Diastereomeric phosphonate ester adducts of chymotrypsin: 31P-NMR measurements

Generation of diastereomeric phosphonate ester adducts of chymotrypsin was evidenced for the first time by ³¹P NMR and spectrophotometric kinetic measurements. ³¹P NMR signals were recorded for 4-nitrophenyl 2-propyl methylphosphonate (IMN) at 32.2 ppm and for its hydrolysis product at 26.3 ppm downfield from phosphoric acid. The inhibition of α-chymotrypsin at pH > 8.0 by the faster reacting enantiomer of IMN or 2-propyl methylphosphonochloridate (IMCl), or other phosphonate ester analogs of these compounds, all caused a ～6.0 ppm downfield shift of the ³¹P signal to the 39–40 ppm region. IMN, when applied below the stoichiometric amount of chymotrypsin, under the same conditions, generated two signals, at 39.0 and at 37.4 ppm. Scans accumulated in hourly intervals showed the decomposition of both diastereomers, with approximate half-lives of 12 h at pH 8.0 and 22°C, into a species with a resonance at 35.5 ppm. The most likely reaction to account for the appearance of this new peak is the enzymic dealkylation of the isopropyl group from the covalently bound phosphonate ester. We base this conclusion mostly on the similarity of the upfield shift to the hydrolysis of phosphonate esters. Contrary to experience with phosphate ester adducts of serine proteases, no signal was detected higher than 25.0 ppm downfield from phosphoric acid for several phosphonate ester adducts of chymotrypsin and in no case did the resonance for the adduct shift further downfield in the course of the experiments. © 1993 Wiley-Liss, Inc.     

Mechanisms of hydration effects on the structural-dynamic and functional characteristics of photosynthetic membranes in various purple bacteria

NMR spectra and T₁, T₂ relaxation times for ¹H, ¹³C and ³¹P nuclei in membranes of R. rubrum and Rb. sphaeroides recorded at different relative humidity, as well as hydration curves and electron transfer efficiency of these membranes and membranes of E. shaposhnikovii, reveal complicated relations between structural-dynamic and functional characteristics. A number of sites of the electron transfer chain are shown to be under the control of structural-dynamic mechanisms. Different parameters characterizing these membranes at low humidity and during hydration have been established. These findings and analysis of the data from model systems reveal four different stages of hydration. Each of them is associated with specific changes in structure, dynamics, and function of photosynthetic membranes and their components. In the first stage the hydration of some polar groups leads to local changes in the dynamics of the protein component and this influences the recombination between photoactive pigment P and intermediate acceptor Q_A. The second stage is induced by incorporation of water molecules into the hydrogen bonds between the polar head groups of the lipids and within macromolecules. This results in changes of the dynamics of the membranes, the efficiency of the electron transfer between the quinones and the efficiency of photooxidation of cytochrome c. In the third stage all polar groups are hydrated owing to the appearance of free water with a high dielectric constant. This makes possible lateral mobility of membrane components and changes in distances between the interacting macromolecular components. Therefore, the regulation of photosynthetic processes can be mediated with the participation of mobile carriers. Finally, in the fourth stage, complete humidification provides conditions for regulation of photosynthesis at the cell level. The mechanisms influencing these processes and the efficiency and regulation of electron transfer in various parts of the photosynthetic chain are discussed. Received: 1 August 1996 / Accepted: 4 July 1997     

NMR of fd coat protein

The conformations of the major coat protein of a filamentous bacteriophage can be described by nuclear magnetic resonance spectroscopy of the protein and the virus. The NMR experiments involve detection of the ¹³C and ¹H nuclei of the coat protein. Both the ¹³C and ¹H nuclear magnetic resonance (NMR) spectra show that regions of the polypeptide chain have substantially more motion than a typical globular protein. The fd coat protein was purified by gel chromatography of the SDS solubilized virus. Natural abundance ¹³C NMR spectra at 38 MHz resolve all of the nonprotonated aromatic carbons from the three phenylalanines, two tyrosines, and one tryptophan of the coat protein. The α carbons of the coat protein show at least two different classes of relaxation behavior, indicative of substantial variation in the motion of the backbone carbons in contrast to the rigidity of the α carbons of globular proteins. The ¹H spectrum at 360 MHz shows all of the aromatic carbons and many of the amide protons. Titration of a ¹H spectra gives the pKas for the tyrosines.     

Regulation of Intracellular pH in Guinea Pig Cerebral Cortex Ex Vivo Studied by 31P and 1H Nuclear Magnetic Resonance Spectroscopy: Role of Extracellular Bicarbonate and Chloride

Abstract: The role of transmembrane processes that are dependent on external anions in the regulation of cerebral intracellular pH (pH_i), high-energy metabolites, and lactate was investigated using ³¹P and ¹H NMR spectroscopy in an ex vivo brain slice preparation. During oxygenated superfusion, removal of external HCO₃^?/CO₂ in the presence of Na⁺ led to a sustained split of the inorganic phosphate (P_i) peak so that the pH_i indicated by one part of the peak was 0.38 pH units more alkaline and by the other part 0.10 pH units more acidic at 5 min than in the presence of HCO₃^?. The pH in the compartment with a higher pH_i value returned to 7.29 ± 0.04 by 10.5 min of superfusion in a HCO₃^?-free medium, whereas the pH_i in an acidic compartment was reduced to 7.02. In the presence of 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid or the absence of external Cl^?, removal of HCO₃^? caused alkalinization without split of the P_i peak. Both treatments reduced the rate of pH_i normalization following alkalinization. Simultaneous omission of external HCO₃^? and Na⁺ did not inhibit alkalinization of the pH_i following CO₂ exit. All these data show that the acid loading mechanism at neutral pH_i is mediated by an Na⁺-independent anion transport. During severe hypoxia, pH_i dropped from 7.29 ± 0.05 to 6.13 ± 0.16 and from 7.33 ± 0.03 to 6.67 ± 0.05 in the absence and presence of HCO₃^?, respectively, in Na⁺-containing medium. Lactate accumulated to 18.7 ± 2.8 and 19.6 ± 1.5 mmol/kg under the respective conditions. In the HCO₃^?-free medium supplemented with 1 mM amiloride, the pH_i fell only to 6.94 ± 0.08 despite the lactate concentration of 18.9 ± 2.4 mmol/kg. Acidification caused by hypoxia was also small in the slice preparations superfused in the absence of both HCO₃^? and Cl^?, as the pH_i was 7.01 ± 0.12 at a lactate concentration of 24.5 ± 2.4 mmol/kg. These data indicate that apart from anaerobic glucose metabolism, separate acidifying mechanisms are functioning during hypoxia under these conditions. Recovery of phosphocreatine levels following reoxygenation was >75% relative to the prehypoxic level in the slice preparations superfused in the absence of HCO₃^? but <47% in those preparations superfused without HCO₃^? and Cl^?. This indicates that either neutral pH_i or absence of Cl^? during hypoxia was deleterious to the energy metabolism. The present data indicate that Cl^?/HCO₃^? exchange mechanisms have distinct roles in cerebral H⁺ homeostasis depending on the level of pH_i and energy state.     

Effect of Hypoglycemic Encephalopathy upon Amino Acids, High-Energy Phosphates, and pHi in the Rat Brain In Vivo: Detection by Sequential 1H and 31P NMR Spectroscopy

Metabolic alterations in amino acids, high-energy phosphates, and intracellular pH during and after insulin hypoglycemia in the rat brain was studied in vivo by 1H and 31P nuclear magnetic resonance (NMR) spectroscopy. Sequential accumulations of 1H and 31P spectra were obtained from a double-tuned surface coil positioned over the exposed skull of a rat while the electroencephalogram was recorded continuously. The transition to EEG silence was accompanied by rapid declines in phosphocreatine, nucleoside triphosphate, and an increase in inorganic orthophosphate in 31P spectra. In 1H spectra acquired during the same time interval, the resonances of glutamate and glutamine decreased in intensity while a progressive increase in aspartate was observed. Following glucose administration, glutamate and aspartate returned to control levels (recovery half-time, 8 min); recovery of glutamine was incomplete. An increase in lactate was detected in the 1H spectrum during recovery but it was not associated with any change in the intracellular pH as assessed in the corresponding 31P spectrum. Phosphocreatine returned to control levels following glucose administration, in contrast to nucleoside triphosphate and inorganic orthophosphate which recovered to only 80% and 200% of their control levels, respectively. These results show that the changes in cerebral amino acids and high-energy phosphates detected by alternating the collection of 1H and 31P spectra allow for a detailed assessment of the metabolic response of the hypoglycemic brain in vivo.     

Existence of lipid microdomains in bilayer of dipalmitoyl phosphatidylcholine (DPPC) and 1-stearoyl-2-docosahexenoyl phosphatidylserine (SDPS) and their perturbation by chlorpromazine: a 13C and 31P solid-state NMR study

The polyunsaturated fatty acid docosahexaenoic acid (DHA, 22 : 6, n-3) is found at a level of about 50% in the phospholipids of neuronal tissue membranes and appears to be crucial to human health. Dipalmitoyl phosphatidylcholine (DPPC, 16 : 0/16 : 0 PC) and the DHA containing 1-stearoyl-2-docosahexenoyl phosphatidylserine (SDPS) were used to make DPPC (60%)/SDPS (40%) bilayers with and without 10 mol% chlorpromazine (CPZ), a cationic, amphiphilic phenothiazine.

Resonances that are present in ¹³C NMR spectrum of the DPPC (60%)/SDPS (40%) sample and that disappear in presence of 10% CPZ most probably are due to the special interface environment, e.g. the hydrophobic mismatch, at the interface of DPPC and SDPS microdomains in the DPPC/SDPS bilayer. In itself the appearance of resonances at novel chemical shift values is a clear demonstration of a unique chemical environment in the DPPC (60%)/SDPS (40%) bilayer. The findings of the study presented here suggest CPZ bound to the phosphate of SDPS will slow down and partially inhibit such a DHA acyl chain movement in the DPPC/SDPS bilayer. This would affect the area occupied by a SDPS molecule (in the bilayer) and probably the thickness of the bilayer where SDPS molecules reside as well. It is quite likely that such CPZ caused changes can affect the function of proteins embedded in the bilayer.     

蟾蜍甾烯14β-artebufogenin的核磁共振光谱信号全归属

本论文利用柱色谱法从中药蟾酥中分离得到一个蟾蜍甾烯化合物,利用二维核磁共振和其它化学及光谱学手段对其结构进行了鉴定,并首次给出了其完整的核磁信号归属.