Purification of high yields of catalytically active lysyl oxidase directly from Escherichia coli cell culture.

Lysyl oxidase is a highly insoluble enzyme requiring high concentrations of urea to solubilize. A method to obtain lysyl oxidase in high yields directly from an Escherichia coli culture without the need for refolding of inclusion bodies has been developed using nutrient rich media. pET21b was used to overexpress the lysyl oxidase enzyme and to introduce a C-terminal 6X histidine tag for purification. Lysyl oxidase yields of 10 mg of active and properly folded enzyme per liter of media have been obtained. Purification was achieved via affinity chromatography using a Ni-NTA column. Copper content was found to be 19%. LTQ cofactor formation in LOX is a self-processing event in the presence of copper. LTQ content was determined to be 24% based on reaction with phenylhydrazine to form a phenylhydrazone adduct. Quantification of this adduct was attained using the previously reported extinction coefficient of 15.4 mM(-1)cm(-1). LTQ presence was also verified by redox cycling. Specific enzymatic activity was measured to be 0.31 U/mg, one of the highest activities reported.

Characterization of recombinant lysyl oxidase propeptide.

Lysyl oxidase enzyme activity is critical for the biosynthesis of mature and functional collagens and elastin. In addition, lysyl oxidase has tumor suppressor activity that has been shown to depend on the propeptide region (LOX-PP) derived from pro-lysyl oxidase (Pro-LOX) and not on lysyl oxidase enzyme activity. Pro-LOX is secreted as a 50 kDa proenzyme and then undergoes biosynthetic proteolytic processing to active approximately 30 kDa LOX enzyme and LOX-PP. The present study reports the efficient recombinant expression and purification of rat LOX-PP. Moreover, using enzymatic deglycosylation and DTT derivatization combined with mass spectrometry technologies, it is shown for the first time that rLOX-PP and naturally occurring LOX-PP contain both N- and O-linked carbohydrates. Structure predictions furthermore suggest that LOX-PP is a mostly disordered protein, which was experimentally confirmed in circular dichroism studies. Due to its high isoelectric point and its disordered structure, we propose that LOX-PP can associate with extracellular and intracellular binding partners to affect its known biological activities as a tumor suppressor and inhibitor of cell proliferation.

Purification of enzymatically active human lysyl oxidase and lysyl oxidase-like protein from Escherichia coli inclusion bodies.

Lysyl oxidase (LOX) is an extracellular copper dependent enzyme catalyzing lysine-derived cross-links in extracellular matrix proteins. Recent molecular cloning has revealed the existence of a LOX family consisting of LOX and four lysyl oxidase-like proteins (LOXLs; LOXL, LOXL2, LOXL3, and LOXL4). Each member of the LOX family contains a copper-binding domain, residues for lysyl-tyrosyl quinone, and a cytokine receptor-like domain. Very recently, novel functions, such as tumor suppression, cellular senescence, and chemotaxis, have been attributed to this family of amine oxidases, but functional differences among the family members have yet to be determined. For efficient expression and purification, we cloned the cDNAs corresponding to proteolytically processed forms of LOX (LOX-p) and LOXL (LOXL-p1 and LOXL-p2) into a bacterial expression vector pET21a with six continuous histidine codons attached to the 3' of the gene. The recombinant proteins were purified with nickel-chelating affinity chromatography and converted into enzymatically active forms by stepwise dialysis in the presence of N-lauroylsarcosinate and Cu2+. The purified LOX-p, LOXL-p1, and LOXL-p2 proteins showed specific amine oxidase activity of 0.097, 0.054, and 0.150 U/mg, respectively, which was inhibited by beta-aminopropionitrile (BAPN), a specific inhibitor of LOX. Availability of these pure and active forms of LOX and LOXLs will be significantly helpful in functional studies related to substrate specificity and crystal structures of this family of amine oxidases.

Crystallization of Pichia pastoris lysyl oxidase.

A copper-containing amine oxidase (PPLO) from the yeast Pichia pastoris has been purified and crystallized in two forms. PPLO is a glycoprotein. The molecular mass from SDS-polyacrylamide gels is 112 kDa, consistent with 20% glycosylation by weight (the calculated molecular weight of the polypeptide is 89.7 kDa). Orthorhombic crystals belonging to space group P2(1)2(1)2(1), with unit-cell parameters a = 163.7, b = 316.1, c = 84.0 A, diffract to 2.65 A resolution. Monoclinic crystals belonging to space group C2, with unit-cell parameters a = 248.4, b = 121.1, c = 151.8 A, beta = 124.6 degrees, diffract to 1.65 A resolution. Native data have been recorded from each crystal form at 100 K using synchrotron radiation. A self-rotation function for the monoclinic crystal form reveals the presence of a non-crystallographic twofold axis perpendicular to the crystallographic twofold axis, consistent with the presence of two dimers in the asymmetric unit.

Lysyl oxidase-like protein from bovine aorta. Isolation and maturation to an active form by bone morphogenetic protein-1.

Recently several cDNAs have been described encoding lysyl oxidase-like proteins. Their deduced amino acid sequences are characterized by a strong similarity in the C-terminal region, corresponding to the lysyl oxidase family catalytic domain, and by marked differences in the N-terminal regions. Different biological functions have been described for lysyl oxidases in addition to their traditionally assumed cross-linking role. To answer the question of whether these different functions are carried out by different lysyl oxidases, purified and active forms of these enzymes are required. At present only the classical form of lysyl oxidase has been purified and characterized. The purpose of this study was to isolate and characterize the lysyl oxidase-like protein. In view of the strong sequence homology with the C-terminal domain of other lysyl oxidases, we chose to purify the protein from bovine aorta using antibodies specific to the N-terminal domain of the proenzyme. We have isolated a 56-kDa protein identified by amino acid sequencing as the bovine lysyl oxidase-like precursor, which is cleaved at the Arg-Arg-Arg sequence at positions 89-91 by a furin-like activity, as revealed after deblocking of the N-terminal residue. The immunopurified protein was largely inactive, but further processing in vitro by bone morphogenetic protein-1 led to an enzyme that was active on elastin and collagen substrates.

Identification of L-amino acid/L-lysine alpha-amino oxidase in mouse brain.

Lysine, an essential amino acid is catabolized in brain through only the pipecolic acid pathway. During the formation of pipecolic acid, alpha-deamination of lysine, and the formation of the alpha-keto acid as well as its cyclized product are pre-requisites. The enzyme mediated alpha-deamination of L-lysine and the formation of the alpha-keto acid and the cyclized product are not demonstrated so far. Both lysine and pipecolic acid are known to increase in brain under the conditions of fasting, studies were therefore undertaken to identify the enzyme responsible for the alpha-deamination of L-lysine in the brain tissue of mice which were fasted. The detection of the alpha-keto acid of L-lysine -alpha-keto-epsilon-amino caproic acid and its cyclized product-delta-piperidine-2-carboxylate was facilitated by the use of L-[U-14C]-lysine as the substrate. The quantitation of the radioactivity in reaction products was done after separation by ion exchange chromatographic methods. The formation of the alpha-keto acid was enzyme mediated, the alpha-keto acid formed was established by reaction with N-methyl benzothiazolinone hydrazone hydrochloride. The cyclized product was accounted in a fraction which matched the resolution of authentic pipecolic acid on the Dowex column, and the cyclized product was confirmed by spectrophotometry. The hitherto undemonstrated alpha-amino deaminating enzyme of L-lysine in brain tissue, the alpha-keto acid of L-lysine and its cyclized product in a mammalian system could thus be demonstrated in the present study. These findings confirm the involvement of L-lysine oxidase/L-amino acid oxidase in the formation of pipecolic acid from L-lysine.

Aminoalkylaziridines as substrates and inhibitors of lysyl oxidase: specific inactivation of the enzyme by N-(5-aminopentyl)aziridine.

The reaction of lysyl oxidase was assessed with members of a series of aminoalkylaziridines in which the primary amino group and the aziridinyl nitrogen were separated by 3-7 methylene carbons. Among these, N-(5-aminopentyl)aziridine proved to be the poorest substrate by far and to inhibit the enzyme activity. Aminoalkylaziridines with chain lengths shorter or longer than five carbons did not inhibit the enzyme. The resulting inhibition was competitive with productive substrates and became irreversible with time, following pseudo first order kinetics with a KI of 0.22 mM. N-(5-aminopentyl)aziridine appears to act as a bifunctional affinity label covalently interacting with the active site of this enzyme.

Identification of lysyl oxidase and TRAMP as the major proteins in dissociative extracts of the demineralized collagen matrix of porcine dentine.

Carbonated apatite (dahllite) is formed within and between collagen fibrils in the mineralization of connective tissues. However, the mechanism of crystal nucleation at these sites has not been resolved. To identify non-collagenous proteins that may be involved in the nucleation process we have utilized a dissociative extraction procedure to isolate proteins associated non-covalently with the de-mineralized collagen matrix of dentine isolated from tooth roots of adult porcine incisors. Following extraction of dentine fragments with 4M GuHCl (G1-extract) and 0.5M EDTA (E-extract), de-mineralized collagen matrix-associated proteins were isolated with a second series of extractions with 4M GuHCl (G2-extract). Analysis of the G2-extracts on SDS-PAGE revealed two major 32 kDa and 24 kDa protein bands, comprising > 80% of the extracted non-collagenous proteins. The 32 kDa protein was purified by FPLC on hydroxyapatite and Mono Q resins, followed by HPLC reverse-phase chromatography. Small amounts of 26 kDa and 6 kDa proteins, which appear to represent proteolytically processed, disulphide-linked fragments of the 32 kDa protein, co-eluted with the major protein. The 32 kDa protein was identified as lysyl oxidase from amino acid sequence analysis of a 13 kDa CNBr peptide obtained from protein purified by preparative electrophoresis on SDS-PAGE. Fractionation of the 24 kDa protein on FPLC Mono Q resin generated < 5 closely eluting protein peaks. The proteins from these peaks were similar in size, staining properties, amino acid composition and CNBr digestion patterns. Each protein was immunoreactive with antibodies raised against a tyrosine-rich acidic matrix protein (TRAMP), reported previously to co-purify with lysyl oxidase. These studies, therefore, show that lysyl oxidase, which is important in collagen cross-link formation, and proteins with properties of TRAMP, a protein that can modulate collagen fibrillogenesis, are the major proteins in dissociative extracts of de-mineralized porcine dentine.

Identification of the quinone cofactor in a lysyl oxidase from Pichia pastoris.

A copper amine oxidase from Pichia pastoris is the only known non-mammalian lysyl oxidase [Tur, S.S. and Lerch, K. (1988) FEBS Lett. 238, 74-76]. Recently, the cofactor in mammalian lysyl oxidase has been identified as a novel lysine tyrosylquinone moiety [Wang, S.X., Mure, M., Medzihradszky, K.F., Burlingame, A.L., Brown, D.E., Dooley, D.M., Smith, A.J., Kagan, H.M. and Klinman, J.P. (1996) Science 273, 1078-1084]. In order to identify the cofactor in P. pastoris lysyl oxidase, we have isolated the phenylhydrazone-derivative of the active-site peptide. This peptide has the active-site sequence conserved among topa quinone containing amine oxidases. The resonance Raman spectra of the phenylhydrazone derivatives of the enzyme, active-site peptide, and a topa quinone model compound are essentially identical. Collectively, these results establish that P. pastoris lysyl oxidase is a topa quinone enzyme.

Expression of active, human lysyl oxidase in Escherichia coli.

Lysyl oxidase (LO) is a copper amine oxidase of the extracellular matrix which initiates covalent cross-linking in collagens and elastin. Human LO was expressed in Escherichia coli. At 37 degrees C, large amounts of protein were obtained, but in the form of insoluble aggregates. Lowering the growth temperature, and reducing the amount of inducer, resulted in the production of soluble LO, which was active on a degrees [3H]lysine-labeled elastin substrate. LO was also targeted to the periplasm as a fusion protein with the pelb signal peptide. The periplasmic enzyme was soluble, active and inhibited by beta-aminopropionitrile. Production of the carbonyl co-factor is therefore not a limitation in the expression of active LO in bacteria.

A crosslinked cofactor in lysyl oxidase: redox function for amino acid side chains.

A previously unknown redox cofactor has been identified in the active site of lysyl oxidase from the bovine aorta. Edman sequencing, mass spectrometry, ultraviolet-visible spectra, and resonance Raman studies showed that this cofactor is a quinone. Its structure is derived from the crosslinking of the epsilon-amino group of a peptidyl lysine with the modified side chain of a tyrosyl residue, and it has been designated lysine tyrosylquinone. This quinone appears to be the only example of a mammalian cofactor formed from the crosslinking of two amino acid side chains. This discovery expands the range of known quino-cofactor structures and has implications for the mechanism of their biogenesis.

Pre- and post-translational regulation of lysyl oxidase by transforming growth factor-beta 1 in osteoblastic MC3T3-E1 cells.

The final enzymatic step required for collagen cross-linking is the extracellular oxidative deamination of peptidyl-lysine and -hydroxylysine residues by lysyl oxidase. A cross-linked collagenous extracellular matrix is required for bone formation. The goals of this study were to compare the transforming growth factor (TGF)-beta 1 regulation of lysyl oxidase enzyme activity and steady state mRNA levels to changes in COL1A1 mRNA levels in MC3T3-E1 osteoblastic cells. TGF-beta 1 increased steady state lysyl oxidase and COL1A1 mRNA levels in a dose- and time-dependent manner. The increase in lysyl oxidase mRNA levels was transient, peaking at 12 h and 8.8 times controls in cells treated with 400 pM TGF-beta 1. COL1A1 steady state mRNA levels increased maximally to 3.5-fold of controls. Development of increased lysyl oxidase enzyme activity was delayed and was of slightly lower magnitude than the increase in its mRNA levels. This suggested limiting post-translational processing of lysyl oxidase proenzyme. Pulse-labeling/immunoprecipitation studies demonstrated slow proenzyme secretion and proteolytic processing. Development and application of an independent assay for lysyl oxidase proenzyme proteolytic processing activity verified its proportionately lower stimulation by 400 pM TGF-beta 1. Thus, lysyl oxidase regulation by TGF-beta 1 in osteoblastic cell cultures occurs at both pre- and post-translational levels. This regulation is consistent with increased production of a collagenous extracellular matrix.

Transient expression of lysyl oxidase by liver myofibroblasts in murine schistosomiasis.

BACKGROUND: Murine schistosomiasis provides an experimental model of reversible fibrosis. The lysyl oxidase catalyzes the first step of collagen and elastin enzymatic cross-linking and appears to be a crucial factor in stabilizing the neosynthesized extracellular matrix in the liver. EXPERIMENTAL DESIGN: A cDNA probe encoding a portion of the murine lysyl oxidase was cloned, and antibodies were raised against the corresponding recombinant peptide expressed as a fusion protein. Both tools were used to examine the expression of the lysyl oxidase mRNAs and peptides, all within granulomas and cells extracted from infected liver. RESULTS: Transient up-regulation of two dominant 4.5 kb and 5.5 kb transcripts was observed among four mRNAs hybridizing with the lysyl oxidase cDNA probe during the development of granulomas. An identical time course was obtained for alpha 1(I) procollagen messenger expression. The lysyl oxidase expression was observed in type I collagen producing cells mainly localized at the periphery of fibroinflammatory granulomas and it disappeared in late granulomas. The lysyl oxidase and type I collagen expressing cells, located within granulomas, exhibited the characteristics of myofibroblasts, as judged by their expression of alpha-smooth muscle actin and desmin and by their ultrastructural morphology. Four antigenically related peptides were immunopurified from an enriched preparation of myofibroblast-like cells extracted from infected mouse liver. Two of these peptides had the molecular weight of prolysyl oxidase (50,000) and activated lysyl oxidase (32,000). The two others (28,000 and 66,000) might correspond to cleavage product or dimeric form respectively. CONCLUSIONS: This study demonstrates that the lysyl oxidase was transiently up-regulated at the transcriptional level parallely to alpha(1)I procollagen within developing granulomas. Myofibroblasts are involved in the expression of the lysyl oxidase which may be secreted as a proenzyme and an activated enzyme.

Tyrosine-rich acidic matrix protein (TRAMP) accelerates collagen fibril formation in vitro.

Tyrosine-rich acidic matrix protein (TRAMP) is a recently discovered protein that co-purifies with porcine skin lysyl oxidase and is equivalent to the M(r) 22,000 extracellular matrix protein from bovine skin that co-purifies with dermatan sulfate proteoglycans (Cronshaw, A. D., MacBeath, J. R. E., Shackleton, D. R., Collins, J. F., Fothergill-Gilmore, L. A., and Hulmes, D. J. S. (1993) Matrix 13, 255-266; Neame, P. J., Choi, H. U., and Rosenberg, L. C. (1989) J. Biol. Chem. 264, 5474-5479). The effect of TRAMP on collagen fibril formation was studied in vitro by reconstitution of fibrils from lathyritic rat skin collagen I. Fibril formation was initiated by the warm start procedure, in which acidic collagen solutions and double strength neutral buffer, both preincubated separately at 34 degrees C, were mixed. When monitored by turbidimetry, TRAMP was found to accelerate collagen fibril formation. Acceleration occurred at sub-stoichiometric molar ratios of TRAMP collagen, and the presence of TRAMP stabilized the fibrils against low temperature dissociation. It was confirmed by centrifugation that the amount of fibrillar collagen formed in the presence of TRAMP was greater than in its absence. By SDS-polyacrylamide gel electrophoresis and scanning densitometry, binding of TRAMP to collagen was detected that approached saturation with a molar ratio of TRAMP to collagen of approximately 1:2. Fibrils formed in the presence of TRAMP were normal when observed by electron microscopy, although fibril diameters were smaller than the controls. TRAMP was found to partially reverse the inhibitory effects of urea and increased ionic strength on the kinetics of fibril formation, although inhibition by glucose was unaffected. TRAMP also accelerated the assembly of pepsin-treated collagen, where the non-helical, telopeptide regions were partially removed. Acceleration of collagen fibril formation by TRAMP is discussed in the light of the known effects of other extracellular matrix components on this process.

Kinetics and stereospecificity of the lysyl oxidase reaction.

The structural specificity of amine oxidation by lysyl oxidase was investigated using kinetic and NMR spectroscopic analyses. Substrate efficiency increased with increasing molecular distance from the alpha-carbon of the aromatic moiety substituted on the aliphatic chains of a series of primary amines. The p-hydroxyl substituent of p-hydroxybenzylamine significantly increased kcat over that of benzylamine, whereas this was not the case when tyramine and phenethylamine were compared. Direct spectrophotometric measurement of p-hydroxybenzaldehyde formation yielded burst kinetics, the second, slower phase of which was eliminated under anaerobic conditions. Thus, enzyme reoxidation is the more rate-limiting of the two half-reactions catalyzed with this substrate by this ping-pong enzyme. 1H NMR spectroscopy of the alcohol reductively derived from the aldehyde product of the lysyl oxidase-catalyzed oxidation of deuterated tyramine indicated that the pro-S but not the pro-R alpha-deuteron was catalytically abstracted. Moreover, lysyl oxidase catalyzed solvent exchange of protons at the C-2 position. Such stereospecificity and proton exchange uniquely differentiates lysyl oxidase from all but an aortic semicarbazide-sensitive amine oxidase among the pro-S-specific copper-dependent amine oxidases analyzed thus far.

TRAMP (tyrosine rich acidic matrix protein), a protein that co-purifies with lysyl oxidase from porcine skin. Identification of TRAMP as the dermatan sulphate proteoglycan-associated 22K extracellular matrix protein.

A protein (M(r)24 K) that co-purifies with porcine skin lysyl oxidase (M(r)34 K) has been isolated and characterised. Five variants of the 24 K protein were identified by Mono Q ion-exchange FPLC, as were four variants of lysyl oxidase. Amino acid analysis and partial sequencing revealed near identity of a 36-residue CNBr peptide from porcine skin lysyl oxidase to corresponding regions of the putative lysyl oxidase precursor derived from rat and human cDNA. The 24 K protein was found to be unrelated to lysyl oxidase, but comparison with a protein sequence database showed it to be the same as a recently described protein from bovine skin that is associated with dermatan sulphate proteoglycans. The 24 K protein is relatively rich in tyrosine, and isoelectric focussing shows it to be acidic, with pI's in the range 4.1 to 4.4. In view of these properties, we propose the name TRAMP (Tyrosine Rich Acidic Matrix Protein) to identify this protein. Though TRAMP appears not to be glycosylated, several experiments indicate the presence of sulphotyrosine residues. When assayed using an elastin substrate, the activity of lysyl oxidase is unaffected by TRAMP.

Oxidation, cross-linking, and insolubilization of recombinant tropoelastin by purified lysyl oxidase.

The use of recombinant human tropoelastin (rTE) and selected variants thereof as substrates for the assay of lysyl oxidase activity in vitro was explored. The possibility was also assessed that an insoluble elastin-like product could be generated from this elastin precursor in the absence of other macromolecules found associated with elastin in vivo. rTE was more efficiently oxidized by lysyl oxidase than the insoluble chick aorta elastin substrate conventionally used. Anionic amphiphilic elastin ligands strongly inhibited rTE oxidation consistent with the importance of electrostatic enzyme-substrate interactions previously noted with the insoluble elastin substrate. An rTE variant, rTE delta 26A, lacking the hydrophilic sequence coded by exon 26A, was a less effective substrate than rTE, largely due to an increase in Km, while the kinetic parameters for the oxidation of rTE delta 36, lacking the C-terminal polybasic sequence coded by exon 36, were quite similar to those for rTE. Incubation of rTE delta 26A with lysyl oxidase not only resulted in the generation of peptidyl alpha-aminoadipic-delta-semialdehyde and lysine-derived cross-linkages, but also yielded a product insoluble in hot 0.1 N NaOH, consistent with the properties of insoluble elastin. Thus, oxidation, cross-linking and insolubilization of elastin substrates by lysyl oxidase can occur in the absence of other macromolecules implicated as being involved in this process in vivo, although such macromolecules may be essential to obtain the proper alignment between tropoelastin units for specifically placed cross-linkages and optimally functional elastic fibers.

Purification, properties and influence of dietary copper on accumulation and functional activity of lysyl oxidase in rat skin.

Lysyl oxidase (protein-lysine 6-oxidase; EC 1.4.3.13) is a copper-containing enzyme that functions extracellularly and catalyses the oxidative deamination of peptidyl lysine. Lysyl oxidase was purified 150-175-fold from urea extracts of rat skin and uteri. Features of the enzyme were similar to those reported previously for lysyl oxidase obtained from rat aorta and bovine ligamenture. However, both approximately 40 and approximately 32 kDa polypeptide chains could be isolated from rat skin with apparent lysyl oxidase activity. Antibodies raised in chickens against the approximately 40 kDa form of lysyl oxidase detected the approximately 32 kDa form in immunoblots. Consequently it is inferred that the approximately 32 kDa form of lysyl oxidase is processed from the approximately 40 kDa form of the enzyme. The antibodies were also used to prepare anti(rat lysyl oxidase) affinity columns to facilitate the separation of lysyl oxidase from other proteins in studies to assess the extent to which lysyl oxidase serves as a reservoir for skin copper. At 16 h after an oral dose of copper, as 67Cu, about 6-8% of the total 67Cu incorporated into rat skin was found in association with lysyl oxidase. The lysyl oxidase concentration in rat skin was 2.5-7.5 nmol/g (determined by e.l.i.s.a.). Changing the copper status of rats by feeding a diet deficient in copper did not appear to influence lysyl oxidase accumulation in skin nor the percentage of incorporation of 67Cu in skin as lysyl oxidase. However, when rats were deprived of copper, the functional activity of lysyl oxidase in skin was one-third to one-half the normal values.

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.