Heiple J M, Ossowski L
J Exp Med. 1986 Sep 1;164(3):826-40. doi: 10.1084/jem.164.3.826.
The subcellular localization of plasminogen activator (PA) in human neutrophils was studied. The cells were disrupted by nitrogen cavitation and fractionated on Percoll density gradients into three major components containing the plasma membranes, the specific granules, and the azurophilic granules. The biochemical markers we used to identify these organelles were alkaline phosphatase, vitamin B12-binding protein, and beta-glucuronidase, respectively. Using the radioactive fibrin plate method, PA activity and plasminogen-independent fibrinolytic activity were measured. In resting neutrophils, PA was associated mainly with the membranes of the specific granules. In five individual experiments the activity of this fraction varied from 79 to 100% of the total; the remaining activity was found to be associated with the plasma membrane, and no activity was present in the azurophilic granules. In neutrophils that were activated by exposure to PMA (20 ng/ml for 15 min at 37 degrees C), the total recoverable PA activity remained unchanged; however, the main peak of activity (85% of total) shifted from the specific granules to the plasma membranes. The magnitude of the reduction of the enzyme in the specific granules paralleled that of vitamin B12-binding protein. PMA-activated, intact neutrophils had approximately 12-fold more surface-bound PA activity than resting cells. Recovery of PA activity from neutrophils was critically dependent on pretreatment of the intact cells with DFP before cavitation; 100-fold more PA activity was detected in DFP-pretreated cells. At the same time, this pretreatment reduced the plasminogen-independent fibrinolytic activity by approximately sevenfold. We determined that PA present in the neutrophils is of the urokinase (UK) type and that the enzyme is produced and stored as a pro-UK, a form insensitive to DFP inhibition. The reduction in the level of proteases (measured as fibrinolytic activity) and the resistance of pro-UK to DFP are most likely the two major reasons for the greatly improved recovery of PA from the DFP-pretreated cells. These findings show that in resting neutrophils PA is stored in the specific granules, and that during activation, it translocates to the outer surface of the plasma membranes, thus equipping the cell with an ecto-proteolytic potential.
研究了纤溶酶原激活剂（PA）在人中性粒细胞中的亚细胞定位。通过氮空化破坏细胞，并在Percoll密度梯度上进行分级分离，得到含有质膜、特异性颗粒和嗜天青颗粒的三个主要组分。我们用于鉴定这些细胞器的生化标志物分别是碱性磷酸酶、维生素B12结合蛋白和β-葡萄糖醛酸酶。采用放射性纤维蛋白平板法测定PA活性和不依赖纤溶酶原的纤溶活性。在静息中性粒细胞中，PA主要与特异性颗粒的膜相关。在五个独立实验中，该组分的活性占总量的79%至100%；其余活性与质膜相关，嗜天青颗粒中无活性。在暴露于佛波酯（PMA，20 ng/ml，37℃孵育15分钟）激活的中性粒细胞中，可回收的PA总活性保持不变；然而，主要活性峰（占总量的85%）从特异性颗粒转移到质膜。特异性颗粒中酶的减少幅度与维生素B12结合蛋白的减少幅度平行。PMA激活的完整中性粒细胞表面结合的PA活性比静息细胞高约12倍。从中性粒细胞中回收PA活性关键取决于在空化前用二异丙基氟磷酸酯（DFP）预处理完整细胞；在DFP预处理的细胞中检测到的PA活性高100倍。同时，这种预处理使不依赖纤溶酶原的纤溶活性降低约7倍。我们确定中性粒细胞中的PA是尿激酶（UK）型，并且该酶以无活性的尿激酶原形式产生和储存，这种形式对DFP抑制不敏感。蛋白酶水平的降低（以纤溶活性衡量）和无活性尿激酶原对DFP的抗性很可能是从DFP预处理的细胞中PA回收率大大提高的两个主要原因。这些发现表明，在静息中性粒细胞中，PA储存在特异性颗粒中，在激活过程中，它转移到质膜的外表面，从而使细胞具有胞外蛋白水解潜能。
