Measurement of free amino acids in human biological fluids by high-performance liquid chromatography

Godel, H.; Graser, Theodor; Földi, Péter; Pfaender, Peter; Fürst, Peter

doi:10.1016/s0021-9673(01)89028-2

Cited by 234 publications

(69 citation statements)

References 31 publications

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“…to elute the amino acids liberated by carboxypeptidase treatment. Subsequently, the eluate was subjected to 0-phthaldialdehyde amino acid analysis [31]. The truncated protein was eluted from the reverse-phase material as described above and again subjected to carboxypeptidase treatment.…”

Section: Protein Chemical Methodsmentioning

confidence: 99%

Maturation of the large subunit (HYCE) of Escherichia coli hydrogenase 3 requires nickel incorporation followed by C‐terminal processing at Arg537

Rossmann¹,

Sauter²,

Lottspeich

et al. 1994

European Journal of Biochemistry

114

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Purification of the large subunit, HYCE, of Escherichia coli hydrogenase 3 revealed that it is a nickel-containing polypeptide, which is subject to C-terminal proteolytic processing. This processing reaction could be performed in vitro with partially purified components, yielding a low-molecular mass C-terminal peptide which was resolved in a Tricine/SDS/polyacrylamide gel. N-terminal sequencing of this peptide revealed that proteolytic cleavage occurred at the C-terminal side of the arginine residue at position 537, which corresponds to the histidine residue in the highly conserved motif, DPCXXCXXH, of other (NiFe) hydrogenases thought to be involved in active site nickel coordination. Nickel-containing HYCE precursor for in vitro processing, was partially purified from strain HD708 (AhycH) in the presence of the reducing agent dithiothreitol. Using 2-mercaptoethanol instead of dithiothreitol provided pure precursor, which was, however, no longer susceptible to in vitro processing ; it proved to be devoid of nickel indicating that nickel incorporation into the HYCE precursor is a prerequisite for processing. This conclusion was supported by the finding that HYCE precursor from strain HD708 (AhycH) chromatographed with radioactivity from 63Ni incorporated in vivo and could be processed in vitro, whereas HYCE precursor from strain BEF314 (AhypB-E) lacking the nickel insertion system appeared to be devoid of nickel and was not sensitive to in vitro processing.According to their different physiological roles in bacterial metabolism, nickel-containing hydrogenases form a very heterogeneous group of enzymes. They differ with respect to their cellular function (H, oxidation or H, production), metal content, subcellular localization, subunit composition and the electron acceptors and donors used [l]. However, one subunit, called the large subunit, is common to all of these hydrogenases. In the amino acid sequence, it is the most conserved subunit and it is thought to ligand the active-site nickel. Two highly conserved cysteine-rich motifs are present in all known large subunits and probably provide the ligands required for the binding of nickel. One motif, RXCXXC, is located in the N-terminal region and the second, DPCXXCXXH, is located at the C-terminus of the polypeptide chain [l, 21. In addition to the genes encoding the hydrogenase subunits, a number of genes have been sequenced which are required for the formation of active hydrogenases [ 3 -101. Such activation of (NiFe) hydrogenases may involve insertion of nickel into the hydrogenase apoprotein [ l l -131, activation of nickel in the active site, membrane integration [3, 141, complex formation, processing of the small subunit carrying a signal peptide at its N-terminus [15] and processing of the large subunit [3,16, 171 which order these processes take place and whether further reactions are involved.The facultative anaerobe Escherichia coli possesses three hydrogenase isoenzymes [ 181. Two of these isoenzymes (hydrogenases 1 and 2) are membrane-bound uptake hy...

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Section: Protein Chemical Methodsmentioning

confidence: 99%

Maturation of the large subunit (HYCE) of Escherichia coli hydrogenase 3 requires nickel incorporation followed by C‐terminal processing at Arg537

Rossmann¹,

Sauter²,

Lottspeich

et al. 1994

European Journal of Biochemistry

114

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“…Then, unlabeled glutamate (either 5 or 100~tM) was added to the medium, followed by incubation at 37°C.Then, aliquots (10 it!) were taken at intervals and the amount of glutamate in the culture medium was determined by reverse-phase HPLC on a RESOLVE C18 column (3.9 X 150 mm) (Waters), with precolumn derivatization with o-phthalaldehyde and fluorescence detection (Godel et al, 1984).…”

Section: Clearance Of Extracellular Glutamatementioning

confidence: 99%

Functional Expression of a GLT‐1 Type Na⁺‐Dependent Glutamate Transporter in Rat Pinealocytes

Yamada¹,

Yatsushiro²,

Yamamoto

et al. 1997

Journal of Neurochemistry

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Pinealocytes, the neuroendocrine cells that produce melatonin, accumulate glutamate in microvesides through a specific vesicular transporter energetically coupled with vacuolar-type proton ATPase. The glutamate is secreted into the extracellular space through microvesicle-mediated exocytosis and then stimulates neighboring pinealocytes, resulting in inhibition of norepinephrine-dependent melatonin synthesis. In this study, we identified and characterized the plasma membranetype glutamate transporter in rat pinealocytes. The [ 3H]-glutamate uptake by cultured pinealocytes was driven by extracellular Nat, saturated with the [3H]glutamate concentration used, and significantly inhibited by L-glutamate, L-aspartate, ß-threo-hydroxyaspartate, pyrrolidine dicarboxylate, and L-cysteine sulfinate, substrates or inhibitors of the plasma membrane glutamate transporter. Consistently, the clearance of extracellular glutamate, as measured by HPLC, was also dependent on Na~and inhibited by /9-threo-hydroxyaspartate and L-cysteine sulfinate. Immunological studies with site-specific antibodies against three isoforms of the Nat-dependent glutamate transporter (GLT-1, GLAST, and EAAC1) revealed the expression of only the GLT-1 type transporter in pineal glands. Expression of the GLT-1 type transporter in pineal glands was further demonstrated by means of reverse transcription-polymerase chain reaction with specific DNA probes. Immunohistochemical analysis indicated that the immunological counterpart(s) of the GLT-1 is localized in pinealocytes. These results suggested that the GLT-1 -type Nat-dependent transporter is expressed and functions as a reuptake system for glutamate in rat pinealocytes. The physiological role of the transporter in the termination of the glutamate signal in the pineal gland is discussed. Key Words: Na-dependent glutamate transporter-Pinealocyte-Pineal gland -L-Glutamate-GLT-1 -Reuptake-Microvesicle-Endocrine cell.

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“…N-terminal sequencing was performed on a gas/liquid-phase sequencer 477A from Applied Biosysterns (Foster City CA, USA) equipped with an on-line 120A phenylthiohydantoin analyser using the conditions given by the manufacturer. Amino acid composition analyses were performed using the o-phthaldialdehyde method [30] in combination with gas-phase hydrolysis [31].…”

Section: Purification Proceduresmentioning

confidence: 99%

Production and characterization of recombinant mouse neurotrophin‐3

Rudolf

Kolbeck

Lottspeich

et al. 1992

European Journal of Biochemistry

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Neurotrophin-3 (NT-3) is a neurotrophic factor that has recently been cloned on the basis of its structural similarity to other members of the nerve growth factor (NGF) gene family. In order to produce this protein, a recombinant vaccinia virus containing the coding region for mouse NT-3 was constructed. Conditioned medium harvested from cells infected with the recombinant vaccinia virus contained biologically active NT-3 that was purified by chromatography on controlled-pore glass and reversed-phase and gel-filtration high-pressure liquid chromatography. Approximately 200 pg purified NT-3 was obtained from a single cell-factory flask (1.6 1 conditioned medium). N-terminal protein sequencing showed that the mature factor was processed from the precursor at the expected site and its amino acid composition agreed with that predicted from the DNA sequence. The biological activity of the recombinant protein was tested on dissociated neurons prepared from chick embryos. Using spinal sensory neurons, the concentration of purified recombinant NT-3 allowing half-maximal survival was determined to be 25 pg/ml.Nerve growth factor (NGF) is the prototypical neurotrophic molecule and its important role in the survival, development and maintenance of some neuronal populations, particularly in the peripheral nervous system, has been well characterized (for review, see [I]). The tissues innervated by NGF-responsive neurons have been shown to contain and synthesize limited amounts of NGF [2-61 supporting the neurotrophic theory. This concept states that embryonic, naturally occurring cell death, involving the loss of a significant number of neurons in a given population, is regulated by neurotrophic molecules produced by the target; competition for the limited amounts of trophic factor allows the survival of only a portion of the neurons (for recent reviews, see [7, 81). The recent cloning of brain-derived neurotrophic factor (BDNF) revealed that it is structurally related to NGF [9]. The knowledge that NGF had a structurally rekated relative allowed the identification of a third member of this gene family, neurotrophin-3 (NT-3; also described as hippocampus-derived neurotrophic factor or NGF-2) [lo -151. These three neurotrophic factors are synthesized as precursors and upon proteolytic cleavage, the mature, biologically active forms are generated. They are basic proteins with a molecular mass of approximately 13.5 kDa and are characterized by six conserved cysteine residues. The cysteine residues have been shown to form three intramolecular disulfide bridges in mouse NGF [16]. In the mouse, 54 residues out of 120 are common to all three neurotrophic proteins.In view of the minute amounts of neurotrophic factors in almost all tissues and the consequently diMicult purification [18] has yielded recombinant proteins with specific biological activities several orders of magnitude lower than that of natural NGF, rendering these expression systems unattractive for the production of other species of NT. In previous experiments, only...

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Measurement of free amino acids in human biological fluids by high-performance liquid chromatography

Cited by 234 publications

References 31 publications

Maturation of the large subunit (HYCE) of Escherichia coli hydrogenase 3 requires nickel incorporation followed by C‐terminal processing at Arg537

Maturation of the large subunit (HYCE) of Escherichia coli hydrogenase 3 requires nickel incorporation followed by C‐terminal processing at Arg537

Functional Expression of a GLT‐1 Type Na⁺‐Dependent Glutamate Transporter in Rat Pinealocytes

Production and characterization of recombinant mouse neurotrophin‐3

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Measurement of free amino acids in human biological fluids by high-performance liquid chromatography

Cited by 234 publications

References 31 publications

Maturation of the large subunit (HYCE) of Escherichia coli hydrogenase 3 requires nickel incorporation followed by C‐terminal processing at Arg537

Maturation of the large subunit (HYCE) of Escherichia coli hydrogenase 3 requires nickel incorporation followed by C‐terminal processing at Arg537

Functional Expression of a GLT‐1 Type Na+‐Dependent Glutamate Transporter in Rat Pinealocytes

Production and characterization of recombinant mouse neurotrophin‐3

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Functional Expression of a GLT‐1 Type Na⁺‐Dependent Glutamate Transporter in Rat Pinealocytes