A Rapid Vapor-Phase Acid (Hydrochloric Acid and Trifluoroacetic Acid) Hydrolysis of Peptide and Protein

Tsugita, Akira; Uchida, Toyoaki; Mewes, Hans‐Werner; Ataka, Tatsuaki

doi:10.1093/oxfordjournals.jbchem.a122209

Cited by 124 publications

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“…Individual amino acids in the DCAA pool were measured after modified vapor-phase hydrolysis (Tsugita et al 1987;Keil and Kirchman 1991). First, 75 l of 12 mol L Ϫ1 HCl were added to 75 l of sample in a small sample vial.…”

Section: Methodsmentioning

confidence: 99%

Enrichment of amino acids in the sea surface microlayer at coastal and open ocean sites in the North Atlantic Ocean

Kuznetsova

Lee

Aller

et al. 2004

Limnology & Oceanography

129

117

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We investigated how regional differences in environmental parameters influenced enrichment of amino acids in the sea surface microlayer relative to underlying bulk seawater. Concentrations and compositions of dissolved free (DFAA), dissolved combined (DCAA), and particulate (PAA) amino acids were measured in the sea surface microlayer and corresponding subsurface waters along a transect from coastal Massachusetts to open ocean waters of the Sargasso Sea. We also measured total bacteria concentrations, the percent bacteria with damaged membranes, and concentrations of virus-like particles. Microlayer samples taken by two different techniques-screen sampler (thickness 200-400 m) and rotating drum (thickness 30-60 m)-were compared. On average, concentrations of bacteria and amino acids in both subsurface and microlayer water were higher in water masses with higher fluorescence, lower temperature and salinity, and historically higher chlorophyll. The microlayer was enriched with amino acids and bacteria and, in most instances, virus-like particles. Consistent microlayer features, such as nonuniform preferential enrichment with DFAA and PAA and a uniform difference in dissolved amino acid composition between microlayer and subsurface water, were unrelated to environmental parameters measured. Wind, humidity, and light also had no apparent effects on amino acid concentration, composition, or enrichment in the microlayer in this study.

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Section: Methodsmentioning

confidence: 99%

Enrichment of amino acids in the sea surface microlayer at coastal and open ocean sites in the North Atlantic Ocean

Kuznetsova

Lee

Aller

et al. 2004

Limnology & Oceanography

129

117

View full text Add to dashboard Cite

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“…DFAA were measured individually by HPLC (Lindroth & Mopper 1979, Cowie & Hedges 1992 or as total dissolved primary amines by fluorometry (Parsons et al 1984). Concentrations of total hydrolysable amino acids (THAA) were measured by HPLC or fluorometry after vapor-phase hydrolysis (Tsugita et al 1987, Keil & Kirchman 1991. DCAA were calculated as the difference between THAA and DFAA.…”

Section: Methodsmentioning

confidence: 99%

Extracellular enzyme activity and uptake of carbon and nitrogen along an estuarine salinity and nutrient gradient

Mulholland¹,

Lee²,

Glibert³

2003

Mar. Ecol. Prog. Ser.

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Amino acid oxidation (AAO) and peptide hydrolysis (PH) are processes affecting the recycling of organic material and nutrients. We compared extracellular AAO and PH rates to C and N uptake rates along estuarine gradients of salinity, nutrients and productivity in the Pocomoke River, a subestuary of the Chesapeake Bay. This estuary is seasonally depleted in inorganic N, and rich in dissolved organic material (DOM) throughout the year. AAO, PH, and N uptake rates measured in 1999 and 2000 were not limited to particular size fractions measured, or to auto-or heterotrophic groups of organisms. At a station near the turbidity maximum, where chlorophyll a biomass was highest, smaller (<1.2 µm) size-fractions contributed < 20% of the AAO in May and up to 80% in August when AAO rates were ~10 times lower. Most PH was in the larger (>1.2 µm) size-fraction, except at the least saline station in August of both years. Rates of AAO and PH were not linearly correlated with each other seasonally or spatially. Uptake of NH 4 + dominated total N uptake (> 50%) at all but the freshwater station, although uptake of organic compounds was measurable at all sites. Rates of dissolved free amino acid uptake, measured using dually labeled compounds, were substantial (up to 11% of the total N uptake) and contributed both C and N for growth. Dual labels unambiguously demonstrated that uptake rates of amino acid C and N were uncoupled; amino acid N was taken up preferentially to amino acid C even when rates were corrected for N uptake from AAO. Conceptual models of DOM cycling should include the realization that enzymatic processes and uptake of DOM occur in both 'microbial' and larger size fractions. Thus, competition between bacteria and phytoplankton mixotrophs may be an important factor determining the relative uptake of C and N from amino acids and other organic substrates.KEY WORDS: Amino acid oxidation · Peptide hydrolysis · DOM cycling · N uptake · C uptake Resale or republication not permitted without written consent of the publisher

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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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A Rapid Vapor-Phase Acid (Hydrochloric Acid and Trifluoroacetic Acid) Hydrolysis of Peptide and Protein

Cited by 124 publications

References 0 publications

Enrichment of amino acids in the sea surface microlayer at coastal and open ocean sites in the North Atlantic Ocean

Enrichment of amino acids in the sea surface microlayer at coastal and open ocean sites in the North Atlantic Ocean

Extracellular enzyme activity and uptake of carbon and nitrogen along an estuarine salinity and nutrient gradient

Production and characterization of recombinant mouse neurotrophin‐3

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