Metabolic Reprogramming: A Cancer Hallmark Even Warburg Did Not Anticipate

The quantitative histochemistry of the retina.

Publisher Summary This chapter is based on the assays of intermediates of the citric acid cycle and related compounds by fluorometric enzyme methods. An eppendorf fluorometer or a metabolite fluorometer are instruments capable of giving a full-scale deflection of the recorder with 0.25μM NADH, with a noise level less than 2%. At such high sensitivities, the full progress of each enzymatic reaction is recorded. The following accounts for the majority of difficulties and inaccuracies commonly encountered with fluorometrie enzyme methods: All solutions should be dust and particle free; the cuvettes should be temperature equilibrated; Particular care should be taken to avoid contamination of solutions with enzymes, or cross-contamination; Fresh enzyme solution must be made each day; Solutions of pyridine nucleotides are best prepared each day and stored on ice. NAD+ and NADP+ are most stable in a slightly acid solution, and may be diluted with distilled water; and all standard solutions should be neutralized, and assayed spectrophotometrically on the day of use. Metabolic intermediates other than reduced pyridine nueleotides, total CoA, fatty acyl-CoA, and fatty acylearnitine compounds are measured in neutralized perchloric acid extracts of tissues. Perchloric acid is generally more convenient to use than trichloroacetic acid for the extraction, because it may be removed by precipitation as the potassium salt.

[65] Assays of intermediates of the citric acid cycle and related compounds by fluorometric enzyme methods

Publisher Summary This chapter focuses on the polymeric reserve materials and thus excludes the consideration of lipids other than poly-β-hydroxybutyrate, and the disaccharide trehalose, which plays an important role in the economy of the yeast cell, but displays certain features that are at variance with the normally accepted behavior of storage compounds. The chapter considers the evidence for the energy-storage roles of glycogen, polyphosphates and poly-p-hydroxybutyrate, and the current state of knowledge concerning the regulation of their biosynthesis and degradation in the microbial cell. The intracellular location of the enzymes concerned with polymer synthesis and degradation is an intriguing problem. The most detailed work has been carried out with PHB granules, which have PHB synthetase and a factor involved in depolymerase activity associated with the membrane that bounds the granule. The depolymerase system is a complex one that also involves a soluble fraction, underlining the importance of reactions at liquid-solid interfaces in the process and emphasizing that relatively little work has been done to isolate polyphosphate or glycogen granules in their native state in order to examine the possibility of regulation by a membranous system.

Joseph R. Stern

Papers

Enzymatic synthesis of citric acid. II. Crystalline condensing enzyme.

Enzymatic synthesis of citric acid: v. reaction of acetyl coenzyme a

Enzymatic synthesis of citric acid. III. Reversibility and mechanism.

Enzymatic synthesis of citric acid by condensation of acetate and oxalacetate.

Enzymatic synthesis of citric acid. I. Synthesis with soluble enzymes.