by high-resolution proton nuclear magnetic resonance spectroscopy at 250 and 360 MHz. By monitoring the intensities of two hyperfine shifted proton resonances (at about -12 and -18 ppm from H(2)O) and two exchangeable proton resonances (at about -6.4 and -9.4 ppm from H(2)O) as a function of oxygenation, the amount of oxygen bound to the alpha and beta chains of a hemoglobin molecule can be determined and the relationship between tertiary and quaternary structural changes under a given set of experimental conditions can be investigated. These results suggest that: (i) in the absence of organic phosphates, there is no preferential O(2) binding to the alpha or beta chains; (ii) in the presence of organic phosphates, the alpha hemes have a higher affinity for O(2) as compared to the beta hemes; (iii) the ligand-induced structural changes in the hemoglobin molecule are not concerted; and (iv) some cooperativity must be present within the deoxy quaternary state during the oxygenation process. The variations of the exchangeable proton resonances as a function of oxygenation strongly suggest that the breaking of one or more inter- or intrasubunit linkages of a ligated subunit can affect similar linkages in unligated subunits within a tetrameric hemoglobin molecule. Thus, the present results show that two-state allosteric models are not adequate to describe the cooperative oxygenation of hemoglobin. In addition, the present results provide direct correlation to the ligand-induced structural changes (such as in the heme pockets and subunit interfaces) observed to occur in the crystals of deoxy- and oxy-like hemoglobin molecules and in the solution state.
Intracellular pH and 2,3-diphosphoglycerate concentration in sickle cell amenia and normal human blood samples were measured by means of phosphorus-31 nuclear magnetic resonance spectroscopy. To monitor the concentrations of various internal phosphorylated metabolites of intact red blood cells, heparinized blood samples were used and were incubated at 37 degrees C with 5.6% C92, 25% O2, and 69.4% N2. The 31P chemical shifts of phosphorylated compounds, such as 2,3-diphosphoglycerate, adenosine 5'-triphosp-ate, and inorganic phosphate, depend on pH, and by using an appropriate calibration curve, the intracellular pH of intact erythrocytes can be obtained. The intracellular pH values in fresh sickl cell blood and normal blood were found to be 7.14 and 7.29, respectively. However, the whole-blood pH, as measured by a standard pH meter, was found to be 7.54 for both types of blood. The initial concentration of 2,3-diphosphoglycerate in sickle cell blood was about 30% higher, but it was depleted much faster during incubation than that in normal blood. The difference in intracellular pH between these two types of blood samples remained constant during incubation, even after depletion of 2,3-diphosphoglycerate. These results suggest that there are differences in intracellular environment between normal and sickle cell blood. Thus, 31P nuclear magnetic resonance spectroscopy provides a fast, direct, continuous, and noninvasive way to monitor the intracellular environment of intact erythrocytes.
The duplication of the genes coding for 5-S RNA has been followed during the S phase of HeLa cell cultures synchronized by mitotic detechment. Replication was analyzed by exposing the cultures to bromodeoxyuridine (BrdUrd) for 3 h at successive intervals and analyzing the DNA product in CsCl density gradients. DNA containing the 5-S genes was detected in the gradients by molecular hybridization using purified 125I-labeled 5 S RNA. In addition, as an internal marker of S phase chronology, late DNA replication was followed by examining the incorporation of [3H]thymidine into repetitive DNA (C0t less than 0.01 fraction). The results indicated that greater than 85% of the cells in the culture were synchronously dividing and that greater than 80% of the 5-S genes replicate in the first half of S phase, whereas the rapidly reassociating fraction of the DNA replicates in the second half of S phase.
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Using variable temperature techniques, the spin label spectral resolution of hemoglobin labeled at the beta93 cysteines with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodonacetamide has been greatly enhanced. The effects of different ligands, inositol hexaphosphate, pH and salt concentration upon spin labeled ferrous and ferric hemoglobin indicate that the beta chain tertiary structure exhibits considerable variability within the oxy and deoxy quaternary structures. From these studies ligand and spin state changes both appear to be of significance in producing structural changes; binding of inositol hexaphosphate then produces further structural changes secondary in amplitude.
Fluorinated fatty acids of the general formula CH3(CH2)13-mCF2(CH2)m-2COOH are informative spectroscopic probes of the gel to liquid-crystalline phase transitions in phospholipid dispersions and in biological membranes. We present theoretical considerations to suggest that the 19F nuclear magnetic resonance line shapes are very different for frozen and fluid lipid regions. Our studies confirm this expectation for mixed phospholipid multilamellar dispersions containing a trace of difluoromyristate. The method correctly measures the onset and completion temperatures of the transition in the well-studied dimyristoylphosphaditylcholine distearoylphosphatidylcholine system and also describes the motional behavior of the solid and fluid phases within the transition. Lipids extracted from Escherichia coli membranes show similar motional phenomena through the transition-temperature range according to 19F nuclear magnetic resonance studies of difluoromyristate biosynthetically incorporated into the K1060B5 strain, an unsaturated fatty acid auxotroph. Intact cells or membrane vesicles show substantially different behavior from extracted lipids, indicating that membrane proteins significantly perturb the phase transition. Evidence presented in this paper also shows that the 19F resonance from Escherichia coli phospholipids is sensitive to various intramembrane interactions. There is a general decrease in restriction of motion due to neutral lipids and an opposite effect due to the architecture of the native membrane. Neither effect is temperature sensitive. However, there are interactions in the intact membrane, affecting the 19F resonance, that are temperature dependent both due to the phase-transition process and due to processes occurring at high temperatures.
High-resolution proton nuclear magnetic resonance studies of hemoglobins Providence-Asn (beta82EF6 Lys replaced by Asn) and Providence-Asp (beta82EF6 Lys replaced by Asp) show that different amino acid substitutions at the same position in the hemoglobin molecule have different effects on the structure of the protein molecule. Hemoglobin Providence-Asp appears to be in a low-affinity tertiary structure in both the deoxy and carbonmonoxy forms. Deoxyhemoglobin Providence-Asn has its beta heme resonance shifted downfield slightly from its position in normal adult hemoglobin; however, the tertiary structures of the heme pocket of hemoglobins A and Providence-Asn are very similar when both proteins are in the carbonmonoxy form. These results are consistent with the oxygen equilibrium measurements of Bonaventura, J., et al. [(1976) J. Biol. Chem. 251, 7563] which show that both Hb Providence-Asn and Hb Providence-Asp have oxygen affinities lower than normal adult hemoglobin, with Hb Providence-Asp having the lowest. Our studies of the effects of sodium chloride on the hyperfine shifted proton resonances of deoxyhemoglobins A, Providence-Asn, and Providence-Asp indicate that the beta82EF6 lysine is probably one, but not the only binding site for chloride ions.
High-resolution proton nuclear magnetic resonance studies of deoxyhemoglobins Osler (beta145HC2 Tyr replaced by Asp) and McKees Rocks (beta 145HC2 Tyr replaced by term) indicate that these hemoglobins are predominately in the oxy quaternary structure in 0.1 M [bis(2-hydroxyethyl)imino]-tris(hydroxymethyl) methane buffer at pH 7. Upon the addition of inositol hexaphosphate, the proton nuclear magnetic resonance spectra of these hemoglobins become similar to those characteristic of a hemoglobin molecule in the deoxy quaternary structure. The exchangeable proton resonance which is found at -6.4 ppm from H2O in the spectrum of normal human adult deoxyhemoglobin is absent in the spectra of these two mutant hemoglobins. Consequently we believe the hydrogen bond between the hydroxyl group of tyrosine-beta145HC2 and the carboxyl oxygen of valine-beta98FG5 gives rise to this resonance. This assignment allows us to use the -6.4ppm resonance as an important tertiary structural probe in the investigation of the cooperative oxygenation of hemoglobin.
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Several fluorinated fatty acids of the general structure CH3(CH2)13--mCF2(CH2)m--2COOH are incorporated biosynthetically as unsaturated fatty acid analogues into the phospholipids of Escherichia coli. Under optimum conditions an unsaturated fatty acid autotroph, K1060B5, can be grown so that 50% of the total phospholipid fatty acids are 8,8-difluoromyristate. Conditions are found for which more than 20% of the fatty acids are fluorinated before a decrease in growth rate is observed. We have used 19F nuclear magnetic resonance to examine membranes isolated from E. coli grown under the latter conditions. A comparison is made with spectra of aqueous dispersions of extracted E. coli phospholipids and model multilayer phospholipid membranes. An explanation of the 19F resonance line shape in these membrane systems and the relationship to a molecular order parameter is given. It is apparent that 19F nuclear magnetic resonance is more sensitive to the degree of ordering or fluidity of phospholipids than spin labels or fluorescent probes. For instance, a dramatic effect of membrane protein on lipid fluidity can be seen. Finally, this method can be used to measure the proportion of frozen and fluid lipid in biological membranes at temperatures within the span of the gel-to-lipid phase transition.
We describe here the isolation, purification, and structural characterization of a lipid A precursor synthesized under nonpermissive conditions by a mutant of Salmonella typhimurium conditionally defective in the synthesis of the 3-deoxy-D-mannoctulosonate (2-keto-3-deoxyoctonate, KDO) region of the lipopolysaccharide. The precursor was isolated free from lipopolysaccharide, murein, and phospholipids by extraction of delipidated cells with 90% phenol/CHCL3/petroleum ether. The molecule was recovered from the phenol phase after precipitation of lipopolysaccharide with H2O and subsequently purified by DEAE-cellulose chromatography. Structural analyses showed that the lipid A precursor is a phosphorylated glucosamine disaccharide containing one ester and two amide-linked residues of beta-hydroxymyristate. In contrast to lipid A, the precursor disaccharide lacks ester-linked 12:0 and 14:0 fatty acids as well as KDO. The molecule contains 2 phosphate residues both of which were identified as phosphomonoesters by 31P NMR spectroscopy. One of the phosphomonoesters is located in position 1 of the reducing terminal glucosamine residue; the location of the other phosphomonoester was not determined. The structure of the precursor provides strong support for the conclusion that KDO incorporation occurs at an early stage in lipid A biosynthesis prior to the incorporation of ester-linked saturated fatty acids.
The histidine-binding protein J of Salmonella typhimurium binds L-histidine as a first step in the high-affinity active transport of this amino acid across the cytoplasmic membrane. High-resolution nuclear magnetic resonance spectroscopy has been used to monitor the conformation of histidine-binding protein J in the presence and absence of substrate. Evidence is presented to show that this binding protein undergoes a conformational change involving a substantial number of amino-acid residues (including tryptophans) in the presence of L-histidine and that this change is specific for L-histidine. In order to monitor the involvement of tryptophan residues in the substrate-induced conformational change, 5-fluorotryptophan has been incorporated biosynthetically into the histidine-binding protein J using a tryptophan autotroph of Salmonella typhimurium. There are no significant differences in the conformation and binding activity between the 5-fluorotryptophan-labeled and the normal histidine-binding protein J. Proton and fluorine-19 nuclear magnetic resonance studies of the 5-fluorotryptophan-labeled binding protein show that at least one (and possibly two) of the tryptophan residues undergo(es) a change toward a more hydrophobic environment in the presence of L-histidine. These observations are supported by fluorescence data and by differences in the reactivity of the tryptophan residues of this protein toward N-bromosuccinimide in the presence and absence of substrate. The present results are consistent with models for the action of periplasmic-binding proteins in shock-sensitive transport systems of gram-negative bacteria which require a substrate-induced conformational change prior to the energy-dependent translocation of substrates.
Hemoglobin M Milwaukee (beta67E11 Val leads to Glu) is a naturally occurring valency hybrid containing two permanently oxidized hemes on the beta chains. In this mutant, the two abnormal beta chains cannot combine with ligands whereas the two alpha chains are normal and can combine with oxygen with a Hill coefficient varying from 1.1 to 1.3 [Udem et al. (1970), J Mol. Biol. 48, 489]. High-resolution proton nuclear magnetic resonance spectroscopy at 250 MHz has been used to investigate the exchangeable, ring-current shifted, ferrous and ferric hyperfine shifted resonances of Hb M Milwaukee in the absence and presence of organic phosphates. The alpha-heme environment, as manifested by the ring-current shifted resonances in the liganded form as well as the ferrous hyperfine shifted resonances in unliganded form, and subunit interactions, as manifested by the exchangeable resonances, are similar in Hb M Milwaukee to those in normal adult human hemoglobin. Organic phosphates can partially or completely inhibit the structural transformation which normally accompanies the binding of oxygen or carbon monoxide to Hb M Milwaukee. Upon stepwise addition of oxygen to deoxy Hb M Milwaukee, the hyperfine shifted resonance spectra of ferric beta chains show features which cannot be attributed to either fully deoxy or oxy species. However, the spectra for partially oxygenated Hb M Milwaukee can be described as an appropriately weighted average of the spectra of sero, singly, and doubly oxygenated species. The ferric hyperfine shifted resonance spectrum of the singly oxygenated intermediate has been calculated by a method employing least-squares analysis of the spectra of partially oxygenated Hb M Milwaukee at several values of oxygen saturation. The spectrum of this intermediate exhibits features which cannot be accounted for by a two-structure model. The present results are consistent with a sequential model for the oxygenation of this mutant hemoglobin. In view of the similarities between normal adult hemoglobin and Hb M Milwaukee, it is suggested that a two-state concerted allosteric model does not provide an adequate description of the structure-function relationship in normal adult hemoglobin.
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