scholarly journals Binding of nicotinamide–adenine dinucleotides to diphtheria toxin

1967 ◽  
Vol 105 (2) ◽  
pp. 635-640 ◽  
Author(s):  
L. Montanaro ◽  
Simonetta Sperti
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Binding Sites ◽  
Diphtheria Toxin ◽  
Dissociation Constants ◽  
Emission Spectra ◽  
Aromatic Amino Acids ◽  
Protein Fluorescence ◽  
Free Nucleotides

1. Changes in protein fluorescence have been utilized in determining the stoicheiometry and dissociation constants of the complexes of diphtheria toxin with NADH2, NAD, NADPH2 and NADP. 2. The binding stoicheiometry is 2moles of NADH2 and 1mole of NADPH2/mole of diphtheria toxin. The binding sites for NADH2 appear to be equivalent and independent. 3. The toxin shows a higher affinity for the reduced than for the oxidized forms of the nucleotides. 4. Dissociation constants at 0·01I, pH7 and 25° are 0·7×10−6m for NADH2 and 0·45×10−6m for NADPH2. Dissociation constants increase with increasing ionic strength, indicating that the binding is mainly electrostatic. 5. Bound NADH2 and NADPH2 may be activated to fluoresce by the transfer of energy from the excited aromatic amino acids of the toxin. Activation and emission spectra of bound and free nucleotides are compared. 6. Since NAD and NADH2 are cofactors specifically required for the inhibition of protein synthesis by diphtheria toxin, the possible role of toxin–nucleotide complexes is discussed in this regard.

scholarly journals BIOSYNTHESIS IN ISOLATED ACETABULARIA CHLOROPLASTS

1972 ◽  
Vol 54 (2) ◽  
pp. 279-294 ◽  
Author(s):  
David C. Shephard ◽  
Wendy B. Levin
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Technical Problems ◽  
Hydrolyzed Protein ◽  
Protein Amino Acids ◽  
Amino Acid Labeling ◽  
Labeling Pattern

The ability of chloroplasts isolated from Acetabulana mediterranea to synthesize the protein amino acids has been investigated. When this chloroplast isolate was presented with 14CO2 for periods of 6–8 hr, tracer was found in essentially all amino acid species of their hydrolyzed protein Phenylalanine labeling was not detected, probably due to technical problems, and hydroxyproline labeling was not tested for The incorporation of 14CO2 into the amino acids is driven by light and, as indicated by the amount of radioactivity lost during ninhydrin decarboxylation on the chromatograms, the amino acids appear to be uniformly labeled. The amino acid labeling pattern of the isolate is similar to that found in plastids labeled with 14CO2 in vivo. The chloroplast isolate did not utilize detectable amounts of externally supplied amino acids in light or, with added adenosine triphosphate (ATP), in darkness. It is concluded that these chloroplasts are a tight cytoplasmic compartment that is independent in supplying the amino acids used for its own protein synthesis. These results are discussed in terms of the role of contaminants in the observed synthesis, the "normalcy" of Acetabularia chloroplasts, the synthetic pathways for amino acids in plastids, and the implications of these observations for cell compartmentation and chloroplast autonomy.

scholarly journals Visualizing Cancer Cell Metabolic Dynamics Regulated With Aromatic Amino Acids Using DO-SRS and 2PEF Microscopy

2021 ◽  
Vol 8 ◽  
Author(s):  
Pegah Bagheri ◽  
Khang Hoang ◽  
Anthony A. Fung ◽  
Sahran Hussain ◽  
Lingyan Shi
Keyword(s):  
Oxidative Stress ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Spatial Distribution ◽  
Lipid Droplets ◽  
De Novo ◽  
Culture Media ◽  
Aromatic Amino Acids ◽  
Unsaturated Lipids ◽  
Metabolic Activities

Oxidative imbalance plays an essential role in the progression of many diseases that include cancer and neurodegenerative diseases. Aromatic amino acids (AAA) such as phenylalanine and tryptophan have the capability of escalating oxidative stress because of their involvement in the production of Reactive Oxygen Species (ROS). Here, we use D2O (heavy water) probed stimulated Raman scattering microscopy (DO-SRS) and two Photon Excitation Fluorescence (2PEF) microscopy as a multimodal imaging approach to visualize metabolic changes in HeLa cells under excess AAA such as phenylalanine or trytophan in culture media. The cellular spatial distribution of de novo lipogenesis, new protein synthesis, NADH, Flavin, unsaturated lipids, and saturated lipids were all imaged and quantified in this experiment. Our studies reveal ∼10% increase in de novo lipogenesis and the ratio of NADH to flavin, and ∼50% increase of the ratio of unsaturated lipids to saturated lipid in cells treated with excess phenylalanine or trytophan. In contrast, these cells exhibited a decrease in the protein synthesis rate by ∼10% under these AAA treatments. The cellular metabolic activities of these biomolecules are indicators of elevated oxidative stress and mitochondrial dysfunction. Furthermore, 3D reconstruction images of lipid droplets were acquired and quantified to observe their spatial distribution around cells’ nuceli under different AAA culture media. We observed a higher number of lipid droplets in excess AAA conditions. Our study showcases that DO-SRS imaging can be used to quantitatively study how excess AAA regulates metabolic activities of cells with subcellular resolution in situ.

scholarly journals Key role of l-alanine in the control of hepatic protein synthesis

1987 ◽  
Vol 241 (2) ◽  
pp. 491-498 ◽  
Author(s):  
D Pérez-Sala ◽  
R Parrilla ◽  
M S Ayuso
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Physiological Role ◽  
Chain Elongation ◽  
Liver Protein ◽  
Isolated Liver Cells ◽  
Metabolic Transformation ◽  
Hepatic Protein Synthesis

We investigated the effects of administration of single amino acids to starved rats on the regulation of protein synthesis in the liver. Of all the amino acids tested, only alanine, ornithine and proline promoted statistically significant increases in the extent of hepatic polyribosome aggregation. The most effective of these was alanine, whose effect of promoting polyribosomal aggregation was accompanied by a decrease in the polypeptide-chain elongation time. The following observations indicate that alanine plays an important physiological role in the regulation of hepatic protein synthesis. Alanine was the amino acid showing the largest decrease in hepatic content in the transition from high (fed) to low (starved) rates of protein synthesis. The administration of glucose or pyruvate is also effective in increasing liver protein synthesis in starved rats, and their effects were accompanied by an increased hepatic alanine content. An increase in hepatic ornithine content does not lead to an increased protein synthesis, unless it is accompanied by an increase of alanine. The effect of alanine is observed either in vivo, in rats pretreated with cycloserine to prevent its transamination, or in isolated liver cells under conditions in which its metabolic transformation is fully impeded.

Glucose-dependent control of leucine metabolism by leucyl-tRNA synthetase 1

Science ◽  
2019 ◽  
pp. eaau2753 ◽  
Author(s):  
Ina Yoon ◽  
Miso Nam ◽  
Hoi Kyoung Kim ◽  
Hee-Sun Moon ◽  
Sungmin Kim ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Energy Production ◽  
Trna Synthetase ◽  
Glucose Starvation ◽  
Catabolic Pathway ◽  
Inhibit Protein Synthesis ◽  
Support Cell ◽  
Save Energy

Despite the importance of glucose and amino acids for energy metabolism, interactions between the two nutrients are not well understood. We provide evidence for a role of leucyl-tRNA synthetase 1 (LARS1) in glucose-dependent control of leucine usage. Upon glucose starvation, LARS1 was phosphorylated by Unc-51 like autophagy activating kinase 1 (ULK1) at the residues crucial for leucine-binding. The phosphorylated LARS1 showed decreased leucine-binding, which may inhibit protein synthesis and help save energy. Leucine, not used to anabolic process, may be available to catabolic pathway for energy generation. The LARS1-mediated changes in leucine utilization might help support cell survival deprived of glucose. Thus, dependent on the availability of glucose, LARS1 may help regulate whether leucine is used for protein synthesis or energy production.

Chain Elongation of Aromatic Amino Acids: the Role of 2-Benzylmalic Acid in the Biosynthesis of a C6C4 Amino Acid and a C6C3 Mustard Oil Glucoside

1974 ◽  
Vol 52 (10) ◽  
pp. 916-921 ◽  
Author(s):  
D. Dörnemann ◽  
W. Löffelhardt ◽  
H. Kindl
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Aromatic Amino Acids ◽  
Chain Elongation ◽  
Mustard Oil ◽  
Barbarea Vulgaris ◽  
Nasturtium Officinale ◽  
A Chain ◽  
Phenylbutyric Acid

A chemical synthesis of specifically 14C-labelled 2-benzylmalic acid, hitherto unknown, was developed. 4-Phenylacetoacetate obtained by condensation of phenylacetyl chloride-1-14C with ethyl acetoacetate yielded 2-benzylmalic acid-2-14C after cyanohydrin reaction and hydrolysis.2-Benzylmalic acid-2-14C, administered to shoots of Nasturtium officinale and Barbarea vulgaris, was shown to be an efficient precursor of the aglucone moiety of the mustard oil glucoside gluconasturtiin. The incorporation of radioactivity agreed well with the values reported for incorporation of 3-benzylmalic acid, but was considerably higher than that obtained after application of L-phenylalanine-U-14C. A conversion of 2-benzylmalic acid into 3-benzylmalic acid and 2-amino-4-phenylbutyric acid could also be demonstrated. These findings provide the final evidence for a chain-lengthening mechanism leading to homologous amino acids as proposed by Underbill and Wetter in 1966.

Role of Acidic and Aromatic Amino Acids inRhodobacter CapsulatusCytochromec1. A Site-Directed Mutagenesis Study†

Biochemistry ◽  
2003 ◽  
Vol 42 (29) ◽  
pp. 8818-8830 ◽  
Author(s):  
Jun Li ◽  
Artur Osyczka ◽  
Richard C. Conover ◽  
Michael K. Johnson ◽  
Hong Qin ◽  
...  
Keyword(s):  
Amino Acids ◽  
Aromatic Amino Acids ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
A Site ◽  
Mutagenesis Study

Effects of branched-chain-enriched amino acids and insulin on forearm leucine kinetics

1999 ◽  
Vol 97 (4) ◽  
pp. 437-448 ◽  
Author(s):  
Michela ZANETTI ◽  
Rocco BARAZZONI ◽  
Edward KIWANUKA ◽  
Paolo TESSARI
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Muscle Protein ◽  
Aromatic Amino Acids ◽  
Leucine Kinetics ◽  
Before And After ◽  
Amino Acid Mixtures ◽  
Leucine Transport ◽  
Branched Chain ◽  
Plasma Leucine

Although amino acid mixtures enriched in branched-chain amino acids (BCAA) and deficient in aromatic amino acids (AAA) are often used together with insulin and glucose in clinical nutrition, their physiological effects on muscle protein anabolism are not known. To this aim, we studied forearm leucine kinetics in post-absorptive volunteers, before and after the systemic infusion of BCAA-enriched, AAA-deficient amino acids along with insulin and the euglycaemic clamp. The results were compared with the effects of insulin infusion alone. A compartmental leucine forearm model was employed at steady state. Hyperaminoacidaemia with hyperinsulinaemia (to ≈ 80–100 μ-units/ml) increased the leucine plasma concentration (+70%; P< 0.001), inflow into the forearm cell (+150%; P< 0.01), disposal into protein synthesis (+100%; P< 0.01), net intracellular retention (P< 0.01), net forearm balance (by ≈ 6-fold; P< 0.01) and net deamination to α-ketoisocaproate (4-methyl-2-oxopentanoate) (+9%; P< 0.05). Leucine release from forearm proteolysis and outflow from the forearm cell were unchanged. In contrast, hyperinsulinaemia alone decreased plasma leucine concentrations (-35%; P< 0.001) and leucine inflow (-20%; P< 0.05) and outflow (-30%; P< 0.01) into and out of forearm cell(s), it increased net intracellular leucine retention (P< 0.03), and it did not change leucine release from forearm proteolysis (-20%; P = 0.138), net leucine deamination to α-ketoisocaproate, leucine disposal into protein synthesis or net forearm protein balance. By considering all data together, leucine disposal into protein synthesis was directly correlated with leucine inflow into the cell (r = 0.71; P< 0.0001). These data indicate that the infusion of BCAA-enriched, AAA-deficient amino acids along with insulin is capable of stimulating forearm (i.e. muscle) protein anabolism in normal volunteers by enhancing intracellular leucine transport and protein synthesis. These effects are probably due to hyperaminoacidaemia and/or its interaction with hyperinsulinaemia, since they were not observed under conditions of hyperinsulinaemia alone.

Control of Muscle Protein Synthesis as a Result of Contractile Activity and Amino Acid Availability: Implications for Protein Requirements

2001 ◽  
Vol 11 (s1) ◽  
pp. S170-S176 ◽  
Author(s):  
Michael J. Rennie
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Muscle Protein ◽  
Contractile Activity ◽  
Muscle Protein Synthesis ◽  
Protein Requirements ◽  
Amino Acid Availability ◽  
Separate Pathway

The major anabolic influences on muscle are feeding and contractile activity. As a result of feeding, anabolism occurs chiefly by increases in protein synthesis with minor changes in protein breakdown. Insulin has a permissive role in increasing synthesis, but the availability of amino acids is crucial for net anabolism. We have investigated the role of amino acids in stimulating muscle protein synthesis, the synergy between exercise and amino acid availability, and some of the signaling elements involved. The results suggest that muscle is acutely sensitive to amino acids, that exercise probably increases the anabolic effects of amino acids by a separate pathway, and that for this reason it is unlikely that accustomed physical exercise increases protein requirements.

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