Ethylene Evolution and Degrading Product from Radioactive ^(14)C-α ketoglutarate in Pseudomonas syringae pv. phaseolicola

Abstract

식물과는 다른 것으로 추정되는 Pseudomonas syringae pv. phaseolicoIa의 Ethylene생성 경로를 알기위해 현재까지 전구체로 밝혀진 α-ketoglutarate를 방사성 동위원소로 표지하여 표지된 에틸렌의 생성과 에틸렌 생성에 관련된 물질을 밝히고자 하였다. Cell-free system을 이용하여 시간에 따라 ethylene과 α-ketoglutarate의 방사능을 측정하고 반응액을 유기산, 케토산, 아미노산 분획으로 분리한 후, 방사능이 높은 분획을 TLC와 Autoradiography로 분리, 확인하였다. 그 결과, 표지된 에틸렌의 생성은 표지된 기질의 감소에 따라 증가하였고 방사능의 변화가 나타난 유기산 분획으로 부터 에틸렌 생성에 관련되는 것으로 보이는 미지의 물질을 분리하였다. 이 물질은 TLC상에서 TCA-cycle의 유기산과는 다른 Rf값을 가지며 UV-spectrum도 다르게 나타났다. [5-^(14)]α-ketoglutarate를 기질로 사용하여 반응시켰을때, 표지된 에틸렌의 생성은 증가하지 않고 ^(14)CO_(2)의생성이 전체 에틸렌 생성에 비례하여 증가하였다. 이러한 결과를 통해 α-ketoglutarate가 P. syringae의 에틸렌 생성의 전구체임을 확인하였고 α-ketoglutarate는 에틸렌 생성과정에서 TCA cycle 유기산과는 다른 유기산으로 전환될 가능성이 큼을 제시하였다. 또 이 과정에서 α-ketoglutarate의 5번째 탄소는 에틸렌으로 전환되지 않고 이산화탄소로 분리되어 나옴을 확인하였다.;The production of ethylene and the metabolic compound from radioactive α-ketoglutarate were studied on the ethylene biosynthetic pathway in Pseudomonas syringae pv. phaseollcola. This bacteria is a pathogen for Puraria lohata(wild) Ohwi (common name, Kudze strain) and is considered to be unique in it's ability to produce very high level of ethylene. With the use of cell-free system, the evolution of labeled ethylene from labeled α-ketoglutarate was investigated at various times. Collected reaction mixtures were fractionated into organic acids, keto acids, and amino acids and metabolic compounds in radioactive fractions were purified by TLC and detected by Autoradiography. Radioactivity of the produced ethylene increased while the radioactivity of the [U-^(14)C] α-ketoglutarate grew decreased. From the fractionation, variations of radioactivities appeared in fractions of organic acid and keto acid and radioactive unknown compound was purified from organic acid fraction by TLC. UV-spectrum and Rf-value in TLC system of this unknown compound were different from the organic acids in TCA-cycle and extracts from this unknown compound on TLC plate can be converted to ethylene in cell-free system. When [5-^(14)C]α-ketoglutarate was used as substrate, ^(14)C-ethylene evolution was not increaed whereas ^(14)CO_(2) Promotion was increased in proportion to total ethylene produced. As the result of that, α-ketoglutarate is a precursor of ethylene biosynthesis in P. syringae and α-ketoglutarate is suggested to beconverted to unknown organic compound, which is not in TCA-cycle, and then convert into ethylene. In process, 5-carbon of α-ketoglutarate is not incorporated into ethylene and seems to be released from α-KG as CO_(2).