Biophysica et Biophysiea Acta~ 493 (1977) 196-200 © Elsevier/North-Holland Biomedical Press

BBA 37699

ISOLATION OF APOPROTEIN FROM BOVINE LU N G SURFACTANT MA- TERIAL BY SODIUM DEOXYCHOLATE

HIDEO SAWADA a, HAJIME YAMABAYASHI a and YOSHIAKI OKAJIMA b a Department oflnternal Medicine, Tokai University School of Medicine, Bohseidai, lsehara 259-11 and b Section of Microbiology, Central Research Laboratory, Tokai University School of Medicine, Bohseidai, Isehara 259-11 (Japan)

(Received November 1 lth, 1976) (Revised manuscript received March 7th, 1977)

SUMMARY

Aqueous solutions of bovine lung surfactant material were solubilized with a high concentration of sodium deoxycholate and the protein moiety could then be separated from the mixed lipid-sodium deoxycholate micelles by gel filtration in the presence of a micellar concentration of sodium deoxycholate of 10 mM. The lipid-free protein showed only one detectable peak in the gel filtrate and the sedimentation rate of this protein was 12.1 S. Sodium dodecyl sulfate disc gel electrophoresis showed essentially the same pattern as did the protein extracted directly from the bovine lung surfactant material with organic solvents, of which the major component has a mole- cular weight of 36 000. This protein should be the main apoprotein of lung surfactant material, and the main band in the sodium dodecyl sulfate disc gel electrophoresis, with a molecular weight of 36 000, would constitute the major protein subunit.

INTRODUCTION

Lung surfactant material has been identified as a lipoprotein, of which apo- protein is the subject of current interest in a few laboratories [1, 2]. King et al. [1] were the first who isolated and identified the apoproteins from sodium dodecyl sulfate (SDS)-treated canine surface active material [1]. SDS usually splits protein into its polypeptide subunits and the possibility remains that the native apoprotein consists of a combination of the subunits identified in SDS disc gel electrophoresis. Hetenius and Simons [3] demonstrated that both sodium deoxycholate and SDS can yield lipid-free protein from human low density lipoprotein by gel filtration, and that sodium deoxycholate does not alter the immunological properties of the resulting soluble apoprotein while SDS does. Applying their method to bovine lung surfactant material, we have tried to obtain the apoprotein free of lipids. The present paper describes the separation of apoprotein from bovine lung surfactant material by gel filtration in the presence of sodium deoxycholate and the sedimentation behavior of this isolated apoprotein.

Abbreviation: SDS, sodium dodecyl sulfate.

197

MATERIALS AND METHODS

Bovine lung surfactant material was isolated from lung washings by Frosolono's method [4]. Samples containing 4-5 mg of protein were dialyzed against 0.05 M Na2CO3 and 0.05 M NaCI (adjusted pH 10 by adding NaHCO3) overnight at 4 °C. In order to prevent protein degradation through the process of solubilization and gel filtration, two kinds of proteinase inhibitors, phenylmethyl sulfonyl fluoride (PheMe- SOzF, Sigma) and tosylamide phenylethyl chloromethyl ketone (TosPheCH2C1, Sigma) were dissolved in dimethyl sulfoxide and were added to the sample solution in a final concentration of 10 -4 M. The mixture was incubated for 15 min at 37 °C. 70 mg of sodium deoxycholate per mg of protein were added to solubilize the sample.

Gel filtration. Swelling, equilibration and packing of the Sephadex and Sepharose gels (Pharmacia) were done as recommended by the manufacturer. The columns were fitted with flow adaptors and run upward with the aid of peristaltic pump connected to the inlet tube. Filtration was performed at room temperature. Blue Dextran (Pharmacia) and pyridoxine (Daiichi) were used to calibrate the columns.

SDS disc gel electrophoresis. This was performed following Fairbanks' method [5] in 7.5 ~o polyacrylamide gel.

Sedimentation measurements. These were carried out using an Hitachi Ana- lytical Ultracentrifuge Model 282, fitted with Schlieren optics.

Protein [6] and phosphorus [7] were determined.

RESULTS

Removal of lipids using sodium deoxycholate. The sample solution was applied to a Sephadex G-75 column (2.5 × 22.5 cm) equilibrated with 10 mM sodium deoxy- cholate, 0.05 M NaC1, 0.05 M Na2CO3, and 1 mM PheMeSOzF and TosPheCH2C1 (adjusted pH 10 by adding NaHCO3). The protein eluted in void volume was a clear solution (Fig. 1). The recovery of protein was 77.8 ~ (mean of five experiments). The protein fraction contained no detectable phospholipids and the phospholipids eluted were in a separate peak as shown in Fig. 1.

Gel filtration of the protein fraction in a Sepharose 6B column. The protein fraction from Sephadex G-75, collected and concentrated by ultrafiltration, was then applied to a Sepharose 6B column (2.5 × 90 cm) equilibrated with the same buffer as used in the Sephadex G-75. Only one detectable peak at 280 nm was observed at Kay 0.3. Because of the difficulty of obtaining optimum protein standards, the deter- mination of molecular weight with the gel filtration technique has not as yet been successful.

SDS disc gel electrophoresis patterns. The protein peak both in Sephadex G-75 and in Sepharose 6B was subjected to SDS disc gel electrophoresis. Both protein fractions showed essentially the same pattern and the major band was calculated to have a molecular weight of 36 000; the pattern was the same as for the protein extracted directly from the lung surfactant material [8].

Sedimentation measurements of the apoproteins. Sedimentation studies on the protein fraction from Sephadex G-75 also showed one detectable peak (Fig. 2). The sedimentation behavior of the protein from Sepharose 6B was checked in comparison with that from Sephadex G-75 using a double cell (Fig. 2) and was judged almost

198

.uMOI

1.0

0 .l= O.

t - O.

Q5

/ I'0 20

PROTEIN

, t .

; \

; \

M '\. 3'0 4'o 5o s'o io

t

o so 9o ,oo

tube no.

Jug

I00

5 0 C

Q

e-i

Fig. 1. Separation of apoprotein and phospholipids from lung surfactant material by gel filtration in the presence of sodium deoxycholate. Column: Sephadex G-75 (2.5 × 22.5 cm). Buffer: 10mM sodium deoxycholate/0.05 M NaC1/0.05 M NazCO3 (adjusted pH 10 by adding NaHCO3). Sample contains 4 mg protein in 2 ml of 0.05 M NaCI/0.05 M Na2CO3 (adjusted to pH 10 by adding NaHCO3), with 280 mg of sodium deoxycholate. Elution volumes of Blue Dextran and pyridoxine are indicated with thick solid arrows.

identical. After several repetitions of the measurement on the protein fraction from Sephadex G-75, the sedimentation rate was determined to be 12.1 S with good repro- ducibility.

DISCUSSION

At the initial stages of our experiments, without adding proteinase inhibitors, the protein peak in void volume from the Sephadex G-75 gel filtration was followed by a long tailing toward total volume. When the protein fraction was concentrated and applied to the same column of Sephadex G-75, the lower peak in void volume and the longer tailing appeared in the subsequent gel filtration. By adding two kinds of proteinase inhibitors, the tailing disappeared and fairly good reproducibility of the protein peak in void volume was obtained. This suggests the existence of certain kinds of proteinase in the lung surfactant material. These would exert their effect on apoprotein which had been split off the lipids by sodium deoxycholate.

The surfactant apoprotein, obtained as described above, consists of a major protein component with 12.1 S. Single measurement of deoxycholic acid was done on the protein peak eluted in void volume of Sephadex G-75 by gas-liquid chromato- graphy according to Grundy et al. [9]. The deoxycholate bound to the protein 'was estimated by subtracting the amount of deoxycholic acid in the elution buffer from that in the fraction of the protein peak. Deoxycholate/protein (w/w) was 0.28. Relatively large amount of deoxycholate was bound to the protein, an evidence sug- gesting that the apoprotein is lipophilic [10]. Molecular weight of this apoprotein

199

Fig. 2. Sedimentation patterns of the apoprotein from lung surfactant material. Upper: the apo- protein from Sephadex G-75 gel filtration. 10 mM sodium deoxycholate/0.05 M Na2CO3/0.05 M NaCl buffer (adjusted pH 10 by adding NaHCO3) at 16 min and 60 000 rev./min. Lower: comparison of sedimentation patterns between the apoprotein from Sephadex G-75 gel filtration (lower) and the apoprotein from Sepharose 6B gel filtration (upper). The same buffer as above at 25 min and 60 000 rev./min.

was estimated about 20. l04, assuming a spherical protein and considering the amount of sodium deoxycholate bound to the protein [11]. The SDS disc gel electrophoresis pattern was essentially the same as that of the protein extracted from lung surfactant material with organic solvent. The major protein band of the latter (70-80 ~ of the total protein content) has molecular weight of 36 000. These findings suggest that the apoprotein isolated by gel filtration in the presence of sodium deoxycholate is an apoprotein of lung surfactant material and that the major band in the SDS disc gel electrophoresis with a molecular weight of 36 000 is its main constituent or its major protein subunit.

ACKNOWLEDGEMENTS

Authors thank Dr. S. Kashiwamata in Aichi Prefectural Colony and Dr. T.

200

Saeki in Depar tment of Biochemistry, Tokai Universi ty School of Medicine for their useful suggestions. They also thank Dr. S. Tazume in Depar tment of Microbiology, Tokai University School of Medicine for the measurement of deoxycholic acid.

REFERENCES

1 King, R. J., Klass, D. J., Gikas, E. G. and Clements, J. A. (1973) Am. J. Physiol. 224, 788-795 2 Klass, D. J. (1973) Am. Rev. Respir. Dis. 107, 784-789 3 Helenius, A. and Simons, K. (1971) Biochemistry 10, 2542-2547 4 Frosolono, M. F., Charms, B. L., Pawlowski, R. and Slivka, S. (1970) J. Lipid Res. 11,439-457 5 Fairbanks, G., Steck, T. L. and Wallach, D. F. H. (1971) Biochemistry 10, 1971-2617 6 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 226, 497-

509 7 Eibl, H. and Lands, W. E. M. (1969) Anal. Biochem. 30, 51-57 8 Sawada, H. and Kashiwamata, S. (1977) Biochim. Biophys. Acta 490, 44-50 9 Grundy, S. M., Ahrens, E. M. H. and Miettinen, T. A. (1965) J. Lipid Res. 6, 397-410

10 Helenius, A. and Simons, K. (1972) J. Biol. Chem. 247, 3656-3661 11 Tanford, C. (1961) Physical Chemistry of Macromolecules, Wiley, New York