Michelis D, Kounnas M Z, Argraves W S, Sanford E D, Borchelt J D, Wright J R
Department of Physiology, University of California, San Francisco.
Am J Respir Cell Mol Biol. 1994 Dec;11(6):692-700. doi: 10.1165/ajrcmb.11.6.7946398.
The goal of the current investigation was to characterize, purify, and identify the proteins that bind surfactant protein A (SP-A). Several polypeptides were purified by SP-A affinity chromatography, and the 200 kD major polypeptide that reacted with SP-A on ligand blots was purified further by preparative SDS-PAGE. Protein sequencing of proteolytically derived subfragments of this polypeptide gave sequences that corresponded completely with nonmuscle (cellular) myosin heavy chain. The 200 kD polypeptide was then found to be immunoreactive with antibodies against cellular myosin. A smaller polypeptide of 135 kD also binds SP-A and appears to be a proteolytic fragment of the 200 kD peptide. The ability of SP-A to bind myosin was confirmed in a microtiter well assay and was found to be concentration dependent. We speculated that the physiologic relevance of the interaction of SP-A with myosin might be to facilitate clearance of myosin from the alveolar subphase following its release during lung injury. In support of this hypothesis, we found that there were detectable levels of myosin in lavage fluid and that SP-A could indeed enhance uptake and degradation of myosin by alveolar macrophages.
当前研究的目的是对与表面活性蛋白A（SP-A）结合的蛋白质进行表征、纯化和鉴定。通过SP-A亲和色谱法纯化了几种多肽，并通过制备性SDS-PAGE进一步纯化了在配体印迹上与SP-A反应的200 kD主要多肽。对该多肽经蛋白酶水解产生的亚片段进行蛋白质测序，得到的序列与非肌肉（细胞）肌球蛋白重链完全一致。然后发现该200 kD多肽与抗细胞肌球蛋白抗体具有免疫反应性。一种较小的135 kD多肽也能结合SP-A，似乎是200 kD肽的蛋白酶水解片段。在微量滴定板试验中证实了SP-A与肌球蛋白结合的能力，且发现其具有浓度依赖性。我们推测，SP-A与肌球蛋白相互作用的生理相关性可能是在肺损伤期间肌球蛋白释放后，促进其从肺泡亚相中清除。为支持这一假设，我们发现灌洗液中存在可检测水平的肌球蛋白，并且SP-A确实可以增强肺泡巨噬细胞对肌球蛋白的摄取和降解。
