Xu S Y, Carlson M, Engström A, Garcia R, Peterson C G, Venge P
Department of Clinical Chemistry, University Hospital, Uppsala, Sweden.
Scand J Clin Lab Invest. 1994 Aug;54(5):365-76. doi: 10.3109/00365519409088436.
A 45 kDa-protein was purified from the granules of human neutrophils. The protein consists of two apparently identical subunits. The isoelectric point was pH 8.40, and the molecular weight 45 kDa (unreduced) or 24 kDa (reduced). Treatment of the protein with Endoglucosidase F resulted in a reduction in the molecular weight to 20 kDa, indicating the presence of N-linked carbohydrate. The extinction coeffient was E1%,1cm = 13.76 at 280 nm. The 60 amino acid sequence revealed up to 65% sequence homology with rat alpha 2-microglobulin-related protein, which belongs to the lipocalin family. The protein co-sedimented with secondary (specific) granule marker proteins and correlated to the neutrophil content of Lactoferrin (r = 0.81, p < 0.001) and was estimated to be 0.59 microgram 10(-6) cells. Release studies showed that the neutrophils released 51.4 +/- 9.0% of the total cellular content of the protein when they were exposed to serum-opsonized particles, which was much higher than the release of Myeloperoxidase (12.7 +/- 3.5%) and Lactoferrin (22.9 +/- 4.7%). The N-terminal and four tryptic fragment amino acid sequence of the protein was identical with an N-formyl peptide binding 24 kDa protein and gelatinase associated protein of human neutrophils. In conclusion, we have purified and characterized a protein, human neutrophil lipocalin (HNL), from the secondary granules of human neutrophils and shown that it is readily mobilized from the neutrophils upon stimulation.
从人中性粒细胞的颗粒中纯化出一种45 kDa的蛋白质。该蛋白质由两个明显相同的亚基组成。其等电点为pH 8.40，分子量为45 kDa（未还原）或24 kDa（还原）。用内切葡糖苷酶F处理该蛋白质后，分子量降至20 kDa，表明存在N-连接的碳水化合物。在280 nm处的消光系数为E1%,1cm = 13.76。60个氨基酸的序列显示与大鼠α2-微球蛋白相关蛋白有高达65%的序列同源性，大鼠α2-微球蛋白相关蛋白属于脂质运载蛋白家族。该蛋白质与二级（特异性）颗粒标记蛋白共沉降，并与乳铁蛋白的中性粒细胞含量相关（r = 0.81，p < 0.001），估计为0.59微克/10(-6)个细胞。释放研究表明，当人中性粒细胞暴露于血清调理颗粒时，它们释放了该蛋白质总细胞含量的51.4 +/- 9.0%，这远高于髓过氧化物酶（12.7 +/- 3.5%）和乳铁蛋白（22.9 +/- 4.7%）的释放量。该蛋白质的N端和四个胰蛋白酶片段氨基酸序列与人类中性粒细胞的一种N-甲酰肽结合24 kDa蛋白质和明胶酶相关蛋白相同。总之，我们从人中性粒细胞的二级颗粒中纯化并鉴定了一种蛋白质，即人中性粒细胞脂质运载蛋白（HNL），并表明它在受到刺激后很容易从中性粒细胞中释放出来。
