Improved solubilization procedures for liquid scintillation counting of biological materials

Hansen, D.; Bush, E.T.

doi:10.1016/0003-2697(67)90016-4

Cited by 134 publications

(27 citation statements)

References 12 publications

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“…The filter discs were counted in a Nuclear Chicago thin-window gas-flow counter, or when it was necessary to count 3H and 14C simultaneously, they were counted in a Packard Tri-Carb Liquid Scintillation Spectrometer. In the latter case NCS Reagent (Nuclear Chicago, Des Plaines, Ill.) (Hansen & Bush, 1967) was used to dissolve the protein precipitates in a toluene-based scintillation fluid. The efficiency of gas-flow counting for 14C was about 25%.…”

Section: Introductionmentioning

confidence: 99%

Levels of Resistance in ribosomes from Genetically Linked, Streptomycin-resistant Mutants of Pneumococcus

Stuart

Ravin

1968

Journal of General Microbiology

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A streptomycin-sensitive strain and six streptomycin-resistant mutants of pneumococcus have been studied. These strains differed in resistance to streptomycin over a 5000-fold range, and the resistance mutations occurred at recombinationally distinct sites of a single genetic locus (the str locus). The effects of streptomycin on cell-free amino acid incorporating systems prepared from each of the strains were studied. Both polyuridylic acid (poly U)-and endogenous mRNA-directed systems were employed. As in Escherichia coli, sensitivity to streptomycin was found to reside in the ribosomal fraction of the amino acid incorporating system.Streptomycin caused both inhibition of l4CC-phenylalanine incorporation and stimulation of 3H-isoleucine incorporation in poly U-directed experiments. The resistance of the amino acid incorporating systems to these effects of streptomycin paralleled the streptomycin resistance of the strain from which the system was derived. In endogenous mRNA-directed systems the incorporation of 14C-valine and 3H-isoleucine was followed simultaneously. Streptomycin caused an inhibition of valine incorporation and a lesser inhibition of isoleucine incorporation. At a higher magnesium concentration streptomycin caused a stimulation of isoleucine incorporation while still inhibiting valine incorporation. The disparate behaviour of isoleucine and valine incorporation in the presence of streptomycin may be due to streptomycin-induced misreading of endogenous mRNA. As in the poly U-directed experiments, the magnitude of the effects of streptomycin on endogenous mRNA directed amino acid incorporation correlated in an inverse fashion with the resistance of the strain from which the system was prepared.These results lead us to conclude that the ribosome is the primary target for streptomycin in pneumococcus. We propose that the affinity of the ribosome for streptomycin at a critical site or sites determines the level of resistance of the bacterium, and that this affinity is affected by mutations in the str locus.

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Section: Introductionmentioning

confidence: 99%

Levels of Resistance in ribosomes from Genetically Linked, Streptomycin-resistant Mutants of Pneumococcus

Stuart

Ravin

1968

Journal of General Microbiology

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“…At least 94% of the 14C accumulated by bacteria remained attached to the filter if it was removed from the scintillant. Nevertheless self-absorption was shown to be negligible by taking a filter whose 14C content had been estimated as above and treating it with hyamine hydroxide (Hansen & Bush, 1967). This dissolved 90% or more of the 14C, which was then estimated in a one-phase system using a I :2 (v/v) mixture of triton X-IOO and the scintillant described above.…”

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confidence: 99%

Methionine Transport in Wild-type and Transport-defective Mutants of Salmonella typhimurium

Ayling

Bridgeland

1972

Journal of General Microbiology

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-2 p~) .Competition studies have shown that the permease has little or no affinity for the other L-amino acids commonly found in proteins. Methionine uptake was competitively inhibited by the growth inhj bitory analogues DLethionine, a-methyl-DL-methionine and DL-methionhe-DL-sulphoximine. Mutants resistant to a-methyl-methionine and methionine sulphoximine have been isolated which were severely defective in the niethionine specific permease. Two of these mutants, metP760 and rnetP761, mapped away from all previously located methionine structural and regulatory genes.

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“…Gel filtration was performed using upward flow at a rate of 0.35 ml per min. Aliquots of fractions were prepared for liquid scintillation counting according to Patterson and Trewavas (23) except that 0.1 ml of a saturated solution of benzoyl peroxide in toluene was added to each vial to bleach pigments (12). The activities of 3H and "4C for each sample were calculated after correction from efficiency calibration curves.…”

Section: Methodsmentioning

confidence: 99%

Light-induced Changes in the Pattern of Protein Synthesis during the Early Stages of Greening of Etiolated Maize Leaves

Kaveh

Harel²

1973

Plant Physiol.

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The effect of light on protein synthesis during the early stages of greening of etiolated maize (Zea mays) leaves was studied using double labeling with leucine and fractionation of proteins by gel filtration and acrylamide gel electrophoresis. The incorporation of labeled leucine into a relatively small number of plastid proteins is effected within the first 30 to 60 minutes of illumination. These proteins do not accumulate with time. When illumination is prolonged, additional proteins are effected.Experiments using inhibitors of protein synthesis suggest that at least some of the proteins effected by 1 hour of illumination might be synthesized in the cytoplasm and not in plastids. Actinomycin D inhibits the incorporation of labeled leucine into some of the protein fractions, but enhances the incorporation into other fractions far above the effect exerted by light.Leaves of dark-grown plants green when they are transferred to light. Greening, which ultimately results in the formation of a functioning photosynthetic apparatus, involves many interrelated processes. These include structural rearrangement, synthesis of RNA and proteins, and a phase of rapid synthesis of chlorophyll which follows a lag period (2-4, 18). Numerous reports suggest that continued synthesis of RNA and proteins is required both for events occurring in the lag phase and for the accumulation of chlorophyll. Most reports on the effect of light on the synthesis of allegedly mRNA and proteins deal with periods of 2 to 6 hr after the beginning of illumination (5,9,16, 18,20,24,31,32). However, several authors reported effects on certain enzymes after much shorter periods (2,6,7, 18 MATERIALS AND METHODSMaize (Zea mays) (WF9 X B37-single cross from Illinois Foundation Seeds Inc.) was grown in vermiculite in the dark at 25 C. Leaves from 8-to 9-day-old plants were harvested under a green safelight, and isotopes were applied as previously described (13). The isotope solutions were brought to identical specific radioactivity with sterilized distilled water and L-leucine solution. The purity of the isotope solutions was examined by paper chromatography and radiochromatography. After a period of uptake in the dark the leaves were either transferred to light (400-600 ft-c) or left in the dark.Isolation of Plastids in a Nonaqueous Medium. Leaves which received various treatments were freeze-dried and 2 g were ground with mortar and pestle in 40 ml of ice cold CCL. The homogenate was filtered through glass wool and 2 ml of n-hexane layered over the filtrate in the centrifugation tube. The tubes were then centrifuged at 13,000g for 15 min in a Sorvall HB-535 rotor. The plastids, which concentrated in the hexane layer at the top, were layered over a mixture of CCl, and n-hexane (100:36) and centrifuged at 13,000g for 15 min. The precipitate consisted mainly of plastids as judged by observations with light and fluorescence microscopes. The precipitate was either extracted with 1% Triton X-100, and the supernatant resulting from centrifuging at 100...

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Improved solubilization procedures for liquid scintillation counting of biological materials

Cited by 134 publications

References 12 publications

Levels of Resistance in ribosomes from Genetically Linked, Streptomycin-resistant Mutants of Pneumococcus

Levels of Resistance in ribosomes from Genetically Linked, Streptomycin-resistant Mutants of Pneumococcus

Methionine Transport in Wild-type and Transport-defective Mutants of Salmonella typhimurium

Light-induced Changes in the Pattern of Protein Synthesis during the Early Stages of Greening of Etiolated Maize Leaves

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