[U-$^{14}C$]-glucose를 사용한 발아 초기 대두씨(Glycine max)에서의 ascorbate 합성 중간물질들을 paper chromatography로 분리하여 그 방사능 분포를 조사한 결과 glucose는 galactose로 전환된 후 L-galactono $gamma$-lactone 중간체를 거쳐 ascorbate 가 되는 것으로 알게되었으며 UDP-galactose 4-epimerase [EC 5.1.3.2] 작용에 의한 glucose의 galactose로의 전환이 확인되었다. UDP-galactose 4-epimerase를 발아 2일째의 대두 추출물로 부터 약 150배 정제한 후 특성을 조사한 결과 UDP-glucose와 UDP-galactose에 대한 Km은 각각 $1.1{ imes}10^{-4}M$, $8{ imes}10^{-5}M$ 이었고, 적정 pH는 9.0, 보조효소인 $NAD^+$와는 비교적 견고하게 결합되어 있는 것으로 판명되었다. 한편 ascorbate 합성단계의 최종 효소로 알려진 L-galactono $gamma$-lactone dehydrogenase [EC 1.3.2.3]의 활성을 조사한 결과 L-galactono $gamma$-lactone에 특이적으로 작용하며 L-gulono $gamma$-lactone과는 거의 반응하지 않았다. 따라서 대두씨에서의 glucose로 부터의 ascorbate의 합성은 주로 glucose가 ralactose로 전환된 후 L-galactono $gamma$-lactone을 거쳐 합성되는 것으로 생각된다.
The biosynthetic pathway of ascorbate from glucose was studied at the early phase of germinating soybean seeds (Glycine max). The homogenate of germinating soybean seeds (2nd day) and [U-$^{14}C$]-glucose were incubated for 3 hours at $37^{circ}C$ and the several intermediates (galactose, L-galactono $gamma$-lactone, L-gulono $gamma$-lactone, UDP-glucose. D-glucuronate. ascorbate, myo-inositol) were separated by paper chromatography. Radioactivity distribution of the above intermediates indicated that ascorbate was synthesized from glucose predominantly via galactose and L-galactono $gamma$-lactone route rather than L-gulono $gamma$-lactone route. This suggested that glucose is epimerized first to galactose by the UDP-galactose 4-epimerase (EC 5.1.3.2) in this germinating soybean seeds. From the homogenate of germinating soybean seeds (2nd day), UDP-galactose 4-epimerase was purified over 150 folds by $MnCl_2$ treatment. ammonium sulfate precipitation, Sephadex G-150 gel filtration, Sephadex G-200 gel filtration and finally calcium phosphate gel adsorption. Km values for UDP-glucose and UDP-galactose, optimum pH were determined and the $NAD^{+}-enzyme$ was investigated. Km for UDP-glucose and UDP-galactose of UDP-galactose 4-epimerase were $1.1{ imes}10^{-4};M$ and $8{ imes}10^{-5};M$respectively. pH optimum was pH 9.0 and $NAD^+$ seemed to be bound to the enzyme relatively strongly. From the above experimental results. it was concluded that the biosynthesis of ascorbate form glucose in germinating soybean seeds seemed to be made predominently via galactose and L-galactono $gamma$-lactone route as other plants.
