Determination of the specific radioactivity of 14C-labeled glutamic acid and glutamine

A new, simple, and accurate method for the sequential determination of the specific radioactivity of [1-¹⁴C]glutamic acid and [1-¹⁴C]glutamine is described. Using this method, radioactivity in H¹⁴CO₃^?, in [¹⁴C]glutamic acid, and in [¹⁴C]-glutamine can be readily determined on a single sample of blood plasma. Radioactivity is released as ¹⁴CO₂ in a stepwise fashion, trapped in the center wells, and counted in a liquid scintillation counter. The applicability of the method is discussed.     

A simple method for determining specific esterase activity in tissue extracts.

A simple procedure for estimating specificB-naphthyl esterase enzyme content and activity in small amounts of crude tissue extract is described. Esterase activity is determined by quantitative densitometry of histochemically stained acrylamide gels. Activity measured this way is linear with the extract volume applied. Esterase content is determined by isotopic labeling with a stoichiometric covalently binding enzyme inhibitor, diisopropylfluorophosphate; followed by electrophoresis and gel slice counting.     

Economical synthesis of 14C-labeled aminolevulinic acid for specific in situ labeling of plant tetrapyrroles

Photosynthesis Research - The application of metabolic radiolabeling techniques to plant tetrapyrroles, i.e., chlorophyll and hemes, is complicated by the difficulty of obtaining sufficient...     

The specific radioactivity of glycolic acid in relation to the specific activity of carbon dioxide evolved in light in photosynthesizing sunflower leaves

Attached leaves of sunflower (Helianthus annuus L.) were exposed to ¹⁴CO₂ during steady-state photosynthesis for 2 to 30 min in 345 l/l CO₂ and 21% O₂ at 29° C and a light intensity of 1300 E m^-2s^-1. Glycolic acid was extracted with water and diethyl ether, and was determined in the aqueous residue by high-pressure liquid column chromatography. The relative specific radioactivity of the glycolic acid synthesized during photosynthesis reached about 100% after 30 min of photosynthesis and was almost equal to that of the CO₂ evolved during photorespiration, their ratio at all times being nearly one. These results provide strong in-vivo evidence that the glycolic acid is the substrate for CO₂ evolved by sunflower leaves in light.     

A new method for the determination of 14C radioactivity in ketone bodies.

A reaction vessel is described consisting of a simple reaction chamber fixed to an ordinary liquid scintillation-counting vial which allows the direct determination of radioactivity in acetone as well as separately in the carboxyl and acetone moieties of acetoacetate and 3-hydroxybutyrate. Radioactivity and its distribution between carbon 1 and carbons 2 through 4 can be determined in total ketone bodies as well as sequentially in acetone, acetoacetate, and 3-hydroxybutyrate. Recoveries with labeled ketone bodies under a variety of analytical conditions are presented.     

A method for determining tissue sulfate

A method for the determination of the inorganic sulfate present in rat liver homogenates has been developed. In order to determine sulfate, a protein-free extract is required. The classical protein precipitation methods of preparing protein-free extracts gave 2.5–40% recovery of added ³⁵SO₄^2?. Separation of the protein by ultrafiltration gave only 29% recovery when 0.15 m KCl was the homogenizing medium. A homogenization medium containing 0.154 m NH₄OH and 20 g EDTA per liter gave 102 ± 11% recovery of added ³⁵SO₄^2? when the protein was separated by ultrafiltration.     

A radio-gas chromatographic method for the determination of 11-oxotestosterone in fish plasma: its use in confirming estimations by radioimmunoassay

A radio-gas chromatographic method has been devised for the estimation of 11-oxotestosterone (17beta-hydroxyandrost-4-ene-3, 11-dione) in fish plasma samples which provides an independent means of validating the radioimmunoassay described earlier. Estimates of the concentration of 11-oxotestosterone in a sample of male rainbow trout plasma by radio-gas chromatography using peak height and peak weight measurements were 6.9 microgram/100 ml and 7.4 microgram/100 ml respectively, in good agreement with that of 7.1 microgram/100 ml determined by radioimmunoassay.     

A novel method for measurement of the specific radioactivity of gamma-32P ATP.

The specific radioactivity of the γ-phosphorus of ATP has been determined by an indirect method. Galactokinase is employed to transfer the terminal phosphate group of [γ-³²P] ATP to [1-³H] galactose. The doubly labeled galactose-1-phosphate is purified by ion exchange chromatography on QAE Sephadex. The specific radioactivity of the phosphorus is calculated from the ${}^{32}\text{P}{}^3\text{H}$ ratio. The method is extremely sensitive, requiring only 0.005 μmoles of ATP with a specific radioactivity of 1 μCi/μmole, and the chromatographic isolation of galactose-1-phosphate is simple and reproducible. The method is directly applicable to the determination of the specific radioactivity of [γ-³²P] ATP in biological samples.     

A method for measurement of the specific radioactivity of the choline moiety of choline phospholipids.

The specific radioactivity of a choline phospholipid has been determined by a double-isotope method. Purified phospholipid was hydrolyzed to release labeled choline, and choline kinase was employed to label the choline with ³²P from [γ-³²P]ATP. The double-labeled phosphorylcholine was purified by ion-exchange chromatography on QAE-Sephadex, and the specific radioactivity of the choline was calculated from the isotope ratio. The method is sensitive, requiring only 5 nmol of choline with a specific radioactivity of 1 μCl/μmol, and the chromatographic isolation of phosphorylcholine is simple and reproducible.     

Randomization of carbon atoms in the glucose molecule and changes of specific radioactivity of14CO2 liberated by the callus tissue ofDaucus carota L. from glucose-6- and -1-14C

On incubation of the callus tissue ofDaucus carota L. in solutions of glucose-6-¹⁴C and -1-¹⁴C the distribution of radioactivity in the molecule of endogenous glucose will change and the ratio of activities of liberated¹⁴CO₂ (C₆/C₁) will rise The limits of possible changes of specific activity of¹⁴CO₂ and of the C₆/C₁ ratio were calculated with respect to the observed randomization and it was shown that the mutual exchange of carbon atoms in the molecules is not the decisive cause of the rise of the ratio. The specific radioactivity of¹⁴C in CO₂ is as much as 12 times higher than that of endogenous glucose and fructose and about twice as high as the theoretical maximum. This might indicate that in addition to the cytoplasmic fraction of glucose the callus cells contain a fraction of low metabolic activity, most likely in the vacuoles, that could account for some of the increase of the C₆/C₁ value. The main reason for the changes in the C₆/C₁ ratio is envisaged in the establishment of isotopic equilibrium between the pentose cycle and glycolysis and other metabolic systems, in particular via triose phosphates, the radioactivity of which can greatly affect the C₆/C₁ ratio, as was shown in a model experiment.     

A method for the estimation of amino acid radioactivity in biological samples

A method for quantitative estimation of total radioactivity present in the free amino acid fraction of tissue samples has been described. Samples deproteinized with cold acetone were extracted, in acidic medium, with ethyl (peroxide free); after centrifugation, the aqueous phase was used for amino acid derivatization at 40°C for 15 h with 1-flouro-2,4-dinitrobenzene in bicarbonate-buffered medium. Aliquots of the derivatized samples were acidified and extracted twice again with ethyl ether. The combined organic phases were placed in glass scintilation vials, dried, and used for the determination of its radiactivity, corresponding to the radioactivity present in the free amino acid fraction of the sample. Deproteinized samples of rat blood plasma, as well as hen egg white and yolk were tested after addition of known quantities of ¹⁴C-labelled amino acids or glucose, for validation of the method. No glucose radioactivity was found in any of the extracted samples. All radioactivity added to the samples in the form of ¹⁴C-labelled alanine, glutamic acid, leucine and phenylalanine was quantitatively recovered in the derivatized fraction; only a fraction of arginine radioactivity was recovered.     

A simple method for determining the boundary layer resistance in leaf cuvettes

Abstract. A new method is described for determining the boundary layer resistance over wet filter paper exposed within a leaf cuvette, based on the energy balance of the filler paper. The boundary layer resistance is calculated by an iterative procedure from measurements of the relative humidity and temperature of the air in the cuvette. Comparisons between the new and the conventional method, involving measurement of the filter paper temperature, show close agreement.
To simplify the method further, a graph has been constructed for the relationship between boundary layer resistance and cuvette relative humidity at temperatures from 15 to 35°C, determined at one value of the ratio of the flow rate through the cuvette to the filter paper area.
An analysis of errors suggests that the new method is less sensitive than the conventional to errors in temperature and humidity.     

Determination of 35S-aminoacyl-transfer ribonucleic acid specific radioactivity in small tissue samples

Rate determination of protein synthesis utilizing tracer amino acid incorporation requires accurate assessment of the specific radioactivity of the labeled precursor aminoacyl-tRNA pool. Previously published methods presumably useful for the measurement of any aminoacyl-tRNA were unsuccessful when applied to [³⁵S]methionine, due to the unique chemical properties of this amino acid. Herein we describe modifications of these methods necessary for the measurement of ³⁵S-aminoacyl-tRNA specific radioactivity from small tissue samples incubated in the presence of [³⁵S]methionine. The use of [³⁵S]methionine of high specific radioactivity enables analysis of the methionyl-tRNA from less than 100 mg of tissue. Conditions for optimal recovery of ³⁵S-labeled dansyl-amino acid derivatives are presented and possible applications of this method are discussed.