【作者】 尹汉萍；

【导师】 徐杰；

【作者基本信息】 华南师范大学 ， 植物学， 2004， 硕士

【摘要】 前人认为乙醇酸氧化酶(EC 1.1.3.15，简称GO)是由一个40kD亚基和黄素单核苷酸(FMN)组成的寡聚酶，但该观点无法解释GO具有从7.1到10.0的近10种等电点(pI)。本实验组徐杰首次提出菠菜GO是一个同工酶，可能含40kD酸性和66kD碱性两种亚基，由于亚基间容易解离，导致等电点的变化。GO同工酶在纯化过程中很不稳定，其全酶分子量(Mr)、活性和pI均会下降，这可能是造成目前报道的GO的Mr和pI等基本参数相差很大的原因。所有纯化后的GO的pI均≤9.6，而粗蛋白中pI＞9.6的GO始终未被纯化出来，这可能与GO同工酶的稳定性差有关。因此，有必要找到一种更简便快速的纯化方法，以便获得pI＞9.6的更接近生理状态的GO同工酶。 本文通过一种改进后的方法，即通过缩短DEAE-cellulose-52柱的长度，以及提高洗脱液的pH，可在9h内从菠菜、菜心和豆角绿叶中纯化GO同工酶。证实该GO同工酶具高活性(90～197．U／mg．min)和高pI(pI＞10.0)。SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)表明，该GO同工酶均有40kD主带。初步证实不同植物中的GO同工酶的亚基M_r是相同的。 多次纯化菜心中的GO同工酶。醋酸薄膜电泳表明其pI＞10.0，而等电聚焦电泳(IEF-PAGE)则表明，该GO同工酶可产生从4.5～10.0的近10种pI。在SDS-PAGE电泳中，GO同工酶始终只存在40kD主带，表明GO同工酶pI的变化与40kD亚基降解无关。 菜心GO同工酶的SDS-PAGE和SDS-毛细管电泳(CE-SDS)显示，该酶除了含40kD主带外，还有很浅的66kD带，和之前我们提出的菠菜GO同工酶含40kD酸性亚基和66kD碱性亚基相似；SOS-PAGE和CE-SDS电泳中，无论加入β-巯基乙醇与否，GO同工酶都只有40kD主带和66kD浅带，表明菜心GO同工酶中40kD酸性亚基和66kD碱性亚基不是以共价二硫键相连；用制备性SDS-PAGE法获得菜心GO同工酶的40kD亚基，并和菜心GO同工酶一起测定其氨基酸组成，该GO同工酶及40kD亚基的碱／酸性氨基酸的比例分别为0.66和0.54，表明40kD亚基可能是个酸性蛋白，而66kD带则是个碱性蛋白。 SDS-PAGE、CE-SDS和氨基酸测定表明，和菠菜GO同工酶类似，菜心GO同工酶也可能含4OkD酸性亚基和66kD碱性亚基。鉴于在醋酸薄膜电泳中菜心G0同工酶的PI>10.0，以及在IEF能聚焦在PI4.5的结果，表明菜心G0同工酶66kD碱性亚基的pl>10.0，而菜心G0同工酶40kD酸性亚基的PI<4 .5。在电场中，非共价相连的G0同工酶两种亚基很容易分离，导致G0同工酶在工EF中出现近10种pl。更多还原

【Abstract】 Glycolate oxidase(EC 1. 1.3.15 GO) was considered a homogeneous enzyme consisted of 40kD subunit and FMN before, but this view could not explain why GO had about ten kinds of isoelectric point(pI) varied from 7.1 to 10. 0; GO from spinach leaves was first reported as an isozyme by Xu Jie in our laboratory, which contained 40kD acidic subunit and 66kD basic subunit. Variation of pI was related to subunits dissociation. GO isozyme was unstable and its molecular weight(Mr), specific activity and pI decreased during purification. All pI of purified GO were smaller than 9. 6 but pI of GO in crude protein greater than 9. 6 had never been purified. This might be related to the unstability of GO during purification. It was necessary to find a simple and fast purification procedure to obtain the GO isozyme with high pI, by wich a physiological GO isozyme can be acquired.In this paper, by shorting the length of DEAE-Cellulose column and increasing the pH of elution solution, one kind of GO isozyme was purified rapidly from green leaves of spanictu Brassica parachinensis Bailey and Vigna unguiculateW. ssp. sesquipedalis(L). Verd in nine hours. GO isozyme displayed high specific activity varied from 90 to 197U/mg. min. and high pI, a value greater than 10.0 assayed by acetate cellulose membrane electrophoresis. 40kD main band appeared when this isozyme preparation was subjected to SDS-PAGE, indicating Mr of GO isozyme subunit from different plants was similar.GO isozyme from Brassica parachinensis Bailey was purified for many times. Its pI was greater than 10. 0 assayed in acetate cellulose membrane electrophoresis, but about ten kinds of pI varied from 4.5 to 10.0 were observed when the same GO isozyme was assayed in IEF. 40kD main band did not alter when GO isozyme was subjected to SDS-PAGE, implying thephenomenon of pI change was not related to 40kD subunit degradability.One 66kD band appeared except 44kD main band when GO isozyme above was subjected to SDS-PAGE and CE-SDS, indicating this GO isozyme was similar to that from spinach leaves which contained 40kD and 66kD simultaneously. Whether B-mercaptoethanol was added or not when GO isozyme was subjected to in SDS-PAGE and CE-SDS, 40kD main band and 66kD band still appeared, indicating two subunits were not linked by covalent disulfide. Amino acid analysis shew that the ratios of basic to acidic amino acid of GO isozyme and its 40kD acidic subunit were 0. 66 and 0. 54, respectively, implying 40kD subunit was a acidic protein, 66kD band was a basic protein.Similar to GO isozyme from spinach leaves, it is most likely that this GO isozyme comprised two noncovalently associated subunits: 66kD basic subunit and 40kD acidic subunit by comparing the differences of electrophoresis pattern in SDS-PAGE and CE-SDS, and by analyzing the amino acid component of GO isozyme and its 40kD subunit. Based on the results in acetate cellulose membrane electrophoresis and IEF, implying the pi of 66kD basic subunit was greater than 10. 0 and that of 40kD acidic subunit was smaller than 4.5. Under electric field, noncovalently associated subunits of GO isozyme dissocated easily which resulted in about ten kinds of pI observed in IEF.更多还原