Surfactant convertase action is not essential for surfactant film formation

N. J. Gross1,2,3, R. Veldhuizen4, F. Possmayer5, and R. Dhand1,2

1 Medical Service, Hines Veterans Affairs Hospital, Hines 60141; Departments of 2 Medicine and 3 Molecular and Cellular Biochemistry, Stritch School of Medicine, Loyola University of Chicago, Maywood, Illinois 60153; and 4 Departments of Medicine and Physiology, Lawson Research Institute and 5 Departments of Obstetrics and Gynaecology and Biochemistry, Medical Research Council Group in Fetal and Neonatal Health and Development, University of Western Ontario, London, Ontario N6A 5A5, Canada

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

A serine-active enzyme, "surfactant convertase," is required for the conversion of surfactant from the tubular myelin (TM) form to the small vesicular (SV) form. This transformation involves at least two steps, the conversion of TM to a surface-active film at the air-fluid interface and the reorientation of the film into the surface-inactive SV form; we asked if convertase was required for the first of these steps. Rat and mouse TMs were pretreated with diisopropyl fluorophosphate (DFP) to inactivate endogenous convertase activity or with vehicle and then were analyzed for their ability to lower surface tension in vitro as an index of the conversion of TM to a surface film. DFP pretreatment did not alter the ability of TM preparations to lower surface tension, as assessed by pulsating bubble, and it did not affect the behavior of TM in a surface balance. In an experiment designed to test the ability of TM to feed a surface film to exhaustion, TMs that had been pretreated with DFP or vehicle performed similarly. These experiments show that convertase activity is not required for the conversion of TM to a monolayer and suggest, instead, that convertase acts at a post surface film stage.

surfactant subtype; surfactant conversion

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

PULMONARY SURFACTANT is secreted from alveolar type II cells as lamellar bodies (LB) and evolves sequentially from this form through the tubular myelin (TM) form of surfactant, a surface film, and a small vesicular (SV) form (1, 9, 12, 16, 17). The mechanisms that control surfactant extracellular metabolism through these subtypes are largely unknown. Although some of these steps appear to proceed spontaneously under appropriate ionic conditions (14), a newly described serine-active enzyme, "surfactant convertase," appears to be required for the conversion of TM through SV (7, 10, 11). The molecular actions of this enzyme and its substrate are unknown. The present study examines its role in surfactant biophysics.

The conversion of TM, which is surface active, to SV, which is much less surface active, can be reproduced in vitro by a procedure that we call "surface cycling" (9). Such conversion requires not only the presence of convertase activity but also the presence of an air-liquid interface (11). The latter requirement suggests that the conversion of TM to SV probably involves at least two steps, 1) conversion of TM to the surface film and 2) conversion of the film to SV, and is consistent with the postulated sequence of surfactant subtype evolution referenced above. The essential role of convertase in the overall conversion of TM to SV has been derived from in vitro experiments (9, 10) in which these two steps are not distinguishable. The aim of the present experiments was to determine if convertase activity was required for the first of these steps, the conversion of TM to the surface film, as inferred by the ability of purified TM to express surface activity in the presence or absence of convertase. We studied the rate of film formation using a pulsating bubble, the minimum surface tension (gamma min) obtained using a pulsating bubble and a modified Kimray-Greenfield surfactometer, and the rate at which a subphase of TM was able to feed surfactant to the surface, in each case comparing purified TM with and without active convertase. We found no differences in surface biophysics in any of these experiments, from which we infer that convertase is not required for the conversion of TM to a surface-active film. This result suggests that convertase is more likely to act at a post surface film stage in the extracellular metabolism of surfactant.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

All reagents were obtained from Sigma Chemical (St. Louis, MO), and solvents were obtained from Fisher Scientific (Fair Lawn, NJ).

Animals and radiolabeling. All experiments were performed on mice or rats. Female CF#1 mice, ~15 g body wt, were obtained from Carworth Farms (Portage, MI); female Sprague-Dawley rats, ~150 g, were obtained from Charles River Laboratories (Wilmington, MA). For experiments in which radiolabeled surfactant was used, groups of three to six mice received 20 µCi of [3H]choline (NEN, Boston, MA) by intraperitoneal injection 18 h before death.

TM preparation. TM was obtained as described previously (8, 10) from bronchoalveolar lavage of rats or mice that had received lethal injections of pentobarbital sodium. [The method described here yields a mixture of tubular myelin and myelin-like membranes, as seen by electron microscopy (8). For convenience, we call this "TM" or "the TM form" of surfactant throughout this paper.] The lungs were lavaged pertracheally with lavage buffer (AF: 0.15 M NaCl, 2 mM CaCl2, and 5 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.4 at 37°C) that was flushed into and out of the lungs three times with five changes. After lavage, the animals with lungs in situ were placed in a 37°C incubator, and a small amount of AF buffer containing 10 µM isoproterenol (as a surfactant secretogogue) was instilled into the lungs. This was flushed into and out of the lungs at 10-min intervals and was removed after 30 min. This process was repeated one time. All lavages, the initial bronchoalveolar lavages and the material obtained from incubated lungs, were combined and divided into two halves. One half was incubated with diisopropyl fluorophosphate (DFP), final concentration 10 mM, at 23°C for 30 min to inhibit endogenous convertase activity. The other half was similarly incubated but without DFP. Treated and untreated preparations were separately centrifuged through a step of 0.1 M sucrose in AF buffer onto a cushion of 0.9 M sucrose in AF buffer in a SW28 rotor at 25,000 revolutions/min (rpm) for 2 h at 4°C. The material that accumulated at the 0.1/0.9 M interface was aspirated, diluted with 5-10 volumes of AF buffer, and sedimented in the SW28 rotor at 25,000 rpm for 2 h. The pellet, called TM preparation, was resuspended in a small volume of AF buffer, dispersed by a single gentle pass through a glass-Teflon Potter homogenizer (size AA; AH Thomas, Philadelphia, PA), and analyzed for surface activity immediately or frozen for later analysis.

Each TM preparation was validated before use. In brief, replicate aliquots of ~100 µg of TM phospholipid were cycled (as previously described, see Ref. 9) for 2 h at 37°C or were not cycled. In each case, the resulting material was analyzed by centrifugation through continuous sucrose gradients, 0.1-0.9 M sucrose in AF buffer, in a SW55 rotor at 45,000 rpm for 18 h at 8°C and was dripped out from the bottom in 18-20 fractions. Phospholipids were purified from the fractions (5) and were assayed for radioactivity, in the case of mouse TM preparations, or phospholipid phosphorus (3), in the case of rat TM preparations. The gradient profiles were analyzed on software (PeakFit; Jandel) that separates overlapping curves and provides estimates of the location and area under each peak. An example of the sucrose gradient profiles that were obtained is shown in Fig. 1, where the buoyant density peak without cycling was 1.050-1.060 g/ml, which is consistent with TM plus LB (8). A small amount (<15% of the total phospholipid) was found at the buoyant density of light or SV subtype, 1.025 g/ml. Cycling of TM preparations that had not been pretreated with DFP showed that about one-half of the phospholipid was translocated to the peak at 1.025 g/ml density, which is consistent with conversion to light or SV subtype (8). Cycling of TM preparations that had been treated with DFP showed that most of the phospholipid remained at the buoyant density of TM. As with uncycled material, a small proportion of phospholipid was found at the buoyant density of SV subtype. This showed that, for each TM preparation, the bulk of the uncycled starting material was of TM or LB subtype and that TM that had not been pretreated with DFP was capable of converting to SV subtype with cycling; however, aliquots that had been pretreated with DFP were incapable of being converted to SV, indicating that endogenous convertase had been inactivated by DFP. TM preparations that did not have these properties were discarded. Two of twelve preparations of rat TM were accordingly discarded. In both cases, this was because the aliquot that had been pretreated with DFP showed substantial conversion to SV buoyant density after cycling. This appears to have been because the DFP itself was not fully active because it did not recur when a new batch of DFP was used. Similarly, occasional preparations of mouse TM were discarded because the starting material contained more than ~20% of phospholipid at SV buoyant density, presumably due to technical faults in the purification of TM. Overall, ~80% of the mouse TM preparations were acceptable.


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Fig. 1.   Validation of a tubular myelin (TM) preparation. Continuous sucrose gradients were centrifuged to equilibrium, and the 3 phospholipid profiles were separated for viewing. Gradient A is an aliquot of TM that was not cycled and shows that most of the phospholipid resolved around a broad peak buoyant density of 1.051 g/ml, which is typical of TM. A minor peak is present at 1.061 g/ml, which is typical of the lamellar body (LB) form. Gradient B is an aliquot of the same preparation that had been cycled for 2 h in vitro and shows that more than one-half of the phospholipid is now present at a peak of buoyant density (1.025 g/ml), which is typical of the "light" or small vesicular (SV) subtype. The rest remained at the buoyant density of TM. Gradient C is of an aliquot of TM that had been pretreated with diisopropyl fluorophosphate (DFP) and subsequently cycled for 2 h. Nearly all of the phospholipid remains at the buoyant density of TM, with only ~15% being found at the density of SV subtype. The LB shoulder at 1.061 g/ml seen in gradient A has disappeared, which is consistent with our previous finding that the conversion of LB to TM is not dependent on the action of convertase (9, 10).

Surface tension analysis. Two methods were employed, a modified Kimray-Greenfield surfactometer method and a pulsating bubble method. The modified Kimray-Greenfield surfactometer (calibrated before each use with weights) was placed in a 37°C incubator that was lined with wet paper to provide a humid environment. The trough was filled with 50 ml of AF buffer as subphase, and the surface was "cleaned" by repeated aspiration of the surface by suction of the surface via a Pasteur pipette positioned at the surface, replenishing the volume aspirated on each occasion. The "cleanness" of the surface was validated by continuous measurement of the surface tension while the surface area was compressed to 15% of the initial surface area without a decrease in the surface tension. When the surface was clean, 250 µg of phospholipid TM preparation, pretreated with either DFP or vehicle, were applied below the surface of the trough subphase. This was stirred with a magnetic stirring bar for 10 min. The surface was then slowly compressed to 15% of its initial area and then reexpanded to initial surface area while surface tension was measured continuously; the cycle time was 180 s. From the surface tension-surface area plots so obtained, we derived the following data: maximum surface tension (gamma max), gamma min, and area of hysteresis, which was obtained by cutting the compression-expansion loop from the surface area-surface tension chart and weighing it.

The second method of analysis employed a pulsating bubble surfactometer (Electronetics, Amherst, NY), as described by Enhorning (4). A bubble was created in a suspension of surfactant (2.5 mg/ml, in the presence of 1.5 mM CaCl2 at 37°C). After 10 s, the bubble was pulsated at 20 pulsations per minute between a maximum bubble radius (Rmax) of 0.55 mm and a minimum radius (Rmin) of 0.4 mm, measuring pressure across the air-liquid interface by a pressure transducer. Surface tension was calculated by the law of Young and Laplace and is presented as a function of the pulsation number.

Surface feeding experiment. The ability of TM surfactant preparations to feed a surface film to exhaustion was determined in the Kimray-Greenfield surfactometer. TM was prepared from mice that had received [3H]choline. In each experiment, an aliquot of 154 µg of TM surfactant phospholipid (a 10-fold excess of that required to provide a monomolecular film to the 57-cm2 surface, assuming an average molecular area at the surface of 48 A2), either pretreated with DFP as above or not, was delivered below the surface while the subphase was continuously stirred by a magnetic stirring bar. The surface was aged for 10 min with continuous stirring of the subphase to allow a surface film to form and then was compressed to 15% of initial surface area with a Teflon dam while surface tension was continuously recorded. At 15% initial surface area, the surface was rapidly aspirated by suction through a Pasteur pipette positioned just above the surface, aspirating 2.0-3.5 ml into a trap close to the pipette tip. The volume of the aspirate was measured (by calibrations on the trap), the pipette was flushed by immediate aspiration of a further 1 ml of water into the trap, and a volume of fresh, warmed, cleaned subphase buffer equal to the volume aspirated from the trough was replaced in the trough. The total radioactivity [in disintegrations/min (dpm)] of each aspirate was measured. The subphase was continuously stirred again for 10 min while the cycle of surface compression, surface aspiration, and replacement of subphase buffer was repeated. These cycles were repeated until surface tension measurements showed negligible residual surface activity, which occurred consistently after 15-18 cycles of spreading and aspiration of the surface film.

The amount of surface-associated phospholipid that was removed with each round of surface spreading and aspiration was calculated as follows. The specific activity of the TM preparation that was used was determined (in dpm/µg phospholipid). The activity of the subphase (in dpm/ml) was measured at the start and after the last cycle; this declined with each cycle of aspiration due to the fact that the subphase was replenished with fresh buffer after each aspiration. We initially calculated the activity of the subphase after each cycle of aspiration and replenishment (a datum that would be required later in the calculation) by the dilutional factor cycle by cycle. However, we found that it was easier and, for practical purposes, just as accurate to interpolate the exponential decline in subphase activity from the initial and final measurements. The volume of the surface film that was aspirated was assumed to be negligible. The absolute radioactivity of the surface itself that was aspirated with each cycle was the total radioactivity in each aspirate minus the activity of the subphase removed with the surface; this later was the product of the total volume of the aspirate and the activity (in dpm/ml) of the aspirate at that cycle number. From the radioactivity of the surface itself, i.e., corrected for the amount of subphase removed with the surface, and the specific activity of TM (assumed to be constant), we calculated the amount of phospholipid that was removed as a surface film with each cycle of film spreading, aging, and aspiration. This is equal to the amount of surface phospholipid delivered to the surface with each round.

Preliminary experiments were performed to determine the optimal time of aging of the surface (as determined by the fall in surface activity and the amount of phospholipid that could be aspirated from the surface); both reached stability at ~10 min. Preliminary experiments also showed that the amount of surface phospholipid that could be aspirated from the surface was independent of the initial amount delivered to the subphase, provided this was greater than ~75 µg of phospholipid as TM. We used two times this amount.

Statistical analysis. Means, SDs, SEs, and Student's t comparisons were performed on a hand-held calculator.

    RESULTS
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Introduction
Methods
Results
Discussion
References

Surface balance study. Three paired experiments were performed. In each experiment, mouse TM preparations that had been pretreated with DFP to inactivate endogenous convertase activity were compared with TM preparations from the same batch that had not been pretreated with DFP. Each batch of TM was analyzed in duplicate, entering the mean of each duplicate as a single datum. The results (see Table 1) show that there were no differences between TM containing active convertase and DFP-inactivated convertase with respect to any of the measured parameters.

                              
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Table 1.   Surface balance study

Pulsating bubble surfactometry. We examined paired untreated and DFP-pretreated TM samples in triplicate (5 experiments). The means from each triplicate determination were entered as a single datum for statistical purposes. The results are shown in Fig. 2. Similar initial surface tensions were observed on the first pulsation after 10 s of adsorption with an Rmin of 37.5 ± 4.0 (SE) mN/m (active) and 34.6 ± 4.2 mN/m (inactive), which were not significantly different. Essentially identical curves were observed for the two groups of TM preparations at Rmax during 100 pulsations, reflecting similar abilities to adsorb to the surface during bubble expansion. In addition, similar rates of decline of surface tension at Rmin were observed over the 100 pulsations, showing that the surface tensions achieved through dynamic compressions during a 50% reduction in bubble surface area were similar. The Rmin values after 100 pulsations were 11.1 ± 3.6 (active) and 8.1 ± 2.4 mN/m (inactive), which were not significantly different.


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Fig. 2.   Pulsating bubble experiments. Surface tensions at maximum and minimum bubble radius (Rmax and Rmin, respectively) as a function of pulsation number on the abscissa. A: 5 separate active samples, i.e., not pretreated with DFP. B: 5 samples pretreated with DFP. Error bars are SE. There was no significant difference between active and inactive at any point.

Feeding of TM to the surface to exhaustion. Three identical paired experiments were performed with separate [3H]choline-labeled mouse TM preparations. Again, each pair was derived from the same group of animals, one half of whose TM had been pretreated with DFP and the other not. Each paired experiment yielded similar results. The results of one experiment are shown in Fig. 3. TM with active convertase is shown in Fig. 3A, and TM with inactive convertase in shown in Fig. 3B, but only the compression phase of each surface tension-surface area cycle is shown. In both, surface tension declined to a minimum value at ~20-25% of the starting surface area during the first six compression-aspiration cycles. Immediately the surface was aspirated, the surface tension returned to a high value (~60-70 mN/m, data not shown). During cycles 7-10, gamma min was reached only at successively smaller surface areas, and during cycles 11-15, the gamma min rose progressively, so that by about cycle 16-18 there was little reduction in surface tension even at minimum surface area. All samples behaved similarly, regardless of whether they contained active or DFP-inactivated convertase.


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Fig. 3.   Surface feeding experiment. Surface tension vs. surface area plots in a Langmuir-type trough of a single pair of TM preparations. Selected compression phases only are shown. Arrows, compression number. A: active TM, i.e., not pretreated with DFP. B: inactive, i.e., pretreated with DFP. Other details as described in text.

The amount of surface phospholipid aspirated, corrected for subphase phospholipid removed with each aspiration, is shown for all experiments in Fig. 4. The data are shown as cumulative amounts of surface phospholipid as a function of the aspiration-respreading cycle number. These data indicate a consistent amount of phospholipid aspirated from the surface in each of approximately the first 8-10 cycles. This amount was 4.0 ± 0.4 (SD) µg/cycle when active convertase was present and 4.0 ± 0.1 µg/cycle when convertase had been inactive by pretreatment with DFP, which is not significantly different. After the first 10-12 cycles, the amount of phospholipid aspirated from the surface decreased progressively, reaching ~1.0 µg/cycle by about cycle 16. The rate and cumulative amount of surfactant recovery from the surface, and therefore delivered to it, was similar for preparations that contained active or inactivated convertase. The overall recovery of initial radioactivity, the sum of aspirated surface plus subphase and residual activity in the trough at the end of the experiment, was similar for both groups, 83 ± 9 (active convertase) vs. 80 ± 14% (inactive convertase). The cumulative amount recovered from the surface alone from the start to the conclusion of the experiment (when surface tension was no longer lowered on surface area compression) was 35.2 ± 6.8 (active convertase) and 35.3 ± 8.6% (inactive convertase) of the amount added to the subphase at the start of the experiments. Thus more than one-third of the surfactant delivered to the subphase as TM was converted to a surface film by successive cycles of surface spreading and aspiration. Most of the remainder, ~40%, was removed as subphase during successive aspirations, and ~6% remained in the subphase at the conclusion of each experiment. We assume the ~20% unaccounted for was lost on the walls of the trough, in the aspiration pipette, or on the platinum flag that was removed, cleaned, and flamed between successive cycles. As the rate of delivery of surfactant from the subphase to the surface was similar whether active or inactive convertase was present, we conclude that the presence of active convertase does not significantly affect the rate at which TM surfactant is delivered to the surface.


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Fig. 4.   Surface feeding experiment. Cumulative phospholipid aspirated from the surface over successive cycles (abscissa) of surface spreading, aging, compression, and aspiration; 3 paired experiments. Active, TM not pretreated with DFP; inactive, TM pretreated with DFP. Symbols are means ± SD; where error bars are not seen, they are smaller than the symbols. There are no significant differences between the 2 slopes or between the corresponding points.

    DISCUSSION
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Introduction
Methods
Results
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References

As stated in the introduction, the action of a serine-active enzyme, surfactant convertase, appears to be required for the conversion of surfactant from the TM form to the SV form (7, 10, 11). However, this transition probably involves at least two steps, the conversion of TM to a surface film and the conversion of this film or fragments extruded by it to the SV subtype. It is therefore of interest to determine at which of these two steps convertase is required. The aim of the present experiments was to determine if convertase was required in the first of these steps by the analysis of the ability of purified TM to express typical surface-active properties in the presence or absence of active convertase.

To perform these studies, we purified surfactant predominantly in the TM form, usually containing a component of the LB form, as validated by the buoyant density of the phospholipid profile on continuous sucrose gradients centrifuged to equilibrium (8, 9). As stated in METHODS, the material that we call TM for convenience is heterogeneous in that it consists of some TM plus myelin-like concentric membranes (8, 10), as well as some LB form. Such preparations contain convertase activity in amounts sufficient to mediate conversion of TM to SV on cycling in vitro, as shown previously (11), and again in the present experiments (Fig. 1). To explore the role of convertase in the expression of surface activity by TM, it was necessary to inactivate endogenous convertase activity. We did this by dividing the surfactant lavaged from rats or mice into two equal portions and treating one with DFP to inactivate its convertase; the other was identically handled but was not exposed to DFP. This yielded paired samples of TM that would be comparable apart from DFP pretreatment. Before use, we validated each paired TM preparation to show that both were almost entirely of the buoyant density typical of TM before cycling and that its TM was capable of converting to the buoyant density of SV on cycling in vitro unless pretreated with DFP (Fig. 1).

As can be seen from the results, the quality of the surfactant preparations was questionable in that the gamma min values obtained were higher and the rate of film formation was slower than with ideal preparations (e.g., Refs. 6 and 13). In part, this may have been due to the need to incubate the surfactants (with DFP or vehicle) before assay. As they nevertheless retain some surface activity, we consider them adequate to answer the question whether convertase is required for the expression of surface activity.

In each of the three groups of experiments we performed, we were unable to detect any differences in the surface-active behavior of TM that contained active or inactivated convertase. The rate of film formation was indistinguishable as measured by the rate of surface tension lowering in a pulsating bubble (Fig. 2). The gamma min achieved by purified TM was also indistinguishable whether measured in a modified Kimray-Greenfield surfactometer (Table 1) or by a pulsating bubble technique (Fig. 2).

Because the results of the above two experiments depend on the ability of quite small amounts of TM to reduce surface tension and because lavaged surfactant contains endogenous convertase, it is conceivable that small amounts of TM could have been "primed" by convertase activity before its exposure to DFP. This might enable TM, even after DFP treatment, to show surface activity even if convertase were, in fact, necessary to feed TM to the surface film.

To exclude this possibility, we devised an additional experiment that would call for a relatively large proportion of the TM in a preparation to be delivered to the surface. The rationale was that if the action of convertase is necessary to convert TM to a surface-active form, TM preparations that lacked active convertase would fail to lower surface tension sooner than those that contained active convertase and also that the cumulative amount of phospholipid that could be aspirated as a surface film would be less. This was not the case (Fig. 4). We calculate that ~35% of the TM applied to the trough was delivered to the surface over the course of 15-20 rounds of TM surfactant spreading and aspiration whether active convertase was present or not. The reason why this figure, ~35%, was not closer to 100% is explained as being due to the fact that most of the TM that was not recovered from the surface itself was coincidentally removed as subphase along with the surface during successive aspirations, ~40% of the total applied to the trough in each case. Thus it was not available for delivery to the surface for reasons related to the experimental technique.

Only ~4 µg of phospholipid were recovered from the surface with each cycle of surface spreading and aspiration. This is less than the 15.4 µg of phospholipid we calculate that the available surface area can accommodate as a monolayer. As the surface tension at maximal area after surface spreading was relatively high (64 mN/m), it is unlikely that a complete surface film was formed. Some "squeeze out" of the surface film may have occurred during the compression that preceded each aspiration. Also, it is possible that not all of the surface was recovered by each aspiration. However, none of these explanations would affect the conclusion that feeding of surfactant to the surface was equivalent whether active or inactive convertase was present.

The results of all of these experiments are consistent and suggest that convertase is not required for the expression of surface activity by TM, tending to exclude a role for convertase in mediating the conversion of TM to a surface-active form of surfactant. This conclusion is also consistent with data from others that show that synthetic TM reconstituted de novo from purified phospholipids and surfactant apoproteins and therefore, presumably devoid of convertase activity, is able to express surface activity (2, 15). By implication, the results of the present experiments and those of previous reports suggest that convertase acts at a post surface film stage, perhaps promoting the reorientation of fragments squeezed out of the film into the SV form.

    ACKNOWLEDGEMENTS

We acknowledge the excellent technical support of James D'Anza, Vija Bublys, and Andelle Teng.

    FOOTNOTES

This work was supported by grants from Veterans Affairs Research (to N. J. Gross and R. Dhand), from the National Heart, Lung, and Blood Institute (HL-45782-01 to N. J. Gross), and from the Medical Research Council of Canada (M268C7 to F. Possmayer).

Address for reprint requests: N. J. Gross, PO Box 1485, Hines, IL 60141.

Received 12 March 1997; accepted in final form 16 July 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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18.   Wright, J. R., R. E. Wager, R. L. Hamilton, M. Huang, and J. A. Clements. Uptake of lung surfactant subfractions into lamellar bodies of adult rat lungs. J. Appl. Physiol. 60: 817-825, 1986[Abstract/Free Full Text].


AJP Lung Cell Mol Physiol 273(5):L907-L912