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Biomedical subjects

H M Kagan

Publications and source records attributed to H M Kagan.

At least 19 recordsLinked to original sources
PubMed1992

Characterization and developmental expression of chick aortic lysyl oxidase.

The complete primary structure of chick lysyl oxidase was determined by recombinant DNA techniques. The nucleotide sequence of contiguous chick lysyl oxidase cDNA clones contained an open reading frame of 1260 bases which encodes a predicted protein of 420 amino acid residues (48,150 Da). In comparison to the deduced primary structure of rat lysyl oxidase, the chick enzyme is larger in size and exhibits a strong conservation of sequence within the latter two thirds of the molecule (92% identity) and a high degree of divergence in the first 150 amino acid residues (60% identity allowing for several insertions in both sequences). The developmental steady-state levels of lysyl oxidase mRNA together with the mRNAs encoding two of the enzyme's substrates (tropoelastin and type I collagen) increased between 8 and 16 days of embryonic development. Although levels of lysyl oxidase mRNA increased during aortic embryogenesis, the specific activity of the enzyme remained fairly constant suggesting that lysyl oxidase activity increases in direct proportion to total protein synthesis and cell number. In situ hybridization showed that the spatial expressions of lysyl oxidase and tropoelastin transcripts differ suggesting that the enzyme and substrate genes are differentially regulated within the cells of the arterial wall.

Amino Acid Sequence↗
PubMed1992

Oxidation of peptidyl lysine by copper complexes of pyrroloquinoline quinone and other quinones. A model for oxidative pathochemistry.

Various o- and p-quinones were assessed as oxidants of peptidyl lysine in elastin and collagen substrates in the presence and absence of divalent copper as paradigms of protein-lysine 6-oxidase (lysyl oxidase) which contains both quinone and copper cofactors. Pyrroloquinoline quinone was among the most active in the absence and the most active of the o- and p-quinones tested in the presence of copper. The optimal rate of elastin oxidation occurred at a 2:1 PQQ/Cu(II) ratio while Cu(II) itself oxidized elastin relatively slightly. Elastin oxidation by 2:1 PQQ/Cu(II) required aerobic conditions consistent with oxygen-dependent turnover of this catalytic pair. Dimethylsulfoxide and catalase individually or in combination inhibited elastin oxidation by PQQ/Cu(II) by approx. 50%, suggesting that oxygen free radical species participate in the reaction. Amino-acid analysis of elastin and collagen substrates oxidized by 2:1 PQQ/Cu and then reduced with borohydride revealed that alpha-aminoadipic-delta-semialdehyde and lesser amounts of covalent cross-linkages were generated by this oxidant. In contrast, lysine oxidase produced aldehydes and significantly greater quantities of cross-linkage products, consistent with the known specificity of the enzyme. These data, thus, indicate the potential for free quinones, such as PQQ, particularly when stimulated by appropriate metal ions, to act as adventitious oxidants of lysine side-chains in proteins.

Anaerobiosis↗
PubMed1992

Post-translational glycosylation and proteolytic processing of a lysyl oxidase precursor.

The synthesis and post-translational processing of a lysyl oxidase precursor protein predicted by the cDNA sequence of rat aorta lysyl oxidase were investigated. In vitro transcription of the cloned lysyl oxidase cDNA and cell-free translation of its mRNA transcript yielded a 47-kDa protein in the absence and a 50-kDa glycosylated protein in the presence of pancreatic membranes, each of which were immunoprecipitated with antibody against the 32-kDa bovine enzyme. Similarly, an N-glycosylated, 50-kDa protein band was synthesized by and immunoprecipitated from cultured neonatal rat aorta smooth muscle cells labeled with [35S]methionine. Pulse-chase studies of proteins newly synthesized by these cells demonstrated that the glycosylated 50-kDa precursor is secreted and that it is processed to the 32-kDa molecular form of lysyl oxidase principally in the medium. The presence of an extracellular processing enzyme activity which converts the 50-kDa precursor to the 32-kDa species was demonstrated by incubating conditioned medium of neonatal rat aorta smooth muscle cell cultures as a source of processing activity with conditioned medium containing 35S-labeled precursor synthesized but not processed by a tumorigenic cell line transfected with a lysyl oxidase expression vector. In contrast to the 50-kDa species, the 32-kDa protein does not appear to be N-glycosylated consistent with the loss of N-linked oligosaccharide units during the processing to the smaller species. The modes of biosynthesis and secretion of lysyl oxidase are discussed in terms of other nonproteolytic activities required for activation of prolysyl oxidase.

Animals↗
PubMed1992

The complete derived amino acid sequence of human lysyl oxidase and assignment of the gene to chromosome 5 (extensive sequence homology with the murine ras recision gene).

Lysyl oxidase catalyzes the oxidation of lysine residues to alpha-aminoadipic-delta-semialdehyde. This is the first step in the covalent cross-linking of collagen and tropoelastin and results in the formation of insoluble collagen and elastic fibers in the extracellular matrix. We have characterized the complete nucleotide sequence of human lysyl oxidase (EC 1.4.3.13) and compared the derived amino acid sequence (417-amino acids) to rat lysyl oxidase and the mouse ras recision gene (rrg). 88% of amino acids and 83% of nucleotides were conserved between human and rat lysyl oxidase. The mouse ras recision gene demonstrated 89% conservation of amino acids with human lysyl oxidase. The sequence conservation was not evenly distributed along the molecule. The carboxy terminus of the protein, which contains the putative copper binding sites and is likely to be the catalytically active domain, was more highly conserved than the amino terminus. The 89% amino acid sequence similarity between the murine ras recision gene and human lysyl oxidase suggests that they are the same gene product. Therefore, in addition to cross linking of extracellular matrix proteins, lysyl oxidase may have a direct role in tumor suppression. Northern blot analysis of poly A+RNA from cultured skin fibroblasts revealed at least three-distinct transcripts, sized 4.8 kb, 3.8 kb and 2.0 kb. In addition, using a panel of human mouse cell hybrids, the lysyl oxidase gene was assigned to human chromosome 5.

Amino Acid Sequence↗
PubMed1991

Specific detection of quinoproteins by redox-cycling staining.

Quinones and related quinonoid substances catalyze redox cycling at an alkaline pH in the presence of excess glycine as reductant. With nitroblue tetrazolium and oxygen present there is concomitant reduction of the tetrazolium to formazan. This property of quinonoid compounds is used for the specific staining of quinoproteins, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose. The dopa-containing vitelline proteins and the 6-hydroxydopa-containing bovine serum amine oxidase are stained with the nitroblue tetrazolium/glycinate reagent. Also, the mammalian quinoproteins, diamine oxidase and lysyl oxidase, purported to contain pyrroloquinoline quinone, tested positive in this procedure. No quinonoid components were detected in three putative pyrroloquinoline quinone-containing quinoproteins, dopamine beta-hydroxylase, lipoxygenase, and peptidylglycine-amidating monoxygenase. Redox-cycling staining therefore confirms the presence of covalently bound quinones in the copper-dependent amine oxidases, but not in two putative quinoprotein oxygenases. Clarification of the biological significance of quinolation should be facilitated by identification of quinoproteins using this approach.

Animals↗
PubMed1991

Properties and function of lysyl oxidase.

Lysyl oxidase catalyzes the oxidation of peptidyl lysine to alpha-aminoadipic-delta-semialdehyde, the precursor to the covalent crosslinkages that stabilize fibers of elastin and collagen. This enzyme contains both copper and a carbonyl cofactor consistent with an o-quinone. The proposed mechanism of action is derived from available kinetic and chemical data and also can account for mechanism-based inhibition of the enzyme by specific monoamines and diamines. Recent evidence for biosynthetic precursors and for the regulation of lysyl oxidase in fibrotic and malignant diseases is discussed.

Animals↗
PubMed1991

Induction of lung lysyl oxidase activity and lysyl oxidase protein by exposure of rats to cadmium chloride: properties of the induced enzyme.

Inspiration of CdCl2 results in a focally fibrotic response in rat lungs and markedly increases the activity of lung lysyl oxidase. Western blot analyses of urea-extractable rat lung proteins revealed that the levels of an immunoreactive, 32,000-Da protein were markedly increased in the cadmium-exposed rat lung tissue, consistent with the induction of lysyl oxidase protein. Anion exchange chromatography revealed low levels of multiple peaks of catalytically functional lysyl oxidase in control rat lung extracts, while the profile of cadmium-exposed rat lung extracts displayed markedly elevated levels of multiple peaks of enzyme activity indicating that the charge heterogeneity is expressed in the activated enzyme. The cadmium-induced enzyme was purified as a species of 32 kDa, without resolving individual ionic variants. The catalytic and physical properties of the isolated enzyme were very similar to those of previously well characterized basal enzyme of bovine aorta, including the presence of a pyrroloquinoline quinone-like carbonyl cofactor. The copper and cadmium content of the cadmium-induced enzyme indicated little if any replacement of tightly-bound copper by cadmium in the exposed lung.

Aminopropionitrile↗
PubMed1990

Structural and catalytic properties of copper in lysyl oxidase.

The spectral and catalytic properties of the copper cofactor in highly purified bovine aortic lysyl oxidase have been examined. As isolated, various preparations of purified lysyl oxidase are associated with 5-9 loosely bound copper atoms per molecule of enzyme which are removed by dialysis against EDTA. The enzyme also contains 0.99 +/- 0.10 g atom of tightly bound copper per 32-kDa monomer which is not removed by this treatment. The copper-free apoenzyme, prepared by dialysis of lysyl oxidase against alpha,alpha'-dipyridyl in 6 M urea, catalyzed neither the oxidative turnover of amine substrates nor the anaerobic production of aldehyde at levels stoichiometric with enzyme active site content, thus contrasting with the ping pong metalloenzyme. Moreover, the spectrum of the apoenzyme was not measurably perturbed upon anaerobic incubation with n-butylamine, while difference absorption bands were generated at 250 and 308 nm in the spectrum of the metalloenzyme incubated under the same conditions. A difference absorption band also developed at 300-310 nm upon anaerobic incubation of pyrroloquinoline quinone, the putative carbonyl cofactor of lysyl oxidase, with n-butylamine. Full restoration of catalytic activity occurred upon the reconstitution of the apoenzyme with 1 g atom of copper/32-kDa monomer, whereas identical treatment of the apoenzyme with divalent salts of zinc, cobalt, iron, mercury, magnesium, or cadmium failed to restore catalytic activity. The EPR spectrum of copper in lysyl oxidase is typical of the tetragonally distorted, octahedrally coordinated Cu(II) sites observed in other amine oxidases and indicates coordination by at least three nitrogen ligands. The single copper atom in the lysyl oxidase monomer is thus essential at least for the catalytic and possibly for the structural integrity of this protein.

2,2'-Dipyridyl↗
PubMed1990

Cloning of rat aorta lysyl oxidase cDNA: complete codons and predicted amino acid sequence.

Lysyl oxidase cDNA clones were identified by their reactivity with anti-bovine lysyl oxidase in a neonatal rat aorta cDNA lambda gt11 expression library. A 500-bp cDNA sequence encoding four of six peptides derived from proteolytic digests of bovine aorta lysyl oxidase was found from the overlapping cDNA sequences of two positive clones. The library was rescreened with a radiolabeled cDNA probe made from one of these clones, thus identifying an additional 13 positive clones. Sequencing of the largest two of these overlapping clones resulted in 2672 bp of cDNA sequence containing partial 5'- and 3'-untranslated sequences of 286 and 1159 nucleotides, respectively, and a complete open reading frame of 1227 bp encoding a polypeptide of 409 amino acids (46 kDa), consistent with the 48 +/- 3 kDa cell-free translation product of rat smooth muscle cell RNA that was immunoprecipitated by anti-bovine lysyl oxidase. The rat aorta cDNA-derived amino acid sequence contains the sequence of each of the six peptides isolated and sequenced from the 32-kDa bovine aorta enzyme, including the C-terminal peptide with sequence identity of 96%. Northern blots screened with lysyl oxidase cDNA probes identified hybridizing species of 5.8 and 4.5 kb in mRNA of rat aorta and lung, while dot blot analyses were negative for lysyl oxidase mRNA in preparations of rat brain, liver, kidney, and heart. A 258-bp segment of the 3'-untranslated region of lysyl oxidase cDNA is 93% identical with a highly conserved region of the 3'-untranslated sequence of rat elastin cDNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗
PubMed1990

Effect of dietary cadmium and/or copper on the bone lysyl oxidase in copper-deficient rats relative to the metabolism of copper in the bone.

Effects of cadmium (Cd) on lysyl oxidase activity and copper (Cu) metabolism in bone were studied using Cu-deficient rats supplemented with Cu and/or Cd in a diet. When fed for 8 weeks on a diet containing 0.3 ppm or less Cu (-Cu diet), weanling rats revealed anemia, and markedly decreased plasma ceruloplasmin activity and serum Cu to less than 15% of normal level, showing features of Cu-deficiency. These rats were divided into four groups and refed for another 2 weeks on the following diets: Group I, -Cu diet; Group II, -Cu diet with 50 ppm Cd (+Cd diet); Group III, -Cu diet supplemented with 15 ppm Cu (+Cu diet); group IV, -Cu diet with both Cu and Cd (+Cu/+Cd diet). After 2 weeks, serum Cu levels of Groups I, II, III and IV were 1.8, 0.8, 78 and 74% of the normal control level (1.438 +/- 0.060 micrograms/ml), respectively. Concentrations of Cu in epi- and metaphyses of the control group, Groups I, II, III and IV were 1.45 +/- 0.20, 0.67 +/- 0.08, 0.76 +/- 0.12, 1.40 +/- 0.31 and 1.22 +/- 0.05 micrograms/g wet tissue, in that order. Concentrations of Cd in epi- and metaphysis increased in only Groups II and IV and were 0.15 +/- 0.03 and 0.18 +/- 0.01 micrograms/g wet tissue, respectively. Thus, having both Cd and Cu supplements in a diet did not inhibit each other's uptake into the tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗
PubMed1979

Changes in lung lysyl oxidase activity in streptozotocin-diabetes and in starvation.

The effects of streptozotocin-induced diabetes and of starvation on the lysyl oxidase activity of rat lung were investigated. Enzyme activity was elevated 2--3 fold in the lungs of streptozotocin-diabetic rats. In contrast, starvation of rats produced a rapid loss of lung lysyl oxidase activity, with levels approximating 25% of control values after 48--72 h of starvation. Enzyme activity was essentially fully restored to control values upon refeeding the 48-h starved animals for 3 h. These studies demonstrate the responsiveness of lysyl oxidase to these physiological states and suggest a component, enzymatic basis of change in lung function known to occur in the diabetic state.

Amino Acid Oxidoreductases↗
PubMed1979

Purification and properties of four species of lysyl oxidase from bovine aorta.

Lysyl oxidase of bovine aorta was resolved into four enzymically active species by elution from DEAE-cellulose with a salt gradient in 6m-urea, consistent with purification results obtained with enzyme of other tissues [Stassen (1976) Biochim. Biophys. Acta438, 49-60]. In the present study, each of the four peaks of activity was purified to apparent homogeneity by subsequent chromatography on gel-filtration media in 6m-urea. Each enzyme is eluted as a species with mol.wt. approx. 30000 under these conditions, although lysyl oxidase polymerizes to a series of multimers with molecular weights ranging up to 1000000 in the absence of urea. The apparent subunit molecular weight of each enzyme species determined by electrophoresis in sodium dodecyl sulphate and 8m-urea is approx. 32000-33000. The amino acid compositions of the purified forms of lysyl oxidase are similar to each other, although sufficient differences exist to conclude that each is a unique molecular species. Incorporation of alpha-toluenesulphonyl fluoride into the purification scheme does not alter the resolution of enzyme into four species, suggesting that proteolysis during isolation is not the basis of the heterogeneity. The similar sensitivities of each form of enzyme to chelating agents and to semicarbazide and isoniazid indicate that each requires the participation of a metal ion, presumably Cu(2+), and of a carbonyl compound for enzyme function. The present study describes a method for the purification of multiple species of lysyl oxidase and reveals that significant chemical differences exist between the different enzyme forms.

Amino Acid Oxidoreductases↗
PubMed1979

A possible role for elastin ligands in the proteolytic degradation of arterial elastic lamellae in the rabbit.

Thoracic aortae of normal rabbits were perfused with pancreatic elastase in vitro at 37 degrees C and 70 mm Hg pressure in the presence or absence of elastin ligands previously shown to stimulate or inhibit the enzymatic degradation of elastin. Perfusion with elastase results in an average of 3.6 lamellae degraded, whereas addition of sodium linoleate before and during the perfusion with elastase increases this value to 7.9 (P less than 0.001). Conversely, perfusion with the cationic detergent, dodecyltrimethylammonium chloride, completely prevents the degradation of elastic lamellae by elastase. These effects do not reflect alterations of the intrinsic catalytic activity of elastase, but apparently indicate the formation of complexes between the elastin ligands and arterial elastic lamellae, as is consistent with prior studies indicating such interactions between fatty acids or detergents and purified elastin. These studies suggest that agents such as fatty acids may significantly alter the metabolic susceptibility of elastin in vivo and possibly contribute to the degradation of elastic lamellae seen in arteries with advanced atherosclerosis.

Animals↗
PubMed1978

Changes in the state of ionization of carboxyl groups in elastin in response to the binding of sodium dodecyl sulfate.

The interaction of sodium dodecyl sulfate with elastin has been studied by complexometric titration. Approximately 1.2 mumoles of protons with a pKapp of 5.45 are taken up by 10 milligrams of insoluble elastin upon the binding of detergent, apparently due to the protonation of normally ionized carboxylate functions in this protein. Since ionized carboxylate functions of elastin are essential for its interaction with elastase and, possibly, metallic cations, these results may have physiological significance in view of the affinity of elastin for lipid-like ligands.

Chemical Phenomena↗