Purification and Some Properties of Tryptophan Synthase Inactivase II from Yeast

Abstract

Tryptophan synthase inactivating enzyme II described earlier has been purified to a homogeneous state. Criteria for homogeneity are (a) a single band in disc electrophoresis at pH 7 and 9.1, and (b) a single sedimentation band in the analyticyl ultracentrifuge. The molecular weight was calculated from sedimentation data to be 82000 ± 6000. Isoelectric focusing indicates an isoelectric point at 5.06. The ultraviolet absorption spectrum of the enzyme at pH 7 shows, in addition to the typical absorption spectrum of proteins, a shoulder at 290 nm. The ratio of absorbance at 280:260 nm was 1.6. pH optimum for the inactivation of yeast tryptophan synthase was 5.5. The tryptophan synthase inactivating enzyme II is very probably a proteinase, because (a) casein and albumin are proteolyzed at a rate comparable to proteolysis by purified “pronase”, (b) the increase in 280-nm-absorbing material in the trichloroacetic-acid-soluble phase parallels the inactivation of tryptophan synthase, and (c) the macromolecular inhibitor from yeast described by Ferguson et al. in this journal last year (32, 444–450) prevents the appearance of 280-nm-absorbing, trichloroacetic-acid-soluble material, i.e. proteolysis, to the same extent that it prevents the inactivation of tryptophan synthase.