Reversible dissociation of tryptophanase into protein subunits

Gopinathan, K. P. ; Demoss, R. D. (1966) Reversible dissociation of tryptophanase into protein subunits PNAS, 56 (4). pp. 1223-1225. ISSN 0027-8424

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Crystalline tryptophanase from Escherichia coli strain B/1t7-A has been shown to behave as a single entity in the analytical ultracentrifuge with an S20, t, value of 9.65, whereas sucrose density gradient centrifugation of crude extracts of the organism showed that tryptophanase sedimented predominantly as a particle with S20, f value of 6.85. Since the crystalline enzyme was isolated in 60 per cent yield, it was suggested that two species of the enzyme coexist in crude extracts and are interconvertible. The sedimentation constant of a purified tryptophanase preparation from E. coli T3, a tryptophan auxotroph of E. coli K12, was determined to be 9.0S. Crude extracts of E. coli T3 also gave two peaks with sedimentation values 9.4S (minor peak) and 6.9S (major peak) by the sucrose density gradient method. A purified tryptophanase from Bacillus alvei has been shown4 to behave as a single peak in the analytical ultracentrifuge; the sedimentation coefficient was calculated to be 10.3S by extrapolation to infinite dilution. All of the sedimentation results cited above can be explained consistently by the observations reported herein. We describe some of our findings on the behavior of tryptophanases from E. coli T3 and B. alvei. Methods: The cell-free extracts, prepared from E. coli by sonic disintegration, and the purified enzyme from B. alvei4 were used as enzyme sources. The sucrose density gradient centrifugation studies were carried out essentially as described by Martin and Ames,5 except that 50 mM potassium phosphate buffer was used in place of 50 mM Tris-Cl wherever indicated. Pyridoxal phosphate was also included in all the gradients to a final concentration of 40 ,μM. Magnesium acetate was included (0.1 mMi) wherever mentioned. Catalase (Worthington Biochemical Corp., N. J.) was used as the reference standard. It was established in separate experiments in the analytical ultracentrifuge that the sedimentation constant of catalase (11.4S) was the same in either 50 mM potassium phosphate buffer or 50 mM Tris- Cl buffer. The tryptophanase and catalase activities were determined as described by Feiss and DeMoss, and Martin and Ames,5 respectively. Results: The purified enzyme from B. alvei sedimented as two peaks, a major peak with sedimentation constant 5.5S and a minor peak at 9.4S in a sucrose density gradient, prepared in 50 mM Tris-Cl. However, when potassium phosphate was used in place of Tris-Cl, the enzyme tended to aggregate predominantly to a larger particle with the sedimentation value of 9.4. The sedimentation behavior of the enzyme from E. coli also was examined in Tris-Cl and in potassium phosphate buffers, and the results with both enzymes are shown in Figures 1-4. The sedimentation constants obtained for the enzymes from E. coli and B. alvei in a series of experiments are presented in Table 1. The presence of Mg++ had no significant influence on the sedimentation constants observed.

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