Holtman W L, Vredenbregt-Heistek J C, Schmitt N F, Feussner I
Center for Phytotechnology RUL-TNO, Department of Plant Biotechnology-TNO, Leiden, The Netherlands.
Eur J Biochem. 1997 Sep 1;248(2):452-8. doi: 10.1111/j.1432-1033.1997.00452.x.
Besides the pre-existing lipoxygenase (LOX-1) present in quiescent grains, a new lipoxygenase (LOX-2) is induced in embryos of germinating barley [Holtman, W. L., Van Duijn, G., Sedee, N. J. A. & Douma, A. C. (1996) Plant Physiol. 111, 569-576]. The fact that LOX-1 and LOX-2 form different products after incubation with linoleic acid, the (9S)- and (13S)-hydroperoxides, respectively [Van Aarle, P. G. M., De Barse, M. M. J., Veldink, G. A. & Vliegenthart, J. F. G. (1991) FEBS Lett. 280, 159-162; Doderer, A., Kokkelink, I., Van der Veen, S., Valk, B. E., Schram, A. W. & Douma, A. C. (1992) Biochim. Biophys. Acta 1120, 97-104], and differ in temporal expression, suggests different physiological functions for the two isoenzymes at the onset of germination. We aimed to obtain more information about these functions by studying the substrate and product specificities of both isoenzymes. Analyses of the products formed from linoleic acid confirmed that LOX-1 oxygenated at C9, and LOX-2 at C13. When testing more complex substrates, it was found that both LOX-1 and LOX-2 were capable of metabolizing esterified fatty acids. Km values from both isoenzymes for free fatty acids were much lower than for esterified fatty acids (7-35-fold for LOX-1 versus 2-8-fold for LOX-2). Interestingly, LOX-1 showed significantly higher Km values for esterified fatty acids than did LOX-2. This was reflected by analyses of the products formed from di- and tri-linoleoylglycerol; LOX-2 formed higher amounts of oxygenated polyunsaturated fatty acids within the esterified lipids than did LOX-1, with a corresponding larger extent of oxygenation. In order to identify potential endogenous substrates, we analyzed free and esterified lipids in total lipid extracts from barley after different periods of germination for LOX-derived products. The results indicated that esterified fatty acids were preferentially metabolized by LOX-2 activity. Analysis of the positional specificity within the lipids after alkaline hydrolysis revealed that only (13S)-hydroxy derivatives were formed, indicating the in vivo action of LOX-2. These data show that LOX-2 is capable of oxygenating storage lipids and suggest that during the onset of germination LOX-2 may be involved in oxygenation of esterified polyunsaturated fatty acids in barley seeds. We suggest that the oxygenation of these lipids precedes the onset of their catabolism and that the degradation product, (9Z,11E,13S)-13-hydroxy-octadecadienoic acid, serves as an endogenous substrate for beta-oxidation and therefore as a carbon source for the growing barley embryo.
除了存在于静止谷物中的预先存在的脂氧合酶（LOX-1）外，在发芽大麦的胚中还诱导产生了一种新的脂氧合酶（LOX-2）[霍尔特曼，W. L.，范杜因，G.，塞德，N. J. A. & 杜马，A. C.（1996）《植物生理学》111，569 - 576]。LOX-1和LOX-2与亚油酸孵育后形成不同产物，分别为（9S）-和（13S）-氢过氧化物[范阿勒，P. G. M.，德巴斯，M. M. J.，韦尔丁克，G. A. & 弗利根哈特，J. F. G.（1991）《欧洲生物化学学会联合会快报》280，159 - 162；多德勒尔，A.，科克莱林克，I.，范德维恩，S.，瓦尔克，B. E.，施拉姆，A. W. & 杜马，A. C.（1992）《生物化学与生物物理学报》1120，97 - 104]，并且在时间表达上存在差异，这表明在发芽开始时这两种同工酶具有不同的生理功能。我们旨在通过研究这两种同工酶的底物和产物特异性来获取更多关于这些功能的信息。对由亚油酸形成的产物的分析证实，LOX-1在C9位氧化，而LOX-2在C13位氧化。当测试更复杂的底物时，发现LOX-1和LOX-2都能够代谢酯化脂肪酸。两种同工酶对游离脂肪酸的Km值远低于对酯化脂肪酸的Km值（LOX-1为7 - 35倍，而LOX-2为2 - 8倍）。有趣的是，LOX-1对酯化脂肪酸的Km值显著高于LOX-2。这通过对二亚油酰甘油和三亚油酰甘油形成的产物的分析得到反映；与LOX-1相比，LOX-2在酯化脂质中形成的氧化多不饱和脂肪酸量更多，且氧化程度相应更大。为了鉴定潜在的内源性底物，我们分析了发芽不同时期大麦总脂质提取物中游离和酯化脂质的LOX衍生产物。结果表明，酯化脂肪酸优先被LOX-2活性代谢。碱性水解后对脂质中位置特异性的分析表明，仅形成了（13S）-羟基衍生物，这表明了LOX-2在体内的作用。这些数据表明LOX-2能够氧化储存脂质，并表明在发芽开始时，LOX-2可能参与大麦种子中酯化多不饱和脂肪酸的氧化。我们认为这些脂质的氧化先于它们的分解代谢开始，并且降解产物（9Z，11E，13S）-13-羟基-十八碳二烯酸作为β-氧化的内源性底物，因此作为生长中的大麦胚的碳源。
