Chander A
University of Pennsylvania School of Medicine, Institute for Environmental Medicine, Philadelphia.
Biochim Biophys Acta. 1992 Jan 24;1123(2):198-206. doi: 10.1016/0005-2760(92)90112-9.
Lung surfactant is synthesized in lung epithelial type II cells and stored in the lamellar bodies prior to its secretion onto the alveolar surface. The lamellar bodies, like other secretory organelles, maintain an ATP-dependent pH gradient that is sensitive to inhibitors of H(+)-ATPase. This report shows that the ATPase activity of lamellar bodies is enriched in a fraction prepared from lamellar bodies that were disrupted after isolation. The apparent Vmax for this enzyme was 150 nmol ATP hydrolyzed per min per mg protein and apparent Km for ATP was approximately 50 microM. The enzyme activity was sensitive to N-ethylmaleimide (NEM), dicyclohexylcarbodiimide (DCCD) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) (all inhibitors of vacuolar-type H(+)-ATPase) and vanadate (inhibitor of phosphoenzyme-type ATPase). Besides, the activity could also be inhibited with diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and Ca2+. Two proteins (of approximately 45 kDa and 17 kDa) of this fraction showed acid-stable phosphorylation with ATP. The labeling of proteins with ATP (-gamma-32P) could be chased with unlabelled ATP, suggesting that phosphorylation and dephosphorylation of these proteins is associated with the ATPase activity. Our results on inhibition characteristics of the enzyme activity suggest that besides a vacuolar type H(+)-ATPase, the lamellar bodies also contain a phosphoenzyme type ATPase that is sensitive to inhibitors of vacuolar type H(+)-ATPase.
肺表面活性物质在肺Ⅱ型上皮细胞中合成，并在分泌到肺泡表面之前储存在板层小体中。与其他分泌细胞器一样，板层小体维持着对H(+)-ATP酶抑制剂敏感的ATP依赖性pH梯度。本报告显示，板层小体的ATP酶活性在从分离后破坏的板层小体中制备的级分中富集。该酶的表观Vmax为每分钟每毫克蛋白质水解150 nmol ATP，ATP的表观Km约为50 μM。酶活性对N-乙基马来酰亚胺（NEM）、二环己基碳二亚胺（DCCD）和7-氯-4-硝基苯并-2-恶唑-1,3-二氮杂茂（NBD-Cl）（均为液泡型H(+)-ATP酶抑制剂）和钒酸盐（磷酸酶型ATP酶抑制剂）敏感。此外，该活性也可被二异硫氰酸根合芪-2,2'-二磺酸（DIDS）和Ca2+抑制。该级分中的两种蛋白质（约45 kDa和17 kDa）显示出与ATP的酸稳定磷酸化。用ATP（-γ-32P）对蛋白质的标记可以被未标记的ATP追踪，表明这些蛋白质的磷酸化和去磷酸化与ATP酶活性相关。我们关于酶活性抑制特性的结果表明，除了液泡型H(+)-ATP酶外，板层小体还含有一种对液泡型H(+)-ATP酶抑制剂敏感的磷酸酶型ATP酶。
