New methods for the colorimetric assay of P IID regulatory protein, uridylyltransferase, and uridylyl-removing enzyme in glutamine synthetase cascade

Abstract

Listen

Translate article

Adenylylation and deadenylylation of glutamine synthetase (GS) are catalyzed by the same adenylyltransferase (ATase). The ability of ATase to catalyze adenylylation is markedly stimulated by the unmodified form of a regulatory protein, P IIA, whereas its capacity to catalyze deadenylylation is stimulated by the uridylylated form (P IID) of the regulatory protein. Interconversion between P IIA and P IID is catalyzed by uridylyltransferase (UTase) and uridylylremoving enzyme (UR). New colorimetric methods were developed for the assays of P IID, UTase, and UR activities. The P IID activity is monitored by its unique ability to stimulate the ATase catalyzed formation of unadenylylated subunits from adenylylated GS. The inerease of unadenylylated subunits is determined by measuring the γ-glutamyltransferase activity of GS under conditions where the activity of an unadenylylated subunit is about 15 times greater than that of an adenylylated subunit (i.e., at pH 8.0 in the presence of Mn 2+). Assays for UTase and UR enzyme are derived by coupling the P IID assay to the UTase and UR reactions. For the UTase reaction, the formation of P IID from P IIA is measured, whereas the decrease in P IID is followed for the UR assay. These assays have been applied to follow the activities of these proteins during their purification procedures, to the mechanistic studies on the deadenylylation reaction, and to determine the activities of these proteins in mutants produced during the genetic study of glutamine synthetase cascade. The problems evolved from these assays are discussed.