Phosphorus nuclear magnetic resonance: a non-invasive technique for the study of muscle bioenergetics during exercise

Phosphorus nuclear magnetic resonance (31P NMR) spectroscopy is a non-destructive analytical laboratory technique that, due to recent technical advances, has become applicable to the study of high-energy phosphate metabolism in both animal and human extremity muscles (in vivo). 31P NMR can assay cellular phosphocreatine, ATP, inorganic phosphate, the phosphorylated glycolytic intermediates, and intra-cellular pH in either resting or exercising muscle, in a non-invasive manner. NMR uses non-perturbing levels of radio-frequency energy as its biophysical probe and can therefore safely study intact muscle in a repeated fashion while exerting no artifactual influence on ongoing metabolic processes. Compared with standard tissue biopsy and biochemical assay techniques, NMR possesses the advantages of being non-invasive, allowing serial in situ studies of the same tissue sample, and providing measurements of only active (unbound) metabolites. NMR studies of exercising muscle have yielded information regarding fatigue mechanisms at the cellular level and are helping resolve long-standing questions regarding the metabolic control of glycolysis, oxidative phosphorylation, and post-exercise phosphocreatine re-synthesis. NMR is also being utilized to measure enzymatic reaction rates in vivo. In the near future, other forms of NMR spectroscopy may also permit the non-invasive measurement of tissue glycogen and lactate content.     

Changes in thyroid state affect pHi and Nai+ homeostasis in rat ventricular myocytes

We have tested the hypothesis that thyroid state may influence both the flow of cellular Ca2+ and the myofilament response to Ca2+ by effects on intracellular pH (pHi) and Na+ (Nai+). Single cardiac myocytes isolated from hypothyroid, euthyroid and hyperthyroid animals were loaded with fura-2/AM (Cai2+ probe), BCECF/AM (pHi probe) or SBFI/AM (Nai+ probe). Compared with hypothyroid animals, myocytes isolated from hyperthyroid rat hearts demonstrated a significant: (1) increase in extent of shortening; (2) decrease in the time to peak contraction; (3) increase in the peak amplitude of the fura-2 fluorescence ratio; (4) decrease in pHi (DeltapHi=0. 19+/-0.05); and (5) increase in Nai+ (DeltaNai+=2.88+/-0.55 mM). We have also compared pHi in Langendorff perfused hypo- and hyperthyroid rat hearts using NMR. We have found that hyperthyroid hearts are 0.15+/-0.03 pH units more acidic than hypothyroid hearts. Analysis of mRNA levels demonstrated that hyperthyroidism increased expression of both the Na+/Ca2+ exchanger and Na+/H+ antiporter, and decreased expression of Na+ channel mRNAs. These changes appear partially responsible for the observed changes in Nai+ and pHi. Our results provide the first evidence that changes in cardiac contractility associated with altered thyroid state not only involve effects on Ca2+, but may also involve changes in the response of the myofilaments to Cai2+mediated by altered pHi and Nai+.     

Evaluation of the electrostatic effect of the 5'-phosphate of the flavin mononucleotide cofactor on the oxidation--reduction potentials of the flavodoxin from desulfovibrio vulgaris (Hildenborough)

Two mutants of the Desulfovibrio vulgaris flavodoxin, T12H and N14H, were generated which, for the first time, place a basic residue within the normally neutral 5'-phosphate binding loop of the flavin mononucleotide cofactor binding site found in all flavodoxins. These histidine residues were designed to form an ion pair with the dianionic 5'-phosphate, either altering its ionization state or offsetting its negative charge to allow evaluation of the magnitude of its electrostatic effect on the redox properties of the cofactor. The midpoint potential for the oxidized/semiquinone couple was not significantly altered in either mutant. However, the midpoint potentials for the semiquinone/hydroquinone couple (Esq/hq) were less negative than that of the wild type, increasing by 28 and 15 mV relative to that of the wild type for the T12H and N14H mutants, respectively, at pH 6. 31P NMR spectroscopy suggests that, just as for wild type, the phosphate group in each mutant does not change its ionization state between pH 6 and 8. Therefore, the small increases in midpoint potential must be linked to the protonation of the histidine residues, either through favorable interactions with the anionic hydroquinone or by the partial compensation of the charge on the 5'-phosphate. Values for the pKa of His12 and His14 in the oxidized flavodoxin were determined by 1H NMR spectroscopy to be 6.71 and 6.93, respectively, which are only modestly elevated relative to the average value for histidines in proteins. This suggests that the histidines do not form strong ion-pairing interactions with the phosphate and/or that the effective charge on the 5'-phosphate may be substantially less than the reported formal dianionic charge. Either way, the data provide evidence for the rather weak electrostatic interaction between a charged group at this site and the anionic flavin hydroquinone. In contrast, Esq/hq reported for the apoflavodoxin-riboflavin complex, which lacks the 5'-phosphate group, is 180 mV less negative than that of the native flavodoxin. The re-evaluation of the redox and cofactor binding properties of the riboflavin complex generated values for the dissociation constants for the riboflavin complex in the oxidized, semiquinone, and hydroquinone oxidation states that are 2100-, 63000-, and 54-fold higher, respectively, than that for the naturally occurring flavin mononucleotide complex. The large redox potential shifts observed for both redox couples in the riboflavin complex are primarily the consequence of a decreased stabilization of the semiquinone rather than the result of the absence of the negative charge of the 5'-phosphate. It is concluded from this study that the negative charge on the phosphate group of the cofactor does not play a disproportionate role in decreasing Esq/hq, at most contributing equivalently with the acidic amino acid residues clustered around the flavin to an unfavorable electrostatic environment for the formation of the flavin hydroquinone anion.     

Catalytic mechanism of the phospholipase D superfamily proceeds via a covalent phosphohistidine intermediate

The phospholipase D (PLD) superfamily includes enzymes of phospholipid metabolism, nucleases, as well as ORFs of unknown function in viruses and pathogenic bacteria. These enzymes are characterized by the invariant sequence motif, H(X)K(X)4D. The endonuclease member Nuc of the PLD family was over-expressed in bacteria and purified to homogeneity. Mutation of the conserved histidine to an asparagine in the endonuclease reduced the kcat for hydrolysis by a factor of 10(5), suggesting that the histidine residue plays a key role in catalysis. In addition to catalyzing hydrolysis, a number of phosphohydrolases will catalyze a phosphate (oxygen)-water exchange reaction. We have taken advantage of this observation and demonstrate that a 32P-labeled protein could be trapped when the enzyme was incubated with 32P-labeled inorganic phosphate. The phosphoenzyme intermediate was stable in 1 M NaOH and labile in 1 M HCl and 1 M hydroxylamine, suggesting that the enzyme forms a phosphohistidine intermediate. The pH-stability profile of the phosphoenzyme intermediate was consistent with phosphohistidine and the only radioactive amino acid found after alkaline hydrolysis was phosphohistidine. These results suggest that the enzymes in the PLD superfamily use the conserved histidine for nucleophilic attack on the substrate phosphorus atom and most likely proceed via a common two-step catalytic mechanism.     

31P NMR identification of metabolites and pH determination in the cyanobacterium Synechocystis sp. PCC 6308

The identity of a number of phosphorus-containing metabolites present in Synechocystis sp. PCC 6308 has been confirmed by 31P NMR spectroscopy. The presence of D-ribulose 1,5-bisphosphate (RuBP); DL-glyceraldehyde 3-phosphate (GlyP); D(-)3-phosphoglyceric acid (3PGA); D-ribulose 5-phosphate (Ru5P);6-phosphogluconic acid (6PGA); phosphoenolpyruvate (PEP); inorganic phosphate (Pi); uridine diphosphoglucose (UDPG); ADP and ATP were demonstrated by the pH dependence of their 31P NMR chemical shifts in spectra of perchloric acid cell extracts. Intracellular pH of cells was determined to be 7.5-7.7.     

Lactate turnover in rat glioma measured by in vivo nuclear magnetic resonance spectroscopy

Elevated tissue lactate concentrations typically found in tumors can be measured by in vivo nuclear magnetic resonance (NMR) spectroscopy. In this study, lactate turnover in rat C6 glioma was determined from in vivo 1H NMR measurements of [3-13C]lactate buildup during steady-state hyperglycemia with [1-13C]glucose. With this tumor model, a narrow range of values was observed for the first-order rate constant that describes lactate efflux, k2 = 0.043 +/- 0.007 (n = 12) SD min-1. For individual animals, the standard error in k2 was small (< 18%), which indicated that the NMR data fit the kinetic model well. Lactate measurements before and after infusing [1-13C]glucose showed that the majority of the tumor lactate pool was metabolically active. Signals from 13C-labeled glutamate in tumors were at least 10-fold smaller than the [3-13C]lactate signal, whereas spectra of the contralateral hemispheres revealed the expected labeling of [4-13C]glutamate, as well as [2-13C] and [3-13C]glutamate, which indicates that label cycled through the tricarboxylic acid cycle in the brain tissue. Lack of significant 13C labeling of glutamate was consistent with low respiratory metabolism in this glioma. It is concluded that lactate in rat C6 glioma is actively turning over and that the kinetics of lactate efflux can be quantified noninvasively by 1H NMR detection of 13C label. This noninvasive NMR approach may offer a valuable tool to help evaluate tumor growth and metabolic responsiveness to therapies.     

A chromatographic method for the preparative separation of phosphohistidines

The driving force behind this new chromatographic technique was to develop a method for purifying preparative quantities of phosphohistidines in a single step that provided good resolution wit eluants that could be easily removed. The current method can provide milligram quantities of each phosphohistidine; however, the later 1H NMR analysis of the dried, individually purified phosphohistidines showed that histidine was present along with each of the individual phosphohistidines. The stability of each phosphohistidine during storage does not appear to be a problem because the amounts of histidine contamination of freshly freeze-dried samples were compared with those of samples stored at -80 degrees C under nitrogen for 2 weeks and were found to be similar (data not shown). Possibly, the lyophilization and preparation of solutions for NMR analysis resulted in a certain amount of hydrolysis of phosphohistidine, particularly with the less stable 1- and 1,3-forms (5, 6). It was noted that when the lyophilized samples were initially dissolved in D2O for NMR analysis, the pH was between 6 and 7; this may have resulted in some hydrolysis of the phosphohistidines. This hydrolysis can be reduced by the addition of 50 mM potassium hydroxide to the pooled fractions collected from the chromatography, so that the alkalinity of the samples is maintained throughout the subsequent processes. The data obtained for the assignment of individual phosphohistidines by 1H and 31P NMR analysis seem consistent with those obtained by other independent studies (6, 10). The NMR analysis was a powerful tool in assigning the identity of each purified phosphohistidine, although caution should be used when considering free phosphohistidines as references for NMR detection of phosphohistidines in native proteins. Lecroisey et al. (10) showed that there were differences between the chemical shifts observed for free phosphohistidine compared to those for phosphohistidine in dipeptides. However, for the purposes of phosphoamino acid analysis, these purified phosphohistidines are used by this group as references in the detection of phosphohistidine in proteins.     

Compartmentation of lactate and glucose metabolism in C6 glioma cells. A 13c and 1H NMR study

13C and 1H NMR spectroscopy was used to investigate the metabolism of L-lactate and D-glucose in C6 glioma cells. The changing of lactate and glucose concentration in the extracellular medium of C6 glioma cells incubated with 5.5 mM glucose and 11 mM lactate indicated a net production of lactate as the consequence of an active aerobic glycolysis. The 13C enrichments of various metabolites were determined after 4-h cell incubation in media containing both substrates, each of them being alternatively labeled in the form of either [3-13C]L-lactate or [1-13C]D-glucose. Using 11 mM [3-13C]L-lactate, the enrichment of glutamate C4, 69%, was found higher than that of alanine C3, 32%, when that of acetyl-CoA C2 was 78%. These results indicated that exogenous lactate was the major substrate for the oxidative metabolism of the cells. Nevertheless, an active glycolysis occurred, leading to a net lactate production. This lactate was, however, metabolically different from the exogenous lactate as both lactate species did not mix into a unique compartment. The results were actually consistent with the concept of the existence of two pools of both lactate and pyruvate, wherein one pool was closely connected with exogenous lactate and was the main fuel for the oxidative metabolism, and the other pool was closely related to aerobic glycolysis.     

Depletion of glucose causes presynaptic inhibition of neuronal transmission in the rat dorsolateral septal nucleus

The present study was performed to further elucidate the mechanism of cadmium (Cd)-induced cytotoxicity in rat hepatocytes focusing on the effects of Cd-induced acidification on cellular production of H2O2 and the integrity of the plasma membrane. Exposure of cells of Cd levels < 50 microM stimulated cellular production of H2O2 in a dose-dependent manner. In cells exposed to 50 microM Cd, generation of the toxic oxygen increased from 5 min after exposure, and reached a plateau at 15 min. The acidic medium at pH 6.5, a value which is corresponding to the cellular pH at maximal acidification induced by Cd, also enhanced production of the active oxygen at almost the same level as 25 microM Cd. These treatments affected permeability barrier of plasma membranes as assessed by nuclear staining with propidium iodide (PI, MW 668) and release of intracellular lactic dehydrogenase (LDH) into surrounding medium. Cd at 50 microM caused nuclear staining by the fluorescent probe, beginning from 15 min at exposure, reaching a peak at 60 min. LDH leakage likewise started from 60 min of Cd exposure onward. The acidic partially prevented by L-ascorbic acid pretreatment. H2O2-induced nuclear staining increased with the increasing pH values from 6.7 to 7.1 Cd at 50 microM lowered the cellular pH within 5 min, but the decreased cellular pH returned to a value near physiological levels 25 min later. Pretreatment with Amiloride, an inhibitor of the Na+/H+ exchange, partially blocked this pH recovery after acidification. The results indicate that Cd caused H2O2 accumulation and H+, Cd and H2O2-related permeability changes of the plasma membrane. This may link to subsequent extensive membrane damage occurring at near physiological cellular pH.     

Anoxidative work and metabolism of isolated perfused rat hearts as influenced by artificially increased heart rate

Under anoxidative conditions the capacity of mechanical activity and the metabolism of isolated rat hearts were studied at various pacemaker-induced heart rates. Isovolumic work decreased to about one third of its initial rate within a 5 min period of anoxidative perfusion and remained almost constant during a further 10 min observation time. Increased heart rate (100, 175, and 250 b.p.m.) was associated with an increase in left ventricular diastolic pressure and with decreases in systolic pressure and maximal dp/dt. Lactate production, tissue creatine phosphate and inorganic phosphate had no consistent relationship to heart rate, whereas tissue ATP decreased with increasing heart rate. It is concluded that the rate of anoxidative isovolumic work cannot be increased by raising the heart rate since anoxidative carbohydrate breakdown cannot be more activated than at the lowest heart rate studied and cannot be more activated than at the lowest heart rate studied and cannot exceed a rate of 30 mumol hexose/min-g dry weight in the perfused heart.     

Calmodulin-dependent nitric-oxide synthase. Mechanism of inhibition by imidazole and phenylimidazoles

Calmodulin-dependent nitric-oxide synthase from bovine brain and GH3 pituitary cells is inhibited by imidazole, 1-phenylimidazole, 2-phenylimidazole, and 4-phenylimidazole, with half-maximal inhibition occurring at 200, 25, 160, and 600 microM concentrations of inhibitor, respectively. Imidazole inhibits the maximal velocity of citrulline formation by the enzyme, but does not alter the concentration of arginine, calmodulin, or (6R)-5,6,7,8,-tetrahydro-L-biopterin required for expression of half-maximal activity. Imidazole, 1-phenylimidazole, 2-phenylimidazole, and 4-phenylimidazole had no effect on calmodulin-dependent reduction of cytochrome c by the enzyme at concentrations up to 50-fold higher than those that inhibited citrulline formation. Imidazole inhibited calmodulin-dependent NADPH consumption by the enzyme with dissolved oxygen as the sole electron acceptor, with half-maximal inhibition occurring at a concentration of 225 microM. These observations are consistent with the proposal that imidazole and phenylimidazoles inhibit citrulline formation and oxygen reduction by acting as a sixth coordination ligand of the heme iron. This interaction prevents the formation of the activated reduced species of oxygen necessary for the formation of citrulline.     

NMR investigation of isotopically labeled cyanide derivatives of lignin peroxidase and manganese peroxidase

The 1H NMR spectroscopy was used to study lignin peroxidase (LiP) and manganese peroxidase (MnP) containing deuterated histidines. LiP and MnP were obtained from a histidine auxotroph of the fungus Phanerochaete chrysosporium grown in the presence of deuterated histidines. The derivatives with deuterated histidines have allowed a firm assignment of the protons of the distal and proximal histidines. We have also found that the LiP from this strain exhibits different orientations of the 2-vinyl group compared to the LiP from the strain previously studied. Mobility of the group has also been detected, thus explaining the apparent inconsistency between X-ray solid-state and NMR solution data. The 15N shift values of 15N-enriched CN- in the cyanide derivatives of LiP and MnP have also been measured. The shift patterns, both for 15N and for the proximal histidine protons of several peroxidases, are consistent with predominant contact shift contributions which reflect the bond strength of the metal-axial ligand. Finally, our results confirm a correlation between shift values of 15N and those of proximal histidine protons and the Fe3+/Fe2+ redox potentials.     

Light-induced photoactivation of hypericin affects the energy metabolism of human glioma cells by inhibiting hexokinase bound to mitochondria

Glucose-dependent energy required for glioma metabolism depends on hexokinase, which is mainly bound to mitochondria. A decrease in intracellular pH leads to a release of hexokinase-binding, which in turn decreases glucose phosphorylation, ATP content, and cell proliferation. Thus, intracellular pH might be a target for therapy of gliomas, and a search for agents able to modulate intracellular pH was initiated. Hypericin, a natural photosensitizer, displays numerous biological activities when exposed to light. Its mechanism and site of action at the cellular level remain unclear, but it probably acts by a type II oxygen-dependent photosensitization mechanism producing singlet oxygen. Hypericin is also able to induce a photogenerated intracellular pH drop, which could constitute an alternative mechanism of hypericin action. In human glioma cells treated for 1 h with 2.5 microg/ml hypericin, light exposure induced a fall in intracellular pH. In these conditions, mitochondria-bound hexokinase was inhibited in a light- and dose-dependent manner, associated with a decreased ATP content, a decrease of mitochondrial transmembrane potential, and a depletion of intracellular glutathione. Hexokinase protein was effectively released from mitochondria, as measured by an ELISA using a specific anti-hexokinase antibody. In addition to decreased glutathione, a response to oxidative stress was confirmed by the concomitant increase in mRNA expression of gamma-glutamyl cysteine synthetase, which catalyzes the rate-limiting step in overall glutathione biosynthesis, and is subject to feedback regulation by glutathione. Hypericin also induced a dose- and light-dependent inhibition of [3H]thymidine uptake and induced apoptosis, as demonstrated by annexin V-FITC binding and cell morphology. This study confirmed the mitochondria as a primary target of photodynamic action. The multifaceted action of hypericin involves the alteration of mitochondria-bound hexokinase, initiating a cascade of events that converge to alter the energy metabolism of glioma cells and their survival. In view of the complex mechanism of action of hypericin, further exploration is warranted in a perspective of its clinical application as a potential phototoxic agent in the treatment of glioma tumors.     

p53 protein expression in breast cancer as related to histopathological characteristics and prognosis

Reaction of Klebsiella aerogenes urease with diethylpyrocarbonate (DEP) led to a pseudo-first-order loss of enzyme activity by a reaction that exhibited saturation kinetics. The rate of urease inactivation by DEP decreased in the presence of active site ligands (urea, phosphate, and boric acid), consistent with the essential reactive residue being located proximal to the catalytic center. The pH dependence for the rate of inactivation indicated that the reactive residue possessed a pKa of 6.5, identical to that of a group that must be deprotonated for catalysis. Full activity was restored when the inactivated enzyme was treated with hydroxylamine, compatible with histidinyl or tyrosinyl reactivity. Spectrophotometric studies were consistent with DEP derivatization of 12 mol of histidine/mol of native enzyme. In the presence of active site ligands, however, approximately 4 mol of histidine/mol of protein were protected from reaction. Each protein molecule is known to possess two catalytic units; hence, we propose that urease possesses at least one essential histidine per catalytic unit.     

Comparison of Aniline Oxidation by Electro-Fenton and Fluidized-Bed Fenton Processes

This study investigated the aniline oxidation at various conditions, namely, pH, H2O2 dose, Fe2+ dose, and aniline concentration, as well as the effects of inorganic ion concentrations on the electro-Fenton and fluidized-bed Fenton processes. Aniline degradation depended on the H2O2 and Fe2+ dose for both processes. The results showed that both processes had the best aniline oxidation efficiency at pH 2.8–3.2. In the electro-Fenton process, approximately 95% of the aniline was removed after 60?min. While the SiO2 carrier contents of fluidized-bed Fenton process was adjusted to 74??g/L, the aniline was gradually removed, and the degradation rate finally reached 83%. The inhibition effect of phosphate ions on aniline oxidation on both processes was more significant than that of chloride ions. Chloride ions have higher inhibition ability on aniline oxidation in the fluidized-bed Fenton process than in the electro-Fenton process. However, the inhibition of phosphate ions on aniline oxidation in the electro-Fenton process was more obvious than in the fluidized-bed Fenton process.     

Aging impairs functional, metabolic and ionic recovery from ischemia-reperfusion and hypoxia-reoxygenation

Functional and metabolic responses to ischemia-reperfusion and hypoxia-reoxygenation were studied in Langendorff perfused hearts from mature (2-4 months) and aged (18-24 months) Wistar rats. Hearts were subjected to 20 min global ischemia or hypoxia followed by 30 min reperfusion or reoxygenation. Cellular metabolism was assessed by 31P-NMR spectroscopy. Normoxic function, phosphate metabolite levels, and cytosolic free energy state (delta GATP) were comparable in both age groups, although free [5'-AMP] and purine efflux were elevated in aged hearts. There were no aging-related differences in phosphate metabolite levels, pH or delta GATP during ischemia or hypoxia. Nevertheless, ischemic and hypoxic contracture tended to be higher in aged hearts. After reperfusion, heart rate x left-ventricular pressure recovered to 55% of pre-ischemia in mature hearts, and only 25% in aged hearts. After reoxygenation, function recovered to 75% in mature hearts and 55% in aged hearts. Recoveries of cellular [ATP], [phosphocreatine], [inorganic phosphate] and [Mg2+] were impaired, and delta GATP was consistently depressed in aged v mature hearts, Impaired recovery of delta GATP was associated with enhanced purine efflux in aged hearts. Post-ischemic Na+ and Ca2+ accumulation was also increased by 30-40% in aged hearts. Tissue damage assessed by post-ischemic creatine kinase efflux was modest in mature hearts (< 2% total tissue activity) and was 2.5-fold higher in aged hearts. The data show that: (i) aging reduces contractile recovery from ischemia/hypoxia; (ii) this is unrelated to the metabolic insult during ischemia/hypoxia, but parallels reduced recovery of delta GATP [inorganic phosphate], [Mg2+]i [Na+] and [Ca2+]; and (iii) increased purine catabolism may contribute to poor metabolic recovery in aged hearts.     

Histidine decarboxylase of Lactobacillus 30a: inactivation and active-site labeling by L-histidine methyl ester

Histidine decarboxylase from Lactobacillus 30a is rapidly and irreversibly inactivated upon incubation with L-histidine methyl ester. The rate of inactivation is first-order with respect to remaining active enzyme and exhibits saturation kinetics with a kinact of 1.2 mM and an apparent first-order rate constant of 0.346 min-1 at pH 4.8 and 25 degrees C. On complete inactivation, 3 mol of [14C]histidine (from L-[14C]histidine methyl ester) and 2 mol of 14C (from L-histidine [14C]methyl ester) are bound in nondialyzable form per mol (190 000 g) of protein inactivated with a corresponding loss of three of the five DTNB-titratable--SH groups that are essential for activity of the native enzyme. Imidazole propionate, a competitive inhibitor of the enzyme, protects against inactivation, loss of --SH groups, and incorporation of radioactivity from both the histidine and the methyl ester moieties of the labeled inhibitor, and kinetic evidence indicates that imidazole propionate and histidine methyl ester compete for binding at the active site of histidine decarboxylase in a mutually exclusive manner. Treatment of the labeled protein with either alkali or hydroxylamine results in the quantitative release of radioactivity. These data suggest that inactivation of histidine decarboxylase by L-histidine methyl ester results from two different modes of interaction between the inhibitor and the active site of histidine decarboxylase; the major interaction involves an essential -SH group.     

In the uncoupling protein (UCP-1) His-214 is involved in the regulation of purine nucleoside triphosphate but not diphosphate binding

The nucleotide binding to uncoupling protein (UCP-1) of brown adipose tissue is regulated by pH. The binding pocket of the nucleotide phosphate moiety has been proposed to be controlled by the protonization of a carboxyl group (pK approximately 4.5) for both nucleoside diphosphates (NDP) and nucleoside triphosphates (NTP) (identified as Glu-190) and of a histidine (pK approximately 7. 2) for NTP only. Here we identify His-214 as a pH sensor specific for NTP binding only. In reconstituted UCP-1 from hamster, DEPC diminishes binding of NTP but not of NDP. It also prevents inhibition of H+ transport by NTP but not by NDP. Hamster UCP-1 expressed in Saccharomyces cerevisiae was mutated to H214N resulting in only moderate change of the binding affinity for NTP (GTP) but a 10-fold affinity decrease with the bulkier substituent in H214W, whereas the affinity for NDP (ADP) was largely unchanged. The steep decrease with pH of the binding affinity for NTP in wild type (from pH 6.0 to 7.5) was much flatter in the mutants. Also, the pH dependence of binding and dissociation rates was diminished in these mutants. The transport of H+ and Cl- was not affected. Thus, His-214 is only involved in nucleotide binding, whereas, as previously shown, His-145 and His-147 are involved only in H+ transport. The results validate the earlier proposal of a histidine regulating the NTP binding in addition to a carboxyl group controlling both NTP and NDP binding. It is proposed that His-214 protrudes into the binding pocket for the gamma-phosphate thus inhibiting NTP binding and that His214H+ is retracted by a background -CO2- group to give way for the gamma-phosphate.     

Hypothalamic amenorrhea and hidden nutritional insults

Three variants of the 57.5 kDa human plasma proteinase inhibitor antithrombin, H1Q, H65C, and H120C, have been expressed in baby hamster kidney cells to permit assignment of the 1H NMR resonances from the three histidines and evaluation of the role of these histidines in heparin binding. The NMR assignments have enabled more definitive interpretation of previous NMR-based studies of human antithrombin to be made. Although resonances of all three histidines are perturbed by heparin binding, only histidine 120 plays a significant role in the heparin binding site. The perturbations of resonances from histidines 1 and 65 indicate proximity to the heparin binding site and consequent sensitivity to the presence of heparin.     

pH in human tumor xenografts and transplanted rat tumors: effect of insulin, inorganic phosphate, and m-iodobenzylguanidine

Various strategies to improve the therapeutic index of anticancer agents aim at inducing, by stimulation of aerobic glycolysis, temporary pH differences between malignant and normal tissues which can be exploited to activate cytotoxic agents selectively in tumors. We have investigated whether the pH reduction induced by glucose, the "drug" commonly used to increase lactic acid production in malignant tissues, can be augmented by pharmacological manipulation of tumor cell glycolysis. At normal plasma glucose concentration (6 +/- 1 mM), inorganic phosphate, a modifier of hexokinase and phosphofructokinase activity, had no effect on pH in two transplanted rat tumors and a human tumor xenograft line (average pH, 6.80; range, 6.65-6.95). When plasma glucose concentration was raised to 30 +/- 3 mM by i.v. infusion of glucose, inorganic phosphate reduced the pH in those tumors which exhibited only a moderate pH response to glucose per se (mean pH, 6.60) to an average value of 6.20 (range, 6.05-6.35). In the same setting, insulin, continuously infused at dose rates up to 600 milliunits/kg body weight/min, did not result in acidification of tumor tissue exceeding that induced by glucose alone. However, the H+ ion activity in both transplanted rat tumors and human tumor xenografts was increased by m-iodobenzylguanidine (MIBG), an inhibitor of mitochondrial respiration. For example, at normoglycemia, MIBG reduced the mean pH in a human mesothelioma xenograft from 6.90 to 6.70. This pH value was further reduced to 6.20 by simultaneous low-dose i.v. glucose infusion (plasma glucose concentration, 14 +/- 3 mM). The acidosis induced by inorganic phosphate and MIBG was tumor specific. Normal tissues of tumor-bearing hosts were only marginally sensitive to hyperphosphatemia or MIBG administration. These results indicate that the known stimulatory effect of exogenous glucose on lactic acid production in malignant tumors in vivo can be further accentuated or, as in the case of MIBG, partially replaced by pharmacological manipulation of aerobic glycolysis using clinically established drugs.