Ortwerth B J, Sharma K K, Olesen P R
Mason Institute of Ophthalmology, University of Missouri, Columbia 65212.
Curr Eye Res. 1994 Aug;13(8):561-7. doi: 10.3109/02713689408999889.
Bovine lens alpha-crystallin inhibited both porcine pancreatic elastase (PPE) and human neutrophil elastase (HNE), but not in the same manner. PPE was immediately inhibited with a stoichiometry of 10 moles of PPE inhibited per mole of alpha-crystallin. The inhibition was markedly decreased by the addition of even low levels of salts. The inhibition was transient, as PPE activity returned to normal with a t1/2 of 30 min even in low salt. HNE required a short preincubation to show maximum inhibition with a stoichiometry of approximately one mole of HNE inhibited per mole of alpha-crystallin. The inhibition of HNE was only slightly decreased by the addition of 0.1 M salt, and HNE activity returned slowly exhibiting a t1/2 of 30 hrs under these conditions. The inhibition of each enzyme by alpha-crystallin was evaluated by Dixon plots giving Ki values of 1.5 nM for PPE and 0.25 nM for HNE. DFP-trypsin was able to compete with PPE for binding to alpha-crystallin and cause the release of PPE already bound to alpha-crystallin. The inhibition of HNE, however, was unaffected by the addition of DFP-trypsin. A mixture of HNE and alpha-crystallin in 0.1 M NaCl was incubated at 25 degrees C for 6 hours. Aliquots showed a slow, continuous cleavage of the alpha-crystallin subunits by SDS-PAGE, but little or no increase in HNE activity.(ABSTRACT TRUNCATED AT 250 WORDS)
牛晶状体α-晶体蛋白对猪胰弹性蛋白酶（PPE）和人中性粒细胞弹性蛋白酶（HNE）均有抑制作用，但作用方式不同。PPE可被立即抑制，其化学计量比为每摩尔α-晶体蛋白抑制10摩尔PPE。即使添加低水平的盐，这种抑制作用也会显著降低。这种抑制是短暂的，因为即使在低盐条件下，PPE活性也会在30分钟的半衰期后恢复正常。HNE需要短暂预孵育才能表现出最大抑制作用，化学计量比约为每摩尔α-晶体蛋白抑制1摩尔HNE。添加0.1M盐只会使HNE的抑制作用略有降低，在这些条件下，HNE活性恢复缓慢，半衰期为30小时。通过Dixon图评估α-晶体蛋白对每种酶的抑制作用，得出PPE的Ki值为1.5 nM，HNE的Ki值为0.25 nM。DFP-胰蛋白酶能够与PPE竞争结合α-晶体蛋白，并导致已与α-晶体蛋白结合的PPE释放。然而，添加DFP-胰蛋白酶对HNE的抑制作用没有影响。将HNE和α-晶体蛋白在0.1M NaCl中的混合物于25℃孵育6小时。通过SDS-PAGE分析等分试样发现，α-晶体蛋白亚基会缓慢、持续地裂解，但HNE活性几乎没有增加或没有增加。（摘要截短于250字）
