2H2O Media Research Articles

Deuteration effects on the in vivo EPR spectrum of the reduced secondary photosystem I electron acceptor A1 in cyanobacteria.

The photoreduction of the secondary PSI electron acceptor A1 in vivo has recently been detected via X-band EPR spectroscopy in intact spinach chloroplasts and in marine cyanobacteria Synechococcus PCC 7002 [Klughammer, C., and Pace, R. J. (1997) Biochim. Biophys. Acta 1318, 133-144]. A further study of the A1- EPR spectrum of Synechococcus PCC 7002 at room temperature with higher-field resolution revealed partially resolved hyperfine structure which was dominated by 0.4 mT splittings of three equivalent protons. The hyperfine splitting was not significantly affected by incubation of the cyanobacteria in 2H2O medium for 20 h, but was absent in fully deuterated cyanobacteria that were grown in 2H2O medium. Anisotropic g-factors consistent with a phylloquinone radical were derived by spectra simulation. Biosynthetic protonation of quinones via the CH3 donor L-methionine in deuterated cells maintained hyperfine structure in the A1- spectrum, indicating the incorporation of CH3 groups in 60% of the deuterated, photoactive A1 molecules. Conversely, biosynthetic quinone deuteration via L-[methyl-d3]methionine in protonated cells led to the loss of the 0. 4 mT splittings in 54% of the A1 molecules. These observations confirm the conclusion of Heathcote et al. [(1996) Biochemistry 35, 6644-6650] of the identity of EPR-detected, photoreduced A1- in vivo with a phylloquinone (vitamin K1) radical in PSI. The partially resolved hyperfine structure of the A1- spectrum indicates an altered spin distribution in the bound vitamin K1- radical in vivo compared to that of unbound vitamin K1- in vitro.

Development of an Isotope Dilution Assay for Precise Determination of Insulin, C-peptide, and Proinsulin Levels in Non-diabetic and Type II Diabetic Individuals with Comparison to Immunoassay

We describe the application of a stable isotope dilution assay (IDA) to determine precise insulin, C-peptide, and proinsulin levels in blood by extraction from serum and quantitation by mass spectrometry using analogues of each target protein labeled with stable isotopes. Insulin and C-peptide levels were also determined by immunoassay, which gave consistently higher results than by IDA, the relative difference being larger at low concentrations. Insulin, C-peptide, and proinsulin levels were all shown by IDA to be higher in type II diabetics than in non-diabetics, with mean values rising from 22 (+/- 2) to 92 (+/- 8), 335 (+/- 11) to 821 (+/- 24), and 6 (+/- 1) to 37 (+/- 3) pM, respectively. Interestingly, the ratio between IDA and immunoassay values for insulin levels increased from 1.3 in non-diabetics to 1.7 in type II diabetics. The ratio between proinsulin and insulin levels by IDA increased from 0.24 in non-diabetics to 0.36 in type II diabetics, whereas the ratio between C-peptide and insulin levels by IDA decreased from 17.6 to 10.7. This disproportionate change in protein levels between different types of individuals has implications for the metabolism of insulin in the diabetics studied (type II) and suggests that C-peptide levels are not always a reliable guide as to pancreatic insulin secretion. In addition, levels of the 33-residue C-peptide (partially trimmed form) were shown to be less than 10% that of the fully trimmed 31-residue C-peptide levels, and we tested IDA in a clinical context by two post-pancreatic graft studies. IDA was shown to give direct, positive identification of the target protein with unrivaled accuracy, avoiding many of the problems associated with present methodology for protein determination.

Small-angle neutron scattering from the reconstituted TF 1 of H +-ATPase from thermophilic bacterium PS3 with deuterated subunits

Subunits alpha, beta and gamma of adenosine triphosphatase (H(+)-ATPase) from the thermophilic bacterium PS3 (TF1) have been over-expressed in Escherichia coli. alpha and beta subunits deuterated to the level of 90% were obtained by culturing E. coli in 2H2O medium. Both the subunits and the reconstituted alpha beta gamma complex, TF1, which contain the deuterated components in various combinations, were studied in solution by small-angle neutron scattering. The individual shapes of the subunits and their organization in the alpha beta gamma-TF1 complex were examined using the techniques of selective deuteration and contrast variation. The alpha and beta subunits are well approximated as ellipsoids of revolution having minor semi-axes of 20.4(+/- 0.4) and 20.0(+/- 0.2) A, and major semi-axes of 53.0(+/- 1.4) and 55.8(+/- 0.9) A, respectively. In the TF1 complex, three beta subunits are aligned to form an equilateral triangle, with their major axes tilted by 35 degrees with respect to the 3-fold axis of the complex. The beta-beta distance is about 53 A. Three alpha subunits are similarly arranged, positioned between the beta subunits, and with their direction of tilt opposite to that of the beta subunits. The centers of the alpha and beta subunits lie in the same plane, forming a hexagon. Adjacent subunits overlap in this model, suggesting that they are not simple ellipsoids of revolution.

Pressure effects on sacroplasmic reticulum: a Fourier transform infrared spectroscpic study

The Ca 2+-ATPase of sacroplasmic reticulum is irreversibly inactivated by exposure to 1.5–2.0 kbar pressure for 30–60 min in a Ca 2+-free medium; mono- or decavanadate (5 mM) or to a lesser extent Ca 2+ (2–20 mM) protect against inactivation (Varga et al. (1986) J. Biol. Chem. 261, 13943–13956). The structural basis of these effects was analyzed by FTIR spectroscopy of sarcoplasmic reticulum in 2H 2O medium. The inactivation of the Ca 2+-ATPase at 1.5–2.0 kbar pressure in a Ca 2+-free medium was accompanied by changes in the Amide II region of the spectrum (1550 cm −1), that are consistent with increased hydrogen-deuterium (H- 2H) exchange, and by the enhancement of a band at 1630 cm −1 in the Amide I region, that is attributed to an increase in β sheet. The frequency of the peak of the Amide 1 band shifted from about 1648 cm −1 at atmospheric pressure to 1642 cm −1 at ≅ 12.5 kbar pressure, suggesting a decrease in α helix, and an increase in β and/or random coil structures. Upon releasing the pressure, the shift of the Amide I band was partially reversed. Vanadate (5 mM), and to a lesser extent Ca 2+ (2–20 mM), protected the Ca 2+-ATPase against pressure-induced changes both in the Amide I and Amide II regions of the spectrum, together with the protection of ATPase activity. These observations establish a correlation between the conformation of the Ca 2+-ATPase and its sensitivity to pressure. The involvement of the ATP binding domain of the Ca 2+-ATPase in the pressure-induced structural changes is suggested by the decreased polarization of fluorescence of fluorescein 5′-isothiocyanate covalently attached to the enzyme.

Infrared spectroscopic studies of detergent‐solubilized uncoupling protein from brown‐adipose‐tissue mitochondria

The uncoupling protein of brown-adipose-tissue mitochondria has been purified in the form of mixed micelles with lipid and reduced Triton X-100. This surfactant has the advantage over conventional Triton X-100, that it does not interfere with amide bands in infrared spectra. The structure of the uncoupling protein in micellar form has been examined by Fourier-transform infrared spectroscopy (FTIR). In order to decompose the amide I contour into its components, band-narrowing (Fourier derivation and deconvolution) and band-decomposition techniques have been used. Combining data from spectra taken in H2O and 2H2O media, the following percentage distribution of secondary structure patterns has been obtained: 50% alpha-helix, 28-30% beta-structure; 13-15% beta-turns and 7% unordered. Thermal denaturation of the uncoupling protein has also been monitored by FTIR. In accordance with previous observations of different proteins, thermal denaturation is marked by a shift in the amide I maximum and the appearance of two new peaks in 2H2O, at around 1620 cm-1 and 1685 cm-1. Denaturation occurs in the 40-50 degrees C temperature range, in agreement with studies of GDP-binding capacity. Cooling down the thermally denatured protein produces a new change in its secondary structure; however, the original conformation is not restored. The uncoupling protein possesses a nucleotide-binding site. On addition of GDP, small changes in protein conformation occur, attributable to changes in tertiary structure. However, no detectable effects are seen in the presence or absence of the other physiological regulators, the free fatty acids. The uncoupling protein shares important similarities in its primary structure with other anion carriers of the mitochondrial membrane; one of these, the adenine-nucleotide translocator, has been used in a comparative study, applying the same FTIR techniques described above for the uncoupling protein. Both proteins have a similar proportion of alpha-helix, probably corresponding to the segments spanning the membrane, but the conformation of the polar domains appears to differ.

A proton nuclear magnetic resonance study of gamma-glutamyl-amino acid cyclotransferase in human erythrocytes.

Spin-echo NMR spectroscopy was shown to be a reliable technique for the monitoring of the in situ cleavage of gamma-Glu-Ala by gamma-glutamyl-amino acid cyclotransferase in whole erythrocytes and hemolysates. Of particular importance was the difference in chemical shifts between peptide resonances and those of the constituent amino acids. Using lysates of varying dilution, it was shown that the specific activity of the enzyme was not concentration-dependent, thus suggesting a lack of cytosolic low-molecular-weight-effectors or enzyme dissociation. Furthermore, the initial velocities of the reaction as a function of substrate concentration obeyed Michaelis-Menten kinetics with a Km = 2.0 +/- 0.3 mmol/l and Vmax = 137 +/- 7 mmol/h/l of cell water in 1H2O medium. Similar analysis in 2H2O medium revealed a solvent kinetic isotope effect of 1.9 +/- 0.4 at low substrate concentrations. The implications of this observation for the mechanism of the reaction are discussed. Cleavage of the peptide by a suspension of intact erythrocytes was at a rate 300 times less than the corresponding lysate flux, thus indicating the rate limitation by transport in the coupled system.

Arachidoyl- and arachidonoyl-coa elongation mechanism in swine cerebral microsomes

Long-chain saturated and polyunsaturated fatty acyl-CoA elongations were studied in swine cerebral microsomes. The elongation of endogenous palmitoyl-CoA to stearate was highly active in both cerebral and liver microsomes, whereas those of arachidoyl-CoA (20:0-CoA) and endogenous arachidonoyl-CoA (20:4-CoA) were high in cerebral microsomes, but negligible in liver microsomes. The elongation of 22:4 to 24:4 was also observed in cerebral microsomes. Both NADPH and NADH at 500 μM were effective in elongation of 16:0-, 20:0- and 20:4-CoA, whereas NADPH was more effective in elongation of 22:4 to 24:4 than NADH. The incorporation of deuterium atoms to the elongated product was detected by the technique of mass fragmentography when the NADPH-dependent elongations of 20:0-CoA and 20:4-CoA were performed 2H 2O medium upon cerebral microsomes. The number of incorporated deuterium atoms into 22:0 elongated from 20:0-CoA was mainly two, and that into 22:4 elongated from 20:4-CoA was mainly three. These results indicated that part of hydrogens in elongated arachidoyl- and arachidonoyl-CoA were transferred from NADPH.

Neutron diffraction studies of oriented photosynthetic membranes

The structures of photosynthetic membranes from chloroplasts of Euglena gracilis and spinach were studied by neutron diffraction from oriented specimens. Orientation of the membranes was achieved in two different ways. (1) Centrifugation and controlled drying; (2) by applying a magnetic field to aqueous suspensions. The low-resolution measurements with different H 2O/ 2H 2O mixtures (contrast variation) support a previously proposed model for the membrane structure, in which the proteins are associated with a compact lipid layer. Membrane arrangements within the chloroplasts, such as membrane pairing, were characterized for several membrane preparations and ionic environments, including one very close to that present “ in vivo”. These membrane arrangements correspond to the presence of grana structure (close contact between exterior faces of membrane sacs) and to different water contents of the sac interior. From the neutron scattering intensity, it is demonstrated that the partition region of the grana structure is hydrophilic. The pair correlation function, calculated directly from the scattering intensity in 2H 2O medium, clearly shows two different separations of membranes within the chloroplast, and gives the disorder of these separations. The scattering is predominantly lamellar, but there is a small contribution from the arrangement of proteins in the plane of the membrane. Degrees of membrane orientation in the magnetic field are measured and related (Appendix) to the optical dichroism measurements of Breton, Michel-Villay & Paillotin.

Further investigation on the lateral and transversal proton currents at the thylakoid membrane level by hydrogen-deuterium exchange

Energetically-coupled processes (electron flow, proton uptake and correlated pH gradient) were investigated on envelope-free chloroplasts of lettuce suspended in 1H 2O or 2H 2O media. Study of the light-intensity and temperature dependencies of these phenomena led to the following observations: 1. At neutral pH, 2H 2O diminishes the transmembrane H + gradient in strong light (chain Photosystem II + Photosystem I) but not in low light; the total H + uptake is increased at all light intensities: the buffering capacity of the inner compartment is increased in heavy water, possibly through enhancement of interactions between membranous titrable groups and the aqueous phase. 2. 2H 2O does not affect the photochemical events of the redox chain, whatever the electron pathway (PSII, PSI or PSII + PSI): only thermal steps are inhibited. The diminution of the apparent quantum yield, sometimes observed, may be ascribed to the dual site of action of the artificial redox carrier (ferricyanide) then used. 3. 2H 2O does not modify the activation energy of the limiting step of the electron flow (PSII + PSI) in uncoupled (44 vs. 47 kJ · mol −1) or — but less clearly — in coupled, i.e., ‘basal’, state (55 vs. 59 kJ · mol −1). 2H 2O does not either change the temperature of the phase transition of the membrane (17°C) for the uncoupled flow. However, a low-temperature transition, observed only for the coupled chain, is slightly increased by 2H 2O; this thermal transition is attributed to the freezing of some bound water near the plastoquinone pool. 4. Δp 2H is smaller than Δp 1H at all temperatures (PSII + PSI chain). ΔpH is quasi-constant from 0°C to 10°C, then decreases when temperature rises. 2H 2O does not change the activation energy of the dark passive H + efflux, which is almost twice that of the coupled electron flow. The phase transition at low temperature suggests that the proton efflux occurs via two parallel pathways, one temperature-dependent and the other temperature-independent. Except for the increase of the internal buffering capacity, the effects of 2H 2O on the membrane conformation seem limited, as shown by the unchanged activation energies of the electron flow and of the H + leakage. The null activation energy observed at low temperature emphasizes the role of the bound water in these processes; however, the different effects of 2H 2O on the transition temperatures indicate that this bound water has different properties when associated with the translocation sites or with the H + leakage ones. This ‘microcompartmentation’ of the membranes is consistent with the concept of lateral pH heterogeneity we have previously suggested (de Kouchkovsky, Y., and Haraux, F. (1981) Biochem. Biophys. Res. Commun. 99, 205–212). The theoretical computations and the experimental results suggest that in the steady state, the internal pH would be several tenths of a ‘unit’ lower near the plastoquinones than near the H + efflux sites (coupling factors); this difference would be increased when 2H + replaces 1H +, owing to the lower mobility of the deuteron. It is concluded that local, and not average, pH (and ΔpH) should be considered for the understanding of the energy transduction processes.

Mechanistic studies of carboxypeptidase Y from Saccharomyces cerevisiae. pH and pD profiles and inactivation at low pH (pD) values.

Steady-state kinetics of carboxypeptidase Y, a proteinase from yeast, were studied by using the reaction of 4-nitrophenyl trimethylacetate as a probe. The pH profile of kcat. is sigmoidal in H2O-based buffers for the carboxypeptidase Y-catalysed hydrolysis of this ester (kcat. referring to the rate of deacylation of trimethylacetyl-carboxypeptidase Y). The corresponding pD profile in 2H2O is doubly sigmoidal, with inflexions at pD approximately 3.8 and approximately 6.8. The ionization of pKDapp. approximately 3.8 is caused by a rapid inactivation in 2H2O media by a process that is only slowly reversed on transfer to pH 7.00 phosphate buffer in H2O. The corresponding inactivation in H2O-based buffers of low pH is considerably slower (approximately 30-fold), follows a first-order rate-dependence and is very strongly pH-dependent, indicating some form of co-operative change in enzyme tertiary structure.

The rate of increase in fluorescence intensity caused by deuteration of tryptophan

Tryptophan was rapidly transferred from 1H 2O into a 2H 2O medium by a stopped-flow device, and a time-dependent fluorescence increase was traced. It has been shown that this fluorescence change is caused by the deuteration of the α-ammonium (—NH + 3) group.

Resonance raman studies of a c type algal cytochrome deuterium shifts and a comparison with mammalian cytochrome c

A c type cytochrome isolated from Synechococcus lividus grown on water and 2H 2O media, has been studied by resonance Raman spectroscopy. The spectra were taken on the oxidized and reduced protein with excitation within the Soret band at 441.6 nm to determine whether individual resonance Raman bands of the heme shift upon deuterium substitution and also to provide a comparison with the spectra of horse heart cytochrome c. Some of the shifts observed with the deuterated heme c are larger than the corresponding shifts in meso-deuterated metalloporphyrins suggesting mixing of peripheral substituent vibrations with the skeletal modes of the porphyrin macrocycle. The algal cytochrome exhibits resonance Raman spectra roughly similar to those of horse heart cytochrome c, consistent with its optical absorption spectra which is typical of c type cytochromes, although a detailed comparison reveals note-worthy differences between the spectra of the two proteins; this may be a reflection of the effect of non-methionine ligands and protein environment of the vibrations of the c type heme in the algal cytochrome.

Essential histidyl residues of octopine dehydrogenase.

Octopine dehydrogenase from muscles of Pecten maximus has been treated by diethylpyrocarbonate at pH 6.0. The enzyme was completely inhibited when 2 histidyl residues were carbethoxylated. In the same conditions rabbit muscle lactate dehydrogenase looses its activity when 12 histidines are modified. The carbethoxylation of both enzymes does not follow a simple first order kinetics. The specificity of the reaction of diethylpyrocarbonate with histidyl residues has been confirmed by the reversal of the inhibition by hydroxylamine, by the lack of reaction with tyrosines and cysteines and by the absence of important structural modifications. The carbethoxylated enzyme gave a normal binary complex with the coenzyme, but the ternary complex formation with substrate analogues is disturbed. The coenzymes or the substrates used separately do not protect the enzyme against diethylpyrocarbonate. In the ternary complexes the reaction with the inhibitor is hindered. The study of the pH effect on the Km of octopine has been made in H2O and 2H2O. One of the downward bends of the curve indicates a group in the free enzyme with a pK of 6.1, which is shifted to 6.6 in 2H2O medium. This confirms the involvement of histidyl residues in the reaction catalysed by octopine dehydrogenase. The number of essential histidines in octopine dehydrogenase is discussed.

Isotope biology of 13C extensive incorporation of highly enriched 13C in the alga Chlorella vulgaris

In contribution of previous work on the consequences of isotopic replacement in living organisms 1, the green alga Chlorella vulgaris has been grown on 13CO 2 (95% 13C) in 1H 2O and 2H 2O media. Cell size distribution was determined and the organisms subjected to cytochemical investigation for nucleic acids, carbohydrates, amino acids, and proteins. Some of the abnormalities noted in 13C 2H organisms are diminished in 13C 2H algae, indicating that the consequences of multiple isotope replacement in living organisms do not necessarily follow a simple additive relationship.

Heavy water ( 2H 2O) as solvent for incubation media in vitro: Effects on two tissues exhibiting significant water shifts: Mammalian cerebral cortex and etiolated pea seedlings

Apical stem segments from 7-day-old etiolated pea seedings when incubated in vitro in the presence of 10 −6 M indolyl-3-acetic acid (IAA) normally exhinit significant elongation and a significantly greater (3-fold) imbibition of fluid than control stem segments. In the presence of an incubation medium made up with 2H 2O (85%, v/v) as solvent, these effects of IAA are completely absent. Swelling of slices of cat cerebral cortex normally encountered upon incubation in vitro was unaffected by incubation in media made up with 2H 2O (99%, v/v) as solvent except for: (1) slices from neonatal kittens, normally resistant to swelling in vitro, where 16% swelling was encountered; and (2) slices from adult cats when incubated in 2H 2O media containing 5 mM K +, where a 2-fold increase in swelling was encountered. In the latter case, tissue fluid spaces accessible to sucrose and to chloride did not change proportionately, suggesting an intracellular locus for the extra fluid of swelling. Contents of electrolytes in control and 2H 2O-treated slices were similar except for K +, which was significantly elevated (+ 18–24 μmoles/g) in slices incubated in 2H 2O media containing either 5 mM or 27 mM K +. The findings are discussed in terms of known effects of 2H 2O on various biological systems, including plant tissues and the mammalian nervous system.

Rate of mutation to phage resistance in 2H 2O medium

The rate of mutation of Escherichia coli B to T1 and T2 phage resistance in 2H 2O medium was compared to the mutation rate in H 2O medium to determine whether the tunneling of a proton from one base to another can account for mutations. The results suggest that proton tunneling, if it causes mutants, is rare when compared with other mechanisms which are responsible for forming mutants.