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1.
Lysyl oxidase is a specific amine oxidase that catalyzes the formation of aldehyde cross-link intermediates in collagen and elastin. In this study, lysyl oxidase from embryonic chick cartilage was purified to constant specific activity and a single protein band on sodium dodecyl sulfate acrylamide gel electrophoresis. This band had an apparent molecular weight of 62,000. The eluted protein cross-reacted with inhibiting antisera developed against highly purified lysyl oxidase. The highly purified enzyme was active with both insoluble elastin and embryonic chick skin or bone collagen precipitated as reconstituted, native fibrils. There was low activity with nonhydroxylated collagen, collagen monomers, or native fibrils isolated from lathyritic calvaria. The maximum number of aldehyde intermediates formed per molecule of collagen that became insoluble was two. These results indicate that lysyl oxidase has maximum activity on ordered aggregates of collagen molecules that may be overlapping associations of only a few collagen molecules across. Formation of aldehyde intermediates and cross-links during fibril formation may facilitate the biosynthesis of stable collagen fibrils and contribute to increased fibril tensile strength in vivo. 相似文献
2.
Aldehyde-deficient non-crosslinked collagen obtained from lathyritic rats and collagen from penicillamine-treated rats, which is not deficient in aldehydes but the crosslinking of which is also inhibited, were implanted into the peritoneal cavity of hypophysectomized rats using the diffusion chamber technique. The enzyme lysyl oxidase which catalyses the aldehyde formation in certain lysyl residues of collagen and elastin was extracted from the skin of hypophysectomized rats. The activity of the enzyme was determined following its incubation with an L-[4,5-3H] lysine-labeled elastin substrate prepared from aortas of 17-day-old chick embryos. The result showed that the aldehyde deficient collagen did not crosslink while in the hypophysectomized animal indicating the lack of active lysyl oxidase in the rats. The enzyme activity in the skin of hypophysectomized animals was markedly reduced as compared with the controls indicating directly the dependance of lysyl oxidase activity on pituitary gland hormones. 相似文献
3.
The generation of covalent cross-linkages in collagen is initiated by the deamination by lysyl oxidase of specific lysine residues in this connective tissue protein. Since lysyl oxidase activity is influenced by ionic ligands bound to its protein substrates, the effect of heparin, an anionic glycosaminoglycan known to bind to collagen, was explored by using collagen and elastin substrates and highly purified lysyl oxidase. Concentrations of heparin up to 1 mg mL-1 had little effect on the enzymatic rate of oxidation if it was added prior to the addition of enzyme to a preformed fibrillar collagen substrate or to an insoluble elastin substrate. However, collagen oxidation was inhibited by 85% if this glycosaminoglycan was present at 0.4 mg mL-1 during collagen fibril formation before addition of the enzyme. Similarly, the rate and extent of collagen fibrillogenesis in the absence of lysyl oxidase were each markedly inhibited in the presence of 0.4 mg mL-1 heparin. Heparin also inhibited the extent of tight binding of lysyl oxidase to preformed fibrils by about 40% under conditions where enzyme activity against preformed fibrils was hardly affected. These results suggest that heparin may modulate the oxidation and thus the insolubilization of extracellular collagen fibers, possibly under conditions where elastin fiber synthesis is not affected, and that the tight binding of lysyl oxidase to collagen is not completely related to the expression of enzyme activity toward this substrate. These results also have mechanistic implications for the retarding effect of heparin on postoperative wound healing. 相似文献
4.
A Fluorometric Assay for Detection of Lysyl Oxidase Enzyme Activity in Biological Samples 总被引:5,自引:0,他引:5
Lysyl oxidase catalyzes the final known enzymatic step required for collagen and elastin cross-linking in the biosynthesis of normal mature functional insoluble extracellular matrices. In addition, lysyl oxidase has been identified as a possible tumor suppressor. Lysyl oxidase activity in biological samples is traditionally and most reliably assessed by tritium release end-point assays using radiolabeled collagen or elastin substrates involving laborious vacuum distillation of the released tritiated water. In addition, a less sensitive fluorometric method exists that employs nonpeptidyl amine lysyl oxidase substrates and measures hydrogen peroxide production with horseradish peroxidase coupled to homovanillate oxidation. The present study describes a more sensitive fluorescent assay for lysyl oxidase activity that utilizes 1,5-diaminopentane as substrate, and released hydrogen peroxide is detected using Amplex red in horseradish peroxidase-coupled reactions. This method allows the detection of 40 ng of enzyme per 2 ml assay at 37 degrees C and is 7.5 times more sensitive than the currently available fluorometric assay for enzyme activity. This method eliminates the interference that occurs in some biological samples and can be successfully used to detect lysyl oxidase activity in cell culture experiments. 相似文献
5.
H M Kagan D M Soucy C G Zoski R J Resnick S S Tang 《Archives of biochemistry and biophysics》1983,221(1):158-167
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. 相似文献
6.
Giampuzzi M Botti G Di Duca M Arata L Ghiggeri G Gusmano R Ravazzolo R Di Donato A 《The Journal of biological chemistry》2000,275(46):36341-36349
Lysyl oxidase is an extracellular enzyme that controls the maturation of collagen and elastin. Lysyl oxidase and collagen III often show similar expression patterns in fibrotic tissues. Therefore, we investigated the influence of lysyl oxidase overexpression on the promoter activity of human COL3A1 gene. Our results showed that when COS-7 cells overexpressed the mature form of lysyl oxidase, the activity of the human COL3A1 promoter was increased up to an average of 12 times when tested by luciferase reporter assay. The effect was specific, because other promoters were not affected. Moreover, lysyl oxidase effect was abolished by beta-aminopropionitrile, a specific inhibitor of its catalytic activity. Electrophoretic mobility shift assay showed a binding activity in the region from -101 to -77 that was significantly increased by lysyl oxidase overexpression. The binding was specifically competed by the cold probe, and the mutagenesis of this region abolished both the binding activity in gel retardation and lysyl oxidase stimulation of COL3A1 promoter in transfection experiments. We identified the binding activity as Ku antigen in its two components: Ku80 and Ku70. This study suggests a new coordinated mechanism by which lysyl oxidase might control the development of fibrosis. 相似文献
7.
Isolation of lysyl hydroxylase, an enzyme of collagen synthesis, from chick embryos as a homogeneous protein. 总被引:4,自引:3,他引:1
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Two procedures are reported for the purification of lysyl hydroxylase, both procedures involving (NH4)2SO4 fractionation, affinity chromatography on concanavalin A-agarose and elution of the column with ethylene glycol. The additional steps in procedure A consist of gel filtration and chromatography on a hydroxyapatite column, and in procedure B of affinity chromatography on collagen linked to agarose and gel filtration. The best preparations obtained with either of the two procedures were pure when examined by sodium dodecyl sulphate-polyacrylamide-disc-gel or slab-gel electrophoresis, but about half of the preparations obtained by procedure A had minor contaminants. The specific activity of a typical preparation purified by procedure B was 13 4000 times that of the 15 000 g supernatant of the chick-embryo homogenate, with a recovery of about 4%. The molecular weight of the pure enzyme was bout 200 000 by gel filtration, and that of the enzyme subunit about 85 000 by sodium dodecyl sulphate/polyacrylamide-disc-gel or slab-gel electrophoresis. It is suggested that the active enzyme is a dimer consisting of only one type of monomer, and that a previously described enzyme form with an apparent molecular weight of about 550 000 is a polymeric form of this dimer. The catalytic-centre activity of the pure enzyme, as determined with a saturating concentration of a synthetic peptide substrate and under conditions specified, was about 3-4 mol/s per mol. 相似文献
8.
Inflammation-associated lysyl oxidase protein expression in vivo, and modulation by FGF-2 plus IGF-1
P. C. Trackman Rudolph J. Graham Howard K. Bittner David L. Carnes James A. Gilles Dana T. Graves 《Histochemistry and cell biology》1998,110(1):9-14
Lysyl oxidase is the extracellular enzyme that catalyzes oxidative deamination of peptidyl-lysine residues in elastin precursors,
and lysine and hydroxylysine residues in collagen precursors to form peptidyl-aldehydes. These aldehydes then spontaneously
condense to crosslink collagen and elastin and thereby allow the formation of a mature and functional extracellular matrix.
In the present study, cryosections made from aseptic immune-induced periapical lesions experimentally generated in laboratory
rats were examined by immunohistochemistry to investigate whether lysyl oxidase protein expression is altered in inflamed
oral tissues. Periapical lesions are experimentally induced endodontic lesions of tooth roots. In addition, the effect of
administration of a mixture of fibroblast growth factor (FGF)-2 and insulin-like growth factor (IGF)-1 into these lesions
on lysyl oxidase expression was determined. Lysyl oxidase expression was found to be increased in non-mineralized connective
tissue adjacent to inflamed lesions. Morphometric analyses indicated that maximum lysyl oxidase expression occurred at a discrete
distance from the lesion not exceeding 350 μm from the inflammatory cells. Staining was associated with mesenchymal cells
with a fibroblastic morphology. No lysyl oxidase staining was found near teeth where no lesion was induced. Application of
a mixture of FGF-2 and IGF-1 resulted in a further twofold increase in lysyl oxidase expression. These results provide a new
in vivo model to study lysyl oxidase regulation, and suggest that inflammatory cells may control lysyl oxidase expression
in oral tissues, possibly by a mechanism involving secretion of cytokines and other factors, probably contributing to the
regulation of extracellular matrix accumulation.
Accepted: 19 December 1998 相似文献
9.
Ultrastructural immunolocalization of lysyl oxidase in vascular connective tissue 总被引:2,自引:0,他引:2
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H M Kagan C A Vaccaro R E Bronson S S Tang J S Brody 《The Journal of cell biology》1986,103(3):1121-1128
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. 相似文献
10.
Lysyl oxidase highly purified from calf aorta was found to be a potent chemotactic agent for unstimulated human peripheral blood mononuclear cells, determined in in vitro assays in Boyden chambers. A typical chemotactic bell-shaped curve was observed, with a maximal migratory response of 237% of control occurring at 10−10 M lysyl oxidase. The chemotactic response was prevented by prior heat inactivation of the enzyme, by treatment of the enzyme with β-aminopropionitrile or ethylenediamine, which are active site-directed inhibitors of lysyl oxidase, and by a competing, lysine-containing peptide substrate of lysyl oxidase. The chemoattractant reponse to lysyl oxidases was characterized by both chemokinetic and chemotactic components. These results raise the possibility that extracellular lysyl oxidase may have important roles to play in biology in addition to its established function in the crosslinking of elastin and collagen. 相似文献
11.
The biological function of the R2a regulatory gene for alkaline phosphatase in Escherichia coli 总被引:4,自引:0,他引:4
S Kida 《Archives of biochemistry and biophysics》1974,163(1):231-237
Previous attempts to purify lysyl oxidase have been frustrated by the failure to recover activity during ion exchange or affinity chromatography. We have found that lysyl oxidase from chick cartilage shows marked stability in buffers containing urea and in these solutions can be recovered in high yield from DKAE-cellulose and collagen-derivatized Sepharose. The purified enzyme was active against both collagen and elastin substrates but devoid of monoamine oxidase activity. An absolute requirement for oxygen for activity was found. 相似文献
12.
The binding of rat liver cytochrome c oxidase to phenyl-Sepharose and various alkyl and omega-aminoalkyl agarose gels has been studied. Deoxycholate-solubilized cytochrome c oxidase was tightly bound to hexyl, octyl, omega-aminohexyl, omega-aminooctyl agarose as well as to phenyl-Sepharose. This hydrophobic interaction was used for the purification of cytochrome c oxidase. The enzyme which was eluted from phenyl-Sepharose was devoid of NADH (NADPH)-acceptor reductase activities. The heme a content was 15.4 nmol per mg of protein. The purified enzyme was resolved into seven polypeptides upon polyacrylamide gel electrophoresis in sodium dodecylsulfate with molecular weights of 40,000, 23,200, 21,500, 14,500, 12,600, 8900, and 4900. Antibodies raised in rabbits against the pure enzyme did not cross-react with cytochrome c oxidases from either beef heart or yeast mitochondria. Cytochrome c oxidase bound to octyl-Sepharose or phenyl-Sepharose exhibited a very low catalytic activity. The possible modes of interaction of cytochrome c oxidase with the hydrophobic ligands are discussed. 相似文献
13.
Changes in collagen cross-linking and lysyl oxidase by estrogen. 总被引:3,自引:0,他引:3
H Sanada J Shikata H Hamamoto Y Ueba T Yamamuro T Takeda 《Biochimica et biophysica acta》1978,541(3):408-413
Dermal collagen solubility and lysyl oxidase activity of bones were measured in DDD mice of advancing age. Insoluble fractions of the dermal collagen increased more rapidly in females than in males after 5 weeks of age. Activity of the lysyl oxidase extracted from bones was higher in females than in males after 4 weeks of age. After sexual maturation, such sex differences were always observed in skin as well as in bone tissue. In other experimental animals, dermal collagen solubility was markedly decreased by estrogen treatment and lysyl oxidase was remarkably activated by estrogen in both skin and bone. Thus it is clear that estrogen stimulates the enzyme activity and accelerates the maturation of collagen and elastin in extracellular space. 相似文献
14.
The molecular state of cytochrome oxidase, complex IV (EC 1.9.3.1), was studied by polyacrylamide gel electrophoresis. Cytochrome oxidase in the presence of non-ionic detergent Emasol 1130 ran as a single band under conditions where there is a small sieving effect as in a 2.5% polyacrylamide gel. This polymeric form is an association of different molecular species that can be dissociated on a preparative electrophoresis system. In such a system, five species of different molecular size with enzymic activity are obtained, though the specific activity is lower in the first running fractions. After treatment in highly dissociating conditions (phenol-acetic acid-water (2:1:1, w/v/v) the polymeric form and the different species present two main fractions on analytical polyacrylamide electrophoresis (7.5% in acrylamide, 35% acetic acid, and 5 m urea). The original form after treatment with 2.5% sodium dodecylsulfate (SDS) also presents two fractions on analytical electrophoresis in the presence of 2.5% sodium dodecylsulfate. These results suggest that the basic subunit structure of the various forms of the cytochrome oxidase consists of two closely related polypeptides. The highest activity was found with the polymeric form of cytochrome oxidase, a fact which may have physiological significance in relation to the natural state of this enzyme in the mitochondrion. 相似文献
15.
Lysyl oxidase catalyzes the oxidation of peptidyl lysine in elastin and collagen and also acts upon nonpeptidyl amines, although the enzyme becomes slowly inactivated while processing nonpeptidyl substrates. In spite of this complexity, it has been possible to devise a continuously monitored peroxidase-coupled fluorometric assay for the oxidation of simple amines by lysyl oxidase. In the present study, optimal assay conditions have been explored and found to include assay temperatures of 50 to 60°C, the presence of urea in the assay, and the use of diaminopentane as substrate. Although the assay is subject to interference by contaminating macromolecules in enzyme fractions, a linear assay response to enzyme concentration is obtained with highly purified lysyl oxidase with a limiting sensitivity of 0.3 μg of enzyme per assay. 相似文献
16.
1. A cyclic nucleotide phosphodiesterase (EC 3.1.4.16) has been partially purified from bovine rod outer segments. The enzyme preparation obtained has a very high specific activity towards cyclic GMP and is still able to hydrolyze cyclic AMP. Upon polyacrylamide gel electrophoresis, one major and three minor protein bands are seen, the enzyme activity being associated with the major band. The enzyme eluted from the gels still hydrolyzes both cyclic nucleotides. At all substrate concentrations tested, cyclic GMP was hydrolyzed at a faster rate. The enzyme eluted from the gel columns migrated as a single band upon electrophoresis in 0.1% sodium dodecyl sulfate-polyacrylamide gels corresponding to a molecular weight of 105 000. 2. A complex kinetic pattern was observed for cyclic GMP hydrolysis: the plot of velocity vs substrate concentration was hyperbolic at low and sigmoidal at higher concentrations. By contrast, simple kinetics were observed for cyclic AMP hydrolysis yielding an apparent Km of 0.1 mM. The unusual kinetics may be implicated in the regulation of cyclic GMP levels in rod outer segments. 3. Cyclic AMP stimulated the hydrolysis of cyclic GMP at low and inhibited it at higher concentrations. Addition of Mg2+ appeared to be necessary for optimum activity. The activity measured in the absence of exogenous Mg2+ was abolished by EDTA. 相似文献
17.
James O. Ochanda Eva A. C. Oduor Rachel Galun Mabel O. Imbuga Kosta Y. Mumcuoglu 《Physiological Entomology》2000,25(3):242-246
The midgut of the human body louse Pediculus humanus humanus contains a thermally stable leucine aminopeptidase, which was detected by agarose gel electrophoresis using l ‐amino oxidase. Midgut extracts were homogenized in saline or in 1% Triton X‐100 and the aminopeptidase was purified by Superose 6 gel filtration chromatography. A peak with enzyme activity that was extracted with or without Triton X‐100 was eluted at a molecular weight 67–69 kDa. Non‐denaturing polyacrylamide gel electrophoresis resolved one band of molecular weight of 69 kDa for samples that were extracted in a saline buffer. Two closely linked bands of molecular weight 67 kDa and 69 kDa were observed in samples that were extracted in 1% Triton X‐100. 相似文献
18.
The synthesis of lysyl oxidase, which initiates the cross-linking of collagen and elastin, was investigated in carbon tetrachloride (CCl4) induced fibrotic liver of rat. Lysyl oxidase activity of the fibrotic liver was 4 times greater than that of normal liver. mRNAs from the livers of normal and CCl4-treated rats were prepared for in vitro protein synthesis and the products were analyzed by immunoprecipitation with a monoclonal antibody against lysyl oxidase. The mRNAs from the fibrotic liver gave more than 3 times higher level of messenger copies for lysyl oxidase than did mRNAs from normal liver. The molecular weight of the nascent lysyl oxidase was 48,000. 相似文献
19.
Latent and active collagenase were extracted from human polymorphonuclear leukocytes. Separation of the two forms of the enzyme was performed by gel filtration on Sepharose 6 B. The latent form of the enzyme was detected from chromatographic fractions after a brief treatment with trypsin or exposure of the fractions to the sulfhydryl reagent phenylmercuric chloride. Latent enzyme eluted before active enzyme from the column, indicating a higher apparent molecular weight. Partially purified latent enzyme exhibited an apparent molecular size of 70-75 kDa as estimated by gel filtration. A value of 50-55 kDa was obtained for active enzyme. Without activation the latent enzyme did not degrade soluble collagen substrate. This was demonstrated by a quantitative viscometric assay and also by sodium dodecyl sulfate polyacrylamide gel electrophoresis, when no typical cleavage products of collagen could be seen. Latent enzyme could not be obtained unless serine protease inhibitors were present during the extraction and purification procedures. The effects of the activators trypsin, phenylmercuric chloride, phenylmethyl sulfonyltrypsin, and N-ethylmaleimide on the latent human polymorphonuclear leukocyte collagenase were studied. Contrary to the suggestion that inactive proteases activate latent human polymorphonuclear leukocyte collagenase, the inactive phenylmethyl sulfonyl-trypsin could not activate latent collagenase. 相似文献