scholarly journals Post-translational regulation of retinal IMPDH1 in vivo to adjust GTP synthesis to illumination conditions

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Anna Plana-Bonamaisó ◽  
Santiago López-Begines ◽  
David Fernández-Justel ◽  
Alexandra Junza ◽  
Ariadna Soler-Tapia ◽  
...  
Keyword(s):  
Translational Regulation ◽  
De Novo ◽  
Bright Light ◽  
Atp Synthesis ◽  
Light Response ◽  
Photoreceptor Cells ◽  
Aggregate Formation ◽  
Rate Limiting Step ◽  
Rate Limiting

We report the in vivo regulation of Inosine-5´-monophosphate dehydrogenase 1 (IMPDH1) in the retina. IMPDH1 catalyzes the rate-limiting step in the de novo synthesis of guanine nucleotides, impacting the cellular pools of GMP, GDP and GTP. Guanine nucleotide homeostasis is central to photoreceptor cells, where cGMP is the signal transducing molecule in the light response. Mutations in IMPDH1 lead to inherited blindness. We unveil a light-dependent phosphorylation of retinal IMPDH1 at Thr159/Ser160 in the Bateman domain that desensitizes the enzyme to allosteric inhibition by GDP/GTP. When exposed to bright light, living mice increase the rate of GTP and ATP synthesis in their retinas; concomitant with IMPDH1 aggregate formation at the outer segment layer. Inhibiting IMPDH activity in living mice delays rod mass recovery. We unveil a novel mechanism of regulation of IMPDH1 in vivo, important for understanding GTP homeostasis in the retina and the pathogenesis of adRP10 IMPDH1 mutations.

Dynamics of hepatic and peripheral insulin effects suggest common rate-limiting step in vivo

Diabetes ◽  
1993 ◽  
Vol 42 (2) ◽  
pp. 296-306 ◽  
Author(s):  
D. C. Bradley ◽  
R. A. Poulin ◽  
R. N. Bergman
Keyword(s):  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

scholarly journals A multi-enzyme cascade for efficient production of d-p-hydroxyphenylglycine from l-tyrosine

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Xu Tan ◽  
Sheng Zhang ◽  
Wei Song ◽  
Jia Liu ◽  
Cong Gao ◽  
...  
Keyword(s):  
Amino Acids ◽  
Specific Activity ◽  
Enantiomeric Excess ◽  
Efficient Production ◽  
Enzyme Cascade ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rotation Strategy ◽  
Rate Limiting

AbstractIn this study, a four-enzyme cascade pathway was developed and reconstructed in vivo for the production of d-p-hydroxyphenylglycine (D-HPG), a valuable intermediate used to produce β-lactam antibiotics and in fine-chemical synthesis, from l-tyrosine. In this pathway, catalytic conversion of the intermediate 4-hydroxyphenylglyoxalate by meso-diaminopimelate dehydrogenase from Corynebacterium glutamicum (CgDAPDH) was identified as the rate-limiting step, followed by application of a mechanism-guided “conformation rotation” strategy to decrease the hydride-transfer distance d(C6HDAP−C4NNADP) and increase CgDAPDH activity. Introduction of the best variant generated by protein engineering (CgDAPDHBC621/D120S/W144S/I169P with 5.32 ± 0.85 U·mg−1 specific activity) into the designed pathway resulted in a D-HPG titer of 42.69 g/L from 50-g/L l-tyrosine in 24 h, with 92.5% conversion, 71.5% isolated yield, and > 99% enantiomeric excess in a 3-L fermenter. This four-enzyme cascade provides an efficient enzymatic approach for the industrial production of D-HPG from cheap amino acids.

scholarly journals Determining the Rate-Limiting Step for Light-Responsive Redox Regulation in Chloroplasts

Antioxidants ◽  
2018 ◽  
Vol 7 (11) ◽  
pp. 153 ◽  
Author(s):  
Keisuke Yoshida ◽  
Toru Hisabori
Keyword(s):  
Redox Regulation ◽  
Reducing Power ◽  
Rubisco Activase ◽  
Power Transfer ◽  
Target Proteins ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

Thiol-based redox regulation ensures light-responsive control of chloroplast functions. Light-derived signal is transferred in the form of reducing power from the photosynthetic electron transport chain to several redox-sensitive target proteins. Two types of protein, ferredoxin-thioredoxin reductase (FTR) and thioredoxin (Trx), are well recognized as the mediators of reducing power. However, it remains unclear which step in a series of redox-relay reactions is the critical bottleneck for determining the rate of target protein reduction. To address this, the redox behaviors of FTR, Trx, and target proteins were extensively characterized in vitro and in vivo. The FTR/Trx redox cascade was reconstituted in vitro using recombinant proteins from Arabidopsis. On the basis of this assay, we found that the FTR catalytic subunit and f-type Trx are rapidly reduced after the drive of reducing power transfer, irrespective of the presence or absence of their downstream target proteins. By contrast, three target proteins, fructose 1,6-bisphosphatase (FBPase), sedoheptulose 1,7-bisphosphatase (SBPase), and Rubisco activase (RCA) showed different reduction patterns; in particular, SBPase was reduced at a low rate. The in vivo study using Arabidopsis plants showed that the Trx family is commonly and rapidly reduced upon high light irradiation, whereas FBPase, SBPase, and RCA are differentially and slowly reduced. Both of these biochemical and physiological findings suggest that reducing power transfer from Trx to its target proteins is a rate-limiting step for chloroplast redox regulation, conferring distinct light-responsive redox behaviors on each of the targets.

scholarly journals The rate-limiting step of protein synthesis in vivo and in vitro and the distribution of growing peptides between the puromycinlabile and puromycin-non-labile sites on polyribosomes

1971 ◽  
Vol 122 (3) ◽  
pp. 267-276 ◽  
Author(s):  
D. C. N. Earl ◽  
Susan T. Hindley
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Donor Site ◽  
Peptide Bond ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Donor And Acceptor ◽  
Rate Limiting

1. At 3 min after an intravenous injection of radioactive amino acids into the rat, the bulk of radioactivity associated with liver polyribosomes can be interpreted as growing peptides. 2. In an attempt to identify the rate-limiting step of protein synthesis in vivo and in vitro, use was made of the action of puromycin at 0°C, in releasing growing peptides only from the donor site, to study the distribution of growing peptides between the donor and acceptor sites. 3. Evidence is presented that all growing peptides in a population of liver polyribosomes labelled in vivo are similarly distributed between the donor and acceptor sites, and that the proportion released by puromycin is not an artifact of methodology. 4. The proportion released by puromycin is about 50% for both liver and muscle polyribosomes labelled in vivo, suggesting that neither the availability nor binding of aminoacyl-tRNA nor peptide bond synthesis nor translocation can limit the rate of protein synthesis in vivo. Attempts to alter this by starvation, hypophysectomy, growth hormone, alloxan, insulin and partial hepatectomy were unsuccessful. 5. Growing peptides on liver polyribosomes labelled in a cell-free system in vitro or by incubating hemidiaphragms in vitro were largely in the donor site, suggesting that either the availability or binding of aminoacyl-tRNA, or peptide bond synthesis, must be rate limiting in vitro and that the rate-limiting step differs from that in vivo. 6. Neither in vivo nor in the hemidiaphragm system in vitro was a correlation found between the proportion of growing peptides in the donor site and changes in the rate of incorporation of radioactivity into protein. This could indicate that the intracellular concentration of amino acids or aminoacyl-tRNA limits the rate of protein synthesis and that the increased incorporation results from a rise to a higher but still suboptimum concentration.

scholarly journals Retrograde trafficking of Argonaute 2 acts as a rate-limiting step for de novo miRNP formation on endoplasmic reticulum–attached polysomes in mammalian cells

2020 ◽  
Vol 3 (2) ◽  
pp. e201800161 ◽  
Author(s):  
Mainak Bose ◽  
Susanta Chatterjee ◽  
Yogaditya Chakrabarty ◽  
Bahnisikha Barman ◽  
Suvendra N Bhattacharyya
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
De Novo ◽  
Mirna Biogenesis ◽  
Argonaute 2 ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Cellular Context ◽  
Subcellular Compartmentalization ◽  
Rate Limiting

microRNAs are short regulatory RNAs in metazoan cells. Regulation of miRNA activity and abundance is evident in human cells where availability of target messages can influence miRNA biogenesis by augmenting the Dicer1-dependent processing of precursors to mature microRNAs. Requirement of subcellular compartmentalization of Ago2, the key component of miRNA repression machineries, for the controlled biogenesis of miRNPs is reported here. The process predominantly happens on the polysomes attached with the endoplasmic reticulum for which the subcellular Ago2 trafficking is found to be essential. Mitochondrial tethering of endoplasmic reticulum and its interaction with endosomes controls Ago2 availability. In cells with depolarized mitochondria, miRNA biogenesis gets impaired, which results in lowering of de novo–formed mature miRNA levels and accumulation of miRNA-free Ago2 on endosomes that fails to interact with Dicer1 and to traffic back to endoplasmic reticulum for de novo miRNA loading. Thus, mitochondria by sensing the cellular context regulates Ago2 trafficking at the subcellular level, which acts as a rate-limiting step in miRNA biogenesis process in mammalian cells.

scholarly journals The effects of a dietary excess of leucine on the synthesis of nicotinamide nucleotides in the rat

1983 ◽  
Vol 49 (3) ◽  
pp. 321-329 ◽  
Author(s):  
Bahieldin I. Magboul ◽  
David A. Bender
Keyword(s):  
Significant Inhibition ◽  
Nucleotide Synthesis ◽  
Tryptophan Oxygenase ◽  
Rate Limiting Step ◽  
Minimum Requirements ◽  
Tissue Homogenates ◽  
Rate Limiting ◽  
Competitive Nature ◽  
Precipitating Factor

1. In order to test the suggestion that a dietary excess of leucine may be a precipitating factor in pellagra, rats were fed on diets that provided 15 g leucine/kg in excess of requirements for 7 weeks from weaning. This led to a significant reduction in the concentrations of nicotinamide nucleotides in liver and blood. The effect was only apparent when the diets provided less than a minimally adequate amount of nicotinamide, so that the animals were dependent on the synthesis of nicotinamide nucleotides from tryptophan to meet all or part of their requirements.2. Urinary excretion of N1-methyl nicotinamide was not a useful indicator of tissue concentrations of nicotinamide nucleotides, and seemed not to be adequately sensitive to differentiate between minimal adequacy and marginal deficiency, as demonstrated by changes in concentrations of nicotinamide nucleotides in liver and blood.3. The addition of leucine to incubation media for the measurement of enzyme activity in tissue homogenates at concentrations within the physiological range, led to a significant activation of tryptophan oxygenase (L-tryptophan: oxygen oxidoreductase (decyclizing), EC 1.13.11.11) and significant inhibition of kynureninase (L-kynurenine hydrolase, EC 3.7.1.3). The effect on tryptophan oxygenase may not be physiologically significant, in view of the considerable range of activity of this enzyme under normal conditions. However, the inhibition of kynureninase, which was primarily competitive with respect to the substrate, probably is physiologically significant, and was enough for this enzyme to become a probable rate-limiting step in tryptophan metabolism and nicotinamide nucleotide synthesis. Other enzymes of the tryptophan – nicotinamide nucleotide pathway were not affected by the addition of leucine to the incubation medium.4. Feeding 15 g leucine/kg diet in excess of minimum requirements had no effect on the activities of tryptophan oxygenase or kynureninase in liver homogenates. This may reflect the reversible competitive nature of the inhibition of kynureninase by leucine, and hence be an artefact of the incubation procedure. Rats fed on the high-leucine diets excreted significantly more kynurenine than did control animals, which is evidence of inhibition of kynureninase in vivo.5. It appears that a dietary excess of leucine, of the order of 15 g/kg above requirements, may be a precipitating factor in pellagra when there is reliance on the synthesis of nicotinamide nucleotides from tryptophan to meet a part or all of the requirements, but not when minimally adequate niacin is available from the diet.

scholarly journals Coassembly of SecYEG and SecA Fully Restores the Properties of the Native Translocon

2018 ◽  
Vol 201 (1) ◽  
Author(s):  
Priya Bariya ◽  
Linda L. Randall
Keyword(s):  
Lipid Bilayers ◽  
Rate Constant ◽  
Apparent Rate Constant ◽  
Membrane Vesicles ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Secretory System ◽  
Rate Limiting

ABSTRACTIn all cells, a highly conserved channel transports proteins across membranes. InEscherichia coli, that channel is SecYEG. Many investigations of this protein complex have used purified SecYEG reconstituted into proteoliposomes. How faithfully do activities of reconstituted systems reflect the properties of SecYEG in the native membrane environment? We investigated by comparing threein vitrosystems: the native membrane environment of inner membrane vesicles and two methods of reconstitution. One method was the widely used reconstitution of SecYEG alone into lipid bilayers. The other was our method of coassembly of SecYEG with SecA, the ATPase of the translocase. For nine different precursor species we assessed parameters that characterize translocation: maximal amplitude of competent precursor translocated, coupling of energy to transfer, and apparent rate constant. In addition, we investigated translocation in the presence and absence of chaperone SecB. For all nine precursors, SecYEG coassembled with SecA was as active as SecYEG in native membrane for each of the parameters studied. Effects of SecB on transport of precursors faithfully mimicked observations madein vivo. From investigation of the nine different precursors, we conclude that the apparent rate constant, which reflects the step that limits the rate of translocation, is dependent on interactions with the translocon of portions of the precursors other than the leader. In addition, in some cases the rate-limiting step is altered by the presence of SecB. Candidates for the rate-limiting step that are consistent with our data are discussed.IMPORTANCEThis work presents a comprehensive quantification of the parameters of transport by the Sec general secretory system in the threein vitrosystems. The standard reconstitution used by most investigators can be enhanced to yield six times as many active translocons simply by adding SecA to SecYEG during reconstitution. This robust system faithfully reflects the properties of translocation in native membrane vesicles. We have expanded the number of precursors studied to nine. This has allowed us to conclude that the rate constant for translocation varies with precursor species.

Regulation of the purine salvage pathway in rat liver

1992 ◽  
Vol 262 (3) ◽  
pp. E344-E352 ◽  
Author(s):  
Y. A. Kim ◽  
M. T. King ◽  
W. E. Teague ◽  
G. A. Rufo ◽  
R. L. Veech ◽  
...  
Keyword(s):  
Rat Liver ◽  
De Novo ◽  
Equilibrium Constants ◽  
Purine Metabolism ◽  
Purine Salvage ◽  
Rate Limiting Step ◽  
Delta G ◽  
Salvage Pathways ◽  
Rate Limiting

The regulation of purine metabolism in rat liver has been examined under conditions that alter the flux through the pathway. Rats were given intraperitoneal injections of ethanol, sodium acetate, or sodium phosphate to attain body water concentrations of approximately 70, 20, and 10 mM, respectively. The livers were freeze-clamped after 30 min, and extracts were made for the analysis of metabolites, cofactors, purine bases, and nucleosides; homogenates were made for the measurement of the activities and kinetic parameters of seven enzymes that participate in purine salvage. The values of the equilibrium constants of nine reactions were determined in vitro and compared with the ratios of the reactants measured in liver. The changes in phosphoribosylpyrophosphate (PRPP), a key intermediate in both the de novo and salvage pathways of purine metabolism, were directly correlated with the changes in ribose 5-phosphate (ribose-5-P); ([PRPP] = 1.7[ribose-5-P] - 7.4 mumol/kg). Ribose-5-P concentrations in turn could be predicted from the liver content of fructose 6-phosphate and glyceraldehyde 3-phosphate by calculation from the known equilibria. The maximum velocities in the tissue of the seven enzymes measured were calculated from the measured substrate values in the liver and with consideration of other effectors of enzyme activity. PRPP synthetase was the least active of the enzymes measured, indicating a possible rate-limiting step. The delta G of the enzyme steps differed from equilibrium values by factors ranging from 4 (nucleoside phosphorylase) to 10(5) (PRPP synthetase and purine transferase reactions). The regulation of purine salvage appeared to depend on the levels of PRPP and ribose-5-P.

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