The Isolation of the Components of the Anthranilate Synthetase Complex from Neurospora crassa

Abstract

Abstract The components in trp 1 and trp 2 mutants of Neurospora crassa which interact in vitro to form an active anthranilate synthetase complex have been purified. The isolated components and the complexes formed by their interaction were characterized enzymatically, and their molecular weights were estimated by gel filtration on Sephadex. The trp 2 gene product (Component I) catalyzed the anthranilate synthetase reaction only with ammonia as the amino donor and had an estimated molecular weight of 80,000. The trp 1 gene product (Component II) occurred as an undissociated or a dissociated form. The undissociated form interacted with Component I to produce glutamine-dependent anthranilate synthetase and had an estimated molecular weight of 200,000. It dissociated to a 30,000 molecular weight subunit (Component IIa) which also interacted with the Component I to produce glutamine-linked anthranilate synthetase and a 160,000 molecular weight subunit (Component IIb) which possessed both N-(5'-phosphoribosyl)anthranilate isomerase and indole-3-glycerol phosphate synthetase activities, but did not interact with Component I. Component I interacted with Component II to form a complex with an estimated molecular weight of 300,000 which possessed glutamine-linked anthranilate synthetase, N-(5'-phosphoribosyl)anthranilate isomerase, and indole-3-glycerol phosphate synthetase activities. Component I interacted with Component IIa to produce a complex which possessed only anthranilate synthetase activity. In contrast to the Component I-Component II complex, the latter complex completely dissociated on Sephadex G-200, but could be stabilized on sucrose gradients as a 6.4 S component.