Is dipalmitoylphosphatidylcholine a substrate for convertase?

Rajiv Dhand1, Jared Young1, Andelle Teng1, Subbiah Krishnasamy1,2, and Nicholas J. Gross1,2

1 Division of Pulmonary and Critical Care Medicine and 2 Cellular and Molecular Biochemistry, Edward Hines Jr. Veterans Affairs Hospital and Loyola University of Chicago Stritch School of Medicine, Hines, Illinois 60141


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Convertase has homology with carboxylesterases, but its substrate(s) is not known. Accordingly, we determined whether dipalmitoylphosphatidylcholine (DPPC), the major phospholipid in surfactant, was a substrate for convertase. We measured [3H]choline release during cycling of the heavy subtype containing [3H]choline-labeled DPPC with convertase, phospholipases A2, B, C, and D, liver esterase, and elastase. Cycling with liver esterase or peanut or cabbage phospholipase D produced the characteristic profile of heavy and light peaks observed on cycling with convertase. In contrast, phospholipases A2, B, and C and yeast phospholipase D produced a broad band of radioactivity across the gradient without distinct peaks. [3H]choline was released when natural surfactant containing [3H]choline-labeled DPPC was cycled with yeast phospholipase D but not with convertase or peanut and cabbage phospholipases D. Similarly, yeast phospholipase D hydrolyzed [3H]choline from [3H]choline-labeled DPPC after incubation in vitro, whereas convertase, liver esterase, or peanut and cabbage phospholipases D did not. Thus convertase, liver esterase, and plant phospholipases D did not hydrolyze choline from DPPC either on cycling or during incubation with enzyme in vitro. In conclusion, conversion of heavy to light subtype of surfactant by convertase may require a phospholipase D type hydrolysis of phospholipids, but the substrate in this reaction is not DPPC.

pulmonary surfactant; surfactant metabolism; surface area cycling; phospholipase D; carboxylesterase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CYCLIC EXPANSION and contraction of the surface (cycling) of heavy buoyant density surfactant in vitro lead to generation of a lighter buoyant density material (light subtype) (12, 26, 31). The composition, morphology, and biophysical activity of the heavy and light subtypes generated by cycling correspond to two major subtypes of surfactant recovered from lung lavage, namely tubular myelin (TM) and small vesicles (SV) (11, 12, 26, 31). A partially purified preparation of convertase promotes conversion of heavy to light subtype on cycling either natural or reconstituted surfactant (6, 10, 19). Although the cycling assay is tedious and semiquantitative, it is the only method to assess convertase activity.

Convertase is a carboxylesterase found in mouse or rat alveolar lavage (2, 3, 13, 14, 19, 20). Recently, convertase activity has also been shown in purified lamellar bodies prepared from the lungs of rabbits (23). The substrate for its action has not been identified, but phospholipids in surfactant are likely candidates. Dipalmitoylphosphatidylcholine (DPPC), the predominant phospholipid in surfactant, plays an important role in reducing surface tension within alveoli (4). We reasoned that DPPC could be a substrate for convertase because the ability to lower surface tension is lost during conversion of the heavy subtype to the light subtype (11, 26, 31). Moreover, the phospholipid molecule is susceptible to hydrolysis by phospholipases at several sites (32). Therefore, we used several esterases and lipases in the cycling assay to determine whether hydrolysis at a specific site is responsible for heavy to light subtype conversion. In previous experiments, we found that plant phospholipases D generated light subtype from heavy subtype on cycling in a manner similar to that observed with convertase (6, 19). Because phospholipase D hydrolyzes the ester link between the base and phosphatidic acid in phospholipids, we measured [3H]choline release to determine whether [3H]choline-labeled DPPC was hydrolyzed during conversion of heavy to light subtype on cycling with convertase. Our findings suggest that DPPC is not hydrolyzed during conversion of heavy to light subtype during cycling with convertase and therefore is unlikely to be a substrate for convertase.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Isotopes were obtained from NEN Life Science Products (Boston, MA) or Amersham Life Science Products (Arlington Heights, IL), reagents from Sigma (St. Louis, MO) or Fisher Chemicals (Itasca, IL), and phospholipids from Avanti Polar Lipids (Alabaster, AL). The synthetic DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) used was 99% pure. All lipids were obtained in chloroform.

Preparation of natural heavy substrate. Mice were injected with 10 µCi of [3H]choline ~18 h before they were killed. Degassed mouse lungs were washed three times with alveolar lavage (AF) buffer (0.15 M NaCl, 5 mM HEPES, 1 mM MgCl2, and 2 mM CaCl2, pH 7.4). The subsequent preparation of surfactant followed the method described previously (14). The phospholipid content of the surfactant was measured (12), and aliquots were stored at -20°C. This surfactant was used as the heavy substrate for the cycling assay.

Preparation of natural surfactant containing [3H]choline- and [14C]palmitate-labeled DPPC. To increase the specific activity of radiolabel in the cycling mixture, we added [1-palmitoyl-14C]DPPC (25 µCi/ml) and [N-methyl-3H]choline DPPC (1 µCi/µl) to nonradiolabeled mouse natural surfactant to a final concentration of 150 dpm/µg phospholipid for each isotope. The surfactant was dried under nitrogen in glass tubes, resuspended in AF buffer by gentle sonication (Sonicator XL2015, Heat Systems, Farmingdale, NY), and frozen in aliquots at -20°C. Immediately before use, the surfactant was homogenized with two strokes in a Potter-type homogenizer. In preliminary experiments (n = 3), we determined that the buoyant density and cycling behavior of this surfactant were similar to those of heavy substrate.

Preparation of convertase. Partially purified convertase was obtained by subjecting mouse or rat alveolar lavage to affinity chromatography with concanavalin A (Con A)-sepharose (6). The yield from the Con A column was then purified by ion-exchange chromatography on a DEAE-sepharose column (27). The active fractions were pooled and concentrated in a Centricon concentrator (Amicon, Beverly, MA) with a 30-kDa molecular-mass cutoff. Proteins from these fractions were separated by 7.5% nondenaturing gel electrophoresis, and convertase was identified by its ability to hydrolyze alpha -naphthyl acetate, a nonspecific substrate for carboxylesterase (19). The eluted band corresponding to convertase in the nondenaturing gel showed one major and three minor bands by SDS-PAGE (Fig. 1). The majority of cycling experiments were performed with the yield from the Con A column as the source of convertase, and in a few experiments, the eluted protein from the nonreducing gel was used to confirm the results.


View larger version (39K):
[in this window]
[in a new window]
 
Fig. 1.   SDS-PAGE of purified mouse convertase. Mouse alveolar lavage was purified by concanavalin A affinity chromatography, concentrated in a Centricon filter with a cutoff at a molecular mass of 30 kDa, and electrophoresed on 7.5% nondenaturing gels. The protein that bound [3H]diisopropylfluorophosphate and possessed esterase activity was eluted from the nondenaturing gel and analyzed by SDS-PAGE (lane 1). Lane 2 shows molecular-mass markers (nos. on right). The 72-kDa protein corresponds to convertase, whereas the 3 minor bands probably represent additional proteins. Other preparations that included an additional step involving ion-exchange chromatography using DEAE-sepharose yielded a similar level of purity.

Other enzymes. Phospholipases A2, B, C, and D (peanut, cabbage, and yeast); porcine liver esterase; and elastase were used. The commercially available porcine liver esterase was purified by electroelution on 7.5% nondenaturing gels (19). Nondenaturing gel (7.5%) electrophoresis of the commercial preparations of yeast phospholipase D revealed two bands. Both bands of yeast phospholipase D were electroeluted from the gels, and each of them hydrolyzed choline from DPPC in vitro (see In vitro assays). The peanut phospholipase D was chromatographically pure.

Cycling. [3H]choline-labeled heavy substrate was incubated with cold diisopropyl fluorophosphate (DFP, 10 mM) at 37°C for 1 h to inhibit endogenous convertase. After centrifugation at 14,000 rpm in a microcentrifuge for 30 min, the pellet was resuspended in AF buffer. For cycling, heavy substrate containing ~100 µg of phospholipid was used in each tube. The purpose of these experiments was to determine the proportion of heavy surfactant that converted to light subtype under various cycling conditions. Cycling was performed in 12 × 75-mm polypropylene tubes (Falcon, Oxnard, CA) for 4 h at 37°C as described earlier (6, 12, 14).

Cycling with various enzymes. An uncycled tube and two tubes that were cycled without enzyme were used as controls. The remaining three tubes were cycled with the enzyme preparation to be tested. Experiments were conducted with ~15 µg/ml of convertase protein (Con A yield) or with 5 U/ml of the following enzymes in the cycling mix: phospholipase A2, phospholipase B, phospholipase C, phospholipase D (peanut), phospholipase D (cabbage), phospholipase D (yeast), porcine liver esterase, and elastase. Three to eight cycling experiments were performed with each enzyme. In some experiments, Orlistat, an inhibitor of mammalian lipases, was added to the cycling mix. In addition, to determine whether the characteristic profile of cycling could be reproduced with very low concentrations of yeast phospholipase D, we repeated the experiments with 0.025, 0.05, 0.1, 0.5, and 1 U/ml of the enzyme in the cycling mixture (n = 3 for each concentration).

In vitro phospholipase D activity. A stock solution of 10 mM DPPC was prepared by mixing [3H]choline-labeled DPPC and cold DPPC to a final radioactivity of 2,000 dpm/nmol, drying the phospholipids in a Savant Speed-Vac concentrator (Savant Instruments, Farmingdale, NY), and resuspending them in 0.05% Triton X-100 by gentle sonication. Phospholipase D activity was assessed by incubation of this substrate with various enzymes and by measuring the amount of [3H]choline released. Various amounts of convertase (5-50 µg of protein), porcine esterase (1-10 units), or phospholipase D (0.05-25 units) were incubated for 40 min with a 1 mM concentration of [3H]choline-labeled DPPC as a substrate in a reaction mixture containing 50 mM sodium dimethylglutarate, pH 6.6, 2 mM sodium oleate, and 2 mM MgCl2 (22). The labeled choline released was extracted by a two-phase separation (22), the aqueous phase was collected, and its radioactivity was quantitated by scintillation counting. The assay was repeated at a range of pH (6.6-7.4) with the dimethylglutarate buffer and with AF buffer. The experiments were also performed with natural heavy surfactant containing [3H]choline- and [14C]palmitate-labeled DPPC.

Because the plant phospholipases D (peanut and cabbage) did not hydrolyze choline in the assay described, we wished to determine whether the enzymes possessed phospholipase D activity. Because phospholipases D are known to have different interfacial requirements for their action, we performed another assay for phospholipase D activity using a modification of the method described by Artiss and colleagues (1). For each test, we took 100 µl of a test mixture containing 96 mM sodium acetate, pH 5.6, 2.4 mM SDS, and 1 mM DPPC mixture (prepared as in the previous assay) and added ethanol to 0.08% (final concentration) with mixing. CaCl2 was added to a final concentration of 50 mM, and the mixture was shaken and equilibrated to 30°C. The samples were transferred to reaction tubes, and the phospholipases D (yeast, cabbage, and peanut) were added. The mixture was incubated for 40 min at 30°C with shaking. The further steps of extraction and determination of choline release were similar to those previously described (22).

Determination of choline release during cycling. We cycled [3H]choline-labeled natural heavy substrate and natural surfactant containing [3H]choline- and [14C]palmitate-labeled DPPC with convertase, peanut phospholipase D, and yeast phospholipase D. After cycling, half of the sample was analyzed for buoyant density. The profiles of radioactivity (14C and 3H) in the heavy and light peaks were plotted. A two-step extraction procedure similar to that previously described (22) was performed on the other half of the cycling mixture to determine the content of free [3H]choline released.

Data analysis. The radioactivity in each fraction was plotted against its buoyant density using an Excel spreadsheet (Microsoft, Redmond, WA). The amount of [3H]choline released (dpm) into the aqueous phase was determined by subtracting the value obtained in parallel controls (no enzyme) from the value obtained with each enzyme. Means ± SD were calculated. Differences in the amount of [3H]choline release were determined by Student's t-test. Statistical significance was established with P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Profiles of light-subtype generation on cycling natural surfactant. Figure 2 shows the profiles of 3H radioactivity on cycling natural surfactant containing [3H]choline-labeled DPPC. Uncycled surfactant had a buoyant density corresponding to heavy subtype (mean 1.057 g/ml, Fig. 2A). Minimal light subtype was generated when this substrate was cycled in the absence of convertase as shown in Fig. 2B. A similar profile was observed on cycling with elastase (5 U/ml) in the cycling mixture. Typical peaks of heavy and light subtype were observed after cycling with convertase (15 µg protein/ml), liver esterase, and peanut and cabbage phospholipases D (5 U/ml of each enzyme). A representative profile on cycling natural surfactant with peanut phospholipase D is shown in Fig. 2C. In contrast, distinct heavy and light peaks were not observed after cycling with phospholipases A2, B, and C and yeast phospholipase D (5 U/ml of each). Instead, there was a broad band of radioactivity across the gradient. A representative profile on cycling natural surfactant with yeast phospholipase D is shown in Fig. 2D. A similar profile was seen with lower concentrations (as low as 0.05 U/ml) of yeast phospholipase D in the cycling mixture.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 2.   Representative profiles after cycling natural heavy substrate containing [3H]choline-labeled dipalmitoylphosphatidylcholine with various enzymes (n = >= 6 experiments). A: uncycled surfactants (control). B: surfactant cycled without enzyme or with elastase (5 U/ml). C: cycling with peanut phospholipase D (5 U/ml). D: cycling with yeast phospholipase D (5 U/ml). Cycling with convertase (15 µg protein/ml), liver esterase, and peanut or cabbage phospholipases D (5 U/ml of each) gave profiles similar to C. In contrast, cycling with phospholipases A2, B, C, and D from yeast (5 U/ml of each) produced profiles similar to that shown in D. Cycling with yeast phospholipase D in concentrations as low as 0.05 U/ml also produced the profile shown in D. H, heavy subtype; L, light subtype.

Similar data were obtained by using [3H]choline- and [14C]palmitate-labeled DPPC in the cycling assay (data not shown). With the concentrations of the enzymes previously indicated, distinct heavy and light peaks were observed after cycling with convertase, liver esterase, and peanut phospholipase D, whereas the yeast phospholipase D had a lipolytic effect.

In vitro assays. Yeast phospholipase D was highly active in releasing [3H]choline from [3H]choline-labeled DPPC in the assay described by Okamura and Yamashita (22). Amounts of yeast phospholipase D as low as 0.05 units produced [3H]choline release that was 8- to 10-fold higher than that produced by 10 units of peanut or 10 units of cabbage phospholipase D (Table 1). Incubation with liver esterase (10 units) or convertase (10 µg) did not produce any detectable [3H]choline release. The assay was repeated with AF buffer, pH 7.4, with identical results. Similar results were also obtained when natural surfactant containing [3H]choline- and [14C]palmitate-labeled DPPC was used as the substrate instead of [3H]choline-labeled DPPC (data not shown).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Release of [3H]choline from [3H]choline-labeled DPPC in vitro

To determine that peanut and cabbage phospholipases D were able to cleave choline from DPPC, we used a modification of the method of Artiss and colleagues (1). Table 1 shows that all three phospholipases D (10 units each) showed significant activity in this assay. However [3H]choline release from [3H]choline-labeled DPPC was two to four times higher with yeast phospholipase D compared with cabbage or peanut phospholipase D.

Measurement of choline release. Table 2 shows [3H]choline release on cycling natural surfactant containing [3H]choline- and [14C]palmitate-labeled DPPC. The amounts of enzymes used corresponded to amounts that were previously shown to produce heavy-to-light subtype conversion on cycling. Only yeast phospholipase D produced significantly greater release of [3H]choline compared with cycling in the absence of enzyme (control, P < 0.001). Orlistat, which inhibits the action of convertase in producing light-subtype generation, had no effect on [3H]choline release during cycling with convertase. Moreover, the [3H]choline release as a percentage of total radioactivity recovered on cycling with yeast phospholipase D (53.5 ± 2.6%) was not affected by Orlistat (58.1 ± 2.4%, P > 0.05).

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   [3H]choline release after cycling natural surfactant containing [3H]choline-labeled DPPC


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Convertase is needed for surfactant conversion. We and others have shown previously that the ability of convertase in converting heavy (TM) to light (SV) subtype was shared by a porcine liver esterase and peanut phospholipase D (3, 6, 19). In the present study, we found that the actions of yeast phospholipase D differed from those of the plant phospholipases D. Interestingly, release of [3H]choline from natural surfactant containing [3H]choline-labeled DPPC was observed during cycling with yeast phospholipase D but not on cycling with convertase, liver esterase, or peanut phospholipase D (Table 2).

Profiles observed on cycling. Potential sites for the action of enzymes on phospholipids are the triglyceride backbone (lipases), the fatty acid substitutions at the C-1 and C-2 position (phospholipases A1, A2, and B), the ester bond with orthophosphoric acid at the C-3 position (phospholipase C), and the ester bond with the base (phospholipase D) (32). Among several enzymes tested, we found that cycling natural surfactant with convertase, a porcine liver esterase, and plant phospholipases D (peanut or cabbage) produced a similar profile of conversion, with distinct peaks corresponding to heavy and light subtypes (Fig. 2). In contrast, no peaks of heavy and light subtypes were observed after cycling heavy substrate with phospholipases A2, B, or C (Fig. 2). With these enzymes, there was a broad band of radioactivity distributed throughout the gradient. Phospholipase activity in human and rodent alveolar lavage has been previously reported to abolish the ability of surfactant to band in sucrose gradients (18). Our findings with phospholipases A2, B, and C, yeast phospholipase D, and pancreatic lipase (10) are consistent with the earlier report. In addition, we found, in accordance with the findings of previous investigators (13, 16), that elastase, a serine protease, did not convert heavy substrate to light subtype on cycling. Thus, in our opinion, hydrolysis of DPPC at any one of several sites produces the "lipolytic" profile after cycling, whereas the formation of heavy and light peaks probably represents an orderly transformation between different structural forms of surfactant (12).

Convertase and liver carboxylesterase. Carboxylesterases are highly conserved in mammals (28). Convertase and porcine liver esterase are closely related carboxylesterases (EC 3.1.1.1) with molecular masses of 72 and 62 kDa on SDS-PAGE, respectively. They are serine- active enzymes that are inhibited by DFP, and they both hydrolyze alpha -naphthyl acetate (19). The carboxylesterases in the lung and liver share considerable sequence homology in various species. For example, mouse convertase is homologous but not identical to mouse liver carboxylesterase (25) and the major mouse serum carboxylesterase Es-N (9, 25). Similarly, the convertase in rat alveolar lavage is highly homologous to ES-2, a carboxylesterase in rat serum that is secreted by the liver (2). We recognize that the convertase used in these experiments was not pure (Fig. 1). However, further purification steps lead to substantial losses of protein, making the yield too low for our cycling experiments. Nevertheless, the finding that pure porcine liver esterase shows convertase activity (19) is strong evidence that esterase activity is required for conversion of heavy substrate to light subtype. Moreover, the similarity in the profile of heavy and light peaks of phospholipids observed after cycling heavy substrate with liver esterase, convertase, and peanut phospholipase D (Fig. 2) suggests that conversion of heavy to light subtype on cycling with these enzymes may involve hydrolysis at the C-3 substitution in phospholipids (phospholipase D-like action). In addition, in common with other phospholipases, the action of convertase occurs at an air-fluid interface, as evidenced by the requirement for repeated expansion and contraction of the surface for conversion to occur. Thus convertase probably belongs to the esterase-lipase superfamily of enzymes, but the natural substrate(s) for convertase is not known.

Is DPPC the substrate? We determined whether hydrolysis of DPPC occurred during conversion of heavy to light subtype. The contrasting profiles observed on cycling natural heavy substrate with convertase, liver esterase, peanut and cabbage phospholipase D vs. with those with yeast phospholipase D (Fig. 2) have already been mentioned. Moreover, choline release was detected when heavy substrate containing [3H]choline-labeled DPPC was cycled with yeast phospholipase D, but not during cycling with peanut phospholipase D, liver esterase, or convertase (Table 2). As little as 0.05 U/ml of yeast phospholipase D in the cycling mixture caused a loss of heavy and light peaks, thereby suggesting that even low levels of hydrolysis of DPPC produce the lipolytic profile (e.g., Fig. 2D). Similarly, the activity of the plant phospholipases D differed from that of yeast phospholipase D in the in vitro assay (Table 1). Diversity in the action of various phospholipases D is well known (21). Several factors, namely, the substrate concentration, state of lipid aggregation, orientation of the phospholipids at the air-fluid interface, and presence of Ca2+ could modify phospholipase D activity (17). For example, SDS at a molar ratio of approximately 2:1 phosphatidylcholine to SDS is a most effective activator of cabbage and peanut phospholipases D (5, 30). Accordingly, cabbage and peanut phospholipases SDS required SDS, ethanol, and a high concentration of Ca2+ for their activity in vitro. Moreover, the broader range of substrate specificities of the plant phospholipases D compared with the yeast enzyme (8, 17) raises the possibility that conversion of heavy to light subtype on cycling with the plant phospholipases D may be due to hydrolysis of other phospholipids in surfactant. Our studies with reconstituted surfactant indicate that phosphatidylglycerol has an important influence on conversion of heavy to light subtype with cycling (7), and further investigations are needed to determine whether hydrolysis of phosphatidylglycerol occurs on cycling heavy subtype with convertase.

The preservation of DPPC during conversion of heavy to light subtype is supported by data from previous investigations. Thus concentrations of DPPC in the light subtype are similar to those found in the heavy subtype (11, 24). DPPC is responsible for the surface tension-lowering property of surfactant (4, 29), and incubation of surfactant with small amounts of phospholipase A2 or C destroys its surface tension-lowering property (18). In contrast, incubation of TM with DFP, an inhibitor of convertase activity, did not alter its biophysical function compared with surfactant that possessed endogenous convertase activity (15). In sum, we found no evidence of hydrolysis of DPPC by convertase, and various enzymes that hydrolyzed DPPC did not produce the characteristic profile of heavy and light peaks on cycling natural surfactant. Therefore, DPPC is probably not a substrate for convertase.

In summary, cycling natural surfactant with liver esterase or plant phospholipases D (peanut or cabbage) produced heavy-to-light subtype conversion similar to that with convertase but did not release choline. In contrast, cycling natural surfactant with yeast phospholipase D released choline but did not convert heavy to light subtype in the manner that the former enzymes did. We conclude therefore that hydrolysis of DPPC is unlikely to explain conversion of heavy natural surfactant to light subtype on cycling with convertase, liver esterase, and peanut or cabbage phospholipase D. Further investigations are needed to determine whether hydrolysis of other phospholipids, such as phosphatidylglycerol, may be responsible for conversion of heavy to light subtypes.


    ACKNOWLEDGEMENTS

We thank Dr. Sumita B. Khattri, Dr. Vijay Sharma, Matthew Kellam, and Syed Irfan for help with the experiments.


    FOOTNOTES

Orlistat used in these experiments was kindly provided by Hoffmann-La Roche, Basel, Switzerland.

This work was supported by a Research Advisory Group and a Merit Review Grant from the Dept. of Veterans Affairs (R. Dhand and N. J. Gross) and by a grant from Chicago Association for Research and Education in Science (R. Dhand).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: R. Dhand, Div. of Pulmonary and Critical Care Medicine, 111 N. Edward Hines Jr. VA Hospital, Hines, IL 60141 (E-mail: dhand{at}research.hines.med.va.gov).

Received 5 April 1999; accepted in final form 26 August 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Artiss, J. D., T. F. Draisey, R. J. Thibert, B. Zak, and K. E. Taylor. The determination of lecithin and total choline-containing phospholipids in amniotic fluid employing enzymes as reagents. Microchem. J. 25: 153-168, 1980[ISI].

2.   Barr, F., H. Clark, and S. Hawgood. Identification of a putative surfactant convertase in rat lung as a secreted serine carboxylesterase. Am. J. Physiol. Lung Cell. Mol. Physiol. 274: L404-L410, 1998[Abstract/Free Full Text].

3.   Clark, H., L. Allen, E. Collins, F. Barr, L. Dobbs, G. Putz, J. Goerke, and S. Hawgood. Localization of a candidate surfactant convertase to type II cells, macrophages and surfactant subfractions. Am. J. Physiol. Lung Cell. Mol. Physiol. 276: L452-L458, 1999[Abstract/Free Full Text].

4.   Clements, J. A. Function of the alveolar lining. Am. Rev. Respir. Dis. 115: 67-71, 1977[ISI][Medline].

5.   Dawson, R. M. C., and N. Hemington. Some properties of purified phospholipase D and especially the effect of amphipathic substances. Biochem. J. 102: 76-86, 1967[ISI][Medline].

6.   Dhand, R., V. K. Sharma, A. L. Teng, S. Krishnasamy, and N. J. Gross. Protein-lipid interactions and enzyme requirements for light subtype generation on cycling reconstituted surfactant. Biochem. Biophys. Res. Commun. 244: 712-719, 1998[ISI][Medline].

7.   Dhand, R., J. Young, S. Krishnasamy, F. Possmayer, and N. J. Gross. Influence of phospholipid composition on the properties of reconstituted surfactants. Lung 177: 127-138, 1999[ISI][Medline].

8.   Exton, J. H. Phospholipase D: enzymology, mechanisms of regulation, and function. Physiol. Rev. 77: 303-320, 1997[Abstract/Free Full Text].

9.   Genetta, T. L., P. D'Eustachio, S. S. Kadner, and T. Finlay. cDNA cloning of esterase 1, the major esterase activity in mouse plasma. Biochem. Biophys. Res. Commun. 151: 1364-1370, 1998.

10.   Gross, N. J. Extracellular metabolism of pulmonary surfactant; the role of a new serine protease. Annu. Rev. Physiol. 57: 135-150, 1995[ISI][Medline].

11.   Gross, N. J., and K. R. Narine. Surfactant subtypes in mice: characterization and quantitation. J. Appl. Physiol. 66: 342-349, 1989[Abstract/Free Full Text].

12.   Gross, N. J., and K. R. Narine. Surfactant subtypes in mice: metabolic relationships and conversion in vitro. J. Appl. Physiol. 67: 414-421, 1989[Abstract/Free Full Text].

13.   Gross, N. J., and R. M. Schultz. Serine proteinase requirement for the extracellular metabolism of pulmonary surfactant. Biochim. Biophys. Acta 1044: 222-230, 1990[ISI][Medline].

14.   Gross, N. J., and R. M. Schultz. Requirements for extracellular metabolism of pulmonary surfactant: tentative identification of serine protease. Am. J. Physiol. Lung Cell. Mol. Physiol. 262: L446-L453, 1992[Abstract/Free Full Text].

15.   Gross, N. J., R. Veldhuizen, F. Possmayer, and R. Dhand. Surfactant convertase action is not essential for surfactant film formation. Am. J. Physiol. Lung Cell. Mol. Physiol. 273: L907-L912, 1997[Abstract/Free Full Text].

16.   Hall, S. B., R. W. Hyde, and M. C. Kahn. Stabilization of lung surfactant particles against conversion by a cycling interface. Am. J. Physiol. Lung Cell. Mol. Physiol. 272: L335-L343, 1997[Abstract/Free Full Text].

17.   Heller, M. Phospholipase D. Adv. Lipid Res. 16: 267-326, 1978[Medline].

18.   Holm, B. A., L. Keicher, M. Liu, J. Sokolowski, and G. Enhorning. Inhibition of pulmonary surfactant function by phospholipases. J. Appl. Physiol. 71: 317-321, 1991[Abstract/Free Full Text].

19.   Krishnasamy, S., N. J. Gross, A. L. Teng, R. M. Schultz, and R. Dhand. Lung "surfactant convertase" is a member of the carboxylesterase family. Biochem. Biophys. Res. Commun. 235: 180-184, 1997[ISI][Medline].

20.   Krishnasamy, S., A. L. Teng, R. Dhand, R. M. Schultz, and N. J. Gross. Molecular cloning, characterization, and differential expression pattern of mouse lung surfactant convertase. Am. J. Physiol. Lung Cell. Mol. Physiol. 275: L969-L975, 1998[Abstract/Free Full Text].

21.   Munnik, T., R. F. Irvine, and A. Musgrave. Phospholipid signalling in plants. Biochim. Biophys. Acta 1389: 222-272, 1998[ISI][Medline].

22.   Okamura, S. I., and S. Yamashita. Purification and characterization of phosphatidylcholine phospholipase D from pig lung. J. Biol. Chem. 269: 31207-31213, 1994[Abstract/Free Full Text].

23.   Oulton, M., E. Edwards, and K. Handa. Convertase activity in alveolar surfactant and lamellar bodies in fetal, newborn, and adult rabbits. J. Appl. Physiol. 86: 71-77, 1999[Abstract/Free Full Text].

24.   Oulton, M., J. MacDonald, D. T. Janigan, and G. T. Faulkner. Mouse alveolar surfactant: characterization of subtypes prepared by differential centrifugation. Lipids 28: 715-720, 1993[ISI][Medline].

25.   Ovnic, M., K. Tepperman, S. Medda, R. W. Elliott, D. A. Stephenson, S. G. Grant, and R. E. Ganschow. Characterization of a murine cDNA encoding a member of the carboxylesterase multigene family. Genomics 9: 344-354, 1991[ISI][Medline].

26.   Putman, E., L. A. J. Creuwels, L. M. G. van Golde, and H. P. Haagsman. Surface properties, morphology and protein composition of pulmonary surfactant subtypes. Biochem. J. 320: 599-605, 1996[ISI][Medline].

27.   Rossomando, E. F. Ion exchange chromatography. Methods Enzymol. 234: 466-468, 1990.

28.   Satoh, T., and M. Hosokawa. The mammalian carboxylesterases: from molecules to functions. Annu. Rev. Pharmacol. Toxicol. 38: 257-288, 1998[ISI][Medline].

29.   Schurch, S., F. Possmayer, S. Cheng, and A. M. Cockshutt. Pulmonary SP-A enhances adsorption and appears to induce surface sorting of lipid extract surfactant. Am. J. Physiol. Lung Cell. Mol. Physiol. 263: L210-L218, 1992[Abstract/Free Full Text].

30.   Tzur, R., and B. Shapiro. Purification of phospholipase D from peanuts. Biochim. Biophys. Acta 280: 290-296, 1972[ISI][Medline].

31.   Veldhuizen, R. A. W., K. Inchley, S. A. Hearn, J. F. Lewis, and F. Possmayer. Degradation of surfactant-associated protein B (SP-B) during in vitro conversion of large to small surfactant aggregates. Biochem. J. 295: 141-147, 1993[ISI][Medline].

32.   Waite, M. Phospholipids. In: Biochemistry of Lipids, Lipoproteins, and Membranes, edited by D. E. Vance, and J. E. Vance. New York: Elsevier, 1996, p. 1-33.


Am J Physiol Lung Cell Mol Physiol 278(1):L19-L24
0002-9513/00 $5.00 Copyright © 2000 the American Physiological Society