Norman H. Giles

Purification and properties of the aromatic (arom synthetic enzyme aggregate of Neurospora crassa

Abstract 1. 1. Procedures are described for the purification of the wild type arom multienzyme complex encoded in the arom gene cluster of Neurospora crassa. 2. 2. The purified aggregate has all five activities present at high levels and there has been no indication that the five activites are separable during purification. 3. 3. The enzyme is readily inactivated by O2 even in the presence of thiols. 4. 4. The molecular weight of the purified aggregate is estimated to be approx. 231 000 on the basis of data from equilibrium sedimentation and amino acid analysis. 5. 5. Experiments on disruption of the purified aggregate (which leads to loss of enzymic activities) and on renaturation (which restores three of the five activities), together with independent studies of allelic complementation, suggest that the complex consists of two identical halves bound together by relatively weak non-peptide bonds. 6. 6. Preliminary evidence has also been obtained for the presence of still smaller components, possibly representing the five different polypeptide chains which are thought to be the basis of subunits of which the aggregate is composed.

Genetic Control of Neurospora malate dehydrogenase and aspartate aminotransferase: I. Mutant selection, linkage, and complementation studies

Abstract A method for the selection of mutants of Neurospora crassa deficient in malate assimilation is described. Six mutants have been localized at either of two unlinked loci in the left arms of two of the seven linkage groups, other than groups I or II. In vivo complementation regularly occurs when heterocaryons are formed between any pair of mutants from different genetic loci. All intragroup (intragenic) complementation tests have yielded negative results. These two distinct genetic loci involved in malate assimilation have been designated ma-1 and ma-2 .

Purification and stability of the multienzyme complex encoded in the arom gene cluster of Neurospora crassa.

Abstract An improved method is described for the purification of the arom multi-enzyme complex from Neurospora crassa . The product of purification is an aggregate of approximately 230 000 mol. wt having five enzyme activities which function in the common pathway in aromatic amoni acid biosynthesis. Conditions promoting dissociation of the enzyme complex has been determined. Alkaline pH values, storage in low ionic-strength buffers, and high temperature all promote dissociation. The aggregate fragments resulting from dissociation are quite labile and have not been thoroughly characterized. However, in all but one of the fragments observed 5-dehydroshikimate reductase (Shikimate: NADP + oxidoreductase, EC 1.1.1.25) and 5-dehydroquinate dehydratase (5-dehydroquinate hydro-lyase, EC 4.2.1.10) activities are associated. These two activities have been found alone or in association with one of the three additional activities. A presumptive dimer of 3-enolpyruvylshikimic acid 5-phosphate synthelase has also been observed. To explain the variety of fragments observed, the minimum number of polypeptides present is postulated to be 4. Thus, the possibility that the 115 000 mol. wt subunits of the complex are single multifunctional polypeptide has been excluded.