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Properties of an Acid Phosphatase in Pulmonary Surfactant (Lung Surfactant/Phosphatidate Phosphatase/Phosphatidylglycerol Phosphate) BRADLEY J

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Properties of an Acid Phosphatase in Pulmonary Surfactant (Lung Surfactant/Phosphatidate Phosphatase/Phosphatidylglycerol Phosphate) BRADLEY J Proc. Natl. Acad. Sci. USA Vol. 77, No. 2, pp. 808-811, February 1980 Biochemistry Properties of an acid phosphatase in pulmonary surfactant (lung surfactant/phosphatidate phosphatase/phosphatidylglycerol phosphate) BRADLEY J. BENSON Cardiovascular Research Institute, and the Department of Biochemistry, University of California, San Francisco, California 94143 Communicated by John A. Clements, November 5, 1979 ABSTRACT Lung surfactant, a lipid-protein complex pu- phatase (phosphatidate phosphohydrolase, EC 3.1.3.4) in in- rified from dog lungs, contains a highly active phosphomo- tracellular lamellar bodies. Spitzer et al. (13) also found the noesterase associated with it. This phosphatase is quite specific enzyme in their preparations of isolated lamellar bodies, for the hydrolysis of phosphatidic acid and 1-acyl-2-lysophos- phatidic acid. The enzyme possesses many of the characteristics whereas Garcia et al. (14) found no phosphatidate phosphatase of the microsomal enzyme, phosphatidate phosphohydrolase in their lamellar body preparations that they could not attribute (EC 3.1.3.4). In addition, we have shown that this enzyme will to microsomal contamination. Recently, however, Spitzer and also convert phosphatidylglycerol phosphate [1(3-sn-phospha- Johnston (15) demonstrated convincingly that the phosphatidate tidyl)sn-glycerol-1-PJ to phosphatidylglycerol [1{3-sn-phos- phosphatase in their lamellar body preparations was not con- phatidyl)sn-glycerolj and Pi. The phosphatidylglycerol phos- tamination from microsomes. Baranska and van Golde (16) phate was made available to the surfactant enzyme in a coupled assay by hydrolysis of cardiolipin [1(3-sn-phosphatidyl)3(3- reported that their preparations of lamellar bodies contained sn-phosphatidyl)sn-glycerolJ by stereospecific cleavage with no phospholipid biosynthetic enzymes that could not be at- phospholipase C (phosphatidylcholine cholinephosphohydro- tributed to microsomal contamination, although their studies lase, EC 3.1.4.3) from Bacillus cereus. This enzyme has been did not measure phosphatidate phosphatase. previously shown to generate the naturally occurring isomer of During further investigations on the function and charac- phosphatidylglycerol phosphate because it has specificity for terization of extracellular SAM proteins, we found an acid the 3(3-sn-phosphatidyl) group of cardiolipin. Other properties of the surfactant enzyme are discussed in relation to its presence phosphomonoesterase that could cleave phosphatidic acid to in lung surface active material. diglyceride and Pi. * This enzyme can also convert the imme- diate precursor of PtdGro, phosphatidylglycerol phosphate Lung surfactant or surface active material (SAM) is a lipid- (PtdGro-P) to PtdGro and Pi. We report here additional protein complex obtained by broncho-alveolar lavage (1). In properties of this enzyme and suggest that the SAM phosphatase vio this material is thought to adsorb to the air/liquid interface may play a role in phosphatidylglycerol biosynthesis. of the terminal air spaces and, by reducing the surface tension at low lung volumes, contribute to the stability of the alveoli (2, MATERIALS AND METHODS 3). Recent studies from this laboratory have indicated that in The sodium salts of phosphatidate (egg PtdCho), cardiolipin, excised rat lungs this acellular lining layer is capable of main- lysophosphatidate (egg PtdCho), p-nitrophenylphosphate, taining the surface tension in the alveoli below 9 mN/m at glucose-6-phosphate, glycerol-3-phosphate, glycerol-2-phos- functional residual volume (4). phate, p-nitrophenyl-D-mannoside, and p-nitrophenyl-N- Chemical analysis of canine surfactant purified by density acetyl-f3-D-glucosaminide were all obtained from Sigma. Di- gradient centrifugation indicates that it is a complex mixture palmitoylphosphatidic acid, diolein, and dioleoylphosphatidic of lipids and proteins (5). There is general agreement that some acid were supplied by Serdary Biochemicals (London, ON 65% of SAM (wt/wt) is phosphatidylcholine (PtdCho) and, of Canada). Silica gel G and H plates were products of Analtech this, dipalmitoyl PtdCho is the principal molecular species. It (Newark, DE). Chloroform and methanol were redistilled be- is this component of lung surfactant to which the unusual sur- fore use and all water used was double distilled. Other reagents face properties of SAM are attributed (6). The next most were of the highest quality obtainable. abundant class of phospholipid by weight is phosphatidylgly- All lipids were checked for purity by thin-layer chroma- cerol (PtdGro) (7, 8), which is found in uniquely high concen- tography prior to use. (When necessary the lipids were purified tration in SAM, 9-10% by weight. The role of this phospholipid by preparative thin-layer chromatography.) Protein concen- in SAM function is unknown. trations were estimated by the method of Lowry et al. (17), as SAM is thought.to be synthesized by the type II epithelial cells modified by Dulley and Grieve (18). Inorganic phosphorus was of the lungs (9, 10). These cells contain organelles, lamellar measured according to Bartlett (19). bodies, that are thought to be the intracellular sites of surfactant Fresh, excised lungs were obtained from normal, healthy storage prior to secretion into the alveoli. These osmiophilic mixed-breed dogs of either sex. The SAM was prepared essen- inclusion bodies have been purified from different species by tially according to the method of King and Clements (1). The density gradient centrifugation after lung homogenization, and surfactant obtained was centrifuged to equilibrium in a con- their phospholipid composition has been shown to be similar tinuous sodium bromide density gradient (1.057-1.124 g./ml). to that of extracellular SAM (8, 11). The surfactant band was removed and the density was deter- Recently, workers in many laboratories have been studying mined in a pycnometer. After dialysis, aliquots were stored at whether lamellar bodies are only a storage organelle of SAM -700 C. or whether they actively participate in phospholipid biosyn- Force/area isotherm measurements were routinely carried thesis or modification. Meban (12), using electron microscopic out on purified SAM preparations. The lung surfactant was histochemistry, reported the presence of phosphatidate phos- Abbreviations: SAM, surface active material; PtdCho, phosphatidyl- The publication costs of this article were defrayed in part by page choline; PtdGro, phosphatidylglycerol; PtdGro-P, phosphatidylglycerol charge payment. This article must therefore be hereby marked "ad- phosphate. vertisement" in accordance with 18 U. S. C. §1734 solely to indicate * Benson, B. J. & Clements, J. A. (1976) Am. Chem. Soc. Abstr. Pap. this fact. (Port City Press, Baltimore, MD) 172, Abstr. 165. 808 Downloaded by guest on September 23, 2021 Biochemistry: Benson Proc. Natl. Acad. Sc. USA 77 (1980) 809 dispersed in 66% (vol/vol) isopropanol and spread onto a sub- by chromatography on silica gel G plates developed with pe- phase of 0.9% NaCi solution buffered to pH 7.4 with Tris'HCI. troleum ether/diethyl ether/acetic acid (80:20:1, vol/vol). The The films were confined in a Teflon trough with a tight-fitting, nonpolar lipid released in the reaction migrated with an RF movable Teflon barrier. Surface tension was monitored by a identical to that of synthetic diolein. Negligible (less than 0.5%) platinum dipping plate-straingauge-amplifier system. water-soluble, esterified phosphate and no inorganic phosphorus Phosphatidate phosphatase activity was measured by a were released during the hydrolysis of cardiolipin. modification of the method of Coleman and Hfibscher (20). Other enzymatic activities assayed (substrates) were: acid Dioleoylphosphatidate (0.3 mM) was suspended in 0.16 ml of phosphatase (EC 3.1.3.2) (p-nitrophenylphosphate) (24); reaction mixture of 50 mM sodium maleate (pH 6.5) containing a-mannosidase (EC 3.2.1.24) (p-nitrophenyl-a-D-mannoside) 5-20 jig of protein. The reaction was carried out at 370C for (25); nonspecific esterase (p-nitrophenylacetate) (26); and 30 or 60 min. The reaction was quenched by addition of tri- f3-N-acetylglucosaminidase (EC 3.2.1.53) (p-nitrophenyl-N- chloroacetic acid. After centrifugation at 8000 X g, Pi in the acetyl-f3-D-glucosaminide) (27). Glucose-6-phosphatase was supernatant was measured by a modification of the method assayed according to Coleman and Bell (28). reported by Baginski et al. (21). All assays were linearly pro- portional to time and protein over the range used in this RESULTS AND DISCUSSION study. Criteria of purity of surfactant PtdGro-P phosphatase activity was measured in a coupled assay system in which PtdGro-P was generated by the action Purified surfactant from canine lungs has a characteristic of phospholipase C according to a modification of the method density of 1.084 g/ml in Tris-buffered saline solutions con- of Cable et al. (22). Cardiolipin (2.5 mg) was incubated with taining sodium bromide to increase the density. Because sur- 1.0 unit of phospholipase C in 0.2 ml of 0.1 M Tris-HCI (pH 7.0) factant is a complex mixture of lipids and proteins, we think it with an aliquot of SAM containing 10,jg of protein. After a 1-hr is important to test each preparation against certain criteria of incubation at 370C, the reaction was stopped by addition of 175 purity before use: the phospholipid-to-protein ratio (wt/wt) Ail of 15% trichloroacetic acid. After centrifugation, the Pi re- should be near 10; the material must possess the antigen pre- leased was then assayed (21). The assay is shown schematically viously shown in this laboratory to be lung- and SAM-specific in Fig. 1. (29); the preparation must be able to form a surface film that, The assay is based on a report by de Haas et al. (23), who on compression, lowers the surface tension of water to below showed that one product of phospholipase C hydrolysis of 9 mN/m (this test is perhaps the most critical because it gives cardiolipin was PtdGro-P and the other product was diacyl- a direct measure of the surfactant "effectiveness" of the ma- glycerol.
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