©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Neutralization of the Positive Charges of Surfactant Protein C
EFFECTS ON STRUCTURE AND FUNCTION (*)

Lambert A. J. M. Creuwels (1), Esther H. Boer (1), Rudy A. Demel (2), Lambert M. G. van Golde (1), Henk P. Haagsman (1)(§)

From the (1)Laboratory of Veterinary Biochemistry and (2)Centre for Biomembranes and Lipid Enzymology, Utrecht University, 3508 TD Utrecht, The Netherlands

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Pulmonary surfactant protein C (SP-C) is a small, extremely hydrophobic peptide with a highly conservative primary structure. The protein is characterized by two adjacent palmitoylated cysteine residues, two positively charged residues (one arginine residue and one lysine residue) in the N-terminal region, and a long hydrophobic stretch. SP-C enhances the adsorption of phospholipids into an air-water interface. To determine the importance of the positively charged residues, we carried out experiments with natural porcine SP-C and modified porcine SP-C (SP-C) in which the positive charges had been blocked by phenylglyoxal. Circular dichroism experiments showed that SP-C had an increased content of -helix. Natural SP-C, but not SP-C, catalyzed insertion of phospholipids into a monolayer at the air-water interface. This reduced insertion was due to a strong reduction of binding of phospholipid vesicles to the monolayer. The insertion catalyzed by the natural porcine SP-C was decreased by an increased pH of the subphase. In contrast to natural SP-C, SP-C induced lipid mixing between phospholipid vesicles. The extent of lipid mixing was a function of the SP-C content. We conclude that the positively charged residues of SP-C are important for the binding of phospholipid vesicles to the monolayer, a process that precedes the insertion of phospholipids into the monolayer.


INTRODUCTION

Pulmonary surfactant, essential for breathing, is a complex mixture of lipids and proteins. It is present at the air-liquid interface of the lung, and its main function is to stabilize the lung by reducing the surface tension. Surfactant consists of 90% lipids and of 8-10% specific proteins(1, 2) . Four surfactant-associated proteins have been described, of which two are hydrophobic. These hydrophobic proteins, surfactant protein (SP)()B (SP-B) and surfactant protein C (SP-C), make up for 13% of the surfactant-specific proteins(3) .

SP-B is essential for the reduction of surface tension in the lung. A recent case report presented clinical evidence that deficiency of this protein is life threatening(4) . If the activity of SP-B was blocked by monoclonal antibodies, an acute inflammatory reaction was seen, together with a decrease in lung-thorax compliance(5) . Evidence has been presented that the activity of SP-B is enhanced by the hydrophilic surfactant protein A(6, 7) .

SP-C, a protein of 35 amino acid residues, is very hydrophobic due to a high content of Val, Ile, and Leu (60% of sequence). The hydrophobic amino acid residues form a long contiguous stretch, reaching from residue 13 to residue 28. The primary structure is highly conservative. In monolayers(8) , as well as in bilayer systems(9, 10, 11) , the secondary structure is mainly -helical. Two palmitoylated cysteine residues can be found on positions 5 and 6(12, 13) , with the exception of canine SP-C, which has only one cysteine residue(14) . The exact function of the acylation is not clear yet, but it has been demonstrated that acylation of SP-C alters structure and physical properties of the protein(15) . A dimeric form of SP-C has been demonstrated, which exhibits surface tension-lowering properties differing from those of monomeric SP-C(16, 17) . SP-C from all known species has two positively charged residues on positions 11 and 12 (a lysine residue and an arginine residue, respectively)(14) , of which the function remains to be elucidated. SP-C, when present in phospholipid vesicles (18, 19) or monolayers(15, 20) , was able to catalyze the insertion of phospholipids into a monolayer or to induce the lipid mixing between neutral phospholipid vesicles and pyrene-PC-labeled vesicles(15) . It also altered the thermodynamic properties of membranes(11, 21) .

To elucidate the importance of the positively charged amino acids for the function or structure of the SP-C, we used the reaction of phenylglyoxal with arginine residues(22, 23) . When proteins are treated with a large excess of phenylglyoxal for a long period, the -amino group of the lysine residue may be modified too, thereby neutralizing all positive charges of SP-C. The reaction is very effective, and the formed derivative is sufficiently stable, especially under mildly acidic conditions(22) . We used the Wilhelmy plate method and circular dichroism and fluorescence experiments to study the role of the positively charged residues of SP-C. The experiments show that these residues are important to bind vesicles prior to the insertion of phospholipids from the vesicles into the monolayer.


EXPERIMENTAL PROCEDURES

Materials

SP-C was isolated from porcine lung lavage. Fresh porcine lungs were lavaged three to five times with a solution of 154 mM NaCl. The proteins were isolated by the method of Hawgood and co-workers(24) . Extraction of pulmonary surfactant was done with n-butanol(25) , which was later removed by rotary evaporation. The residue was dissolved in chloroform/methanol/0.1 M HCl (1:1:0.05, v/v/v), and the solution was centrifuged to remove the insoluble particles. Isolation and separation of SP-B and SP-C from the lipids was done by Sephadex LH-60 chromatography. The proteins were stored in a mixture of chloroform/methanol (1:1, v/v) at -20 °C. Fluorescamine was used to determine the concentration of the proteins(26) . The content of arginine and lysine amino acid residues was determined by amino acid analysis. Phospholipid phosphorus was measured with the method of Bartlett(27) . L--Dipalmitoylphosphatidylcholine (DPPC) was bought from Avanti Polar Lipids, Inc. (Alabaster, AL). L--Phosphatidyl-DL-glycerol and L--phosphatidylcholine were obtained from Sigma. L--Phosphatidylcholine 1-palmitoyl-2-(1-pyrenedecanoyl (pyrene-PC) was purchased from Molecular Probes.

Modification of SP-C

To modify SP-C, 300 µg of porcine SP-C was dried under nitrogen and redissolved in 50 µl of a mixture of N-ethylmorpholine (final concentration 0.2 M) in water and n-butanol (1:9, v/v), pH 8.0. After sonification for 15 s in a water bath sonicator, 50 µl of a 6% phenylglyoxal hydrate solution dissolved in the same buffer was added, and the sample was vortexed. After 60 h (25 °C), the buffer was replaced by 200 µl of chloroform/methanol/0.1 M HCl (1:1:0.05, v/v/v). The reagents were removed from the protein by passage of the reaction mixture through a Sephadex G-25 column. The modified protein was stored under mildly acidic conditions to prevent the decomposition of the derivative(22) .

SDS-Polyacrylamide Gel Electrophoresis

Electrophoresis of the protein was performed by one-dimensional Tricine/SDS-polyacrylamide gel electrophoresis(28) . Staining of the protein was done with silver stain (Bio-Rad).

HPLC

SP-C was analyzed on a reverse phase C column as described earlier(13, 15) .

Monolayer Studies

Lipids (DPPC/PG, 7:3 (w/w)), dissolved in chloroform/methanol (1:1, v/v), were dried under a continuous stream of nitrogen at room temperature. The remaining lipid film was hydrated in 25 mM Hepes (pH 7.0) or 25 mM Tris (pH 7, 9, or 11) and vortexed for 1 min. Sonication with a Branson B12 sonifier with a 0.5-inch flat-top disrupter tip for 5 20 s (10-s intervals) at 30 watts and at a temperature of 55 °C resulted in small unilamellar vesicles (SUV). The vesicles were kept at 37 °C and used the same day at which they were prepared. Measurements were done by the Wilhelmy plate method, using a Cahn 2000 electrobalance, in a temperature-controlled box at 37 °C. For the experiments with a constant surface area, a 5.5-ml Teflon trough was filled with subphase (Hepes buffer or Tris buffer) and stirred continuously. To determine the amount of lipid associated with the monolayer, a trace amount of [C]DPPC was added to the lipid vesicles. For these measurements, a 20-ml Teflon trough (5.3 5.7 cm) was used. Lipids (DPPC/PG or DPPC/PC, 7:3 (w/w)) were mixed with SP-C in various amounts (lipid to protein ratio 5:1 or 10:1 (w/w)). The mixture was spread on the subphase to a surface pressure of 20.0 ± 0.1 mN/m. SUV (end concentration, 10-20 nmol/ml) made of DPPC/PG (7:3, w/w) were injected through an injection hole into the subphase. Lipid insertion from SUV took place without further addition or was initiated after the injection of CaCl (final concentration of the subphase, 3 mM) through the injection hole. The surface pressure was recorded for maximally 45 min. The surface radioactivity was detected using a gas-type detector. After the experiment was ended and a sample of the subphase was taken, the subphase was flushed for 10 min with 25 mM Hepes containing 10 mM EGTA (pH 7.0), with a flow of 10 ml/min. The radioactivity that was not bound to the monolayer was removed; the surface pressure was unaffected by this procedure. Afterward, the monolayers were collected. The interface radioactivity was corrected for the radioactivity of collected subphase(20, 29) .

CD Measurements

Secondary structure of SP-C was analyzed by use of a CD meter as described before(15) .

CD Spectra Analysis

CD spectra were analyzed with the protein secondary structure program obtained from the Japan Spectroscopic Co., Ltd (Tokyo). A r.m.s. error was obtained and gave an indication of the validity of the prediction of the secondary structure. The lower the value, the more reliable the prediction. If the r.m.s. value was higher than 10, the result was not considered as reliable.

Lipid-mixing Experiments

The method used was a modification of the method previously described(30) . All experiments were done at 37 °C. SUV (15 nmol of lipid and DPPC/PG/pyrene-PC or DPPC/PC/pyrene-PC, 63:27:10 (w/w/w)), containing various amounts of protein, were mixed with SUV (300 nmol of lipid, DPPC/PG, 7:3 (w/w)). Hepes (25 mM, pH 7.0) with EGTA (0.2 mM) was used as a buffer. CaCl was added to a final concentration of 3 mM. Fluorescence measurements were done on a Perkin-Elmer luminescence spectrometer (LS50), linked to a personal computer, under continuous stirring. Fluorescence emission spectra were recorded (excitation wavelength, 343 nm; emission wavelength, 360-550 nm) immediately after the initiation of the experiment. The process of lipid mixing was at that time completed. The monomer fluorescence maximum was found at 377 nm and the excimer fluorescence maximum at 475 nm. The excimer-monomer ratio was calculated and used to express the extent of lipid mixing.

Pulsating Bubble Measurements

Lipids (DPPC/PG, 7:3 (w/w)) were mixed with 2% SP-C (by weight), and dried under a stream of nitrogen. The film was stored overnight in an exsiccator. The next day, the sample was rehydrated while shaking for 10 min in 200 µl of 0.9% NaCl with 1.5 mM CaCl at 60 °C. To ensure that all lipids were in suspension, the glass tube was held twice in a sonicator bath for 15 s. In all cases, before samples were taken for assay, the tubes were vortexed for a short time. Surface tension was measured with a pulsating bubble surfactometer (Electronetics Corp., Amherst, NY) at 37 °C. The surface tension of a bubble with a radius of 0.4-0.55 mm was recorded for 10 pulsations (20 pulsations/min).


RESULTS

The proteins were separated from the lipids by Sephadex LH-60 chromatography after butanol extraction of pulmonary surfactant. The fractions were analyzed by Tricine/SDS-polyacrylamide gel electrophoresis under non-reducing conditions and colored by silver staining. The SP-C fractions were pooled and, after modification, analyzed on a reverse phase C column. The modified SP-C exhibited a peak at 45 min, whereas the natural SP-C fraction came off the column at 37 min (Fig. 1). Amino acid analysis showed that 94.7% of the arginine residues and 24.2% of the lysine residues were modified. Circular dichroism measurements showed a difference in secondary structure between the natural porcine SP-C and SP-C collected from the interface (). Due to the modification of the natural SP-C, an increase in the -helix content from 63.5 to 73.6% was observed. The content of random coil decreased from 36.4 to 26.3%. There was no -structure found in these proteins.


Figure 1: HPLC chromatograms for normal and neutralized porcine SP-C.



We followed the calcium-dependent insertion of phospholipids from SUV (10 nmol/ml) into protein-containing monolayers (lipid to protein ratio, 5:1 (w/w)) as a function of time by monitoring the increase in surface pressure and surface radioactivity. The maximum surface (equilibrium) pressure was 48.0-48.5 mN/m. Natural SP-C catalyzed the insertion of phospholipids into a negatively charged monolayer in a protein-dependent way, which agreed with our earlier studies (8, 15) (Fig. 2A, insertion rate 2.25 mN/m/min). If SP-C was used, the insertion rate of phospholipids into the monolayer was strongly reduced (Fig. 2B, insertion rate 0.46 mN/m per min). In the absence of negative charges in the monolayer, the insertion of phospholipids into the monolayer catalyzed by the natural SP-C was faster (Fig. 2, AversusC; insertion rates 5.17 and 0.64 mN/m/min for SP-C and SP-C, respectively). In the absence of protein, no insertion of phospholipids into the monolayer was observed (not shown). The increase in surface radioactivity showed the same pattern as the increase in surface pressure (Fig. 2). The subphase was flushed with a buffer containing 10 mM EGTA to remove all vesicles that were not bound to the monolayer. While flushing, there was no visible effect on the surface pressure. The radioactivity in the subphase dropped to nearly background values. The amount of radioactivity at the interface was just slightly diminished after the flushing, indicating that phospholipids had been inserted into the monolayer, or phospholipid vesicles were firmly bound to the monolayer.


Figure 2: Effect of modification of SP-C on binding to and insertion of phospholipids into preformed SP-C-containing monolayers. A monolayer of DPPC/PG or DPPC/PC and SP-C was spread on a subphase of 25 mM Hepes (pH 7.0) to an initial surface pressure of 20 mN/m. The surface pressure (uppercurve) and the surface radioactivity (lowercurve) were measured simultaneously with time. After 5 min, vesicles (SUV of DPPC/PG, containing trace amounts of [C]DPPC; final concentration, 10 nmol of lipid/ml) and Ca, to a final concentration of 3 mM, were injected into the subphase. A, monolayer of DPPC/PG and natural SP-C; B, monolayer of DPPC/PG and modified SP-C; C, monolayer of DPPC/PC and natural SP-C; D, monolayer of DPPC/PC and modified SP-C.



If instead of a Hepes buffer a Tris buffer was used for the subphase (both of pH 7.0), there was no difference in insertion rate. The calcium-dependent insertion rate was reduced if a subphase with a pH higher than 7 was used (Fig. 3A, insertion rates of 10.98, 6.02, and 1.49 mN/m/min at pH 7.0, 9.0, and 11.0, respectively). If no negative charges were present in the monolayer, the insertion of phospholipids proceeded in the absence of calcium. In this case, the insertion was also pH-dependent; if the pH was higher than 7, a lower insertion rate was observed (1.85 and 0.10 mN/m/min at pH 9.0 and 11.0, respectively, compared with 9.00 mN/m/min at pH 7.0). After 15 min, calcium was added to the subphase. This enhanced the insertion rate (to 6.38 and 6.13 mN/m/min at pH 9.0 and 11.0, respectively), although the insertion rate leveled off quickly at pH 11.0. After the injection of calcium into the subphase, a small decrease in surface pressure was observed. The higher the pH of the subphase, the less increase there was in surface pressure (Fig. 3B).


Figure 3: Effect of pH on phospholipid insertion from SUV into preformed SP-C-containing monolayers. SUV of DPPC/PG (7:3, w/w) were injected into the subphase of 25 mM Tris (pH 7, 9, 11; 20 nmol of lipid/ml) underneath a preformed monolayer of DPPC/PG or DPPC/PC and natural porcine SP-C (20 mN/m). Lipid to protein ratio in the preformed monolayer is 10:1 (w/w). A, monolayer of DPPC/PG; at time 0, Ca was injected to a final concentration of 3 mM to start the insertion. B, monolayer of DPPC/PC; Ca was added after 15 min.



The surface properties of the surfactant proteins can also be measured with a pulsating bubble surfactometer(18) . The minimum surface tension of the pulsating bubble immediately fell to ±5 mN/m with the natural protein. Samples containing SP-C showed a reduction of the surface tension to only 10 mN/m (Fig. 4).


Figure 4: Comparison by pulsating bubble surfactometer of the surface tension lowering properties of natural and modified SP-C. Results are a mean of at least five measurements. The maximum and minimum surface tensions (mean ± S.D., n 5) are indicated by open and closedsymbols, respectively. , natural SP-C; , modified SP-C.



To investigate whether SP-C was able to induce lipid mixing, the protein was incorporated in pyrene-PC-containing vesicles. No protein-induced lipid mixing was observed if DPPC/PG/pyrene-PC or DPPC/PC/pyrene-PC vesicles containing natural SP-C were mixed with DPPC/PG vesicles (Fig. 5). If the vesicles contained SP-C, protein-induced lipid mixing was observed, both in the presence of DPPC/PG and DPPC/PC vesicles. The extent of lipid mixing was a function of the SP-C content.


Figure 5: Lipid mixing as a function of SP-C concentration. Pyrene-PC-labeled vesicles of (A) DPPC/PG (30 mol% PG) or (B) DPPC/PC (30 mol% PC) containing SP-C were mixed with DPPC/PG vesicles (30 mol% PG). The excimer/monomer ratio is a parameter of lipid mixing. The experiment was done at 37 °C. The average and S.D. of three experiments are shown. , vesicles containing natural SP-C and in the absence of CaCl; , vesicles containing natural SP-C and in the presence of 3 mM CaCl; , vesicles containing modified SP-C and in the absence of CaCl; , vesicles containing modified SP-C and in the presence of 3 mM CaCl.




DISCUSSION

In this study, we investigated the role of the positively charged residues in the SP-C protein. It has been proposed that the positive charges play a role in the determination of the transmembrane orientation(31) . Two positive charges, one lysine residue and one arginine residue, are found on positions 11 and 12, respectively, in a variety of species(14) . In this study, the positive charges in the SP-C molecule were neutralized by a modification of the methods described by Takahashi(22) . With this method, all arginine is changed (23) into a sufficiently stable non-charged derivative of arginine. As phenylglyoxal is somewhat less selective for arginine than is e.g. butanedione, also the lysine residue will be (partly) modified(32) . Amino acid analysis and HPLC analysis proved that the majority of the arginine residues and part of the lysine residues were modified. We show by analysis on a reverse phase C column that SP-C was more hydrophobic than the natural porcine SP-C. Circular dichroism measurements demonstrated that SP-C had an increased amount of -helix. Both experiments are in line with the expected neutralization of the positive charges of SP-C.

The insertion rate of phospholipids from DPPC/PG (7:3, w/w) SUV into a monolayer of the same composition containing natural porcine SP-C was calcium and protein dependent. If negatively charged phospholipids were absent in the monolayer, the insertion rate of lipids catalyzed by natural porcine SP-C in the presence of calcium was much higher, due to an increased association of vesicles with the monolayer. This causes smaller distances, and as vesicle monolayer interactions are partially determined by electrostatic forces, Van der Waals and hydration forces will become more important(33) . With the use of C-labeled vesicles, we showed that binding of the negatively charged vesicles to the monolayer by the SP-C was decreased, and consequently a reduced insertion was obtained. There was no indication of a reduced insertion of phospholipids into the monolayer.

As the pK of the guanido group of arginine is about 12.5 (32), the positive charge of the porcine SP-C can be altered if the pH of the subphase is changed in Wilhelmy plate experiments. The highest insertion rate of lipids from negatively charged vesicles was found if a subphase was used with a pH of 7.0. With increasing pH, the insertion rate decreased.

The reduced insertion had to be the result of a reduced function of the SP-C after changing the positive charges of the protein. Insertion of phospholipids was possible, even at a pH of 11.0. This insertion was induced by SP-C and started after the addition of calcium ions. This indicated that there was no denaturation of SP-C. The expected increase in -helix can possibly contribute to the insertion. At the moment that Ca was added, a small dip in the surface pressure was visible (Fig. 3), which could be explained by the condensing effect of the Ca ions on the (already inserted) PG in the monolayer(8) . With the pulsating bubble surfactometer, cyclic adsorption measurements were done, which also showed the reduced function of the neutralized SP-C.

It has been shown earlier that SP-C did not catalyze mixing of lipids of negatively charged lipid vesicles(30) . If 300 nmol of non-charged vesicles containing SP-C were mixed with pyrene-PC-labeled negatively charged vesicles, SP-C-induced lipid mixing was possible(15) . In the present paper, SP-C was present in negatively charged or neutral vesicles. When 15 nmol of these were mixed with 300 nmol of negatively charged vesicles, the relatively small quantity of SP-C was not able to induce lipid mixing. The modified protein, however, was able to induce lipid mixing, even in the absence of calcium. The mechanism by which this process is possible is not yet understood.

In all experiments after the addition of calcium, a decrease in excimer/monomer ratio was observed. This indicates that the positive charge of Ca alone is sufficient to overcome partly the repulsive forces between the vesicles.

In conclusion, the positive charges of the pulmonary surfactant protein C are important both for structure and function of SP-C. The positive charges are responsible for binding of the PG-containing small unilamellar vesicles to the monolayer, a process that precedes the insertion of phospholipids into the monolayer.

  
Table: Comparison of the secondary structures of natural and modified SP-C at the air-water interface, as determined by circular dichroism

Samples were collected at a surface pressure of 30 mN/m. Average numbers (± S.D.) derived from spectra of two or three different monolayer preparations are given as percentages of total structure.



FOOTNOTES

*
This work was supported by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for Scientific Research (NWO). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: the Laboratory of Veterinary Biochemistry, P. O. Box 80.176, 3508 TD Utrecht, the Netherlands. Tel.: 31-30-535378.

The abbreviations used are: SP, surfactant protein; DPPC, dipalmitoylphosphatidylcholine; PC, phosphatidylcholine; PG, phosphatidylglycerol; SUV, small unilamellar vesicles; HPLC, high performance liquid chromatography; N, newton.


REFERENCES
  1. Haagsman, H. P., and van Golde, L. M. G. (1991) Annu. Rev. Physiol.53, 441-464 [CrossRef][Medline] [Order article via Infotrieve]
  2. Keough, K. M. W. (1992) in Pulmonary Surfactant From Molecular Biology to Clinical Practice (Robertson, B., van Golde, L. M. G., and Batenburg, J. J., eds) 2nd Ed, pp. 110-164, Elsevier Science Publishers, Amsterdam
  3. Phizackerley, P. J. R., Town, M.-H., and Newman, G. E. (1979) Biochem. J.183, 731-736 [Medline] [Order article via Infotrieve]
  4. Nogee, L. M., deMello, D. E., Dehner, L. P., and Colten, H. R. (1993) N. Engl. J. Med.328, 406-410 [Free Full Text]
  5. Robertson, B., Kobayashi, T., Ganzuka, M., Grossmann, G., Li, W.-Z., and Suzuki, Y. (1991) Pediatr. Res.30, 239-243 [Abstract]
  6. Hawgood, S., Benson, B. J., Schilling, J., Damm, D., Clements, J. A., and White, R. T. (1987) Proc. Natl. Acad. Sci. U. S. A.84, 66-70 [Abstract]
  7. Chung, J., Yu, S.-H., Whitsett, J. A., Harding, P. G. R., and Possmayer, F. (1989) Biochim. Biophys. Acta1002, 348-358 [Medline] [Order article via Infotrieve]
  8. Oosterlaken-Dijksterhuis, M. A., Haagsman, H. P., van Golde, L. M. G., and Demel, R. A. (1991) Biochemistry30, 10965-10971 [Medline] [Order article via Infotrieve]
  9. Pastrana, B., Mautone, A. J., and Mendelsohn, R. (1991) Biochemistry30, 10058-10064 [Medline] [Order article via Infotrieve]
  10. Vandenbussche, G., Clercx, A., Curstedt, T., Johansson, J., Jörnvall, H., and Ruysschaert, J.-M. (1992) Eur. J. Biochem.203, 201-209 [Abstract]
  11. Shiffer, K., Hawgood, S., Haagsman, H. P., Benson, B., Clements, J. A., and Goerke, J. (1993) Biochemistry32, 590-597 [Medline] [Order article via Infotrieve]
  12. Curstedt, T., Johansson, J., Persson, P., Eklund, A., Robertson, B., Löwenadler, B., and Jörnvall, H. (1990) Proc. Natl. Acad. Sci. U. S. A.87, 2985-2989 [Abstract]
  13. Stults, J. T., Griffin, P. R., Lesikar, D. D., Naidu, A., Moffat, B., and Benson, B. J. (1991) Am. J. Physiol.261, L118-L125 [Medline] [Order article via Infotrieve]
  14. Johansson, J., Persson, P., Löwenadler, B., Robertson, B., Jörnvall, H., and Curstedt, T. (1991) FEBS Lett.281, 119-122 [CrossRef][Medline] [Order article via Infotrieve]
  15. Creuwels, L. A. J. M., Demel, R. A., van Golde, L. M. G., Benson, B. J., and Haagsman, H. P. (1993) J. Biol. Chem.268, 26752-26758 [Abstract/Free Full Text]
  16. Baatz, J. E., Smyth, K. L., Whitsett, J. A., Baxter, C., and Absolom, D. R. (1992) Chem. Phys. Lipids63, 91-104 [CrossRef][Medline] [Order article via Infotrieve]
  17. Creuwels, L. A. J. M., Demel, R. A., van Golde, L. M. G., and Haagsman, H. P. (1995) Biochim. Biophys. Acta, 1254, 326-332 [Medline] [Order article via Infotrieve]
  18. Takahashi, A., and Fujiwara, T. (1986) Biochem. Biophys. Res. Commun.135, 527-532 [Medline] [Order article via Infotrieve]
  19. Warr, R. G., Hawgood, S., Buckley, D. I., Crisp, T. M., Schilling, J., Benson, B. J., Ballard, P. L., Clements, J. A., and White, R. T. (1987) Proc. Natl. Acad. Sci. U. S. A.84, 7915-7919 [Abstract]
  20. Oosterlaken-Dijksterhuis, M. A., Haagsman, H. P., van Golde, L. M. G., and Demel, R. A. (1991) Biochemistry30, 8276-8281 [Medline] [Order article via Infotrieve]
  21. Pérez-Gil, J., Nag, K., Taneva, S., and Keough, K. M. W. (1992) Biophys. J.63, 197-204 [Abstract]
  22. Takahashi, K. (1968) J. Biol Chem.243, 6171-6179 [Abstract/Free Full Text]
  23. Takahashi, K. (1977) J. Biochem. (Tokyo) 81, 395-402 [Abstract]
  24. Hawgood, S., Benson, B. J., and Hamilton, R. L. (1985) Biochemistry24, 184-190 [Medline] [Order article via Infotrieve]
  25. Haagsman, H. P., Hawgood, S., Sargeant, T., Buckley, D., White, R. T., Drickamer, K., and Benson, B. J. (1987) J. Biol. Chem.262, 13877-13880 [Abstract/Free Full Text]
  26. Böhlen, P., Stein, S., Dairman, W., and Udenfriend, S. (1973) Arch. Biochem. Biophys.155, 213-220 [Medline] [Order article via Infotrieve]
  27. Bartlett, G. R. (1959) J. Biol. Chem.234, 466-468 [Free Full Text]
  28. Schägger, H., and von Jagow, G. (1987) Anal. Biochem.166, 368-379 [Medline] [Order article via Infotrieve]
  29. Sasaki, T., and Demel, R. A. (1985) Biochemistry24, 1079-1083 [Medline] [Order article via Infotrieve]
  30. Oosterlaken-Dijksterhuis, M. A., van Eijk, M., van Golde, L. M. G., and Haagsman, H. P. (1992) Biochim. Biophys Acta1110, 45-50 [Medline] [Order article via Infotrieve]
  31. Keller, A., Eistetter, H. R., Voss, T., and Schäfer, K.-P. (1991) Biochem. J.277, 493-499 [Medline] [Order article via Infotrieve]
  32. Riordan, J. F. (1979) Mol. Cell. Biochem.26, 71-92 [Medline] [Order article via Infotrieve]
  33. Lis, L. J., McAlister, M., Fuller, N., Rand, R. P., and Parsegian, V. A. (1982) Biophys. J.37, 657-666 [Abstract]

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