High-Speed Microdialysis–Capillary Electrophoresis Assays for Measuring Branched Chain Amino Acid Uptake in 3T3-L1 cells

ABSTRACT Amino acid uptake by Rhizobium leguminosarum is dominated by two ABC transporters, the general amino acid permease (Aap) and the branched-chain amino acid permease (BraRl). Characterization of the solute specificity of BraRl shows it to be the second general amino acid permease of R. leguminosarum. Although BraRl has high sequence identity to members of the family of hydrophobic amino acid transporters (HAAT), it transports a broad range of solutes, including acidic and basic polar amino acids (l-glutamate, l-arginine, and l-histidine), in addition to neutral amino acids (l-alanine and l-leucine). While amino and carboxyl groups are required for transport, solutes do not have to be α-amino acids. Consistent with this, BraRl is the first ABC transporter to be shown to transport γ-aminobutyric acid (GABA). All previously identified bacterial GABA transporters are secondary carriers of the amino acid-polyamine-organocation (APC) superfamily. Also, transport by BraRl does not appear to be stereospecific as d amino acids cause significant inhibition of uptake of l-glutamate and l-leucine. Unlike all other solutes tested, l-alanine uptake is not dependent on solute binding protein BraCRl. Therefore, a second, unidentified solute binding protein may interact with the BraDEFGRl membrane complex during l-alanine uptake. Overall, the data indicate that BraRl is a general amino acid permease of the HAAT family. Furthermore, BraRl has the broadest solute specificity of any characterized bacterial amino acid transporter.

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Removal of infused amino acids by splanchnic and leg tissues in humans

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1986.250.4.e407 ◽

1986 ◽

Vol 250 (4) ◽

pp. E407-E413 ◽

Cited By ~ 11

Author(s):

R. A. Gelfand ◽

M. G. Glickman ◽

R. Jacob ◽

R. S. Sherwin ◽

R. A. DeFronzo

Keyword(s):

Skeletal Muscle ◽

Amino Acids ◽

Amino Acid ◽

Amino Acid Uptake ◽

Branched Chain Amino Acids ◽

Acid Uptake ◽

Acid Infusion ◽

Amino Acid Infusion ◽

Significant Uptake ◽

Branched Chain

To compare the contributions of splanchnic and skeletal muscle tissues to the disposal of intravenously administered amino acids, regional amino acid exchange was measured across the splanchnic bed and leg in 11 normal volunteers. Postabsorptively, net release of amino acids by leg (largely alanine and glutamine) was complemented by the net splanchnic uptake of amino acids. Amino acid infusion via peripheral vein (0.2 g X kg-1 X h-1) caused a doubling of plasma insulin and glucagon levels and a threefold rise in blood amino acid concentrations. Both splanchnic and leg tissues showed significant uptake of infused amino acids. Splanchnic tissues accounted for approximately 70% of the total body amino acid nitrogen disposal; splanchnic uptake was greatest for the glucogenic amino acids but also included significant quantities of branched-chain amino acids. In contrast, leg amino acid uptake was dominated by the branched-chain amino acids. Based on the measured leg balance, body skeletal muscle was estimated to remove approximately 25-30% of the total infused amino acid load and approximately 65-70% of the infused branched-chain amino acids. Amino acid infusion significantly stimulated both the leg efflux and the splanchnic uptake of glutamine (not contained in the infusate). We conclude that when amino acids are infused peripherally in normal humans, splanchnic viscera (liver and gut) are the major sites of amino acid disposal.

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Amino acid metabolism by hepatocytes in a hybrid liver support bioreactor

The International Journal of Artificial Organs ◽

10.1177/039139889401701208 ◽

1994 ◽

Vol 17 (12) ◽

pp. 663-669 ◽

Cited By ~ 12

Author(s):

M. Fuchs ◽

J. Gerlach ◽

J. Encke ◽

J. Unger ◽

M. Smith ◽

...

Keyword(s):

Amino Acid ◽

Acid Metabolism ◽

Amino Acid Uptake ◽

Acid Uptake ◽

Liver Support ◽

Volume Increase ◽

Liver Support System ◽

Amino Acid Turnover ◽

Acid Release ◽

Branched Chain

The amino acid patterns of medium perfusate in a liver cell bioreactor developed for a hybrid liver support system have been measured. There were considerable changes in the concentrations of glutamic acid, glutamine, alanine, arginine, ornithine and branched chain amino acids during the first 10 days which is indicative of dynamic cellular metabolism. From day 15, steady state conditions of nitrogen metabolism are reflected by stable amino acid turnover. Monitoring of urea, K+, and P-450 activity suggests that hepatocytes have switched to a stable protein synthesis with a general amino acid uptake and keto acid release following cell volume increase

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Analysis of the Pseudomonas putida CA-3 proteome during growth on styrene under nitrogen-limiting and non-limiting conditions

Microbiology ◽

10.1099/mic.0.031153-0 ◽

2009 ◽

Vol 155 (10) ◽

pp. 3348-3361 ◽

Cited By ~ 36

Author(s):

Jasmina Nikodinovic-Runic ◽

Michelle Flanagan ◽

Aisling R. Hume ◽

Gerard Cagney ◽

Kevin E. O'Connor

Keyword(s):

Amino Acid ◽

Pseudomonas Putida ◽

Outer Membrane ◽

Nitrogen Limitation ◽

Nitrogen Assimilation ◽

Nucleotide Metabolism ◽

Acid Uptake ◽

Branched Chain Amino Acid ◽

Branched Chain ◽

Limiting Condition

Pseudomonas putida CA-3 is a styrene-degrading bacterium capable of accumulating medium-chain-length polyhydroxyalkanoate (mclPHA) when exposed to limiting concentrations of a nitrogen source in the growth medium. Using shotgun proteomics we analysed global proteome expression in P. putida CA-3 supplied with styrene as the sole carbon and energy source under N-limiting (condition permissive for mclPHA synthesis) and non-limiting (condition non-permissive for mclPHA accumulation) growth conditions in order to provide insight into the molecular response of P. putida CA-3 to limitation of nitrogen when grown on styrene. A total of 1761 proteins were identified with high confidence and the detected proteins could be assigned to functional groups including styrene degradation, energy, nucleotide metabolism, protein synthesis, transport, stress response and motility. Proteins involved in the upper and lower styrene degradation pathway were expressed throughout the 48 h growth period under both nitrogen limitation and excess. Proteins involved in polyhydroxyalkanoate (PHA) biosynthesis, nitrogen assimilation and amino acid transport, and outer membrane proteins were upregulated under nitrogen limitation. PHA accumulation and biosynthesis were only expressed under nitrogen limitation. Nitrogen assimilation proteins were detected on average at twofold higher amounts under nitrogen limitation. Expression of the branched-chain amino acid ABC transporter was up to 16-fold higher under nitrogen-limiting conditions. Branched chain amino acid uptake by nitrogen-limited cultures was also higher than that by non-limited cultures. Outer membrane lipoproteins were expressed at twofold higher levels under nitrogen limitation. This was confirmed by Western blotting (immunochemical detection) of cells grown under nitrogen limitation. Our study provides the first global description of protein expression changes during growth of any organism on styrene and accumulating mclPHA (nitrogen-limited growth).

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Sequential amino acid measurements during experimental diabetic ketoacidosis

American Journal of Physiology-Legacy Content ◽

10.1152/ajplegacy.1975.228.1.205 ◽

1975 ◽

Vol 228 (1) ◽

pp. 205-211 ◽

Cited By ~ 15

Author(s):

PJ Blackshear ◽

KGMM Alberti

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Whole Blood ◽

Insulin Injection ◽

Amino Acid Uptake ◽

Progressive Increase ◽

Second Phase ◽

Acid Uptake ◽

Male Wistar Rats ◽

Branched Chain

This study was designed to investigate the sequential amino acid response to acute insulin deprivation. Male Wistar rats were made severly diabetic by the intravenous injection of streptozotocin, 150 mg/kg, and maintained on insulin for 7 days. Insulin was then withheld, and measurememts of whole blood and plasma amino acid concentrations were made at 24-h intervals until 120 h, by which time animal mortality was 80%. Alanine and the other gluogenic amino acids displayed a biphasic response to insulin deprivation, decreasing in plasma and whole blood until 72 h after the last insulin injection then increasing in concentration until 120 h. The branched-chain amino acids valine, leucine, and isoleucine remained constant until 72 h after insulin, after which time their concentrations increased dramatically. It was concluded that the first phase represented enhanced gluconeogenesis and in the second phase amino acid uptake for gluconeogenesis was exceeded by net peripheral amino acid release. Enzymatic measurements showed a progressive increase in erythrocyte;plasma distribution ratios for glutamate in ketoacidosis.

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Metabolic Fate of the Increased Yeast Amino Acid Uptake Subsequent to Catabolite Derepression

Journal of Amino Acids ◽

10.1155/2013/461901 ◽

2013 ◽

Vol 2013 ◽

pp. 1-7 ◽

Cited By ~ 7

Author(s):

John S. Hothersall ◽

Aamir Ahmed

Keyword(s):

Amino Acid ◽

Protein Expression ◽

Citrate Synthase ◽

Tca Cycle ◽

Amino Acid Uptake ◽

Acid Uptake ◽

Leucine Oxidation ◽

The Tca Cycle ◽

Branched Chain ◽

Ketoacid Dehydrogenase

Catabolite repression (CCR) regulates amino acid permeases in Saccharomyces cerevisiae via a TOR-kinase mediated mechanism. When glucose, the preferred fuel in S. cerevisiae, is substituted by galactose, amino acid uptake is increased. Here we have assessed the contribution and metabolic significance of this surfeit of amino acid in yeast undergoing catabolite derepression (CDR). L-[U-14C]leucine oxidation was increased 15 ± 1 fold in wild type (WT) strain grown in galactose compared to glucose. Under CDR, leucine oxidation was (i) proportional to uptake, as demonstrated by decreased uptake and oxidation of leucine in strains deleted of major leucine permeases and (ii) entirely dependent upon the TCA cycle, as cytochrome c1 (Cyt1) deleted strains could not grow in galactose. A regulator of amino acid carbon entry into the TCA cycle, branched chain ketoacid dehydrogenase, was also increased 29 ± 3 fold under CCR in WT strain. Protein expression of key TCA cycle enzymes, citrate synthase (Cs), and Cyt1 was increased during CDR. In summary, CDR upregulation of amino acid uptake is accompanied by increased utilization of amino acids for yeast growth. The mechanism for this is likely to be an increase in protein expression of key regulators of the TCA cycle.

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