RADIOMETRIC ASSAY OF TOTAL TRYPTOPHAN HYDROXYLATION BY INTACT CULTURED PINEAL GLANDS

Abstract— A new method is described for the analysis of total tryptophan hydroxylation by intact tissue in culture. This method differs from classical measurements of tryptophan hydroxylase because it allows the detection of changes in tryptophan hydroxylation due to alterations in tryptophan transport, cofactor regeneration and protein synthesis. The rat pineal gland was studied. It was found to hydroxylate tryptophan linearly for a 48-h period. A direct proportional relationship between the concentration of tryptophan in the culture medium and the amount of tryptophan hydroxylated persists between 0.05 and 0.5 m m . As the medium concn of tryptophan is increased above 0.5 m m the amount of tryptophan hydroxylated decreases. Tryptophan hydroxylation is substantially inhibited by 1 m m p-chlorophenylalanine and 0.03 m m cycloheximide. No difference in the total amount of tryptophan hydroxylated was detected between male and female rat pineal glands, between chronically denervated and normal rat pineal glands, or between control glands and glands treated with 0.1-0.001 m m norepinephrine, 0.01 m m isoproterenol, or 0.01 m m mescaline.