Citric acid labelled with

Little was known of the mechanisms of action of aconitase because all attempts at purification failed until it was demonstrated that the enzyme requires iron and cysteine for activity. It has since become evident that the mechanism of the reaction catalyzed by aconitase is extremely complex. C/.y-aconitate was thought to be an intermediate, but the compound could not be isolated. This led Speyer and Dickman [78] to suggest that a common intermediate exists between citrate on the one hand, and aconitate and isocitrate on the other (see Fig. 1-17). These authors found that when citric acid labeled with heavy water was used as a substrate, the isocitrate was extensively labeled, while only traces of deuterium were found in cw-aconitate. This suggested that cw-aconitate is not on the pathway leading from isocitric to citric acid. To explain these results, the authors postulated an intermediate common to c/.y-aconitate and isocitrate consisting of a tricarboxylic acid forming a complex with iron and cysteine. Such a complex would then be capable of intramolecular hydrogen transfer between the carbonium... 

We have considered typical examples of lanthanide and actinide solvent extraction by chelate formation, involving complexes with citric acid and with TTA, to prove that the labelling of a stable element by one of its radioactive isotopes can help to produce accurate data on the stability constants for complex formation. The method is applicable to elements with radioisotopes having a half-life allowing an ion concentration of 10 6m or less. Other methods of partition such as radiopolarography and radio-coulometry also result in accurate thermodynamical data when the same procedure of labelling is used (29). 

Bailey lA, Gadian DG, Mathews PM, Radda GK, Seeley PJ (1981) Studies of metabolism in the isolated perfused rat heart using NMR. FEBS Lett 123 315-318 Bergmeyer UH (1974) Methods of enzymatic analysis. Academic Press New York Chance EM, Seeholzer SH, Kobayashi K, Williamson JR (1983) Mathematical analysis of isotope labeling in the citric acid cycle with applications to NMR studies in perfused rat hearts. J Biol Chem 258 13785-13794... 

Lorber V, Utter MF, Rudney. H, Cook M (1950) The enzymatic formation of citric acid studied with C -labelled oxalacetate. J Biol Chem 185 689-699... 

First obtain the complete titration curve for the sample. This will involve cautious addition of the NaOH around the inflection points so that you can carefully track the sharp change in the pH at these points. Alternatively, you may choose to use a computer for data acquisition, as in Experiment 18, Part B. Phosphoric acid (H3P04) has three hydrogens to be neutralized. The third hydrogen is lost with only a slight change in pH, however, so you may only see two inflection points. If citric acid is also present (check the can label), there maybe other inflection points as well. 

When compounds enriched in the heavy-carbon isotope 13C and the radioactive carbon isotopes nC and 14C became available about 60 years ago, they were soon put to use in tracing the pathway of carbon atoms through the citric acid cycle. One such experiment initiated the controversy over the role of citrate. Acetate labeled in the carboxyl group (designated [1-14C] acetate) was incubated aerobically with an animal tissue preparation. Acetate is enzymatically converted to acetyl-CoA in animal tissues, and the pathway of the... 

C]Glucose Catabolism An actively respiring bacterial culture is briefly incubated with [1-14C] glucose, and the glycolytic and citric acid cycle intermediates are isolated. Where is the 14C in each of the intermediates listed below Consider only the initial incorporation of 14C, in the first pass of labeled glucose through the pathways. 

CoA labeled with 14C (radioactive carbon) at the carboxyl group, CH3—14C—SCoA, into the citric acid cycle, the 2-oxopentanedioate acid (2-ketoglutarate) formed in the fourth step of the cycle would have all of the 14C in the carboxylate group farthest away from the ketone carbonyl group. For some years, this result was used to argue that citric acid itself could not be an intermediate in the formation of 2-oxopentanedioate. Review Section 19-8 and explain how, in stereospecific enzyme-induced reactions, citric acid could be an intermediate in the formation of 2-oxopentanedioate even if the 14C would not appear equally in both carboxylic carbons of the product. 

Triplicate aliquots (0.75 ml) of standard solutions, blanks, and soil extracts are pipetted into labeled 20 ml thick-wall test tubes (15 mm diameter). Citric acid buffer (1.75 ml 0.2 M) is pipetted into each tube. Ninhydrin reagent (1.25 ml) is then pipetted slowly into each tube and vortex-mixed for two seconds. The top of each test tube is loosely covered with aluminum foil and placed in a vigorously boiling water bath for 25 minutes. There should be sufficient water in the bath so that the water level is always above the level of the solutions in the test tubes. The water should return to boiling within 2 minutes of the tubes being placed in the bath. After 25 minutes, the test tube... 

Citric acid, II, 148 III, 238, 242, 248 Iahelled with C11, III, 238 labelled with isotopic C, III, 241 from molasses, IV, 336 from sucrose, IV, 322, 324 Citric acid cycle, in carbohydrate oxidation, III, 238... 

Phytanic Acid Metabolism When phytanic acid uniformly labeled with 14C is fed to a mouse, radioactivity can be detected in malate, a citric acid cycle intermediate, within minutes. Draw a metabolic pathway that could account for this. Which of the carbon atoms in malate would contain 14C label ... 

Symmetry problems. In experiments carried out in 1941 to investigate the citric acid cycle, oxaloacetate labeled with 14c in the carboxyl carbon atom farthest from the keto group was introduced to an active preparation of mitochondria. 

The formulator needs to know the properties of each antioxidant. For example, most of the antioxidants are easily distilled by heat or steam. Propyl gallate will form strong purple complexes with iron. The preservative effects of antioxidants also can be enhanced by addition of metal chelating agents like citric acid. Furthermore, many crude feedstuffs of plant origin, including soybean meal, crude soybean oil, and lecithin, contain a variety of natural quinone-type compounds with beneficial antioxidant properties that do not require labeling. 

As for human cells or enkaryotes, the metabolic path or seqnence involves the conversion of glucose by what is labeled glycolysis. In a series of ten steps there is first produced a compound called pyruvic acid or pyruvate (the latter designating a so-called salt or componnd of pyruvic acid). In the presence of oxygen, this is fnrther converted to carbon dioxide and water in what is variously called the tricarboxylic acid cycle, or citric acid cycle, or Krebs cycle. The overall operation may be conveniently called aerobic glycolysis, with each step requiring a particnlar enzyme, and most usually requiring snpportive reactions. 

To avoid complication from extensively labeled amino acids from either [l-I3C]glucose or [2-13C]glycerol, Hong and Jakes utilized growth media supplemented with unlabeled amino acid products of the citric acid cycle in order to obtain simplified l3C NMR spectra of the colicin la channel domain.8111... 

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