Structure and dynamics of plasmalogen model membranes containing cholesterol: a deuterium NMR study

Deuterium nuclear magnetic resonance (²H-NMR) was used to investigate the structure and dynamics of the sn-2 hydrocarbon chain of semi-synthetical choline and ethanolamine plasmalogens in bilayers containing 0, 30, and 50 mol% cholesterol. The deuterium NMR spectra of the choline plasmalogen yielded well-resolved quadrupolar splittings which could be assigned to the corresponding hydrocarbon chain deuterons. The sn-2 acyl chain was found to adopt a similar conformation as observed in the corresponding diacyl phospholipid, however, the flexibility at the level of the C-2 methylene segment of the plasmalogen was increased. Deuterium NMR spectra of bilayers composed of the ethanolamine plasmalogen yielded quadrupolar splittings of the C-2 segment much larger than those of the corresponding diacyl lipids, suggesting that the sn-2 chain is oriented perpendicular to the membrane surface at all segments. Cholesterol increased the ordering of the choline plasmalogen acyl chain to the same extent as in diacyl lipid bilayers. T₁ relaxation time measurements demonstrated only minor dynamical differences between choline plasmalogen and diacyl lipids in model membranes.     

Microwave-assisted methylation of cassava starch with dimethyl carbonate

A novel and environmentally friendly process for the methylation of cassava starch with dimethyl carbonate (DMC) could be accelerated by employing a combined strategy: using disodium hydrogen phosphate (Na₂HPO₄) as the catalyst (chemical means) and microwave irradiation as the energy source (physical means). By varying the volume of 5% sodium chloride aqueous solution between 50 and 150 mL, the amount of Na₂HPO₄ between 0 and 1.25 g, the volume of DMC between 75 and 200 mL, and the microwave time from 5 to 20 min, methyl cassava starch with degree of substitution (DS) values in the range of 0.033 and 1.087 was prepared. The chemical structure of methyl cassava starch was analyzed by ¹H NMR spectroscopy.     

Rapid homogeneous lauroylation of wheat straw hemicelluloses under mild conditions

Hemicellulose-based hydrophobic biomaterials with degrees of substitution ranging from 0.46 to 1.54 were synthesized under mild conditions in homogeneous media (N,N-dimethylformamide-lithium chloride) by reacting the native wheat straw hemicellulosic polymers with lauroyl chloride using 4-dimethylaminopyridine as a catalyst. Other catalysts such as N-bromosuccinimide, N-methyl pyrrolidine, N-methyl pyrrolidinone, and pyridine were also investigated. Under optimum reaction conditions (2 equiv of lauroyl chloride and triethylamine per hydroxyl group, 5% 4-dimethylaminopyridine, 40 °C, 35 min), a high DS value of 1.54 was obtained. The biomaterials were characterized by FT-IR spectroscopy and ¹³C NMR spectroscopy as well as by thermal analysis. The results showed that the lauroylation occurred preferably at the C-3 hydroxyl group of β-d-Xylp units in the hemicelluloses, and the thermal stability of the hydrophobic polymers increased by esterification.     

C, N CP MAS and high resolution multinuclear NMR study of methyl

The relative proportion of L-iduronic acid (IdoA) and D-glucuronic acid (GlcA) is of great importance for the structure–function relationship of chondroitin sulfate (CS)/dermatan sulfate (DS). However, determination of the isotypes of uronic acid residues in CS/DS is still a challenge, due to the instability of free uronic acid released by chemical degradation and its conversion to unsaturated uronic acid by digestion with bacterial eliminase. ¹H-Nuclear magnetic resonance (NMR) spectroscopy is a promising tool with which to address this issue, but the traditional method based on the assignment of the ring proton signals of IdoA and GlcA residues still has drawbacks such as the serious overlap of signals in the ¹H-NMR spectrum of CS/DS polysaccharides. We found that the proton signals of the N-acetyl group of N-acetyl-D-galactosamines in CS and DS could be clearly distinguished and accurately integrated in the one-dimensional (1D) ¹H-NMR spectrum. Based on this finding, here we report a novel, sensitive, and nondestructive 1D ¹H-NMR-based method to determine the proportion of IdoA and GlcA residues in CS/DS hybrid chains. The contributions of Fuchuan Li and Shuhei Yamada should be considered equal.     

Determination of iduronic acid and glucuronic acid in sulfated chondroitin/dermatan hybrid chains by <Superscript>1</Superscript>H-nuclear magnetic resonance spectroscopy

The relative proportion of L-iduronic acid (IdoA) and D-glucuronic acid (GlcA) is of great importance for the structure–function relationship of chondroitin sulfate (CS)/dermatan sulfate (DS). However, determination of the isotypes of uronic acid residues in CS/DS is still a challenge, due to the instability of free uronic acid released by chemical degradation and its conversion to unsaturated uronic acid by digestion with bacterial eliminase. ¹H-Nuclear magnetic resonance (NMR) spectroscopy is a promising tool with which to address this issue, but the traditional method based on the assignment of the ring proton signals of IdoA and GlcA residues still has drawbacks such as the serious overlap of signals in the ¹H-NMR spectrum of CS/DS polysaccharides. We found that the proton signals of the N-acetyl group of N-acetyl-D-galactosamines in CS and DS could be clearly distinguished and accurately integrated in the one-dimensional (1D) ¹H-NMR spectrum. Based on this finding, here we report a novel, sensitive, and nondestructive 1D ¹H-NMR-based method to determine the proportion of IdoA and GlcA residues in CS/DS hybrid chains. The contributions of Fuchuan Li and Shuhei Yamada should be considered equal.     

Interaction of local anesthetics with small phospholipid vesicles investigated by proton NMR spectroscopy

The effects of the local anesthetics tetracaine, procaine (both charged at pH 6), and benzocaine (uncharged) on phospholipid liposomes have been investigated by 500 MHz ¹H NMR Spectroscopy. All the drugs reverse the Pr³⁺ induced shifts of phospholipid resonances in the same sequence as they are shifted by addition of Pr³⁺: choline POCH₂- > choline-CH₂N > choline-N(CH₃)₃ > glycerol > glycerol > acyl C₂ > acyl C₃. The drug effects result from incorporation of positive charges (tetracaine and procaine) and from the induction of a conformational change of the phospholipid head group via an action on the lipid glycerol backbone (benzocaine). From titration experiments with tetracaine on liposomes containing Pr³⁺ inside and outside is derived that the drug passes the bilayer by transverse diffusion. Tetracaine partitions outside/inside at a ratio of 21. Changes in linewidths of the drug resonances when incorporated into the liposomes allow the conclusion that the tetracaine molecule is located in an elongated way between the lipid acyl chains with its nitrogen group near the glycerol backbone. Benzocaine, showing strong effects on the line shapes of the protons on C₂ and C₃ of the lipid acyl chains is also located near the glycerol backbone, the region with the strongest hydrophobic forces.This work was supported by the Deutsche Forschungsgemeinschaft (SFB 30), Cardiology.     

Assimilation of nod gene inducer 14C-naringenin and the incorporation of labelled carbon atoms into the acyl side chain of a host-specific Nod factor produced by Rhizobium leguminosarum bv. viciae

The fate of ¹⁴C-naringenin during its specific activation of nod genes in Rhizobium leguminosarum bv. viciae was examined. After incubation with either strain RBL5560 or its pSym-cured derivative in a medium supplemented with ¹⁴C-naringenin at nod gene-inducing concentrations of 2 nM (ca. 12.5 kBq) plus cold acetate (0.5 M), a radiocarbon inventory for the cells and supernatant extracts was obtained. The level of ¹⁴C-label incorporation was also determined in the fractionated cellular components. Using ¹⁴C-acetate at 0.5 M (1036 kBq) and cold naringenin (2 nM) in incubations with strain RBL5560 as a separate treatment, the Nod metabolites were detected by thin layer and high performance liquid chromatographic methods and the data provided the basis for identification of the Nod factors from the supernatant obtained from ¹⁴C-naringenin treatments. Subsequent radio-biochemical and chemical analyses revealed that RBL5560 cells assimilated ¹⁴C-naringenin during the activation of nod genes. Our analysis also showed that labelled carbon atoms from the ¹⁴C-naringenin were incorporated into the acyl moiety of a lipo-oligosaccharide Nod factor, NodRlv IV, present in the culture supernatants of RBL5560. The pSym-cured derivative failed to synthesize any Nod metabolites in a ¹⁴C-naringenin supplemented medium. The tracing of flavonoid-derived carbon atoms to the acyl chain of a host-specific Nod factor, a moiety that defines host specificity for this Rhizobium, adds a new dimension to the signalling function of flavonoids in legume-Rhizobium interactions.Abbreviations Ac acyl chain - ca calculated approximately - dpm disintegrations per minute - HPLC High Performance Liquid Chromatography - pSym symbiotic plasmid - R. Rhizobium - TLC Thin Layer Chromatography     

Bradykinin-induced potassium current in cultured bovine aortic endothelial cells

Summary Bovine aortic endothelial cells (BAECs) respond to bradykinin with an increase in cytosolic-free Ca²⁺ concentration, [Ca²⁺] _i , accompanied by an increase in surface membrane K⁺ permeability. In this study, electrophysiological measurement of K⁺ current was combined with⁸⁶Rb⁺ efflux measurements to characterize the K⁺ flux pathway in BAECs. Bradykinin- and Ca²⁺-activated K⁺ currents were identified and shown to be blocked by the alkylammonium compound, tetrabutylammonium chloride and by the scorpion toxin,noxiustoxin, but not by apamin or tetraethylammonium chloride. Whole-cell and single-channel current analysis suggest that the threshold for Ca²⁺ activation is in the range of 10 to 100nm [Ca²⁺] _i . The whole-cell current measurement show voltage sensitivity only at the membrane potentials more positive than 0 mV where significant current decay occurs during a sustained depolarizing pulse. Another K⁺ current present in control conditions, an inwardly rectifying K⁺ current, was blocked by Ba²⁺ and was not affected bynoxiustoxin or tetrabutylammonium chloride. Efflux of⁸⁶Rb^– from BAEC monolayers was stimulated by both bradykinin and ionomycin. Stimulated efflux was blocked by tetrabutyl- and tetrapentyl-ammonium chloride and bynoxiustoxin, but not by apamin or furosemide. Thus,⁸⁶Rb⁺ efflux stimulated by bradykinin and ionomycin has the same pharmacological sensitivity as the bradykinin- and Ca²⁺-activated membrane currents. The results confirm that bradykinin-stimulated⁸⁶Rb⁺ efflux occurs via Ca²⁺-activated K⁺ channels. The blocking agents identified may provide a means for interpreting the role of the Ca²⁺-activated K⁺ current in the response of BAECs to bradykinin.     

Disulfiram lowers Ca2+, Mg2+-ATPase activity of rat brain synaptosomes

The chronic administration of disulfiram (DS) to rats resulted in significant decrease of synaptosomal Ca²⁺, Mg²⁺-ATPase activity. In vitro studies indicated that DS (ID₅₀=20 M) produced a dose-dependent inhibition of Ca²⁺, Mg²⁺-ATPase. However, diethyldithio-carbamate, a metabolite of DS, failed to modify Ca²⁺, Mg²⁺-ATPase activity, implying that the decrease in ATPase activity in DS administered rats was due to the effect of parent compound. The DS-mediated inhibition (48%) of ATPase activity was comparable with a similar degree of inhibition (49%) achieved by treating the synaptosomal membranes with N-ethylmaleimide (ID₅₀=20 M) in vitro. Furthermore, the inhibition by DS was neither altered by washing the membranes with EGTA nor reversed by treatment with sulfhydryl reagents such as GSH or dithiothreitol. About 74% and 68% decrease of synaptosomal Ca²⁺, Mg²⁺-ATPase specific activity was observed when treated with DS (30 M) and EGTA (100 M) respectively. The remaining 25–30% of total activity is suggested to be of Mg²⁺-dependent ATPase activity. This indicates that both these drugs may act on a common target, calmodulin component that represents 70–75% of total Ca²⁺, Mg²⁺-ATPase activity. Therefore, DS-mediated modulation of synaptosomal Ca²⁺, Mg²⁺-ATPase activity could affect its function of maintaining intracellular Ca²⁺ concentration. This could contribute to the deleterious effects on CNS.     

New ultrasensitive 32P-postlabelling method for the analysis of 3,N4-etheno-2′-deoxycytidine in human urine

Abstract

Etheno–DNA adducts are generated from exogenous carcinogens such as vinyl chloride and urethane and also from endogenous lipid peroxidation products such as trans-4-hydroxy-2-nonenal (HNE). The present authors and others have established that 1,N⁶-ethenodeoxyadenosine (εdA) and 3,N⁴-ethenodeoxycytidine (εdC) are present in human urine and could be explored as biomarkers for monitoring whole-body oxidative stress. The present study reports on a new ultrasensitive ³²P-postlabelling/thin-layer chromatography (TLC) method for the analysis of εdC as deoxynucleoside in human urine. The urine samples were purified and enriched on a solid-phase silica C-18 column followed by a semi-preparative reverse-phase high-performance liquid chromatography. The purified sample was labelled with a multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster (Dm-dNK) in the presence of 5′-bromo-2′-deoxyuridine (BrdU) as internal standard. The absolute sensitivity of the method was 0.1 fmol εdC detectable in 500 µl of human urine. The analysis of human urine samples from 15 healthy volunteers revealed a mean εdC level of 2.49±1.76 (SD) fmol µmol^?1 creatinine (range 0.66–6.42). By this non-invasive method, εdC in human urine could be explored as a biomarker for oxidative stress-related human diseases.     

Interactions of D-cellobiose with p-toluenesulfonic acid in aqueous solution: a C NMR study

The effects of adding D₂SO₄, and p-toluenesulfonic acid-d to D-cellobiose dissolved in D₂O were investigated at 23 °C by plotting ¹³C NMR chemical shift changes (Δδ) against the acid to D-cellobiose molar ratio. ¹³C Chemical shifts of all 18 carbon signals from α and β anomers of D-cellobiose showed gradual decreases due to increasing acidity in aqueous D₂SO₄ medium. The C-1 of the α anomer showed a slightly higher response to increasing D⁺ concentration in the surrounding. In the aqueous p-toluenesulfonic acid-d medium, C-6′ and C-4′ carbons of both α, and β anomeric forms of D-cellobiose are significantly affected by increasing the sulfonic acid concentrations, and this may be due to a 1:1 interaction of p-toluenesulfonic acid-d with the C-6′, C-4′ region of the cellobiose molecule.     

Ultraviolet Photoalteration of Late Na+ Current in Guinea-pig Ventricular Myocytes

UV irradiation has multiple effects on mammalian cells, including modification of ion channel function. The present study was undertaken to investigate the response of membrane currents in guinea-pig ventricular myocytes to the type A (355, 380 nm) irradiation commonly used in Ca²⁺ imaging studies. Myocytes configured for whole-cell voltage clamp were generally held at −80 mV, dialyzed with K⁺-, Na⁺-free pipette solution, and bathed with K⁺-free Tyrode’s solution at 22°C. During experiments that lasted for ≈ 35 min, UVA irradiation caused a progressive increase in slowly-inactivating inward current elicited by 200-ms depolarizations from −80 to −40 mV, but had little effect on background current or on L-type Ca²⁺ current. Trials with depolarized holding potential, Ca²⁺ channel blockers, and tetrodotoxin (TTX) established that the current induced by irradiation was late (slowly-inactivating) Na⁺ current (I_Na). The amplitude of the late inward current sensitive to 100 μM TTX was increased by 3.5-fold after 20–30 min of irradiation. UVA modulation of late I_Na may (i) interfere with imaging studies, and (ii) provide a paradigm for investigation of intracellular factors likely to influence slow inactivation of cardiac I_Na.     

The effects of experimentally altered gill chloride cell surface area on acid-base regulation in rainbow trout during metabolic alkalosis

To evaluate the role of the gill chloride cells in regulating metabolic alkalosis in rainbow trout (Oncorhynchus mykiss), the surface area of branchial chloride cells was altered experimentally using combined cortisol/ovine growth hormone injections. Long-term (10-day) treatment of fish with cortisol/ovine growth hormone caused an increase in the two-dimensional chloride cell fractional surface area when compared to uninjected fish (from 8.4 to 29.7%). This was the combined result of an increase in the size of individual cells (from 34.6 to 59.2 m²) and increased numbers of cells (from 2368 to 5006 cells · mm^-2). Metabolic alkalosis was induced by intra-arterial infusion of 140 mmol · l^-1 NaHCO₃; control fish were infused with 140 mmol · l^-1 NaCl. Blood pH and plasma [HCO₃ ^-] increased in both the untreated and the cortisol/ovine growth hormone-treated fish. However, the increases in pH (from 8.05 to 8.53) and [HCO₃ ^-] (from 5.9 to 22.2 mmol · l^-1) in the untreated fish were significantly greater than in the cortisol/ovine growth hormone-treated fish (pH increased from 7.78 to 8.11; [HCO₃ ^-] increased from 5.5 to 13.9 mmol · l^-1). In all fish, NaHCO₃ infusion elicited an increase in the rate of branchial basic equivalent excretion (acidic equivalent uptake) which, in turn, was caused by decreases and increases in branchial Na⁺ uptake and Cl^- uptake, respectively. In the untreated fish, there was a pronounced increase (75%) in chloride cell surface area during NaHCO₃ infusion. The attenuation of the metabolic alkalosis during HCO₃ ^- infusion in the cortical/ovine growth hormone-treated fish was caused, at least in part, by an enhancement of branchial basic equivalent excretion. In these fish that already displayed a proliferation of chloride cells, there was no further increase in chloride cell surface area. The changes in Na⁺ influx and Cl^- influx were quantitatively similar during NaHCO₃ infusion in both groups. This suggests that the greater rate of base excretion in the cortisol/ovine growth hormone-treated fish was caused by a greater percentage of Cl^- uptake being coupled to HCO₃ ^- excretion and less to Cl^- excretion (Cl^- exchange diffusion).Abbreviations Amm total ammonia - bw body weight - CC chloride cell - CCFA chloride cell fractional area - cort/oGH cortisol/ovine growth hormone - dpm disintegrations per minute - J ^Amm net flux of total ammonia - J _in unidirectional influx - J _inCl^- chloride ion uptake - J _inNa⁺ sodium ion uptake - J _netH⁺ net acidic equivalent flux - J ^TA net flux of titrable alkalinity - MS 222 ethyl-m-aminobenzoate - oGH ovine growth hormone - PVC pavement cell - SEM scanning electron microscope - TA titrable alkalinity     

Cellular and transepithelial responses of goldfish intestinal epithelium to chloride substitutions

Summary In goldfish intestine chloride was substituted by large inorganic anions (gluconate or glucuronate) either mucosally, serosally or bilaterally. Changes in intracellular activities of chloride (a _i Cl^–), sodium (a _i Na⁺) and potassium (a _i K⁺), pH_i, relative volume, membrane and transepithelial potentials, transepithelial resistance and voltage divider ratio were measured. Control values were:a _i Cl^–=35 meq/liter, a _i Na⁺=11 meq/liter and a _i K⁺=95 meq/liter. During bilateral substitution the latter two did not change while a _i Cl^– dropped to virtually zero.Mucosal membrane potentials (_ms) were: control,-53 mV; serosal substitution,-51 mV; bilateral substitution,-66 mV; while during mucosal substitution a transient depolarization occurred and the final steady state _ms was-66 mV.During control and bilateral substitution the transepithelial potentials (_ms) did not differ from zero. During unilateral substitutions _ms was small, in the order of magnitude of the errors in the liquid junction potentials near the measuring salt bridges.During bilateral substitution pH _i increased 0.4 pH units. Cellular volume decreased during mucosal substitution to 88% in 40 min; after serosal substitution it transiently increased, but the new steady-state value was not significantly above its control value.Three minutes after mucosal substitution ana _i Cl^– of approx. 10 meq/liter was measured.Chemical concentrations of Na, K and Cl were determined under control conditions and bilateral substitution. Cl concentrations were also measured as a function of time after unilateral substitutions.The data indicate an electrically silent chloride influx mechanism in the brush border membrane and an electrodiffusional chloride efflux in the basolateral membrane. A substantial bicarbonate permeability is present in the basolateral membrane. The results are in agreement with the observed changes in membrane resistances, volume changes and pH changes.     

Characterisation of oligosaccharides from the chondroitin/dermatan sulphates: H and C NMR studies of oligosaccharides generated by nitrous acid depolymerisation

The polymers chondroitin sulphate and dermatan sulphate have been fragmented by an anhydrous hydrazine/nitrous acid procedure. The resulting disaccharides from the polymer repeat sequences were reduced with NaBH₄ and purified by ion exchange chromatography. Whereas enzymatic depolymerisation leads to the loss of the distinction between glucuronic and iduronic acids of CS and DS in the resultant disaccharides, nitrous acid depolymerisation retains these structures. Complete ¹H and ¹³C NMR data have been derived for the major components which were shown to have the structures: GlcA-(β1→3)-anTal6S-ol (I) and l-IdoA-(α1→3)-anTal4S-ol (II), where anTal-ol represents (2,5)anhydro-d-talitol and 6S/4S represent O-ester sulphate groups at C-6/C-4 sites.     

Properties of an anion/H+ contransport system in L1210 cells that utilizes phthalate as a nonphysiological substrate

Summary [¹⁴C]Phthalate is transported into L1210 cells via two separate routes, an anion exchange system whose primary substrates are folate compounds, and a second less active system which is sensitive to bromosulfophthalein. When the principal uptake component was blocked by a specific irreversible inhibitor of this system, the remaining route (at pH 7.4) appeared to be saturable and was inhibited by several anions in addition to bromosulfophthalein (K _i =2 m), including 8-anilino-1-naphthalein sulfonate (K _i =25 m), unlabeled phthalate (K _i =500 m), and chloride (K _i =3500 m). A pronounced effect by pH was also observed. Influx and total uptake of phthalate both increased progressively with decreasing pH and reached values that were 20-fold higher at pH 6.0, compared with pH 7.4. This pH-dependent increase could be blocked, however, by the addition of compounds (nigericin and carbonylcyanidem-chlorophenylhydrazone) which, in combination, collapse proton gradients. Phthalate efflux was relatively insensitive to changes in extracellular pH but could be inhibited (up to 90%) by bromosulfophthalein. Several other anions also inhibited efflux, but to a lesser extent, while chloride, phthalate, lactate, glycolate and acetate enhanced efflux up to 1.8-fold. Efflux also increased at pH 6.0, but not at pH 7.5, upon addition of nigericin and carbonylcyanidem-chlorophenylhydrazone. These results suggest that phthalate is a nonphysiological substrate for a carrier system which mediates transport via an anion/H⁺ symport mechanism. This system is not the lactate/H⁺ symport carrier of L1210 cells since: (A) phthalate and lactate influx were inhibited to differing degrees by various anions; and (B) lactic anhydride inhibited the influx and efflux of lactate but had no effect on the transmembrane movement of phthalate. The specificity of this system suggests that its primary anion substrate may be chloride.     

Chloride transport activation by plasma osmolarity during rapid adaptation to high salinity of Fundulus heteroclitus

Transition from low salt water to sea water of the euryhaline fish, Fundulus heteroclitus, involves a rapid signal that induces salt secretion by the gill chloride cells. An increase of 65 mOsm in plasma osmolarity was found during the transition. The isolated, chloridecell-rich opercular epithelium of sea-water-adapted Fundulus exposed to 50 mOsm mannitol on the basolateral side showed a 100% increase in chloride secretion, which was inhibited by bumetanide 10^–4 m and 10^–4 m DPC (N-Phenylanthranilic acid). No effect of these drugs was found on apical side exposure. A Na⁺/H⁺ exchanger, demonstrated by NH₄Cl exposure, was inhibited by amiloride and its analogues and stimulated by IBMX, phorbol esters, and epithelial growth factor (EGF). Inhibition of the Na⁺/H⁺ exchanger blocks the chloride secretion increase due to basolateral hypertonicity. A Cl^–/HCO ₃ ^– exchanger was also found in the chloride cells, inhibited by 10^–4 m DIDS but not involved in the hyperosmotic response. Ca²⁺ concentration in the medium was critical for the stimulation of Cl^– secretion to occur. Chloride cell volume shrinks in response to hypertonicity of the basolateral side in sea-water-adapted operculi; no effect was found on the apical side. Freshwater-adapted fish chloride cells show increased water permeability of the apical side. It is concluded that the rapid signal for adaptation to higher salinities is an increased tonicity of the plasma that induces chloride cell shrinkage, increased chloride secretion with activation of the Na⁺K⁺2Cl^– cotransporter, the Na⁺/H⁺ exchanger and opening of Cl^– channels.The work was supported by the National Institutes of Health, Research Grant EYO1340 to J.A.Z. Part of this research was performed while Dr. Zadunaisky was a Scholar In Residence at the Fogarty International Center of The National Institutes of Health in Bethesda, Maryland. Ms. Dawn Roberts was a fellow of the Grass Foundation and Pew Foundation during this work. Grants from the National Science Foundation and the National Institutes of Health to the Mount Desert Island Biological Laboratory also provided assistance for this research.     

Purification and characterization of a cytoplasmic enzyme component of the Na+-activated malonate decarboxylase system of Malonomonas rubra: acetyl-S-acyl carrier protein: malonate acyl carrier protein-SH transferase

Malonate decarboxylation by crude extracts of Malonomonas rubra was specifically activated by Na⁺ and less efficiently by Li⁺ ions. The extracts contained an enzyme catalyzing CoA transfer from malonyl-CoA to acetate, yielding acetyl-CoA and malonate. After about a 26-fold purification of the malonyl-CoA:acetate CoA transferase, an almost pure enzyme was obtained, indicating that about 4% of the cellular protein consisted of the CoA transferase. This abundance of the transferase is in accord with its proposed role as an enzyme component of the malonate decarboxylase system, the key enzyme of energy metabolism in this organism. The apparent molecular weight of the polypeptide was 67,000 as revealed from SDS-polyacrylamide gel electrophoresis. A similar molecular weight was estimated for the native transferase by gel chromatography, indicating that the enzyme exists as a monomer. Kinetic analyses of the CoA transferase yielded the following: pH-optimum at pH 5.5, an apparent K_m for malonyl-CoA of 1.9mM, for acetate of 54mM, for acetyl-CoA of 6.9mM, and for malonate of 0.5mM. Malonate or citrate inhibited the enzyme with an apparent K_i of 0.4mM and 3.0mM, respectively. The isolated CoA transferase increased the activity of malonate decarboxylase of a crude enzyme system, in which part of the endogenous CoA transferase was inactivated by borohydride, about three-fold. These results indicate that the CoA transferase functions physiologically as a component of the malonate decarboxylase system, in which it catalyzes the transfer of acyl carrier protein from acetyl acyl carrier protein and malonate to yield malonyl acyl carrier protein and acetate. Malonate is thus activated on the enzyme by exchange for the catalytically important enzymebound acetyl thioester residues noted previously. This type of substrate activation resembles the catalytic mechanism of citrate lyase and citramalate lyase.Abbreviations DTNB 5,5 Dithiobis (2-nitrobenzoate) - MES 2-(N-Morpholino)ethanesulfonic acid - TAPS N-[Tris(hydroxymethyl)-methyl]-3-aminopropanesulfonic acid - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Platelet Ca2+ handling in essential hypertension: Role of a plasma ouabain-like factor

A humoral ouabain-like plasma factor has been observed in patients with essential hypertension (EHT). In the present study, we hypothesized that this humoral factor might be responsible for the elevated cytosolic free calcium concentrations [Ca²⁺]_i seen in these patients. Patients with mild to moderate EHT and their normotensive first degree blood relatives (NTBR) participated in the study. Platelet Na⁺, K⁺-ATPase activity was assayed in EHT patients and their NT first-degree relatives. To confirm the ouabain-like activity in plasma from EHT patients, control platelets were incubated with EHT and NTBR plasma and their Na⁺, K⁺-ATPase activity was measured. In addition, the effect of EHT plasma on platelet⁴⁵Ca-uptake was studied. Thein vitro effects of ouabain (10 ΜM) on (i)⁴⁵Ca-uptake and (ii) [Ca²⁺]_i response in control platelets were also observed. A decreased Na⁺K⁺-ATPase activity (P< 0.05) was observed in platelet membranes from EHT patients. Incubation of control platelets with EHT plasma decreased their Na⁺, K⁺-ATPase activity (P< 0.01) and increased their⁴⁵Ca-uptake (P< 0.05). C-18 Sep-Pak filtered hypertensive plasma extracts (containing the ouabain-like fraction) also decreased Na⁺, K⁺-ATPase activity (P< 001) in control platelet membranes.In vitro incubation of control platelets with ouabain increased⁴⁵Ca-uptake (P< 005) and [Ca²⁺]_i response (P< 0.05) in these platelets. Thus it appears that an ouabain-like factor in the EHT plasma may contribute to the elevated platelet [Ca²⁺]_i observed in EHT patients.     

NMR analysis of plant nitrogen metabolism

The analysis of primary and secondary nitrogen metabolism in plants by nuclear magnetic resonance (NMR) spectroscopy is comprehensively reviewed. NMR is a versatile analytical tool, and the combined use of ¹H, ²H, ¹³C, ¹⁴N and ¹⁵N NMR allows detailed investigation of the acquisition, assimilation and metabolism of nitrogen. The analysis of tissue extracts can be complemented by the in vivo NMR analysis of functioning tissues and cell suspensions, and by the application of solid state NMR techniques. Moreover stable isotope labelling with ²H-, ¹³C- and ¹⁵N-labelled precursors provides direct insight into specific pathways, with the option of both time-course and steady state analysis increasing the potential value of the approach. The scope of the NMR method, and its contribution to studies of plant nitrogen metabolism, are illustrated with a wide range of examples. These include studies of the GS/GOGAT pathway of ammonium assimilation, investigations of the metabolism of glutamate, glycine and other amino acids, and applications to tropane alkaloid metabolism. The continuing development of the NMR technique, together with potential applications in the emerging fields of metabolomics and metabolic flux analysis, leads to the conclusion that NMR will play an increasingly valuable role in the analysis of plant nitrogen metabolism.