Subunit structure of anthranilate synthetase from Neurospora crassa.
Freshly purified preparations of anthranilate synthetase complex from Neurospora crassa appeared to be homogeneous on polyacrylamide disc gels and were composed of two distinct subunits, 94,000 and 70,000 daltons, respectively, as determined by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Carboxymethylation of the complex or treatment with guanidine hydrochloride and urea before sodium dodecyl sulfate treatment did not alter the subunit pattern. When the purified complex was iodinated with 125I- or methylated with [14C]dimethylsulfate, no labeled components other than the two subunits stained with Coomassie blue were detected after electrophoresis in the presence of sodium dodecyl sulfate. Although some purified preparations were stable, most were unstable upon storage. Analysis of the unstable preparations on nondenaturing and sodium dodecyl sulfate polyacrylamide disc gels revealed that the complex in these preparations was progressively fragmented to smaller components and subunits upon repeated freeze-thaw treatment or prolonged incubation at or above 4 degrees. Distinct fragments were generated ranging in size down to 25,000 daltons, and some fragments retained some of the activities associated with the anthranilate synthetase complex. On the basis of these and earlier studies, we conclude that anthranilate synthetase from Neurospora crassa is composed of two distinct subunits in an alpha2beta2 structure; one subunit is a trifunctional peptide which contains the catalytic sites for the phosphoribosylanthranilate isomerase and indoleglycerol phosphate synthetase reactions, and associates with the second subunit to form glutamine-dependent anthranilate synthetase. The smaller subunits and components previously reported for this complex are apparently due to protease activity present in purified preparations.
D000878:Anthranilate Synthase / Q000302:isolation & purification; D001665:Binding Sites; D004589:Electrophoresis, Disc; D006146:Guanidines; D046911:Macromolecular Substances; D008626:Mercuribenzoates; D008970:Molecular Weight; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D011485:Protein Binding; D014508:Urea
D006146:Guanidines; D046911:Macromolecular Substances; D008626:Mercuribenzoates; D014508:Urea; D000878:Anthranilate Synthase