Pulmonary Surfactant" Distribution of Lipids, Proteins and Surface Activity in Ultracentrifugation of Rabbit Pulmonary Washing and Derived Fractions GIUSEPPE COLACICCO, APURBA K. RAY, and A.R. BUCKELEW, Jr., Departments of Pathology and Pediatrics, Albert Einstein College of Medicine, Bronx, NY 10461

ABSTRACT

Lavage from normal adult rabbit lung and two known derived fractions, Fraction T and Fraction S, were subjected to either differential ultracentrlfugation in 1.090 g/ml KBr or sucrose density gradie n t u l t r a c e n t r i f u g a t i o n ; the surface activity of the lipid extract of selected fractions was measured. In differential ultracentrifugation, the three starting materials yielded a pellicle containing > 85% of the phospholipid with 8 0 % of the total sIgA (protein T) and up to 60% of the total phospholipid, whereas all the albumin and IgG were found at very low densities, 1.010 and 1.025, respectively; Fraction T, having nearly equal weights of one single protein, sIgA, and phospholipid, produced two contiguous bands at densities 1.059 and 1.078, totalling >85% of its phospholipid and < 2 5 % of its protein, the balance of which was found free of phospholipid at densities 1.020 to 1.050; comprising >80% of the phospholipid and < 2 0 % of the protein of pulmonary washing, Fraction S yielded two small bands at densities 1.028 and 1.044 and a major band at d = 1.059. In surface activity measurements: when the total lipid extract of the bands from the sucrose density gradient ultracentrifugation was spread as a film, in spite of similarly high dipalmitoyl lecithin contents (about 70% palmitoyl residue), the lipid of the band of Fraction T and that of the high density band of Fraction S were very active (~min = 0); whereas the lipid of the band of pulmonary washing and that of the lowest density band of Fraction S were not active, ]tmi n being 18 dyne/cm and 21 dyne/cm,

respectively. This work brings forth three major conclusions. First, under conditions which are used to isolate serum lipoproteins, no lipoprotein was obtained from either of the three surfactant fractions and most of the lipid was found virtually free of protein. Second, the isopicnic equilibrium of a given ultracentrlfugation fraction varied with the molecular structure of its constituents and could not be accounted for by the latter's average densities; instead, major roles must be played by particle geometry and their water contents. Third, although the various lipid samples contained the same quantities of palmitoyl residues (70%), the surface activities varied with the physical state of the lipid, method of assay, and some other undefined factors. INTRODUCTION

Although it is recognized that dipalmitoyl lecithin (DPL) is the essential component of pulmonary surfactant (1-4), there is no evidence as yet for the origin, state, and role of certain major proteins (albumin, IgG, and slgA) that were found with the lipid in rabbit pulmonary washing and in two derived surfactant fractions: Fraction T, which contains slgA as the only major protein, and Fraction S, which comprises most of the phospholipid of pulmonary washing and small quantities o f albumin, IgG, and slgA (5,6). Several surfactant fractions have been derived from pulmonary washing, a surfactant preparation by itself (2). Different procedures yielded highly surface active fractions with little protein from dog (7) and rabbit (2) lung washings. In contrast, the surfactant fraction obtained by King and Clements (8) from dog tracheal lavage contained appreciable amounts of a small MW protein (about 10,000 dalton), which was identified as lung specific and part of the lung surfactant system (8-10). Two other proteins of MW 62,000 and 36,000, found in the pulmonary washings of various species, have also been related to pulmonary surfactant (9,11-13). Another surface active preparation 879

G. COLACICCO, A.K. RAY, AND A.R. BUCKELEW, JR.

880

was isolated from homogenate as well as pulmonary wasb.hag of dog lung; this fraction which was erroneously thought to be a specific lipoprotein surfactant, consisted of about 80% lipid and 20% protein (14); the latter had the electrophoretic mobility of protein T (2) or slgA (6). Recent studies (6,15) have established that protein (14) to be also IgA, which is present in the pulmonary washing of several species (15,16), is not an alveolar protein (17) and cannot therefore be a component of alveolar surfactant. The relative quantities of proteins as well as llpids were made to vary with the methods o f lung lavage (I 8). Two phenomena are evident from a rapid examination of this background. First is the enormous diversity in the preparative procedures, diversity which must be considered when one is trying to account for the marked differences in yield, composition, and surface activity of the given fractions; this phenomenon was briefly referred to by King and Clements (Ref. 19, p. 724). Second, most of the cited methods considered only a selected, relatively small fraction, either in a band in the density gradient, in a pellicle floating on heavy medium, or in a pellet, whereas most of the phospholipid surfactant was either discarded (13,14) or lost in the many-step procedures, including dialysis (8). Using differential ultracentrifugation and primarily sucrose density gradient ultracentrifugation, we studied the behavior of rabbit pulmonary washings and Fractions T and S (5,6). Unlike previous reports (8,13,14), we examined the lipid and protein composition of all the aliquots of the density gradient instead of an arbitrarily selected band. MATERIALS AND METHODS Pulmonary Washing

This was obtained from freshly excised lungs of New Zealand white rabbits (3 to 4 kg) by the following method. The rabbit was anesthetized with 15 mg/kg pentobarbital and then sacrificed by transection of the abdominal aorta. The trachea was clamped during inhalation, and the lungs were removed en block and put on crushed ice. Saline, at room temperature, 10 ml/g wet lung, was introduced with a syringe via trachea, aspirated, and pushed back gently at the rate of 0.20 cycle/min; at the end of the second cycle, the milky white lavage was collected (60 to 70 ml from 100 ml of starting saline). The crude pulmonary washing was centrifuged at 1,000 x g, 0 C, for 20 min, in order to remove cells (mostly macrophages) and other debris; centrifugation at 750 x g for 10 rain (13) yielded identical results. LIPIDS, VOL. 12, NO. 11

Fraction T (FxT) and Fraction S (FxS)

The supernatant of the first (low speed) centrifugation, or cell-free PW, was concentrated 20 x by vacuum dialysis at 4 C; 10 ml of concentrated PW, equivalent to 200 ml of original PW, was applied to a Sephadex G-200 column that was equilibrated with 1 mM trisHC1 in 1 M NaC1, containing 1 mM EDTA, pH 7.5. After the usual elution (11), the fraction collected in the void volume (Fraction I) was dialyzed against 0.15 M NaC1 and then centrifuged at 49,000 x g for 1 hr at 4 C. The supernatant, referred to as F x T contained most of protein T and a nearly equal weight of phospholipid. The pellet, FxS, contained most of the lipid of dialyzed Fraction I and a mixture of three proteins, serum albumin, 3,-globulin, and slgA (6). The lipid and protein compositions were similar to those already published for both fractions (5,6). The phospholipid/prorein ratio varied from 0.5 to 1.5 in FxT and 4 to 8 in FxS. Protein and Lipid Standards

Fibrinogen and thyrogiobulin were obtained f r o m Sigma Chemical Co., St.Louis, MO; lysozyme from Worthington Biochemical Co., Milburn, NJ; and ovalbumin from ScliwarzMann Laboratories, New York, NY. Rabbit s e r u m albumin and "/-globulin were from Pentex, Kankakee, IL. Lipid standards were purchased from Supelco (BeUefonte, PA). Chromatographically homogeneous methyl esters of 10:0, 12:0, 14:0, 14:1, 16:0, 16:1, 18:0, 18:1, 18:2, and 20:0 fatty acids were products of Applied Science Laboratory, State College, PA. Aqueous dispersions of lipid, protein, and lipid-protein systems were attained by a 5 rnin ultrasonic irradiation in ice at 80 watts in a sonifier eel/ disruptor (Model W 185D, Branson Sonic Power Co., Plainview, Long Island, NY). Thin Layer Chromatography (TLC) of Lipifls

The lipids of given fractions were extracted into chloroform by shaking the aqueous fraction once with 4 vol of chloroform:methanol, 2:1, and a second time with 4 vol of chloroform. The two phases were separated by centrifugation in the cold. The lipids were then fractionated and identified by their migration on TLC plates precoated with Silica Gel G (Analtech, Newark, DE), using chloroform: m e t h a n o l : c o n e , ammonia:water, 70:30:2:4, and hexane:ether:acetic acid, 80:30:1, mixtures as developing solvent systems for the separation of phospholipids and neutral lipids (20), respectively. The lipid spots were shown either by staining with 12 vapor or by charring

PULMONARY SURFACTANT FRACTIONS after spray with sulfuric acid. The phospholipids were detected by m o l y b d e n u m trioxide spray (21). Ninhydrin spray (0.2% in 95% ethanol, acidified with glacial HAc) was used to reveal free amino groups on the TLC plates. The lipid composition was determined by densitometry and gas liquid chromatography against appropriate standards. The phospholipid and neutral lipid spots were scraped off the TLC plates and extracted with chloroform: methanol:ether, 1 : 1 : 1 , a n d chloroform: methanol:water, 1:2:0.8, respectively (22). The recovery was better than 95% when tritiated DPL and 14C-palmitic acid were used as markers. The lipid residue was subjected to phosphorus analysis in the case of phospholipids and to methylation and gas liquid chromatography (GLC) for all the lipids, except for cholesterol, which was estimated by densitometry of the charred spot after spraying with 50% H2SO 4. Quantitation of the lipids by GLC was performed by adaptation of the method described in Kales (22). Protein and Phosphorus Determinations

Protein concentration was estimated by the method of Lowry et al. (23) using 0.2% SDS as a dispersing agent and bovine serum albumin as a standard. Phosphorus was determined according to a modification of the method of Beveridge and Johnson (24), using perchloric acid for digestion; the phospholipid content was obtained by multiplying the phosphorus content by 25. Disc Gel Electrophoresis

881

Density Gradient Ultracentrifugation

The aqueous sample, 1 to 3 ml, was layered on top of a linear gradient which was prepared in 38.5 ml cellulose nitrate tube (Beckman) by mixing 0.1 M and 1.0 M sucrose (grade I, Sigma) in appropriate gradient maker. A 2 ml 2 M sucrose cushion was used. The gradients were centrifuged in SW 27 rotor at 80,000 x g for 18 hr at 4 C in a Beckman L2-65B preparative ultracentrifuge. Aliquots and/or visible bands were collected from the bottom using a piercing device and a photoelectric volumetric dispensor (Biichler Instruments, Fort Lee, NJ). The gradient was first divided in 16 aliquots, and, after analyses, for convenience the aliquots were combined in pairs to make the eight fractions that are described in tables and figures. The same technique was used to study density graclients in electrolyte solutions between 0.15 M and 1.75 M NaBr or KBr. Differential Oltracentrifugation

The cell-free pulmonary washing in 0.15 M NaC1 (d = 1.006) was made to density 1.090 by addition of solid KBr at 15 C and centrifuged in polycarbonate tubes of 38.5 ml capacity in a fixed angle rotor (Beckman Model 50.1) at 110,000 x g for 24 hr at 15 C in a preparative ultracentrifuge, Beckman Model L2-65B. The lipid pellicle was separated from the infranatant and analyzed for lipids and proteins. The differential ultracentrifugation was carried out at 15 C, since this temperature was used in the isolation of serum lipoproteins (2,26). Dispersions of concentrated Fraction T and Fraction S were diluted to contain 200/~g/ml phospholipid in 0.15 M NaC1, were then sonicated 15 min in ice (above), and finally subjected to ultracentrifugation. Sonication of the samples had no effect on the results. With selected samples, the ones containing unusually large protein contents, we used centrifugations in sequential density steps, i.e., 1.006, 1.063, and 1.210 g/ml KBr (2,26) with the view to ascertain if lipoproteins were present in the pulmonary washing.

Polyacrylamide, 7.5%, was used in the presence of 0.2% SDS according to the method of Weber and Osborn (25), except that no reducing agent was used~ Proteins of known MW served as standards; the protein bands were stained with Coomassie blue and quantitated densitometrically at 550 nm. To verify the identity of the proteins that we have always referred to as serum albumin and IgG (5,6), the two proteins were separated by preparative electrophoresis of pulmonary Gas Liquid Chromatography (GLC) washing on slab gel (7.5% polyacrylamide), GLC was performed in a Barber-Colman using tris-glycine buffer, pH 8.8. The proteins were extracted from the gel with small amounts (Chicago, IL) apparatus. To the dry lipid of 0.1% SDS in 0.05 M a m m o n i u m bicarbonate, sample, I ml mixture of methanol:boron tridialyzed against water, and lyophilized. Of fluoride:benzene, 3.5:3.5:3.0, was added, and equal aliquots of each protein, 50/ag each, one- methylation was carried out according to Morhalf were incubated with 2% /3-mercaptoeth- rison and Smith (27). The fatty acid esters were anol, 3 hr at 3 7 C and then 2 min at 100C, extracted in hexane, and the resulting solution using rabbit serum albumin and IgG as stan- was injected into the GLC column. The latter, 6 dards. Samples of the reduced and nonreduced ft long, consisted of B2-ethylene glycol succiproteins from pulmonary washing were run side nate 15% on anachrom ABS 90/IG0 mesh P. Temperature of the column was 170 C, and by side in slab gel electrophoresis as before.

LIPIDS, VOL. 12, NO. 11

882

G. C O L A C I C C O , A.IC R A Y , A N D A.R. B U C K E L E W , JR.

4O

RESULTS

0 Protem

/~

Although each experiment was repeated two to four times, each set of data represents one single experiment; the trends in all similar experiments were consistent.

2O co ._c

Lipid and Protein Composition of Rabbit Pulmonary Washing

10

o

"a g 4(

r

-5

,.Q

"~ 30 i:5 20 10 BOT I OW

I

I

I

I0

20

30

ml

I

40 IOPOF TUBE

FIG. 1. Percent distribution of phosphotipid (PL) (e) and protein (o) in Linear NaBr End KBr density gradients of rabbit pulmonary washing.

flow rate of the carrier gas, argon, was 60 ml/min. Swurface Activity

The dynamic surface tension, in continuous cyclic compression and decompression of the surfactant film, was determined by a modified Langmuir-Wilhelmy method according to the procedure described by Clements and Tierney (28). The trough, having 65 cm2 (100%) useful area, was carved out of a heavy Teflon block; the film was contained by a winding Teflon strip guided by a light Teflon piston, which was c o n n e c t e d to the automatic compressiondecompression mechanism. Surface tension was measured by a platinum plate suspended from a Cahn electrobalance. The amplified output of the electromechanical transducer and the area changes between 100% and 20% were fed, respectively, into the Y and X axes of an X-Y recorder, (Esterline Angus, Model XY-8511, Indianapolis, IN). The films were prepared by spreading the given fraction from either aqueous or organic medium on the aqueous hypophase, which consisted of 0.15 M NaCI, at 25 C. The solutions of reagent grade salts were prepared in water that was distilled twice (once over alkaline permanganate) and were foamed in order to remove surface active contaminants (29). L I P I D S , V O L . 12, NO. 11

Since the lipid composition of rabbit pulmonary washing is known (30) and the major lipids are unmistakably identifiable by migration on TLC plates, we did not seek new identification, which is also beyond the scope of this communication. Using the same lipids, we are only comparing the lipid patterns of the various centrifugation fractions. As already reported by us (5) and others (3 I), the PL and protein contents of individual rabbit pulmonary washings varied between 100 and 600 /ag/ml each. Such variations became smaller when the washings of 6 to 24 rabbits were pooled; then, the phospholipid and protein contents ranged from 250 to 350 /a/ml each, with most of the values between 300 and 350 /ag/ml. Although most of the individual lavages had a phospholipid/protein ratio near unity, we have seen preparations in which that ratio was as high as 3.0 and as low as 0.3. Itowever, in all cases, although the quantities of each lipid and protein could vary, there were no new lipids or proteins beside the ones that have always appeared in the lavage of normM rabbit lung, as in the following patterns: phosphatidyl choline >> cholesterol /> phosphatidyl ethanolamine -~ phosphatidyl glycerol > sphingomyelin i> cholesteryl ester > free fatty acid ~> phosphatidyl serine; albumin >~ sIgA > IgG. Occasionally we saw PW preparations with protein pattern slgA ~> albumin, especially when the lavage was prepared with ice cold saline in ice cold lung, or after the lung had been perfused with 1 /aM ritodrine, a ~ m i m e t i c (18). In line with previous results from gel filtration of normal rabbit pulmonary washing (5), disc gel electrophoresis of the latter showed an average protein distribution of 60% serum albumin, 20% sIgA (protein T), 10% IgG, and 10% minor proteins; among these, invariably appeared a protein of small MW, about 10,000. For simplicity, in the disc gel electrophoresis patterns we do not show the other minor proteins. The unstained albumin-like and IgGlike bands were eluted from the gel; after reduction with /3-mercaptoethanol, they yielded the expected products as revealed by gel electrophoresis and immunodiffusion, thus identifying the two proteins as albumin and IgG. The identify of IgA was reported elsewhere (6).

PULMONARY SURFACTANT FRACTIONS The lipid composition of PW had the following pattern: 70% phospholipid and 30% nonphospholipid or neutral lipid. The latter consisted of cholesterol (50%), cholesteryl ester (30%) and free fatty acid (20%); there were only traces of triglyceride. More than 70% of the phospholipid was lecithin, which, by GLC, had >I 70% palmitoyl residue; the lecithin and otherlipids for the GLC analysis were recovered trom the TLC plates (see Methods).

883 PW

TOP

BOTTOM

i

SDS-DISC ~,iL:: ;:!

PW

, ~i,i:lilt .163

/ / /

1040

Differential

Ultracentrifugation

After 24 hr centfifugation in 1.090 g/ml KBr at 110,000 x g, 15 C, no pellet resulted from either the cell-free pulmonary washing, Fraction T or Fraction S. The pellicle contained more than 80% of the phospholipid and virtually no protein, in a phospholipid to protein ratio of about 100/1; the trace amounts of protein in the pellicle consisted of albumin and slgA, both of which could be nonspecificaUy entrapped in the lipid particles. All the protein (albumin, IgG and slgA) was found in the infranatant, as indicated by protein analysis (Lowry) and by densitometry of the electrophoresis gels. Density Gradient Ultracentrifugation

The results of two types of experiments are presented: one with ~ a d i e n t s of NaBr and KBr in Figure 1 and one with gradients of sucrose in Figure 2 and Table I.

1,012

]JN L

Q o

PE PC

;qqo

I E

WI~

TLC

o

Fx.__~T

~

TOP

BOTTOM

T

-DISC

FxT

Pulmonary washing in NaBr and KBr density gradients (Fig. 1): The density profiles of these two gradients are similar to those of the corresponding sucrose gradients (below). In all the gradients, the phospholipid peaked sharply with the band at density near 1.040. The protein peaked either with the phospholipid, as in KBr, or at a slightly lower density, as in NaBr and in sucrose. In general, the protein was distributed in the low density regions of the gradient as shown also with the sucrose density gradient of pulmonary washing (below). Sucrose density gradients: A graphic representation of gradient and distribution of proteins in disc gels and lipids on TLC plates is shown in Figure 2 for pulmonary washing and Fraction T. Fraction S was omitted for simplicity, since the protein and lipid patterns were similar to those of pulmonary washings. The percent distribution of phospholipid and protein of the various gradient regions and bands is 9summarized in Table I for pulmonary washing, Fraction T, and Fraction S. (a) Pulmonary washing (Fig. 2, Table I): A single band appeared at density 1.040, containing most of slgA (T) and about 33% of the total phosphollpid; a considerable quantity of phos-

o

1.012

NL

~"~'

~' ~

PE

PC

~

*

g 0

"~ ~

~

TLC

g C)

9 FIG. 2. Disc gel electrophoresis pattern of proteins and thin layer chromatography pattern of lipids from all the aliquots of the sucrose density gradient ultracentrifugation of rabbit pulmonary washing and Fraction T. pholipid centrifuged together with the balance of protein T on the high density side of the visible band, i.e., in tubes 4, 5, and 6. In this particular sample, sIgA represented about 35%, as opposed to an average 20% of the total protein content of pulmonary washing. The abundance of sIgA was confirmed by the amount of this protein in tubes 3, 4, 5, and 6. The protein in tubes 7 and 8 was essentially the low MW protein mentioned above. T h e p h o s p h o l i p i d / p r o t e i n ratios of the banded material varied between 1.0 and 3.0 (1.71 in Table I). But after 48 hr dialysis at 4 C, LIPIDS, VOL. 12, NO. 11

G. COLACICCO, A.K. RAY, AND A.R. BUCKELEW, JR.

884

TABLE I Phospholipid (PL) and Protein Compositions of all the Aliquots from Sucrose Density Gradient Ultracentrifugations of (a) Rabbit Pulmonary Washing, (b) Fraction T and, (c) Fraction S Density

Tube

Protein %

PL %

PL/Protein

(a) 1.012 1.025 1.040 (band) 1.057 1.074 1.091 1.117 1.162 (b) 1.020 1.028 1.043 1.059 (band) 1.078 (band) 1.097 1.123 1.169

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8

21.0 26.2 24.6 7.0 5.5 3.0 4.4 8.7 3.4 27.9 35.4 17.0 8.8 1.7 1.7 4.1

9.3 14.1 32.5 27.0 13.0 2.0 0.8 1.7 0.0 3.8 5.7 43.9 42.5 1.1 1.4 1.4

0.57 0.70 1.71 5.16 3.00 0.89 0.25 0.25 0.00 0.14 0.17 2.77 5.17 0.70 0.86 0.35

(c) 1.016 1.028 (band I) 1.044 (band II) 1.059 (band III) 1.075 1.091 1.115 1.161

1 2 3 4 5 6 7 8

3.7 12.4 17.2 19.6 13.3 10.0 8.0 15.4

3.1 7.5 21.1 50.1 13.8 3.4 0.7 0.6

5.00 3.74 7.65 15.66 6.42 2.08 0.35 0.19

t h e ratio decreased c o n s i d e r a b l y (e.g., f r o m 1.71 to 1.07), because of loss of p h o s p h o l i p i d d u r i n g dialysis. The o t h e r t w o m a j o r p r o t e i n s , a l b u m i n a n d IgG, were f o u n d in t h e u p p e r r e g i o n s o f t h e g r a d i e n t w i t h ~< 23% p h o s p h o l i p i d a n d appreciable q u a n t i t i e s of n e u t r a l lipid ( c h o l e s t e r o l c h o l e s t e r y l ester). Unlike t h e p h o s p h o l i p i d s , t h e n e u t r a l lipids were d i s t r i b u t e d n e a r l y e v e n l y t h r o u g h o u t the gradient; all t h e free f a t t y acids were in t h e a l b u m i n f r a c t i o n . S t r i k i n g was t h e a p p e a r a n c e o f t h e small m o l e c u l a r w e i g h t p r o t e i n in all t h e g r a d i e n t f r a c t i o n s , p a r t i c u l a r l y in t h e h i g h d e n s i t y regions. The same p a t t e r n o f lipid a n d p r o t e i n d i s t r i b u t i o n in t h e g r a d i e n t was o b s e r v e d w h e n 1 0 x c o n c e n t r a t e d r a b b i t p u l m o n a r y w a s h i n g was used.

(c) Fraction S (Table 1): T h r e e b a n d s were o b s e r v e d , a m a j o r o n e ( B a n d III) at d = 1.059 a n d t w o m i n o r b a n d s at densities 1.044 a n d 1.028. Most o f t h e p h o s p h o l i p i d ( 5 0 % ) was in t h e h i g h density b a n d t o g e t h e r w i t h 2 0 % o f t h e p r o t e i n in a p h o s p h o l i p i d / p r o t e i n r a t i o o f 16; t h e r e m a i n i n g p r o t e i n was d i s t r i b u t e d m o r e or less evenly t h r o u g h o u t t h e g r a d i e n t . Disc gel electrophoresis showed three proteins, albumin a n d T-globulin in t h e u p p e r regions o f t h e gradient, a n d p r o t e i n T m a i n l y at densities 1.044, in a p a t t e r n t h a t was very similar t o t h e o n e o b s e r v e d w i t h p u l m o n a r y washing, in spite of t h e very large l i p i d / p r o t e i n r a t i o i n F r a c t i o n S.

(b) Fraction T (Fig. 2, Table 1): T w o wide b a n d s were seen, a h e a v y o n e at d = 1.078 a n d a light b a n d at d = 1.059. T h e c o m b i n e d b a n d s contained 86% of the total phospholipid of F r a c t i o n T a n d o n l y 26% o f t h e p r o t e i n . Most o f t h e p r o t e i n , sIgA (63%), was f o u n d in t w o l o w e r d e n s i t y regions, 1.028 a n d 1.043, w i t h little p h o s p h o l i p i d a n d some n e u t r a l lipid (see TLC data). T h e small MW p r o t e i n was also p r o m i n e n t in various regions o f t h e g r a d i e n t , a n d was c o n c e n t r a t e d in t h e h i g h e r d e n s i t y band.

B e c a u s e of k n o w n c o r r e l a t i o n s b e t w e e n f a t t y acyl chain c o m p o s i t i o n a n d surface t e n sion l o w e r i n g ability of l e c i t h i n s ( 2 , 3 2 - 3 4 ) , t h e t o t a l lipid was e x t r a c t e d f r o m e a c h of t h e ultrac e n t r i f u g a t i o n b a n d s o f p u l m o n a r y washing, F r a c t i o n T ( c o m b i n e d b a n d s ) a n d F r a c t i o n S, t h e f a t t y acyl c o m p o s i t i o n was d e t e r m i n e d b y gas liquid c h r o m a t o g r a p h y ( T a b l e II), a n d t h e d y n a m i c surface t e n s i o n was m e a s u r e d at 25 C w i t h films t h a t were spread f r o m e i t h e r t h e i n t a c t a q u e o u s f r a c t i o n or its t o t a l lipid e x t r a c t . The surface t e n s i o n vs. p e r c e n t area data are

LIPIDS, VOL. 12, NO. 11

Surface Activity

PULMONARY SURFACTANT FRACTIONS

885

TABLE II Fatty Acid Composition of Total Lipid Extracts of the Visible Bands from the Sucrose Density Gradient Ultracentrifugations of Pulmonary Washing, Fraction T and Fraction S (As Well as Lecithin Isolated from Rabbit Pulmonary Washing, in Extreme Left Column, PC). I

Fatty acid

FxS Bands II d=1.044

III d=1.059

PC a

PW

FxT

d=1.028

%

%

%

%

--2.4 0.3 68.3 7.3 1.6 12.5 7.6 10.5

2.2 2.6 0.9 67.4 4.6 2.9 14.2 5.1 18.0

. . . . . . . . . 2.8 9.7 0.4 1.4 69.7 69.6 4.0 3.2 2.5 --14.1 1.8 5.7 14.5 Zero 21.0

2.2 0.4 58.7 3.7 2.1 23.5 9.4 18.0

3.3 1.8 1.1 67.4 5.1 3.4 14.6 3.4 Zero

69.9

70.3

72.2

69.6

60.8

"/0.8

(Total) 30.1 (?) Not identified.

29.7

27.8

30.4

39.2

29.2

12:1(2) 14:0 14:1 16:0 16:1 18:0 18:1 18:x b ~/min Factors % Positive (C16:0 + C18:0) Negative

%

%

aPC, phosphatidyl choline isolated from rabbit PW (Ref. 2). bprobaly the cis-5-octadecenoic acid described by King et al. in dog lung (Ref. 43) and seen also by Colacicco and Scarpelli in rabbit pulmonary washing (Ref. 2). p r e s e n t e d in Figure 3. T h e surface t e n s i o n of saline, 72 d y n e / c m , was n o t a l t e r e d w h e n t h e a q u e o u s dispersion o f t h e i n t a c t b a n d s , c o n t a i n i n g 2 0 /ag to 90 /ag p h o s p h o l i p i d was a p p l i e d t o t h e saline's surface; this m e a n t t h a t n o lipid or p r o t e i n s p r e a d as a film f r o m t h e a q u e o u s samples. H o w e v e r , w h e n t h e same q u a n t i t y o f p h o s p h o l i p i d was a p p l i e d f r o m t h e s o l u t i o n o f t h e t o t a l lipid e x t r a c t o f t h e b a n d in organic s o l v e n t , t h e surface t e n s i o n was l o w e r e d t o s a t u r a t i o n values o f 18 d y n e / c m for pulmonary washing, zero f o r F r a c t i o n T a n d 21 d y n e / c m , 18 d y n e / c m a n d zero, respectively, for t h e l o w d e n s i t y ( 1 . 0 2 8 ) , i n t e r m e d i a t e density (1.044), and high density (1.059) bands o f F r a c t i o n S. DISCUSSI ON

In t h e light of t h e f o r e g o i n g data, we w i s h t o f o c u s o u r a t t e n t i o n o n five p h e n o m e n a , w h i c h s h o u l d be c o n s i d e r e d in t h e s t u d y o f (a) t h e m e c h a n i s m s o f lipid a n d p r o t e i n d i s t r i b u t i o n in the preparation and ultracentrifugation of various s u r f a c t a n t f r a c t i o n s a n d (b) t h e p h y s i cochemicalnature and behavior of pulmonary s u r f a c t a n t . T h e five p h e n o m e n a are: T h e lipoprotein nature of pulmonary surfactant, the

i d e n t i t y of t h e p r o t e i n s of p u l m o n a r y washing, t h e isopicnic e q u i l i b r i u m of t h e m a j o r p r o t e i n s o f p u l m o n a r y washing, t h e small m o l e c u l a r weight p r o t e i n , a n d t h e surface a c t i v i t y o f t h e lipid e x t r a c t of selected s u r f a c t a n t f r a c t i o n s . Lipoprotein Nature of Pulmonary Surfactant

U n d e r c o n d i t i o n s in w h i c h l i p o p r o t e i n s are i s o l a t e d f r o m s e r u m ( 2 , 2 6 ) , virtually n o p r o t e i n was f o u n d in t h e lipid pellicle t h a t f l o a t e d o n 1.090 g / m l KBr in d i f f e r e n t i a l u l t r a c e n t r i f u g a t i o n o f e i t h e r p u l m o n a r y wash/ng, F r a c t i o n T a n d F r a c t i o n S. Since less t h a n 1% p r o t e i n was f o u n d in t h e lipid pellicle a n d t h e p r o t e i n consisted o f traces o f s e r u m a l b u m i n a n d slgA, we also c o n c l u d e t h a t n o s e r u m l i p o p r o t e i n was p r e s e n t in r a b b i t p u l m o n a r y washing. Our results are d i f f e r e n t f r o m t h o s e of King a n d C l e m e n t s ( 8 , 1 9 ) , w h o , u s i n g a similar b u t not identical technique, found appreciable q u a n t i t i e s ( 7 t o 10%) o f a small MW p r o t e i n in t h e lipid pellicle. As it was p o i n t e d o u t b y t h e o t h e r a u t h o r s (Ref. 19, p. 724), s u c h v a r i a t i o n s c o u l d b e due t o large as well as s m a l l differe n c e s in t h e e x p e r i m e n t a l m e t h o d s . T w o specul a t i o n s can be o f f e r e d t o e x p l a i n t h e o b s e r v e d v a r i a t i o n s . First is t h e c o m p o s i t i o n o f t h e lavage LIPIDS, VOL. 12, NO. 11

G. COLACICCO,A.K. RAY,AND A.R. BUCKELEW,JR.

886 72

-

lipoprotein nature of such an association. First, it was recently found that protein T is an immunoglobulin A (6). Second, by immunofluorescence, the protein was not found in the alveolar lining layer (17). Third, a lipoprotein with a fixed stoichiometry would have banded in the gradient at a fixed density. Instead, with the same protein, densities and stoichiometries of the protein-lipid band were different, d = 1.040 and d = 1.070, depending on whether the starting material was pulmonary washing or Fraction T, respectively. Our failure to obtain a specific lung lipoprorein in rabbit pulmonary washing does not negate the possible presence of lipoprotein in washings that were obtained and processed by different techniques. However, the identity of such lipoproteins (8,12,13,19) with pulmonary surfactant must still be demonstrated.

PW d = 1.040

72 FRACTION T

E u

c:

-.~ 72r l

Fx

S

BAND I d=1.028

7

Identity of the Major Proteins of Pulmonary Washing and Fraction T and S o O~ ~)

Fx S

~

1

8

72

100

BAND /I d = 1.044

Fx S

%AREA

BAND 1Tf

20

FIG. 3. Dynamic surface tension (7)-axea curves of spread films of total lipid extracts of the ultracentrifugation bands of pulmonary washing, Fraction T (combined bands; Fig. 2 and Table I), and Fraction S (Table I). Subphase: 0.15 M NaC1, 25 C. medium; the presence of ions other than 0.15 M NaC1 (8) could cause release of the specific pulmonary lipoprotein, which we left behind when the lavage medium was simply 0.15 M NaC1. Second, the various manipulations (repeated ultracentrifugations and dialyses; Ref. 8) could result either in the concentration of a given protein because of loss of lipid during dialysis or in the formation of an artifactual lipoprotein particle (31). In the present study, the protein that could have been suspected to be associated with the phospholipid surfactant was protein T (5). However, several observations speak against the

LIPIDS,VOL. 12, NO. 11

We have shown beyond doubt (above and Ref. 6) that the three major proteins of pulmonary washing (A, G, and T, in Fig. 2) are serum albumin, IgG, and IgA. We did not find the proteins of MW 36,000 or 62,000 which Bhattacharyya et al. described in rabbit pulmonary washing (13). It should be noted that the method used by the other authors (13) in preparing the lung lavage was quite different from ours. They discarded the washing that we collected in 0.15 M NaC1, and instead they studied a lavage that was obtained (after the 0.15 M NaC1 washing) in hypotonic buffer; this medium conceivably dislodged (from the lung structures) constituents that we left behind. For similar reasons, we may not be able to collect the fraction that was described by King et al. (11) and which contained a lung specific protein of MW 34,000. In the preparation of pulmonary washing, those authors used 5 mM tris and a few millimoles of CaC12 and MgC12 in their saline (0.15 M NaC1) buffered at pH 7.3 (8,9,11) as opposed to 0.15 M NaC1, pH " 5 . 6 in our experiments. Isopicnic Equilibrium of the Major Proteins of Pulmonary Washing

Very striking was the position of serum albumin, IgG, and IgA at very low densities in the sucrose gradients of either pulmonary washing, Fraction T or Fraction S (Fig. 2 and Table I). Such a position cannot be accounted for simply by the average densities of the constituents. Consider, for example, two lipid-rich fractions, namely the band of pulmonary washing at d = 1.040 and the low density band

PULMONARY SURFACTANT FRACTIONS

887

of Fraction T at d = 1.059. Using the data in Table I and Figure 2 for those bands and assigning a density of 1.35 g[ml to the hydrated prorein (35) and 1.000 to 1.060 to the aqueous lipid (36-38), we obtain average densities (for the two bands) greater than 1.10 g/ml, not near the above values of 1.040 and 1.059. Furthermore, the tendency of proteins (albumin > IgG > IgA) to equilibrate in discrete regions of the sucrose gradient at densities smaller than 1.040 (Fig. 2 and Table I) must be accounted for by a new phenomenon. We propose a model in which the presence of small quantities of lipid causes the protein to attain conformations that make up an osmometer permeable to water.

(2,34,40), since it is conceivable that a large reservoir of lung lecithin in the subphase can provide a ready supply of molecular DPL to t h e film adsorbed at the interface, whereas in the spread film at 25 C most of the surface active DPL is eliminated after the first few compressions into surface structures that do not release molecular DPL (2,41). Other factors, however, play a role; the molecular structure of lecithin is one. In the analysis of the activity of spread films of lecithin as compared to the other lipid extracts shown in Table II and Figure 3, we like to d i s t i n g u i s h between positive and negative factors. Positive are the palmitoyl (16:0) anff stearoyl (18: 0) residues, since dipalmitoyl and The Small MW Protein distearoyl lecithins lower surface tension to Negligible quantities of the protein of ca. zero at 25 C as well as at 37 C (33,34). Negative 10,000 dalton were seen in the SDS disc gel are cholesterol above 20 mole % (40) and all electrophoresis of either pulmonary washing or the unsaturated fatty acyl chains and the satuFraction T. Large amounts of this protein, how- rated ones with less than 16 C atoms. Since the cholesterol content in all the ever, appeared in the SDS gel of several aliquots from the sucrose density gradient ultracentrif- examined fractions was very low (10 dyne/cm), Except for band II of Fraction S, which had pulmonary washing or Fraction T (Fig. 2). The data suggest that either the protein is not part the lowest palmitoyl (58.7%) and the highest of a native lipoprotein, or the latter's architec- oleyl 23.5%) contents, all the other fractions ture and composition are altered during the contained about 70% positive factors, most of various manipulations of the surfactant frac- which was palmitoyl. Since the quantitative differences among the total positives or among tions (5,31,39). The identity of the protein remains to be the total negatives in the active (~/min = zero) established. Its size suggests some similarity and inactive (~/min > 10 dyne/cm) fractions are with the lung specific protein isolated by King very small, a qualitative factor must be sought. et al. (9) from dog lung. However, another This could be either the distribution of the acyl point should be made; if this protein is part of a chains (dipalmitoyl vs. mixed palmitoyl-, oleyl, lipoprotein in the band at density 1.040 in the etc.), the specific effect of one unsaturated sucrose gradient of pulmonary washing (Fig. 2, residue such as the cis-5-octadecenoyl (43) or upper panel), it could not be the same lipo- some y e t imponderable contaminant. Except protein described by King and Clements (8) at for the surface activity effect of the mixed density 1.089, unless the two are altered differ- chain palmitoyl-myristoyl lecithin (42), little is ently in the two different procedures. The known to answer the above questions. The question will not be resolved until we verify the negative effect of free fatty acids on the surface activity of DPL (44) must be excluded for two !dentity of the two proteins. reasons; first, the ultracentrifugation bands did Surface Activity not contain free fatty acids, all of which were The lecithin isolated from rabbit pulmonary in the albumin fractions, second, the isolated washing was very active (')'rain = 0) when films lecithin (Table II and Ref. 2 and 30) which did were adsorbed from dispersions of 200 gg/ml in not contain free fatty acids, was not active. 0.15 M NaC1 (2) and much less active (~/min > 10 dyne/cm) when the film was spread from ACKNOWLEDGMENTS organic solvent (2,30). This difference could be This w o r k was supported by a NIH grant from the caused by the physical state of the lipid LIPIDS, VOL. 12, NO. 11

888

G. COLACICCO, A.K. RAY, AND A.R. BUCKELEW, JR.

Heart, Lung and Blood Institute. 23. REFERENCES 24. 1. Brown, E.S., Am. J. Physiol. 207:402 (1964). 2. Colacicco, G., and E.M. ScarpeUi, in "Biological Horizons in Surface Science," Edited by L.M. Prince and D.F. Sears, Academic Press, New York, NY, 1973, pp. 367-425. 3. King, R.J., Fed. Proc. 33:2238 (1974). 4. Goerke, J., Biochim. Biophys. Acta 344:241 (1974). 5. Colacicco, G., A.R. Buckelew, Jr., and E.M. Scarpelli, J. Appl. Physiol. 34:743 (1973). 6. Colacicco, G., A.IC Ray, H.R. Hendrickson, A.R. Buckelew, Jr., and E.M. Scarpelli, Prep. Biochem. 6:443 (1976). 7. Steim, J . M . R . A . Redding, C.T. Hauck, and M. Stein, Biochem. Biophys. Res. Commun. 34:434 (1969). 8. King, R.J., and J.A. Clements, Am. J. Physiol. 223:707 (1972). 9. King, R.J., D.J. Klass, E.G. Gikas, and J.A. Clements, Ibid. 224:788 (1973). 10. Klass, D.J., Am. Rev. Respir. Dis. 107:784 (1973). 110 King, 1LJ., E.G. Gikas, J. Ruch, and J.A. Clements. Ibid. 110:273 (1974). 12. Passero, M.A., R.W. Tye, KoHo Kilburn, and W.S. Lynn, Proc. Natl. Acad. Sci., USA 70:973 (1973). 13. Bhattacharyya, S.N., M.A. Passero, R.P. DiAugustine, and W.S. Lynn, J. Clino Invest. 55:914 (1975). 14. Frosolono, M.F., B.L. Charms, R. Pawlowski, and S. Slivka, J. Lipid Res. 11:439 (1970). 15. Colacicco, G., A.IC Ray, and C.F. Gillman, "Proteins of Pulmonary Washing of Various Animal Species," in preparation. 16. Reynolds, H.Y., and H.H. Newball, J. Lab. Clin. Med. 84:559 (1974). 17. Colacicco, G., CoF. Gillman, _A_.IC Ray, and R.M. Rosenbaum, Immunol. Communo (In press). 18. Colacicco, G., "Effects of Lung Perfusion with Beta-Mimetics and Prostaglandins on the Protein Composition of Rabbit Pulmonary Washing," in preparation~ 19. King, R.Jo, and J.A. Clements, Am. J. Physiol. 223:715 (1972). 20. Pfleger, R.C., N.G. Anderson, and F. Snyder, Biochemistry 7:2826 (1968). 21. Dittmer, J.C., and R.L. Lester, J. Lipid Res. 5:126 (1964). 22~ Kates, M~ "Techniques of Lipidology," North

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40. 41. 42. 43. 44.

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[ Revision receive d April 2 7, 19 7 7 ]