scholarly journals Rate-limiting factors in urate synthesis and gluconeogenesis in avian liver

1978 ◽  
Vol 172 (2) ◽  
pp. 193-203 ◽  
Author(s):  
James P. Mapes ◽  
Hans A. Krebs
Keyword(s):  
Amino Acids ◽  
Limiting Factors ◽  
Cyclic Process ◽  
Urea Synthesis ◽  
Phosphoribosyl Pyrophosphate ◽  
Metabolic Characteristics ◽  
Glucose Synthesis ◽  
Glutamine Synthesis ◽  
Purine Ring ◽  
Rate Limiting

1. Urate synthesis and other metabolic characteristics of isolated chicken hepatocytes were studied. 2. The distinction is made between immediate precursors of the purine ring (glycine, glutamine, aspartate, formyltetrahydrofolate, bicarbonate) and ultimate precursors from which the immediate precursors are formed in the liver. 3. In hepatocytes from well-fed chickens the rate of urate synthesis was not greatly increased by the addition of amino acids or NH4Cl, but in hepatocytes from 72h-starved chickens the rate was much increased when alanine or asparagine was added as the only substrate. Other amino acids, when added alone, did not affect the rate. The exceptional effect of alanine and asparagine is due to the ready formation of the immediate precursors. 4. Conditions are described under which glutamine, serine, glycine plus formate, ribose and glucose increased the rate of urate synthesis. 5. At 1mm-NH4Cl (a concentration not much higher than that of blood plasma) the rate of urate synthesis in the presence of lactate was increased, but higher concentrations inhibited urate synthesis in the presence of lactate or alanine; with alanine even 1mm-NH4Cl was inhibitory. 6. Glucose synthesis from lactate, alanine or dihydroxyacetone was also inhibited by 1mm-NH4Cl. 7. NH4Cl inhibition of urate and glucose synthesis was paralleled by an increased rate of glutamine synthesis. Thus in the presence of NH4Cl the gluconeogenic precursors are diverted from the pathway of gluconeogenesis to that of glutamate and glutamine synthesis. This implies that the synthesis of these amino acids is the primary process in the detoxication of ammonia in the avian liver. 8. Urate synthesis, like urea synthesis, can be looked on as a cyclic process with either phosphoribosyl pyrophosphate or ribose acting as the carrier on which the purine ring is assembled. 9. The energy requirements of urate synthesis depend on whether phosphoribosyl pyrophosphate is regenerated from IMP by pyrophosphorylase or by phosphorylation and pyrophosphorylation of ribose. It is 6 or 9 pyrophosphate bonds of ATP respectively.

scholarly journals Is the adenine nucleotide translocator rate-limiting for oxidative phosphorylation?

1978 ◽  
Vol 172 (2) ◽  
pp. 333-342 ◽  
Author(s):  
Marion Stubbs ◽  
Pierre V. Vignais ◽  
Hans A. Krebs
Keyword(s):  
Phosphoenolpyruvate Carboxykinase ◽  
Adenine Nucleotide ◽  
Phosphoglycerate Kinase ◽  
Isolated Hepatocytes ◽  
Urea Synthesis ◽  
Intermediary Metabolites ◽  
Glucose Synthesis ◽  
High Concentrations ◽  
K Concentration ◽  
Rate Limiting

1. The effects of atractyloside and carboxyatractyloside (between 5 and 40μm) on O2 uptake, glucose synthesis, urea synthesis, the adenine nucleotide content and the intracellular K+ concentration were measured in isolated hepatocytes. 2. Urea synthesis was much less inhibited than glucose synthesis by both atractylosides. Measurements of intermediary metabolites of carbohydrate metabolism in freeze-clamped liver after injection of atractyloside into rats indicate that inhibition of gluconeogenesis is due to interference at the cytosolic reactions requiring ATP (phosphoenolpyruvate carboxykinase and 3-phosphoglycerate kinase). 3. The decrease in [ATP]/[ADP]×[Pi] after addition of atractyloside or carboxyatractyloside was restricted to the cytosol. 4. Dihydroxyacetone can be converted either into glucose with the consumption of 2mol of ATP (per mol of glucose) or into lactate with the production of 2mol of ATP. In the presence of high concentrations of atractyloside and carboxyatractyloside more ATP was produced than was used for the synthesis of glucose from dihydroxyacetone, probably for the maintenance of intracellular [K+]. 5. When the rates of respiration were altered by changing substrates, the degrees of inhibition of respiration and translocation by a given concentration of the atractylosides were the same, whereas at a given concentration of HCN the degree of inhibition was high at higher initial rates, and low at lower initial rates. 6. Inhibition of a complex series of reactions by atractyloside does not necessarily indicate that the translocator is a rate-limiting step in that sequence as Th. P. M. Akerboom, H. Bookelman & J. M. Tager [(1977) FEBS. Lett.74, 50–54] assume. This point is discussed.

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.

Kinetic model of hybridoma cultures for the identification of rate limiting factors and process optimisation

1996 ◽  
Vol 42 (2-3) ◽  
pp. 197-205
Author(s):  
A. Lourenço da Silva ◽  
A. Marc ◽  
J.M. Engasser ◽  
J.L. Goergen
Keyword(s):  
Kinetic Model ◽  
Limiting Factors ◽  
Process Optimisation ◽  
Rate Limiting

scholarly journals A Critical Role of Glutamine and Asparagine γ-Nitrogen in Nucleotide Biosynthesis in Cancer Cells Hijacked by an Oncogenic Virus

mBio ◽  
2017 ◽  
Vol 8 (4) ◽  
Author(s):  
Ying Zhu ◽  
Tingting Li ◽  
Suzane Ramos da Silva ◽  
Jae-Jin Lee ◽  
Chun Lu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cancer Cells ◽  
Metabolic Pathway ◽  
Tca Cycle ◽  
Glutamine Metabolism ◽  
Transformed Cells ◽  
Oncogenic Virus ◽  
Phosphoribosyl Pyrophosphate ◽  
Rate Limiting

ABSTRACT While glutamine is a nonessential amino acid that can be synthesized from glucose, some cancer cells primarily depend on glutamine for their growth, proliferation, and survival. Numerous types of cancer also depend on asparagine for cell proliferation. The underlying mechanisms of the glutamine and asparagine requirement in cancer cells in different contexts remain unclear. In this study, we show that the oncogenic virus Kaposi’s sarcoma-associated herpesvirus (KSHV) accelerates the glutamine metabolism of glucose-independent proliferation of cancer cells by upregulating the expression of numerous critical enzymes, including glutaminase 2 (GLS2), glutamate dehydrogenase 1 (GLUD1), and glutamic-oxaloacetic transaminase 2 (GOT2), to support cell proliferation. Surprisingly, cell crisis is rescued only completely by supplementation with asparagine but minimally by supplementation with α-ketoglutarate, aspartate, or glutamate upon glutamine deprivation, implying an essential role of γ-nitrogen in glutamine and asparagine for cell proliferation. Specifically, glutamine and asparagine provide the critical γ-nitrogen for purine and pyrimidine biosynthesis, as knockdown of four rate-limiting enzymes in the pathways, including carbamoylphosphate synthetase 2 (CAD), phosphoribosyl pyrophosphate amidotransferase (PPAT), and phosphoribosyl pyrophosphate synthetases 1 and 2 (PRPS1 and PRPS2, respectively), suppresses cell proliferation. These findings indicate that glutamine and asparagine are shunted to the biosynthesis of nucleotides and nonessential amino acids from the tricarboxylic acid (TCA) cycle to support the anabolic proliferation of KSHV-transformed cells. Our results illustrate a novel mechanism by which an oncogenic virus hijacks a metabolic pathway for cell proliferation and imply potential therapeutic applications in specific types of cancer that depend on this pathway. IMPORTANCE We have previously found that Kaposi’s sarcoma-associated herpesvirus (KSHV) can efficiently infect and transform primary mesenchymal stem cells; however, the metabolic pathways supporting the anabolic proliferation of KSHV-transformed cells remain unknown. Glutamine and asparagine are essential for supporting the growth, proliferation, and survival of some cancer cells. In this study, we have found that KSHV accelerates glutamine metabolism by upregulating numerous critical metabolic enzymes. Unlike most cancer cells that primarily utilize glutamine and asparagine to replenish the TCA cycle, KSHV-transformed cells depend on glutamine and asparagine for providing γ-nitrogen for purine and pyrimidine biosynthesis. We identified four rate-limiting enzymes in this pathway that are essential for the proliferation of KSHV-transformed cells. Our results demonstrate a novel mechanism by which an oncogenic virus hijacks a metabolic pathway for cell proliferation and imply potential therapeutic applications in specific types of cancer that depend on this pathway.

scholarly journals The Effect of Glycomacropeptide versus Amino Acids on Phenylalanine and Tyrosine Variability over 24 Hours in Children with PKU: A Randomized Controlled Trial

Nutrients ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 520 ◽  
Author(s):  
Anne Daly ◽  
Sharon Evans ◽  
Satnam Chahal ◽  
Saikat Santra ◽  
Alex Pinto ◽  
...  
Keyword(s):  
Amino Acids ◽  
Controlled Trial ◽  
Target Range ◽  
Blood Spots ◽  
Meal Plan ◽  
Significant Difference ◽  
Randomised Controlled ◽  
Limiting Amino Acids ◽  
The Impact ◽  
Rate Limiting

Introduction: In phenylketonuria (PKU), evidence suggests that casein glycomacropeptide supplemented with rate-limiting amino acids (CGMP-AA) is associated with better protein utilisation and less blood phenylalanine (Phe) variability. Aim: To study the impact of CGMP-AA on blood Phe variability using 3 different dietary regimens in children with PKU. Methods: This was a 6-week randomised controlled cross-over study comparing CGMP-AA vs. Phe-free l-amino acids (l-AA) assessing blood Phe and tyrosine (Tyr) variability over 24 h in 19 children (7 boys) with PKU, with a median age of 10 years (6–16). Subjects were randomised to 3 dietary regimens: (1) R1, CGMP-AA and usual dietary Phe (CGMP + Phe); (2) R2, CGMP-AA − Phe content of CGMP-AA from usual diet (CGMP − Phe); and (3) R3, l-AA and usual dietary Phe. Each regimen was administered for 14 days. Over the last 48 h on days 13 and 14, blood spots were collected every 4 h at 08 h, 12 h, 16 h, 20 h, 24 h, and 04 h. Isocaloric intake and the same meal plan and protein substitute dosage at standardised times were maintained when blood spots were collected. Results: Eighteen children completed the study. Median Phe concentrations over 24 h for each group were (range) R1, 290 (30–580), R2, 220 (10–670), R3, 165 (10–640) μmol/L. R1 vs. R2 and R1 vs. R3 p < 0.0001; R2 vs. R3 p = 0.0009. There was a significant difference in median Phe at each time point between R1 vs. R2, p = 0.0027 and R1 vs. R3, p < 0.0001, but not between any time points for R2 vs. R3. Tyr was significantly higher in both R1 and R2 [70 (20–240 μmol/L] compared to R3 [60 (10–200) μmol/L]. In children < 12 years, blood Phe remained in the target range (120–360 μmol/L), over 24 h, for 75% of the time in R1, 72% in R2 and 64% in R3; for children aged ≥ 12 years, blood Phe was in target range (120–600 μmol/L) in R1 and R2 for 100% of the time, but 64% in R3. Conclusions: The residual Phe in CGMP-AA increased blood Phe concentration in children. CGMP-AA appears to give less blood Phe variability compared to l-AA, but this effect may be masked by the increased blood Phe concentrations associated with its Phe contribution. Reducing dietary Phe intake to compensate for CGMP-AA Phe content may help.

Is Cognitive Psychopathology Plausible? Illustrations from Memory Research

1996 ◽  
Vol 41 (7) ◽  
pp. S5-S13 ◽  
Author(s):  
Jean-Marie Danion ◽  
Herbert Weingartner ◽  
Leonard Singer
Keyword(s):  
Cognitive Remediation ◽  
Preliminary Evidence ◽  
Limiting Factors ◽  
Specific Rate ◽  
Memory Impairments ◽  
Central Processing ◽  
Elementary Computation ◽  
The One ◽  
Functional Mechanisms ◽  
Rate Limiting

Objective: To examine the strengths and weaknesses of cognitive psychopathology through the specific examples of the memory impairments associated with the administration of benzodiazepines, with schizophrenia, and with depression. Method: These examples are analyzed with reference to a model of memory based on the principle of division between specialized and central processing structures. A basic contention is that it is useful to consider 2 broad classes of processes—automatic, associative, or sensory/perceptual processes on the one hand and intentional, strategic, or reflective processes on the other hand—as being separate. Results: The functional mechanisms of the memory impairments associated with these conditions are beginning to be identified, and there is preliminary evidence that a deficit in an elementary computation may have dramatic consequences on highest cognitive functions. There is also evidence that certain memory impairments are linked to specific dysfunctional outcomes in everyday life. By showing that specific rate-limiting factors of cognitive performance can be identified and are amenable to cognitive interventions, existing data open the door for theoretically and empirically based cognitive remediation of mental disorders. Conclusion: The bulk of available evidence (albeit limited) makes the enterprise of cognitive psychopathology quite plausible and convincing.

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 Real-time study of the urea cycle using 15N n.m.r. in the isolated perfused rat liver

1992 ◽  
Vol 287 (3) ◽  
pp. 813-820 ◽  
Author(s):  
A Geissler ◽  
K Kanamori ◽  
B D Ross
Keyword(s):  
Rat Liver ◽  
Urea Cycle ◽  
Isolated Perfused Rat Liver ◽  
15N Recovery ◽  
Urea Synthesis ◽  
Perfused Liver ◽  
Biochemical Assays ◽  
Low Concentrations ◽  
15N Urea ◽  
Rate Limiting

1. Isolated rat liver was perfused with 10 mM-15NH4Cl, 5 mM-lactate and 1 mM-ornithine, or with 3 mM-[15N]alanine and 1 mM-ornithine, in haemoglobin-free medium. The liver was physiologically stable for over 3 h and synthesized urea at the rate of 1.15 mumol.min-1.g of liver-1 (15NH4(+)-perfused) or 0.41 mumol.min-1.g-1 ([15N]alanine-perfused). 2. The perfused liver was continuously monitored by 15N n.m.r. spectroscopy at 20.27 MHz for 15N. Well-resolved 15N resonances of precursors and intermediates of the urea cycle, present at tissue concentrations of 0.2-3.0 mumol/g, were observed from the intact liver in 5-40 min of acquisition. Key metabolites in liver extract and the final perfusion medium were analysed by n.m.r. and by biochemical assays to determine fractional 15N enrichment and the total 15N recovery. 3. In 15NH4(+)-perfused liver (n = 6), 15N incorporation into glutamate and alanine (1.0-1.3 mumol/g), as well as progressive formation of [15N2]urea, was observed during the first 2 h of perfusion. In the second and third hour, hepatic concentrations of [omega-15N]citrulline and [omega, omega'-15N]argininosuccinate increased to n.m.r.-detectable levels (0.3-0.9 mumol/g). The [15N]aspartate pool was large in the absence of added ornithine, but on its addition was rapidly incorporated into argininosuccinate (n = 3). 4. In [15N]alanine-perfused liver, major metabolites were [15N]glutamate, [gamma-15N]glutamine and [15N]urea. Urea-cycle intermediates were undetectable. 5. The results suggest that, in intact liver provided with excess ammonia, low concentrations of cytosolic argininosuccinate synthetase and argininosuccinate lyase limited the rate of metabolite flux in the urea cycle. By contrast, in alanine-perfused liver at a physiological rate of urea synthesis, mitochondrial carbamoylphosphate synthetase was rate-limiting. 6. The potential utility of 15N n.m.r. for study of metabolite channelling through urea-cycle enzymes in intact liver is discussed.

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