油菜叶片硝酸还原酶同功酶的纯化与特性

Purification and Characterization of Nitrate Reductase Isozymes from Brassica campestris Leaves

通过对硝酸还原酶（ＮＲ）亲和层析洗脱过程的部分改进，从油菜叶片中分离纯化到诱导型（ｉＮＲ）及组成型（ｃＮＲ）两种硝酸还原酶同功酶。电泳分析表明两者均达到银染单带纯。ｃＮＲ分子量（ＭＷ）为４５０ｋＤ．ｉＮＲＭＷ为２２０ｋＤ；两者亚基ＭＷ均为１１０ｋＤ，但亚基数目不一样，氨基酸组成也有差异。ｉＮＲ与ｃＮＤ的等电点ｐＨ不同，分别为４．４及６．０，免疫交叉反应显示ｃＮＲ的抗原性为ｉＮＲ的７５％。

It has been known that there are mainly two types of nitrate reductase (NR) isozymes, inducuible NR (iNR) and constitutive NR (cNR), in hihger plants. Although iNR has been thoroughly studied, there has been little known about cNR. For this reason,Brassica campetris was used as the material in our case for investigation on the purification and characterization of NR isozymes. For the purification of cNR,the original elution procedure of Blue Dextran Sepharose 4B affinity chromatography was slightly modified. After the removal of nonspecific proteinsby loading buffer, an additional step was inserted for further washing some stably adsorbed proteins with 0.1 mol/ L KC1, then iNR and cNR were specifically eluted by 50 Umol/ L NADH and NADPH seperately. By this simple improvement, iNR and cNR were simultaneously purified, and reached the purity of electrophoretic monoband developed by silver staining.Analysed by non-denaturing gradient (7.4%-20% ) polyacrylamidegel electrophoresis (PAGE), the molecular weights (MW) of iNR and cNR were found to be 220 kD and 450 kD respectively. When isozymes were denatured and identified by gradient (10%～20% ) SDS-PAGE, the subunit comPOsitions of iNR and cNR were discovered to be similar, whose MWs were both at 110 kD. Furthermore, isoelectric focusing result showed that the pI of iNR was at pH 4. 4, whereas cNR' s was at pH 6. 0.Comparing with other results reported by literatures, cNR was likely to resemble nitrate reductuses from lower plants, such as fungi and algae, indicating itS earlier origin in evolution.Finally, the antigenicity of cNR was assayed to be 75% of iNR' s, implyingthat the antigenic determinant of cNR was somewhat different from that of iNR. Desipte of the same reaction catalysed by these two isozymes, there might exist some difference in their comformation, which was preliminarily supPOrted by the active staining of NR. Inferred from this result, the microheterogeneity of cNR appeared to bemore evident than that of iNR.