Osborne J C, Bengtsson-Olivecrona G, Lee N S, Olivecrona T
Biochemistry. 1985 Sep 24;24(20):5606-11. doi: 10.1021/bi00341a048.
Sedimentation equilibrium analysis demonstrated that preparations of bovine lipoprotein lipase contain a complex mixture of dimers and higher oligomers of enzyme protein. Enzyme activity profiles from sedimentation equilibrium as well as from gel filtration indicated that activity is associated almost exclusively with the dimer fraction. To explore if the enzyme could be dissociated into active monomers, 0.75 M guanidinium chloride was used. Sedimentation velocity measurements demonstrated that this treatment led to dissociation of the lipase protein into monomers. Concomitant with dissociation, there was an irreversible loss of catalytic activity and a moderate change in secondary structure as detected by circular dichroism. The rate of inactivation increased with decreasing concentrations of active lipase, but addition of inactive lipase protein did not slow down the inactivation. This indicates that reversible interactions between active species precede the irreversible loss of activity. The implication is that dissociation initially leads to a monomer form which is in reversible equilibrium with the active dimer, but which decays rapidly into an inactive form, and is therefore not detected as a stable component in the system.
沉降平衡分析表明，牛脂蛋白脂肪酶制剂包含酶蛋白二聚体和更高聚体的复杂混合物。沉降平衡以及凝胶过滤的酶活性谱表明，活性几乎完全与二聚体部分相关。为了探究该酶是否能解离成有活性的单体，使用了0.75M的氯化胍。沉降速度测量表明，这种处理导致脂肪酶蛋白解离成单体。伴随着解离，催化活性出现不可逆丧失，并且通过圆二色性检测到二级结构有适度变化。失活速率随着活性脂肪酶浓度的降低而增加，但添加无活性的脂肪酶蛋白并不会减缓失活。这表明活性物种之间的可逆相互作用先于不可逆的活性丧失。这意味着解离最初导致一种单体形式，它与活性二聚体处于可逆平衡，但会迅速衰变成无活性形式，因此在系统中未被检测为稳定成分。
