The subunit structure of the arom multienzyme complex of Neurospora crassa. A possible pentafunctional polypeptide chain

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Abstract
A new procedure for the purification of the arom multienzyme complex from N. crassa is presented. Important factors are the inactivation of proteinases by phenylmethanesulfonyl fluoride and the use of cellulose phosphate as an affinity adsorbent. A homogeneous enzyme, with a specific shikimate dehydrogenase activity of 70 units/mg of protein, is obtained in 25% yield. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate, combined with cross-linking studies using dimethyl suberimidate, suggest that the complex is composed of 2 subunits of MW 165,000. Glycerol-density-gradient centrifugation indicates a MW for the intace complex of about 270,000. Evidence for the effects of proteolysis, both during the preparation and on storage of the purified complex, is presented, and previous reports in the literature of the occurrence of multiple subunits are discussed in this light.