Saura Sahu

Structural studies on a glycoprotein isolated from alveoli of patients with alveolar proteinosis

A major glycoprotein 36 000 molecular weight) has been isolated from lung lavage of patients with alveolar proteinosis and found to contain five residues of hydroxyproline, fifty residues of glycine, three residues of methionine, 3 mol of sialic acid, 4.4 mol of mannose, 4.0 mol of galactose, 6.0 mol of glucosamine, and 1 mol of fucose. Cyanogen bromide (CNBr) treatment of the glycoprotein resulted, as expected, in four peptides of apparent molecular weights of 18 000, 12 000, 5000 and 1000, respectively. The chemical compositions of the CNBr peptides indicate the presence of hydroxyproline and high amounts of glycine in all but one of the peptides; two of the four CNBr peptides contain carbohydrate. Gel filtration, acrylamide gel electrophoresis and end-group analyses of the native glycoprotein and its CNBr peptides indicate that the peptides are homogeneous. End-group analyses of the CNBr cleavage products assign the 18 000 molecular weight peptide to the NH2-terminal portion and the 1000 molecular weight peptide to the COOH-terminal portion of the native glycoprotein molecule. Pronase digestion of the 36 000 molecular weight glycoprotein, followed by gel filtration and cation exchange chromatography, resulted in two fractions. One fraction was acidic and contained all the carbohydrate, a high content of aspartic acid and no hydroxyproline. The other fraction was basic and contained 8.4% hydroxyproline, 14% proline, 28% glycine and no carbohydrate, suggesting the presence of collagen-like sequence in the peptide chain. Paper electrophoresis of the basic fraction demonstrated two components, the amino acid compositions of which are identical to those of collagen. Partial amino-terminal sequence analysis of one of the CNBr peptides (18 000 molecular weight) indicated the presence of -Fly-Pro-HyP-Gly-sequence in the peptide chain, which confirms our suggestion that collagen-like regions are present in the native glycoprotein molecule. Limited acid hydrolysis of the acidic fraction and subsequent fractionation of the acid hydrolysate using Dowex column yielded a fraction which produced brown colour with ninhydrin reagent. Paper chromatography of this fraction demonstrated a large component which also stained brown with ninhydrin reagent. After acid hydrolysis, this component was found to consist of equal amounts of asparitic acid and glucosamine, indicating that the N-acetylglucosamine of the oligosaccharides is linked to the asparagine residue of the peptide. No serine or threonine linkages are present.

Characterization of phospholipase a from pulmonary secretions of patients with alveolar proteinosis

Phospholipase A2 (EC 3.1.1.4) from the insoluble pulmonary secretions that accumulate in the lungs of patients with alveolar proteinosis has been purified. The pure enzyme gives a single sharp band upon sodium dodecyl sulfate polyacrylamide gel electrophoresis. Amino acid analysis of the protein shows high content of cystine, aspartic acid, glutamic acid, serine, glycine, leucine and lysine. Only one N-terminal residue, alanine, can be detected. Gel filtration as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis indicate an apparent molecular weight of 75 000 for the enzyme. The enzyme activity has a pH optimum between 7.5 and 8.5 and is stimulated by sodium deoxycholate and CaCl2.

Hyaluronic acid. An indicator of pathological conditions of human lungs

Hyaluronic acid was the only glycosaminoglycan found in detectable amounts in the pulmonary secretions of patients with cystic fibrosis. The compound gave a hexuronate/hexosamines molar ratio of approximately 1. Glucosamine represented over 98% of the total hexosamines, the remainder being galactosamine. No hexoses or sulfate could be detected. It moved as a single spot with the mobility of standard hyaluronic acid on cellulose acetate electrophoresis and this spot disappeared after digestion with testicular hyaluronidase. It was associated with trace amounts of protein, the major amino acids of which are aspartic acid, glutamic acid, glycine, and alanine.

Lipid composition of human alveolar macrophages

Lipids from human alveolar macrophages were characterized. The cells were obtained from a patient with pulmonary fibrosis and alveolar monocytosis. Phospholipids accounted for 80% of the total lipids, with the remainder neutral lipids. Phosphatidylcholine was the major phosphatide. Phosphatidylethanolamine, sphingomyelin, phosphatidylglycerol, and lyso-(bis)phosphatidic acid were the other phosphatides present. Arachidonic acid, especially in phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol, was present in significantly high amounts. The lipids from the human cells were compared with those obtained from normal rabbit alveolar macrophages. Five major differences are observed between the lipids from these two species. First, phosphatidylcholine and sphingomyelin from human cells contained predominantly palmitic acid, whereas those from rabbit cells consisted mainly of fatty acids with 18 carbon atoms. Second, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol from the patient underwent partial oxidation, whereas the lipids of rabbit macrophages were not oxidized. Third, the lyso(bis)phosphatidic acid from the human cells contained significantly less arachidonic acid than that from rabbit cells. Fourth, all phosphatides, except phosphatidylglycerol, in human macrophages contained significantly less linoleic acid compared with those from rabbit cells. In the case of phosphatidylglycerol, the reverse was true. Fifth, free fatty acids were present only in the cells obstained from the patient.

Hyaluronic acid in the pulmonary secretions of patients with alveolar proteinosis

Hyaluronic acid was the only glycosaminoglycan found in the pulmonary secretions of patients with alveolar proteinosis. The compound gave a hexouronate/hexosamine molar ratio of about 1∶1. Glucosamine constituted over 98% of the hexosamines, the remaining being galactosamine. It moved as a single spot with the mobility of standard hyaluronic acid on cellulose acetate electrophoresis, and this spot disappeared after digestion with hyaluronidase. It was associated with small amounts of proteins, the major amino acids of which are aspartic acid, glutamic acid, glycine, alanine, and leucine.

Phospholipase a in pulmonary secretions of patients with alveolar proteinosis

This report presents evidence for the presence of phospholipase A2 (EC 3.1.1.4) activity in the insoluble pulmonary secretions of patients with alveolar proteinosis. The enzyme activity has a pH optimum between 7.5 and 8.5 and is stimulated by deoxycholate and Ca2+.

Lipid chemotaxins isolated from culture filtrates ofEscherichia coli and from oxidized lipids

Lipid extracts of sterile culture filtrates ofEscherichia coli were shown to contain approximately 75% of the chemotactic activity for human polymorphonuclear leukocytes and rabbit alveolar macrophages. Fractionation and purification of these lipids revealed the presence of many unknown lipids of widely different properties, but all were anionic and at very low concentrations, chemotactic. The only one of active molecules that could be identified was an unsaturated ultraviolet-absorbing hydroxy fatty acid, which, following catalytic reduction with hydrogen, was found to be hydroxyeicosanoic acid. This fatty acids Chromatographic behavior was very similar to that of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE), which is a potent chemotaxin for polymorphonuclear leukocytes and macrophages. Unknown chemotaxins could be generated by the oxidation of known unsaturated lipids. Prostaglandins A2 and E2 produced potent chemotaxins upon aerobic oxidation. Malonaldehyde, a peroxidation product of unsaturated lipids, when reacted with phosphatidylethanolamine in aerobic conditions, also produced strong chemotactic agents. The chemotactic activity of these products could be destroyed by catalytic reduction with hydrogen and by methylation with dry methanolic HCl. These data indicate that the nonenzymatic oxidation of unsaturated lipids generates some products that are potent chemotaxins for mammalian inflammatory cells.

Metabolism of arachidonic acid in rabbit alveolar macrophages.

Rabbit alveolar macrophages incorporated radioactive arachidonic acid primarily into phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and triglycerides. When these cells, so labelled, were washed and treated with polystyrene beads, there was a distinct and reproducible decrease in the radioactivity of phosphatidylcholine, phosphatidylethanolamine, and triglyceerides. This decrease was accounted for by the appearance of two major prostaglandins, PGE2 and PGF2a, and uncharacterized hydroxy fatty acids, which may be 12-hydroxy-5,8,10,14-eicosatetraenoic acid, 5-hydroxy-6,8,11, 14-eicosatetraenoic acid, and/or 8-hydroxy-9,11,14-eicosatrienoic acid.

A high-molecular-weight alveolar glycoprotein in the cell-free culture medium of human fetal lung type II pneumocytes.

A glycoprotein of an apparent molecular weight of 250,000 has been purified from the cell-free culture medium of human fetal lung Type II pneumocytes. Its amino acid composition shows the presence of trace amounts of hydroxyproline but no hydroxylysine. It is relatively rich in aspartic acid, glutamic acid, glycine and leucine. It contains about 6.0% hexose, 2.7% sialic acid and 3.7% glucosamine. The hexoses are galactose, mannose and fucose. No glucose or galactosamine can be detected. This high-molecular-weight glycoprotein cross-reacts with an antiserum prepared in rabbits against a glycoprotein of similar size and composition isolated from the alveolar secretions of patients suffering from pulmonary alveolar proteinosis [20]. It appears that the high-molecular-weight alveolar glycoprotein is a secretory product of the alveolar Type II cells.

Lipid composition of sputum from patients with asthma and patients with cystic fibrosis

Lipids from the sputum of patients with asthma and with cystic flbrosis were isolated and characterized. In both cases, lipids constituted approximately 30% of the dry material. Phosphatidylcholine was the most abundant lipid. Significant amounts of phosphatidylethanolamine and phosphatidylglycerol were present. Hexosyl ceramides, sphingomyelin, phosphatidylinositol, lysophosphatidylcholine, and lysophosphatidylethanolamine were present as minor lipid components. Appreciable quantities of neutral lipids were present, of which triglycerides and cholesterol were the main constituents. Phosphatidylcholine, sphingomyelin, and phosphatidylglycerol were highly saturated. Large amounts of phosphatidylcholine containing mostly palmitic acid, particularly in the asthmatic sputum, suggests that this highly saturated phospholipid is synthesized in the upper airways for reasons other than its beneficial surface-active properties in the alveoli.