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Functional Amino Acid Residues of Recombinant Tobacco Acetolactate Synthase
Chong, Chom-Kyu,Chang, Soo-Ik,Choi, Jung-Do Korean Society for Biochemistry and Molecular Biol 1998 Journal of biochemistry and molecular biology Vol.31 No.3
Acetolactate synthase (ALS) is the common enzyme in the biosynthetic pathways leading to leucine, valine, and isoleucine. Tobacco ALS was expressed in E. coli and purified to homogeneity. The recombinant tobacco ALS was inactivated by thiol-specific reagents, N-ethylmaleimide (NEM) and 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB). Inactivation of the ALS by NEM followed pseudo-first order kinetics and was first order with respect to the modifier. The substrate pyruvate protected the enzyme against the inactivation by NEM and DTNB. Extrapolation to complete inactivation of the enzyme by DTNB showed modification of approximately 2 out of 4 total cysteinyl residues (or 2 cysteinyl and 1 cysteinyl residues), with approximately 1 residue protected by pyruvate. The tobacco ALS was also inactivated by the tryptophanspecific reagent, N-bromosuccinimide (NBS), and was similarly protected by pyruvate. The kinetics of the inactivation was first-order with respect to NBS. The present data suggest that cysteinyl and tryptophanyl residues play a key role in the catalytic function of the enzyme.
Purification and Characterization of Acetolactate Synthase from Barley
Chong, Chom-Kyu,Chang, Soo-Ik,Choi, Jung-Do Korean Society for Biochemistry and Molecular Biol 1997 Journal of biochemistry and molecular biology Vol.30 No.4
Acetolactate synthase (ALS) catalyzes the first common step in the biosynthesis of branchedchain amino acids, valine, leucine, and isoleucine. ALS is the target site for several structually diverse classes of herbicides including sulfonylureas, imidazolinones. and triazolopyrimidines. We have purified ALS from etiolated barley shoots to homogeneity. The five major purification steps are ammonium sulfate fractionation, DEAE anion exchange, hydroxylapatite, Bio-Gel A gel filtration, and low pressure Mono-Q chrornatoqraphy. Approximately 170-fold purification was achieved and the yield was 0.45% of initial activity in the crude extract. Both SDS-PAGE and Western blot analysis showed a single polypeptide of ALS with an apparent molecular mass of 64 kDa. The result of nondenaturing gel electrophoresis with activity staining indicated that the molecular mass of its native form is approximately 225 to 250 kDa. The values of $K_m$ for pyruvate. pl. and optimum pH of ALS were determined to be 2.0 mM, 5.2. and 7.0. respectively Feedback inhibition studies showed that ALS is more susceptible to leucine than valine. And $IC_{50}$ value of Cadre, a class of irnidazolinones, is about $1.5\mu{M}$ for ALS.
Purification and Characterization of Acetolactate Synthase from Barley
Chong, Chom Kyu,Chang, Soo Ik,Choi, Jung Do 생화학분자생물학회 1998 BMB Reports Vol.30 No.4
Acetolactate synthase (ALS) catalyzes the first common step in the biosynthesis of branchedchain amino acids, valine, leucine. and isoleucine. ALS is the target site for several structually diverse classes of herbicides including sulfonylureas, imidazolinones, and triazolopyrimidines. We have purified ALS from etiolated barley shoots to homogeneity. The five major purification steps are ammonium sulfate fractionation, DEAE anion exchange, hydroxylapatite, Bio-Gel A gel filtration, and low pressure Mono-Q chromatography. Approximately 170-fold purification was achieved and the yield was 0.45% of initial activity in the crude extract. Both SDS-PAGE and Western blot analysis showed a single polypeptide of ALS with an apparent molecular mass of 64 kDa. The result of nondenaturing gel electrophoresis with activity staining indicated that the molecular mass of its native form is approximately 225 to 250 kDa. The values of K_m for pyruvate, pl. and optimum pH of ALS were determined to be 2.0 mM, 5.2. and 7.0, respectively. Feedback inhibition studies showed that ALS is more susceptible to leucine than valine. And IC_50 value of Cadre, a class of imidazolinones, is about 1.5 μM for ALS.