Behal R H, Oliver D J
Department of Botany, Iowa State University, Ames 50011, USA.
Arch Biochem Biophys. 1997 Dec 1;348(1):65-74. doi: 10.1006/abbi.1997.0359.
A cDNA EST clone encoding the C-terminal portion of Arabidopsis thaliana fumarase was identified by homology analysis. A fragment of cDNA encoding the N-terminal region of fumarase was amplified from a cDNA library using PCR and cloned. Genomic DNA corresponding to the coding region of fumarase was amplified and cloned. Arabidopsis fumarase was expressed as a chimeric fusion protein and polyclonal antibodies were generated. Fumarase was purified to near-homogeneity (over 600-fold) from etiolated Pisum sativum mitochondria. The identification of fumarase was confirmed by a combination of immunoblot and N-terminal amino acid sequencing. Kinetic analysis of highly purified fumarase yielded a KM(malate) of 0.45 mM and a Vmax(malate) of 650 mumol of fumarate/min/ mg. The pea fumarase was inhibited by the alpha-keto acids pyruvate and alpha-ketoglutarate at low millimolar concentrations. Adenylates were highly inhibitory; the degree of this inhibition was reduced in the presence of Mg2+, suggesting that uncomplexed adenylates are the inhibitory species.
通过同源性分析鉴定出一个编码拟南芥富马酸酶C末端部分的cDNA EST克隆。使用PCR从cDNA文库中扩增并克隆了编码富马酸酶N末端区域的cDNA片段。扩增并克隆了与富马酸酶编码区域相对应的基因组DNA。将拟南芥富马酸酶表达为嵌合融合蛋白并产生多克隆抗体。从黄化豌豆线粒体中纯化富马酸酶至接近均一性（超过600倍）。通过免疫印迹和N末端氨基酸测序相结合的方法确认了富马酸酶的鉴定。对高度纯化的富马酸酶进行动力学分析，得出苹果酸的Km值为0.45 mM，富马酸的Vmax值为650 μmol/分钟/毫克。豌豆富马酸酶在低毫摩尔浓度下受到α-酮酸丙酮酸和α-酮戊二酸的抑制。腺苷酸具有高度抑制作用；在Mg2+存在下这种抑制程度降低，表明未复合的腺苷酸是抑制性物质。
