Fluorescently labeled pulmonary surfactant protein C in spread phospholipid monolayers

Nag, Kaushik; Pérez‐Gil, Jesús; Cruz, Antonio; Keough, K. M. W.

doi:10.1016/s0006-3495(96)79221-4

Cited by 72 publications

(76 citation statements)

References 61 publications

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“…The difference might be explained by an effect of SP-B and SP-C in Curosurf1. This hypothesis is supported by the experiments of NAG et al [19] who showed that the addition of SP-C to phospholipids modifies the transition of LE-to LC-phases. Enigmatic, however, are the differences in surface topography between porcine lipid extract (Curosurf1) films containing SP-B and SP-C on the one hand, and bovine lipid extract surfactant films with different concentrations of SP-B and SP-C.…”

Section: Discussionmentioning

confidence: 56%

Structures of surfactant films: a scanning force microscopy study

Grunder¹,

Gehr²,

Bachofen³

et al. 1999

Eur Respir J

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The alveolar lining layer is thought to consist of a continuous duplex layer, i.e., an aqueous hypophase covered by a thin surfactant film which is a monolayer with dipalmitoyl-phosphatidylcholine (DPPC) as its most important component. Findings obtained by electron microscopy and results from in vitro experiments suggest, however, that the structure and hence the structure-function relations of surfactant films are more complex. In order to better define their structures films of surfactants were studied by scanning force microscopy.Four different surfactants were spread on a Langmuir-Wilhelmy balance, and then transferred onto a solid mica plate by the Langmuir-Blodgett technique, under various states of film compression. Imaging of the films by scanning force microscopy was performed in the contact (repulsive) mode in air.The scanning force micrographs revealed that surfactant films are not homogeneous, but rather undergo phase transitions depending on the surface pressures. Even at comparable surface pressures different surfactants show quite different surface patterns. Differences in surface structure can even be observed in films containing surfactant proteins (SP)-B and SP-C.These observations give further evidence that the widely accepted hypothesis of a regular monolayer of phospholipids governing the surface tension probably does not hold true, but that the structure-function relationship of surface active surfactant films is even more complex than hitherto thought. Eur Respir J 1999; 14: 1290±1296. According to a widely accepted hypothesis the alveolar lining layer has the following structural properties: it is a continuous duplex layer consisting of an aqueous hypophase covered by a thin surfactant film, and the surfactant film is a monolayer with dipalmitoyl-phosphatidylcholine (DPPC) as its most important component [1±4].There are numerous studies of the structure of the lining layer, all of which are fragmentary. Indeed, the sample preparation and preservation of this delicate structure for microscopy is extremely difficult; there is no standard to distinguish between facts and artefacts [5], and it is not easy to conceive of how monolayers of saturated phospholipids can be preserved and visualized by the usual fixatives [6]. As to the first condition, i.e., the existence and continuity of a duplex layer, GIL and WEIBEL [7] were the first to convincingly demonstrate a two-phase lining layer by transmission electron microscopy. However, the film was often fragmented and disposed in patches, and could not be detected on flat parts of the alveolar walls. Although studies of freeze-fractured preparations suggested its continuity [8], the exact structure of the lining layer has remained a matter of debate. More recently, BAS-TACKY et al. [9] have clearly demonstrated the continuity of the lining layer by low-temperature electron microscopy, and improved tissue preparations have also resulted in a better visualization of the film such that all of the evidence now available suggests a continuum...

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Section: Discussionmentioning

confidence: 56%

Structures of surfactant films: a scanning force microscopy study

Grunder¹,

Gehr²,

Bachofen³

et al. 1999

Eur Respir J

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“…Derivatization of amine groups in SP-C requires treatment of the protein at mild alkaline pH, to ensure deprotonation and reactivity of the amine functions (21)(22)(23)(24). On the other hand, incubation of the protein at basic pH may produce cleavage of the labile thioesther bonds that attach palmitic chains to the two cysteines in the N-terminal segment of the protein (2, 13).…”

Section: Resultsmentioning

confidence: 99%

“…In the past we used amine-reactive reagents to introduce different groups in SP-C, such as visible-emission fluorescent (22,23) or spin (24) probes. Derivatization of amines is critically dependent on pH as they are good nucleophiles only in their nonprotonated state.…”

Section: Discussionmentioning

confidence: 99%

“…We have labeled amines in SP-B and SP-C by adding traces of buffer to the protein solution in chloroform/methanol, so allowing a certain control of apparent pH (22)(23)(24). However, alkaline pH can produce cleavage of the labile thioesther bonds which maintain the two cysteines of SP-C palmitoylated (2,22) and, in fact, we could not avoid total or partial depalmitoylation of the protein in previous studies. A proper characterization of the structure and lipid-protein interactions of the N-terminal segment of SP-C requires maintaining the protein in the palmitoylated form.…”

Section: Discussionmentioning

confidence: 99%

“…An alternative is the site-selective introduction of an extrinsic fluorescent probe in SP-C, which may allow identification of structural features that could be directly assigned to local regions in the protein (21). Characterization of lipid-protein interactions of SP-C by other techniques such as epifluorescence microscopy (22,23) or electron spin resonance (24) has also required introduction of extrinsic probes in proper locations of the protein, without producing significant alterations of the native conformation. However, the procedure to derivatize SP-C in organic solutions has not been optimized, specially to ensure preservation of the palmitic chains bound to the structure of SP-C via thioesther linkages, and also to produce incorporation of single groups in selective sites.…”

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confidence: 99%

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