Purification and Characterization

نویسندگان

  • Ross S. FELDBERG
  • Prasanta DATTA
چکیده

A procedure for the purification of threonine deaminase from Rhodospirillum rubrum is described. The 2200-fold purified enzyme was judged to be pure by disc-gel electrophoresis and ultracentrifugation. Sedimentation velocity centrifugation yielded an s : , , ~ value of 8.1 S and a D ~ O , ~ of 4.65 x 10-7 em2 x see-l. Assuming a partial specific volume of 0.74 g/ml the molecular weight of 164000 was calculated from the Svedberg equation. However, from calibrated Sephadex G-200 columns, a D ~ O , ~ of 4.15 x 10-7 em2 x sec-l was obtained and using this equation, the molecular weight was calculated to be 180000. Electrophoresis of the enzyme on polyacrylamide gels in the presence of sodium dodecyl sulfate revealed that the native enzyme was composed of four subunits, each of about 46000 molecular weight. In addition to the native tetrameric molecule, an active octameric species was detected in polyacrylamide gel electrophoresis. The spectrum of the pure enzyme showed absorption maxima a t 279nm and 412nm, characteristic of pyridoxal 5’-phosphate-containing enzymes. Analysis of the cofactor content revealed the presence of 4.2 moles of pyridoxal phosphate per 180000 g of protein. The enzyme displayed a normal Michaelis-Menten substrate saturation curve in the absence of isoleucine. High concentrations of isoleucine inhibited the enzyme activity somewhat only at low levels of the substrate ; the inhibition was weak as compared to all other biosynthetic threonine deaminases. No other amino acid or any nucleotide tested produced any significant activation or inhibition. In assays at pH values greater than 8.4, a transient build up of a 245 nm absorbing species was observed. It is suggested that this species corresponds to one of the two intermediates of the deamination reaction, a-aminocrotonate or a-iminobutyrate, and that a t high pH the ratelimiting step in the reaction is the hydrolysis of the intermediate to the final products, 2-0x0butyrate and ammonia.

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تاریخ انتشار 2004