Enzymes participating in urea-utilizing system in Perilla frutescens

Regarding the utilization of urea by plant, urease, ammonia dehydrogenase system and postulated urea dehydrogenase were investigated in Perilla frutescens. Urease activity in the enzyme solution prepared by usual procedure was extremely low. However, a higher activity was confirmed when the enzyme solution was prepared with great care, for example, under protecting SH-group with mercaptoethanol or by extracting the enzyme from quickly frozen leaf with liquid nitrogen. Oxidation and reduction systems between ammonia and nitrate were investigated in Perilla leaf and root. Four dehydrogenases of ammonia, hydroxylamine, hyponitrite and nitrite, and four reductases of nitrate, nitrite, hyponitrite and hydroxylamine were established in leaf and root of Perilla. It was found that the activity of these enzymes is dependent on the kind of nitrogen source supplied to the plant, suggesting that the enzymes have the close relationship with utilization of nitrogen. Each dehydrogenase and reductase were inhibited specifically by various inhibitors. Formation of hydroxylamine, nitrite and nitrate was confirmed as the oxidation products by the dehydrogenases, and nitrite, hyponitrite, hydroxylamine and ammonia were determined as the reduction products by the reductases. Enzyme which catalyses the reduction of NAD or NADP in presence of urea was discovered in leaf of Perilla. The activity was confirmed in leaf of the plant supplied with urea or ammonia but not with nitrate. The inhibiting rate of various inhibitors on urea-enzyme was differ from that of the same reagent on ammonia dehydrogenase. The maximum activity was obtained at 10^-3M urea, ten times higher concentration than that with ammonia. Non-urea 14C-compounds were formed from ^14C-urea by the enzyme.シソの葉のウレアーゼ活性をC^14-尿素から生成されるC^14O_2を追及することによつて測定した.活性は酵素液の調製中に失活し易く常法によつて調製した酵素液についてはほとんど検出されなかつたが液体窒素で迅速に凍結しその状態で抽出すればかなりの活性が認められた.しかしメルカプトエタノールを添加してSH基を保護しながら調製するとさらに強力なウレアーゼ活性が確認された.アンモニア酸化系は葉および根に証明された.アンモニア-,ヒドロキシルアミン-,次亜硝酸-および亜硝酸脱水素酵素の特異的な4酵素によつて逐次酸化系が構成されていることが阻害剤の影響および反応生成物の測定によつて明らかにされた.葉の脱水素酵素はNADおよびNADPの両者と共軛しアンモニアまたは尿素を窒素源として生育したシソとくに後者で強く,硝酸投与区のものでは微弱であつた.これに対して根の酵素はNADにのみ特異的であつてアンモニア区および尿素区で認められるがとくに前者で強い.硝酸還元系も葉に検出された.これもアンモニア酸化系の場合と同様に特異的な4種のNADH2-硝酸-,亜硝酸-,次亜硝酸-およびヒドロキシルアミン還元酵素によつて構成され,しかも窒素源依存性があつて硝酸区のシソの葉に強くアンモニアおよび尿素投与区のものは微弱であつた.尿素の存在でNADあるいはNADPを還元する酵素が尿素およびアンモニア投与区の葉に証明されたが硝酸区のものには認められなかつた.本酵素はアンモニア脱水素酵素と異ることが阻害剤の影響等から確かめられ尿素:NAD(P)oxidoreductaseと推定される.本酵素の作用によつてC^14-尿素からC^14O_2とは異なり,またウレアーゼによつても分解されないC^14-化合物が生成されることが確かめられた