Absence Of External Substrate Research Articles

New Scandium Borylimido Chemistry: Synthesis, Bonding, and Reactivity.

We report a combined synthetic, mechanistic, and theoretical study of the first borylimido complex of a rare earth metal, (NacNacNMe2)Sc{NB(NAr'CH)2} (25, Ar' = 2,6-C6H3iPr2, NacNacNMe2 = Ar'NC(Me)CHC(Me)NCH2CH2NMe2). Thermolysis of the methyl-borylamide (NacNacNMe2)Sc(Me){NHB(NAr'CH)2} (18) generated transient imide 25 via rate-determining, first-order methane elimination (KIE ≈ 8.7). In the absence of external substrate, 25 underwent a reversible cyclometalation reaction (sp3 C-H bond addition to Sc═Nimide) with a methyl group of the NacNacNMe2 ligand forming {MeC(NC6H3iPrCH(Me)CH2)CHC(Me)NCH2CH2NMe2}Sc{NHB(NAr'CH)2} (21). In the presence of pyridine or DMAP, reversible sp2 C-H bond activation occurred, forming orthometalated complexes (NacNacNMe2)Sc{NHB(NAr'CH)2}(η2-4-NC5H3R) (R = H or NMe2). In situ reaction of 25 with HCCTol gives irreversible sp C-H bond activation under kinetic control, and with MeCCPh [2+2] cycloaddition to Sc═Nimide takes place. These reactions represent the first substrate activation processes for any metal-bound borylimide. The bonding in 25 and the mechanism and thermodynamics of the reactions have been studied using density functional theory (DFT), supported by quantum theory of atoms in molecules and natural bond orbital analysis. Although the borylimido and arylimido dianions studied here are formally isoelectronic and possess comparable frontier molecular orbitals, the borylimido ligand is both a better π-donor and σ-donor, forming stronger and shorter metal-nitrogen bonds with somewhat reduced ionicity. Despite this, reactions of these types of borylimides with C-H or C≡C bonds are all more exothermic and more strongly activating than for the corresponding arylimides. DFT calculations on model systems of differing steric bulk unpicked the underlying thermodynamic factors controlling the reactions of 25 and its reaction partners, and a detailed comparison was made with the previously described arylimido homologues.

Effects of heavy metal cations on the mitochondrial ornithine/citrulline transporter reconstituted in liposomes

The effect of heavy metal cations on the mitochondrial ornithine/citrulline transporter was tested in proteoliposomes reconstituted with the protein purified from rat liver. The transport activity was measured as [(3)H]ornithine uptake in proteoliposomes containing internal ornithine (ornithine/ornithine antiport mode) or as [(3)H]ornithine efflux in the absence of external substrate (ornithine/H(+) transport mode). 0.1mM Cu(2+), Pb(2+), Hg(2+), Cd(2+) and Zn(2+) strongly inhibited (more than 85%) the antiport; whereas Mn(2+), Co(2+) and Ni(2+) inhibited less efficiently (25, 47 and 69%, respectively). The IC(50) values of the transporter for the different metal ions ranged from 0.71 to 350μM. Co(2+) and Ni(2+) also inhibited the [(3)H]ornithine efflux whereas Cu(2+), Pb(2+), Hg(2+), Cd(2+) and Zn(2+) stimulated the [(3)H]ornithine efflux. The stimulation of the [(3)H]ornithine efflux by Cu(2+) and Cd(2+) (as well as by Pb(2+), Hg(2+) and Zn(2+)) was not prevented by NEM and was reversed by DTE. These features indicated that the inhibition of the antiport was due to the interaction of the Cu(2+), Pb(2+), Hg(2+), Cd(2+) and Zn(2+) with a population of SH groups, of the transporter, responsible for the inhibition of the physiological function; whereas the stimulation of [(3)H]ornithine efflux was due to the induction of a pore-like function of the transporter caused by interaction of cations with a different population of SH groups. Differently, the inhibition of the ornithine transporter by Ni(2+), Co(2+) or Mn(2+) was caused by interaction with the substrate binding site, as indicated by the competitive or mixed inhibition.

Substrate‐ and Ubiquitin‐Dependent Trafficking of the Yeast Siderophore Transporter Sit1

Eukaryotic plasma membrane transporters are subjected to a tightly regulated intracellular trafficking. The yeast siderophore iron transporter1 (Sit1) displays substrate-regulated trafficking. It is targeted to the plasma membrane or to a vacuolar degradative pathway when synthesized in the presence or absence of external substrate, respectively. Sorting of Sit1 to the vacuolar pathway is dependent on the clathrin adaptor Gga2, and more specifically on its C-GAT subdomain. Plasma membrane undergoes substrate-induced ubiquitylation dependent on the Rsp5 ubiquitin protein ligase. Sit1 is also ubiquitylated in an Rsp5-dependent manner in internal compartments when expressed in the absence of substrate. In several rsp5 mutants including cells deleted for RSP5, Sit1 expressed in the absence of substrate is correctly targeted to the endosomal pathway but its sorting to multivesicular bodies (MVBs) is impaired. Consequently, it displays endosome to plasma membrane targeting, with kinetics similar to those observed in vps mutants defective for MVB sorting. Plasma membrane Sit1 is modified by Lys63-linked ubiquitin chains. We also show for the first time in yeast that modification by this latter type of ubiquitin chains is required directly or indirectly for efficient MVB sorting, as it is for efficient internalization at the plasma membrane.

Fast, Triangular Voltage Clamp for Recording and Kinetic Analysis of an Ion Transporter Expressed in Xenopus Oocytes

We present a procedure for determination of 11 system parameters of an ion transporter expressed in Xenopus oocytes. The experiments consist of fast triangular voltage-clamp experiments in the presence and absence of external substrate. A four-state enzymatic cycle operating between an external and an internal section of electrodiffusion is used for analysis. The explicit example treats experiments with the fungal 2 H + - N O 3 − symporter EnNRT, a member of the major superfamily transporters. The results comprise a density of ≈150 fmol functional transporter molecules per oocyte, a gross charge number z E ≈ −0.3 of the empty binding site of the enzyme, individual rate constants for reorientation of the empty and occupied binding site in the range of 5–500 s −1, electrical access sections between bulk solutions and reaction cycle of ∼3% inside and 15% outside, an increase of internal N O 3 − at the plasma membrane from ∼0.5 to ∼2 mM during exposure to external N O 3 − , and K D ≈ 0.3 μM 3 inside and K D ≈ 3 μM 3 outside in binding the triplicate substrate ( 2 H + + N O 3 − ). The results compare well with the known structure of the lactose permease, another major superfamily transporter.

The human tumour suppressor gene SLC5A8 expresses a Na+-monocarboxylate cotransporter.

The orphan cotransport protein expressed by the SLC5A8 gene has been shown to play a role in controlling the growth of colon cancers, and the silencing of this gene is a common and early event in human colon neoplasia. We expressed this protein in Xenopus laevis oocytes and have found that it transports small monocarboxylic acids. The electrogenic activity of the cotransporter, which we have named SMCT (sodium monocarboxylate transporter), was dependent on external Na(+) and was compatible with a 3 : 1 stoichiometry between Na(+) and monocarboxylates. A portion of the SMCT-mediated current was also Cl(-) dependent, but Cl(-) was not cotransported. SMCT transports a variety of monocarboxylates (similar to unrelated monocarboxylate transport proteins) and most transported monocarboxylates demonstrated K(m) values near 100 microm, apart from acetate and d-lactate, for which the protein showed less affinity. SMCT was strongly inhibited by 1 mm probenecid or ibuprofen. In the absence of external substrate, a Na(+)-independent leak current was also observed to pass through SMCT. SMCT activity was strongly inhibited after prolonged exposure to high external concentrations of monocarboxylates. The transport of monocarboxylates in anionic form was confirmed by the observation of a concomitant alkalinization of the cytosol. SMCT, being expressed in colon and kidney, represents a novel means by which Na(+), short-chain fatty acids and other monocarboxylates are transported in these tissues. The significance of a Na(+)-monocarboxylate transporter to colon cancer presumably stems from the transport of butyrate, which is well known for having anti-proliferative and apoptosis-inducing activity in colon epithelial cells.

Response of a strict anaerobe to oxygen: survival strategies in Desulfovibrio gigas.

The biochemical response to oxygen of the strictly anaerobic sulfate-reducing bacterium Desulfovibrio gigas was studied with the goal of elucidating survival strategies in oxic environments. Cultures of D. gigas on medium containing lactate and sulfate were exposed to oxygen (concentration 5-120 micro M). Growth was fully inhibited by oxygen, but the cultures resumed growth as soon as they were shifted back to anoxic conditions. Following 24 h exposure to oxygen the growth rate was as high as 70 % of the growth rates observed before oxygenation. Catalase levels and activity were enhanced by exposure to oxygen whereas superoxide-scavenging and glutathione reductase activities were not affected. The general pattern of cellular proteins as analysed by two-dimensional electrophoresis was altered in the presence of oxygen, the levels of approximately 12 % of the detected proteins being markedly increased. Among the induced proteins, a homologue of a 60 kDa eukaryotic heat-shock protein (Hsp60) was identified by immunoassay analysis. In the absence of external substrates, the steady-state levels of nucleoside triphosphates detected by in vivo (31)P-NMR under saturating concentrations of oxygen were 20 % higher than under anoxic conditions. The higher energy levels developed under oxygen correlated with a lower rate of substrate (glycogen) mobilization, but no experimental evidence for a contribution from oxidative phosphorylation was found. The hypothesis that oxygen interferes with ATP dissipation processes is discussed.

Citrin and aralar1 are Ca(2+)-stimulated aspartate/glutamate transporters in mitochondria.

The mitochondrial aspartate/glutamate carrier catalyzes an important step in both the urea cycle and the aspartate/malate NADH shuttle. Citrin and aralar1 are homologous proteins belonging to the mitochondrial carrier family with EF-hand Ca(2+)-binding motifs in their N-terminal domains. Both proteins and their C-terminal domains were overexpressed in Escherichia coli, reconstituted into liposomes and shown to catalyze the electrogenic exchange of aspartate for glutamate and a H(+). Overexpression of the carriers in transfected human cells increased the activity of the malate/aspartate NADH shuttle. These results demonstrate that citrin and aralar1 are isoforms of the hitherto unidentified aspartate/glutamate carrier and explain why mutations in citrin cause type II citrullinemia in humans. The activity of citrin and aralar1 as aspartate/glutamate exchangers was stimulated by Ca(2+) on the external side of the inner mitochondrial membrane, where the Ca(2+)-binding domains of these proteins are localized. These results show that the aspartate/glutamate carrier is regulated by Ca(2+) through a mechanism independent of Ca(2+) entry into mitochondria, and suggest a novel mechanism of Ca(2+) regulation of the aspartate/malate shuttle.

Simultaneous storage and degradation of phb and glycogen in activated sludge cultures

Bacteria in activated sludge are subjected to periods of substrate availability and absence of external substrates. The response of bacteria to such dynamic conditions was studied in a 2 L sequencing batch reactor (SBR) by subjecting a mixed microbial population to successive periods of external substrate availability (feast period) and no external substrate availability (famine period). In previous studies, acetate or glucose was used as single substrate leading to the storage of polyhydroxybutyrate or glycogen, respectively. In this study, a mixture of acetate and glucose was used. It appeared that both substrates were consumed simultaneously. The relative contribution of growth and storage processes was in these experiments similar as in the systems fed with a single substrate only. The ratio of substrate uptake over substrate storage was 0.6 Cmol/Cmol for both substrates. The uptake rate of acetate was not influenced by the simultaneous uptake of glucose. The degradation kinetics and rates of the storage compounds were the same as for the systems in which only one compound was stored in the activated sludge. The global performance of the culture grown on mixed substrates could therefore be described as the sum of the conversions observed in cultures fed with the individual substrates.

Arg-425 of the Citrate Transporter CitP Is Responsible for High Affinity Binding of Di- and Tricarboxylates

The citrate transporter of Leuconostoc mesenteroides (CitP) catalyzes exchange of divalent anionic citrate from the medium for monovalent anionic lactate, which is an end product of citrate degradation. The exchange generates a membrane potential and thus metabolic energy for the cell. The mechanism by which CitP transports both a divalent and a monovalent substrate was the subject of this investigation. Previous studies indicated that CitP is specific for substrates containing a 2-hydroxycarboxylate motif, HO-CR(2)-COO(-). CitP has a high affinity for substrates that have a "second" carboxylate at one of the R groups, such as divalent citrate and (S)-malate (Bandell, M., and Lolkema, J. S. (1999) Biochemistry 38, 10352-10360). Monovalent anionic substrates that lack this second carboxylate were found to bind with a low affinity. In the present study we have constructed site-directed mutants, changing Arg-425 into a lysine or a cysteine residue. By using two substrates, i.e. (S)-malate and 2-hydroxyisobutyrate, the substrate specificity of the mutants was analyzed. In both mutants the affinity for divalent (S)-malate was strongly decreased, whereas the affinity for monovalent 2-hydroxyisobutyrate was not. The largest effect was seen when the arginine was changed into the neutral cysteine, which reduced the affinity for (S)-malate over 50-fold. Chemical modification of the R425C mutant with the sulfhydryl reagent 2-aminoethyl methanethiosulfonate, which restores the positive charge at position 425, dramatically reactivated the mutant transporter. The R425C and R425K mutants revealed a substrate protectable inhibition by other sulfhydryl reagents and the lysine reagent 2,4,6-trinitrobenzene sulfonate, respectively. It is concluded that Arg-425 complexes the charged carboxylate present in divalent substrates but that is absent in monovalent substrates, and thus plays an important role in the generation of the membrane potential.

Stoichiometry and Kinetics of the High-affinity H+-coupled Peptide Transporter PepT2

Proton-coupled peptide transporters mediate the absorption of a large variety of di- and tripeptides as well as peptide-like pharmacologically active compounds. We report a kinetic analysis of the rat kidney high-affinity peptide transporter PepT2 expressed in Xenopus oocytes. By use of simultaneous radioactive uptake and current measurements under voltage-clamp condition, the charge to substrate uptake ratio was found to be close to 2 for both D-Phe-L-Ala and D-Phe-L-Glu, indicating that the H+:substrate stoichiometry is 2:1 and 3:1 for neutral and anionic dipeptides, respectively. The higher stoichiometry for anionic peptides suggests that they are transported in the protonated form. For D-Phe-L-Lys, the charge:uptake ratio averaged 2.4 from pooled experiments, suggesting that Phe-Lys crosses the membrane via PepT2 either in its deprotonated (neutral) or its positively charged form, averaging a H+:Phe-Lys stoichiometry of 1.4:1. These findings led to the overall conclusion that PepT2 couples transport of one peptide molecule to two H+. This is in contrast to the low-affinity transporter PepT1 that couples transport of one peptide to one H+. Quinapril inhibited PepT2-mediated currents in presence or in absence of external substrates. Oocytes expressing PepT2 exhibited quinapril-sensitive outward currents. In the absence of external substrate, a quinapril-sensitive proton inward current (proton leak) was also observed which, together with the observed pH-dependent PepT2-specific presteady-state currents (Ipss), indicates that at least one H+ binds to the transporter prior to substrate. PepT2 exhibited Ipss in response to hyperpolarization at pH 6.5-8.0. However, contrary to previous observations on various transporters, 1) no significant currents were observed corresponding to voltage jumps returning from hyperpolarization, and 2) at reduced extracellular pH, no significant Ipss were observed in either direction. Together with observed lower substrate affinities and decreased PepT2-mediated currents at hyperpolarized Vm, our data are consistent with the concept that hyperpolarization exerts inactivation effects on the transporter which are enhanced by low pH. Our studies revealed distinct properties of PepT2, compared with PepT1 and other ion-coupled transporters.

Maintenance, endogeneous respiration, lysis, decay and predation

In activated sludge processes an increased sludge age is associated with a decreased sludge production. This phenomenon is generally interpreted as a result of endogenous respiration processes. In the activated sludge models cell lysis (or decay) is incorporated. The lysis is modelled such that it leads to generation of particulate substrate, which by a hydrolysis process is converted into soluble substrate. The substrate is then converted to biomass again by growth processes. In this manner a good description of activated sludge processes is obtained, however this does not mean that the proposed mechanism is microbiologically correct. The lysis/decay model mechanism is a strongly simplified representation of reality. This paper tries to review the processes grouped under endogenous respiration in activated sludge models. Mechanisms and processes such as maintenance, lysis, internal and external decay, predation and death-regeneration are discussed. From recent microbial research it has become evident that cells do not die by themselves. Bacteria are however subject to predation by protozoa. Bacteria store reserve polymers that in absence of external substrate are used for growth and maintenance processes. This conversion will in general be denoted as endogenous respiration. Based on the literature review the phenomena are discussed and organised, in order to create a working platform for discussing more detailed activated sludge models, one of which is being sketched.

Maintenance, endogeneous respiration, lysis, decay and predation

Research Article| January 01 1999 Maintenance, endogeneous respiration, lysis, decay and predation Mark C. M. Van Loosdrecht; Mark C. M. Van Loosdrecht *Kluyver Institute for Biotechnology, TU-Delft, Julianalaan 672628 BC Delft, The Netherlands Search for other works by this author on: This Site PubMed Google Scholar Mogens Henze Mogens Henze **Department of Environmental Science and Engineering, Building 115, Technical University of Denmark, DK-2800 Lyngby, Denmark Search for other works by this author on: This Site PubMed Google Scholar Water Sci Technol (1999) 39 (1): 107–117. https://doi.org/10.2166/wst.1999.0024 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Share Icon Share Twitter LinkedIn Tools Icon Tools Cite Icon Cite Permissions Search Site Search Dropdown Menu nav search search input Search input auto suggest search filter All ContentAll JournalsThis Journal Search Advanced Search Citation Mark C. M. Van Loosdrecht, Mogens Henze; Maintenance, endogeneous respiration, lysis, decay and predation. Water Sci Technol 1 January 1999; 39 (1): 107–117. doi: https://doi.org/10.2166/wst.1999.0024 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex In activated sludge processes an increased sludge age is associated with a decreased sludge production. This phenomenon is generally interpreted as a result of endogenous respiration processes. In the activated sludge models cell lysis (or decay) is incorporated. The lysis is modelled such that it leads to generation of particulate substrate, which by a hydrolysis process is converted into soluble substrate. The substrate is then converted to biomass again by growth processes. In this manner a good description of activated sludge processes is obtained, however this does not mean that the proposed mechanism is microbiologically correct. The lysis/decay model mechanism is a strongly simplified representation of reality. This paper tries to review the processes grouped under endogenous respiration in activated sludge models. Mechanisms and processes such as maintenance, lysis, internal and external decay, predation and death-regeneration are discussed. From recent microbial research it has become evident that cells do not die by themselves. Bacteria are however subject to predation by protozoa. Bacteria store reserve polymers that in absence of external substrate are used for growth and maintenance processes. This conversion will in general be denoted as endogenous respiration. Based on the literature review the phenomena are discussed and organised, in order to create a working platform for discussing more detailed activated sludge models, one of which is being sketched. Maintenance, activated sludge, decay, endogenous respiration, lysis, predation, modelling, ASM1, ASM2, ASM3 This content is only available as a PDF. © IWA Publishing 1999 You do not currently have access to this content.

Differential Contribution of Oxidative and Glycolytic Energy to Thrombin-Induced Platelet Functions

SummaryWhen washed rabbit-blood platelets were preincubated in an artificial medium in the absence of external substrates, they aggregated in response to a low concentration of thrombin. The aggregation was completely inhibited after the preincubation with respiratory inhibitors. When glucose together with the respiratory inhibitors was added during the incubation, the aggregation was accelerated, whilst it was counteracted when Krebs-cycle substrates were added.ATP was generated actively during the incubation in the absence of external substrates, as well as in the presence of succinate. The ATP-generation was extremely inhibited by oligomycin. When glucose was added during the incubation with the respiratory inhibitor, the comparable amount of ATP with those in the oxidative systems was generated. Metabolic ADP was accumulated in the oxidative systems, particularly in the presence of succinate, in contrast to its low level in the glucose + oligomycin system. The results suggest that the counteraction of the aggregation by the Krebs-cycle substrate is attributed to the low adenylate energy charge. It is suggested that anaerobic glycolysis creates favorable energy condition for aggregation as compared with oxidative phosphorylation, although the washed platelets can be energized to a level above threshold of the aggregation when either one of the two energy generating systems is exerted.After the incubation of the platelets in the presence of thrombin, a higher level of metabolic ATP was observed under glycolytic condition, than under oxidative condition. Pulse-labelling experiments showed that ADP produced during the aggregation was rephosphorylated in a later part of the incubation in the glucose + KCN-fortified system. In the succinate-fortified system, the re-phosphorylation was very slow. The results suggest that oxidative phosphorylation is reduced in platelet aggregates treated with thrombin.

The Effects of Temperature and Some Inhibitors an the Carnitine Exchange System of Heart Mitochondria

1. 11-Trimethylamino-undecanoyl-L-carnitine (Ki = 56 muM) and 6-trimethylaminohexanoyl-L-carnitine (Ki = 1.3 mM) are competitive inhibitors of the ox heart mitochondrial carnitine exchange system. The latter is itself a poor substrate, with a V about 0.7% of that for carnitine. These compounds have been used as 'stop inhibitors' in a study of the effect of temperature on the carnitine exchange. 2. The carnitineout in equilibrium carnitinein exchange has a temperature coefficient (Q10) of 8.5 and an activation energy of 176 kJ/mol which is constant from 0 degrees - 18 degrees C. The Km for L-carnitine (5.3 mM) is also constant over this temperature range. 3. The slow efflux (leak) of mitochondrial L-[14C]carnitine in the absence of external substrate has a similar temperature dependence and is also inhibited by 11-trimethylamino-undecanoyl-L-carnitine. It is probably mediated by the same system as the very much faster exchange with external carnitine.

In vitro and in vivo germination of spores of Marssonina brunnea (Ell. et Ev.) P. Magn

A water soluble inhibitor of germination is present on the surfaces of freshly collected spores of Marssonina brunnea (Ell et Ev.) P. Magn. Spores of this pathogen have a high potential for germination even in the absence of external substrates. Among the poplar clones examined there was no correlation between spore germination on leaf discs and the disease reaction of the discs under controlled conditions. However under these conditions the disease development on leaf discs taken from clone I-69/55 was correlated with the high resistance of this clone to Marssonina leaf spot in the field.

Oxidative and biosynthetic utilization by human spermatozoa of a metabolite of the female reproductive tract.

In vitro investigations of the metabolism of mammalian spermatozoa have amply demonstrated their ability to utilize a variety of glycolysable and oxidizable substrates1. No information is, however, available on the metabolism of spermatozoa within the female reproductive tract. If, as is probable, the epithelial cells use up not only the glucose supplied by the circulatory system, but also oxidizable intermediates such as pyruvate and lactate, the spermatozoa in contact with those cells would have to oxidize their endogenous reserves of lipid material, provided that the oxygen tension in the various regions of tho tract is adequate to support the aerobic metabolism of the spermatozoa. In the absence of external substrates the conditions in the female genital tract would not be favourable for the survival of spermatozoa for prolonged periods of time. Whereas this may not affect fertility in animal species in which mating occurs at the time of oestrus, with rapid ascent of an adequate number of spermatozoa, a limited survival capacity due to the lack of substrates may affect the prospects of fertilization in man.

Endogenous alcoholic fermentation in yeast induced by 2, 4-dinitrophenol

1. 1. Although well-starved yeast, under anaerobic conditions, in the absence of external substrate, produces no readily measurable quantity of CO 2, the addition of appropriate concentrations of 2, 4-dinitrophenol (DNP) or of azide can induce an appreciable CO 2 production. 2. 2. The DNP-induced CO 2 production is associated with the production of ethyl alcohol, which was specifically identified by the melting point of its p-nitrobenzoyl derivative. At least one-half equivalent of alcohol was recovered for each equivalent of CO 2 produced. 3. 3. At least one-third of the substrate for the DNP-induced fermentation is glycogen. Almost one-half is acid-hydrolyzable, reducible, carbohydrate reserve. 4. 4. It is concluded that glycogen can be made available as a substrate for alcoholic fermentation, by the action of DNP or azide on an “uncoupling mechanism” which, in the normal cell, prevents the fermentation of glycogen.