Jin L, Lee Y P, Jonas A
Department of Biochemistry, University of Illinois at Urbana-Champaign 61801, USA.
J Lipid Res. 1997 Jun;38(6):1085-93.
We established a Chinese hamster ovary cell line that constitutively expresses up to 5 mg/L of human recombinant lecithin: cholesterol acyltransferase (rLCAT). We purified the rLCAT to > 96% purity, and characterized it along with plasma LCAT (pLCAT) biochemically and biophysically. The recombinant enzyme is more heavily glycosylated and more heterogeneous in its carbohydrate content than the plasma enzyme, as revealed by differences in molecular weight and pI isoforms, determined by mass spectrometry and isoelectric focusing. Recombinant LCAT is half as active enzymatically as pLCAT. The difference in activity is due to differences in the catalytic rates rather than in the apparent K(m) values, suggesting that the binding of the rLCAT to interfaces is not altered by its different glycosylation pattern. Despite these differences, rLCAT has essentially the same intrinsic tryptophan fluorescence emission spectrum and far-UV CD spectrum as pLCAT, indicating that the tertiary and secondary structures of both enzyme forms are very similar. Both enzyme forms have a propensity to self-associate, and their multimers appear resistant to dissociation by SDS and dilution. The free energies of unfolding (delta G(H2O)) of rLCAT and pLCAT are 3.4 +/- 0.2 and 3.2 +/- 0.2 kcal/mol, respectively, as determined by guanidine hydrochloride denaturation monitored by fluorescence. These relatively low delta G(H2O) values support the notion that LCAT is capable of undergoing major conformational changes upon interaction with interfacial substrates.
我们建立了一种中国仓鼠卵巢细胞系，该细胞系可组成型表达高达5 mg/L的人重组卵磷脂：胆固醇酰基转移酶（rLCAT）。我们将rLCAT纯化至纯度大于96%，并对其以及血浆LCAT（pLCAT）进行了生化和生物物理特性分析。通过质谱和等电聚焦测定的分子量和pI同工型差异表明，重组酶比血浆酶的糖基化程度更高，且碳水化合物含量更不均一。重组LCAT的酶活性是pLCAT的一半。活性差异是由于催化速率不同，而非表观K(m)值不同，这表明rLCAT与界面的结合并未因其不同的糖基化模式而改变。尽管存在这些差异，但rLCAT与pLCAT的内在色氨酸荧光发射光谱和远紫外圆二色光谱基本相同，这表明两种酶形式的三级和二级结构非常相似。两种酶形式都有自我缔合的倾向，并且它们的多聚体似乎对SDS和稀释诱导的解离具有抗性。通过荧光监测盐酸胍变性测定，rLCAT和pLCAT的解折叠自由能（delta G(H2O)）分别为±0.2 kcal/mol。这些相对较低的delta G(H2O)值支持了LCAT在与界面底物相互作用时能够发生重大构象变化的观点。
