甘藷蘋果酸去氫酶之生化學研究

Abstract

蘋果酸去氫酶 (malate dehydrogenase, MDH EC 1.1.1.37) 廣泛存在生物體中。負責可逆性催化oxaloacetate 和malate間的轉換。蘋果酸去氫酶在高等植物中有多種異構酶，存在不同的胞器中，使用不同的輔酶，對於催化之生理途徑有其專一性。 本實驗室之前於綠竹cDNA庫篩選細胞分裂素氧化酶/去氫酶時，意外篩到了一可能為綠竹gMDH的基因，因此對MDH產生極大興趣，本實驗進行甘藷MDH之純化、生化性質及酵素動力學研究。 從甘藷中純化MDH，經緩衝液粗抽、硫酸銨分劃、膠體過濾法 (Sephacryl S-300) 及親和層析法 (Blue Sepharose¬¬¬ CL-4B) 得到的純化蛋白質，其反應最適反應溫度為45 ℃，最適pH值為10~10.5；金屬離子會提高MDH活性；重金屬離子以Hg2+抑制MDH活性最甚；而化學修飾劑DEPC可抑制MDH活性，推測MDH之活性區可能含有histidine；MDH所參與之生理途徑之下游產物皆對MDH有抑制作用。

在酵素動力學中，MDH對malate之Vmax值為1.12 nmol / min，Km值為0.21 mM；對NAD+之Vmax值為4.41 nmol/min，Km值為0.18 mM。從膠體過濾法及原態膠體電泳可推測MDH原態分子量約為70 kD；從SDS-PAGE得知MDH單體約為34 kD，推測甘藷MDH應為同質二元體。

Malate dehydrogenase (MDH EC 1.1.1.37) is a ubiquitous enzyme in all kinds of living organisms. MDH catalyzes the interconversion of oxaloacetate and malate. Higher plants contain multiple forms of MDH that differ in co-enzyme specificity, subcellular localization and physiological function. Unexpectedly, a putative mdh cDNA was screened with the specific probe of cytokinin from the cDNA library of Bambusa oldhamii in our laboratory. For comparing the MDH of bamboo, we continue studying about MDH in sweet potato. We purified MDH from sweet potato through buffer extraction, ammonium sulfate precipitation, gel filtration (Sephacryl S-300) and affinity chromatography (Blue Sepharose CL-4B). The optimal temperature of MDH is 45 ℃; the optimal pH is 10-10.5; MDH activity was benefited by metal ions, but heavy metal ions inhibited its activity. DEPC inhibited MDH activity through modifying histidine. About the MDH enzyme kinetics, the Vmax of malate is 1.12 nmol / min and the Km is 0.21 mM; the Vmax of NAD+ is 4.41 nmol / min and the Km is 0.18 mM. The molecular weight of native MDH was estimated to be 70 kD and the subunit of MDH was about 34 kD. It is estimated that MDH might be a homodimeric enzyme.