Shou-Hwa Yu

Role of bovine pulmonary surfactant-associated proteins in the surface-active property of phospholipid mixtures.

The surfactant-associated proteins, SP-A, SP-B and SP-C have been isolated from bovine pulmonary surfactant. The biophysical roles of SP-B and SP-C in reconstituted surfactants, with various phospholipid mixtures subjected to different thermal treatments, have been examined using a pulsating bubble surfactometer. The phospholipid mixtures were: (A) dipalmitoylphosphatidylcholine (DPPC)/egg phosphatidylcholine (PC)/egg phosphatidylglycerol (PG) (6:2:2, w/w); (B) DPPC/PG (9:1); and (C) DPPC/PG (7:3). Thermal treatments involved mixing SP-B or SP-C, at room temperature, with lipids in chloroform/methanol (9:1, v/v) and removing the solvent under N2 by (1) evaporation at room temperature; (2) evaporation at 45 degrees C; or (3) incubation at 45 degrees C overnight prior to evaporation at 45 degrees C. In all cases, 45 degrees C solvent evaporation was the most effective treatment. DPPC/egg PG (7:3) was the most favourable lipid composition. With either a static or a pulsating bubble, SP-C promoted a rapid decrease in surface tension with little change thereafter. This implies that SP-C is effective in enhancing phospholipid adsorption but does not play an important role in the removal of non-DPPC lipid from the monolayer. While SP-B was not as effective in facilitating phospholipid absorption, samples containing this protein could achieve near zero surface tension upon pulsation. A very low surface tension could also be attained during the initial pulsation of DPPC/PG plus SP-B mixtures which had been allowed to adsorb until equilibrium. This observation indicates that SP-B promotes the removal of PG from the monolayer. SP-A alone had only a slight effect on the surface activity of the DPPC/PG (7:3) mixture, and did not accelerate adsorption of samples containing SP-C. However, SP-A facilitated phospholipid adsorption and may also enhance the removal of PG from monolayers in the presence of SP-B.

Effect of pulmonary surfactant protein B (SP-B) and calcium on phospholipid adsorption and squeeze-out of phosphatidylglycerol from binary phospholipid monolayers containing dipalmitoylphosphatidylcholine.

The pulsating bubble technique was used to study the surface activity of binary phospholipid mixtures containing dipalmitoyl-phosphatidylcholine (DPPC) and an unsaturated acidic phospholipid such as egg phosphatidylglycerol (egg PG), 1-palmitoyl-2-oleoyl-PG (POPG) or egg phosphatidic acid (egg PA) in the presence of surfactant-associated protein B (SP-B) and calcium. The relative surface activities were DPPC/egg PG/SP-B (7:3:1%) greater than DPPC/POPG/SP-B (7:3:1%) greater than DPPC/egg PA/SP-B (7:3:1%). The Wilhelmy surface plate technique was utilized to investigate the interaction between pure SP-B in the bulk phase (0.9% NaCl/1.5 mM CaCl2) and preformed DPPC or phosphatidylglycerol (PG) monolayers. Although SP-B injected into the bulk phase reduces the surface tension of a clean surface, no evidence was obtained for the insertion of SP-B into surface monolayers at equilibrium surface tension. Surface radioactivity measurements and the Wilhelmy surface plate technique were also used to study the potential interactions between liposomes of DPPC/POPG (7:3) with or without SP-B and surface monolayers of [14C]DPPC or [14C]POPG. No exchange of phosphatidylcholine (PC) or PG was found between the monolayer and liposomes. We also compared the adsorption of pure POPG or 1-palmitoyl-2-oleoyl- phosphatidylcholine (POPC) and binary mixed liposomes with DPPC in the presence or absence of SP-B and calcium. DPPC/POPG/SP-B (7:3:1%) was found to be more surface active than pure POPG plus 1% SP-B in the presence of calcium. Injection of SP-B into the bulk phase promoted the adsorption of DPPC/POPG liposomes to a greater extent than POPG liposomes. The enhanced adsorption was dependent on the presence of calcium. In contrast to PG, DPPC/POPC/SP-B (7:3:1%) was less surface active than pure POPC plus 1% SP-B either in the presence or absence of calcium. Our findings suggested that the molecular composition and organization of mixed monolayers play an important role in the surface activity of the surfactant.

Effect of surfactant-associated protein-A (SP-A) on the activity of lipid extract surfactant.

The properties of natural bovine surfactant and its lipid extract have been examined with a pulsating bubble surfactometer which assesses the ability of surfactant lipids to adsorb to the air/liquid interface and reduce the surface tension to near 0 dynes/cm during dynamic compression. Studies conducted at 1 mg/ml phospholipid revealed that the surface activity (i.e., the ability to produce low surface tensions) of lipid extracts could be enhanced by incubating the sample at 37 degrees C for 120 min or by addition of CaCl2. In contrast, incubation at 37 degrees C only slightly improved the biophysical activity of natural surfactant and the addition of CaCl2 had a more modest effect than with lipid extracts. With 20 mM CaCl2, the surfactant activity of lipid extract surfactant was similar to that of natural surfactant. Incubation with EDTA reduced the biophysical activity of natural surfactant. Experiments in which increasing amounts of lipid extract were replaced by natural surfactant revealed that small amounts of natural surfactant enhanced the surfactant activity of lipid extract. The biophysical activity of lipid extract surfactant was also increased by the addition of soluble surfactant-associated protein-A (SP-A) (28-36 kDa) purified from natural bovine surfactant. These results indicate that SP-A (28-36 kDa) improves the surfactant activity of lipid extracts by enhancing the rate of adsorption and/or spreading of phospholipid at the air/liquid interface resulting in the formation of a stable lipid monolayer at lower bulk concentrations of either phospholipid or calcium.

Adsorption, compression and stability of surface films from natural, lipid extract and reconstituted pulmonary surfactants

A pulsating bubble surfactometer was used to study the surface activities and surface film stabilities of bovine pulmonary surfactants (10 mg/ml) and a reconstituted surfactant (10 mg/ml). Pulmonary surfactants were natural surfactant (NS), lipid extract surfactant [LES(chol)] and lipid extract surfactant without neutral lipids (LES). NS is composed of phospholipids, neutral lipids and surfactant-associated proteins (SP-A, SP-B and SP-C). Both LES(chol) and LES are organic solvent extracts of NS. LES(chol) retains all the components of NS except SP-A. Reconstituted surfactant was dipalmitoylphosphatidylcholine (DPPC): 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG): SP-B/7:3:1%. All three pulmonary surfactants attained the equilibrium surface tension almost instantaneously at 37 degrees C. The adsorption rates of NS and LES(chol) at 24 degrees C were similar to those at 37 degrees C, while LES exhibited a lower adsorption rate at 24 degrees C. Reconstituted surfactant adsorbed slower than any of the pulmonary surfactants. Film stability was studied by recording the spontaneous increase in the pressure gradient of a static bubble at the minimum size (Rmin) once near zero surface tension was attained. The order of surface film stabilities were: reconstituted surfactant > > NS > LES > LES(chol). Surface films of NS and LES could be stabilized by prolonged pulsation, while film stability of LES(chol) was only moderately affected by pulsation. These results indicate that SP-A in NS promotes formation of some unique structure, possibly tubular myelin, which induces selective adsorption of lipids into the surface.

Effect of pulmonary surfactant protein A (SP-A) and calcium on the adsorption of cholesterol and film stability

The effect of exogenous cholesterol on the stability of surface films at 37 degrees C from various surfactants was studied with the pulsating bubble surfactometer. Addition of cholesterol (5%, w/w) to bovine lipid extract surfactant (bLES) or mixtures of dipalmitoylphosphatidylcholine/1-palmitoyl-2-oleoyl-phosphatidylglycerol /SP-B (7:3:1%) dispersed in 1.5 mM CaCl2/0.9% NaCl resulted in unstable surface films. Although 10% cholesterol only partially impaired the surface activity of bLES, it virtually abolished that of the reconstituted surfactant. The inhibitory effects of cholesterol were significantly repressed by SP-A (10%, w/w of lipid) and 3 mM CaCl2 or 5 mM CaCl2 without SP-A. Adsorption of cholesterol from various surfactants into the air/water interface was examined by measuring the surface radioactivity of [14C]cholesterol. Cholesterol alone dispersed in 1.5 mM CaCl2/0.9% NaCl could not adsorb to the interface, but it adsorbed readily when mixed with bLES. Cholesterol adsorption was markedly suppressed by SP-A in 3 mM CaCl2/0.9% NaCl or 5 mM CaCl2/0.9% NaCl without SP-A. Electron microscopy revealed striking ultrastructural differences between bLES/5% cholesterol/10% SP-A in 3 mM CaCl2/0.9% NaCl and bLES/5% cholesterol in 3 or 5 mM CaCl2/0.9% NaCl. The former exhibited large multilayer and small unilamellar vesicles, while the latter displayed condensed patches of aggregates. Adsorption studies showed aggregated patches adsorbed more rapidly than vesicles but attained lower equilibrium surface pressures. These results indicate SP-A and calcium limit the adsorption of surfactant cholesterol to the air-water interface.

Artificial pulmonary surfactant: Potential role for hexagonal HII phase in the formation of a surface-active monolayer

Natural surfactant possesses the ability to rapidly reduce the surface tension of a bubble pulsating at 20 cycles per min at 37°C to less than 30 dyn/cm at maximum radius and to less than 1 dyn/cm at minimum radius. The preparation of two artificial surfactant systems, containing either dipalmitoylphosphatidylcholine (DPPC) and phosphatidylethanolamine (PE) (5:5), or DPPC plus PE and phosphatidylglycerol (5:4:1 or 6:3:1), is described. Formation of artificial surfactants which mimic the essential properties of natural surfactant was correlated with the appearance of particles of aggregated lipids. The effects of lipid composition, calcium ion concentration, pH, temperature and mechanical agitation were determined. It is proposed that these artificial surfactant systems may produce a surface-active monolayer through the involvement of nonbilayer structures with properties similar to hexagonal HII phase.

Structural relationship between the two small hydrophobic apoproteins in bovine pulmonary surfactant

Lipid extracts of bovine pulmonary surfactant contain two very hydrophobic surfactant-associated proteins (SP) designated SP-B (15 kDa nonreduced) and SP-C (3.5 kDa). These two low molecular weight apoproteins were delipidated and purified on silica SEP-PAK cartridges using various reagents. Dansylation studies revealed that the 15 kDa apoprotein has three N-termini: Phe, Leu and Ile, while the 3.5 kDa apoprotein has two N-termini: Leu and Ile. In either protein, only a very small amount of N-Ile is present. Quantitative N-terminal dansylation analysis of the 15 kDa protein indicated that Phe and Leu (plus Ile) are present in a 1:1 ratio. Carboxy-terminal analysis showed that the 15 kDa protein contains C-terminal Gly, and the 3.5 kDa protein contains C-terminal Leu. Gas-phase amino terminal sequencing of the 15 kDa protein revealed almost exclusively the Phe-polypeptide (SP-B). These results suggest that the 15 kDa apoprotein is not an oligomer of SP-B and SP-C. The reason that analysis of SP-B reveals N-terminal Leu and Ile by dansylation which cannot be confirmed by amino acid sequencing is not known.

Isolation and further characterization of the anti-encephalitogenic rat spinal cord protein from brain, spinal cord and peripheral nerves.

Abstract After fresh rat nervous tissue was delipidated with acetone, all the immunoreactive rat spinal cord protein (RSCP) in brain (RB-SCP), spinal cord (RSCP) and peripheral nerve (RSCP-PN) could be extracted with 0.05 M sodium acetate buffer, pH 5.0. The extracts were purified by absorption on CM-52 cellulose at pH 5.0 and by gel filtration on Sephadex G-50. At this stage, RSCP-PN formed one band when analyzed by SDS-polyacrylamide gel electrophoresis. In contrast, preparations of RB-SCP and RSCP contained two components of similar molecular size. Only the larger component was present in the specific precipitates formed by mixing anti-RSCP antibody with RB-SCP or RSCP, clearly indicating that the smaller component was a contaminant. Thus, the final purification step for RB-SCP and RSCP consisted of absorption on cyanogen bromide-activated Sepharose-4B coupled to anti-RSCP antibody followed by elution of immunoreactive RSCP at pH 2.5. RB-SCP, RSCP and RSCP-PN isolated from delipidated nervous tissue were identical by immunodiffusion analysis. RSCPs have a molecular weight of 11 000 ± 500, as estimated by gel exclusion chromatography and by SDS-polyacrylamide gel electrophoresis. They have the electrophoretic mobility in agarose at pH 8.6 of a serum β-globulin and an isoelectric point of pH 4.5. RB-SCP and RSCP contain respectively about 17, 24 and 42% of basic, acidic and hydrophobic amino acids. RSCP-PN has a somewhat higher content of acidic amino acids, (31%) and a lower proportion of hydrophobic amino acids (32%). All RSCPs lack cystine and contain only 0.2% of tyrosine. Immunodiffusion analyses showed that at the antibody level there is no immunochemical relation between bovine SCP (BSCP) and purified RB-SCP, RSCP or RSCP-PN. However, there is a protein in rat peripheral nerve that is similar but not identical to BSCP. This protein is not present in extracts of brain or in extracts of spinal cord from which the spinal nerves have been removed.

Pulmonary surfactant-associated proteins: their role in surface tension reduction

It is generally, albeit not universally [1,2], accepted that pulmonary surfactant stabilizes the lung by lowering the surface tension at the air-water interface of the alveoli. The requirement for pulmonary surfactant is particularly critical at birth when the newborn infant must clear its lungs of fetal pulmonary fluid and establish regular air breathing. Indeed, as outlined in detail in other chapters of this book, treatment of prematurely delivered infants with extracts of bovine or porcine surfactant leads to a significant reduction in the incidence and in the intensity of the respiratory distress syndrome of the neonate [3–5].