The concentration of amino acids by yeast cells depleted of adenosine triphosphate.
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ABSTRACT:
SUBMITTER: Eddy AA
PROVIDER: S-EPMC1179680 | biostudies-other | 1970 Dec
REPOSITORIES: biostudies-other
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Project description:1. Despite the depletion of both their content of exchangeable endogenous amino acids and reserves of ATP, starved hypo-osmotically shocked preparations of the tumour cells accumulated relatively large amounts of (14)C-labelled 2-aminoisobutyrate, l-alanine, glycine, l-leucine, l-methionine, l-phenylalanine and l-serine, against their respective concentration gradients, by a process apparently driven by the spontaneous flow of Na(+) ions into the cellular phase. Dependent on (a) which compound was used, (b) its concentration and (c) the direction of the Na(+) ion gradient, the peak value of the ratio of the cellular to extracellular amino acid concentration varied from about 0.4 to 7. 2. The extent to which ATP increased the ratio was defined for l-methionine. 3. Chemical analysis of the cellular amino acid content showed that this increased in parallel with the absorption of (14)C. 4. The accumulation of l-methionine and of glycine, against their own concentration gradients, continued in the presence of either 0.3mm-ouabain or 10mug of oligomycin/ml. Thus the sodium pump was probably not involved in the process when ATP was lacking. 5. l-Leucine caused 0.72+/-0.12 (s.e.m.; 6) extra equivalents of Na(+) to enter the shocked starved tumour cells in parallel with the uptake of leucine itself. Only a small loss of K(+) was induced. 6. The influx and efflux of l-methionine in preparations depleted of ATP were both markedly accelerated by the presence of Na(+) ions. 7. The observations provide further examples of the application of the ion-gradient hypothesis, according to which Na(+) ions act as co-substrates of the amino acid pump. The quantitative importance of parallel Na(+)-independent systems was studied with a new mathematical model.
| S-EPMC1177783 | biostudies-other
Project description: Not available
| S-EPMC6263008 | biostudies-literature
Project description:1. At pH 4.5 and 30degreesC, yeast preparations depleted of ATP in the presence of antimycin and deoxyglucose spontaneously lost K+, gaining roughly an equivalent amount of H+. 2. Five proton conductors including azide and 2,4-dinitrophenol accelerated this process, as did [14C]glycine, which was absorbed with two extra equivalents of H+. 3. The rate of glycine uptake at pH 4.5 diminished fourfold when cellular K+ fell by 20%. 4. The distribution of [14C]propionate indicated that the intracellular pH fell from 6.2 to 5.7 when the cellular content of K+ fell by 30%. 5. Glycine uptake from a 5 muM solution was about 400 times faster at pH 4.5 than it was at pH 7.4 with 100mM-KC1 present ostensibly to lower the membrane potential. 6. Yeast preparations containing 2mM-[14C]glycine absorbed a further amount from a 0.1 muM solution at pH 4.5. After about 10 min a net movement of [14C]glycine out of the yeast occurred. The ratio of the cellular [14Ia1glycine concentration to the concentration outside the yeast reached 4 X 10(4) in these assays, whereas at pH 7.4 in the presence of 100mM-KC1 it did not exceed 15 in 3h. Dimitrophenol lowered the accumulation ratio at pH 4.5, apparently by causing proton conduction. 7. The observations are consistent with the notion that glycine uptake is driven by a proton symport mechanism. 8. Possible factors governing the strikingly low rate of glycine efflux as opposed to its optimum rate of influx are discussed.
| S-EPMC1172769 | biostudies-other
Project description:PurposeTo test whether adenosine triphosphate (ATP) release links cytoskeletal remodeling with release of matrix metalloproteinases (MMPs), regulators of outflow facility and intraocular pressure.MethodsATP release was measured by luciferin-luciferase. Ecto-ATPases from transformed human trabecular meshwork (TM) cells (TM5) and explant-derived TM cells were identified by RT-PCR. Actin was visualized by phalloidin staining. Cell viability was assayed by lactate dehydrogenase and thiazolyl blue tetrazolium bromide methods and propidium iodide exclusion, gene expression by real-time PCR, and MMP release by zymography. Cell volume was monitored by electronic cell sorting.ResultsHypotonicity (50%) and mechanical stretch increased ATP release with similar pharmacologic profiles. TM cells expressed ecto-ATPases E-NPP1-3, E-NTPD2, E-NTPD8, and CD73. Prolonged dexamethasone (DEX) exposure (? 2 weeks), but not brief exposure (3 days), increased cross-linked actin networks and reduced swelling-triggered ATP release. Cytochalasin D (CCD) exerted opposite effects. Neither DEX nor CCD altered the cell viability, gene expression, or pharmacologic profile of ATP-release pathways. DEX accelerated, and CCD slowed, the regulatory volume decrease after hypotonic exposure. Activating A(1) adenosine receptors (A(1)ARs) increased total MMP-2 and MMP-9 release. DEX reduced total A(1)AR-triggered MMP release, and CCD increased the active form of MMP-2 release. The A(1)AR agonist CHA and the A(1)AR antagonist DPCPX partially reversed the effects of DEX and CCD, respectively.ConclusionsCytoskeletal restructuring modulated swelling-activated ATP release, in part by changing the duration of cell swelling after hypotonic challenge. Modifying ATP release is expected to modulate MMP secretion by altering ecto-enzymatic delivery of adenosine to A(1)ARs, linking cytoskeletal remodeling and MMP-mediated modulation of outflow facility.
| S-EPMC3220413 | biostudies-literature
Project description:The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) (CRISPR/Cas) systems have recently emerged as powerful molecular biosensing tools based on their collateral cleavage activity due to their simplicity, sensitivity, specificity, and broad applicability. However, the direct application of the collateral cleavage activity for in situ intracellular detection is still challenging. Here, we debut a CRISPR/Cas-assisted nanoneedle sensor (nanoCRISPR) for intracellular adenosine triphosphate (ATP), which avoids the challenges associated with intracellular collateral cleavage by introducing a two-step process of intracellular target recognition, followed by extracellular transduction and detection. ATP recognition occurs by first presenting in the cell cytosol an aptamer-locked Cas12a activator conjugated to nanoneedles; the recognition event unlocks the activator immobilized on the nanoneedles. The nanoneedles are then removed from the cells and exposed to the Cas12a/crRNA complex, where the activator triggers the cleavage of an ssDNA fluorophore-quencher pair, generating a detectable fluorescence signal. NanoCRISPR has an ATP detection limit of 246 nM and a dynamic range from 1.56 to 50 μM. Importantly, nanoCRISPR can detect intracellular ATP in 30 min in live cells without impacting cell viability. We anticipate that the nanoCRISPR approach will contribute to broadening the biomedical applications of CRISPR/Cas sensors for the detection of diverse intracellular molecules in living systems.
| S-EPMC10623508 | biostudies-literature
Project description:Yeast assimilable nitrogen (YAN) is essential for yeast growth and metabolism during apple (Malus x domestica Borkh.) cider fermentation. YAN concentration and composition can impact cider fermentation kinetics and the formation of volatile aroma compounds by yeast. The YAN concentration and composition of apples grown in Virginia, USA over the course of two seasons was determined through analysis of both free amino nitrogen (FAN) and ammonium ion concentration. FAN was the largest fraction of YAN, with a mean value of 51 mg N L-1 FAN compared to 9 mg N L-1 ammonium. Observed YAN values ranged from nine to 249 mg N L-1, with a mean value of 59 mg N L-1. Ninety-four percent of all samples analyzed in this study contained <140 mg N L-1 YAN, a concentration generally considered the minimum level needed in grape-based wines for yeast to fully utilize all of the fermentable sugars. FAN concentration was correlated with total YAN concentration, but ammonium concentration was not. Likewise, there was no correlation between FAN and ammonium concentration.
| S-EPMC5778214 | biostudies-literature
Project description:Adenosine triphosphate phosphoribosyltransferase (ATP-PRT) catalyzes the first step in histidine biosynthesis, a pathway essential to microorganisms and a validated target for antimicrobial drug design. The ATP-PRT enzyme catalyzes the reversible substitution reaction between phosphoribosyl pyrophosphate and ATP. The enzyme exists in two structurally distinct forms, a short- and a long-form enzyme. These forms share a catalytic core dimer but bear completely different allosteric domains and thus distinct quaternary assemblies. Understanding enzymatic transition states can provide essential information on the reaction mechanisms and insight into how differences in domain structure influence the reaction chemistry, as well as providing a template for inhibitor design. In this study, the transition state structures for ATP-PRT enzymes from Campylobacter jejuni and Mycobacterium tuberculosis (long-form enzymes) and from Lactococcus lactis (short-form) were determined and compared. Intrinsic kinetic isotope effects (KIEs) were obtained at reaction sensitive positions for the reverse reaction using phosphonoacetic acid, an alternative substrate to the natural substrate pyrophosphate. The experimental KIEs demonstrated mechanistic similarities between the three enzymes and provided experimental boundaries for quantum chemical calculations to characterize the transition states. Predicted transition state structures support a dissociative reaction mechanism with a DN*AN‡ transition state. Weak interactions from the incoming nucleophile and a fully dissociated ATP adenine are predicted regardless of the difference in overall structure and quaternary assembly. These studies establish that despite significant differences in the quaternary assembly and regulatory machinery between ATP-PRT enzymes from different sources, the reaction chemistry and catalytic mechanism are conserved.
| S-EPMC6693643 | biostudies-literature
Project description:BackgroundHuman PMS2 (hPMS2) homologues act to nick 5' and 3' to misincorporated nucleotides during mismatch repair in organisms that lack MutH. Mn(++) was previously found to stimulate the endonuclease activity of these homologues. ATP was required for the nicking activity of hPMS2 and yPMS1, but was reported to inhibit bacterial MutL proteins from Thermus thermophilus and Aquifex aeolicus that displayed homology to hPMS2. Mutational analysis has identified the DQHA(X)(2)E(X)(4)E motif present in the C-terminus of PMS2 homologues as important for endonuclease activity.Methodologies/principal findingsWe examined the effect ATP had on the Mn(++) induced nicking of supercoiled pBR322 by full-length and mutant A. aeolicus MutL (Aae MutL) proteins. Assays were single time point, enzyme titration experiments or reaction time courses. The maximum velocity for MutL nicking was determined to be 1.6+/-0.08x10(-5) s(-1) and 4.2+/-0.3x10(-5) s(-1) in the absence and presence of ATP, respectively. AMPPNP stimulated the nicking activity to a similar extent as ATP. A truncated Aae MutL protein composed of only the C-terminal 123 amino acid residues was found to nick supercoiled DNA. Furthermore, mutations in the conserved C-terminal DQHA(X)(2)E(X)(4)E and CPHGRP motifs were shown to abolish Aae MutL endonuclease activity.ConclusionsATP stimulated the Mn(++) induced endonuclease activity of Aae MutL. Experiments utilizing AMPPNP implied that the stimulation did not require ATP hydrolysis. A mutation in the DQHA(X)(2)E(X)(4)E motif of Aae MutL further supported the role of this region in endonclease activity. For the first time, to our knowledge, we demonstrate that changing the histidine residue in the conserved CPHGRP motif abolishes endonucleolytic activity of a hPMS2 homologue. Finally, the C-terminal 123 amino acid residues of Aae MutL were sufficient to display Mn(++) induced nicking activity.
| S-EPMC2744016 | biostudies-literature
Project description:The structural constituents of the large eukaryotic ribosomal subunit are 3 ribosomal RNAs, namely the 25S, 5.8S and 5S rRNA and about 46 ribosomal proteins (r-proteins). They assemble and mature in a highly dynamic process that involves more than 150 proteins and 70 small RNAs. Ribosome biogenesis starts in the nucleolus, continues in the nucleoplasm and is completed after nucleo-cytoplasmic translocation of the subunits in the cytoplasm. In this work we created 26 yeast strains, each of which conditionally expresses one of the large ribosomal subunit (LSU) proteins. In vivo depletion of the analysed LSU r-proteins was lethal and led to destabilisation and degradation of the LSU and/or its precursors. Detailed steady state and metabolic pulse labelling analyses of rRNA precursors in these mutant strains showed that LSU r-proteins can be grouped according to their requirement for efficient progression of different steps of large ribosomal subunit maturation. Comparative analyses of the observed phenotypes and the nature of r-protein-rRNA interactions as predicted by current atomic LSU structure models led us to discuss working hypotheses on i) how individual r-proteins control the productive processing of the major 5' end of 5.8S rRNA precursors by exonucleases Rat1p and Xrn1p, and ii) the nature of structural characteristics of nascent LSUs that are required for cytoplasmic accumulation of nascent subunits but are nonessential for most of the nuclear LSU pre-rRNA processing events.
| S-EPMC2788216 | biostudies-literature
Project description:The chemical synthesis of a adenosine triphosphate analogue, 1-adenosin-5'-yl 3-(3-methylbut-2-enyl) triphosphoric acid diester (ApppD), is described. ApppD is known to be an active metabolite of the mevalonate pathway in the human body like its structural isomer isopentenyl ester of ATP (ApppI). Very recently, ApppI has been found to possess novel function(s); now it will also be possible to examine the effects of ApppD more precisely because it can be synthesized in reasonable amounts. 1-Adenosin-5'-yl 3-(3-methylbut-2-enyl) diphosphoric acid diester (AppD; a adenosine diphosphate analogue) was also isolated from the synthesis mixture. Both ApppD and AppD were characterized by 1H, 13C, 31P NMR and mass spectrometry methods.
| S-EPMC6044877 | biostudies-literature