Human Placental Lysyl Oxidase - The Journal of Biological Chemistry

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 259, No. 11, Issue of June 10,pp. 6996-7002.1984 Printed in U.S.A.

8 1984 by The American Society of Biological Chemists. Inc

Human Placental Lysyl Oxidase

(Received for publication, December 1, 1983)

Helena Kuivaniemi, Eeva-Riitta Savolainen,and KariI. KivirikkoS From the Collagen Research Unit, University of Oulu, Department of Medical Biochemistry, SF-90220Oulu 22, Finland

Lysyl oxidase from human placentas gave four catalytically active forms on DEAE-cellulose chromatography in 6 M urea. The first two of these were combined to form pool I and the remaining two to form pool 11. Pool I was purified to homogeneity, while the final pool I1 enzyme usually had one minor contaminant. The molecular weight of both enzyme pools was identical, 6 M urea and by being about 30,000 by gel filtration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No distinct differences were found between the two pools in amino acid composition, specific activity, or the use of various substrates. Two antisera were prepared, one to the total enzyme protein (pools I and 11) and the other to pool I. Both antisera inhibited and precipitated crude placental lysyl oxidase, the two enzyme pools, and crude human skin fibroblast enzyme, there being no differences between the various enzyme forms. Both antisera alsostained the twoenzyme pools in immunoblotting of denatured proteins. The data suggest that there are no major catalytic, molecular, or immunological differencesbetweenthe multiple forms of human lysyl oxidase. An antiserum prepared to any of theenzyme forms can, therefore,probably be used to study the total enzyme protein.

ontheproperties of thenormalhuman enzyme and the availability of assays for the enzyme protein. Increases in lysyl oxidase activity have been reported during thedevelopment of experimental fibrosis in several animal tissues (e.g. Ref. X ) , such findings suggesting that measurements of the enzyme might give useful information in human fibrotic disorders. Various tissues contain potentlysyl oxidase inhibitors (1,16), however, and thusassays of the enzyme protein would seem to be more reliable than assays of enzyme activity. In the work to be reported here we purified and partially characterized lysyl oxidase from human placental tissue and prepared two specific antisera to theenzyme. EXPERIMENTALPROCEDURES

Materials-Substrates of [6-3H]lysine-laheled purified chick embryo calvaria collagen (17) and crude chick embryo aorta elastin (12) were prepared as described elsewhere, except that 500 pCi of the isotope were used per 200 16-day chick embryo calvaria or 200 pCi/ 30 aortas. A [6-3H]lysine-labeled chick embryo tendon procollagen substrate was prepared in freshly isolated chick embryo tendon cells and purified through the (NHASO, precipitation step as described elsewhere for procollagen NH2-terminal proteinase (18), except that the incubation medium contained 50 pg/ml of 8-aminopropionitrile and 250 pCi/lOs cells of [6-3H]lysine and the sample was finally dialyzed against 0.40 M NaCl in 50 mM Hepes’ buffer, pH 7.5.A collagen-agarose affinity column was prepared by coupling denatured (8) citrate-soluble collagen from the skin of lathyritic (19) rats to 4% Lysyl oxidase initiates the cross-linking of collagens and agarose (Sepharose 4B),as described for the purification of galactoelastin by catalyzing oxidative deamination of the €-amino sylhydroxylysyl glucosyltransferase (20), except that the column was with 2 M urea in 50 mM Tris-HC1 buffer, pH 7.8,a t 4 ”C. group in certainlysine and hydroxylysine residuesof collagens equilibrated [6-3H]Lysine (17.3 Ci/mmol) was purchased from New England Nuand lysineresidues of elastin (1). The enzyme has been clear and 0-aminopropionitrile fumarate from Sigma. DEAE-cellulose purified to near homogeneity or homogeneity from chick and (DE52) was from Whatman, and Sepharose 4B and Sephacryl S-200 bovine aorta and cartilage and from bovine ligamentum nu- from Pharmacia. All other chemicals were at least of analytical grade. chae (2-10). Preparations of lysyl oxidase from all these The urea solutions were freshly deionized by shaking with Amberlite sources are heterogeneous, with multiple peaks of activity in MB-3 from BDH Chemicals, United Kingdom. of Lysyl Oxidase-The procedures used for purifying DEAE-cellulose chromatography,the molecular weight of thePurification enzyme from embryonic chick cartilage (5, 6) and chick ( 2 ) and most species being about 30,000 or a multiple thereof (1-10). bovine (8) aortas were adopted and modified for the isolation of lysyl The reasons for the multiple forms are unknown, but recent oxidase from human placental tissues. All procedures were carried data indicate that there are definite structural and catalytic out at 0-4 “C. Five separate batches of two placentas each were homogenized, similarities between the four forms of the enzyme in bovine extracted, and takenthrough the first chromatographystep (collagenaorta and cartilage(10). agarose) within 5 days. The two placentas (about 1 kg, wet weight) No data are currently available on the purification and were rinsed on a cheesecloth with cold 0.15 M NaCl in 0.1 M sodium properties of lysyl oxidase from any humansource. Deficiency phosphate, pH 7.8, minced with scissors, and homogenized in the in this enzyme activity has recently been found in several above solution (2 ml of solution/g of tissue) within about halfan hour human heritable connective tissue disorders associated with of delivery in a Waring blender at full speed for two bursts of 30 s abnormalities in copper metabolism (11-14). Studies on the with a 1-min interval. The homogenate was centrifuged at 10,000 X mechanisms of these abnormalitieswould require information g for 20 min and the residue suspended in a new hatch of the same solution (2 ml/g) and centrifuged as above. This residue was then suspended in 4.3 M urea containing 50 mM Tris-HC1, pH 7.8 (2 ml/ *This work was supported in part by grants from the Medical g), stirred for 1 h, and centrifuged as above. The supernatant was Research Council of the Academy of Finland. Thecosts of publication saved, and the residue was extracted overnight with 4 M urea in 50 of this article were defrayed in part by the payment of page charges. ______ This article must therefore be hereby marked“aduertisement” in The abbreviations used are: Hepes, 4-(2-Hydroxyethyl)-l-piperaccordance with 18 U.S.C. Section 1734 solely to indicate this fact. azineethanesulfonic acid; SDS, sodium dodecyl sulfate. $ To whom correspondence should be addressed. ___^

’

6996

Human Placental Lysyl Oxidase mM Tris-HC1, pH 7.8 (2 ml/g), and centrifuged at 10,000 X g for 30 min. This supernatantwas pooledwith the previous one and thepool diluted with an equal volume of 50 mM Tris-HC1, pH 7.8, to reduce the molarity of the urea to about 2 M. About 700 ml of collagen-agarose in 2 M urea containing 50 mM Tris-HC1, pH 7.8, was added to the diluted enzyme pool, and the mixture was slowly stirred for about 10-15 h. The collagen-agarose was allowed to settle, the supernatant removed, and the collagenagarose then suspended in about 2000 ml of 2 M urea in 50 mM TrisHCI and allowed to settle again. The collagen-agarosewas transferred to a column of 5-cm diameter, and the washing with 2 M urea in 50 mM Tris-HC1 was continued until the AZWnm of the effluent with a 1-cm lightpath was below 0.005. The column was then washed with 300 mlof 1 M NaCl in 50 mM Tris-HC1 buffer, pH 7.8, and finally eluted with 6 M urea in 10 M sodium phosphate, pH 7.8. Fractions of 15 mlwere collected and pooled as indicated in Fig. iA under “Results.” Five collagen-agarose enzyme pools were combined and applied to a DEAE-cellulose column (5 X 50 cm) equilibrated with 6 M urea in 10 mM sodium phosphate, pH 7.8. The column was eluted with a 1000-ml linear gradient of 0 to 0.5 M NaCl in 6 M urea and 10 mM sodium phosphate, pH 7.8, followed by 1000 ml of 0.5 M NaCl in the 6 M urea-phosphate solution. Fractions of 15 ml were collected and two enzyme pools formed as shown in Fig. 1B under “Results.” The two pools were concentrated to about 1 ml in an Amicon ultrafiltration cell with a PM-10 membrane and applied separately to a Sephacryl S-200 column (1.5 x 90 cm) equilibrated and eluted with 6 M urea in 50 M Tris-HC1, pH 7.8. Fractions of 1.5 ml were collected, and those containing most of the enzyme activity were combined to constitute the final enzyme pools I and 11. Purification of the Contaminant Present in Lysyl Oxidase PoolIIFractions representing the descending side of the lastenzyme activity peak in the DEAE-cellulose chromatography (fractions 156-165 in Fig. 1B under “Results”) were combined and this pool concentrated to about 1ml in an ultrafiltration cell with a PM-10 membrane. Since lysyl oxidase polymerizes in the absence of urea (8), the pool was dialyzed against 0.15 M NaCl in 0.1 M sodium phosphate, pH 7.8. It was then applied to a Sephacryl S-200 column (0.8 X 70 cm) equilibrated and eluted with the same solution. The polymerizedlysyl oxidase protein was in part lost during this gel filtration and in part eluted as a broad peak in the void volume. The only major protein peak eluting close to thetotal column volume constituted the purified contaminant. Preparation of Antisera to Lysyl Oxidase-An antiserum against the total enzyme protein was prepared by combining about 20 pg of lysyl oxidase pool I with 20 pg of pool 11. The 40 pg of protein were then concentrated to about 0.2 ml in an ultrafiltration cell with a PM-10 membrane. This sample was diluted to 1ml with 0.15 M NaCI, mixed with 1 ml of complete Freund‘s adjuvant, and injected intradermally at 10 sites in the back of a rabbit. 0.5 mi of crude Bordetella pertussis vaccine was injected subcutaneously at aseparate site. Further injections of 40 pg of lysyl oxidase mixed with incomplete Freund’s adjuvant, as above, were given 2, 4,6, and 8 weeks later. The blood was obtained by heart puncture under anesthesia 3 weeks after the last injection, and the serum was stored at -20 “C. An antiserum to the pool I enzyme was prepared as above, except that the rabbit was injected with 40 pg of pool I enzyme protein. Immunoadsorption of the Antibodies on the Contaminant Linked to Agarose-An immunoaffinity column was prepared by coupling 0.5 mg of the purified contaminant to 2 ml of 4% agarose (Sepharose 4B) by the CNBr activation technique (21). The column was equilibrated and eluted with 0.15 M NaCl in 50 mM Tris-HC1, pH 7.5, Aliquots of 1-2 ml of the antisera were passed through this column at a flow rate of 1 ml/h, and the effluents were recycled twice. After each chromatography the column was regenerated with 3 M NaSCN and equilibrated as above. Immunoprecipitation-Lysyl oxidase was incubated with varying amounts of the antiserum in a final volume of 0.4 ml of 0.15 M NaCl in 0.1 M sodium phosphate, pH 7.8, at 4 “C for 1b. Anti-rabbit serum, 50 rl, was then added and the incubation was continued for 12 h. After centrifugation a t 12,000 X g for 20 min, aliquots of the supernatants were tested for the enzyme activity. SDS-Polyacrylamide Gel Electrophoresis-This was carried out using either disc (22) or slab (23) gels that were stained with Coomassie brilliant blue. Standard proteins used for calibration were bovine serum albumin (molecular weight 67,000),ovalbumin (45,000),

6997

aldolase (40,000), pepsin (35,000), trypsin inhibitor (21,500), and cytochrome c (11,700). Immunoblotting-After SDS-polyacrylamide slab gel electrophoresis, the proteins were transferred to nitrocellulose sheets (24) and were stained by a heparin-toluidine method (25). The nitrocellulose sheets were destained with a solution of 8% acetic acid and 50% methanol in HzO, and immunological staining was then performed using a Vectastain ABC kit (Vector Laboratories) based on the formation of an avidin-biotin-peroxidase complex. Assay of Lysyl Oxidase Activity-Theenzyme activity was measured in a final volume of 0.6 ml with 600,000 dpm of [6-3H]lysine-labeled purified chick embryo calvaria collagen as substrate (17). The substrate was preincubated a t 37 “C for 60 min to promote fibril formation (17), and the incubation time with lysyl oxidase was then 10 h. In some experiments t,he collagen substrate was replaced by 2,000,000 dpm of [6-3H]lysine-labeled chick aorta elastin or chick embryo tendon procollagen. Samples of the enzyme were dialyzed against 0.15 M NaCl in 0.1 M sodium phosphate, pH 7.8, at 4 “C for 4 h before assay to remove urea, except in cases when high enzyme concentrations made it possible to use such small aliquots that the urea concentration in the assay mixture was less than 50 mM, a concentration which inhibits the enzyme activity by less than 5% (8). Amino Acid Analysis-Protein samples of about 20 pg were concentrated to about 0.5 ml by ultrafiltration with a PM-IO membrane and dialyzed exhaustively against distilled water. The protein was then hydrolyzed for 20 h at 110 “C in 1 ml of 6 M HCl in tubes sealed under N,. The sample was evaporated to dryness, redissolved in the analysis buffer, and assayed on a Kontron Liquimat 111amino acid analyzer. Other Assays-The protein content of crude enzyme samples was measured using a Bio-Rad protein assay kit (Bio-Rad) and that of partially purified and pure preparations by determining A2w nm or nm with bovine serum albumin as a standard. The specific activities of the purified chick embryo calvaria collagen substrates were measured by hydrolyzing aliquots for 20 h at 120 “C in 6 M HC1 and measuring the hydroxyproline content (26). The collagen content was then calculated on the basis of the hydroxyproline content (27) of the three polypeptide chains in the triple helical molecule. RESULTS

Purification of Lysyl Oxidase from Human Placental Tissue-Initial experiments indicated that the specific activity of lysyl oxidase in urea extractsof human placental homogenates is relatively low, being only about 5% of that in urea extracts of human skinhomogenates when expressedper unit of tissue, wet weight. Nevertheless, it was apparentthat placentas are theonly tissue which can be obtained in quantities large enough to purify andcharacterizethehuman enzyme. In the final procedure, 5 kg of the wet tissue was used as the starting materialfor each preparation. The urea extract contained only about 11%of the total placental protein, the bulk of the protein, but no lysyl oxidase activity, being found in the first phosphate-buffered NaCl extract. The first step may, therefore, involve an about 9-fold purification that is notincluded in the calculationsshown in TableI. The actual purification obtained in the whole procedure may thus be over 500,000-fold. Due to the large volume of the urea extract, the initial extractions and the collagen agarose chromatography were carried out infive separate batches,which were pooled before DEAE-cellulosechromatography. The largevolumes also made it necessary to perform most of the collagen-agarose step in a bucket, only part of washing and the elution (Fig. 1A) being carried out ina column. In theDEAE-cellulose chromatography mostof the protein passed through the column, and lysyl oxidase could then be eluted with a linear gradient of 0 to 0.5 M NaCl in 6 M urea (Fig. 1 B ) . In accordance with data on theenzyme from chick embryo (6) and bovine (10)cartilage andbovine aortic tissue (8, lo), the activity was consistently found in four separate

Human Placenta!1 Lysyl Oxidase

6998 TABLEI

Purification of two lysyl oxidase pools from a urea extract of human placental tissue The startinematerial was 5 ke of human Dlacental tissue. Purification Total lO-'.total lO-'.specific step

Urea extract Collagen-agarose DEAE-cellulose Pool I Pool I1 Pool I + I1 Sephacryl S-200 of pool I of pool I1 Pools I + I1

protein

activity

activity

mg

dpm

dpmlmg

107,000 154

56,380 13,920

6210 1.08 3290 4850 1.17 2570 2.25

0.53 90.39

-fold

1 171

3550 3010 6560

61,260 32,470 2760 0.085 50,450 26,740 2300 0.086 5060 0.171

FIG. 2. Gel filtration of the human placental lysyl oxidase pools I (A) and I1 ( B )on a Sephacryl S-200 column. Conditions as described under "Experimental Procedures." M, enzyme acprotein. tivity; -,

' 4 '

0

z-

I'

11

"

b67K

E

f2.0

L G

4 0 K -

c2

t, 1.0 U

-2l.5K

W

F N

z

W

0

Lo

2

6o

2

io'

-1lJK

FRACTION NUMBER

FIG. 1. Affinity chromatography ( A ) and DEAE-cellulose chromatography ( B ) of human placental lysyl oxidase. Both chromatographies were carried out as described under "Experimental Procedures." A, only the washing with 1 M NaCl (first protein peak) and the elution are shown. Fractions 46-59 were pooled and purified further. B, the arrow indicates the start of the gradient. Two enzyme enzyme activity; pools (I and 11) were formed as indicated. U, -, protein.

FIG.3. SDS-polyacrylamide disc gel electrophoresis of the human placental lysyl oxidase pools I (A) and I1 ( B )and the purified contaminantof poolI1 (C).The acrylamideconcentration was 10%.

I1 enzyme usually had one major band and one minorband, termed the contaminant and representing 10-30% of the total protein (Fig. 3B). peaks (Fig. 1B).In order to obtain enough material for further Molecular Properties of the Two Enzyme Pools-The mocharacterization, thefour peaks were not purified separately, lecular weight of lysyl oxidase in both pools was identical, but peaks 1and 2 were combined to form enzyme pool I, and being about 30,000 both by gel filtration on Sephacryl S-200 peaks 3 and 4 to form enzyme pool I1 (Fig. 1B). in 6 M urea and by SDS-polyacrylamide gel electrophoresis In the final gel filtration both enzyme pools gave only a with or withoutreduction with mercaptoethanol. The molecsingle peak of activity, the elutionposition of this peak being ular weight of the contaminant presentin the pool I1 enzyme identical for both enzyme pools (Fig. 2, A and B ) . The enzyme was about 23,000 by SDS-polyacrylamide gel electrophoresis. in pool I was usually entirely pure when studied by SDSSome experiments showed an additionalvery weak band in polyacrylamide gel electrophoresis (Fig. 3A), whereas the pool both enzyme pools, the mobility of which corresponded to a

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TABLEI1 molecular weight of about 60,000. The intensity of this band Amino acid composition of human placental lysyl oxidase and a varied inrepeated SDS-polyacrylamide gel electrophoresis contaminating protein experiments using the same enzyme preparations, suggesting The values for the pool I enzyme and the contaminant are given that it does not represent a contaminant but is due to the as means k S.D. of assays on 5 and 6 separate preparations, respecformation of small amounts of the enzyme dimerduring tively, while those for the pool I1 enzyme are expressed as mean electrophoresis. This suggestion is supported by the fact that deviation of assays on two separate preparations.The values are not the fractions pooled after the gel filtration (apparent molec- correctedfor losses during hydrolysis.The values reported previously ular weight of about 30,000) did not contain any bovine serum for lysyl oxidase from chick embryo cartilage(6) and bovine aorta(8) albumin (molecular weight 67,000) when protein standards are also shown. The values for the bovine aortic enzyme were originallyreportedseparatelyforallfourpeaksobtained in DEAEwere used. chromatography (8) butarenow shown as the meansof When the lysyl oxidase preparations were stored at 4 "cin ceIIuIose those for Deaks I and I1 (0001I) and ueaks 111 and IV (pool 11). 6 M urea for several weeks SDS-polyacrylamide gel electroHuman placental ConamBovine aortic Chick phoresis showed an additional weak band, the mobility of enzyme inant enzyme (8) cartiAmino which corresponded to a molecular weight of about 22,000. A lage acid of pool similar band with a molecular weight of about 24,000 has Pool I Pool I1 11 Pool I Pool I1 (6) previously been found in old preparations of bovine aortic residues/1OOo lysyl oxidase andhas been thought to be due to a slow Asx 125 f 10 123 f 15 113 & 8 124 108 136 proteolysis of the enzyme protein (10). The possibility was Thr 59 f 5 55 f 3 54 1 52 53 56 considered, therefore, that not only the present M,= 22,000 113 82 Ser 98 f 6 101 f 10 84 f 4" 95 band but also the M, = 23,000 contaminant of the pool I1 Glx 133 f 5 130+ 5 175 % 5b 124 130 106 enzyme might be related to lysyl oxidase. TOstudy this, the Pro 57 -+ 3 5 8 f 3 47 & lb 55 50 58 M,= 23,000 contaminant was purified to homogeneity (Fig. 114 f 16 111 f 7 114 & 8 104 142 97 Gly Ala 77 f 8 75 f 2 56 76 84 66 3b 3C), as described under "Experimental Procedures," and was Val 51 f 4 48 1 51 -+- 3 41 33 39 analyzed for its amino acid composition. ND' ND 25 16 30 ND CYS The amino acid compositions of the four forms of lysyl Met ND ND ND 16 16 15 oxidase obtained by DEAE-cellulose chromatography of the Ile 33 C 2 33 .+ 3 33 f 1 30 29 40 bovine aortic enzyme have been reported to be very similar, Leu 73 f 4 17 & 1 33 f 1' 72 75 67 but sufficiently different to suggest that all forms are distinct 24 +. 7 2 2 + 1 67 f 13b 28 20 65 Tyr Phe 31 & 2 34 + 0 43 2b 29 25 27 molecular species (8). The present data, however, do not His 27 k 4 29 f 1 20 k 3 32 25 29 indicate the existence of any definite differences in amino 46 f 2 47 + 2 38 & 4a 33 32 31 Lys acid composition between the two pools of the human placen52 -+ 4 5 7 + 1 72 -t l b 53 58 59 Arg tal enzyme, the mean values for both being essentially iden" p< 0.01 uersu pool I enzyme. tical (Table 11). The amino acid composition of the human 'p < 0.001 uersus pool I enzyme (Student's t test). placental enzyme was found to resemble those of the bovine ND, not determined. aortic (8) and chick embryo cartilage (6) lysyl oxidases, although some differences were also observed, such as thehigher TABLE111 concentrations of valine and lysine in the human enzyme Substrate swcificity of the two human lysyl oxidase w o k (Table 11). The contaminant of the pool I1 enzyme clearly Tritium released differed from lysyl oxidase in its amino acid composition, the Substrate Pool I Pool II Pool 1/11 most distinct differences being found in the contentsof gluctamic acid plus glutamine, proline, alanine, leucine, tyrosine, dPm phenylalanine, and arginine present (Table 11). collagen" calvaria Chick 2330 1870 Catalytic Properties of the Two Enzyme Pools-The specific 2360 2070 1.19 elastinb 1210 1220 activity of the pool I1 lysyl oxidase was usually 70-90% ofaortaChick 1440 1220 1.09 that of the pool I enzyme (Table I), the magnitude of this Chick procollagen' tendon 0 0 difference corresponding to the amountof contaminant pres0 10 ent in pool 11. It thus seems likely that the true specific * [6-3HJLysine-Iabeled purified chick embryo calvaria collagen, activity of the enzyme in both pools is the same. 600,000 dpm. Both enzyme pools utilized chick calvaria collagen and '[6-3H]Lysine-labeledchick embryo aortaelastin, 2,000,000 dpm. chick aorta elastin as substrates, and therewere no significant [6-3H]Lysine-labeledprocollagenfrom chick embryo tendon cells, differences between them in this respect (Table 111). Further- 2,000,000 dpm. more, no difference was found between the two pools in the K , for chick calvaria collagen (Fig. 4), the value being about difference in the amounts of antiserum required for pools I 2.5 X M inboth cases. No reaction took place when the and I1 (Fig. 5s).Antiserum I likewise inhibited and precipisubstrate was chick tendon procollagen (Table 111). tated theactivity of both lysyl oxidase pools (Fig. 6),although Immunological Characterization of the Enzyme-Two dif- it was less effective than antiserum T (compare Figs. 5 and ferent antiserawere prepared to human lysyl oxidase, one to 6). Again no significant difference was found in the volumes total enzyme protein, i.e. pools 1 and II together, termed here of antiserum required for a similar degree of inhibition or antiserum T, and the other to pool the I enzyme only, referred precipitation of pools I and I1 (Fig. 6), even though the to as antiserumI. preparation had been made as an antiserum to the pool I Antiserum T inhibited all the activity of crude lysyl oxidase enzyme only. Both antisera also inhibited and precipitated and both enzyme pools, there being no significant difference the activity of lysyl oxidase from the medium of cultured in the volumes of the antiserum required for a50%inhibition human skin fibroblasts (as shown for precipitation by antiof the same amount of activity units of the various enzyme serum T in Fig. 7). No significant difference was found in the forms (Fig. 5A). Thisantiserum also precipitated all the volumes of the antisera required for a 50% inhibition or activity of both enzyme pools, there again being no significant precipitation of the same amount of activity units in the case

*

~~

*

*

*

7000

Human Placental Lysyl Oxidase I

01

02

03

COLLAGEN ( r n l )

FIG. 4. Effect of the concentration of purified chick calvaria collagen on the reaction with human placental lysyl oxidase pools I and 11. The enzyme activity was measured in a final volume of 0.6 ml with 0.05 r g of purified pool I or I1 enzyme and varying concentrations of the collagen substrate. One ml of the substrate contained 1.11 nmol of collagen with a specific activity of 5.2 X 1OI5 dpm/mol. The X, thus corresponds to 0.147 nmol/0.6 ml. o“---o, poo! I enzyme; c-“., pool I1 enzyme.

of the crude skin fibroblast enzyme and thepurified placental enzyme pools I and I1 when assayed in the same experiment. Both antisera stained pool I and pool I1 lysyl oxidases in immunoblottingexperimentsafter SDS-polyacrylamide gel electrophoresis of the denatured proteins (Fig. 8). Although antiserum I was less effective than antiserum T in inhibiting and precipitating the nativeenzyme, it was consistently the more effective of the two in staining the denatured enzyme protein under the conditions used. No difference was found in the staining of the pool I and pool I1 enzymes. In those experiments in which the additional M, = 60,000 band was seen in the pure enzyme preparations this band was also stained by both antisera, and the M,= 22,000 band present in old enzyme preparations was likewise stained. All these experiments were carried out with antisera that had been immunoadsorbed on a column containing the purified M , = 23,000 contaminant linked toagarose, and no stainingof this contaminant was seen in any enzyme preparation or even when the SDS-polyacrylamide slab gels were overloaded with the purified contaminant. When not immunoadsorbed, however, both antisera also stained theM , = 23,000 contaminant presentinthe pool I1 enzyme, antiserum T stronglyand antiserum I very weakly.

I

I

I

2000}

SERUM ( p i )

FIG. 5. Inhibition ( A )and precipitation ( B )of various forms of human placental lysyl oxidase by antiserum T. Conditions as described under “Experimental Procedures.” Aliquots of 0-10 p l of nonimmune serum gave no inhibition or precipitation, whereas 30 pl gave inhibition and precipitation of about 15%.A-A, crude placental lysyl oxidase purified through the collagen-agarose affinity chromatography step; .”--., purified pool I enzyme; w, purified pool I1 enzyme.

results indicate a molecular weight of about 30,000 for the enzyme from human placental tissue, but an additional minor species of about 60,000 was seen insome SDS-polyacrylamide gel electrophoresis experiments, but not in themajority. The formation of such polymers,probablydimers, even in the DISCUSSION presence of SDS may explain the contradictory values reHuman lysyl oxidase was isolated here as a homogeneous ported previously. In agreement with data on lysyl oxidase protein for the first time. The human placental enzyme resem- from bovine aortic tissue (lo), the purified human placental bled those from chick embryo (6) and bovine (10) cartilage enzyme was found tobe slowly converted duringstorage to a formwithamolecular weight about 7000 lower than the and also from bovine aorta (8, 10) and ligamentum nuchae (7), in that the activity was consistently found in DEAE- original. Although the specific activities of the four species of the cellulose chromatography asfour separate species, allof which be different (8),the had an identical molecular weight. Previous data concerning bovine aortic lysyl oxidase are reported to the molecular weight of lysyl oxidase are conflicting, as some present results suggest that the truespecific activities of the studies have reported avalue of about 30,000 for all four various forms of the human placental enzyme are identical. species of the enzyme from chick embryo and bovine tissues In agreement with data on lysyl oxidase from chick aorta (1) (6-8, lo), while others have reported a value of about 60,000 and cartilage (6) and bovine aorta (lo), no differences were foundbetweenpools I and I1of the human enzyme with for enzyme fromthesamesources ( 2 , 4, 5 ) . The present

Human Placental Lysyl Oxidase I

7001

I

A*

2000

FIG.7. Precipitation of crude lysyl oxidase from cultured human skin fibroblasts by antiserum T. Confluent human skin fibroblasts were cultured for 24 h in a serum-free medium supplemented with 5 mg/ml of bovine serum albumin (14), and aliquots of the medium corresponding to 250,000 cells were then used as the source of crude lysyl oxidase. Aliquots of 0-15 pl of nonimmune serum gave no precipitation, whereas 50 pl gave a precipitation of about 15%.

FIG.6. Inhibition ( A )and precipitation ( B )of purified pool I and pool I1 lysyl oxidases by antiserum I. Aliquots of 0-10 pl of nonimmune serum gave no inhibition or precipitation, whereas 30 pl gave an inhibition of about 15% and a precipitation of about 10% and 100 pl an inhibition of about 20%. W,pool I enzyme; U, pool I1 enzyme. respect to the use of collagen and elastin as substrates. Furthermore, the present study indicates that there are no differences between enzyme pools I and I1 in their K,,, values for chick calvaria collagen. The K,,, of 2.5 X M determined here for both pools is slightly lower than the values of 9.5 x lo-' M (17) and 2.4 x lo6 M (6) previously reported for the enzyme from chick embryo cartilage. The failure to observe any activity with either of the human enzyme pools whenthe substrate was chick embryo tendon procollagen agrees with data which indicate that lysyl oxidase preferentially acts on collagen in native-type fibrils (1,5, 17) and suggests that the reaction with the enzyme inthe biosynthesis of type I collagen in vivo may not take place until theremoval of at least one of the two propeptides. The present and previous (1, 6, 8, 10) data on the substrate requirements of lysyl oxidase suggest that themultiple enzyme forms may not have any functional differences. The two pools of human placental lysyl oxidase were also very similar, if not identical, in their amino acid composition.

FIG.8. Analysis of lysyl oxidase pools I and II by immunoblotting after SDS-polyacrylamide slab gel electrophoresis. The acrylamide concentration was 12%. To demonstrate the specificity of the staining, the pool I and pool I1 enzymes were purified only through the DEAE-cellulose chromatography step. The antisera had been immunoadsorbed on a column containing the purified pool I1 contaminant linked to agarose and were used at dilutions of 1:16 for antiserum T and 1:50 for antiserum I. Experiment I: A, pool I enzyme, antiserum T; B, pool I1 enzyme, antiserum T. Experiment II: C,pool I enzyme, antiserum I; D, pool I1 enzyme, antiserum I. The arrows indicate the position of the pure pool I enzyme in a nitrocellulose sheet that was stained by a heparin-toluidine method (25). Neither were any differences found between the four formsof the bovine aortic enzyme in the peptide maps produced by digestion with Staphylococcus aureus V8 protease (10). The existence of the multiple lysyl oxidase formsis probably not explained by carbamylation of amino groups on the enzyme due to cyanate derived from urea, as the four forms of the bovine aortic enzyme retained their original chromatographic

7002


REFERENCES behavior on DEAE-cellulose with time (IO), a finding that was confirmed here with the human placental lysyl oxidase 1. Siegel, R. C. (1979) Znt. Reu. Connect. Tissue Res. 8, 73-118 2. Harris, E. D., Gonnerman, W. A., Savage, J. E., and ODell, B. (details not shown). Furthermore, amino acid analyses of acid L. (1974) Biochim. Biophys. Acta 341,332-344 hydrolysates of the four aortic enzyme species did not contain 3. Narayanan, A. S., Siegel, R. C., and Martin, G. R. (1974) Arch. any detectable amounts of homocitrulline, the carbamylated Biochem. Biophys. 162,231-237 derivative of lysine (8). Thenature of the multiple lysyl 4. Vidal, G. P., Shieh, J. J., and Yasunobu, K. T. (1975) Biochem. Biophys. Res. Commun. 64,989-995 oxidase forms remains to be determined. The great similarities 5. Siegel, R. C., and Fu, J. C. C. (1976) J. Biol. Chem. 2 5 1 , 5779found here and earlier (1,6-8,lO) between the various enzyme 5785 species by a number of criteria suggest that the differences 6. Stassen, F. L. H. (1976) Biochim. Biophys. Acta 438,49-60 may exist at thelevel of some post-translational modification 7. Jordan, R. E., Milbury, P., Sullivan, K. A., Trackman, P. C., and of the enzyme protein. The possibility is not excluded, howKagan, H. M. (1977) Adu. Exp. Biol. Med. 7 9 , 531-542 8. Kagan, H. M., Sullivan, K. A., Olsson, T. A.,111, and Cronlund, ever, that the multiple enzyme forms represent products of A. L. (1979) Biochem. J. 177,203-214 four very similar but not identical genes that are expressed in 9. Han, S., and Tanzer, M.L. (1979) J. Biol. Chem. 254, 10438a number of different tissues. 10442 All the immunological properties studied werelikewise 10. Sullivan, K.A., and Kagan, H. M. (1982) J. Biol. Chem. 2 5 7 , 13520-13526 identical in the two humanplacental lysyl oxidase pools. Antiserum I, which had been prepared to pool I enzyme only, 11. Byers, P. H.,Siegel, R. C., Holbrook, K. A., Narayanan, A. S., Bornstein, P., and Hall, J. G. (1980) N . Engl. J . Med. 303,61inhibited and precipitated both enzyme pools and stained 65 both in immunoblotting, no differences being found between 12. Royce, P. M., Camakaris, J., and Danks, D. M. (1980) Biochem. the two pools in any of these properties. Interestingly, antiJ. 192,579-586 serum T was more effective than antiserum I in inhibiting 13. Kuivaniemi, H., Peltonen, L., Palotie, A., Kaitila, I., and Kivirikko, K. I. (1982) J. Clin. Znuest. 6 9 , 730-733 and precipitating the native enzyme, whereas antiserum I was 14. Peltonen, L., Kuivaniemi, H., Palotie, A., Horn, N., Kaitila, I., more effective in staining theenzyme in immunoblottingafter and Kivirikko, K. I. (1983) Biochemistry 22,6156-6163 SDS-polyacrylamide slab gel electrophoresis. This suggests 15. Carter, E. A., McCarron, M. J., Alpert, E., and Isselbacher, K. J. (1982) Gastroenterology 82, 526-534 that antiserum T may primarily recognize some determinants present only in thenative enzyme, while antiserum I primarily 16. Rowe, D. W., McGoodwin, E. B., Martin, G. R., and Grahn, D. (1977) J. Biol. Chem. 252,939-942 recognizes those present in the denatured protein. Both anti- 17. Siegel, R. C. (1974) Proc. Natl. Acad. Sci. U. S. A . 71,4826-4830 sera also inhibited and precipitated the lysyl oxidase activity 18. Prockop, D.J., and Tuderman, L. (1982) Methods Enzymol. 8 2 , present in the medium of cultured human skin fibroblasts, 305-319 and no differences were found between this crude enzyme and 19. Bornstein, P., and Piez, K. A. (1966) Biochemistry 6,3460-3473 the two purified placental lysyl oxidase pools in this respect. 20. Kivirikko, K. I., and Myllylii, R. (1982) Methods Enzyrnol. 82, 245-304 These findings suggest that the human skin enzyme does not 21. Cuatrecasas. P.. and Anfinsen, C.B. (1971) Methods Enzymol. have any catalytically active isozymes that differ from those 22,345-358 of the human placental lysyl oxidase. Taken as a whole, the 22. Weber. K.. and Osborn. M. (1969) J. Biol. Chem. 244,4406-4412 data suggest that there are no major immunological differ- 23. King, J., and Laemrnli; U. K. (1971) J. Mol. Biol. 62, 465-477 H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acod. ences between the multiple lysyl oxidase forms, and thus an 24. Towbin, Sci. U. S. A. 76, 4350-4354 antiserum prepared to any one of the enzyme species can 25. Vartio, T., and Vaheri, A. (1981) J. Biol. Chem. 256, 13085probably be used to study the total enzyme protein. 13090 '

Acknowledgment-We gratefully acknowledge the expert technical assistance of Aila Jokinen.

26. Kivirikko, K. I., Laitinen, O., and Prockop, D. J. (1967) Anal. Biochem. 1 9 , 249-255 27. Miller, E. J., and Gay, S. (1982) Methods Enzymol. 82,3-32