An ultramicro method of amino acid analyses: Application to studies of protein metabolism in cultured cells

Airhart, J; Kelley, Jason; Brayden, Joseph E.; Low, Robert B.; Stirewalt, William S.

doi:10.1016/0003-2697(79)90552-9

Cited by 101 publications

(45 citation statements)

References 18 publications

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“…These determinations were performed as previously described by Airhart et al (2) with 3H-labeled dansyl chloride (5-dimethylamino-l-naphthalenesulfonyl chloride, Amersham). The specific radioactivity of the dansyl chloride used was determined by reaction with standard samples of ["4C]leucine and ['4C]proline.…”

Section: Methodsmentioning

confidence: 99%

“…Subsequently, most of the dansyl hydroxide formed was eluted with 5 x 1 ml 5% v/v aqueous acetic acid and the dansyl derivatives were eluted with 3 x 1 ml 80%o v/v aqueous acetone. The acetone eluate was taken to dryness in a stream of nitrogen, dissolved in an appropriate volume of 1% triethylamine in ethanol, and a fraction (about 50%o) subjected to TLC on polyamide plates (F 1700 from Schleicher and Schuell, Keene, NH) (2,17). Combinations of the solvent systems described in the lieterature permitted separation of the derivatives of all the amino acids studied.…”

Section: Methodsmentioning

confidence: 99%

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Amino Acid Synthesis in Photosynthesizing Spinach Cells

Larsen¹,

Cornwell²,

Gee³

et al. 1981

Plant Physiol.

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Isolated cells from leaves of Spincia oleracea have been maintaied in a state capable of high rates of pbotosynthetic CO2 fixatin for more than 60 hours. The incorporation of 14CO2 under saturating CO2 conditions into carbohydrates, carboxylic acids, and amino acids, and the effect of ammonia on this incorporation have been studied. Total incorporation, specific radioactivty, and pool size have been determined as a function of time for most of the protein amino acids and for y-aminobutyric acid. The measurements of specific radio-activities and of the approaches to "C "saturation" of some amino acids indicate the presence and relative sizes of metabolically active and passive pools of these amino acids.Added ammonia decreased carbon fixation into carbohydrates and increased fixation into carboxylic acids and amino acids. Different amino acids were, however, affected in different and highly specific ways. Ammonia caused large stimulator effects in incorporation of 'C into glutamine (a factor of 21), aspartate, asparagine, valine, alanine, arginie, and histidine. No effect or slght decreases were seen in glycine, serine, phenylalanine, and tyrosine labeling. In the case of glutamate, "4C labeling decreased, but specific radioactiity increased. The production of labeled -aminobutyric acid was virtuafly stopped by ammonia.The results indicate that added ammonia stimulates the reactions mediated by pyruvate kinase and phosphoenolpyruvate carboxylase, as seen with other plant systems. The data on the effects of added ammonia on total labelin, pool sizes, and specific radioactivities of several amino acids provides a number of indications about the intracellular sites of principal synthesis from carbon skeletons of these amino acids and the selective nature of effects of increased intracellular ammonia concentration on such synthesis.In recent years, the production of photosynthetically active cells from leaf tissue has been reported for a number of plant species, including Papaver somniferum (opium poppy) (22), spinach (31), and cotton (28). Such cells afford possibilities for studying plant metabolism in a simple but intact system. Effects on poppy cell metabolism of ammonia (24), sulfite (23), and 2,4-D (25) have been reported from this laboratory, and others have described effects of ammonia and other nitrogen compounds on metabolism in isolated spinach cells (31,32 incorporation into amino acids has been studied to provide information about amino acid biosynthesis and the provision of carbon skeletons for this purpose. For the amino acids occurring in the free state, values have been obtained for total 14C labeling, specific radioactivities, and, by combination of these data, pool sizes. The methods used have permitted some measurements for all the protein amino acids except cysteine/cystine and methionine, and thus provide a more detailed and complete picture of amino acid formation from CO2 in leaf cells than previously available.Previous work from this and other laboratories has demonstrated that ammoni...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Amino Acid Synthesis in Photosynthesizing Spinach Cells

Larsen¹,

Cornwell²,

Gee³

et al. 1981

Plant Physiol.

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“…R. Low and W. Stirewalt (University of Vermont, Burlington, VT, U.S.A.) using a procedure involving dansylation of the amino acid followed by t.l.c. [19].…”

Section: Cell Culturementioning

confidence: 99%

Inhibition of cell proliferation by interferons. Relative contributions of changes in protein synthesis and breakdown to growth control of human lymphoblastoid cells

McNurlan¹,

Clemens²

1986

Biochemical Journal

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Treatment of the Daudi line of human lymphoblastoid cells with concentrations of human interferons within the physiological range progressively inhibits cell proliferation over 1-4 days. Rigorous measurement of the overall rate of protein synthesis during this period, using a concentration of [3H]phenylalanine sufficient to equalize the specific radioactivity of intracellular and extracellular precursor pools, shows that protein synthesis becomes progressively inhibited as the growth inhibition develops. There is a strong correlation between inhibition of amino acid incorporation and inhibition of cell proliferation. In contrast, we find no evidence for any increase in protein degradation rate under these conditions. These results suggest that interferon treatment of susceptible cells can inhibit protein synthesis even in the absence of virus infection and that this inhibition is of a sufficient magnitude to account for the anti-proliferative effect. INTRODUCTIONThe ability of interferons to inhibit viral replication in infected cells through effects on [4][5][6]. On the other hand,-inhibition of incorporation of labelled amino acids has been observed in some systems in which cell proliferation is inhibited [7][8][9]. The human lymphoblastoid Daudi cell line is extremely sensitive in exhibiting a marked suppression of growth when human interferons are added in culture [10][11][12]. We have investigated, using this system, the extent to which the changes in growth rate can be correlated with changes in protein synthesis. In order to do this it is necessary to quantify both processes rigorously. Such an approach also allows indirect estimates to be made of the rate of protein breakdown, and of the relative contributions of changes in synthesis versus breakdown to the inhibition of cell growth caused by interferon treatment.The rate of incorporation of a radioactive amino acid into protein reflects both the actual rate at which protein is synthesized and the specific radioactivity of the precursor which is being incorporated. If the values of the latter are different between two populations of cells then spurious conclusions can be drawn. In an attempt to overcome any such problems we have adapted procedures developed for measurement of protein synthesis rates in perfused organs [13] and in tissues of

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“…Specific radioactivity of proline in fibroblast intracellular free amino acid pool and amino acyl-transfer RNA (tRNA) pool were measured by a method described by Airhart et al (13) and modified by Hildebran et al (14). In these experiments, confluent fibroblasts were cultured in P-60 culture dishes (Falcon Labware) with 2 layer was precipitated with trichloroacetic acid; the supernatant containing the intracellular free amino acid pool was analyzed as described below to determine intracellular free proline-specific radioactivity.…”

Section: Introductionmentioning

confidence: 99%

Bleomycin-induced pulmonary fibrosis in hamsters. An alveolar macrophage product increases fibroblast prostaglandin E2 and cyclic adenosine monophosphate and suppresses fibroblast proliferation and collagen production.

Clark¹,

Kostal²,

Marino³

1983

J. Clin. Invest.

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A B S T R A C T Bleomycin-induced pulmonary fibrosis in hamsters is associated with collagen accumulation that results from increased lung collagen synthesis rates. However, 1-2 wk after intratracheal instillation of bleomycin, lung collagen synthesis rates decline toward control values. To evaluate the potential role of the bronchoalveolar macrophage in regulating lung collagen production, we studied the effects of macrophages from normal and bleomycin-treated hamsters upon fibroblasts in vitro. We observed: (a) Medium from macrophage cultures decreased fibroblast [3H]thymidine incorporation and nondialyzable [3H]hydroxyproline production in a dose-dependent manner. Fibroblast cell counts were decreased in exposed cultures, and fibroblast viability was unchanged. Procollagen prolyl hydroxylation and prolyl-transfer RNA-specific activity were not altered by macrophage medium; this indicates that [3H]hydroxyproline reflects collagen production rate under the experimental conditions. (b) The suppressive effect of macrophage medium was selective for collagen since collagen production decreased more than noncollagen protein production. (c) Medium from bleomycin-treated hamster macrophages suppressed fibroblast proliferation and collagen production to a greater degree than medium from normal hamster macrophages. (d) Fibroblast suppression by macrophage medium was associated with increased fibroblast endogenous prostaglandin E2 production and intracel-A preliminary report of these studies was presented at the

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An ultramicro method of amino acid analyses: Application to studies of protein metabolism in cultured cells

Cited by 101 publications

References 18 publications

Amino Acid Synthesis in Photosynthesizing Spinach Cells

Amino Acid Synthesis in Photosynthesizing Spinach Cells

Inhibition of cell proliferation by interferons. Relative contributions of changes in protein synthesis and breakdown to growth control of human lymphoblastoid cells

Bleomycin-induced pulmonary fibrosis in hamsters. An alveolar macrophage product increases fibroblast prostaglandin E2 and cyclic adenosine monophosphate and suppresses fibroblast proliferation and collagen production.

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