Dansylation of amino acids for high-performance liquid chromatography analysis

Tapuhi, Yitzhak; Schmidt, Donald E.; Lindner, Wolfgang; Karger, Barry L.

doi:10.1016/0003-2697(81)90534-0

Cited by 381 publications

(119 citation statements)

References 8 publications

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“…Ten species from Panama were analyzed by HPLC-ESI-MS for protein/nonprotein amino/imino acid content. Metabolites having primary or secondary amines were first derivatized (48). Tertiary amines were analyzed directly.…”

Section: Methodsmentioning

confidence: 99%

The evolution of antiherbivore defenses and their contribution to species coexistence in the tropical tree genusInga

Kursar

Dexter²,

Lokvam

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

271

353

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Plants and their herbivores constitute more than half of the organisms in tropical forests. Therefore, a better understanding of the evolution of plant defenses against their herbivores may be central for our understanding of tropical biodiversity. Here, we address the evolution of antiherbivore defenses and their possible contribution to coexistence in the Neotropical tree genus Inga (Fabaceae). Inga has >300 species, has radiated recently, and is frequently one of the most diverse and abundant genera at a given site. For 37 species from Panama and Peru we characterized developmental, ant, and chemical defenses against herbivores. We found extensive variation in defenses, but little evidence of phylogenetic signal. Furthermore, in a multivariate analysis, developmental, ant, and chemical defenses varied independently (were orthogonal) and appear to have evolved independently of each other. Our results are consistent with strong selection for divergent defensive traits, presumably mediated by herbivores. In an analysis of community assembly, we found that Inga species co-occurring as neighbors are more different in antiherbivore defenses than random, suggesting that possessing a rare defense phenotype increases fitness. These results imply that interactions with herbivores may be an important axis of niche differentiation that permits the coexistence of many species of Inga within a single site. Interactions between plants and their herbivores likely play a key role in the generation and maintenance of the conspicuously high plant diversity in the tropics. plant defenses ͉ community assembly ͉ phylogenetic signal ͉ herbivory ͉ tropical diversity

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Section: Methodsmentioning

confidence: 99%

The evolution of antiherbivore defenses and their contribution to species coexistence in the tropical tree genusInga

Kursar

Dexter²,

Lokvam

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

271

353

View full text Add to dashboard Cite

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“…After the mixtures had been incubated for 2 h at 37 uC, the enzymes were removed using a Microcon YM-10 filter (10 kDa cutoff; Amicon). The ultrafiltration products were dansylated as previously described (Tapuhi et al, 1981). Aliquots (20 ml) of each sample were applied onto an XTerra RP 18 column (4.66150 mm; Waters) by injection.…”

Section: Methodsmentioning

confidence: 99%

Production of hydrogen sulfide by two enzymes associated with biosynthesis of homocysteine and lanthionine in Fusobacterium nucleatum subsp. nucleatum ATCC 25586

et al. 2010

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Fusobacterium nucleatum produces a large amount of the toxic metabolite hydrogen sulfide in the oral cavity. Here, we report the molecular basis of F. nucleatum H2S production, which is associated with two different enzymes: the previously reported Cdl (Fn1220) and the newly identified Lcd (Fn0625). SDS-PAGE analysis with activity staining revealed that crude enzyme extracts from F. nucleatum ATCC 25586 contained three major H2S-producing proteins. Two of the proteins with low molecular masses migrated similarly to purified Fn0625 and Fn1220. Their kinetic values suggested that Fn0625 had a lower enzymic capacity to produce H2S from l-cysteine (∼30 %) than Fn1220. The Fn0625 protein degraded a variety of substrates containing βC–S linkages to produce ammonia, pyruvate and sulfur-containing products. Unlike Fn0625, Fn1220 produced neither pyruvate nor ammonia from l-cysteine. Reversed-phase HPLC separation and mass spectrometry showed that incubation of l-cysteine with Fn1220 produced H2S and an uncommon amino acid, lanthionine, which is a natural constituent of the peptidoglycans of F. nucleatum ATCC 25586. In contrast, most of the sulfur-containing substrates tested, except l-cysteine, were not used by Fn1220. Real-time PCR analysis demonstrated that the fn1220 gene showed several-fold higher expression than fn0625 and housekeeping genes in exponential-phase cultures of F. nucleatum. Thus, we conclude that Fn0625 and Fn1220 produce H2S in distinct manners: Fn0625 carries out β-elimination of l-cysteine to produce H2S, pyruvate and ammonia, whereas Fn1220 catalyses the β-replacement of l-cysteine to produce H2S and lanthionine, the latter of which may be used for peptidoglycan formation in F. nucleatum.

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“…The pooled extract was air dried and dissolved in a 2-ml 40 mm lithium carbonate (pH 9.5). The procedure of Tapuhi et al (17) was used to separate dansylated amino acids on an HPLC column, with the following modifications: the column consisted of 250 x 4.6 mm 4,um C18 spherisorb ODS. A linear gradient ramp was employed, where methanol was increased from 40 to 75% during a 50-min sample separation, at a constant flow rate of 1.4 ml/min.…”

Section: Methodsmentioning

confidence: 99%

Physiological Changes in Cultured Sorghum Cells in Response to Induced Water Stress

Bhaskaran¹,

Smith²,

Newton³

1985

Plant Physiol.

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ABSTRACITen varieties of Sorghum bicolor (L.) Moench were grown as callus cultures under conditions of water stress, which was induced by addition of polyethylene glycol (molecular weight 8000) in the medium. Growth and free proline were estimated in the control and water-stressed cultures. In all varieties, proline levels were low in the absence of water stress and the levels increased in response to water stress. However, the magnitude of these increases were not correlated with stress tolerance of the individual varieties in culture. Thus increase in proline seems to be an incidental consequence of stress in vitro and not an adaptive response to combat water stress in sorghum.Drought stress is very common in the sorghum-growing regions of the world, and several distinct varieties have evolved with varying degrees ofdrought tolerance (1). There are different mechanisms by which plants cope with water stress. One of the physiological responses to drought is the accumulation of intracellular solutes (16,18). These are generally low mol wt metabolic products such as carbohydrates, amino acids, sugar phosphates, etc. The extent to which each of these plays a role in osmotic adjustment varies with the species. Accumulation of solutes in response to water deficits has been observed in leaves ofsorghum (2,8,9). A similar phenomenon was also observed when plant cells were cultured under conditions of induced water stress using PEG (5). It was further shown that PEG itself did not contribute significantly to the osmotic adjustment (4).There is virtually no information about the physiology of sorghum callus grown in vitro under osmotic stress. Recently, we have been able to show a certain degree of correlation between drought responses in the field and response to water stress in culture (manuscript in preparation). In this study, an attempt was made to analyze free proline levels in sorghum when grown as callus cultures with and without addition ofPEG in the culture medium. The rationale was that this approach would enable us to look for any inherent differences at the cellular level that make some varieties better than others in terms of drought tolerance. advantage to any variety to cope with water stress. Such knowledge is fundamental in selecting genotypes for breeding for drought tolerance. MATERIALS AND METHODSCallus Cultures. Callus cultures were initiated from sorghum seeds using the procedure of Smith et al. (14). Seeds of Sorghum bicolor(L.) Moench varieties B35, RTx7078, RTx7000, RTx432, BTx3197, BTx623, 1790E, RTx430, R9188, and BTx378 were used in our experiments. Callus was subcultured at 4-week intervals until enough callus material was obtained to start them on the stress media. All calli were less than 6 months old when they were subjected to osmotic stress.Water Stress. PEG, mol wt 8000, was added to the growth medium at concentrations of 5, 10, 15, 20, and 25% (w/v) before adjusting the pH of the medium to 5.7. Twenty-five ml of medium was distributed into each culture tube and Heller supports ...

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Dansylation of amino acids for high-performance liquid chromatography analysis

Cited by 381 publications

References 8 publications

The evolution of antiherbivore defenses and their contribution to species coexistence in the tropical tree genusInga

The evolution of antiherbivore defenses and their contribution to species coexistence in the tropical tree genusInga

Production of hydrogen sulfide by two enzymes associated with biosynthesis of homocysteine and lanthionine in Fusobacterium nucleatum subsp. nucleatum ATCC 25586

Physiological Changes in Cultured Sorghum Cells in Response to Induced Water Stress

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