Synthesis of experimentally induced glutamine synthetase (glutamotransferase activity) in embryonic chick retina in vitro

Abstract

The phenomenon of precocious appearance and rapid enhancement of glutamotransferase activity of embryonic chick neural retina in response to explanation in vitro has been subjected to more detailed scrutiny. On the basis of cofactor requirements, pH optima, response to specific inhibitors, and ratio of transferase to synthetase activity, the enzyme undergoing change was classified as a glutamine synthetase. Tests for the presence of enzyme activators and/or inhibitors undergoing change during increase in enzyme activity were negative, suggesting that increase in activity was due to synthesis of new enzyme. This was substantiated by the finding that low levels of puromycin blocked completely and reversibly increases in enzyme activity under culture conditions; and that actinomycin D similarly blocked such increases irreversibly. Preexisting levels of activity appeared stable in the presence of either inhibitor. These data were interpreted to mean that enhancement of glutamotransferase activity which occurs in culture is due to synthesis of new enzyme; it also suggested a lability of the RNA involved and consequently a rather direct genomic control over the rate of synthesis of the enzyme in early ontogenesis. Pigmented epithelium of embryonic eye, vitreous humor, and embryo extract showed no effect on the increase in glutamine synthetase activity in the explanted retina; this was interpreted as diminishing but not excluding the possibility that a stable, diffusible systemic factor is responsible for control of early ontogenesis of this enzyme in the embryo. Glutamine, glutamate, and γ-aminobutyrate were found to lower synthesis of the enzyme in culture when added to the medium, but none of these amino acids appeared capable of total repression at concentrations approaching physiological values. Glutathione (oxidized and reduced), asparagine, aspartate, and glucose all had no demonstrable affect upon the growth of the enzyme in culture.