Sathe S, Sakata M, Beaton A R, Sack R A
SUNY, College of Optometry, NY, USA.
Curr Eye Res. 1998 Apr;17(4):348-62. doi: 10.1080/02713689808951215.
Previous work identified polymorphonuclear leukocyte (PMN) elastase as the major caseinolytic entity in tears collected after overnight eye closure. This study was designed to identify the principal serine protease inhibitors (serpins) in tears and to determine their function in the regulation of PMN cell proteases on eye closure.
Reflex and closed eye tear samples were collected by microcapillary tube and centrifuged. After reflex and closed eye supernatants (R and C) were fractionated by HPLC, samples were subjected to casein zymography and reverse zymography. Western blots were utilized to screen tears and HPLC fractions for elastase, cathepsin G and proteinase-3 and to obtain semi-quantitative data on alpha 1-protease inhibitor (alp1), alpha 1-antichymotrypsin (alpha 1-Achy), secretory leukocyte protease inhibitor (SLPI), elafin and alpha 2-macroglobulin (alpha 2-M) as well as associated complexes and products. To confirm specificity of reactivity, samples were immunoprecipitated for a given protease or serpin and screened for the coprecipitation of interacting species.
Although R fluid contains no caseinolytic activity, it contains low levels of serpin-like activity principally in the form of SLPI (5-10 ng/microliter). Lesser amounts of alpha 2-M, alpha 1-Achy and alp1 (approximately < 1-3 ng/microliter) are also evident. C fluid is associated with very high levels of PMN cell proteases along with a approximately 5-20-fold increase in the concentrations of all of the above inhibitors. Trace levels of elafin were also detected. The concentrations of rapid reacting inhibitors exceeded that of proteases, with SLPI, alpha 1-Achy and alp1 being the principal functional entities. In atypical samples, complexes of elastase and alpha 2-M were also encountered.
SLPI, a known antimicrobial agent and an elastase and cathepsin G inhibitor, is the principal serpin in R fluid. C fluid is associated with a marked increase in the concentrations of an array of rapid reacting serpins capable of inhibiting all known PMN cell serine proteases. In the normal closed eye, the concentration of rapid reacting inhibitors always exceeds that of proteases with C fluid also containing a functional reserve of the slow reacting inhibitor alpha 2-M.
先前的研究确定多形核白细胞（PMN）弹性蛋白酶是闭眼过夜后收集的泪液中主要的酪蛋白水解成分。本研究旨在确定泪液中的主要丝氨酸蛋白酶抑制剂（丝氨酸蛋白酶抑制剂），并确定它们在闭眼时对PMN细胞蛋白酶调节中的作用。
用微量毛细管收集反射性泪液和闭眼泪液样本并离心。反射性泪液和闭眼泪液上清液（R和C）经高效液相色谱（HPLC）分离后，进行酪蛋白酶谱分析和反向酶谱分析。利用蛋白质免疫印迹法筛选泪液和HPLC组分中的弹性蛋白酶、组织蛋白酶G和蛋白酶-3，并获得关于α1-蛋白酶抑制剂（alp1）、α1-抗糜蛋白酶（α1-Achy）、分泌型白细胞蛋白酶抑制剂（SLPI）、弹性蛋白和α2-巨球蛋白（α2-M）以及相关复合物和产物的半定量数据。为了确认反应的特异性，对给定的蛋白酶或丝氨酸蛋白酶抑制剂进行免疫沉淀，并筛选相互作用物质的共沉淀。
尽管R液不具有酪蛋白水解活性，但它含有低水平的丝氨酸蛋白酶抑制剂样活性，主要形式为SLPI（5-10 ng/微升）。也可明显检测到少量的α2-M、α1-Achy和alp1（约<1-3 ng/微升）。C液与非常高水平的PMN细胞蛋白酶相关，同时上述所有抑制剂的浓度增加约5-20倍。还检测到痕量水平的弹性蛋白。快速反应抑制剂的浓度超过蛋白酶，其中SLPI、α1-Achy和alp1是主要的功能成分。在非典型样本中，还发现了弹性蛋白酶与α2-M的复合物。
SLPI是一种已知的抗菌剂，也是弹性蛋白酶和组织蛋白酶G的抑制剂，是R液中的主要丝氨酸蛋白酶抑制剂。C液与一系列能够抑制所有已知PMN细胞丝氨酸蛋白酶的快速反应丝氨酸蛋白酶抑制剂的浓度显著增加有关。在正常闭眼情况下，快速反应抑制剂的浓度总是超过蛋白酶，C液还含有缓慢反应抑制剂α2-M的功能储备。
