400 MHz two-dimensional nuclear Overhauser spectroscopy on anesthetic interaction with lipid bilayer

Interaction between a volatile anesthetic, methoxyflurane, and dipalmitoylphosphatidylcholine (DPPC) vesicle membrane was analyzed by nuclear Overhauser effect (NOE) difference spectroscopy and two-dimensional nuclear Overhauser spectroscopy (NOESY). The NOE difference spectra were obtained by selectively irradiating methoxy protons (hydrophobic end) of the anesthetic: a negative nuclear Overhauser effect of -2.94% was observed with the choline methyl protons of DPPC. The NOESY spectra revealed a cross-peak between the anesthetic methoxy protons and the choline methyl protons. A dipole-dipole interaction exists between the hydrophobic end of the anesthetic and the hydrophilic head group of DPPC. No other cross-peaks were observed. The anesthetic orients itself at the membrane/water interface by interacting with the hydrophilic surface of the DPPC membrane, leaving the hydrophilic end of the anesthetic molecule in the aqueous phase. The preferred residence site of dipolar volatile anesthetics is the membrane/water interface.     

Insulin-degrading enzyme is capable of degrading receptor-bound insulin

In the investigation of the intracellular sites of insulin degradation, it might be important whether receptor-bound insulin could be a substrate for insulin-degrading enzyme (IDE). Insulin receptor and IDE were purified from rat liver using a wheat germ agglutinin column and monoclonal anti-IDE antibody affinity column, respectively. [125I]insulin-receptor complex was incubated with various amounts of IDE at 0 degree C in the presence of disuccinimidyl suberate and analyzed by reduced 7.5% SDS-PAGE and autoradiography. With increasing amounts of IDE, the radioactivity of 135 kd band (insulin receptor alpha-subunit) decreased, whereas that of 110 kd band (IDE) appeared then gradually increased, suggesting that IDE could bind to receptor-bound insulin. During incubation of insulin-receptor complex with IDE at 37 degrees C, about half of the [125I]insulin was dissociated from the complex. However, the time course of [125I]insulin degradation in this incubation was essentially identical to that of free [125I]insulin degradation. Cross-linked, non-dissociable receptor-bound [125I]insulin was also degraded by IDE. Rebinding studies to IM-9 cells showed that the receptor binding activity of dissociated [125I]insulin from insulin-receptor complex incubated with IDE was significantly (p less than 0.001) decreased as compared with that without the enzyme. These results, therefore, show that IDE could recognize and degrade receptor-bound insulin, and suggest that IDE may be involved in insulin metabolism during receptor-mediated endocytosis through the degradation of receptor-bound insulin in early neutral vesicles before their internal pH is acidified.     

Covalent linkage of 125I-insulin to a cytosolic insulin-degrading enzyme

Cytosol extracts high in insulin-degrading activity were cross-linked to 125I-insulin with the bifunctional cross-linker disuccinimidyl suberate. With cytosols from either rat muscle, liver, kidney or brain or human erythrocytes, only a single protein (Mr = 110,000) was specifically labeled. Three different lines of evidence indicated that this labeled protein is insulin-degrading enzyme, a cysteine protease which accounts for most of the insulin-degrading activity in cell extracts. Firstly, the cross-linking of 125I-insulin to this protein is inhibited by unlabeled insulin over the same concentration range of insulin which inhibits degradation. Separated insulin A and B chain were less potent at inhibiting cross-linking, whereas bovine serum albumin and cytochrome c were without effect. Secondly, antibodies to purified insulin-degrading enzyme precipitated the labeled protein in parallel with their ability to precipitate the insulin-degrading activity of the extracts. Thirdly, when the insulin-degrading activity was purified 40,000-fold from erythrocytes, this Mr 110,000 protein co-purified. These results indicate that cross-linking 125I-insulin may be a convenient method for labeling the insulin-degrading enzyme.     

Interfacial preference of anesthetic action upon the phase transition of phospholipid bilayers and partition equilibrium of inhalation anesthetics between membrane and deuterium oxide

The half-height linewidth (v 1/2) of the 1H-NMR spectra of dipalmitoylphosphatidylcholine vesicles changes abruptly at the phase transition temperature. In the absence of inhalation anesthetics, proton signals from the choline head group (hydrophilic interface) and acyl-chain tails (lipid core) change at the same temperature of 39.6 degrees C. The present study compared the effect of four inhalation anesthetics, i.e., methoxyflurane, chloroform, halothane and enflurane, upon the ligand-induced phase transition of phosphatidylcholine vesicle membranes at 37 degrees C. The anesthetics showed differential action upon the phase transition of the phospholipid vesicle membranes between the lipid core and the hydrophilic interface. The concentrations of anesthetics which induced the phase transition of the lipid core were about 2-fold greater than those required for the phase transition of the interfacial choline head groups. From the area under the proton signals of inhalation anesthetics in the NMR spectra, the maximum solubilities of methoxyflurane, chloroform and halothane in 2H2O at 37 degrees C were determined to be 0.671 . 10(-4), 2.637 . 10(-4) and 1.398 . 10(-4) (expressed as mole fractions), or 3.35, 13.17 and 6.98 mmol/1000 g 2H2O, respectively. The solubilities of the anesthetic vapor in 2H2O expressed as mole fractions according to Henry's law ere 9.586 . 10(-4), 6.432 . 10(-4) and 2.311 10(-4)/atm (1.013 . 10(5) Pa) partial pressure, respectively. The presence of phospholipid vesicles in 2H2O increased the solubility of the inhalation anesthetics. From difference between solubility in 2H2O and a dipalmitoylphosphatidylcholine vesicle suspension, the partition coefficients of methoxyflurane, chloroform and halothane between the phospholipid vesicle membranes and 2H2O were estimated. These values, calculated from the mole fractions, were 3364, 1660 and 3850, respectively at 37 degrees C.     

Basic pH-induced modification of excitation-energy dynamics in fucoxanthin chlorophyll a/c-binding proteins isolated from a pinguiophyte,Glossomastix chrysoplasta

Photosynthetic organisms have diversified light-harvesting complexes (LHCs) to collect solar energy efficiently, leading to an acquisition of their ecological niches. Herein we report on biochemical and spectroscopic characterizations of fucoxanthin chlorophyll a/c-binding protein (FCP) complexes isolated from a marine pinguiophyte Glossomastix chrysoplasta. The pinguiophyte FCP showed one subunit band in SDS-PAGE and one protein-complex band with a molecular weight at around 66 kDa in clear-native PAGE. By HPLC analysis, the FCP possesses chlorophylls a and c, fucoxanthin, and violaxanthin. To clarify excitation-energy-relaxation processes in the FCP, we measured time-resolved fluorescence spectra at 77 K of the FCP adapted to pH 5.0, 6.5, and 8.0. Fluorescence curves measured at pH 5.0 and 8.0 showed shorter lifetime components compared with those at pH 6.5. The rapid decay components at pH 5.0 and 8.0 are unveiled by fluorescence decay-associated (FDA) spectra; fluorescence decays occur in the 270 and 160-ps FDA spectra only at pH 5.0 and 8.0, respectively. In addition, energy-transfer pathways with time constants of tens of picoseconds are altered under the basic pH condition but not the acidic pH condition. These findings provide novel insights into pH-dependent energy-transfer and energy-quenching machinery in not only FCP family but also photosynthetic LHCs.     

Regulation of excitation energy in Nannochloropsis photosystem II

Photosynthesis Research - Recently, we isolated a complex consisting of photosystem II (PSII) and light-harvesting complexes (LHCs) from Nannochloropsis granulata (Umetani et al. Photosynth Res...     

Alterations in photosynthetic pigments and amino acid composition of D1 protein change energy distribution in photosystem II

The marine cyanobacterium Prochlorococcus marinus accumulates divinyl chlorophylls instead of monovinyl chlorophylls to harvest light energy. As well as this difference in its chromophore composition, some amino acid residues in its photosystem II D1 protein were different from the conserved amino acid residues in other photosynthetic organisms. We examined PSII complexes isolated from mutants of Synechocystis sp. PCC 6803, in which chromophore and D1 protein were altered (Hisashi Ito and Ayumi Tanaka, 2011) to clarify the effects of chromophores/D1 protein composition on the excitation energy distribution. We prepared the mutants accumulating divinyl chlorophyll (DV mutant). The amino acid residues of V205 and G282 in the D1 protein were substituted with M205 and C282 in the DV mutant to mimic Prochlorococcus D1 protein (DV-V205M/G282C mutant). Isolated PSII complexes were analyzed by time-resolved fluorescence spectroscopy. Energy transfer in CP47 was interrupted in PSII containing divinyl chlorophylls. The V205M/G282C mutation did not recover the energy transfer pathway in CP47, instead, the mutation allowed the excitation energy transfer from CP43 to CP47, which neighbors in the PSII dimer. Mutual orientation of the subcomplexes of PSII might be affected by the substitution. The changes of the energy transfer pathways would reduce energy transfer from antennae to the PSII reaction center, and allow Prochlorococcus to acquire light tolerance.     

Excitation energy relaxation in a symbiotic cyanobacterium, Prochloron didemni, occurring in coral-reef ascidians, and in a free-living cyanobacterium, Prochlorothrix hollandica

The marine cyanobacterium Prochloron is a unique photosynthetic organism that lives in obligate symbiosis with colonial ascidians. We compared Prochloron harbored in four different host species and cultured Prochlorothrix by means of spectroscopic measurements, including time-resolved fluorescence, to investigate host-induced differences in light-harvesting strategies between the cyanobacteria. The light-harvesting efficiency of photosystems including antenna Pcb, PS II-PS I connection, and pigment status, especially that of PS I Red Chls, were different among the four samples. We also discuss relationships between these observed characteristics and the light conditions, to which Prochloron cells are exposed, influenced by distribution pattern in the host colonies, presence or absence of tunic spicules, and microenvironments within the ascidians' habitat.     

A strong exonic splicing enhancer in dystrophin exon 19 achieve proper splicing without an upstream polypyrimidine tract

Proper splicing is known to proceed under the control of conserved cis-elements located at exon-intron boundaries. Recently, it was shown that additional elements, such as exonic splicing enhancers (ESEs), are essential for the proper splicing of certain exons, in addition to the splice donor and acceptor site sequences; however, the relationship between these cis-elements is still unclear. In this report, we utilize dystrophin exon 19 to analyse the relationship between the ESE and its upstream acceptor site sequences. Dystrophin exon 19, which maintains adequate splicing donor and acceptor consensus sequences, encodes exonic splicing enhancer (dys-ESE19) sequences. Splice pattern analysis, using a minigene reporter expressed in HeLa cells, showed that either a strong polypyrimidine tract (PPT) or a fully active dys-ESE19 is sufficient for proper splicing. Each of these two cis-elements has enough activity for proper exon 19 splicing suggesting that the PPT, which is believed to be an essential cis-element for splicing, is dispensable when the downstream exon contains a strong ESE. This compensation was only seen in living cells but not in 'in vitro splicing'. This suggests the possibility that the previous splicing experiments using an in vitro splicing system could underestimate the activity of ESEs.     

Solvent effects on excitation relaxation dynamics of a keto-carotenoid, siphonaxanthin

Solvent effects on relaxation dynamics of a keto-carotenoid, siphonaxanthin, were investigated by means of the femtosecond time-resolved fluorescence spectroscopy. After excitation to the S2 state of siphonaxanthin, the S2-->1(n, pi*) internal conversion occurred with a time constant of 30-35 fs, followed by the 1(n, pi*)-->S1 internal conversion in 180-200 fs. Solvent dependence of the internal conversions was small, however intensities of the S1 fluorescence with its lifetime of longer than 10 ps were enhanced in methanol. These were explained by displacement of the potential surfaces and interaction through the hydrogen-bond between the C=O group of siphonaxanthin and solvents.