An effielent seintillation radioassay system and sensitive protein assay for submilligram quantities of subcellular membranes

A simple combination of commercially available vials has been devised to reduce both the system volume (2–4 ml) and the consumption of scintillation chemicals for radioassay. Counting efficiency is improved by adding a transparent liquid between the vials. The estimation of protein in the samples either before or after admixture with scintillation fluid is possible with a simple application of the trinitrobenzenesulfonic acid method, following removal of nonprotein trinitrophenylated material by a butanol extraction.     

Pseudomonas aeruginosa Chemotaxis Associated with Blooms of N(2)-Fixing Blue-Green Algae (Cyanobacteria)

Pseudomonas aeruginosa (Schroeter) Migula, a numerically significant bacterium found during N(2)-fixing blooms of the blue-green algae (cyanobacteria) Anabaena sp. in the Chowan River, North Carolina, was chemotactically attracted to amino acids when tested in a radioassay. The bacterium was labeled with P(i), and the disintegrations per minute determined by liquid scintillation counting were proportional to the number of cells accumulating in microcapillaries containing amino acids. Positive chemotaxis was observed toward all of the amino acids tested, although the degrees of response varied. Since many nitrogen-fixing blue-green algae secrete nitrogenous compounds, this attraction may be instrumental in establishing a symbiotic relationship between this bacterium and blue-green algae in freshwater.     

Pseudomonas aeruginosa Chemotaxis Associated with Blooms of N2-Fixing Blue-Green Algae (Cyanobacteria)

Pseudomonas aeruginosa (Schroeter) Migula, a numerically significant bacterium found during N₂-fixing blooms of the blue-green algae (cyanobacteria) Anabaena sp. in the Chowan River, North Carolina, was chemotactically attracted to amino acids when tested in a radioassay. The bacterium was labeled with ³²P_i, and the disintegrations per minute determined by liquid scintillation counting were proportional to the number of cells accumulating in microcapillaries containing amino acids. Positive chemotaxis was observed toward all of the amino acids tested, although the degrees of response varied. Since many nitrogen-fixing blue-green algae secrete nitrogenous compounds, this attraction may be instrumental in establishing a symbiotic relationship between this bacterium and blue-green algae in freshwater.     

Technique-dependent variations in cerebral microvessel blood volumes and hematocrits in the rat.

To quantitate small parenchymal microvessel blood volumes in the brain, the distribution spaces of radiolabeled red blood cells (RBC) and serum albumin (RISA) were assessed in rats by different methods of tissue sampling and radioassay. Three minutes after intravenous administration of 55Fe-RBCs and/or 125I-RISA, the rats were decapitated. The brain was either immediately frozen within the skull and later removed (head-frozen group) or rapidly removed from the skull and then frozen (brain-frozen group). Radioactivity was measured either by liquid scintillation counting of tissue pieces, which contained pial plus large and small parenchymal microvessels, or by quantitative autoradiography (QAR) of tissue sections, which indicated small parenchymal microvessel blood only. In 12 of 15 areas, the RISA, RBC, and blood volumes determined by liquid scintillation counting of head-frozen tissue pieces were equal to or greater than those of brain-frozen tissue; this indicated less than or equal to 25% greater blood retention in pial and parenchymal microvessels with head freezing. At the parenchymal microvessel level (QAR assay), the distribution volumes of RBCs, RISA, and blood were similar with the two freezing techniques; hence with QAR either freezing procedure can be used to assess small parenchymal microvessel blood volumes.     

Phosphate uptake in Saccharomyces cerevisiae Hansen wild type and phenotypes exposed to space flight irradiation.

Rates of phosphate uptake were approximately twice as great for Saccharomyces cerevisiae single-cell phenotypic isolates exposed to space parameters as for the wild-type ground control. Quantitative determination of 32P was performed by liquid scintillation spectrometry utilizing Cerenkov radiation counting techniques.     

Phosphate uptake in Saccharomyces cerevisiae Hansen wild type and phenotypes exposed to space flight irradiation.

Rates of phosphate uptake were approximately twice as great for Saccharomyces cerevisiae single-cell phenotypic isolates exposed to space parameters as for the wild-type ground control. Quantitative determination of 32P was performed by liquid scintillation spectrometry utilizing Cerenkov radiation counting techniques.     

New assay technique for reactions that produce radioactive gases

A new technique is described for the radioassay of gas-producing reactions. The technique is simple and rapid and is useful for the determination of enzyme activities on a routine basis. The technique involves a closed reaction system and gas-solid scintillation counting with scintillator plastic.     

Quantification of 32P-labeled samples in gel fragments using the flat-bed liquid scintillation counter

Quantification of 32P in bands after gel electrophoresis was performed using the flat-bed scintillation counter (Betaplate). The most convenient system involved placing fragments of dried gel between two glass fiber sheets, each previously sealed in a thin plastic bag with liquid scintillant. Good pulse-height spectra and counting efficiencies were obtained with low cross talk and background. The method has been used to quantify mRNA in RNA antisense-protection assays that were linear over a wide range (1-20000 cpm). Cross talk and background could be reduced further by an alternative technique utilizing plastic trays with shallow wells in which a solid scintillant had been melted. Fragments were immersed in the molten scintillant (90 degrees C), which was allowed to solidify, by cooling, before counting.     

Measurement of 32P following colorimetric phosphorus analysis

A procedure for the determination of both ³²P and phosphate contents on the same sample has been described. Following phosphate estimation by the method of Bartlett (5), the solution containing the blue reduced phosphomolybdate complex is decolorized at high pH. ³²P is measured in this transparent aqueous solution with a liquid scintillation spectrometer, without the addition of scintillators, by utilizing the ?erenkov radiation.     

A hexokinase-coupled radioassay for pyruvate kinase

A procedure for the assay of low activities of pyruvate kinase (0.01 to 4 mIU) is described. The method consists of coupling the formation of ATP by the pyruvate kinase reaction to hexokinase in the presence of uniformly labeled [¹⁴C]glucose. The labeled glucose 6-phosphate thus formed is easily separated from the unreacted glucose using small columns of Dowex 1-X8 formate and detected by liquid scintillation spectrometry. Chromatographic patterns of pyruvate kinases from 25 mg of rat liver, 3.5 mg of frog oocyte, and 0.5 mg of the whole body of the fruit fly Drosophila melanogaster are presented as illustrations of the sensitivity of the radioassay.     

Studies on the activating enzyme for iron protein of nitrogenase from Rhodospirillum rubrum

Removal of ADP-ribose from the iron protein of nitrogenase by activating enzyme resulted in the activation of the inactive iron protein. A radioassay that directly measured the initial velocity of the activation was developed using iron protein radiolabeled with either [8-3H]- or [G-32P]ADP-ribose. The release of radiolabeled ADP-ribose by activating enzyme was linearly correlated with the increase in the specific activity of the iron protein as measured by acetylene reduction. Both ATP and MnCl2 were required for the activation of inactive iron protein. The optimal ratio of [MnCl2]/[ATP] in the radioassay was 2:1, and the optimal concentrations were 4 mM and 2 mM for [MnCl2] and [ATP], respectively. The Km for inactive iron protein was 74 microM and the Vmax was 628 pmol of [32P] ADP-ribose released min-1 microgram of activating enzyme-1. Adenosine, cytidine, guanosine, or uridine mono-, di-, or triphosphates did not substitute for ATP in the activation of native iron protein. Activating enzyme removed ADP-ribose from oxygen-denatured iron protein in the absence of ATP. ADP, ADP-ribose, pyrophosphate, and high concentrations of NaCl inhibited activating enzyme activity.     

Liquid scintillation counting of energetic beta-emitting isotopes on solid supports: problems arising from particle range

If emitters of energetic β-particles (such as ³²P) are counted on solid supports in liquid scintillation systems, considerable distortion of the apparent energy spectrum of the β-particles can result if the material is placed on the bottoms or against the walls of vials. This results if the path length in the scintillation fluid available to a substantial proportion of the β-particles is short compared with their range. The phenomenon leads to reduced and variable efficiency in normal counting channels and to difficulties in double-labeling.     

Low background scintillation counting

Counting radioactive samples with Beckman Instrument's Ready Caps, using a restricted energy window, LL-UL = 400-1000, resulted in machine backgrounds of under 2 cpm and efficiencies of counting relative to liquid scintillation cocktails (LSC) of 51%, 65%, 57%, 62%, and 1% for 32P, 125I, 14C, 35S and 3H, respectively. Signal-to-noise ratios from a quantitative molecular hybridization technique were increased 8-10 fold. There may be a general application for this product in experiments yielding low amounts of radioactivity in liquid samples.     

Differential interactions of beta particles with liquid enantiomers and internal timing of chiral molecules

The Cerenkov and liquid scintillation technique was applied to detect differential interactions between beta particles and chiral molecules. The highly helical beta emitting 32P and the barely helical beta emitting 3H were dissolved in the liquid enantiomers of 2-phenylbutyric acid. The interactions of the beta particles with the enantiomers were compared. Differential interactions were observed in case of 32P. The stopping power of R enantiomers was greater than that of the S enantiomer. The apparent decay of 32P was faster in the S enantiomer than in the R enantiomer. It is not clear whether this is due to some mistake in measurement or reflects the postulated difference of internal timing of the two enantiomers. Since, in spite of all efforts, contamination could not totally be excluded and data are significant only to one sigma, results must be considered preliminary. The method, however, seems to be more sensitive than all previously used methods. The differential effect was always in the same direction in at least 5 replicas.     

大鼠单根近端肾小管Na+-K+-ATPase活性测定方法的改进

本研究旨在对Doucet等报道的定量检测大鼠单根近端肾小管Na^＋-K^＋-ATPase活性方法进行改进。取经过Ⅱ型胶原酶消化的大鼠肾脏皮质组织,在体视显微镜下手工分离单根近端肾小管,并测量其长度,经低渗和冻融处理后与[γ-^32P]ATP共同孵育,液闪法检测从[γ-^32P]ATP解离出的^32Pi,采用修正后的公式计算Na^＋-K^＋-ATPase活性。改良法与Doucet等的方法比较,测定单根近端肾小管Na^＋-K^＋-ATPase活性无显著性差异（P〉0．05）。改进后的方法节省试剂,操作简便、省时。     

A new isotopic assay for purine nucleoside phosphorylase

We have developed a new assay for purine nucleoside phosphorylase which is based on the release of tritium when [2-3H]inosine is used as the substrate and the reaction is coupled with xanthine oxidase. After the reaction is terminated, residual [2-3H]inosine is adsorbed on charcoal and the supernatant solution is assayed for radioactivity by liquid scintillation spectrometry. The new method gave results indistinguishable from those obtained by spectrophotometric determination of uric acid produced by the phosphorylase-xanthine oxidase-coupled reaction or by radioassay of chromatographically isolated [8-14C]hypoxanthine when [8-14C]inosine was used as substrate. The new method is faster than those involving chromatographic isolation of products. In comparison with spectrophotometric methods, it not only requires less manual time, but it also has the advantage that it can be used to study inhibitors whose ultraviolet absorption might interfere with spectrophotometric determination of uric acid.     

Use of scintillometric quantitation of unscheduled DNA synthesis in isolated rat hepatocytes for the screening of genotoxic agents

The induction of unscheduled DNA synthesis has been considered as a suitable endpoint for the screening of genotoxic agents. Experimentally, unscheduled DNA synthesis is most frequently measured by autoradiography. The purpose of this report was to examine the usefulness of the liquid scintillation counting technique in measuring unscheduled DNA synthesis response in isolated rat hepatocytes. The various liquid scintillation counting-based unscheduled DNA synthesis assay procedures were examined according to the following groupings: (1) procedures based on the acid precipitation of cellular macromolecules, (2) procedures based on isopycnic gradient centrifugation of solubilized cells, (3) procedures based on nuclei isolation in conjunction with other DNA purification methods, and (4) procedures based on the selective retention of hepatocellular DNA. Limited cases in which test chemicals gave positive unscheduled DNA synthesis response in liquid scintillation counting-based assays and negative unscheduled DNA synthesis response in autoradiography-based assays are presented. It is concluded that liquid scintillation counting-based unscheduled DNA synthesis assays represent an appropriate system for inclusion in carcinogenicity and mutagenicity testing programs.Abbreviations 2-AAF 2-acetylaminofluorene - 2-AF 2-aminofluorene - AFB₁ aflatoxin B₁ - ARG autoradiography - DMN dimethylnitrosamine - LSC liquid scintillation counting - MMS methyl methanesulfonate - MNNG N-methyl-N-nitro-N-nitrosoguanidine - 4-NQO 4-nitroquinoline-1-oxide - PCA perchloric acid - TCA trichloroacetic acid - UDS unscheduled DNA synthesis     

Phosphorylation assay in liquid scintillation counter using C̆erenkov radiation of P: Application to photophosphorylation

A simple procedure is described for the assay of phosphorylation using C?erenkov radiation to detect ³²P in a liquid scintillation counter. Unreacted ³²P_i is first removed from the reaction mixture as the phosphomolybdate complex by butanol/benzene extraction. Addition of ammonium hydroxide to the remaining aqueous fraction avoids color quenching, phase separation, and instability in the counting rate during measurement of ³²P. Application of this procedure to several photophosphorylation systems is included.     

Rapid protein kinase assay using phosphocellulose-paper absorption.

Protein kinase (EC 2.7.1.37) catalyzes the phosphorylation of serine and threonine residues of a number of proteins. Histone is widely used as an acceptor substrate in measuring the activity of this enzyme isolated from a variety of sources. We have devised a rapid procedure for resolving phosphohistone from ATP and its metabolites based on the specific absorption of phosphorylated histone onto phosphocellulose paper. Using [γ-³²P]ATP as the phosphoryl donor, aliquots of the protein kinase assay mixture are applied to phosphocellulose-paper disks that are then immersed in water which elutes [γ-³²P]ATP and metabolites. After brief organic solvent extraction and drying, bound radioactivity is measured by liquid scintillation spectrometry.     

Bacterial growth kinetics: modelling and evaluation of two-compartment radioassay

Quantitative measurements of bacterial growth may be made using a radioassay technique. This method measures, by scintillation counting, the 14CO2 derived from the bacterial metabolism of a 14C-labeled substrate. Mathematical growth models may serve as reliable tools for estimation of the generation rate constant (or slope of the growth curve) and provide a basis for evaluating assay performance. Two models, i.e., exponential and logistic, are proposed. Both models yielded an accurate fit to the data from radioactive measurement of bacterial growth. The exponential model yielded high precision values of the generation rate constant, with an average relative standard deviation of 1.2%. Under most conditions the assay demonstrated no changes in the slopes of growth curves when the number of bacteria per inoculation was changed. However, the radiometric assay by scintillation method had a growth-inhibiting effect on a few strains of bacteria. The source of this problem was thought to be hypersensitivity to trace amounts of toluene remaining on the detector.