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Biomedical subjects

H M Kagan

Publications and source records attributed to H M Kagan.

At least 73 records · Page 4Linked to original sources
PubMed1986

Oxidation of lysine side-chains of elastin by the myeloperoxidase system and by stimulated human neutrophils.

Exposure of [3H]-lysine labeled elastin to either purified myeloperoxidase plus H2O2 and halides or human neutrophils plus phorbol myristate acetate resulted in oxidation of lysine side chains quantitated as 3H2O release. In both the enzyme and cell system oxidation was blocked by azide, cyanide or catalase, but not by beta-aminopropionitrile, an inhibitor of lysyl oxidase. Myeloperoxidase-deficient neutrophils were ineffective unless exogenous myeloperoxidase was added. These data provide a biochemical basis for inflammatory changes in connective tissue proteins mediated by oxidant secretory products of neutrophils.

Animals↗
PubMed1986

Ultrastructural changes in hamster lung 15 min to 3 hr after exposure to pancreatic elastase.

A single endotracheal instillation of porcine pancreatic elastase into hamster lungs induces morphological changes that can be detected as early as 15 min after the instillation of enzyme. Lung tissue from animals sacrificed at six time points between 15 min and 3 hr after enzyme instillation was examined for ultrastructural alterations. There were few of these alterations and they were highly focal, but they included damaged epithelial cells and partially digested elastic fibers that occurred both in the parenchyma and the pleura. A cytochemical technique employing N-t-Boc-L-alanine-p-nitrothiophenyl ester as a substrate for elastase-like enzymes was also used in an effort to locate pancreatic elastase shortly after instillation into the lungs. Reaction product was observed on the alveolar surface and in pinocytotic vesicles of alveolar type I cells, in connective tissue areas, in fibroblasts, and in pinocytotic vesicles of capillary endothelial cells. The location of reaction product coupled with ultrastructural alterations in the tissue suggests that pancreatic elastase instilled into the trachea may reach the interstitium in two ways: First, transport may take place across intact alveolar type I cells via pinocytotic vesicles, and second, a small amount of enzyme may gain access to the connective tissue after the disruption of epithelium in a few highly focal areas.

Animals↗
PubMed1986

Ultrastructural immunolocalization of lysyl oxidase in vascular connective tissue.

The localization of lysyl oxidase was examined in calf and rat aortic connective tissue at the ultrastructural level using polyclonal chicken anti-lysyl oxidase and gold conjugated rabbit anti-chicken immunoglobulin G to identify immunoreactive sites. Electron microscopy of calf aortic specimens revealed discrete gold deposits at the interface between extracellular bundles of amorphous elastin and the microfibrils circumferentially surrounding these bundles. The antibody did not react with microfibrils which were distant from the interface with elastin. There was negligible deposition of gold within the bundles of amorphous elastin and those few deposits seen at these sites appeared to be associated with strands of microfibrils. Lysyl oxidase was similarly localized in newborn rat aorta at the interface between microfibrils and nascent elastin fibers. Gold deposits were not seen in association with extracellular collagen fibers even after collagen-associated proteoglycans had been degraded by chondroitinase ABC. However, the antibody did recognize collagen-bound lysyl oxidase in collagen fibers prepared from purified collagen to which the enzyme had been added in vitro. No reaction product was seen if the anti-lysyl oxidase was preadsorbed with purified lysyl oxidase illustrating the specificity of the antibody probe. The present results are consistent with a model of elastogenesis predicting the radial growth of the elastin fiber by the deposition and crosslinking of tropoelastin units at the fiber-microfibril interface.

Amino Acid Oxidoreductases↗
PubMed1986

Comparison of lysyl oxidase from bovine lung and aorta.

A prior report had concluded that bovine lung lysyl oxidase displayed an unusual resistance to inhibition by beta-aminopropionitrile (BAPN) in contrast to the enzyme isolated from other connective tissues. Therefore, lysyl oxidase was purified from fetal bovine lung and from aorta of young calves by parallel procedures, and key chromatographic and catalytic properties of these enzymes were directly compared. The enzymes prepared from both tissues each demonstrated the same multiplicity of enzyme species which resolve on DEAE-cellulose and otherwise demonstrated the same chromatographic behavior on gel exclusion media and on collagen-Sepharose and Cibacron blue-Sepharose columns. The activities of the unresolved but partially purified enzyme species of lung and of aortic lysyl oxidase were each fully inhibitable by approximately the same low (mu molar) concentrations of BAPN. Thus, the enzymes of both tissues were found to be very similar to each other by several criteria.

Amino Acid Oxidoreductases↗
PubMed1985

Assessment of lysyl oxidase variants by urea gel electrophoresis: evidence against disulfide isomers as bases of the enzyme heterogeneity.

Methods for the copurification and rapid assessment of the protein profiles corresponding to the multiple variants of bovine aortic lysyl oxidase are described. The individual variants do not resolve from each other by electrophoresis in sodium dodecyl sulfate but are resolved by gel electrophoresis in 8 M urea, thus providing a new method for their detection independent of enzyme assay. Alkylation of the purified mixture of the variants with iodoacetamide after reduction with dithiothreitol identified three disulfides per 32,000-Da monomer. Urea gel electrophoresis revealed that the heterogeneity of lysyl oxidase persists after reduction and alkylation, indicating that disulfide isomers are not the bases of the enzyme heterogeneity.

Amino Acid Oxidoreductases↗
PubMed1985

Immunological characterization of bovine lysyl oxidase.

Antibodies to homogeneously purified bovine aortic lysyl oxidase were prepared in chickens. The chicken anti-lysyl oxidase antiserum effectively inhibited bovine aortic lysyl oxidase activity. Non-immune antiserum from chickens, goats and humans was found to enhance bovine aortic lysyl oxidase activity, while non-immune rabbit serum inhibited enzyme activity. A competitive ELISA was developed to monitor immunoreactive lysyl oxidase during purification. Chromatography of bovine lysyl oxidase on Sephacryl S-200, the final step in purification, revealed two peaks of immunoreactive lysyl oxidase. The large molecular weight peak appears to contain inactive multimeric forms of the enzyme.

Amino Acid Oxidoreductases↗
PubMed1985

Binding of lysyl oxidase to fibrils of type I collagen.

The binding of highly purified bovine aortic lysyl oxidase to native fibrils of type I collagen has been measured by assay of unbound lysyl oxidase activity in the supernatants of enzyme-collagen mixtures after centrifugation. The apparent binding affinity of lysyl oxidase for native fibrils is quite similar to that for fibrils prepared from pepsin- or chymotrypsin-digested type I collagen, demonstrating that the enzyme binds to the triple-helical portion of collagen molecules. The data also indicate that the enzyme binds predominantly to the fibrillar surface. The results suggest that lysyl oxidase initiates crosslink formation at an early stage in collagen fibrillogenesis.

Amino Acid Oxidoreductases↗
PubMed1984

Influence of sequence and charge on the specificity of lysyl oxidase toward protein and synthetic peptide substrates.

Lysyl oxidase initiates the covalent cross-linking of elastin and collagen by oxidizing lysine residues in these proteins to alpha-aminoadipic-delta-semialdehyde. Sequences surrounding susceptible lysines in elastin are considerably different from those in collagen and yet the same enzyme can oxidize both substrates. Possible bases of the specificity have been explored assaying for H2O2 release accompanying the oxidation of synthetic peptide and protein substrates. Rates of oxidation of random co-polymers were maximal with (Ala,Lys)n and decreased in the order (Val,Lys)n greater than (Leu,Lys)n greater than (Lys)n greater than (Phe,Lys)n greater than (Tyr,Lys)n. The ordered polymer (Ala-Lys-Glu)n was oxidized at only 3% of the rate of (Ala,Lys)n, implying inhibition by peptidyl glutamate. Consistent with this conclusion, kinetic analyses using ordered oligopeptides revealed that, relative to Ala-Ala-Lys-Ala-Ala, Km is increased 9.3-fold for lysine in Ala-Ala-Lys-Glu-Ala-Ala, 2.5-fold in Ala-Ala-Lys-Arg-Ala-Ala, and 1.8-fold in Ala-Ala-Glu-Lys-Ala-Ala. Tyrosine C-terminal to lysine in such peptides also increases Km 5-fold. In addition, lysyl oxidase oxidized lysine in various proteins with basic isoelectric points and was much less or not active against various acidic proteins. Lysyl oxidase was inactive against native bovine serum albumin but effectively oxidized albumin if albumin carboxyl functions were first amidated by chemical modification. These results suggest that peptides bind to lysyl oxidase in a preferred directional sense and indicate-that net anionic character as well as the specific position of anionic residues in substrates can selectively effect substrate potential. Implications of these results for the oxidation of elastin and collagen are discussed.

Amino Acid Oxidoreductases↗
PubMed1984

Beta-substituted ethylamine derivatives as suicide inhibitors of lysyl oxidase.

Lysyl oxidase initiates the covalent cross-linking of elastin and collagen, converting lysyl residues in these proteins to peptidyl aldehyde residues. The present study explored structural and electron withdrawing features required to generate mechanism-based inhibitors of this enzyme with antifibrotic potential. It was found that the electron withdrawing nitrile moiety of beta-aminopropionitrile (BAPN), a naturally occurring syncatalytic inhibitor of lysyl oxidase, can be replaced by chlorine, bromine, or the nitro function to yield 2-haloamines or nitroethylamine compounds which also act as mechanism-based irreversible inhibitors of this enzyme. BAPN and 2-bromo- and 2-chloroethylamine exhibit similar KI values of 6-10 microM. However, the enzyme becomes irreversibly inactivated significantly faster by either of the 2-haloamines than by BAPN. 2-Nitroethylamine has by far the poorest affinity for the enzyme and inactivates much more slowly than the other amines of this series, consistent with interference with optimal enzyme-inhibitor interactions by the anionic nitro group. Unlike BAPN, 2-bromoethylamine is processed to a detectable aldehyde product upon incubation with enzyme, showing a partition ratio of 1.2 mol of acetaldehyde formed per mol of 2-bromo-ethylamine which becomes covalently incorporated in the enzyme. The results are consistent with the processing of 2-bromo-ethylamine to an enzyme-ethyleneamine Schiff base subject to hydrolysis to acetaldehyde or to covalent attack at carbon 2 by an enzyme nucleophile. Thus, beta-haloamines represent a new series of suicide inhibitors of lysyl oxidase which can inactivate the enzyme faster than BAPN and hence may have antifibrotic potential.

Amino Acid Oxidoreductases↗
PubMed1984

Alterations in collagen biosynthesis and in metallothionein in lungs of rats acutely or repeatedly exposed to cadmium chloride aerosol.

Comparisons were made between morphologic and biochemical changes occurring in the lungs of rats receiving a single exposure or repeated 2-h exposures of aerosolized 0.1% cadmium chloride, simulating varied exposure conditions potentially experienced by humans. Lung lysyl oxidase activity increases in both models, although less so with repeated exposures. Pulsing lung tissue in vitro with 3H-proline indicated that rates of collagen biosynthesis were elevated preferentially over rates of noncollagen protein synthesis in both exposure models. Lung metallothionein increased nearly linearly over 21 days of repeated exposure. Histologic examination revealed scarred lesions distorting alveolar structure and bronchioles in both models. However, scarring and cell exudation into the airways and interstitium was less in the repeatedly exposed model. The results indicate the activation of a connective tissue repair reaction in both models. Increased levels of metallothionein after repeated exposure may both sequester cadmium and reduce pools of copper available for lysyl oxidase synthesis, thus limiting the fibrotic response.

Aerosols↗
PubMed1983

Histone H1 is a substrate for lysyl oxidase and contains endogenous sodium borotritide-reducible residues.

Incubation of purified bovine aortic lysyl oxidase with rat liver or calf thymus H1 histone results in the catalytic formation of hydrogen peroxide, indicating the substrate potential of H1 for this connective tissue enzyme. Sodium borotritide-reducible residues consistent with aminoadipic semialdehyde and the lysinonorleucine crosslinkage were generated in H1 by incubation with lysyl oxidase. H1 histone also contains endogenous reducible functions including an unidentified prominent tritiated peak eluting near tyrosine as well as other lesser peaks, one of which is consistent with lysinonorleucine.

Amino Acid Oxidoreductases↗
PubMed1983

Reaction of aortic lysyl oxidase with beta-aminopropionitrile.

beta-Aminopropionitrile (BAPN) is a potent irreversible inhibitor of lysyl oxidase, the enzyme which initiates cross-linkage formation in elastin and collagen. The initial interaction of BAPN with aortic lysyl oxidase is competitive with elastin or alkyl amine substrates. Irreversible inhibition develops in a time- and temperature-dependent fashion upon incubation of enzyme with BAPN in the absence of substrate with a limiting inactivation rate constant of 0.16 min-1 and a KI of 6 microM at 37 degrees C. The labeled carbons of [1,2-14C]BAPN and [3-14C]BAPN covalently bind to the enzyme to equivalent extents and in parallel with the development of inactivation, negating the possibility that the nitrile moiety is eliminated from BAPN by enzymatic action. The copper content of the enzyme is not significantly altered upon interaction with BAPN. The extent of labeling by [14C]BAPN is reduced by prior treatment of the enzyme with carbonyl-modifying reagents, suggesting the possibility of enzyme-inhibitor Schiff base formation. However, BAPN is not processed to a free aldehyde product upon incubation with lysyl oxidase. A mechanism of inhibition is postulated which involves the formation of a covalent bond between an enzyme nucleophile and a ketenimine formed from BAPN by enzyme-assisted beta-proton abstraction.

Amino Acid Oxidoreductases↗
PubMed1983

Multiple modes of catalysis-dependent inhibition and inactivation of aortic lysyl oxidase.

Lysyl oxidase purified from bovine aorta can oxidize simple alkyl mono- and diamine substrates yielding the respective aldehyde, H2O2, and ammonia as products. The oxidation of such substrates is limited to approximately 100 catalytic turnovers per enzyme molecule since lysyl oxidase is syncatalytically and irreversibly inactivated in the course of oxidation of these amines. The present study reveals that addition of oxidant scavengers protects significantly against inactivation of lysyl oxidase and that the ammonia product is a reversible competitive inhibitor of amine oxidation. Further, the enzyme becomes covalently labeled by the amine substrate or its enzyme-processed derivative during catalysis. Thus, lysyl oxidase appears subject to multiple modes of catalysis-dependent inhibition or inactivation. Syncatalytic inactivation of lysyl oxidase might represent a means of restricting the activity of this enzyme toward its elastin and collagen substrates in vivo.

Amines↗
PubMed1983

Myocardial healing and repair after experimental infarction in the rabbit.

Adequacy of healing after acute myocardial infarction may determine the incidence of postmyocardial infarction rupture and ventricular aneurysm. Accordingly, in 36 rabbits, from 1 to 8 days after coronary ligation, and in 18 shams, we measured collagen formation and mechanical resistance of the infarcted left ventricle to stretch and rupture. Prolyl hydroxylase, an intracellular enzyme of collagen synthesis, increased from control activity of 3970 +/- 431 to 9224 +/- 643 counts/min per mg (cpm/mg) extractable protein (P less than 0.01) at 48 hours and was nearly maximal at 3 days postmyocardial infarction (14,518 +/- 2,030 cpm/mg, P less than 0.01). Lysyl oxidase, an extracellular collagen cross-linkage enzyme, increased from control activity of 29.6 +/- 4.8 to 74.7 +/- 18.8 cpm/mg extractable protein (P less than 0.01) at 72 hours and peaked at 121.5 +/- 7.3 (P less than 0.01) 4-6 days postmyocardial infarction. Hydroxyproline, a measure of collagen content, increased from control of 2.8 +/- 0.2 to 5.3 +/- 0.6 mg/g dry weight (P less than 0.05) at 72 hours and continued to increase at 8 days postmyocardial infarction (14.5 +/- 1.7 mg/g dry weight; P less than 0.01). When enzyme activities and hydroxyproline content were expressed relative to other reference bases, including DNA, tissue protein, dry weight, and total left ventricle, similar results were obtained. The mechanical properties of the infarcted left ventricle were determined by filling a balloon in the excised left ventricle until rupture. The rupture threshold in the normal left ventricle, [664 +/- 43 mm Hg (n = 16)], was not significantly different from that of the infarcted left ventricle on days 1-8 postmyocardial infarction. However, left ventricular rupture occurred more often through the myocardial infarction on days 1-4 postmyocardial infarction (59%) than on days 6 and 8 (18%; P = 0.03) when collagen content had significantly increased. Wall stress at the point of rupture in left ventricles from shams and normals was 30 +/- 2 g/mm2; tensile strength in isolated left ventricle muscle strips was 25 +/- 4 g/mm2 and in isolated scar strips at 7 days postmyocardial infarction was 59 +/- 7 g/mm2. The passive stiffness of the infarcted left ventricle increased from control of 61 +/- 5 to 94 +/- 6 mm Hg/100 microliters (P less than 0.05) at 4 days and 100 +/- 7 mm Hg/100 microliters (P less than 0.01) at 6 days postmyocardial infarction. Stiffness correlated with hydroxyproline content over the 8 days postmyocardial infarction (r = 0.599; P less than 0.001). Thus, the acutely infarcted ventricle was highly resistant to rupture during the initial 48 hours postmyocardial infarction, before any increase in collagen occurred. This result suggests that the preinfarction collagen content has an important role in preventing rupture. After 72 hours postmyocardial infarction, collagen synthesis appeared to be a determinant of infarct stiffness and resistance of the infarcted ventricle to rupture.

Animals↗
PubMed1983

Cigarette smoke blocks cross-linking of elastin in vitro.

Water-soluble components of the gas phase of filtered cigarette smoke inhibit formation of covalent desmosine cross-links during conversion of tropoelastin to elastin in vitro. These same smoke components also suppress lysyl-oxidase-catalyzed oxidation of lysine epsilon-amino groups in tropoelastin (the chemical step preceding formation of all elastin cross-links, including desmosine) in a dose-dependent fashion. However, gas phase smoke does not block the oxidation of diaminopentane by lysyl oxidase. Thus, gas phase cigarette smoke may possess substrate-directed (rather than enzyme-directed) inhibitory components capable of interfering with elastin cross-linking in vitro. Similar effects occurring in smokers' lungs could impede elastin repair and contribute to the development of pulmonary emphysema.

Animals↗
PubMed1982

Evidence for structural similarities in the multiple forms of aortic and cartilage lysyl oxidase and a catalytically quiescent aortic protein.

Lysyl oxidase purified from urea extracts of various connective tissues resolves into multiple catalytically functional species upon chromatography on DEAE-cellulose in 6 M urea. The four enzyme species of bovine aorta retain their original chromatographic behavior on DEAE with time of storage and after purification to homogeneity by gel exclusion chromatography. The peptide maps of each aortic enzyme partially digested by STaphylococcus aureus V8 protease are very similar to each other, as are the peptide maps of complete tryptic digests of each enzyme. Such similarity also exists between the peptide maps of the aortic enzyme and the urea-extractable lysyl oxidase of bovine cartilage, as well as with the peptide maps of a catalytically quiescent protein resolved from the aortic enzyme by gel exclusion chromatography. The substrate activity profiles of the multiple aortic enzyme species are also extremely similar. Although the origin of the enzyme multiplicity remains to be established, there is evident structural and catalytic similarities between the enzyme forms.

Amino Acid Oxidoreductases↗