Crystallization and characterization of 1-pyrroline-5-carboxylate dehydrogenase from Bacillus sphaericus.

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1-Pyrroline-5-carboxylate dehydrogenase was purified and crystallized from Bacillus sphaericus. The crystalline preparation gave a single band on polyacrylamide slab gel electrophoresis. The molecular weight of the enzyme was determined to be about 100, 000 by gel filtration. The enzyme consists of two subunits which are identical in molecular weight (50, 000), as judged on SDS slab gel electrophoresis. The enzyme shows an optimum pH of 6.5 to 7.0. Its activity was 8.1 times higher with NADP+ than with NAD+, and the enzyme was stabilized by NADP+. The apparent Km values for L-l-pyrroline-5-carboxylate, NADP+ and NAD+ are 4.2×10-5M (with NADP+), 9.5×10-6M and 2.5×10-3M, respectively. The enzyme reaction is irreversible. A simple method for the determination of L-ornithine involving ornithine δ-aminotransferase and 1-pyrroline-5-carboxylate dehydrogenase from B. sphaericus was developed. A linear relationship was found between the absorbance at 340nm and the amount of L-ornithine (50-400nmol), and between the fluorescence and the amount of L-ornithine (0.2-10nmol).
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