Isolation and characterization of proteoglycans from porcine lungs.

Proteoglycans were extracted from porcine lungs with 4 M guanidinium chloride. The extract was subjected to associative density gradient centrifugation, and four equal fractions, labeled A1 through A4 from the bottom to the top of the gradient, were obtained. The pooled A1 fractions containing proteoglycan aggregates were further fractionated by dissociative density gradient centrifugation to yield four equal fractions labeled A1D1 through A1D4 from the bottom to the top of the gradient. These fractions were analyzed for their protein, uronic acid, glucosamine, galactosamine, hexose, and sialic acid content. The fraction A1D1 with the highest buoyant density had the highest content of uronic acid and galactosamine, and lowest content of protein, indicating the enrichment of proteoglycan monomers at the bottom of the dissociative density gradient. As the density of the gradient decreased, the protein, hexoses, and sialic acid content increased, whereas uronic acid and galactosamine content decreased. The amino acid analysis showed similar composition for all four fractions with aspartic acid, serine, glutamic acid, proline, glycine, alanine, valine, and leucine as the major constituent amino acids. No hydroxyproline was detected in any of the fractions. As the buoyant density of the fractions decreased, the aspartic acid content increased and glycine content decreased.


Proteoglycans
were extracted from porcine lungs with 4 M guanidinium chloride.
The extract was subjected to associative density gradient centrifugation, and four equal fractions, labeled A1 through Ad from the bottom to the top of the gradient, were obtained. 'Ihe pooled A1 fractions containing proteoglycan aggregates were further fractionated by dissociative density gradient centrifugation to yield four equal fractions labeled AIDI through AID4 from the bottom to the top of the gradient.
These fractions were analyzed for their protein, uranic acid, glucosamine, galactosamine, hexose, and sialic acid content.
The fraction AIDI with the highest buoyant density had the highest content of uranic acid and galactosamine, and lowest content of protein, indicating the enrichment of proteoglycan monomers at the bottom of the dissociative density gradient.
As the density of the gradient decreased, the protein, hexoses, and sialic acid content increased, whereas uranic acid and galactosamine content decreased.
The amino acid analysis showed similar composition for all four fractions with aspartic acid, serine, glutamic acid, proline, glycine, alanine, valine, and leutine as the major constituent amino acids. No hydroxyproline was detected in any of the fractions. As the buoyant density of the fractions decreased, the aspartic acid content increased and glycine content decreased.
Proteoglycans, like collagen and elastic fibers, are a major component of all connective tissues. In recent years, proteoglycans of cartilage from various sources have been isolated and characterized (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). Results of these studies indicate that the cartilage proteoglycans are large aggregates composed of up to 140 proteoglycan subunits linked noncovalently to a linear molecule of hyaluronic acid in association with two link proteins. According to the current structural model, the proteoglycan monomer consists of approximately 110 chondroitin sulfate and 50 keratan sulfate side chains attached covalently to a linear core protein . The average molecular weights of the  core protein, chondroitin  sulfate, and keratan sulfate are about  200,000,20,000 and 5,000, respectively. The average molecular weight of the proteoglycan monomer is approximately 2 to 3 x 10" (3,13,14). The proteoglycan monomer can be divided into three distinct portions: a smaller nonvariable hyaluronic acid-binding region with a few or no polysaccharide side chains at one end, and a much larger variable segment containing chondroitin sulfates on the other end, with a keratan sulfate-rich region in the middle of the molecule. The variation in the size of the proteoglycan molecule is mainly due to the variation in the size of the chondroitin sulfate-enriched region (12,15,16).
In terms of the overall composition by weight, the

AND DISCUSSION
The lung tissue used in the present study was free from bronchi, large blood vessels, and trachea. Light micrographs of this tissue taken from random samples show the smooth muscle around arterioles, veins, and terminal bronchi and demonstrate the absence of cartilage ( Figs. 1 and 2). Proteoglycans were extracted from the lung tissue in 4 M guanidinium chloride, pH 5.8, which solubilized approximately 60 to 70% of the total glycosaminoglycans as measured by uranic acid. The major glycosaminoglycans extracted by this dissociative solvent were hyaluronic acid, chondroitin sulfate, and heparin sulfate (Fig. 3). When this proteoglycan extract was subjected to the density gradient centrifugation under associative conditions (20), four equal fractions labeled A, through Aq from the bottom to the top of the gradient were obtained. The distribution of uranic acid and protein in these fractions is shown in Fig. 4. The pools of Fractions Al through Aq contained approximately 75, 14, 9, and 2% of the total uranic acid, respectively.
The material recovered as a surface gel at the top of the associative gradient accounted for approximately 2 to 3% of the applied sample, based on the dry weight, and contained less than 1% of the total uranic acid. To ensure removal of extraneous proteins, the A, samples were recycled through a second associative density gradient, and the bottom one-half of these samples were pooled. These recycled A, samples containing proteoglycan aggregates (5) were subjected to the dissociative density gradient centrifugation (20), and four equal fractions labeled A,D, through AID4 from the bottom to the top of the gradient were obtained. The relative amounts and composition of these four fractions are given in Table I. Approximately 4 to 6 mg of proteoglycans were obtained from 1 g of wet tissue. The proteoglycans recovered at the highest density had the lowest protein and highest uranic acid and galactosamine content. Chondroitin sulfates were the only detectable glycosaminoglycans present in this fraction (Fig. 3). Their identification was confirmed by the disappearance of the band corresponding to chondroitin sulfates on the cellulose acetate electrophoresis after digestion with chondroitinase AC and chondroitinase ABC. With the decrease in the density of the dissociative gradient the protein, hexoses, as well as the sialic acid content increased, whereas uranic acid and galactosamine decreased. AID2 and AlDs fractions contained mostly hyaluronic acid and small amounts of heparin sulfate (Fig. 3). After digestion with Streptomyces hyaluronidase the major band corresponding to hyaluronic acid on the cellulose acetate paper disappeared, but a minor band corresponding to heparin sulfate could be detected. Heparin sulfate was further confirmed by its susceptibility to deamination cleavage (30). The AID4 fraction was rich in protein, glucosamine, hexoses, and sialic acid. This fraction was found to contain heparin sulfate (Fig. 3) which could be degraded by the action of nitrous acid. The SDS polyacrylamide gel electrophoresis of AID.+ fraction showed two protein bands with apparent molecular weights of 50,009 and 65,000 (Fig. 5). These two proteins may be compared with the two hyaluronic acid-binding proteins of apparent molecular weights 40,000 and 65,006 isolated by Hascall and Heinegard (6) (4), dermatan sulfate (5), keratan sulfate (S), and heparin (7). dalton protein present in the AID4 fraction (Fig. 5) represents the hyaluronic acid-binding region of the proteoglycan monomer as described by Hascall and Heinegard (6) is not known. Gel chromatography (Fig. 6) was used to assess the size of the proteoglycans as well as the effect of combining samples from the top and middle of the dissociative gradient on the proteoglycan monomers. Most of the proteoglycan aggregates, AI, were excluded from the Bio-Gel A-50m column, whereas most of the monomers, AIDI, were included in the column. However, a small fraction of AI was included in the column, and a small portion of AIDI was excluded from the column. The fractions from the middle of the dissociative gradient increased the size of the proteoglycan monomers (Fig. 6, f  proteoglycans very little by itself (Fig. 6h), although in the presence of the middle fractions, AID2 and AIDS, it did increase the size of the proteoglycan monomers (Fig. 6, i and j) . It, therefore, appears that an active component was present in the middle fractions, which contain hyaluronic acid. The amino acid composition of four fractions, AlDl through A1D4, are somewhat similar but not identical (Table II). The major constituent amino acids are aspartic acid, serine, glutamic acid, proline, glycine, alanine, valine, and leucine. No hydroxyproline or hydroxylysine was detected in any of these fractions. The lung AIDl fraction differs in its amino acid composition from that of the cartilage AIDI fraction (5) especially in its higher content of aspartic acid and alanine and lower content of serine and proline. The amino acid composition of the AID4 fraction is quite different from those of collagen and elastin. It is similar to the amino acid composition of the "structural glycoproteins" present in the connective tissue matrix (41). Our results agree with the previously published reports that the content of aspartic acid increases and that of glycine decreases with the decrease in buoyant density of the proteoglycan fractions (7,42,43). The proteoglycans isolated from the lung tissue are comparable to those of aorta (44). The 4 M guanidinium chloride extracts of bovine aorta contained chondroitin sulfates, hyaluronic acid, and heparin sulfate. The proteoglycans of aorta interacted with hyaluronic acid and cardiovascular connective tissue proteins to form aggregates.
The results described in this report clearly demonstrate that cartilage-free lung tissue contains cartilage-like proteo-4266 Lung Proteoglycans