Masters Theses

Date of Award

12-1993

Degree Type

Thesis

Degree Name

Master of Science

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

John W. Koontz

Committee Members

Wesley Wicks, Jorge Churchich

Abstract

Two observations in the literature have led us to believe that glucocorticoids may affect the metabolism of the vitamers of pyridoxal-5-phosphate (PLP). The steroid induces many PLP dependent enzymes in the liver and it has been suggested that PLP regulates steroid hormone action. We have examined the effect of the synthetic glucocorticoid, dexamethasone, on vitamin B6 metabolism in a rat hepatoma cell line, KRC-7. Previous experiments, which are verified herein, have demonstrated that this cell line elicits a typical sensitivity to dexamethasone. Pyridoxal kinase and pyridoxine-5-phospate or pyridoxamine-5-phospate oxidase (the two most important enzymes in the biosynthesis of the physiologically active form of the vitamer PLP) were measured in cells following treatment with dexamethasone. The level of 4-pyridoxic acid in the culture medium was assayed as a measure of the rate of catabolism of the vitamers. The activities of pyridoxal kinase, PNP-oxidase, or the formation of 4-pyridoxic acid do not change after cells are treated with 10-6M dexamethasone for a period of 24 hours. These results indicate that the glucocorticoid does not alter the metabolism of the vitamers. A novel fluorometric assay was proposed to measure pyridoxal kinase activity, an enzyme which phosphorylates pyridoxal to form pyridoxal-5-phospate. The basis of this assay relies on th reaction of hydroxylamine with the aldehyde groups of both pyridoxal (PL) and pyridoxal-5-phosphate (PLP) to form PL-oxime and PLP-oxime. Addition of hydroxylamine to either PL or PLP increases the fluorescence at 450 nm upon excitation at 380 nm. Reaction kinetics indicate that PLP reacts much faster with hydroxylamine than does PL. Therefore, the fluorescence due to the PLP-oxime can easily be distinguished from the fluorescence due to the PL-oxime. We demonstrate that this assay satisfies the requirements for being both time and concentration dependent using both purified pyridoxal Idnase as well as enzyme activity detected in crude extracts of cultured hepatoma cells. Separately, I have utilized culture conditions which have been reported to deplete the intracellular stores of PLP. One would predict that under these constrained conditions the cells would be more acutely sensitive to the potential effects of dexamethasone on vitamer metabolism. I measured not only pyridoxal Idnase, the rate limiting enzyme in PLP biosynthesis, but also the holoenzyme to total enzyme ratios of two PLP dependent enzymes, aspartate aminotransferase and tyrosine aminotransferase. This gives an indication of whether these culture conditions have lead to depletion of PLP in the cells. Finally, I also measured the induction of tyrosine aminotransferase enzyme activity by dexamethasone under these conditions. Under the initial constraints imposed upon the cell, i.e. treatment of non-growing cells with 5mM 4-deoxypyridoxine or incubation of non-growing cells in pyridoxal deficient medium, there was no effect on basal TAT activity, TAT holoenzyme to total enzyme ratio, or TAT fold induction by dexamethasone. Basal AAT activity did not change nor did its holoenzyme to total enzyme ratio. These initial results differed from previously published reports. Therefore, I carried out the same experiments using the conditions designed to deplete PLP using growing cells. Under these circumstances, the basal TAT activity increases, the TAT holoenzyme to total enzyme ratio decreases and TAT induction by dexamethasone does not change. AAT basal activity decreases and the holo to total enzyme ratios decrease. The changes in basal TAT activity, the holo/total enzyme activity and the loss in basal AAT corroborate previous reports. The decrease in the holo/total activity ratios for both TAT and AAT and the loss of basal AAT indicate that the cells are being depleted of intracellular PLP under these conditions. Thus, while attempts to deplete PLP in nongrowing, resting cells were unsuccessful, it was readily accomplished in growing, dividing cells.

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