PURIFICATION OF SOLUBLE GUANYLYL CYCLASE FROM BOVINE LUNG BY A NEW IMMUNOAFFINITY CHROMATOGRAPHIC METHOD
PURIFICATION OF SOLUBLE GUANYLYL CYCLASE FROM BOVINE LUNG BY A NEW IMMUNOAFFINITY CHROMATOGRAPHIC METHOD
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DOI：
10.1111/j.1432-1033.1990.tb15572.x
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发表时间：
1990-06-20
期刊：
影响因子：
--
通讯作者：
BOHME, E
中科院分区：
文献类型：
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作者：
HUMBERT, P;NIROOMAND, F;BOHME, E
可溶性鸟苷酸环化酶从牛肺纯化的免疫亲和层析方法使用IgG组分的抗血清对合成肽的70 kDa的亚基的酶的C-末端。在阴离子交换层析后，将酶结合到免疫亲和柱上，并用合成肽洗脱。该方法允许从40 g环溶质蛋白中方便地分离2 mg明显均质的酶。该酶具有约150 kDa的表观分子量，并由两个亚基（70 kDa和73 kDa），通过凝胶渗透快速蛋白质液相色谱和SDS/PAGE测定。在Mg ~（2+）和Mn ~（2+）存在下测定的基础活性分别为10-20 nmol·cm·min ~（-1）·cm ~（-1）·mg ~（-1）和80-100 nmol·cm·min ~（-1）·mg ~（-1）。该酶表现出血红素蛋白典型的紫外-可见吸收光谱，在430 nm处具有Soret带。用含NO的化合物刺激纯化的酶。在硝普钠存在下测得的最大酶活性为1.2-2.4 μ mol·min-1·mg-1（硝普钠在1.3-1.9 μ M时的半数最大效应）和0.9-1.8 μ mol·cm·min-1·cm·mg-1（在0.28-0.41 μ M硝普钠下的半最大效应）。该方法开发的大规模纯化的可溶性鸟苷酸环化酶的免疫亲和层析，使用合成肽的洗脱酶，似乎是上级以上描述的方法。由于可以容易地获得针对对应于各蛋白质的推导氨基酸序列的合成肽的抗体，所述方法可以适用于各种蛋白质的方便的大规模纯化。
Soluble guanylyl cyclase was purified from bovine lung by an immunoaffinity chromatographic method using IgG fractions of antisera against a synthetic peptide of the C-terminus of the 70-kDa subunit of the enzyme. After anion-exchange chromatography, the enzyme was bound to an immunoaffinity column and was eluted with the synthetic peptide. This method allowed the convenient isolation of 2 mg of apparently homogeneous enzyme from 40 g cyclosolic proteins. The enzyme had an apparent molecular mass of about 150 kDa and consisted of two subunits (70 kDa and 73 kDa) as determined by gel permeation fast protein liquid chromatography and SDS/PAGE. The basal activities determined in the presence of Mg2+ and Mn2+ were 10-20 nmol.cntdot.min-1.cntdot.mg-1 and 80-100 nmol.cntdot.min-1.cntdot.mg-1, respectively. The enzyme exhibited an ultraviolet-visible absorption spectrum typical for hemoproteins, with a Soret band at 430 nm. The purified enzyme was stimulated by NO-containing compounds. Maximal enzyme activities measured in the presence of sodium nitroprusside were 1.2-2.4 .mu.mol.cntdot.min-1.cntdot.mg-1 (half-maximal effect of sodium nitroprusside at 1.3-1.9 .mu.M) and 0.9-1.8 .mu.mol.cntdot.min-1.cntdot.mg-1 (half-maximal effect at 0.28-0.41 .mu.M sodium nitroprusside) in the presence of Mg2+ and Mn2+, respectively. The method developed for the large-scale purification of soluble guanylyl cyclase by immunoaffinity chromatography, using synthetic peptides for the elution of the enzyme, appears to be superior to previously described methods. As antibodies against synthetic peptides corresponding to deduced amino acid sequences of the respective protein are easily obtained, the described method may be suitable for a convenient large-scale purification of various proteins.