Microbial fermentative preparation of l-[15N2]lysine and its tracer: Application to serum amino acid kinetic studies

Little is known concerning the expression of amino acid transporters during intestinal epithelial cell differentiation. The transport mechanism ofl-glutamate and its regulation during the differentiation process were investigated using the human intestinal Caco-2 cell line. Kinetic studies demonstrated the presence of a single, high-affinity,d-aspartate-sensitive l-glutamate transport system in both confluent and fully differentiated Caco-2 cells. This transport was clearly Na+ dependent, with a Hill coefficient of 2.9 ± 0.3, suggesting a 3 Na+-to-1 glutamate stoichiometry and corresponding to the well-characterized XA,G − system. The excitatory amino acid transporter (EAAT)1 transcript was consistently expressed in the Caco-2 cell line, whereas the epithelial and neuronal EAAT3 transporter was barely detected. In contrast with systems B0 and y+, which have previously been reported to be downregulated when Caco-2 cells stop proliferating, l-glutamate transport capacity was found to increase steadily between day 8 and day 17. This increase was correlated with the level of EAAT1 mRNA, which might reflect an increase in EAAT1 gene transcription and/or stabilization of the EAAT1 transcript.

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Leucine kinetics from [2H3]- and [13C]leucine infused simultaneously by gut and vein

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1991.260.1.e111 ◽

1991 ◽

Vol 260 (1) ◽

pp. E111-E117 ◽

Cited By ~ 12

Author(s):

R. A. Hoerr ◽

D. E. Matthews ◽

D. M. Bier ◽

V. R. Young

Keyword(s):

Amino Acid ◽

Human Subjects ◽

Kinetic Studies ◽

Tracer Study ◽

Experimental Diet ◽

Fed State ◽

Leucine Kinetics ◽

Tracer Kinetic ◽

Plasma Leucine ◽

Plasma Compartment

In amino acid tracer kinetic studies of the fed state, ingested amino acid may be taken up during its initial transit through splanchnic tissues and thus not enter the plasma compartment where tracer is infused. To investigate this possibility, adult human subjects received simultaneous intravenous (iv) and intragastric (ig) leucine tracer infusions, first during a postabsorptive (PA) 4-h primed continuous ig infusion of L-[1-13C]-leucine and L-[5,5,5-2H3]leucine iv, followed on a separate day by a fed infusion, in which an ig infusion of a liquid formula was started 2 h before the tracer infusion and continued throughout the tracer study. Subjects were accustomed to a constant experimental diet supplying 1.5 g protein.kg-1.day-1 and 41-45 kcal.kg-1.day-1 for 7 and 12 days before the PA and fed studies, respectively. For the PA study, plasma enrichment for the ig tracer was 3.34 +/- 0.27 (SE) mol + excess and for the iv tracer it was 4.18 +/- 0.10 (P less than 0.02). Enrichments of alpha-keto-isocaproic acid (KIC) were 3.24 +/- 0.16 (ig) and 3.02 +/- 0.14 (iv), respectively [not significant (NS)]. For the fed study, plasma leucine enrichment for the ig tracer was 2.15 +/- 0.14 and for the iv tracer was 2.84 +/- 0.09 (P less than 0.02). KIC enrichments were 2.02 +/- 0.08 (ig) and 2.24 +/- 0.08 (iv), respectively (NS). In the PA study, the ratio of the plasma leucine enrichments for the ig and iv tracers was 0.80 +/- 0.06 and in the fed experiment, 0.76 +/- 0.05, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

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Invasive Techniques to Evaluate Protein and Amino Acid Metabolism: Regional and Whole Body Amino Acid Kinetic Studies

Protein and Amino Acid Metabolism in Cancer Cachexia - Medical Intelligence Unit ◽

10.1007/978-3-662-22346-8_3 ◽

1996 ◽

pp. 33-49

Author(s):

Russell S. Berman

Keyword(s):

Amino Acid ◽

Amino Acid Metabolism ◽

Acid Metabolism ◽

Kinetic Studies ◽

Whole Body ◽

Invasive Techniques

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Kinetic Studies on the Interaction of D-Amino Acid Oxidase with Benzoate and o-Aminobenzoate

The Journal of Biochemistry ◽

10.1093/oxfordjournals.jbchem.a129660 ◽

1971 ◽

Vol 70 (3) ◽

pp. 457-465 ◽

Cited By ~ 5

Author(s):

Morimitsu NISHIKIMI ◽

Mayumi OSAMURA ◽

Kunio YAGI

Keyword(s):

Amino Acid ◽

Kinetic Studies ◽

Acid Oxidase ◽

Amino Acid Oxidase

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Molecular Analysis of the Rebeccamycin l-Amino Acid Oxidase from Lechevalieria aerocolonigenes ATCC 39243

Journal of Bacteriology ◽

10.1128/jb.187.6.2084-2092.2005 ◽

2005 ◽

Vol 187 (6) ◽

pp. 2084-2092 ◽

Cited By ~ 69

Author(s):

Tomoyasu Nishizawa ◽

Courtney C. Aldrich ◽

David H. Sherman

Keyword(s):

Amino Acid ◽

Biochemical Characterization ◽

Kinetic Studies ◽

Biochemical Properties ◽

Protein Product ◽

Acid Oxidase ◽

Amino Acid Oxidase ◽

Left Hand ◽

Key Enzyme ◽

Hand Region

ABSTRACT Rebeccamycin, a member of the tryptophan-derived indolocarbazole family, is produced by Lechevalieria aerocolonigenes ATCC 39243. The biosynthetic pathway that specifies biosynthesis of this important metabolite is comprised of 11 genes spanning 18 kb of DNA. A presumed early enzyme involved in elaboration of the rebeccamycin aglycone is encoded by rebO, located at the left-hand region of the reb gene cluster. The deduced protein product, RebO (51.9 kDa), is an l-amino acid oxidase (l-AAO) that has 27% identity to an l-AAO from Scomber japonicus (animal, mackerel) and is a member of the family of FAD-dependent oxidase enzymes. In order to study the biochemical properties of this key enzyme, the rebO gene was overexpressed and purified from Escherichia coli. Biochemical characterization showed that RebO is dimeric, with a molecular mass of approximately 101 kDa. Further analysis revealed that the enzyme contains a noncovalently bound FAD cofactor and is reoxidized at the expense of molecular oxygen by producing one molecule of hydrogen peroxide. Based on kinetic studies, RebO shows significant preference for 7-chloro-l-tryptophan, suggesting its likely role as the natural early pathway substrate. Furthermore, the native RebO enzyme has evident, albeit limited, flexibility as shown by bioconversion studies with unnatural substrates. This work provides the first analysis of a structural enzyme involved in construction of this important class of indolocarbazole natural products.

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Recycling of an amino acid label with prolonged isotope infusion: implications for kinetic studies

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1985.248.4.e482 ◽

1985 ◽

Vol 248 (4) ◽

pp. E482-E487 ◽

Cited By ~ 4

Author(s):

W. F. Schwenk ◽

E. Tsalikian ◽

B. Beaufrere ◽

M. W. Haymond

Keyword(s):

Amino Acids ◽

Steady State ◽

Amino Acid ◽

Infusion Rate ◽

Kinetic Studies ◽

Intracellular Pool ◽

Group I ◽

Group Ii ◽

Plasma Leucine ◽

Amino Acid Label

To investigate whether recycling of a labeled amino acid would occur after 24 h of infusion, two groups of normal volunteers were infused with [3H]leucine and alpha-[14C]-ketoisocaproate for 4 h and [2H3]leucine for either 4 or 24 h (groups I and II, respectively). Entry of [2H3 )leucine at steady state into the plasma space was indistinguishable from its infusion rate for group I but 30% higher (P less than 0.001) than this rate for group II, demonstrating significant recycling of label. After discontinuation of the infusions, isotope disappearance from the plasma space was followed for 2 h. The 3H and 14C decay data for both groups suggest that plasma leucine and alpha-ketoisocaproate are derived from a single intracellular pool in the postabsorptive state. In group I, the 3H and 2H labels decayed identically; whereas, in group II, the decay of [2H3]-leucine and alpha-[2H3]ketoisocaproate was slower (P less than 0.01) than the decay of [3H]leucine and alpha-[3H]ketoisocaproate, confirming re-entry of label after a 24-h infusion. Therefore kinetic values calculated from models assuming no recycling of labeled amino acids are most likely not quantitative and must be interpreted with care when flux does not change or decreases.

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