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1.
Proliferation and differentiation of epithelial cells are thought to be regulated by soluble factors in extracellular fluid and insoluble components of the extracellular matrix. We have examined the combined effects of soluble factors and an extracellular matrix (EHS matrix) on DNA synthesis, cell proliferation, and surfactant protein gene expression in primary cultures of alveolar type II epithelial cells. Cells on EHS matrix cultured in DMEM containing insulin, cholera toxin, EGF, aFGF, 5% rat serum, and 15-fold concentrated bronchoalveolar lavage fluid (D-GM) formed larger aggregates than cells cultured on the same substratum in DMEM containing 5% rat serum (D-5). Cells cultured in D-GM on EHS matrix incorporated more [3H]-thymidine than cells on the same substratum in D-5, with an eight-fold increase seen on day 4 of culture. This increase in [3H]-thymidine incorporation was accompanied by a labeling index of greater than 65% of the cells. Cell counts showed that exposure of type II cells on EHS matrix to D-GM resulted in increased cell number on day 4 of culture. [3H]-thymidine autoradiography combined with immunostaining with anti-cytokeratin, anti-SP-A, and anti-vimentin antibodies demonstrated that the proliferating cells were epithelial cells that contained SP-A. Type II cells cultured on plastic in D-GM also showed increased [3H]-thymidine incorporation compared to cells cultured in D-5. The level of [3H]-thymidine incorporation by cells on plastic, however, was significantly less than that seen in cells cultured in the same medium on EHS matrix. Type II cells cultured on EHS matrix in D-GM had a decreased abundance of mRNAs for SP-A and SP-C than cells cultured on EHS matrix in D-5 as determined by Northern analysis. This inhibition was reversed by switching from D-GM to D-5 on day 4 and culturing the cells for an additional 4 days. In contrast, SP-B mRNA was increased in response to D-GM. This increase was not reversed by switching from D-GM to D-5 on day 4. These results suggest that the interaction of soluble factors and extracellular matrix components has a strong influence on type II cell proliferation, which were partially associated with the reversible inhibition of lung tissue-specific protein mRNAs. Their dynamic interplay among the type II cell, the extracellular matrix, and growth factors may determine multicellular functions and play an important role in normal lung development and in the repair of the lung epithelium following injury. 相似文献
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ZC Chroneos R Abdolrasulnia JA Whitsett WR Rice VL Shepherd 《Canadian Metallurgical Quarterly》1996,271(27):16375-16383
In the present report we have characterized the binding of surfactant protein A (SP-A) to bone marrow-derived macrophages, U937 cells, alveolar macrophages, and type II epithelial cells. The binding of SP-A to all cell types is Ca2+-dependent and trypsin-sensitive, but type II cells express distinct Ca2+-independent binding sites. The binding of SP-A to macrophages is independent of known cell surface carbohydrate-specific receptors and of glycoconjugate binding sites on the surface of the cells and is distinct from binding to C1q receptors. Based on ligand blot analysis, both type II cells and macrophages express a 210-kDa SP-A-binding protein. The 210-kDa protein was purified to apparent homogeneity from U937 macrophage membranes using affinity chromatography with noncovalently immobilized surfactant protein A, and was purified from rat lung by differential detergent and salt extraction of isolated rat lung membranes. Polyclonal antibodies against the rat lung SP-A-binding protein inhibit binding of SP-A to both type II cells and macrophages, indicating that the 210-kDa protein is expressed on the cell surface. The polyclonal antibodies also block the SP-A-mediated inhibition of phospholipid secretion by type II cells, indicating that the 210-kDa protein is a functional cell-surface receptor on type II cells. In a separate report we have determined that antibodies to the SP-A receptor block the SP-A-mediated uptake of Mycobacterium bovis, indicating that the macrophage SP-A receptor is involved in SP-A-mediated clearance of pathogens. 相似文献
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BACKGROUND: There are disparate reports concerning the presence of surfactant proteins in the airways of lung. The recent finding of SP-A in tracheobronchial epithelium and submucosal glands in lungs from second trimester humans has renewed interest in potential new functions of surfactant in lung biology. METHODS: In situ hybridization studies were done to determine the distribution of SP-A, SP-B, and SP-C in baboon lung specimens from 60, 90, 120, 140, 160, and 180 (term) days of gestation and adults. Lungs from gestation controls were obtained at the time of hysterotomy and adult lungs at necropsy. Riboprobes used for in situ hybridization contained the entire coding regions for human SP-A, SP-B, and SP-C. RESULTS: At 60 days, SP-C mRNA expression was evident in focal portions of primitive tubular epithelium but not bronchi. This distal pattern of SP-C mRNA expression persisted and was present in some epithelial cells of respiratory bronchioles at term. At 90 days, SP-A mRNA expression was present in the epithelium of trachea and large bronchi. SP-B mRNA expression was found in small bronchi, bronchioles, and distal tubular epithelium at 120 days of gestation. SP-A mRNA bronchiolar localization became evident at 140 days of gestation and alveolar type 2 cellular expression at 160 days of gestation. Abrupt transitions of surfactant protein expression were identified (e.g., SP-A mRNA-positive cells in the epithelium of large bronchi with adjoining SP-B mRNA expression in small bronchi and bronchioles). CONCLUSIONS: Findings in the baboon indicate that there are well-delineated sites of surfactant protein mRNA expression in bronchial and bronchiolar epithelia. mRNA expressions of SP-A and SP-B are present in both bronchial and bronchiolar epithelium but at different sites, whereas SP-C expression is seen in loci of epithelial cells in respiratory bronchioles. 相似文献
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TR Korfhagen V Sheftelyevich MS Burhans MD Bruno GF Ross SE Wert MT Stahlman AH Jobe M Ikegami JA Whitsett JH Fisher 《Canadian Metallurgical Quarterly》1998,273(43):28438-28443
Surfactant protein D (SP-D) is a 43-kDa member of the collectin family of collagenous lectin domain-containing proteins that is expressed in epithelial cells of the lung. The SP-D gene was targeted by homologous recombination in embryonic stem cells that were used to produce SP-D (+/-) and SP-D (-/-) mice. Both SP-D (-/-) and SP-D (+/-) mice survived normally in the perinatal and postnatal periods. Whereas no abnormalities were observed in SP-D (+/-) mice, alveolar and tissue phosphatidylcholine pool sizes were markedly increased in SP-D (-/-) mice. Increased numbers of large foamy alveolar macrophages and enlarged alveoli were also observed in SP-D (-/-) mice. Phospholipid composition was unaltered in SP-D (-/-) mice, but surfactant morphology was abnormal, consisting of dense phospholipid membranous arrays with decreased tubular myelin. The pulmonary lipoidosis in the SP-D (-/-) mice was not associated with accumulation of surfactant proteins B or C, or their mRNAs, distinguishing the disorder from alveolar proteinosis syndromes. Surfactant protein A mRNA was reduced and, SP-A protein appeared to be reduced in SP-D (-/-) compared with wild type mice. Targeting of the mouse SP-D gene caused accumulation of surfactant lipid and altered phospholipid structures, demonstrating a previously unsuspected role for SP-D in surfactant lipid homeostasis in vivo. 相似文献
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FX McCormack 《Canadian Metallurgical Quarterly》1998,1408(2-3):109-131
Surfactant protein A (SP-A) is a highly ordered, oligomeric glycoprotein that is secreted into the airspaces of the lung by the pulmonary epithelium. The in vitro activities of protein suggest diverse roles in pulmonary host defense and surfactant homeostasis, structure and surface activity. Functional mapping of SP-A using directed mutagenesis has identified domains which interact with surfactant phospholipids, alveolar type II cells and microbes. Recently developed genetically manipulated animal models are beginning to clarify the critical physiological roles for SP-A in the normal lung, and in the pathophysiology of pulmonary disease. 相似文献
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N Toki K Sueishi M Minamitani H Maeda H Nakano Y Suzuki 《Canadian Metallurgical Quarterly》1995,26(11):1252-1259
Forty-three patients with nonimmunologic hydrops fetalis (NIHF), including 32 patients (74%) with hypoplastic lung, were immunohistochemically examined for the expression of surfactant apolipoproteins (SPs), using anti-gamma G immunoglobulins against human SP-A with a molecular weight (MW) of 35 K and SP-B with a MW of 5 K compared with that in 59 patients in a control group and 45 patients with hypoplastic lung induced by causes other than NIHF. In the control group, SP-A was expressed in the lungs from 23 gestational weeks and became more numerous and intense in alveolar type II cells after 31 gestational weeks, whereas SP-B began to be expressed from 20 gestational weeks, and almost all patients showed a diffuse positivity after 26 gestational weeks. In the NIHF group, SP-A expression was generally weak, even after 31 gestation weeks. Moreover, most of the patients showing a weak expression of SP-A were also associated with hypoplastic lung and had a clinical history of persistent intrauterine pleural effusion of more than 2 weeks. Conversely, the immunoreactivity of SP-B was well preserved in NIHF cases either with or without hypoplastic lung. These results suggest that in the NIHF lung, there is a possible delay in the functional maturation or development of SP-A synthesis by alveolar type II cells, and this retardation of the functional maturation in type II cells also participates in the postnatal respiratory insufficiency in NIHF. 相似文献
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A Meneghetti WV Cardoso JS Brody MC Williams 《Canadian Metallurgical Quarterly》1996,44(10):1173-1182
Explants of embryonic lung are often used to characterize lung growth, bronchial tree pattern, and cell differentiation. Most investigators culture lungs for 3-7 days in defined media lacking, e.g., added growth factors or hormones. If growth and differentiation are comparable to that in vivo, these cultures show considerable promise for identifying developmental regulatory molecules and target genes, and for elucidating molecular responses. We used in situ hybridization and RT-PCR to compare times and sites of expression of mRNAs of six epithelial genes in cultured and uncultured fetal rat lungs. These genes, expressed in distal lung of adult rats, are surfactant proteins (SP) A, B, and C; LAR, a receptor-type tyrosine phosphatase; Clara cell secretory protein (CC10, CCSP); and T1alpha. SP-A, SF-B, LAR, and CC10 are expressed by both Clara and Type II cells in adult animals. SP-C and T1alpha are unique markers for Type II and Type I cells, respectively. SP-C, LAR, and T1alpha are expressed before the lung is explanted (Day 13.5); SP-A, -B, and CC10 mRNAs are first detected later. The onset of expression is similar in vivo and in vitro. Although the patterns of expression differ for each mRNA, their sites of expression in culture match those in vivo relative to the bronchial tree. The explanted embryonic lung appears to be an excellent experimental model. 相似文献
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The abundant and restricted expression of surfactant proteins SP-A and SP-D within the lung makes these collectins specific markers for lung diseases. The measurement of SP-A and SP-D in amniotic fluids and tracheal aspirates reflects lung maturity and the production level of the lung surfactant in infants with respiratory distress syndrome (RDS). The SP-A concentrations in bronchoalveolar lavage (BAL) fluids are significantly decreased in patients with acute respiratory distress syndrome (ARDS) and also in patients at risk to develop ARDS. The prominent increase of these proteins in BAL fluids and sputum is diagnostic for pulmonary alveolar proteinosis (PAP). The concentrations of SP-A and SP-D in BAL fluids from patients with idiopathic pulmonary fibrosis (IPF) and interstitial pneumonia with collagen vascular diseases (IPCD) are rather lower than those in healthy controls and the SP-A/phospholipid ratio may be a useful marker of survival prediction. SP-A and SP-D appear in the circulation in specific lung diseases. Their serum concentrations significantly increase in patients with PAP, IPF and IPCD. The successive monitoring of serum levels of SP-A and SP-D may predict the disease activity. The serum SP-A levels increase in patients with ARDS. SP-A is also a marker for lung adenocarcinomas and can be used to differentiate lung adenocarcinomas from other types and metastatic cancers from other origins, and to detect metastasis of lung adenocarcinomas. 相似文献
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D Ohmer-Schr?ck C Schlatterer H Plattner J Schlepper-Sch?fer 《Canadian Metallurgical Quarterly》1993,26(5):374-380
We analyzed the binding mechanism of human recombinant lung surfactant protein A (SP-A) to rat alveolar macrophages using anti-SP-A antiserum and protein A coated onto gold particles. Results were compared with our recent data on binding and uptake of SP-A-coated colloidal gold particles. The rationale for the current approach was to avoid any possible steric effects on SP-A binding to the cell surface. Binding of unlabeled SP-A depends on the presence of calcium ions in the medium and involves a mannose-specific mechanism. Binding is partly inhibited by the collagenase-resistant fragment of SP-A, representing mainly the globular part of SP-A. Taken together, these facts indicate binding of SP-A via the carbohydrate binding site on the globular region of SP-A. On the other hand, a partial inhibition of SP-A binding by fragments of C1q (representing the collagenous region of C1q) indicates a second binding site for SP-A by the collagen-like portion to the C1q receptor of macrophages. We conclude that two different mechanisms are probably involved in SP-A binding to alveolar macrophages. Specificity of the binding was shown with fluorescein-labeled SP-A. Binding was inhibited by an excess of unlabeled SP-A. Binding and uptake of SP-A are seen only with alveolar macrophages and not with other macrophage populations isolated from rat, such as liver macrophages (Kupffer cells), resident peritoneal macrophages, and peritoneal macrophages activated by Corynebacterium parvum. Therefore, binding sites for SP-A occur exclusively on alveolar macrophages.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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In the present study, we characterized surfactant protein (SP)-A messenger RNA (mRNA) in mid-trimester human fetal trachea and bronchi. SP-A protein was localized by immunocytochemistry to scattered epithelial cells in the airway surface epithelium and in submucosal glands of the fetal trachea and bronchi. SP-A mRNA (2.2 kb) was detected by Northern blot analysis in human fetal trachea, as well as in primary and more distal bronchi. The levels of detectable SP-A mRNA were highest in the upper airways and were decreased in smaller bronchi in comparison. SP-A mRNA was barely detectable in the distal fetal lung tissue. In contrast, SP-A mRNA was abundant in cultured explants of distal human fetal lung tissue. SP-A1 and SP-A2 mRNA were detected by primer extension analysis in adult human lung tissue and in cultured human fetal lung explants. Only SP-A2 mRNA was detected in RNA isolated from human fetal trachea and bronchi. SP-A mRNA was localized by in situ hybridization in the fetal trachea and bronchi in scattered cells in the surface epithelium and, most prominently, in submucosal glands. Our results suggest that SP-A2, and not SP-A1, is produced in the human fetal tracheal and bronchial epithelium and in submucosal glands. 相似文献
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S Pattanajitvilai Y Kuroki W Tsunezawa FX McCormack DR Voelker 《Canadian Metallurgical Quarterly》1998,273(10):5702-5707
In previous studies, tandem mutagenesis of Glu195 and Arg197 of surfactant protein A (SP-A) has implicated both residues as critical participants in the interaction of the molecule with alveolar type II cells and phospholipids. We substituted Ala, Lys, His, Asp, and Asn mutations for Arg to evaluate the role of a basic amino acid at position 197 in SP-A action. Unexpectedly, Ala197 retained complete activity in the SP-A functions of carbohydrate binding, type II cell binding, inhibition of surfactant secretion, lipid binding, lipid aggregation, and lipid uptake by type II cells. The results unambiguously demonstrate that Arg197 is not mechanistically essential for SP-A function. The Lys197 mutation displayed all functions of the wild type protein but exhibited a 2-fold increase in lipid uptake activity. The His197 mutation displayed all SP-A functions studied except for lipid uptake. The results obtained with the His197 mutation clearly demonstrate that lipid aggregation alone by SP-A is insufficient to promote lipid uptake by type II cells. These findings indicate that specific interactions between type II cells and SP-A are involved in the phospholipid uptake processes. 相似文献
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H Arai H Hirano S Mushiake M Nakayama G Takada K Sekiguchi 《Canadian Metallurgical Quarterly》1997,151(2):403-412
Cell-matrix interactions have been shown to regulate the development of the lung, particularly airway branching and alveolarization. Fibronectin is the major constituent of pulmonary extracellular matrix and exists in multiple isoforms arising from alternative RNA splicing. EDA and EDB are the two major alternatively spliced segments, the expression of which is regulated in a spatiotemporal and oncodevelopmental manner. In this study, we investigated immunohistochemically the distribution of the EDA- and EDB-containing fibronectin isoforms (referred to as EDA+ fibronectin and EDB+ fibronectin, respectively) in normal and hypoplastic human lungs at different gestational ages to explore the role of these fibronectin isoforms in alveolarization. EDA+ fibronectin was expressed around the distal airspaces throughout the development of both normal and hypoplastic lungs. In contrast, the expression of EDB+ fibronectin was restricted to the lung with morphologically immature acinar complex, typically observed in normally developing lungs of < 30 gestational weeks or in hypoplastic lungs. To further confirm the restricted expression of EDB+ fibronectin in immature acinar complex, we examined the correlation of EDB+ fibronectin expression with that of the surfactant protein SP-A, a biochemical marker for the differentiated type II pneumocytes. A clear inverse relationship between the immunoreactivities for EDB+ fibronectin and SP-A was observed in both control and hypoplastic lungs. Given the proposed importance of fibronectins in the differentiation of alveolar epithelial cells, our results suggest that the EDB segment plays a regulatory role in the differentiation of immature acinar epithelial cells into type II pneumocytes. The EDB segment may also serve as a new histochemical marker for the functional maturity of fetal lung tissues. 相似文献
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Pulmonary surfactant is a complex mixture of lipids and proteins that functions to keep alveoli from collapsing at the end of expiration. Dipalmitoylphosphatidylcholine has been identified as the most important component for lowering surface tension at the air-liquid interface. Hydrophobic surfactant apoproteins, SP-B and SP-C, play essential roles in the biophysical functions of the surfactant phospholipids. Hydrophilic surfactant apoproteins (SP-A and SP-D) that are members of C-type lectin superfamily, interact with phospholipids and glycolipids and modulate host defense functions in the lung. SP-A also plays an important role in regulating phospholipid homeostasis in the alveolar spaces. Recent advances in genetics and molecular biology have clarified the structure-function relationship of surfactant apoproteins. 相似文献
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We examined the synthesis, transport, and localization of surfactant protein A (SP-A) in primary cultures of alveolar type II cells. In type II cells maintained in culture for 6 h, 39% of the SP-A pool detected with an enzyme-linked immunosorbent assay (ELISA) was found in lamellar bodies (LBs). After 24 h in culture, 53% of the cellular SP-A pool was found in LBs. The absolute amount of SP-A in the LB compartment was almost identical at 6 and 24 h of culture. In contrast to the results obtained with ELISA, 35S labeling of newly synthesized SP-A revealed that less than 7% of the cellular SP-A pool was in LBs at either 6 or 24 h of culture. In the 6-h cultures, 17% of the total (i.e., cells and media) [35S]SP-A pool was extracellular. In the 24-h cultures, 70% of the [35S]SP-A pool was extracellular. The secretion of [35S]SP-A was blocked by brefeldin A at all times. When medium containing newly secreted [35S]SP-A was incubated with alveolar type II cells maintained in culture for 24 h, the protein was taken up and incorporated into the LB fraction. More than 80% of the internalized SP-A was associated with the LB compartment after a 6 h incubation. The uptake of [35S]SP-A was blocked at 4 degrees C and was promoted by addition of unlabeled SP-A at 37 degrees C. These findings support a pathway of extracellular routing of SP-A prior to its accumulation in LBs in cultured type II cells. 相似文献
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R Koslowski LG Dobbs KW Wenzel D Schuh M Müller M Kasper 《Canadian Metallurgical Quarterly》1998,12(6):1397-1403
After lung injury, the epithelial cells lining the alveolar surface in rat lung show an altered distribution of several membrane proteins. Pulmonary fibrosis was induced by intratracheal administration of bleomycin into the lung of rats and the distribution of RTI40, a recently detected alveolar epithelial type I cell antigen, was examined, as well as the relationship between RTI40 and a type I cell-specific antigen recognized by the monoclonal antibody MEP-1 and the type I cell-binding lectin Bauhinia purpurea in serial sections and double stainings. Loss of RTI40 protein was observed in fibrotic lungs, particularly in areas with obliteration of alveoli. Pre-embedding immunoelectron microscopy confirmed this observation by detection of RTI40 protein in the alveolar lumen. Western blot analysis revealed elevated levels of RTI40 in the bronchoalveolar fluid of bleomycin-treated rats with a maximum at day 7 after treatment. Twenty-eight days after bleomycin application, the bronchoalveolar fluid contained three times the amount of RTI40 x mg protein(-1) of control lungs, as determined by semiquantitative dot blot. These results suggest RTI40 as a tool for the evaluation of alveolar epithelial type I cell behaviour during re-epithelialization processes. 相似文献
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Y Kuroki S Tsutahara N Shijubo H Takahashi M Shiratori A Hattori Y Honda S Abe T Akino 《Canadian Metallurgical Quarterly》1993,147(3):723-729
An enzyme-linked immunosorbent assay using monoclonal antibodies to human lung surfactant protein A (SP-A) was applied to sera from patients with lung diseases. We examined whether SP-A appears in the sera of patients with diseases that are known to cause alterations in surfactant composition in bronchoalveolar lavage fluids, and we characterized the SP-A that was found. The level of SP-A in sera from 57 healthy volunteers was 45 +/- 3 ng/ml (mean +/- SEM). The levels in patients with idiopathic pulmonary fibrosis (IPF) (205 +/- 23 ng/ml, n = 32) and pulmonary alveolar proteinosis (PAP) (285 +/- 23 ng/ml, n = 6) were significantly higher than those in healthy control subjects (p < 0.01), whereas those of sarcoidosis (n = 16), pneumonia (n = 14), and tuberculosis (n = 14) were 52 +/- 27 ng/ml, 65 +/- 11 ng/ml, and 49 +/- 23 ng/ml, respectively. Electrophoresis and immunoblotting analysis demonstrated that the fraction isolated from serum of a patient with PAP or IPF by anti-SP-A immunoaffinity column chromatography consisted chiefly of human IgG and IgM, and that it also contained SP-A. Furthermore, IgG was found in preparation of purified human SP-A. SP-A was demonstrated to bind to nonimmune IgG coated onto microtiter wells. Gel filtration analysis revealed that serum SP-A was eluted at fractions of larger molecular size than was the purified SP-A. These findings suggest that SP-A appears in the bloodstream as a complex with immunoglobulin in IPF and in PAP. 相似文献
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AB Fisher C Dodia A Chander MF Beers SR Bates 《Canadian Metallurgical Quarterly》1994,1211(3):256-262
A marked sequence homology has been noted between lung surfactant protein A (SP-A) and an inhibitor of phospholipase A2 (PLA2) isolated from the serum of Trimeresurus flavoviridis (Habu snake). This study evaluated the effect of SP-A on PLA2 activity from several sources. SP-A was isolated from bovine or rat lung surfactant by extraction with 1-butanol and octyl beta-D-glucopyranoside. The addition of SP-A produced a concentration-dependent inhibition of T. flavoviridis PLA2 that indicated non-competitive kinetics with Ki 5 micrograms/ml. Inhibition was reversed by heat inactivation, disulfide bond reduction or alkylation of SP-A, or by the presence of anti-SP-A antibody. Treatment of SP-A with endoglycosidase F or the presence of variation monosaccharides or lectins did not alter SP-A inhibition. Binding of PLA2 to SP-A was shown by ultrafiltration and was abolished by SP-A alkylation or the presence of SDS. The SP-A/PLA2 complex recovered from the ultrafilter had essentially no enzymatic activity, but activity was restored by treatment with mercaptoethanol. SP-A had no effect on activity of PLA2 from Naja naja, Crotalus atrox, or bovine pancreas. These results indicate that surfactant protein A selectively inhibits Trimeresurus phospholipase A2 activity and suggest that binding to the enzyme is the mechanism for inhibition. 相似文献