A procollagen C-proteinase inhibitor diminishes collagen and lysyl oxidase processing but not collagen cross-linking in osteoblastic cultures

The deposition of insoluble functional collagen occurs following extracellular proteolytic processing of procollagens by procollagen N- and C-proteinases, fibril formation, and lysyl oxidase dependent cross-linking. Procollagen C-proteinases in addition process and activate lysyl oxidase. The present study evaluates a possible role for procollagen C-proteinases in controlling different aspects of collagen deposition in vitro. Studies determine whether inhibition of procollagen C-proteinase activity with a specific BMP-1 inhibitor results in perturbations in lysyl oxidase activation, and in collagen processing, deposition, and cross-linking in phenotypically normal cultured murine MC3T3-E1 cells. Data show that BMP-1 Inhibitor dose dependently inhibits lysyl oxidase activation by up to 50% in undifferentiated proliferating cells. In differentiating cultures, BMP-1 inhibitor decreased collagen processing but did not inhibit the accumulation of mature collagen cross-links. Finally, electron microscopy studies show that collagen fibril diameter increased. Thus, inhibition of procollagen C-proteinases results in perturbed collagen deposition primarily via decreased collagen processing.     

A role for lysyl oxidase regulation in the control of normal collagen deposition in differentiating osteoblast cultures

Differentiation of phenotypically normal osteoblast cultures leads to formation of a bone-like extracellular matrix in vitro. Maximum collagen synthesis occurs early in the life of these cultures, whereas insoluble collagen deposition occurs later and is accompanied by a diminished rate of collagen synthesis. The mechanisms that control collagen deposition seem likely to include regulation of extracellular collagen biosynthetic enzymes, but expression patterns of these enzymes in differentiating osteoblasts has received little attention. The present study determined the regulation of lysyl oxidase as a function of differentiation of phenotypically normal murine MC3T3-E1 cells at the level of RNA and protein expression and enzyme activity. In addition, the regulation of BMP-1/mTLD mRNA levels that encodes procollagen C-proteinases was assayed. The role of lysyl oxidase in controlling insoluble collagen accumulation was further investigated in inhibition studies utilizing beta-aminopropionitrile, a specific inhibitor of lysyl oxidase enzyme activity. Results indicate that lysyl oxidase is regulated as a function of differentiation of MC3T3-E1 cells, and that the maximum increase in lysyl oxidase activity precedes the most efficient phase of insoluble collagen accumulation. By contrast BMP-1/mTLD is more constitutively expressed. Inhibition of lysyl oxidase in these cultures increases the accumulation of abnormal collagen fibrils, as determined by solubility studies and by electron microscopy. Taken together, these data support that regulation of lysyl oxidase activity plays a key role in the control of collagen deposition by osteoblast cultures.     

Multiple bone morphogenetic protein 1-related mammalian metalloproteinases process pro-lysyl oxidase at the correct physiological site and control lysyl oxidase activation in mouse embryo fibroblast cultures

Lysyl oxidase catalyzes the final enzymatic step required for collagen and elastin cross-linking in extracellular matrix biosynthesis. Pro-lysyl oxidase is processed by procollagen C-proteinase activity, which also removes the C-propeptides of procollagens I-III. The Bmp1 gene encodes two procollagen C-proteinases: bone morphogenetic protein 1 (BMP-1) and mammalian Tolloid (mTLD). Mammalian Tolloid-like (mTLL)-1 and -2 are two genetically distinct BMP-1-related proteinases, and mTLL-1 has been shown to have procollagen C-proteinase activity. The present study is the first to directly compare pro-lysyl oxidase processing by these four related proteinases. In vitro assays with purified recombinant enzymes show that all four proteinases productively cleave pro-lysyl oxidase at the correct physiological site but that BMP-1 is 3-, 15-, and 20-fold more efficient than mTLL-1, mTLL-2, and mTLD, respectively. To more directly assess the roles of BMP-1 and mTLL-1 in lysyl oxidase activation by connective tissue cells, fibroblasts cultured from Bmp1-null, Tll1-null, and Bmp1/Tll1 double null mouse embryos, thus lacking BMP-1/mTLD, mTLL-1, or all three enzymes, respectively, were assayed for lysyl oxidase enzyme activity and for accumulation of pro-lysyl oxidase and mature approximately 30-kDa lysyl oxidase. Wild type cells or cells singly null for Bmp1 or Tll1 all produced both pro-lysyl oxidase and processed lysyl oxidase at similar levels, indicating apparently normal levels of processing, consistent with enzyme activity data. In contrast, double null Bmp1/Tll1 cells produced predominantly unprocessed 50-kDa pro-lysyl oxidase and had lysyl oxidase enzyme activity diminished by 70% compared with wild type, Bmp1-null, and Tll1-null cells. Thus, the combination of BMP-1/mTLD and mTLL-1 is shown to be responsible for the majority of processing leading to activation of lysyl oxidase by murine embryonic fibroblasts, whereas in vitro studies identify pro-lysyl oxidase as the first known substrate for mTLL-2.     

Intracellular distribution of the lysyl oxidase propeptide in osteoblastic cells

Lysyl oxidase plays a critical role in the formation of the extracellular matrix, and its activity is required for the normal maturation and cross-linking of collagen and elastin. An 18-kDa lysyl oxidase propeptide (LOPP) is generated from 50-kDa prolysyl oxidase by extracellular proteolytic cleavage during the biosynthesis of active 30-kDa lysyl oxidase enzyme. The fate and the functions of the LOPP are largely unknown, although intact LOPP was previously observed in osteoblast cultures. We investigated the spatial localization of molecular forms of lysyl oxidase, including LOPP in proliferating and differentiating osteoblasts, by using confocal immunofluorescence microscopy and Western blots of cytoplasmic and nuclear extracts. In the present study, a stage-dependent intracellular distribution of LOPP in the osteoblastic cell was observed. In proliferating osteoblasts, LOPP epitopes were principally associated with the Golgi and endoplasmic reticulum, and mature lysyl oxidase epitopes were found principally in the nucleus and perinuclear region. In differentiating cells, LOPP and mature lysyl oxidase immunostaining showed clear colocalization with the microtubule network. The subcellular distribution of LOPP and its temporal and physical association with microtubules were confirmed by Western blot and far Western blot studies. We also report that N-glycosylated and nonglycosylated LOPP are present in MC3T3-E1 cell cultures. We conclude that LOPP has a stage-dependent intracellular distribution in osteoblastic cells. Future studies are needed to investigate whether the LOPP associations with microtubules or the osteoblast nucleus have functional effects for osteoblast differentiation and bone formation. microtubules; confocal immunofluorescence microscopy; extracellular matrix; osteoblast differentiation     

Stepwise proteolytic activation of type I procollagen to collagen within the secretory pathway of tendon fibroblasts in situ

Proteolytic cleavage of procollagen I to collagen I is essential for the formation of collagen fibrils in the extracellular matrix of vertebrate tissues. Procollagen is cleaved by the procollagen N- and C-proteinases, which remove the respective N- and C-propeptides from procollagen. Procollagen processing is initiated within the secretory pathway in tendon fibroblasts, which are adept in assembling an ordered extracellular matrix of collagen fibrils in vivo. It was thought that intracellular processing was restricted to the TGN (trans-Golgi network). In the present study, brefeldin A treatment of tendon explant cultures showed that N-proteinase activity is present in the resulting fused ER (endoplasmic reticulum)-Golgi compartment, but that C-proteinase activity is restricted to the TGN in embryonic chick tendon fibroblasts. In late embryonic and postnatal rat tail and postnatal mouse tail tendon, C-proteinase activity was detected in TGN and pre-TGN compartments. Preventing activation of the procollagen N- and C-proteinases with the furin inhibitor Dec-RVKR-CMK (decanoyl-Arg-Val-Lys-Arg-chloromethylketone) indicated that only a fraction of intracellular procollagen cleavage was mediated by newly activated proteinases. In conclusion, the N-propeptides are removed earlier in the secretory pathway than the C-propeptides. The removal of the C-propeptides in post-Golgi compartments most probably indicates preparation of collagen molecules for fibril formation at the cell-matrix interface.     

Interaction properties of the procollagen C-proteinase enhancer protein shed light on the mechanism of stimulation of BMP-1

Procollagen C-proteinase enhancer (PCPE) is an extracellular matrix glycoprotein that binds to the C-propeptide of procollagen I and can enhance the activities of procollagen C-proteinases up to 20-fold. To determine the molecular mechanism of PCPE activity, the interactions of the recombinant protein with the procollagen molecule as well as with its isolated C-propeptide domain were studied using surface plasmon resonance (BIAcore) technology. Binding required the presence of divalent metal cations such as calcium and manganese. By ligand blotting, calcium was found to bind to the C-propeptide domains of procollagens I and III but not to PCPE. By chemical cross-linking, the stoichiometry of the PCPE/C-propeptide interaction was found to be 1:1 in accordance with enzyme kinetic data. The use of a monoclonal antibody directed against the N-terminal region of the C-propeptide suggested that this region is probably not involved in binding to PCPE. Association and dissociation kinetics of the C-propeptide domains of procollagens I and III on immobilized PCPE were rapid. Extrapolation to saturation equilibrium yielded apparent equilibrium dissociation constants in the range 150-400 nM. In contrast, the association/dissociation kinetics of intact procollagen molecules on immobilized PCPE were relatively slow, corresponding to a dissociation constant of 1 nM. Finally, pN-collagen (i.e. procollagen devoid of the C-terminal propeptide domain) was also found to bind to immobilized PCPE, suggesting that PCPE binds to sites on either side of the procollagen cleavage site, thereby facilitating the action of procollagen C-proteinases.     

PCOLCE2 encodes a functional procollagen C-proteinase enhancer (PCPE2) that is a collagen-binding protein differing in distribution of expression and post-translational modification from the previously described PCPE1

The procollagen COOH-terminal proteinase enhancer (PCPE) is a glycoprotein that binds the COOH-terminal propeptide of type I procollagen and potentiates its cleavage by procollagen C-proteinases, such as bone morphogenetic protein-1 (BMP-1). Recently, sequencing of a human expressed sequence tag, which maps near the primary open angle glaucoma region on chromosome 3q21, showed it to encode a novel protein with only 43% identity with PCPE but with a similar domain structure. Here we show this novel protein to be a functional procollagen COOH-terminal proteinase enhancer with activity comparable with that of PCPE and thus propose the designations PCPE2 and PCPE1, respectively. PCPE2 is shown to have a much more limited distribution of expression than does PCPE1, with strong expression primarily in nonossified cartilage in developing tissues and at high levels in the adult heart. PCPE2 is shown to be a glycoprotein that differs markedly in the nature of its glycosylation from that of PCPE1. PCPE2 is also shown to have markedly stronger affinity for heparin than PCPE1, which may account for higher affinities for cell layers. Unexpectedly, both PCPE1 and PCPE2 were found to be collagen-binding proteins, capable of binding at multiple sites on the triple helical portions of fibrillar collagens and also capable of competing for such binding with procollagen C-proteinases. The latter observations may provide insights into the ways PCPEs affect the kinetics of the C-proteinase reaction and into the physical interactions that occur between procollagen C-proteinases and their substrates.     

Mechanical Load Enhances Procollagen Processing in Dermal Fibroblasts by Regulating Levels of Procollagen C-Proteinase

Mechanical forces are emerging as key regulators of cell function. We hypothesize that mechanical load may influence dermal fibroblast activity. We assessed the direct effects of mechanical load on human dermal fibroblast procollagen synthesis and processing in vitro. Cells were loaded in a biaxial loading system (Flexercell 3000). Hydroxyproline levels were measured in the medium and cell layer as an estimate of procollagen synthesis and processing to insoluble collagen. Mechanical load (in the presence of serum or TGF-beta) enhanced procollagen synthesis by 45 +/- 3% (P < 0.001), and 38 +/- 4% (P < 0.001), respectively, over unloaded growth factor controls after 48 h. Insoluble collagen deposition was enhanced in the same cultures by 115 +/- 8% (P < 0.01) and 72% +/- 9% (P < 0.01), respectively. This effect was inhibited using l-arginine suggesting that procollagen C-proteinase, the enzyme which directly cleaves the C-terminal propeptide of procollagen to form insoluble collagen, is required for the fiber formation observed. Procollagen mRNA levels in loaded samples increased by more than two-fold in both serum and TGF-beta-treated cultures at 48 h. Procollagen C-proteinase mRNA levels were also enhanced by a similar magnitude, although the increase was observed at 24 h. Procollagen C-proteinase protein levels were also increased at this time. Protein and mRNA levels of the procollagen C-proteinase enhancer protein, which binds the C-terminal propeptide of procollagen to enhance the rate of peptide cleavage, were unaffected by mechanical load. This study demonstrates that mechanical load promotes procollagen synthesis in dermal fibroblasts by enhancing gene expression and posttranslational processing of procollagen.     

Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase

The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites.     

Type I procollagen C-proteinase from mouse fibroblasts. Purification and demonstration of a 55-kDa enhancer glycoprotein

The enzyme procollagen C-proteinase removes the carboxy-terminal propeptide from procollagen. In the present study we describe an improved procedure for the purification of this enzyme. From the medium of cultured mouse fibroblasts, consisting of ammonium sulfate precipitation, gel filtration and affinity chromatography on a lysyl-Sepharose column, followed by chromatography on a column of Sepharose coupled to the carboxy-terminal propeptide of type I procollagen (PP-Sepharose). This procedure yielded a practically homogeneous, 18,500-fold-purified enzyme preparation and the molecular mass of the purified C-proteinase as determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis was 80 kDa. The lysyl-Sepharose step separated the enzyme from the majority of the contaminating proteins, including a 55-kDa protein which was further purified by PP-Sepharose chromatography and identified as an additional form of the 36-kDa and 34-kDa procollagen C-proteinase enhancer proteins described before [Adar et al. (1986) Collagen Relat. Res. 6,267-277]. It enhanced the C-proteinase activity, bound to the carboxyl propeptide of type I procollagen, cross-reacted immunologically with the 36-kDa as well as the 34-kDa enhancer proteins, and in common with the latter proteins, it was glycosylated. In the course of PP-Sepharose chromatography, a large proportion of the 55-kDa protein disappeared with the concomitant appearance of the smaller enhancer proteins. All these findings suggest that the 55-kDa protein is a precursor of the low molecular mass enhancer proteins. Also suggested from this study is that lysyl-Sepharose chromatography is a highly beneficial purification step which may find use in the purification of the C-proteinase from other sources as well.     

Dried plum polyphenols attenuate the detrimental effects of TNF-alpha on osteoblast function coincident with up-regulation of Runx2, Osterix and IGF-I

Previous studies have demonstrated that dried plums which contain high amounts of polyphenols can restore bone mass and structure, and significantly increase indices of bone formation. The purpose of this study was to determine how dried plum polyphenols influence osteoblast activity and mineralized nodule formation under normal and inflammatory conditions. MC3T3-E1 cells were plated and pretreated with dried plum polyphenols (0, 2.5, 5, 10 and 20 microg/ml) and 24 h later stimulated with TNF-alpha (0 or 1.0 ng/ml). The 5, 10 and 20 microg/ml doses of polyphenols significantly increased intracellular ALP activity under normal conditions at 7 and 14 days, and restored the TNF-alpha-induced suppression of intracellular ALP activity by 14 days (P<.001). Polyphenols also increased mineralized nodule formation under normal and inflammatory conditions. In the absence of TNF-alpha, 5 microg/ml of polyphenols significantly up-regulated the growth factor, IGF-I, compared to controls, and the 5 and 10 microg/ml doses increased the expression of lysyl oxidase involved in collagen crosslinking. TNF-alpha decreased the expression of Runx2, Osterix and IGF-I, and polyphenols restored their mRNA levels to that of the controls. Although TNF-alpha failed to alter lysyl oxidase at 18 h, the polyphenols up-regulated its expression (P<.05) in the presence of TNF-alpha. As expected, TNF-alpha up-regulated RANKL mRNA and polyphenols suppressed RANKL expression without altering OPG. Based on these findings, we conclude that dried plum polyphenols enhance osteoblast activity and function by up-regulating Runx2, Osterix and IGF-I and increasing lysyl oxidase expression, and at the same time attenuate osteoclastogenesis signaling.     

beta-catenin signaling and regulation of cyclin D1 promoter in NRK-49F cells transformed by down-regulation of the tumor suppressor lysyl oxidase

Lysyl oxidase is the enzyme that is essential for collagen and elastin cross-linking. Previous investigations showed that lysyl oxidase is down-regulated in many human tumors and ras-transformed cells. Recently, we proved that antisense down-regulation of lysyl oxidase in NRK-49F cells induced phenotypic changes and oncogenic transformation, characterized by p21(ras) activation and beta-catenin/cyclin D1 up-regulation. In the present paper, we examined beta-catenin intracellular distribution and its association with E-cadherin. We observed an increased association between E-cadherin and beta-catenin in the lysyl-oxidase down-regulated cells during serum starvation. Moreover, we found that beta-catenin cytoplasmic and nuclear levels were increased, suggesting a failure of its down-regulation by the APC-GSK-3beta system, in particular the GSK-3beta phosphorylation of ser-33/37 and thr-41 of beta-catenin. Finally, we investigated the mechanisms leading to the observed cyclin D1 up-regulation. We showed that in the antisense lysyl oxidase cells the cyclin D1 promoter was activated through the LEF and the ATF/CRE sites in the proximal promoter. While the promoter activation through LEF is compatible with beta-catenin signaling, we investigated the possibility that the CRE-dependent activation might be linked to the down-regulation of lysyl oxidase. In fact, up-regulation of lysyl oxidase in a COS-7 cell model showed a significant diminution of the CREB protein binding to the cyclin D1 promoter, leading to a dramatic inhibition of its activity and a significant down-regulation of cyclin D1 protein level in vivo. Finally, our study describes some major anomalies occurring in lysyl oxidase down-regulated fibroblasts, related to beta-catenin signaling and cyclin D1 expression.     

Expression of lysyl oxidase isoforms in MC3T3-E1 osteoblastic cells

Covalent intermolecular cross-linking of collagen is initiated by the action of lysyl oxidase (LOX) on the telopeptidyl lysine and hydroxylysine residues. Recently, several LOX isoforms, i.e., LOX-like proteins 1-4 (LOXL1-4), have been identified but their specific tissue distribution and functions are still largely unknown. In this study, mRNA expression of LOX and LOXL1-4 in MC3T3-E1 osteoblastic cells was screened by RT-PCR and quantitatively analyzed by real-time PCR during cell differentiation and matrix mineralization. The results demonstrated that LOX and all LOXLs, except LOXL2, were expressed in this cell line and that the expression pattern during cell differentiation and matrix mineralization was distinct from one another. This indicates that the expression of LOX and its isoforms is highly regulated during osteoblast differentiation, suggesting their distinct roles in collagen matrix stabilization and subsequent mineralization.     

NMR structure of the netrin-like domain (NTR) of human type I procollagen C-proteinase enhancer defines structural consensus of NTR domains and assesses potential proteinase inhibitory activity and ligand binding

Procollagen C-proteinase enhancer (PCOLCE) proteins are extracellular matrix proteins that enhance the activities of procollagen C-proteinases by binding to the C-propeptide of procollagen I. PCOLCE proteins are built of three structural modules, consisting of two CUB domains followed by a C-terminal netrin-like (NTR) domain. While the enhancement of proteinase activity can be ascribed solely to the CUB domains, sequence homology of the NTR domain with tissue inhibitors of metalloproteinases suggest proteinase inhibitory activity for the NTR domain. Here we present the three-dimensional structure of the NTR domain of human PCOLCE1 as the first example of a structural domain with the canonical features of an NTR module. The structure rules out a binding mode to metalloproteinases similar to that of tissue inhibitors of metalloproteinases but suggests possible inhibitory function toward specific serine proteinases. Sequence conservation between 13 PCOLCE proteins from different organisms suggests a conserved binding surface for other protein partners.     

T3 affects expression of collagen I and collagen cross-linking in bone cell cultures

Thyroid hormones (T3, T4) have a broad range of effects on bone, however, its role in determining the quality of bone matrix is poorly understood. In-vitro, the immortalized mouse osteoblast-like cell line MC3T3-E1 forms a tissue like structure, consisting of several cell layers, whose formation is affected by T3 significantly. In this culture system, we investigated the effects of T3 on cell multiplication, collagen synthesis, expression of genes related to the collagen cross-linking process and on the formation of cross-links.T3 compared to controls modulated cell multiplication, up-regulated collagen synthesis time and dose dependently, and stimulated protein synthesis. T3 increased mRNA expressions of procollagen-lysine-1,2-oxoglutarate 5-dioxygenase 2 (Plod2) and of lysyloxidase (Lox), both genes involved in post-translational modification of collagen. Moreover, it stimulated mRNA expression of bone morphogenetic protein 1 (Bmp1), the processing enzyme of the lysyloxidase-precursor and of procollagen. An increase in the collagen cross-link-ratio Pyr/deDHLNL indicates, that T3 modulated cross-link maturation in the MC3T3-E1 culture system. These results demonstrate that T3 directly regulates collagen synthesis and collagen cross-linking by up-regulating gene expression of the specific cross-link related enzymes, and underlines the importance of a well-balanced concentration of thyroid hormones for maintenance of bone quality.     

Inhibition of bone morphogenetic protein 1 by native and altered forms of alpha2-macroglobulin

The four mammalian bone morphogenetic protein 1 (BMP1)-like proteinases act to proteolytically convert procollagens to the major fibrous components of the extracellular matrix. They also activate lysyl oxidase, an enzyme necessary to the covalent cross-linking that gives collagen fibrils much of their tensile strength. Thus, these four proteinases are attractive targets for interventions designed to limit the excess formation of fibrous collagenous matrix that characterizes fibrosis. Although it has previously been reported that the serum protein alpha(2)-macroglobulin is unable to inhibit the astacin-like proteinases meprin alpha and meprin beta, we herein demonstrate alpha(2)-macroglobulin to be a potent inhibitor of the similar BMP1-like proteinases. BMP1 is shown to cleave the alpha(2)-macroglobulin "bait" region, at a single specific site, which resembles the sites at which BMP1-like proteinases cleave the C-propeptides of procollagens I-III. alpha(2)-Macroglobulin is an irreversible inhibitor that is shown to bind bone morphogenetic protein 1 in a covalent complex. It is also demonstrated that genetically modified alpha(2)-macroglobulin, in which the native bait region is replaced by sequences flanking the probiglycan BMP1 cleavage site, is enhanced approximately 24-fold in its ability to inhibit BMP1, and is capable of inhibiting the biosynthetic processing of procollagen I by cells. These findings suggest possible therapeutic interventions involving ectopic expression of modified versions of alpha(2)-macroglobulin in the treatment of fibrotic conditions.     

A Fluorometric Assay for Detection of Lysyl Oxidase Enzyme Activity in Biological Samples

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.