FATTY ACID UPTAKE IS THE RATE LIMITING STEP IN THE DE NOVO PHOSPHOLIPID SYNTHESIS BY TUMOR CELLS GROWING IN VIVO

1981 ◽  
Vol 9 (2) ◽  
pp. 293P-293P
Author(s):  
K. Fonck ◽  
A. W. T. Konings
Keyword(s):  
Fatty Acid ◽  
Tumor Cells ◽  
De Novo ◽  
Fatty Acid Uptake ◽  
Acid Uptake ◽  
Phospholipid Synthesis ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

Fatty acid uptake in Escherichia coli: regulation by recruitment of fatty acyl-CoA synthetase to the plasma membrane

1993 ◽  
Vol 71 (1-2) ◽  
pp. 51-56 ◽  
Author(s):  
Dev Mangroo ◽  
Gerhard E. Gerber
Keyword(s):  
Escherichia Coli ◽  
Plasma Membrane ◽  
Fatty Acid ◽  
Fatty Acid Uptake ◽  
Fatty Acyl ◽  
Acid Uptake ◽  
Bacterial Membranes ◽  
Rate Limiting Step ◽  
Triton X ◽  
Rate Limiting

Fatty acid uptake in Escherichia coli has been shown to be inhibited by starvation and to be reversed by a short preincubation of the starved cells with D- or L-lactate, succinate, and acetate; these effects on oleate uptake were due to regulation of the rate-limiting step which involves fatty acyl-CoA synthetase. Investigation into the mechanism of regulation of fatty acyl-CoA synthetase showed that D-lactate did not affect the activity of the enzyme directly. Fatty acyl-CoA synthetase was found to be activated by about 20-fold by Triton X-100 and by another 4-fold by the addition of bacterial membranes. D-Lactate treatment was shown to result in coisolation of fatty acyl-CoA synthetase with the plasma membrane; these results are consistent with the interpretation that recruitment of the enzyme to the plasma membrane by D-lactate results in its activation and consequently in the increased level of fatty acid uptake.Key words: fatty acid, uptake, regulation, recruitment, fatty acyl-CoA synthetase, Escherichia coli, plasma membrane.

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 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.

Sex differences in hepatic fatty acid uptake reflect a greater affinity of the transport system in females

1992 ◽  
Vol 263 (3) ◽  
pp. G380-G385 ◽  
Author(s):  
D. Sorrentino ◽  
S. L. Zhou ◽  
E. Kokkotou ◽  
P. D. Berk
Keyword(s):  
Plasma Membrane ◽  
Fatty Acid ◽  
Transport System ◽  
Fatty Acid Uptake ◽  
Surface Expression ◽  
Female Rats ◽  
Acid Uptake ◽  
Fatty Acid Binding ◽  
Male And Female Rats

In this study, we examined the hypothesis that the reported sex difference in hepatic free fatty acid (FFA) uptake involves the putative FFA transport system, the plasma membrane fatty acid binding protein (FABPpm). In hepatocytes isolated from both male and female rats, initial [3H]oleate uptake velocity reflected transmembrane influx and not subsequent metabolism and was a saturable function of the unbound oleate concentration. Although Vmax values were similar (61 +/- 2 vs. 65 +/- 5 pmol.min-1.5 x 10(4) cells-1 for females and males, respectively), the apparent Km was significantly smaller in females (40 +/- 4 vs. 90 +/- 11 nM; P less than 0.05), reflecting faster influx velocities in female cells over a range of unbound oleate concentrations. The oleate efflux rate constant was also greater in females (0.280 +/- 0.014 vs. 0.198 +/- 0.020 min-1; P less than 0.05) despite their greater hepatic content of cytosolic FABP. Finally, despite the greater rates of transmembrane FFA flux in female hepatocytes, the surface expression of FABPpm was virtually identical in the two sexes (2.5 +/- 0.5 vs. 2.4 +/- 0.4 microgram/10(6) cells). Collectively, these data indicate that at FFA-to-albumin ratios occurring in vivo the plasma membrane of female hepatocytes transports oleate bidirectionally at a greater rate than that of male hepatocytes. A sex-related difference in the functional affinity of FABPpm for FFA appears the most likely explanation for the greater oleate uptake in females.

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