Distal ligand reactivity and quaternary structure studies of proximally detached hemoglobins.

The linkage between the proximal histidines and the proximal polypeptide in normal adult human hemoglobin (Hb A) has been proposed to play a major role in transmitting allosteric effects between oxygen binding sites [Perutz, M. F. (1970) Nature 228, 726-734]. Here we present circular dichroism (CD), (1)H NMR, analytical ultracentrifugation, and stopped-flow kinetic data to better define the quaternary structure of hemoglobins in which the linkage between the proximal histidines and the polypeptide backbone has been broken [Barrick et al. Nat. Struct. Biol. 4, 78-83 (1997)] and to characterize the distal ligand binding properties of these proximally detached Hbs. CD spectroscopy indicates that rHb (alphaH87G) and rHb (alphaH87G/betaH92G) retain at least partial T-quaternary structure with distal ligand bound, whereas rHb (betaH92G) does not, consistent with (1)H NMR spectra. Analytical ultracentrifugation reveals significant tetramer dissociation in rHb (betaH92G) to be the likely cause of loss of T-state markers. These quaternary structure studies indicate that in distally liganded Hb, the T-state is compatible with proximal linkages in the beta- but not the alpha-chains. (1)H NMR titrations of rHb (alphaH87G) with n-butyl isocyanide demonstrate the alpha-chains to be of high affinity as compared with the beta-chains. Comparing ligand association and dissociation rates between the rHb (alphaH87G) variant with the T- and R-states of wild-type Hb A indicates that at the alpha-chains, carbon monoxide affinity is modulated entirely by the proximal linkage, rather than from distal interactions. Some residual allosteric interactions may remain operative at the beta-chains of rHb (alphaH87G).

An additional H-bond in the alpha 1 beta 2 interface as the structural basis for the low oxygen affinity and high cooperativity of a novel recombinant hemoglobin (beta L105W).

Site-directed mutagenesis has been used to construct three recombinant mutant hemoglobins (rHbs), rHb(beta L105W), rHb(alpha D94A/betaL105W), and rHb(alpha D94A). rHb(beta L105W) is designed to form a new hydrogen bond from beta 105Trp to alpha 94Asp in the alpha(1)beta(2) subunit interface to lower the oxygen binding affinity by stabilizing the deoxy quaternary structure. We have found that rHb(beta L105W) does indeed possess a very low oxygen affinity and maintains normal cooperativity (P(50) = 28.2 mmHg, n(max) = 2.6 in 0.1 M sodium phosphate at pH 7.4) compared to those of Hb A (P(50) = 9.9 mmHg, n(max) = 3.2 at pH 7.4). rHb(alpha D94A/beta L105W) and rHb(alpha D94A) are expressed to provide evidence that rHb(betaL 105W) does form a new H-bond from beta 105Trp to alpha 94Asp in the alpha(1)beta(2) subunit interface of the deoxy quaternary structure. Our multinuclear, multidimensional nuclear magnetic resonance (NMR) studies on (15)N-labeled rHb(beta L105W) have identified the indole nitrogen-attached (1)H resonance of beta 105Trp for rHb(beta L105W). (1)H NMR studies on Hb A and mutant rHbs have been used to investigate the structural basis for the low O(2) affinity of rHb(beta L105W). Our NMR results provide evidence that rHb(beta L105W) forms a new H-bond from beta 105Trp to alpha 94Asp in the alpha(1)beta(2) subunit interface of the deoxy quaternary structure. The NMR results also show that these three rHbs can switch from the R quaternary structure to the T quaternary structure in their ligated state upon addition of an allosteric effector, inositol hexaphosphate. We propose that the low O(2) affinity of rHb(beta L105W) is due to the formation of a new H-bond between alpha 105Trp and alpha 94Asp in the deoxy quaternary structure.

NMR reveals hydrogen bonds between oxygen and distal histidines in oxyhemoglobin.

Compared with free heme, the proteins hemoglobin (Hb) and myoglobin (Mb) exhibit greatly enhanced affinity for oxygen relative to carbon monoxide. This physiologically vital property has been attributed to either steric hindrance of CO or stabilization of O(2) binding by a hydrogen bond with the distal histidine. We report here the first direct evidence of such a hydrogen bond in both alpha- and beta-chains of oxyhemoglobin, as revealed by heteronuclear NMR spectra of chain-selectively labeled samples. Using these spectra, we have assigned the imidazole ring (1)H and (15)N chemical shifts of the proximal and distal histidines in both carbonmonoxy- and oxy-Hb. Because of their proximity to the heme, these chemical shifts are extremely sensitive to the heme pocket conformation. Comparison of the measured chemical shifts with values predicted from x-ray structures suggests differences between the solution and crystal structures of oxy-Hb. The chemical shift discrepancies could be accounted for by very small displacements of the proximal and distal histidines. This suggests that NMR could be used to obtain very high-resolution heme pocket structures of Hb, Mb, and other heme proteins.

Chain-selective isotopic labeling for NMR studies of large multimeric proteins: application to hemoglobin.

Multidimensional, multinuclear NMR has the potential to elucidate the mechanisms of allostery and cooperativity in multimeric proteins under near-physiological conditions. However, NMR studies of proteins made up of non-equivalent subunits face the problem of severe resonance overlap, which can prevent the unambiguous assignment of resonances, a necessary step in interpreting the spectra. We report the application of a chain-selective labeling technique, in which one type of subunit is labeled at a time, to carbonmonoxy-hemoglobin A (HbCO A). This labeling method can be used to extend previous resonance assignments of key amino acid residues, which are important to the physiological function of hemoglobin. Among these amino acid residues are the surface histidyls, which account for the majority of the Bohr effect. In the present work, we report the results of two-dimensional heteronuclear multiple quantum coherence (HMQC) experiments performed on recombinant (15)N-labeled HbCO A. In addition to the C2-proton (H epsilon(1)) chemical shifts, these spectra also reveal the corresponding C4-proton (H delta(2)) resonances, correlated with the N epsilon(2) and N delta(1) chemical shifts of all 13 surface histidines per alpha beta dimer. The HMQC spectrum also allows the assignment of the H delta(1), H epsilon(1), and N epsilon(1) resonances of all three tryptophan residues per alpha beta dimer in HbCO A. These results indicate that heteronuclear NMR, used with chain-selective isotopic labeling, can provide resonance assignments of key regions in large, multimeric proteins, suggesting an approach to elucidating the solution structure of hemoglobin, a protein with molecular weight 64.5 kDa.

Assessment of roles of surface histidyl residues in the molecular basis of the Bohr effect and of beta 143 histidine in the binding of 2,3-bisphosphoglycerate in human normal adult hemoglobin.

Site-directed mutagenesis has been used to construct two mutant recombinant hemoglobins (rHbs), rHb(betaH116Q) and rHb(betaH143S). Purified rHbs were used to assign the C2 proton resonances of beta116His and beta143His and to resolve the ambiguous assignments made over the past years. In the present work, we have identified the C2 proton resonances of two surface histidyl residues of the beta chain, beta116His and beta143His, in both the carbonmonoxy and deoxy forms, by comparing the proton nuclear magnetic resonance (NMR) spectra of human normal adult hemoglobin (Hb A) with those of rHbs. Current assignments plus other previous assignments complete the assignments for all 24 surface histidyl residues of human normal adult hemoglobin. The individual pK values of 24 histidyl residues of Hb A were also measured in deuterium oxide (D(2)O) in 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) buffer in the presence of 0.1 M chloride at 29 degrees C by monitoring the shifts of the C2 proton resonances of the histidyl residues as a function of pH. Among those surface histidyl residues, beta146His has the biggest contribution to the alkaline Bohr effect (63% at pH 7.4), and beta143His has the biggest contribution to the acid Bohr effect (71% at pH 5.1). alpha20His, alpha112His, and beta117His have essentially no contribution; alpha50His, alpha72His, alpha89His, beta97His, and beta116His have moderate positive contributions; and beta2His and beta77His have a moderate negative contribution to the Bohr effect. The sum of the contributions from 24 surface histidyl residues accounted for 86% of the alkaline Bohr effect at pH 7.4 and about 55% of the acid Bohr effect at pH 5.1. Although beta143His is located in the binding site for 2,3-bisphosphoglycerate (2,3-BPG) according to the crystal structure of deoxy-Hb A complexed with 2, 3-BPG, beta143His is not essential for the binding of 2,3-BPG in the neutral pH range according to the proton NMR and oxygen affinity studies presented here. With the accurately measured and assigned individual pK values for all surface histidyl residues, it is now possible to evaluate the Bohr effect microscopically for novel recombinant Hbs with important functional properties, such as low oxygen affinity and high cooperativity. The present study further confirms the importance of a global electrostatic network in regulating the Bohr effect of the hemoglobin molecule.

Structural consequences of anesthetic and nonimmobilizer interaction with gramicidin A channels.

Although interactions of general anesthetics with soluble proteins have been studied, the specific interactions with membrane bound-proteins that characterize general anesthesia are largely unknown. The structural modulations of anesthetic interactions with synaptic ion channels have not been elucidated. Using gramicidin A as a simplified model for transmembrane ion channels, we have recently demonstrated that a pair of structurally similar volatile anesthetic and nonimmobilizer, 1-chloro-1,2,2-trifluorocyclobutane (F3) and 1,2-dichlorohexafluorocyclobutane (F6), respectively, have distinctly different effects on the channel function. Using high-resolution NMR structural analysis, we show here that neither F3 nor F6 at pharmacologically relevant concentrations can significantly affect the secondary structure of the gramicidin A channel. Although both the anesthetic F3 and the nonimmobilizer F6 can perturb residues at the middle section of the channel deep inside the hydrophobic region in the sodium dodecyl sulfate micelles, only F3, but not F6, can significantly alter the chemical shifts of the tryptophan indole N-H protons near the channel entrances. The results are consistent with the notion that anesthetics cause functional change of the channel by interacting with the amphipathic domains at the peptide-lipid-water interface.

Characterization of the lipid-carrier involved in the synthesis of enterobacterial common antigen (ECA) and identification of a novel phosphoglyceride in a mutant of Salmonella typhimurium defective in ECA synthesis.

The polysaccharide chains of enterobacterial common antigen (ECA) consist of linear trisaccharide repeat units with the structure -->3)-alpha-d-Fuc4NAc-(1-->4)-beta-d-ManNAcA-(1--> 4)-alpha-d-GlcNAc-(1-->, where Fuc4NAc is 4-acetamido-4, 6-dideoxy-d-galactose, ManNAcA is N -acetyl-d- mannosaminuronic acid, and GlcNAc is N -acetyl-d-glucosamine. The major form of ECA (ECAPG) consists of polysaccharide chains that are believed to be covalently linked to diacylglycerol through phosphodiester linkage; the phospholipid moiety functions to anchor molecules in the outer membrane. The ECA trisaccharide repeat unit is assembled as a polyisoprenyl-linked intermediate which has been tentatively identified as Fuc4NAc-ManNAcA-GlcNAc-pyrophosphorylundecaprenol (lipid III). Subsequent chain-elongation presumably occurs by a block-polymerization mechanism. However, the identity of the polyisoprenoid carrier-lipid has not been established. Accordingly, the current studies were conducted in an effort to structurally characterize the polyisoprenyl lipid-carrier involved in ECA synthesis. Isolation and characterization of the lipid carrier was facilitated by the accumulation of a ManNAcA-GlcNAc-pyrophosphorylpolyisoprenyl lipid (lipid II) in mutants of Salmonella typhimurium defective in the synthesis of TDP-Fuc4NAc, the donor of Fuc4NAc residues for ECA synthesis. Analyses of lipid II preparations by fast atom bombardment tandem mass spectroscopy (FAB-MS/MS) resulted in the identification of the lipid-carrier as the 55-carbon polyisoprenyl alcohol, undecaprenol. These analyses also resulted in the identification of a novel glycolipid which copurified with lipid II. FAB-MS/MS analyses of this glycolipid revealed its structure to be 1,2-diacyl- sn -glycero-3-pryophosphoryl-GlcNAc-ManNAcA (DGP-disaccharide). An examination of purified ECAPGby phosphorus-31 nuclear magnetic resonance spectroscopy confirmed that the polysaccharide chains are linked to diacylglycerol through phosphodiester linkage. Thus, DGP-disaccharide does not appear to be an intermediate in ECAPGsynthesis. Nevertheless, although the available evidence clearly indicate that lipid II is a precursor of DGP-disaccharide, the function of this novel glycolipid is not yet known, and it may be an intermediate in the biosynthesis of a molecule other than ECAPG.

1H, 13C, and 15N NMR backbone assignments and chemical-shift-derived secondary structure of glutamine-binding protein of Escherichia coli.

1H, 13C, and 15N NMR assignments of the backbone atoms and beta-carbons have been made for liganded glutamine-binding protein (GlnBP) of Escherichia coli, a monomeric protein with 226 amino acid residues and a molecular weight of 24935 Da. GlnBP is a periplasmic binding protein which plays an essential role in the active transport of L-glutamine through the cytoplasmic membrane. The assignments have been obtained from three-dimensional triple-resonance NMR experiments on a 13C, 15N uniformly labeled sample as well as specifically labeled samples. Results from the 3D triple-resonance experiments, HNCO, HN(CO)CA, HN(COCA)HA, HNCA, HN(CA)HA, HN(CA)CO, and CBCA(CO)NH, are the main sources used to make the resonance assignments. Other 3D experiments, such as HNCACB, COCAH, HCACO, HCACON, and HOHAHA-HMQC, have been used to confirm the resonance assignments and to extend connections where resonance peaks are missing in some of the experiments mentioned above. We have assigned more than 95% of the polypeptide backbone resonances of GlnBP. The result of the standard manual assignment is in agreement with that predicted by an automated probailistic method developed in our laboratory. A solution secondary structure of the GlnBP-Gln complex has been proposed based on chemical shift deviations from random coil values. Eight alpha-helices and 10 beta-strands are derived using the Chemical Shift Index method.

A test of the role of the proximal histidines in the Perutz model for cooperativity in haemoglobin.

Human haemoglobin has long been a paradigm for cooperative ligand binding and allostery. Through analysis of the crystal structures of deoxyhaemoglobin and liganded haemoglobin, Perutz proposed a model for cooperativity in which the bond between the proximal histidine and the protein couples haem rearrangements to protein structure rearrangements. Here we test this model by deleting the bonds between the histidine imidazole side chain and the polypeptide. This detachment method allows us to determine directly the contribution of proximal histidine coupling to cooperativity of distal ligand binding. Proximal detachment significantly increases ligand affinity, reduces cooperativity, and prevents quaternary structure switching, in accord with the Perutz model. Residual cooperativity indicates that additional haem communication pathways exist that do not involve the proximal histidine coupling mechanism.

Production of human normal adult and fetal hemoglobins in Escherichia coli.

A hemoglobin expression system in Escherichia coli is described. In order to produce authentic human hemoglobin, we need to co-express both methionine aminopeptidase and globin genes under the control of a strong promoter. We have constructed three plasmids, pHE2, pHE4 and pHE7, for the expression of human normal adult hemoglobin and a plasmid, pHE9, for the expression of human fetal hemoglobin, in high yields. The globin genes can be derived from either synthetic genes or human globin cDNAs. The extra amino-terminal methionine residues of the expressed globins can be removed by the co-expressed methionine aminopeptidase. The heme is inserted correctly into the expressed alpha-globin from our expression plasmids. A fraction (approximately 25%) of the heme is not inserted correctly into the expressed beta- or gamma-globin. However, the incorrectly inserted hemes can be converted into the correct conformation by carrying out a simple oxidation-reduction process on the purified hemoglobin molecule. We have investigated the functional properties of the expressed hemoglobins by measuring their oxygen-binding properties and their structural features by obtaining their 1H-NMR spectra. Our results show that authentic human normal adult and fetal hemoglobins can be produced from our expression plasmids in E. coli and in high yields. Our expression system allows us to design and to produce any recombinant hemoglobins needed for our research on the structure-function relationship in hemoglobin.

A 19F-NMR study of the equilibrium unfolding of membrane-associated D-lactate dehydrogenase of Escherichia coli.

Partially folded protein intermediates have been observed by 19F-NMR spectroscopy during the equilibrium unfolding of the membrane-associated D-lactate dehydrogenase (D-LDH) of Escherichia coli by a denaturant, guanidine hydrochloride (Gdn.HCl). The results from 19F-NMR and circular dichroism spectroscopic studies suggest that the intermediates observed at low Gdn.HCl concentrations (< 3.5 M) exhibit features similar to "molten globules" that contain considerable amounts of secondary and tertiary structure. The results of 19F-NMR studies on 5F-Trp-labeled D-LDH, such as the chemical shift changes, nuclear Overhauser effect, and solvent-induced isotopic shift effect, show that different regions of D-LDH unfold nonuniformly in Gdn.HCl in the presence of lysophosphatidylcholine. The polypeptide appears to unfold in a general order from the carboxyl end to the amino end, in agreement with previous findings from our laboratory that the carboxyl-terminal region of D-LDH is largely exposed to the solvent while the amino-terminal region is buried in the protein core. The structure of the partially unfolded intermediate forms of D-LDH is stabilized in the presence of lipid-like detergents, such as lysophosphatidylcholine.

1H-NMR investigation of the oxygenation of hemoglobin in intact human red blood cells.

Using improved selective excitation methods for protein nuclear magnetic resonance (NMR), we have conducted measurements of the oxygenation of hemoglobin inside intact human red blood cells. The selective excitation methods use pulse shape-insensitive suppression of the water signal, while producing uniform phase excitation in the region of interest and, thus, are suitable for a wide variety of applications in vivo. We have measured the areas of 1H-NMR resonances of the hyperfine-shifted, exchangeable N delta H protons of the proximal histidine residues of the alpha- and beta-chains in deoxyhemoglobin (63 and 76 ppm downfield from the proton resonance of 2,2-dimethyl-2-silapentane-5-sulfonate (DSS), respectively), which are sensitive to the paramagnetic state of the iron, and for which the alpha- and beta-chain resonances are resolved, and from the ring current-shifted gamma 2-CH3 protons of the distal valine residues in oxyhemoglobin (2.4 ppm upfield from DSS), which are sensitive to the conformation of the heme pocket in the oxy state. We have found that the proximal histidine resonances are directly correlated with the degree of oxygenation of hemoglobin, whereas the distal valine resonances appear to be correlated with the conformation in the heme pocket that occurs after the binding of oxygen, in both the presence and absence of 2,3-diphosphoglycerate. In addition, from the proximal histidine resonances, we have observed a preference for the binding of oxygen to the alpha-chain (up to about 10%) of hemoglobin over the beta-chain in both the presence and absence of 2,3-diphosphoglycerate. These new results obtained in intact erythrocytes are consistent with our previous 1H-NMR studies on purified human normal adult hemoglobin. A unique feature of our 1H-NMR method is the ability to monitor the binding of oxygen specifically to the alpha- and beta-chains of hemoglobin both in solution and in intact red blood cells. This information is essential to our understanding of the molecular basis for the hemoglobin molecule serving as the oxygen carrier in vertebrates.

Liver regeneration after partial hepatectomy in the rat. Sequential events monitored by 31P-nuclear magnetic resonance spectroscopy and biochemical studies.

BACKGROUND: Little data exist wherein both the 31P nuclear magnetic resonance (NMR) signals and biochemical changes associated with hepatic regeneration after a 70% hepatic resection have been assessed simultaneously. EXPERIMENTAL DESIGN: Two groups of rats were used: one group underwent a 70% partial hepatectomy and the second underwent a sham operation. Both groups were followed sequentially for 192 hours by in vivo serial 31P-NMR spectroscopy of the liver and its phospholipid extracts. Liver injury and function were assessed by biochemical means. RESULTS: After surgery, a significant reduction in ATP and an increase in the phosphomonoester signal for the hepatectomized animals were noted as compared with the controls (p < 0.05). The phosphodiester content of the liver in the hepatectomized rats declined to nonmeasurable amounts in vivo. The nadir of ATP occurred 72 hours after surgery. The area of the phosphomonoester relative to an external reference of methylenediphosphonic acid peak increased steadily over the first 96 hours, whereas that of the area ratio of the inorganic phosphate/methylenediphosphonic increased over the first 72 hours posthepatectomy. The intracellular pH declined sharply in the first 3 days, followed by a gradual recovery over the next 5 days. Little change in the intracellular pH was observed for the control animals. A significant increase in the area of the phosphorylethanolamine relative to an internal reference of methylenediphosphonic and a reduction in the glycerophosphorylethanolamine and glycerophosphorylcholine peaks were noted during the first four post-hepatectomy days as measured by 31P-NMR of perchloric acid liver extracts (p < 0.05). CONCLUSIONS: It has been found that a concerted reduction in the intracellular ATP and intracellular pH coupled with an increase in inorganic phosphate and high levels of phosphorylethanolamine occur as a result of hepatic regeneration and the physiologic changes induced. These data demonstrated that a coordinated pattern of biochemical changes occur with and after hepatic regeneration. Moreover, NMR spectroscopy demonstrates an increase in phosphomonoesters and a decline in phosphodiesters during hepatic regeneration. These measures and, more specifically, the ratio of these two lipid classes may provide a biochemical snapshot of the regeneration status of the liver.

Production of unmodified human adult hemoglobin in Escherichia coli.

We have constructed a plasmid (pHE2) in which the synthetic human alpha- and beta-globin genes and the methionine aminopeptidase (Met-AP) gene from Escherichia coli are coexpressed under the control of separate tac promoters. The Hbs were expressed in E. coli JM109 and purified by fast protein liquid chromatography, producing two major components, a and b. Electrospray mass spectrometry shows that at least 98% and about 90% of the expressed alpha and beta chains of component a, respectively, have the expected masses. The remaining 10% of the beta chain in component a corresponds in mass to the beta chain plus methionine. In component b, both alpha and beta chains have the correct masses without detectable N-terminal methionine (< 2%). These results have been confirmed by Edman degradation studies of the amino-terminal sequences of the alpha and beta chains of these two recombinant Hb (rHb) samples. rHbs from components a and b exhibit visible optical spectra identical to that of human normal adult Hb (Hb A). Component a and Hb A have very similar oxygen-binding properties, but component b shows somewhat altered oxygen binding, especially at low pH values. 1H-NMR spectra of component a and Hb A are essentially identical, whereas those of component b exhibit altered ring current-shifted and hyperfine-shifted proton resonances, indicating altered heme conformation in the beta chain. These altered resonance patterns can be changed to those of Hb A by converting component b to the ferric state and then to the deoxy state and finally back to either the carbonmonoxy or oxy form. Thus, our E. coli expression system produces native, unmodified Hb A in high yield and can be used to produce desired mutant Hbs.

Measurement of fluxes through the pentose phosphate pathway in erythrocytes from individuals with sickle cell anemia by carbon-13 nuclear magnetic resonance spectroscopy.

Erythrocytes from individuals with sickle cell anemia have previously been shown to have increased levels of intracellular oxidants and increased oxidative damage. Oxidative damage has been implicated in the events leading to the painful crises and hemolytic anemia found in sickle cell anemia. Since the pentose phosphate pathway (PPP) is an important source of reducing capacity in erythrocytes, we have investigated the fluxes through the PPP in normal and sickle cell erythrocytes using [2-13C]D-glucose and carbon-13 nuclear magnetic resonance (NMR) spectroscopy. Our results indicate that sickle cell erythrocytes have a flux through the PPP of 0.13 +/- 0.02 mumol/h per ml erythrocytes that is comparable to that in normal erythrocytes, 0.21 +/- 0.02 mumol/h per ml erythrocytes. However, when stimulated with methylene blue, sickle cell erythrocytes show a decreased response, 0.59 +/- 0.10 mumol/h per ml erythrocytes, compared to normal erythrocytes, 1.64 +/- 0.10 mumol/h per ml erythrocytes. When homogeneous populations of sickle cell erythrocytes are isolated by density gradient centrifugation, the rate of flux through the PPP in methylene blue-stimulated sickle cell erythrocytes, 1.16 +/- 0.16 mumol/h per ml erythrocytes, approaches that in methylene blue-stimulated normal erythrocytes. In addition, by analyzing the dose response to methylene blue, we have found that the decreased stimulation of the PPP by methylene blue in heterogeneous populations of sickle cell erythrocytes is a failure of methylene blue to stimulate the PPP rather than a deficiency in the PPP in sickle cell erythrocytes.

A carbon-13 nuclear magnetic resonance investigation of the metabolic fluxes associated with glucose metabolism in human erythrocytes.

We have used [2-13C]D-glucose and carbon-13 nuclear magnetic resonance (NMR) spectroscopy to investigate metabolic fluxes through the major pathways of glucose metabolism in intact human erythrocytes and to determine the interactions among these pathways under conditions that perturb metabolism. Using the method described, we have been able to measure fluxes through the pentose phosphate pathway, phosphofructokinase, the 2,3-diphosphoglycerate bypass, and phosphoglycerate kinase, as well as glucose uptake, concurrently and in a single experiment. We have measured these fluxes in normal human erythrocytes under the following conditions: (1) fully oxygenated; (2) treated with methylene blue; and (3) deoxygenated. This method makes it possible to monitor various metabolic effects of stresses in normal and pathological states. Not only has 13C-NMR spectroscopy proved to be a useful method for measuring in vivo flux through the pentose phosphate pathway, but it has also provided additional information about the cycling of metabolites through the non-oxidative portion of the pentose phosphate pathway. Our evidence from experiments with [1-13C]-, [2-13C]-, and [3-13C]D-glucoses indicates that there is an observable reverse flux of fructose 6-phosphate through the reactions catalyzed by transketolase and transaldolase, even in the presence of a net flux through the pentose phosphate pathway.

Multidimensional 1H and 15N NMR investigation of glutamine-binding protein of Escherichia coli.

Specific and uniform 15N labelings along with site-directed mutagenesis of glutamine-binding protein have been utilized to obtain assignments of the His156, Trp32 and Trp220 residues. These assignments have been made not only to further study the importance of these 3 amino acid residues in protein-ligand and protein-protein interactions associated with the active transport of L-glutamine across the cytoplasmic membrane of Escherichia coli, but also to serve as the starting points in the sequence-specific backbone assignment. The assignment of H epsilon 2 of His156 refines the earlier model where this particular proton forms an intermolecular hydrogen bond to the delta-carbonyl of L-glutamine, while assignments of both Trp32 and Trp220 show the variation in local structures which ensure the specificity in ligand binding and protein-protein interaction. Using 3D NOESY-HMQC NMR, amide connectivities can be traced along 8-9 amino acid residues at a time. This paper illustrates the usefulness of combining 15N isotopic labeling and multinuclear, multidimensional NMR techniques for a structural investigation of a protein with a molecular weight of 25,000.

Cyclosporine and liver regeneration studied by in vivo 31P nuclear magnetic resonance spectroscopy.

The changes in fructose-1-phosphate (F-1-P), intracellular pH, and ATP content of the liver after a fructose challenge were investigated noninvasively in vivo using phosphorus-31 nuclear magnetic resonance spectroscopy of dog liver four days after a portacaval shunt (PCS) with or without portal venous infusion of cyclosporin (CsA). The F-1-P metabolism was slower in PCS dogs (N = 2) as compared to either the normal (N = 2) or PCS + CsA-treated dogs (N = 3) (P less than 0.05). The intracellular pH temporarily decreased from 7.3 +/- 0.05 to 7.0 +/- 0.05 during the fructose challenge. The regenerative indexes were increased in the PCS + CsA group (P less than 0.01). These data obtained in vivo using 31P-NMR spectroscopy in the liver following a portacaval shunt, suggest that: (1) the energy status of the liver and the metabolic response to fructose are reduced in PCS compared to normal animals and (2) CsA treatment enhances the regenerative response of the liver and prevents the reduction in hepatic function associated with portacaval shunting.

Effect of cyclosporine on hepatic energy status and on fructose metabolism after portacaval shunt in dog as monitored by phosphorus-31 nuclear magnetic resonance spectroscopy in vivo.

The effect of cyclosporin A on the hepatic energy status and intracellular pH of the liver and its response to a fructose challenge has been investigated using in vivo phosphorus-31 nuclear magnetic resonance spectroscopy in dogs. Three experimental groups were studied: (a) control dogs (n = 5), (b) dogs 4 days after the creation of an end-to-side portacaval shunt (n = 5), and (c) dogs 4 days after portacaval shunt and continuous infusion of cyclosporin A (4 mg/kg/day) by way of the left portal vein (portacaval shunt plus cyclosporin A, n = 5). The phosphorus-31 nuclear magnetic resonance spectra were obtained at 81 MHz using a Bruker BIOSPEC II 4.7-tesla nuclear magnetic resonance system equipped with a 40-cm horizontal bore superconducting solenoid. The phosphomonoesters (p less than 0.01), inorganic phosphate and ATP levels (p less than 0.05) were decreased significantly in portacaval shunt-treated and in portacaval shunt-plus-cyclosporin A-treated dogs compared with unshunted control dogs. After a fructose challenge (750 mg/kg body wt, intravenously), fructose-1-phosphate metabolism was reduced in portacaval shunt-treated dogs compared with either the normal or portacaval shunt-plus-cyclosporin A-treated dogs (p less than 0.05). Both portacaval shunt- and portacaval shunt-plus-cyclosporin A-treated dogs demonstrated a reduced decline in ATP levels after fructose infusion when compared with the controls (p less than 0.05). Immediately after the fructose challenge, the intracellular pH decreased from 7.30 +/- 0.03 to 7.00 +/- 0.05 in all animals (p less than 0.01) and then gradually returned to normal over 60 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain pH in acute isocapnic metabolic acidosis and hypoxia: a 31P-nuclear magnetic resonance study.

It is well known that brain pH changes rapidly in acute hypercapnia or hypocapnia. The effect of acute isocapnic metabolic acid-base change on brain pH is less certain. To study this problem, acute isocapnic metabolic acidosis was induced by HCl or lactic acid infusions in rats, and recovery from acidosis was accomplished by NaHCO3 infusion. Brain pH was measured by 31P-nuclear magnetic resonance. Despite decreases in blood pH of 0.34 and 0.36 units, respectively, in less than 1 h of acid infusion and rapid recovery during bicarbonate infusion, brain pH was unaffected (ranging between 7.08 and 7.11) and was uncorrelated with blood pH. The blood pH minus brain pH gradient was eliminated by the acidosis. By contrast, hypoxia-induced endogenous lactic acidosis lowered blood and brain pH equivalently, but the fall in brain pH preceded that in blood. During normoxic recovery, brain pH overshot and became alkaline when blood pH was still significantly reduced and blood lactate levels were markedly elevated. Presumably, this is due to stimulated active H+ transport. The results demonstrate that brain pH is affected differently in metabolic, respiratory, and endogenous acid-base disturbances. Thus brain pH cannot be predicted solely from blood pH values.