Year: 1984
Type: article
Abstract: The goal of this thesis was to study the compartmentation of phosphorylated glycolytic intermediates in intact Chicken Embryo Fibroblasts (CEFs) using 3'p NMR at 109 MHz. 1 Because glycolysis is regulated differently in normal and virally transformed CEFs, NMR experiments were performed on both types of cells.A technique for maintaining functional cells at high densities in an NMR magnet is described.Dialysis fibers were imbedded in the cell pellet and medium was pumped through the tubing, perfusing the cells with oxygen, glucose and buffer, and removing waste products.Signals were detected from cytoplasmic inorganic phosphate (Pi)' AT?, NAD, NADH, phosphorylcholine and phosphorylethanolamine.The effect of external glucose on cytoplasmic pools of phosphates was studied.When cells were perfused with glucose-free medium the rate of glycolysis decreased, the amplitudes of the AT? resonances decreased, and the ?intensi ty increased.The quanti ty of NMR-1 detectable Pi produced was significantly greater than the quantity of NMR-detectable ATP which was lost.When the amount of glucose in the perfusate was increased trom 1 mg/ml to either 10 mg/ml or 40 mg/ml 2 the amplitude of the signal from Pi decreased, without a corresponding increase in the amplitudes of the ATP resonances.However, experiments with 32p labeled Pi showed that as the concentration of glucose in the medium was increased, the amount of phosphate sequestered in the cells increased.We conclude that there is a pool of Pi which is not detected by high resolution NMR and that the size of this pool increases as the rate of glycolysis increases.These effects were found only in cultured cells; the data for transformed and normal cells were similar; .Longtitudinalrelaxation times of intracellular phosphates in normal, transformed, and primary CEFs were measured.Relaxation times were significantly shorter in primary cells than in normal and transformed cells.These results demonstrate that relaxation times of phosphates are sensitive to structural and metabolic changes which occur when cells are grown in culture.
Author: Gregory S. Karczmar
Language: English
Subfield: Spectroscopy
Field: Chemistry
Domain: Physical Sciences
Open Access status: green
Funder: U.S. Department of Energy
Awards: G1398330795, G6636457713
OpenAlex ID: W1481256379