The effects of deuterium oxide (2H2O) on the polymerization of tubulin in vitro

The initial rate and final extent of polymerization of both bovine brain tubulin and sea urchin egg tubulin were enhanced in the presence of ²H₂O. The yields were increased in association with the elevation of the ²H₂O concentration. ²H₂O also reduced the critical concentration for polymerization of brain tubulin. Thermodynamic analysis was attempted using the temperature dependence of the critical concentration for polymerization in the presence of ²H₂O. We obtained linear van 't Hoff plots and calculated thermodynamic parameters which were positive and were increased with the elevation of the ²H₂O concentration. The enhancement of the polymerization of tubulin by ²H₂O could, therefore, be the result of the strenghening of intra-and/or inter-molecular hydrophobic interactions of the tubulin molecules. We believe that the increase in lenghth and number of microtubules of the mitotic spindles in the dividing cells of the eukaryotes with ²H₂O may be caused by the direct involvement of ²H₂O in the polymerization of tubulin.     

Kinetic β-deuterium isotope effects suggest a covalent mechanism for the protein folding enzyme peptidylprolyl cis/trans-isomerase

The cis/trans interconversion of Glt-Ala-Ala-Pro-Phe-4-nitroanilide and Glt-Ala-Gly-Pro-Phe-4-nitroanilide was studied both enzymatically and nonenzymatically by measuring kinetic β-deuterium isotope effects. The hydrogen atom at the α-carbon atom of the Xaa residue within the Xaa-Pro moiety was substituted by deuterium. In the nonenzymatic case the transition state of rotation is reflected by k_H/k_D > 1. When catalysed by 17 kDa PPIase the same bond rotation is characterized by k_H/k_D < 1. This suggests a covalent mechanism of catalysis which involves an approximately tetravalent carbon of the prolyl imidic bond for the transition state of reaction.     

The use of isotope tracers for identifying populations of migratory birds

 To determine whether stable isotopes can be used for identifying the geographic origins of migratory bird populations, we examined the isotopic composition of hydrogen (deuterium, δD), carbon (δ¹³C), and strontium (δ⁸⁷Sr) in tissues of a migratory passerine, the black-throated blue warbler (Dendroica caerulescens), throughout its breeding range in eastern North America. δD and δ¹³C values in feathers, which are grown in the breeding area, varied systematically along a latitudinal gradient, being highest in samples from the southern end of the species’ breeding range in Georgia and lowest in southern Canada. In addition, δD decreased from east to west across the northern part of the breeding range, from New Brunswick to Michigan. δ⁸⁷Sr ratios were highest in the Appalachian Mountains, and decreased towards the west. These patterns are consistent with geographical variation in the isotopic composition of the natural environment, i.e., with that of precipitation, plants, and soils for δD, δ¹³C, and δ⁸⁷Sr, respectively. Preliminary analyses of the δD and δ¹³C composition of feathers collected from warblers in their Caribbean winter grounds indicate that these individuals were mostly from northern breeding populations. Furthermore, variances in isotope ratios in samples from local areas in winter tended to be larger than those in summer, suggesting that individuals from different breeding localities may mix in winter habitats. These isotope markers, therefore, have the potential for locating the breeding origins of migratory species on their winter areas, for quantifying the degree of mixing of breeding populations on migratory and wintering sites, and for documenting other aspects of the population structure migratory animals – information needed for studies of year-round ecology of these species as well as for their conservation. Combining information from several stable isotopes will help to increase the resolution for determining the geographic origins of individuals in such highly vagile populations. Received: 24 April 1995 / Accepted: 2 June 1996     

Metabolic activation of unsaturated derivatives of valproic acid. Identification of novel glutathione adducts formed through coenzyme A-dependent and -independent processes

The ability of 2-n-propyl-4-pentenoic acid (Δ⁴-VPA) and 2-n-propyl-2(E)-pentenoic acid ([E]-Δ²-VPA), two unsaturated metabolites of valproic acid (VPA), to form reactive intermediates, deplete hepatic glutathione (GSH) and cause accumulation of liver triglycerides was investigated in the rat. With the aid of ionspray liquid chromatography-tandem mass spectrometry (LC-MS/MS), three GSH adducts were detected in the bile of Δ⁴-VPA-treated animals and were identified as 4-hydroxy-5-glutathion-S-yl-VPA-γ-lactone, 5-glutathion-S-yl-(E)-Δ³-VPA and 3-oxo-5-glutathion-S-yl-VPA. A fourth conjugate was identified tentatively as 4-glutathion-S-yl-5-hydroxy-VPA. Quantitative analysis of the corresponding N-acetylcysteine (NAC) conjugates in urine indicated that metabolism of Δ⁴-VPA via the GSH-dependent pathways accounted for approximately 20% of an acute dose (100 mg kg⁻¹ i.p.). In contrast, when rats were given an equivalent dose of (E)-Δ²-VPA, only one GSH adduct (5-glutathion-S-yl-(E)-Δ³-VPA) was detected at low concentrations in bile. In vitro experiments with rat liver mitochondria demonstrated that Δ⁴-VPA undergoes coenzyme A- and ATP-dependent metabolic activation in this organelle via the β-oxidation pathway to intermediates which bind covalently to proteins. When liver homogenates and hepatic mitochondria from rats injected with Δ⁴-VPA, (E)-Δ²-VPA or VPA were analyzed for GSH content, it was found that only Δ⁴-VPA depleted GSH pools significantly. Treatment of rats with Δ⁴-VPA and (to a lesser extent) VPA led to an accumulation of liver triglycerides, whereas (E)-Δ²-VPA had no measurable effect. It is concluded that Δ⁴-VPA undergoes metabolic activation by both microsomal cytochrome P-450-dependent and mitochondrial coenzyme A-dependent processes, and that the resulting electrophilic intermediates, which are trapped in part by GSH, may mediate the hepatotoxic effects of this compound. In contrast, (E)-Δ²-VPA is not transformed to any appreciable extent to reactive metabolites, which thus accounts for the apparent lack of hepatotoxicity of this positional isomer in the rat.     

Tracing water uptake by jarrah (Eucalyptus marginata) trees using natural abundances of deuterium

 Seasonal change in the δ²H content of water from twig sap, soil, rainfall and groundwater were measured to determine the water sources accessed by jarrah (Eucalyptus marginata) trees at three sites in Western Australia with differing soils and depths to water table. During winter and spring the main contributor to the water uptake of the trees was stored water in the surface layers of the soil replenished by predominantly winter rainfall. With the onset of summer drought jarrah became more reliant on water from deeper down the profile. There was no clear evidence that jarrah could tap water from groundwater more than 14 m deep in deep sands. Defining the source of water for trees in deep lateritic soils using stable isotopes is hampered by the uniform deuterium profiles down most of the unsaturated zone and into the groundwater. There was a limited response in the δ²H values of sapwater in twigs to changes in the δ²H of the upper layers of the deep sand following input of rainfall in autumn. The damped response was related to the small variation in the δ²H composition of rainfall in most events during the year and the mixing in the tree of water extracted from different locations in the soil profile. Received: 21 August 1995 / Accepted: 3 December 1995     

Relationships between carbon and hydrogen isotope ratios and nitrogen levels in leaves ofClusia species and two other Clusiaceae genera at various sites and different altitudes in Venezuela

Samples of the Clusiaceae generaClusia, Oedematopus andDystovomita were collected at various sites and different altitudes in northern and south-western Venezuela. Analyses of stable isotopes of carbon and hydrogen and of leaf-nitrogen levels were performed on the dried samples. Correlations among these variables, i.e. carbon isotope discrimination (), hydrogen isotope ratio (D) and N-levels, and with altitude were assessed. In the samples, where values of above 15 indicate predominant performance of C₃ photosynthesis, there were slight tendencies of increasing , D and N-levels with increasing altitude and of increasing with increasing N. Although these correlations taken separately were not statistically significant, they support each other and indicate increasing transpiration and increased leaf-nutrient supply at increasing altitude. Performance of crassulacean acid metabolism (CAM) in species ofClusia appears to be restricted to altitudes below 1500 m a.s.l. There was a significant negative correlation of with altitude in the samples, where values of below 10 indicated predominant performance of CAM. This suggests that phases II and IV of CAM are progressively suppressed towards the upper altitudinal limit of CAM inClusia in northern Venezuela. It is concluded that among the large number of environmental factors and combinations thereof, which determine the expression of CAM inClusia and trigger C₃-CAM transitions in C₃/CAM intermediate species, low availability of water is the most important.     

High-level 2H/13C/15N labeling of proteins for NMR studies

Summary The protein human carbonic anhydrase II (HCA II) has been isotopically labeled with ²H, ¹³C and ¹⁵N for high-resolution NMR assignment studies and pulse sequence development. To increase the sensitivity of several key ¹H/¹³C/¹⁵N triple-resonance correlation experiments, ²H has been incorporated into HCA II in order to decrease the rates of ¹³C and ¹H_N T₂ relaxation. NMR quantities of protein with essentially complete aliphatic ²H incorporation have been obtained by growth of E. coli in defined media containing D₂O, [1,2-¹³C₂, 99%] sodium acetate, and [¹⁵N, 99%] ammonium chloride. Complete aliphatic deuterium enrichment is optimal for ¹³C and ¹⁵N backbone NMR assignment studies, since the ¹³C and ¹H_N T₂ relaxation times and, therefore, sensitivity are maximized. In addition, complete aliphatic deuteration increases both resolution and sensitivity by eliminating the differential ²H isotopic shift observed for partially deuterated CH_nD_m moieties.     

The mechanism of deuterium oxide-induced protein degradation in Lemna minor

Transfer of Lemna minor fronds to culture medium containing 50% (v/v) deuterium oxide induces a large increase in the rate of protein breakdown, which is not due to an increase in the activity of acidic or neutral proteolytic enzymes or peptidases. Biochemical and ultrastructural evidence indicates that deuterium oxide affects the properties of certain membranes, particularly the tonoplast, and allows vacuolar proteolytic enzymes to pass into the cytoplasm and cause the increased protein breakdown.Abbreviations BAPA benzylarginine-p-nitroanilide - LPA leucine-p-nitroanilide - TCA trichloroacetic acid     

Low hydration phase properties of phospholipid mixtures

An experimental investigation of the low hydration phase properties of phospholipid mixtures is described. ²H (D₂O) NMR, X-ray diffraction and differential scanning calorimetry have been used to elucidate the phase properties of mixtures of the mixed chain phospholipids palmitoyloleoylphosphatidylcholine (POPC) and palmitoyloleoylphosphatidylethanolamine (POPE). At 10% hydration pure POPE exhibited a H_II phase above 330 K, a fluid lamellar phase below 315 K, and a minimally hydrated crystalline phase below 300 K. For the 1:1 mixture, the samples exhibited only gel or fluid phases between 270 K and 360 K for hydrations in the range 15% to 30%. Below 15% hydration the mixture exhibited two fluid phases with different repeat spacings, as predicted previously.     

Norepinephrine Metabolism in Man Using Deuterium Labeling: Turnover of 4-Hydroxy-3-Methoxymandelic Acid

Abstract: 4-Hydroxy-3-methoxymandelic acid (HMMA; VMA) labeled with three deuterium atoms was used to study the turnover and fate of HMMA following intravenous injection. Five healthy men were given a pulse dose of 5.0 μmol of labeled HMMA. Plasma and urinary levels of both endogenous and labeled HMMA were subsequently followed by gas chromatography-mass spectrometry using selected ion detection. The kinetic parameters were determined both with and without compensation for the pool expansion caused by the injection of labeled HMMA. The urinary recovery of labeled HMMA was 85 × 10% (mean ± SD). No conversion of HMMA t o 4-hydroxy-3-methoxyphenyl glycol (HMPG) occurred. The biological half-life of HMMA was 0.54 ± 0.22 h. The apparent volume of distribution was 0.36 ± 0.11 L/kg. The production rate or body turnover was 1.27 ± 0.51 μmol HMM/h and urinary excretion rate was 0.82 ± 0.22 μmol/h. These results show that HMMA is turning over rapidly in a relatively small volume of distribution and that, unlike HMPG, it is an end metabolite of norepinephrine in man.