Liquid Scintillation Counting of Carbon-14.  Use of Ethanolamine-Ethylene Glycol Monomethyl Ether-Toluene

Jeffay, Henry; Alvarez, Julian

doi:10.1021/ac60172a041

Cited by 434 publications

(84 citation statements)

References 9 publications

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“…At designated times the apple tissue was removed from duplicate flasks, separated from the incubation medium via filtration, washed on the filter with 5 ml of fresh medium, oven-dried, and oxidized by dry combustion. The CO, was collected in ethanolamine and counted by liquid scintillation (6).…”

Section: Methodsmentioning

confidence: 99%

Inhibition of Ethylene Production by Rhizobitoxine

1971

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Rhizobitoxine, an inhibitor of methionine biosynthesis in Salmonella typhimurium, inhibited ethylene production about 75% in light-grown sorghum seedlings and in senescent apple tissue. Ethylene production stimulated by indoleacetic acid and kinetin in sorgh-um was similarly inhibited. With both apple and sorghum, the inhibition could only be partially relieved by additions of methionine. A methionine analogue, a-keto-ymethylthiobutyric acid, which has been suggested as an intermediate between methionine and ethylene, had no effect on the inhibition.Incorporation of "4C from added methionine-'4C into ethylene was curtailed by rhizobitoxine to about the same extent as was ethylene production. These results suggest that rhizobitoxine interferes with ethylene biosynthesis by blocking the conversion of methionine to ethylene and not indirectly by inhibiting the biosynthesis of methionine. Ethylene production by Pemicillium digitatum, a fungus which produces ethylene via pathways not utilizing methionine as a precursor, was not affected by rhizobitoxine.Two model systems for the generation of ethylene in plant tissues have been described by Lieberman and co-workers, one utilizing methionine as a substrate (8), and the other utilizing linolenate (1 1). In addition, methionine can serve as a precursor of ethylene in plant tissues (2, 7). To help assess the physiological importance of methionine as an ethylene precursor, a specific inhibitor of methionine biosynthesis was sought. Rhizobitoxine appeared to offer that potential.Rhizobitoxine is a phytotoxin produced by certain strains of the soybean root nodule bacterium Rhizobium japonicum (15).It inhibits greening of new leaf tissue of many plants and causes the main visual symptom of the disease in soybean known as rihizobial-induced chlorosis (14). The precise structure of rhizobitoxine remains to be elucidated; however, it is known to be a basic sulfur-containing amino acid which yields an ether derivative of homoserine upon desulfurization (13). Rhizobitoxine inhibits the growth of Salmonella typhimurium by inhibiting /3-cystathionase, an enzyme in the methionine biosynthetic pathway (12). It also irreversibly inactivates /Bcystathionase isolated from spinach leaves (4); however, the physiological effect of this lesion on the biosynthesis of methionine in spinach has yet to be assessed. We report here that rhizobitoxine inhibits ethylene biosynthesis in sorghum seedlings and in senescent apple tissue by the unexpected mechanism of blocking the conversion of methionine to ethylene. Hegari were surface-sterilized by wetting with ethanol and then immersing in an aqueous solution of 0.2% HgCl2 + 1% HCl for 2 min. After rinsing well, the seeds were germinated on moist filter paper in a Petri dish at 27 C in the dark. Two days after imbibition, the seedlings were transplanted to 50-ml Erlenmeyer flasks constructed with a side arm to collect CO2. Six seedlings per flask (about 300 mg fresh wt) were supported on a nylon mesh screen held 1.0 cm above the flask bottom...

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Section: Methodsmentioning

confidence: 99%

Inhibition of Ethylene Production by Rhizobitoxine

1971

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“…CO 2 was absorbed from the bags of expired air in a mixture of ethanolamine:methylcellosolve 1:2 [10] using a modification of the procedure described by FREDRICKSON and ONO [7]. Each bag was connected to a flow system which proceeded first through a 70 ml side-arm test tube in an acetone dry ice mixture, in order to trap water, and then through a series of 30 ml screw cap test tubes placed inside 70 ml side-arm test tubes.…”

Section: Methodsmentioning

confidence: 99%

“…After a bag was emptied, the inner test tubes were removed and their screw caps applied tightly to await assay of the contents. The amount of absorbed 14 CO 2 was determined by counting 3.0 ml from each tube in a mixture of toluene, methylcellosolve and 2,5-diphenyloxazole (PPO) [10] using a liquid scintillation spectrometer. The CO 2 content of the absorbed 14 CO 2 was determined by the method of VAN SLYKE [25] using concentrated lactic acid and a 0.2 ml aliquot of trapping solution.…”

Section: Methodsmentioning

confidence: 99%

“…The specific activities of the serine isolated from plasma after the injection of glycine- [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C are shown in table II. In the control subjects, in the absence of a glycine-infused state, the specific activities of serine decreased sharply from maxima at the earliest times studied.…”

Section: Conversion Of Glycine To Serinementioning

confidence: 99%

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Metabolism of Glycine in the Nonketotic Form of Hyperglycinemia

et al. 1968

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Hyperglycinemia is a disorder of amino acid metabolism characterized by the presence of increased concentrations of glycine in the blood, urine, and cerebrospinal fluid. It is now recognized that there are two forms of hyperglycinemia each representing distinct diseases. These studies were designed to assess the metabolism of glycine in the nonketotic form of hyperglycinemia. Isotope content was assessed in respiratory CO 2 and in glycine, serine and the /? carbon of serine of plasma after the separate intravenous injections of glycine-1-14 C and glycine-2-14 C. The specific activities of 14 CO 2 isolated from expired air after the injection of glycine-1-14 G ( fig. 2) declined in control subjects from peak values at 10 to 15 minutes in nearly linear fashion over a 2-hour period. In contrast, curves obtained in the patients were rather flat, rising slowly after injection to highest values at about 60 minutes. At 15 minutes, values for the control individuals exceeded those of the patients by a factor of 5-to 10-fold. These data indicate a defect in the formation of 14 CO 2 from the first carbon of glycine. When the control subjects were infused with nonisotopic glycine to produce pools comparable to those found in the patient, the specific activities of the serine isolated from plasma after the injection of glycine-1-14 C (table II) were virtually the same in both groups. The rate of conversion of glycine-2-14 C to serine ( fig. 3) in the patients was, however, considerably slower than it was in the control subjects for at least the first 30 minutes, and the curves were flat throughout. Degradation of the serine isolated from plasma and precipitation of the /S carbon as formaldemethone indicated that the incorporation of the a carbon of glycine into the /? carbon of serine was much higher in the controls than in the patients (fig. 4). The curves for the patients approximated the abscissa indicating virtually no conversion.These data indicate that in nonketotic hyperglycinemia there is a defect in the oxidation of carbon 1 of glycine to CO 2 and in the conversion of carbon 2 of glycine to carbon 3 of serine. This is consistent with a defect in an enzyme catalyzing the transformation of glycine to CO 2 , NH 3 and hydroxymethyltetrahydrofolate. SpeculationThe data obtained indicate that patients with nonketotic hyperglycinemia are unable in vivo to convert the first carbon of glycine directly to CO 2 and the second carbon of glycine to the third carbon of serine. This is consistent with a genetic defect in an enzyme which catalyzes decarboxylation and formation of hydroxymethyl tetrahydrofolate from glycine. It should be possible to document such a defect at a cellular and subcellular level.

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“…All of the material was combusted but if the dry weight of any organ exceeded 200 mg it was divided into components not exceeding 200 mg. The released carbon dioxide was trapped as ethanolamine carbonate in a 1 : 9 (v/v) ethanolaminemethylcellosolve mixture (Jeffray and Alvarez 1961). An aliquot of the carbonate solution was added to a scintillant of toluene-p-terphenyl-dimethyl-POPOP (867: 8 : 0·2 by weight) and counted in a Tri-carb spectrometer (efficiency of 80%).…”

Section: (C) Sampling and Measurementmentioning

confidence: 99%

Distribution of Assimilate During Stem Elongation in Wheat

Patrick¹

1972

Aust. Jnl. Of Bio. Sci.

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During the phase of stem extension in plants of Triticum aestivum L. cv. Stewart, the distribution of assimilated 14C appeared to be related to sink size, proximity to the source, and a canalizing effect imposed by the vascular system on the movement between leaves. Evidence was found of a greater resistance to export from a leaf in the upward than in the downward direction and this is consistent with the observed arrangement of the sieve elements linking the bundles at the nodes. The cross· sectional area of the phloem did not appear to impose a limitation on the amount of material transported to the apex. The bulk of carbon imported by a growing leaf was consistently transported from the second lamina below. Import from other leaves continued after the emergence of a lamina and accounted for some 80% of its final dry weight and 50% of that in the attached sheath. The elongating internodes 81ther side of the leaf formed large sinks for its photosynthate. Ear growth, prior to its emergence, was supported by the upper three leaves. After emergence the flag leaf was the main supplier.

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Liquid Scintillation Counting of Carbon-14. Use of Ethanolamine-Ethylene Glycol Monomethyl Ether-Toluene

Cited by 434 publications

References 9 publications

Inhibition of Ethylene Production by Rhizobitoxine

Inhibition of Ethylene Production by Rhizobitoxine

Metabolism of Glycine in the Nonketotic Form of Hyperglycinemia

Distribution of Assimilate During Stem Elongation in Wheat

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