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Three human basement membranes, glomerular basement membrane (GBM) from renal cortex, alveolar basement membrane (ABM) from lung parenchyma and trophoblast basement membrane (TBM) from the terminal villi of placenta have been isolated by sieving and sonication techniques. Canine GBM and ABM were also prepared. There were marked differences among the membranes from human tissues. Compared to GBM, TBM had very little collagen but contained high concentrations of charged amino acids. ABM was intermediate in composition between GBM and TBM and contained desmosine and isodesmosine indicative of the presence of elastin. Canine ABM (c-ABM) did not contain desmosine or isodesmosine. In the canine system an antigen was detected in ABM which was not present in GBM. The membrane preparations were analyzed for fibronectin content using a specific antiserum to fibronectin. This glycoprotein could not be detected in GBM whereas it was present in ABM in amounts up to 0.8% and in TBM in amounts as high as 7.2%. All the membranes induced the formation of precipitating antibodies in rabbits. Soluble material obtained from the membranes by alkali extraction, reduction of disulfide bonds, enzymatic digestion with elastase, plasmin or collagenase provided immunologically reactive fragments. These soluble fragments gave reactions of identity among the three basement membranes in immunodiffusion reactions in gels with antisera raised to all three BMs. The finding that plasmin digests basement membranes suggests that it may play a role in connective tissue remodeling. The fact that elastase degrades basement membranes provides an endogenous system for injury which may be triggered by infections.

A sensitive, reproducible, and rapid radioimmunoassay was developed to determine clastin peptide content of human serum in nanogram amounts. Significant differences have been determined between normal volunteers, both smokers and nonsmokers, and patients wth chronic obstructive pulmonary disease. The average concentration of clastin peptides in 9 normal nonsmokers was 19.88 ± 10.46 μg/ml serum, indistinguishable from that in 7 normal smokers which was 17.64 ± 8.54 μg/ml. Average concentration of clastin peptides in 17 patients with lung disease was 53.38 ± 22.46 μg/ml, significantly different (P

Fibronectin (Fn), a high molecular weight glycoprotein, was found to constitute 0.43% of the normal adult beagle dog lung. The tissue Fn (TFn) was solubilized by sequential chemical extractions and quantified by ELISA (enzyme-linked immunoabsorbent assay). Subsequent plasmin digestion did not appear to solubilize significantly more Fn. Since 70% of the lung tissue was solubilized by the extractions and plasmin digestions, the TFn quantified represented the bulk of lung Fn. The TFn was identical to plasma fibronectin in the ELISA and one can infer that the Fn molecule is not significantly altered as it is incorporated into the lung connective tissue matrix. Lungs from beagles in which fibrosis had been induced with bleomycin contained 0.99% Fn, more than a twofold increase over normal. In the ELISA TFn from fibrotic lungs gave an inhibition curve of the same shape as did TFn from normal lungs. Thus, Fn from fibrotic lungs is not different qualitatively from Fn from normal lungs in any way detectable with this antiserum. The TFn content of plasmin digests of intact lung was less than that of extracts, which was the converse of results obtained on placenta (B. A. Bray (1985) Biochem. J. 226, 811–815). This difference between lung TFn and placental TFn may be due to differences in degree of glycosylation, which determines susceptibility to proteases.

Mature, crosslinked elastin has been isolated from 4 human and 12 monkey uterine cervices. A modification of previous methods for determination of elastin content was devised to quantitate the low amounts of elastin in the crude connective tissue of uterine cervices. The percentage of elastin was found to range between 0.9 and 2.4% and did not appear to change at various stages of gestation.

A peptide of 6,700–8,000 daltons has been isolated from an ethanolic KOH digest of bovine ligamentum nuchae elastin by a combination of ion exchange chromatography and gel filtration techniques. The peptide is non-polar and is composed almost entirely of glycine, valine, and proline (or hydroxyproline) in the ratio 2:2:1. Edman degradation of the first 17 residues gave the sequence: NH 2 -Gly-Phe-Pro-Gly-Val-Gly-Val-Pro-Gly-Val-Gly-Val-Pro-Gly-Val-Gly-Val. These results suggest that the peptide is made up of repeating pentapeptide units with the sequence: Pro-Gly-Val-Gly-Val.

Two methods are described for the purification of Clostridium histolyticum collagenase. One method involves gel filtration on columns of Sephadex G-200, the other, ion-exchange chromatography on DEAE Sephadex A-50. In both cases, calcium-containing buffers were found necessary for good recoveries of enzyme. Yields of collagenase activity up to 94%, free of nonspecific proteolytic activities, were consistently obtained.

Bovine ligamentum nuchae elastin was partially hydrolyzed by ethanolic KOH. Fractionation of the dialyzed product on a column of SE-Sephadex yielded eight fractions which were characterized by amino acid analysis as well as hexose and lipid contents. The approximate molecular weight or size range of each of the fractions was determined by Sephadex gel filtration. Study of the amino acid distribution among these fractions revealed the presence of three classes of peptide. The first peak eluted from the SE-Sephadex was found to be a mixture of acidic glyco-peptides, possibly derived from a structural glycoprotein component of elastin. A second type of peptide was represented by the second and third peaks which contained almost exclusively the neutral amino acids glycine, valine and proline. Fraction II was a monodisperse peptide of about 10,000 molecular weight, containing these three amino acids in the approximate ratio of 2:2:1. The third general group of peptide isolated, fractions IV through VIII, was a...

A modification of the Bratton-Marshall test (10) developed by Goldbarg and Rutenburg (9) for the assay of amino peptidase has been adapted to the determination of naphthylamine released from the specific trypsin substrate benzoylarginine-β-naphthylamide (3). Advantages of this method over others used in similar procedures include specificity, sensitivity, good reproducibility, easy colorimetric assay, lack of interference by contaminants present in crude enzyme preparations, tissues, and fluids, and the possibility of using the same substrate for parallel histochemical tests. The procedure has been applied to the determination of trypsin itself, other enzymes with similar specificity for arginine side chains, and the trypsin-like cathepsin B enzymes of liver, kidney, spleen, muscle, and other tissues. It has also been used to assay trypsin inhibitors, in particular those found in tissues.

The enzymatic degradation of purified blood group substances represents an increasingly fruitful approach to the elucidation of their immunochemical specificity, and progress to date has recently been reviewed (Kabat, 1956). A number of enzymes from different microorganisms have been described which split one or more of the blood group substances with resultant changes in serological specificity. Stack and Morgan (1949) have investigated enzymes, first studied by Schiff (1935), of Clostridium welchii, which decompose the A, B and O(H) substances. Watkins (1953) described enzymes in cell free extracts of the protozoan Trichomonas foetus which inactivated the A, B, O(H), and Lewis blood group substances; and Watkins and Morgan (1954) have shown that partially purified enzyme preparations from the same source destroyed the O(H), M and N substances on intact erythrocytes without affecting the A, B, P or S antigens. Besides inactivating the specific antigens mentioned, the enzymes of T. foetus rendered all erythrocytes nonspecifically agglutinable (panagglutinable), rendered D-positive cells agglutinable by incomplete anti-D, and inactivated the receptor sites for the hemagglutinin of influenza virus type A (PR8). Cell free extracts of Lactobacillus bifidus (var. penn) have been shown by Gy6rgy, et al. (1952) to possess enzymatic activity against human and animal blood group A, B, and 0(H) substances, being most active against the latter. Iseki and Tsunoda (1952) have reported that cell free extracts of Bacillu-s fulminans, an aerobic spore bearer, show enzymatic

A bacterial elastase has been isolated from a flavobacterium species and freed from contaminating peptidase and unspecific proteinase activity. Some properties of this new enzyme, such as pH, stability, effect of metals and other potential inhibitors, kinetics, and specificity are described. The bacterial enzyme is more specific than pancreatic elastase, has a lower pH optimum, and is not affected by 1% NaCl and serum inhibitors.

Continuing our studies of the antigenicity of papillary cystadenocarcinoma of the ovary we have investigated the production of antibodies in rabbits immunized with a tissue lyophilisate. Ouchterlony double diffusion showed 4 precipitin lines, of which one cross-reacted with normal ovarian tissue, one was identical with the line developed with tissue culture cell sonicate antigen, and 2 additional lines were found only with the tumor homogenate. Immunoelectrophoresis indicated that one antigenic component had a mobility corresponding to α-globulin and one line appeared in the β-globulin region. Gel filtration on Sephadex G-200 yielded 6 fractions of which Fraction 1 and Fraction 3 were tumor specific. Fraction 1 gave one precipitin line against nonadsorbed serum to unfractionated tumor homogenate, Fraction 3 gave 2 nonidentical and distinct lines.

Purified insoluble native Achilles tendon collagen was digested with Clostridium histolyticum collagenase. The N-terminal sequence of the mixed peptides to position 3 was determined by a modification of the Edman degradation procedure, C-terminal amino acids by hydrazinolysis. Experimental losses and non-specific cleavage of the peptides were estimated from model compounds and appropriate corrections made. At the N-terminus 182 residues out of 200 were glycine, 10 were alanine, while isoleucine, phenylalanine, and valine accounted for the remainder. In the second position 77 residues out of 200 were proline, but significant amounts of other amino acids were present showing that the specificity requirement of the enzyme is less rigorous than originally suspected. Ten residues of hydroxyproline in this position were unexpected. In the third N-terminal position the distribution was even more heterogenous, but hydroxyproline and alanine, mostly in tripeptides, predominated. C-terminally, 58 residues were hydroxyproline, 42 alanine, 29 arginine, with other amino acids in considerably lower amounts. No glycine was detected at the C-terminus. Attempts were made to deduce the distribution of third position residues between tripeptides and longer chain peptides and comparisons made with results obtained by others with different collagens.

Bovine ligamentum nuchae elastin, after solubilization by pancreatic elastase, was dialyzed and fractionated on a column of DEAE-Sephadex A-50. The resulting fractions were analyzed for their component amino acids. The first fraction eluted had the greatest average peptide chain length and 87% of its residues as the three nonpolar amino acids-glycine, valine, and proline in the ratio 2:2:1, with insignificant amounts of the polar amino acids and cross-linking agents. It was found not to have any antigenic sites. Another fraction which was the major component of the digest contained the cross-links in an amount three times that found in complete elastin but was only weakly antigenic. The fraction with the greatest antigenicity was found to be enriched in polar amino acids and tyrosine; it also contained 7% carbohydrate as glucose and 50% lipid.

Amiodarone, a cardiac antiarrhythmic agent, has been associated with the development of interstitial pulmonary fibrosis in patients receiving prolonged therapy with the drug. To further assess the toxic effects of amiodarone on lung tissue, Syrian hamsters were given a single intratracheal insufflation of the agent and evaluated for histologic evidence of lung injury. Control animals received intratracheal insufflations of the vehicle in which amiodarone was dissolved. After an initial, transient alveolitis in both experimental and control animals, the amiodarone-treated lungs developed increased interstitial thickening due to fibrinous exudates, alveolar epithelial hyperplasia, inflammatory cell infiltrates, and marked deposition of collagen manifested on trichrome staining. Controls, in contrast, showed nearly complete resolution of the initial alveolitis. An unusual feature of the amiodarone-induced lung injury was reemergence of the alveolitis between 5 and 14 days, which included a marked influx of eosinophils into the lung. Although the precise mechanism of the lung injury is not known, the persistence of the acute inflammatory cells as well as the presence of eosinophils suggests a hypersensitivity-type reaction. Furthermore, the progression of lung injury to fibrosis after a single insult with the drug suggests that mere discontinuation of amiodarone therapy in humans may not reverse the disease process, but that corticosteroid therapy may also be required. Amiodarone appears to be a useful agent to induce diffuse fibrotic reactions in the lung that morphologically resemble idiopathic pulmonary fibrosis in humans.

The glycosaminoglycans produced by a mixed cell line of normal adult rat lung and an endothelial clone derived from this line were isolated and examined. Cellulose acetate electrophoresis of media and cells before and after digestion with specific enzymes indicated that all the major glycosaminoglycans except keratan sulfate were synthesized by both cultures. Heparan sulfate and dermatan sulfate were found only in the cell fraction while hyaluronic acid was found in both the medium and the cell fractions. The chondroitin sulfates were isolated from the medium.The endothelial clone produced a 4: 1 ratio of glucosamine to galactosamine in the medium from the fifth through thirteenth months of culture. The medium of the mixed cell line initially contained glycosaminoglycans with a glucosamine to galactosamine ratio of 2:1 but after approximately one year of culture, the ratio had changed to 4.6: 1 suggesting that the culture contained predominantly endothelial cells.

Several methods are described which employ thin layer chromatography and electrophoresis for investigations into the structure and synthesis of crosslinked elastin. These procedures are more sensitive and involve less elaborate equipment than separation on paper. The methods described include an electrophoretic procedure for detecting the cross-linking amino acids in elastin hydrolysates, a method for completely separating desmosine, isodesmosine and merodesmosine in these hydrolysates, and a method for producing two-dimensional peptide maps of elastase digests of elastin.

Antibodies were raised in rabbits against human lung elastic fiber and peptides derived from human lung elastin and microfibrils. Elastic fiber produced antibodies with very low liter. Elastin peptides had low immunogenicity and multiple injections of at least 100 mg per rabbit per injection were required to produce antibodies. Microfibril peptides had a much higher immunogenicity and produced antibodies with multiple injections of only 10 mg per rabbit per injection.Anti-elastic fiber serum did not react with peptides derived from microfibrils or elastin at concentrations of 5 mg or 10 mg per ml solution. At 25 mg/ml solution microfibril peptides gave a faint precipitin line in Ouchterlony double diffusion tests, but elastin peptides did not react. There was no cross reactivity between microfibril peptides and anti-elastin serum or elastin peptides and anti-microfibril serum at low concentration. At higher concentration, i.e. 25 mg/ml, some cross reactivity was observed with both antigens and both antise...

Summary Synthesis of crosslinked elastin by a major lung cell has not previously been reported. Elastin production by an established clone of rat lung endothelial cells was detected by two separate, highly sensitive methods. The first procedure involved isolation and identification of the labelled, elastin-specific crosslinking amino acids desmosine and isodesmosine by thin layer electrophoresis and radioautography. The second procedure involved detection of elastin by immunofluorescence, using anti-rat lung elastin peptide serum.

Elastin peptides were fractionated on a SE-Sephadex C-50 column by sequential elution with the following buffers: a) 0. 01 N acetic acid, b) linear gradient from 0. 05 N sodium acetate buffer, pH 4. 0 to 0. 4 N sodium acetate buffer, pH 4. 6, c) 0. 4 N sodium acetate buffer, pH 4. 6, d) 0. 5 N sodium hydroxide. Based on the elution profile, tubes were pooled into 12 fractions. Amino acid analysis of these fractions showed that the first 7 fractions contained mostly nonpolar peptides which lacked desmosine and isodesmosine residues. The other 5 fractions contained more polar peptides with desmosine and isodesmosine. Only polar fractions containing desmosine and isodesmosine proved to be antigenic when assayed by double immunodiffusion test. These antigenic peptide fractions were subjected to further analysis. Separation by gel filtration on Bio-Gel P-150 showed that each of the antigenic fractions consisted of at least 3 polypeptides with apparent molecular weights of 57, 000, 46, 000 and 9, 000. The two h...

A simple method for the preparation of 5-phospho- d -arabonic acid by oxidation of d -fructose 6-phosphate is described. The significance of such an easily accessible phosphorylated product of the carbohydrate series obtainable in excellent yield is discussed.

A group of enzymes, fractionally precipitated from Cl. histolyticum filtrates at high ammonium sulfate concentrations, was found capable of hydrolyzing a wide variety of simple peptide substrates. The peptidases have been characterized; their pH optima, stability, behavior toward metal ions and various inhibitors, kinetics, and specificity were studied and comparisons were made with other known exopeptidases. It was found that a number of different enzymes was involved and that their ratio varied considerably in different preparations. The enzymes are not identical with any known group, including previously reported Cl. histolyticum enzymes.

Two peptides valyl-valine and leucyl-valine were isolated from 18 hour HCL hydrolyzates of bovine ligamentum nuchae elastin by a combination of gel filtration on Bio-Gel P-2 and partition chromatography on Sephadex G-25. The amounts of both peptides recovered from elastin were approximately 0.5%. The extreme resistance of these peptides to acid hydrolysis may be attributed to steric effects produced by their valine residue(s). These effects may play a role in producing the observed property of elasticity of this protein by interfering with the usual hydrogen bonding in the peptide α-helices. Such a condition may lead to a reduced rigidity, or greater extensibility of the peptide chains.

The Bratton-Marshall test for naphthylamine has been modified and adapted to determine the naphthylamine released on hydrolysis of the specific chymotrypsin substrate glutaryl- l -phenylalanine-β-naphthylamide. This substrate shows advantages over others used in similar procedures because it combines satisfactorily solubility, specificity, and sensitivity with the release of naphthylamine upon hydrolysis. The procedure has been applied to the determination of chymotrypsin itself and to other enzymes with similar specificities such as the chymotrypsinlike cathepsin C of liver, kidney, tumors, and other tissues. It has been used also to assay chymotrypsin inhibitors, particularly those found in tissues.

Summary Up till now readily formed derivatives from which the free sugars could be easily recovered were lacking in the case of several carbohydrates of biochemical interest. 2,5-Dichlorophenylhydrazones satisfy these requirements. Their preparation and characteristics are described. Asymmetrical diphenylhydrazine is preferable as a reagent for the separation of arabinose and ribose.

The specific collagenase present in Cl. histolyticum filtrates has been further purified by continuous electrophoresis of enzyme preparations obtained by ammonium sulfate fractionation. Its specificity was confirmed by its inability to attack a variety of protein and synthetic substrates. The effect of metal ions, various inhibitors, different buffers, pH, and heat was investigated. Some applications and the general importance of the enzyme are discussed.

When guinea pigs instead of rabbits were used as the host animals, 8--16 times higher antibody titers against human lung elastin peptides were produced with only 1/20 the amount of antigen per unit body weight. This corresponds to a 200-fold enhancement of the immune response.

Incorporation of [35]S-sulfate into hepatic glycosaminoglycans (GAGs) is affected by intravenous administration of endotoxin. There are significant increases in total labeled glycosaminoglycans and in the percentage of labeled dermatan sulfate 72 h post-endotoxin.

It is established that the product of oxidative degradation of d -fructose 6-phosphate is d -arabonic acid 5-phosphate.