Richard J. King

Aspects of secondary and quaternary structure of surfactant protein A from canine lung

The results of a large number of studies indicate that pulmonary surfactant contains a unique protein whose principal isoform has a molecular weight of about 30,000, and whose presence in surfactant is associated with important metabolic and physicochemical properties. This protein, SP-A, as isolated from canine surfactant, contains a domain of 24 repeating triplets of Gly-X-Y, similar to that found in collagens. These studies were undertaken to determine whether SP-A forms a collagen-like triple helix when in solution, and to describe certain aspects of its size and shape. Our experiments were done on SP-A extracted by two different methods from canine surfactant, and on SP-A produced by molecular cloning. The results from all three preparations were similar. The circular dichroism of the complete protein was characterized by a relatively large negative ellipticity at 205 nm, with a negative shoulder ranging from 215 to 230 nm. There was no positive ellipticity, and the spectrum was not characteristic of collagen. Trypsin hydrolysis resulted in a fragment with peak negative ellipticity at about 200 nm, without the negative shoulder. Further hydrolysis of this fragment with pepsin resulted in a CD spectrum similar to that of collagen. The spectrum of the collagen-like fragment was reversibly sensitive to heating to 50 degrees C, and was irreversibly lost after treatment with bacterial collagenase. SP-A migrated on molecular sieving gels with an equivalent Stokes radius of 110 to 120 A, and had a sedimentation coefficient of 14 S. Using these data we calculate a molecular weight of about 700,000. The hydrodynamic characteristics can be approximated as a prolate ellipsoid of revolution having an axial ratio of about 20. We conclude that SP-A aggregates into a complex of 18 monomers, which may form six triple-helices. The shape of the complex is considerably more globular than collagen and is not consistent with end-to-end binding of the helices to form fibrous structures.

Lipid-Apolipoprotein Interactions in Surfactant Studied by Reassembly

Recent evidence suggests that the structure of tubular myelin, an extracellular form of pulmonary surfactant, is dependent on the interaction of lipids with certain proteins specific for this material and with calcium ions. In order to investigate how protein and calcium may affect the surfactant complex, we studied the composition and properties of reassembly materials formed with a major surfactant apolipoprotein (35,000-38,000 molecular weight) and the principal lipids found in the natural material. We were interested in three questions: 1) Does this apolipoprotein preferentially associate with certain of the lipids in surfactant? 2) What forces are involved in the binding? 3) Does the interaction result in changes in the physical state of the lipid? We found that this apolipoprotein binds phosphatidylcholines that are in a gel phase with much greater affinity than it does phosphatidylcholines that are liquid-crystalline. However, maximum binding does not occur with the pure phosphatidylcholines but rather with mixtures of phosphatidylcholines and 15% phosphatidylglycerol. Calcium ions have no effect on the amount of apolipoprotein that is bound, but they do modify the physical state of the reassembly lipoprotein and the stoichiometry of lipid to protein. These results indicate that the binding of the apolipoprotein to the lipid does not primarily involve ionic bonds. However, apolar interactions, which are influenced by the state of the lamellar phospholipid, appear to be important. Small amounts of phosphatidylglycerol and other glycolipids, which probably disrupt the regularity of a gel-phase lamellar structure when mixed with saturated phosphatidylcholines, may provide binding sites favoring the interaction. Indirect evidence, based on thermodynamic analyses, suggests that phosphatidylcholines may be partially immobilized about the protein in the formation of the complex. This conclusions is reinforced by the preliminary findings obtained from the differential scanning calorimetry of the reassembly materials.

Interaction between the 35 kDa apolipoprotein of pulmonary surfactant and saturated phosphatidylcholines. Effects of temperature.

We studied the interaction between the 35 kDa apolipoprotein of canine pulmonary surfactant (SP 35) and five saturated phosphatidylcholines: distearoyl (DSPC), diheptadecanoyl (DHPC), dipalmitoyl (DPPC), dimyristoyl (DMPC), and dilauroyl (DLPC); and two monoenoic unsaturated phosphatidylcholines: dioleoyl (DOPC) and dielaidyl (DEPC), using temperatures at which all of the phospholipids except DOPC were in both the gel and liquid-crystalline states. The experiments were carried out in a buffer without Ca2+. The amount of apolipoprotein which was bound by both small unilamellar and multilayered vesicles of these lipids decreased as the temperature was increased. Moreover, near the temperatures of the phase transitions of all lipids except DLPC, there was an abrupt and marked reduction in binding of protein, in that over a 3-4 degree change in temperature there was an abrupt decrease in bound apolipoprotein. A similar change in binding occurred using DLPC, although the relatively large changes in bound protein occurred at about 10 and 20 degrees C, temperatures which are above the phase transition temperature of this lipid. Experiments using DOPC were limited to temperatures above the phase transition, and apolipoprotein binding was low. Experiments monitoring the intrinsic fluorescence of the protein, and the fluorescence of bis-1-anilino-8-naphthalene sulfonic acid bound to the protein, revealed a possible conformational change at about 40 degrees C. Measurement of intrinsic fluorescence provided the same result whether or not the protein was associated with lipid. DSC of the apolipoprotein indicated that this change was not associated with a measurable thermogenic process. We found that the interaction with DPPC was reversible at 42 degrees C, and we measured the thermodynamic parameters of the interaction at this temperature. These were: delta G0 = -8.0 kcal/mol apolipoprotein; delta H0 = -88 kcal/mol; delta S0 = -254 cal/Cdeg per mol. We conclude that the interaction between SP 35 and saturated phosphatidylcholines is temperature sensitive, and this probably reflects differences in the ability of gel and liquid-crystalline phospholipids to bind this protein. Both the delta H0 and delta S0 of the interaction are negative, and may reflect an immobilization of phospholipid around the apolipoprotein to form a boundary layer. This hypothesis is consistent with the findings obtained by DSC, in which the enthalpy of the phase transition of DMPC in lipid-apolipoprotein recombinants was found to be about 60% of that expected for a pure and unperturbed multilamellar dispersion.

Metabolism of platelet-activating factor (alkylacetylphosphocholine) by type-II epithelial cells and fibroblasts from rat lungs.

The uptake and metabolism of 3H-labeled platelet-activating factor by interstitial and epithelial cells from rat lungs was investigated. The uptake of 1-O-[3H]octadecyl-2-acetyl-sn-glycero-3-phosphocholine (3H-AGEPC) by alveolar type-II cells was linear with time from 5 to 60 min, with an average rate of 660 and 450 fmol/10(6) cells for cells in primary culture for 48 to 72 h, respectively. AGEPC was rapidly metabolized and by 10 min 60% of AGEPC was converted into long-chain acylphosphatidylcholine (PC) (50%) and 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (lyso-GEPC) (10%). By 60 min radioactivity in AGEPC was less than 10% of the total intracellular activity. Lyso-GEPC remained at about 10% throughout the incubation period. The uptake of 3H-AGEPC by fibroblasts was very similar to type II cells, but the rate of metabolism was slower. AGEPC in fibroblasts constituted 85% of the cellular counts after 10 min of incubation, and 50% by 60 min. After 60 min only 30% of the AGEPC was converted to alkylacyl-PC. Characterization of the fatty acids in the alkylacyl-PC of both the type-II cells and lung fibroblasts indicated that arachidonic acid was preferentially (more than 90%) inserted at the 2-position.