Isolation and characterization of membrane-associated proteoglycans from normal and malignant human mammary epithelial cells

The plasma membrane-associated proteoglycans of a malignant human breast cell line (MDA-MB-231) were compared with the corresponding proteoglycans from a normal cell line (HBL-100). The labeled proteoglycans were isolated from the plasma membranes of cells grown in the presence of [³H]glucosamine and [³⁵S]Na₂SO₄ by extraction with guanidine hydrochloride and subsequently purified by DEAE-ion exchange chromatography. Their structural properties were established by treatment with nitrous acid, heparitinase and chondroitinase ABC, and by gel filtration before and after alkaline -elimination. About 18% of the proteoglycans synthesized by these cell lines were associated with the plasma membranes. The HBL plasma membranes contained 80% heparan sulfate and 20% chondroitin sulfate proteoglycans whereas MDA plasma membranes had 50% heparan sulfate and 50% chondroitin sulfate proteoglycans. The MDA plasma membrane contained two heparan sulfate proteoglycans, both having nearly the same molecular size as the two species secreted into the medium by these cells. The HBL plasma membrane also contained two hydrodynamic size heparan sulfate proteoglycans. The larger hydrodynamic size species has a slightly lower molecular size than that secreted into the medium, and the smaller hydrodynamic size species was not detectable in the medium. Even though the major chondroitin sulfate proteoglycans from MDA plasma membranes were smaller in size than those from HBL plasma membrane, a larger proportion of the glycosaminoglycan chains of the former were bigger than those from the latter.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate - Di-OS 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-d-galactose - Di-4S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-4-O-sulfo-d-galactose - Di-6S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-6-O-sulfo-d-galactose - Gdn-HCl guanidine hydrochloride - WGA wheat germ agglutinin     

Chromate resistance and reduction in Pseudomonas fluorescens strain LB300

Pseudomonas fluorescens LB300 is a chromateresistant strain isolated from chromium-contaminated river sediment. Chromate resistance is conferred by the plasmid pLHB1. Strain LB300 grew in minimal salts medium with as much as 1000 g of K₂CrO₄ ml^–1, and actively reduced chromate to Cr(III) while growing aerobically on a variety of substrates. Chromate was also reduced during anaerobic growth on acetate, the chromate serving as terminal electron acceptor. P. fluorescens LB303, a plasmidless, chromatesensitive variant of P. fluorescens LB300, did not grow in minimal salts medium with more than 10 g of K₂CrO₄ ml^–1. However, resting cells of strain LB303 grown without chromate reduced chromate as well as strain LB300 cells grown under the same conditions. Furthermore, resting cells of chromate-sensitive Pseudomonas putida strain AC10, also catalyzed chromate reduction. Evidently chromate resistance and chromate reduction in these organisms are unrelated. Comparison of the rates of chromate reduction by chromate grown cells and cells grown without chromate indicated that the chromate reductase activity is constitutive. Studies with cell-free extracts show that the reductase is membrane-associated and can mediate the transfer of electrons from NADH to chromate.     

Factors affecting chloride conductance in apical membrane vesicles from human placenta

Summary Apical membrane vesicles from human term placenta were isolated using a magnesium precipitation technique, and the purity of the vesicles was assessed morphologically using scanning and transmission electron microscopy, and biochemically, using marker enzymes. The vesicles were found to be morphologically intact and significantly enriched in enzymes associated with apical membranes. ³⁶Cl^– uptake into these vesicles was studied in the presence of an outwardly directed Cl^– gradient. This uptake was found to be time dependent, with an initial rapid uptake tending to peak between 10 and 20 min and thereafter decline. Uptake was found to be voltage dependent since 5 m valinomycin caused a decrease in uptake. The effects of N-phenylanthranilic acid (NPA) and 4,4-diisothiocyanostilbene-2,2-disulphonic acid (DIDS) and bumetanide on the initial rate of Cl^– were examined in the presence and absence of 5 m valinomycin. NPA and DIDS inhibited isotope uptake strongly with IC₅₀ values of 0.83±0.35 m and 3.43±0.37 m, respectively, in the absence of valinomycin. Although valinomycin reduced ³⁶Cl^– uptake by about 80% when added before the isotope, DIDS reduced the uptake which remained in a concentration-dependent fashion with an IC₅₀ of 5.6±2.1 m. Under these conditions, NPA was without effect at concentrations below 100 m. Bumetanide was without effect at the concentrations used in the absence of valinomycin. However, following valinomycin pretreatment, bumetanide reduced ³⁶Cl^– uptake significantly at 100 m concentration. Vesicle diameter, as assessed by flow cytometry, did not change under the conditions employed.The effects of some fatty acids were also investigated. Arachidonic acid and linoleic acid inhibited Cl^– uptake with IC₅₀ values of 37.6±14.9 m and 4.59±0.51 m, respectively. Arachidonyl alcohol and elaidic acid were found to be without effect. These studies show that human placental brush border membrane vesicles possess a chloride conductance channel, the activity of which can be measured in the presence of an outwardly directed Cl^– gradient and this channel is sensitive to Cl^– channel inhibitors, especially N-phenylanthranilic acid, and can be inhibited by unsaturated fatty acids such as arachidonic acid and linoleic acid.This work was supported in part by the Cystic Fibrosis Association of Ireland and Eolas, The Irish Science and Technology Agency. The technical assistance of Mr. Cormac O' Connell in the preparation of the electron micrographs and of Mr. Roddy Monks in the flow cytometric analysis is gratefully acknowledged.     

Aerobic chromate reduction by Bacillus subtilis

We have studied the reduction of hexavalent chromium (chromate) to the less toxic trivalent form by using cell suspensions and cell-free extracts from the common soil bacterium, Bacillus subtilis. B. subtilis was able to grow and reduce chromate at concentrations ranging from 0.1 to 1 mM K₂CrO₄. Chromate reduction was not affected by a 20-fold excess of nitrate-compound that serves as alternate electron acceptor and antagonizes chromate reduction by anaerobic bacteria. Metabolic poisons including sodium azide and sodium cyanide inhibited chromate reduction. Reduction was effected by a constitutive system associated with the soluble protein fraction and not with the membrane fraction. The reducing activity was heat labile and showed a K_m of 188 m CrO₄ ^2-. The reductase can mediate the transfer of electrons from NAD(P)H to chromate. The results suggest that chromate is reduced via a detoxification system rather than dissimilatory electron transport.     

Chemoenzymatic galactosialylation with integrated cofactor regeneration

As a precursor for the chemical synthesis of sialylated oligosaccharides, the trisaccharide glycoside Neu5Ac (2-8)Gal(1-4)GlcNAc(1-O)-pent-4-ene was synthesized starting from GlcNAc(1-O)-pent-4-ene, UDP-glucose andN-acetylneuraminic acid in a one pot reaction employing galactosyltransferase and (2-6)sialyl-transferase in a complete cofactor regeneration system.Abbreviations Neu5Ac N-acetylneuraminic acid - CMP-Neu5Ac cytidine 5-monophosphosialate - CMP cytidine 5-monophosphate - CDP cytidine 5-diphosphate - CTP cytidine 5-triphosphate - Gal galactose - GlcNAc N-acetylglucosamine - UDP uridine 5-diphosphate - UDP-Glc uridine-5-diphosphoglucose - UDP-Gal uridine-5-diphosphogalactose - PEP phosphoenolpyruvate     

Malate regulation of Mg2+-ATPase of chloroplast coupling factor 1

The regulatory effects of malate on chloroplast Mg²⁺-ATPase were investigated and the mechanism was discussed. Malate stimulated methanol-activated membrane-bound and isolated CF₁ Mg²⁺-ATPase activity. The subunit of CF₁ may be involved in malate regulation of the enzyme function. Modification of subunit at one site of the peptide by NEM may affect malate stimulation of ATPase while at another site may have no effect. The effect of malate on the Mg²⁺-ATPase was also controlled by the Mg²⁺/ATP ratio in the reaction medium. The enhancing effect of malate on Mg²⁺-ATPase activity depended on the presence of high concentrations of Mg²⁺ in the reaction mixture. Kinetic study showed that malate raised the V_max of catalysis without affecting the K_m for Mg²⁺ ATP. The experiments imply that the stimulation of Mg²⁺-ATPase by malate is probably correlated with the P_i binding site on the enzyme. The regulation of ATPase activity by malate in chloroplasts may be relevant to its function in vivo.Abbreviations CF₁ chloroplast coupling factor 1 - CF₁ (-) and CF₁ (-) CF₁ deficient in the and subunit - MF₁ mitochondria coupling factor 1 - NEM N-ethylmaleimide - PMS phenazine methosulfate - OG n-octyl--d-glucopyranoside     

Effects of Cr on proton extrusion, potassium uptake and transmembrane electric potential in maize root segments

Abstract Proton extrusion of maize root Zea mays segments, was inhibited by the presence of Cr (o.n. + 6; present in solution as CrO₄^2-, Cr₂O₇^2-) in the incubation medium: the minimum inhibiting concentration was 2 × 10^?3 mol m^?3 and the inhibition progressively increased with Cr concentration. Cr inhibited proton extrusion. Also, when this activity was stimulated by the presence of K⁺ or fusicoccin (FC) in the incubation medium, the K⁺ and FC stimulating effect was still present when proton extrusion was inhibited by Cr. In addition, Cr inhibited K⁺ uptake. This inhibition was higher (50%) at K⁺ concentrations up to 1 mol m^?3 lower (15%) at higher K⁺ concentrations. This result indicates that the system responsible for K⁺ uptake operating at low K⁺ concentrations is more sensitive to Cr inhibition. Cr had no effect on transmembrane electric potential (PD). The depolarizing and hyper-polarizing effect of K+ and FC, respectively, were not affected by Cr; but Cr enhances the depolarizing effect of the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCP). These results indicate that Cr inhibited the proton translocating mechanism coupled with K⁺ uptake, but did not change the net transport of charges through the plasmalemma. The Cr effect is discussed, taking into account the possibility of a direct effect of Cr at the membrane level or, alternatively, of an effect on some metabolic processes controlling membrane function.     

Ribulose bisphosphate carboxylase from Thiobacillus A2. Its purification and properties

Ribulose bisphosphate carboxylase (EC 4.1.1.39) from Thiobacillus A2 has been purified to homogeneity on the basis of polyacrylamide gel electrophoresis and U.V. analysis during sedimentation velocity studies. The enzyme had an optimum pH of about 8.2 with Tris-HCl buffers. The molecular weight was about 521000 with an S _rel. of 16.9. K _m for RuBP was 122 M, for total CO₂ it was 4.17 mM, and for Mg²⁺ 20.0 M. The absolute requirement for a divalent cation was satisfied by Mg²⁺ which was replaceable to a certain extent by Mn²⁺. Activity was not significantly affected by SO ₄ ^2- , SO ₃ ^2- , or S₂O ₃ ^2- at 1.0 mM. At this concentration S^2- caused a 27% stimulation. All mercurials tested were inhibitory. pHMB was the most potent causing about 60% inhibition at 0.01 mM. This inhibition was reversible by low concentrations of cysteine. Cyanide was also inhibitory. Its mode of inhibition with respect to RuBP was un-competitive and with a K _i of 20 M. Lost activity could be restored partially by GSH or Cu²⁺. Although azide at the concentration tested had no significant effect on enzyme activity, 2,4-dinitrophenol at 1.0 mM caused 91% inhibition. Finally, activity was also affected by energy charge.Abbreviations ATP adenosine-5-triphosphate - GAPDH glyceraldehyde phosphate dehydrogenase - GSH (reduced) glutathione - G6P glucose-6-phosphate - NAD⁺ nicotinamide adenine dinucleotide - NADP⁺ nicotinamide adenine dinucleotide phosphate - pHMB parahydroxymercuribenzoate - 6PG 6-phosphogluconate - 3-PGA 3-phosphoglycerate - PGK phosphoglyceratekinase - RuBP ribulose-1,5-bisphosphate     

Chromate tolerance in strains of Rhodosporidium toruloides modulated by thiosulfate and sulfur amino acids

Cr(VI) tolerance was studied in four strains of Rhodosporidium toruloides and compared with that of a fifth strain, DBVPG 6662, isolated from metallurgical wastes and known to be Cr(VI) resistant. Tolerance was studied in relation to different species of sulfur (sulfates, thiosulfates, methionine, cysteine) at different concentrations. Djenkolic acid, a poor source of sulfur and an activator of sulfate transport, was also considered. In synthetic medium all strains except the Cr(VI)-resistant one started to be inhibited by 10 g ml^– (0.2 mm) Cr(VI) as K₂Cr₂O₇. DBVPG 6662 was inhibited by 100 g ml^– (2.0 mm) Cr(VI). In Yeast Nitrogen Base without amino acids (minimal medium), supplemented with varying concentrations of chromate, all Cr(VI)-sensitive strains accumulated concentrations of total chromium (from 0.8 to 1.0 g mg^– cell dry wt) after 18 h of incubation at 28 °C. In minimal medium supplemented with 10 g ml^– Cr(VI), the addition of sulfate did not significantly improve the yeast growth. Cysteine at m levels increased tolerance up to 10 g ml^–, whereas methionine only reduced the Cr(VI) toxicity in the strain DBVPG 6739. Additions of djenkolic acid resulted in increased Cr(VI) sensitivity in all strains. The best inorganic sulfur species for conferring high tolerance was thiosulfate at concentrations up to 1 mm. In all cases increased Cr(VI) tolerance was due to a significantly reduced uptake in the oxyanion by the cells and not to the chemical reduction of Cr(VI) to Cr(III) by sulfur compounds.     

Heavy-metal ion uptake by plants from nutrient solutions with metal ion,plant species and growth period variations

Summary Rape, cucumber, wheat, oats and tomato were grown for one to two weeks in nutrient solutions with heavy metals added. Of the metal ions tested (Cr³⁺, Cu²⁺, Co²⁺, CrO₄ ^2-, Ni²⁺, Cd²⁺, Pb²⁺, Mn²⁺, Zn²⁺ and Ag⁺), manganese, nickel and lead exhibited the greatest mobility in cucumber plants, which resulted in the highest shoot/root concentration ratio. Silver was not translocated to the shoots of cucumber plants in measurable amounts.When the plants were grown with 1.0, 10 and 100 M cadmium or nickel in the solution, the shoot and root concentration increased 5–10 times if the metal ion concentration of the solution was increased 10 times.The plants showed great differences in cadmium and nickel uptake. In the shoot, the cadmium concentration increased in the order: oats = wheat < cucumber = rape < tomato, and in the root in the order: oats = wheat < cucumber = rape < tomato. The great uptake of cadmium and nickel by tomato is notable and agrees with other reports.The nickel, and especially the cadmium, concentration in roots and shoots increases with the age of the plant.The results are discussed and related to other investigations. The need for research on the uptake mechanisms of non-essential heavy metals is emphasized. re]19750415     

2,3-Dimercaptopropanol Inhibits Ca2+ Transport in Microsomes from Brain But Not from Fast-Skeletal Muscle

Ca²⁺ is involved in the regulation of a variety of physiological processes, but a persistent increase in free cytosolic Ca²⁺ concentrations may contribute to cell injury. Dimercaprol (BAL) is a compound used in the treatment of mercury intoxication, but presents low therapeutic efficacy. The molecular mechanism responsible for the BAL toxicity is poorly known. In the present study, the effect of BAL and inorganic and organic mercury on Ca²⁺ transport by Ca²⁺-ATPases located in the sarco/endoplasmic reticulum of fast-skeletal muscle and brain was examined. Ca²⁺ uptake by brain and fast-skeletal muscle microsomes was inhibited in a dose-dependent manner by Hg²⁺. The calculated IC₅₀ for Ca²⁺ uptake inhibition by HgCl₂ was 1.05 ± 0.09 M (n = 8) for brain and 0.72 ± 0.06 M (n = 9) for muscle. The difference was significant at p < 0.01 (data expressed as mean ± SD). At a low concentration (1 M), 2,3-dimercaptopropanol had no effect on Ca²⁺ uptake by brain or muscle vesicles and did not abolish the inhibition caused by Hg²⁺. A high concentration of BAL (1 mM) nearly abolished the inhibition caused by 1.75 M HgCl₂ or 6 M CH₃HgCl in skeletal muscle. Surprisingly, at intermediate concentrations (40–100 M) BAL partially inhibited Ca²⁺ transport in brain but had no effect on muscle. Furthermore, ATP hydrolysis by brain or muscle microsomes was not inhibited by BAL. These results suggest that in brain microsomes BAL affects in a different way Ca²⁺ transport and ATP hydrolysis. The increase in BAL concentration observed after toxic administration of this compound to experimental animals may contribute to deregulate Ca²⁺ homoeostasis and, consequently, to the neurotoxicity of BAL.     

The effect of copper toxicity on the contents of nitrogen compounds in Silene vulgaris (Moench) Garcke

The effect of copper on the uptake of nitrogen and the tissue contents of inorganic nitrogen, amino acids and proteins were studied in cooper-sensitive Silene vulgaris (Moench) Garcke, grown at different nitrogen sources (NH₄ ⁺ or NO₃ ^-). All the toxic copper levels tested, i.e. 4, 8, 16 M Cu²⁺, strongly inhibited the uptake of nitrogen, especially of NO₃ ^-, and decreased the content of NO₃ ^-, amino acids and proteins. Especially at 4 and 8 M Cu²⁺, NH₄ ⁺ accumulated in the plants, suggesting that the conversion of NH₄ ^- into amino acids was inhibited.     

Charge ordering in the metal–insulator transition of V-doped CrO<Subscript>2</Subscript> in the rutile structure

Electronic, magnetic, and structural properties of pure and V-doped CrO₂ were extensively investigated utilizing density functional theory. Usually, pure CrO₂ is a half-metallic ferromagnet with conductive spin majority species and insulating spin minority species. This system remains in its half-metallic ferromagnetic phase even at 50% V-substitution for Cr within the crystal. The V-substituted compound Cr_0.5V_0.5O₂ encounters metal–insulator transition upon the application of on-site Coulomb repulsion U?=?7 eV preserving its ferromagnetism in the insulating phase. It is revealed in this study that Cr³⁺-V⁵⁺ charge ordering accompanied by the transfer of the single V-3d electron to the Cr-3dt_2g orbitals triggers metal–insulator transition in Cr_0.5V_0.5O₂. The ferromagnetism of Cr_0.5V_0.5O₂ in the insulating phase arises predominantly due to strong Hund’s coupling between the occupied electrons in the Cr-t_2g states. Besides this, the ferromagnetic Curie temperature (T_c) decreases significantly due to V-substitution. Interestingly, a structural distortion is observed due to tilting of CrO₆ or VO₆ octahedra across the metal–insulator transition of Cr_0.5V_0.5O₂.

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Graphical abstract The V-doped compound Cr_0.5V_0.5O₂ is found a half-metallic ferromagnet (HMF) in the absence of on-site Coulomb interaction (U). This HMF behavor maintains up to U?=?6 eV. Eventually, this system encounters metal-insulator transition (MIT) upon the application of U?=?7 eV with a band gap of E_g ~ 0.31 eV. Nevertheless, applications of higher U widen the band gaps. In this figure, calculated total (black), Cr-3d (red), V-3d (violet), and O-2p (blue) DOS of Cr_0.5V_0.5O₂ for U?=?8 eV are illustrated. The system is insulating with a band gap of E_g ~ 0.7 eV.

     

Interspecies differences in the preference of ammonium and nitrate in vascular plants

Three solution experiments were performed to test the importance of NH ₄ ⁺ versus NO ₃ ^- +NH ₄ ⁺ to growth of 23 wild-forest and open-land species, using field-relevant soil solution concentrations at pH 4.5. At N concentrations of 1–200 M growth increased with increasing N supply in Carex pilulifera, Deschampsia flexuosa, Elymus caninus and Bromus benekenii. Geum urbanum was the most N demanding species and had little growth below 200 M. The preference for NH ₄ ⁺ or NO ₃ ^- +NH ₄ ⁺ was tested also at pH 4.0; no antagonism was found between NH ₄ ⁺ and H⁺, as indicated by similar relative growth in both of the N treatments at both pH levels. Growth in solution with NH ₄ ⁺ relative to NO ₃ ^- +NH ₄ ⁺ , 200 M, was negatively related to the mean pH of the field occurrence of the species tested; acid-tolerant species grew equally well with only NH ₄ ⁺ as with NO ₃ ^- +NH ₄ ⁺ (Oxalis acetosella, Carex pilulifera, Festuca gigantea, Poa nemoralis, Deschampsia flexuosa, Stellaria holostea, Rumex acetosella), while species of less acid soils were favoured by NO ₃ ^- +NH ₄ ⁺ (Urtica dioica, Ficaria verna, Melandrium rubrum, Aegopodium podagraria, Geum urbanum, Bromus benekenii, Sanguisorba minor, Melica ciliata, Silene rupestris, Viscaria vulgaris, Plantago lanceolata). Intermediate species were Convallaria majalis, Elymus caninus, Hordelymus europaeus and Milium effusum. No antagonism between NH ₄ ⁺ and Ca²⁺, Mg²⁺ and K⁺ was indicated by the total uptake of the elements during the experiment.     

Comparative studies of chromosomal aberration and mutagenicity of trivalent and hexavalent chromium

The comparative cytogenetic and mutagenic effects between trivalent and hexavalent chromium were investigated. Five chromium compounds, K₂Cr₂O₇ and K₂CrO₄ containing Cr⁶⁺, and Cr(CH₃COO)₃, Cr(NO₃)₃ and CrCl₃ containing Cr³⁺, were examined for their ability to induce chromosomal damage in cultures of human leukocytes, for their reactivity with DNA by a rec-assay system and for mutagenicity in the E. coli Hs30R test system.Chromosome-breaking activity was significantly higher for the compounds with hexavalent than trivalent chromium, the efficiency being in the decreasing order K₂Cr₂O₇ > K₂CrO₄ >> Cr(CHCOO)₃ > Cr(NO₃)₃, CrCl₃. In the rec-assay and mutation assay, hexavalent (K₂Cr₂O₇ and K₂CrO₄) and trivalent Cr(CH₃COO)₃) compounds gave positive results, their mutagenic potential being higher in the same order of clastogenic magnitude.     

Biosorption of chromium to fungi

Eighteen fungal strains were isolated from water and soil samples and tested for their ability to enrich chromium. The microorganism with the highest enrichment capacity, a zygomycete (Mucor hiemalis MP/92/3/4), was chosen for detailed investigations. Some basic tests such as the pH-dependence, the kinetics of the enrichment and the metal selectivity were carried out with the two most frequent oxidation states of chromium, the trivalent cation (Cr³⁺) and the hexavalent anion (CrO₄ ^2–). With Cr³⁺ the enrichment showed a saturation kinetic reaching 70% of the maximum capacity after about 30 min, whereas with CrO₄ ^2– a linear time course with a much lower metal enrichment was observed. The highest level of enrichment for Cr³⁺ was observed at pH 5.5 (21.4 mg/g dry wt), and for CrO₄ ^2– at pH 1 (4.3 mg/g dry wt). Investigations concerning the metal enrichment selectivity resulted in the following series of decreasing ion uptake: Cr³⁺ > Cu²⁺ > Pb²⁺ > Ag⁺ > Al³⁺ > Co²⁺ > Zn²⁺ > Ni²⁺ > Fe²⁺ > Mo⁵⁺ > Cd²⁺ > ^2– > CrO₄ ^2– > VO^3–, calculated on a molar basis. Trivalent chromium caused a staining of the outer cell wall region in transmission electron microscopy. The localization of chromium in the stained outer layers of the cell wall could be verified by electron energy loss spectroscopy. The enrichment of Cr³⁺ by M. hiemalis seemed to be mainly a passive biosorption to the cell wall, whereas for the uptake of CrO₄ ^2– intracellular accumulation as well as biosorption is possible.     

On the biosynthesis of 5-hydroxybenzimidazolylcobamide (vitamin B12-factor III) in Methanosarcina barkeri

Exogenous 5-hydroxy-[2-¹⁴C]benzimidazole was transformed by Methanosarcina barkeri into 5-hydroxy-[2-¹⁴C]benzimidazolylcobamide. Thereby the endogenous biosynthesis of 5-hydroxybenzimidazole was completely blocked.Benzimidazole and 5,6-dimethylbenzimidazole were used by M. barkeri to form benzimidazolylcobamide respectively 5,6-dimethylbenzimidazolylcobamide (vitamin B₁₂), but in these cases the endogenous biosynthesis of factor III was not completely suppressed.With [2-¹⁴C]benzimidazole it was demonstrated that this base as well as the benzimidazolylcobamide formed thereof are no precursors in the biosynthesis of 5-hydroxybenzimidazolylcobamide.Glycine instead was found to be a building block for the biosynthesis of 5-hydroxybenzimidazole, since radioactivity from [1-¹⁴C] and [2-¹⁴C]glycine was incorporated, into the base moiety of factor III, but not into its corrin moiety. With [1-¹³C]glycine and ¹³C-NMR-spectroscopy it was shown that C-1 of glycine gets C-3a of 5-hydroxybenzimidazole.[1-¹³C]glycine also led to a single prominent signal in the ¹³C-NMR-spectrum of coenzyme F₄₂₀, this was assigned to C-10a.Thus C-1 of glycine was incorporated into the hydroxybenzene part of 5-hydroxybenzimidazole, whereas it was not incorporated into this part of coenzyme F₄₂₀, indicating that the hydroxybenzene part of these two compounds is not formed from a common intermediate. L-[U-¹⁴C]glutamate led to the exclusive labeling of the corrin ring of factor III, showing that the corrin precursor 5-aminolevulinic acid is formed by the C-5 pathway in M. barkeri.These experiments indicate that the biosynthesis of factor III in the archaebacterium M. barkeri is similar to the corrinoid biosynthesis in the anaerobic eubacteria Eubacterium limosum, Clostridium barkeri, and Clostridium thermoaceticum.     

Nuclear Overhauser effect investigation on GM1 ganglioside containingN-glycolyl-neuraminic acid (II3Neu5GcGgOse4Cer)

The conformational properties of the oligosaccharide chain of GM1 ganglioside containingN-glycolyl-neuraminic acid, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer, were studied through NMR nuclear Overhauser effect investigations on the monomeric ganglioside in dimethylsulfoxide, and on mixed micelles of ganglioside and dodecylphosphocholine in water. Several interresidual contacts for the trisaccharide core--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-were found to fix the relative orientitation of the three saccharides, while the glycosidic linkage of the terminal -Gal-was found to be quite mobile as the -Gal-(1-3)--GalNAc-disaccharide exists in different conformations. These results are similar to those found for two GM1 gangliosides containingN-acetyl-neuraminic acid and neuraminic acid [1].Abbreviations Ganglioside nomenclature is in accordance with Svennerholm [23] and the IUPAC-IUB Recommendations [24] GM3(Neu5Ac) II³Neu5AcLacCer, -Neu5Ac-(2-3)--Gal-(1-4)--Glc-(1-1)-Cer - GM3(Neu5Gc) II³Neu5GcLacCer, -Neu5Gc-(2-3)--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu5Ac) II³Neu5AcGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu5Gc) II³Neu5GcGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu) II³NeuGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu-(2-3)]--Glc-(1-1)-Cer - GD1a IV³Neu5AcII³Neu5AcGgOse₄Cer, -Neu5Ac-(2-3)--Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GalNAc-GD1a IV⁴GalNAcIV³Neu5AcII³Neu5AcGgOse₄Cer, -GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - Neu neuraminic acid - Neu5Ac N-acetyl-neuraminic acid - Neu5Gc N-glycolyl-neuraminic acid - Cer ceramide     

Mechanistic origin of the kinetic cooperativity for the ATPase activity of sarcoplasmic reticulum

The (Ca²⁺-Mg²⁺)-ATPase from sarcoplasmic reticulum presents negative cooperativity for the hydrolysis of Mg²⁺-ATP at different concentration ranges of this substrate. A kinetic model is proposed according to which Mg²⁺-ATP may bind to three different enzymatic species present during the catalytic cycle, E (K ₁=1 µM), EP.Ca₂ (K ₉=500 µM) and *EP (K ₇=20 µM), accelerating the release of P_i. The fact that each of these species has a different affinity for Mg²⁺-ATP allows a significant enhancement of the rate of P_i release to the medium at the different ranges of Mg²⁺-ATP concentration where the enzyme shows a kinetic cooperativity. The kinetic analysis of this mechanism yields an equation which is a ratio of two cubic polynomials (3:3 rate equations) with respect to Mg²⁺-ATP and which may explain the negative cooperativity of the enzyme at different concentration ranges of Mg²⁺-ATP.Abbreviations: EGTA, ethylene glycol bis(-aminoethylether)-N,N,N,N-tetraacetic acid; I.U., international units; piruvate kinase (EC 2.7.1.40); lactate dehydrogenase (EC 1.1.1.27); ATP phosphohydrolase (EC 3.8.1.3).     

Uptake of piperidine and pipecolic acid by synaptosomes from mouse brain

Piperidine is actively transported into the synaptosomal fraction of adult mouse brain. The transport mechanism appears to be Na⁺ independent but is temperature dependent and sensitive to ouabain. Analysis of kinetic experiments indicates only a low-affinity transport system to be present. By contrast the uptake ofD,L-[³H]pipecolic acid at a concentration of 4×10^–7 M was temperature and Na⁺ dependent, ouabain sensitive, and revealed a two-component system with aK _m =3.9±0.17×10^–6 M,V _max=129±6 pmol/mg protein/3 min for the high-affinity system and aK _m =90.2±4.3×10^–6 M,V _max=2.45±0.19 nmol/mg protein/3 min for the low-affinity system. Compounds structurally related to pipecolic acid such as glycine,l-proline, 4-amino-n-butyric acid, and 5-amino-n-valeric acid showed an inhibitory effect on uptake at a concentration of 10^–4 M. The demonstration of biosynthesis of pipecolic acid in mouse brain and the presence of a high-affinity sodium-dependent uptake system suggest a physiological role of this substance in the central nervous system.