THE JOURNAL OF BIOLOC~CAL CHEMISTRY Vol. 251, No. 18, Issue of September 25, pp. 5779-5785, 1976

Printed m U.S.A.

Collagen Cross-linking

PURIFICATION AND SUBSTRATE SPECIFICITY OF LYSYL OXIDASE*

(Received for publication, March 4, 1976)

ROBERT C. SIEGEL+ AND JOSEPH C. C. Fu

From the Departments of Medicine and Orthopaedic Surgery, University of California, San Francisco, California 94143

Lysyl oxidase is a specific amine oxidase that catalyzes the formation of aldehyde cross-link intermediates in collagen and elastin. In this study, lysyl oxidase from embryonic chick cartilage was purified to constant specific activity and a single protein band on sodium dodecyl sulfate acrylamide gel electrophoresis. This band had an apparent molecular weight of 62,000. The eluted protein cross-reacted with inhibiting antisera developed against highly purified lysyl oxidase. The highly purified enzyme was active with both insoluble elastin and embryonic chick skin or bone collagen precipitated as reconstituted, native fibrils. There was low activity with nonhydroxylated collagen, collagen monomers, or native fibrils isolated from lathyritic calvaria. The maximum number of aldehyde intermediates formed per molecule of collagen that became insoluble was two. These results indicate that lysyl oxidase has maximum activity on ordered aggregates of collagen molecules that may be overlapping associations of only a few collagen molecules across. Formation of aldehyde intermediates and cross-links during fibril formation may facilitate the biosynthesis of stable collagen fibrils and contribute to increased fibril

tensile strength in duo.

Lysyl oxidase is a specific amine oxidase (1-5) that catalyzes collagen substrates was significantly increased when collagen the formation of c-aldehydes in collagen and elastin from was precipitated from solution as reconstituted fibrils (10). In certain lysyl and hydroxylysyl residues (Fig. 1) (6). These the present study, the activity of highly purified chick cartilage aldehydes, allysine’ and hydroxyallysine, are intermediates in lysyl oxidase has been compared with both collagen and elastin formation of intra- and intermolecular cross-links. In collagen, substrates and the nature of the optimal collagen substrate allysine and hydroxyallysine form Schiff base intermolecular further defined.

cross-links bv condensing with the t-amino grouns of certain lysyl and hydroxylysyl residues (7,B). Two allysyl residues may EXPERIMENTAL PROCEDURES

also form cross-links by aldol condensation in both collagen Materials and elastin (9). After aldehyde formation, further reactions in cross-link biosynthesis are believed to proceed spontaneously.

b [4,5-3H]Lysine (60 Ci/mmol), DL [6-3H]lysine (5 Ci/mmol), L [‘%]lysine (224 mCi/mmol) were obtained from New England Nu-

Lysyl oxidase was originally detected by a tritium release clear. Reagent grade urea was obtained from Sigma Chemical Co.

assay in which chick aorta elastin labeled with [6-3H]lysine in Amino acid analyzer resin was purchased from Medical and Research

organ culture was used as a substrate (1). Tritium was released Service, Menlo Park, Ca.

from position 6 as aldehydes formed (Fig. 1) under the ,Mothnda

influence of lysyl oxidasel The reaction was inhibited by micromolar concentrations of P-aminopropionitrile (1). Ini-

Preparation of Lysyl Ox&se-Femoral and tibia1 epiphyseal carti-

tially, enzyme activity with elastin substrates was found to be lage from 17.day-old chick embryos was dissected free of surrounding t’ Issues and homogenized in 0.15 M NaCl, 0.10 M Na2HP0,, pH 7.8 (2

much higher than that with similarly labeled collagen sub- ml/g of tissue) at 4”, as previously described (1, 3). Following strates (6). This was presumed due to the greater number of centrifugation at 17,000 x g for 10 min, the pellet was again

lysyl residues in elastin that participated in cross-linking. homogenized in the same buffer and recentrifuged. The supernatant

However, recently we found that lysyl oxidase activity with from these two homogenizations was discarded. The pellet was then homogenized in 6 M urea, 0.05 M Tris, pH 7.6 (2 ml/g of original tissue) at 25”,

*This research was supported by National Institutes of Health and centrifuged at 40,000 x g for 60 min. The pellet was

Grants AM-16424, AM-09406, and AM-18237. re-extracted with the 6 M urea buffer and recentrifuged. The supema-

$ Recipient of National Institutes of Health Research Career Devel- tants from both urea extracts were pooled and used for subsequent studies.

opment Award K04 AM-00114. A portion of this work was done when R. C. S. was a fellow of the Helen Hay Whitney Foundation.

The pooled urea extract was loaded directly onto a DEAE-52 cellulose column (2.5 x 18 cm) previously equilibrated with 6 M urea,

’ The abbreviations used are: allysine, 2-aminoadipaldehydic acid; 0.05 M Tris, pH 7.6 at 25”. After loading, the column was washed with hydroxyallysine, 2-amino&hydroxyadipaldehydic acid; PBS, phos- this buffer until the absorbance at 280 nm had returned to the initial phate-buffered saline. value. Elution was achieved in 6 M urea, 0.05 M Tris, pH 7.6, using a

5779

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5780 Substrate Specificity of Lysyl Oxidase

linear gradient of NaCl from 0 to 0.5 M. The total volume of the gradient was 400 ml. Two distinct peaks of enzyme activity were routinely found. Fractions from each peak were dialyzed overnight at 4” against PBS and pooled. They were then stirred for 3 h at 4” with a Sepharose 4B “affinity” resin which had been prepared by coupling lathyritic rat skin collagen to Sepharose 4B after cyanogen bromide activation (10). The resin was then eluted sequentially at 25” with 0.05 M Tris, pH 7.6; 1 M NaCl, 0.05 M Tris, pH 7.6; and 6 M urea, 0.05 M Tris, pH 7.6. Enzyme activity from both activity peaks was eluted in the 6 M urea, 0.05 M Tris fraction. This fraction was loaded onto a DEAE-cel- lulose column (1.5 x 10 cm) at 25”. Elution was achieved in 6 M urea, 0.05 M Tris, pH 7.6, using a linear gradient of NaCl from 0 to 0.5 M. The total volume of the gradient was 200 ml. Fractions were again dialyzed against the phosphate/saline buffer and the tube with highest activity used for subsequent studies. This fraction, purified from the second peak of activity on the initial DEAE-chromatogram had protein content (11) of approximately 2 pglml and is designated “highly purified” lysyl oxidase in subsequent studies. For some experiments the tubes with highest activity were purified by another cycle of absorption to and elution from the affinity resin and rechromatography on DEAE.

Enzyme fractions were analyzed by sodium dodecyl sulfate acrylam- ide gel electrophoresis as described by Weber and Osborn (12) with 5% acrylamide gels. Individual fractions were concentrated in a Minicon concentrator or by dialysis against distilled water and lyophilization. All fractions were run in the presence of mercaptoethanol and then stained with Coomassie blue (12).

Preparation -of Substrates-The elastin substrate labeled with L- [4,5-3H]lysine was prepared from 17.day-old chick embryo aortas as previously described (1, 13).

Chick calvaria collagen substrates labeled with L- [6-3H]lysine were prepared from Ill-day-old chick embryo calvaria as previously described (10, 13). Amino acid analysis (14) of the purified material indicated it was pure collagen (Table I) (15). The specific activity of the [6-“Hllysine collagen substrate varied slightly with each prepara-

NH2 tH2

“:=O

RI-:-H - R’-:H

I Lysyl Oxidose (;“2j2

(f”2j2 -NH-CH-C-

-NH-C”-:- 0:

0 ~~~~~ (R,=H) or d Aminoadipic acid

Hydroxylyryl (R,=OH) Residue b semioldehyde (R,=H; Allysine) or b- Hydroxy, d Aminoodipic acid

b remialdehyde (R,=OH;

Hydroxyollysine) Residue

FIG. 1. Enzymatic formation of aldehyde cross-link intermediates in collagen.

TABLE I

Amino acid analysis of purified chick calvaria collagen substrate”

4-Hydroxyproline

Aspartic acid

Threonine Serine

Glutamic acid

Proline Glycine

Alanine

Valine Methionine

Isol,eucine Leucine Tyrosine

Phenylalanine Hydroxylysine

Lysine

Histidine

Arginine

D Residues/lOOO.

103

47

18 26

75 114

331

117

21 5.5

9

22 3.3

16

9

28 5.2

50

tion but was approximately 3.50 x lOI dpm/mol of collagen as mea- sured by determination of hydroxyproline on amino acid analysis. Over 99% of the radioactivity was present initially as hydroxylysyl and lysyl residues. The ratio of radioactive hydroxylysyl to lysyl residues was 0.42 to 0.46 in most experiments.

Unlabeled 17.day-old chick embryo calvaria collagen was prepared by injecting the yolk sacs of 15-day-old eggs with 20 mg of fl-aminopro- pionitrile in sterile saline. The eggs were harvested at 17 days, the calvaria homogenized in 1 M NaCl, 0.05 M Tris, pH 7.4 at 4”, and purified by 20% NaCl precipitation as described previously with radioactive calvaria (10, 13).

Chick embryo skin collagen was prepared by incubating 17.day-old chick embryo skin in organ culture with [6-3H]lysine as described for the bone collagen (10, 13). Purification was also done similarly. The material obtained had approximately 35% of the radioactivity present as collagen based on hydroxyproline content on amino acid analysis.

Assays-Assays with the elastin substrate were done as previously described (1, 13). Total volume of the incubation mixture was 1.5 ml. One milliliter of the tritium water isolated by vacuum distillation was analyzed with 10 ml of Aquasol in a Packard liquid scintillation spectrometer. Counting efficiency was 30%.

Assays with tritium-labeled collagen substrates were also done as previously described. In each assay tube, 0.5 to 0.6 nmol of collagen was used. The substrate was precipitated as reconstituted fibrils by incubation at 37” for 60 min. Specific enzyme fractions, 0.5 ml, were then added and incubations usually done for 2 h. Total volume of the incubation mixture was 0.8 ml. After distillation 0.5-ml aliquots were analyzed similarly to the elastin assay.

In some experiments, the collagen substrate was replaced by either intact chick calvaria that had been labeled with Db[6-3H]lysine in organ culture under the same conditions used to prepare collagen substrates or homogenates of these bones suspended in 0.02 M NaH2P0,, pH 7.4, to maintain the newly synthesized collagen as native fibrils (16). In addition nonhydroxylated collagen labeled with [6-3H]lysine was prepared from chick calvaria as previously de- scribed (17) and used as substrate.

Following incubation in some experiments, the mixture was dis- solved in 6 M urea, 0.05 M Tris, pH 7.5, with addition of 5 mg of unlabeled chick calvaria collagen and the solution dialyzed against 0.06 M sodium acetate at 4”. After dialysis, the sample was clarified by filtration through glass wool. An aliquot was taken to determine the per cent of radioactivity that remained soluble.

Preparation and Assay of Antisera to Highly Purified Chick Lysyl Oxidase-Preparations of highly purified chick lysyl oxidase were dialyzed against distilled water, concentrated by lyophilization, and then redissolved in water and mixed with an equal volume of complete Freund’s adjuvant. Rabbits were immunized with 0.2 mg of enzyme injected intradermally at several sites. Four weeks later, the rabbits were injected with 0.1 mg of enzyme mixed with incomplete Freund’s adjuvant. Beginning 2 weeks after the second injection rabbits were bled at weekly to biweekly intervals by ear vein puncture. The sera were stored at -20” until use. Antisera were assayed for inhibitory activity by incubating 10 to 50 rl with 1 pg of purified lysyl oxidase and 0.1 mg of bovine serum albumin for 15 min. The mixture was then added to the collagen substrate after it had been preincubated at 37” for 1 h and assayed as described above.

Electron microscopy of reconstituted fibrils was done with a Zeiss EMSA electron microscope. The collagen substrate was incubated for 1 h at 37”, stained with 2% sodium phosphotungstate and then placed on Formvar-coated grids and allowed to dry.

RESULTS

Pooled urea extracts of embryonic cartilage were used as an enzyme source for further purification since they contained 70% of the total enzyme activity and were initially 7-fold more pure than the phosphate/saline extracts (Table II). With DEAE-chromatography (Fig. 2), two peaks of enzyme activity eluted from the column. These had similar activity profiles with either the collagen or elastin substrate. The second activity peak contained more than twice the total enzyme activity as the first and was approximately twice as pure (Table II). Consequently, most studies were done with this preparation after it had been further purified by affinity purification and a second DEAE-chromatography procedure

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Substrate Specificity of Lysyl Oxidase

TABLE II

Purification of lysyl oridase

5781

Substrate preincubated at 37” for 1 h prior to addition of enzyme fraction. Incubations at 37” for 2 h. Activity expressed as net SH

release/ml/lOB cpm of chick calvarium collagen substrate, specific activity lo6 cpm/nmol, multiplied by the total volume of the fraction.

Purification factor

Total activity Tl release” Total protein Specific activity Yield uer.suS initial

phosphate extract

wr.sUS initial urea extract

cw w 'H cpmlmg protein 90 Initial phosphate 309,000 624 482 1

extract

Initial urea 718,000 200 3592 100 7.46

extract

DEAE No. 1

1st activity peak 163,000 1.44 113,006 22.1 235

2nd activity peak 356,090 1.62 220,006 49.6 457

Affinity eluate of DEAE No. 1

2nd activity peak 235,000 0.250 940,000 32.7 1950

DEAE No. 2 212,000 0.060 3,530,oOO 29.4 7320

DEAE No. 3 90,000 0.025 3,580,OOO 12.5 7440

“Fractions (0.5 ml) assayed with [6-3H]lysine chick collagen substrate, 5 x lo5 cpm of substrate per assay tube.

0.134

1

31.6

61.2

262 982

998

05 0 loo 200 300

ELUTION VOLUME (ml)

FIG. 2. DEAE-cellulose chromatography of 6 M urea extract of 17.day chick embryo epiphyseal cartilage. Eighty milliliters of extract were applied to a DEAE-cellulose column (2.5 x 18 cm) in 6 M urea, 0.05 M Tris, pH 7.6 at 25”. Elution was achieved in 6 M urea, 0.05 M Tris, pH 7.6, using a gradient (- -1 of NaCl from 0 (1) to 0.5 M (1) in a total volume of 400 ml. The absorbance was continuously monitored at 280 nm; flow rate was 4.5 ml/min and g-ml fractions were collected. Fractions were dialyzed against 0.15 M NaCl, 0.1 M NaH2P0,, pH 7.8, overnight. Aliquots (0.5 ml) were assayed with either [6-‘H]lysine collagen substrate (U--Xl) or [4,5-3H]lysine elastin substrate (0. .O). Incubations were done for 2 h at 37”. Activity is expressed as net 3H release/500,000 ‘H cpm of collagen substrate or l,OOO,OOO “H cpm of elastin substrate.

(Fig. 3). The activity eluted from the second DEAE-column as a single peak with activity for both substrates although the relative activity for the collagen substrate was greater than on the initial, DEAE-chromatogram. The first activity peak from the initial DEAE-chromatogram was also purified by affinity chromatography and subsequent DEAE-chromatography. It emerged from the DEAE-column as a single peak of activity (not shown) but again eluted at lower ionic strength than did the second peak. Recycling enzyme from the second peak through a second affinity step and third DEAE-procedure did not increase the specific activity significantly above that after the second DEAE-chromatogram (Table II). The purification

ELUTKIN VOLUME (ml)

FIG. 3. DEAE-cellulose chromatography of partially purified lysyl oxidase. The 6 M urea, 0.05 M Tris, pH 7.6, eluate from an “affinity” resin consisting of Sepharose 4B resin with lathyritic rat skin collagen coupled to it by CNBr activation was loaded directly onto a DEAE-52 column (1.5 x 10 cm) previously equilibrated with the same buffer. Elution was achieved in 6 M urea, 0.05 M Tris, pH 7.6, using a linear gradient of NaCl from 0 to 0.5 M in a total volume of 200 ml. Flow rate was at 4.5 ml/min with g-ml fractions collected and continuous monitoring at 280 nm. Assays with [6-3H]lysine collagen (0. ‘0) or [4,5-3H]lysine elastin (O- - -0) were done as described in Fig. 2.

was followed with the collagen substrate instead of the elastin substrate used in previous studies (3, 4, 13) since the collagen substrate is more sensitive than the elastin substrate and requires shorter incubation times. Previous purifications have been expressed in terms of an enzyme unit equal to 100 cpm of tritium released from 600,000 cpm of chick aorta elastin substrate in 8 h. One elastin unit is equivalent to 35 cpm of tritium released from lo-’ mol of collagen substrate in 2 h. The

highly purified lysyl oxidase preparation after the second DEAE-chromatogram has 80 elastin units/Kg of protein or 1 elastin unit is equivalent to 1.3 x IO-@ g of protein. This material has not lost significant enzyme activity when frozen at -20” for 1 year.

Analysis of the individual fractions by sodium dodecyl sulfate acrylamide gel electrophoresis indicated that there was a single protein band after chromatography for the second time on DEAE (Fig. 4). This band had an apparent molecular weight of 62,000. Sodium dodecyl sulfate-acrylamide gel elec- trophoresis of the first peak of activity from the initial DEAE run also gave a protein band with apparent molecular weight 62,000 (not shown). Attempts to elute the purified enzyme

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5782 Substrate Specificity of Lysyl Oxidase

ABCDEF FIG. 4. Sodium dodecyl sulfate acrylamide gel electrophoresis of

enzyme fractions after various steps in purification. Five per cent acrylamide gels run at 8 ma/tube for 4 h as described in text. All samples applied after reduction with mercaptoethanol. A, initial urea extract (Table I); B, first peak of lysyl oxidase activity on DEAE chromatography (Fig. 2); C, second peak of lysyl oxidase activity on DEAE chromatography (Fig. 2); D, second peak of lysyl oxidase activity on DEAE (Gel C) after further purification by absorption to and elution from Sepharose 4B/rat skin collagen affinity resin (Table I); E, fraction with maximum lysyl oxidase activity after chromatogra- phy on DEAE a second time (Fig. 3); F, fraction with maximum enzyme activity after recycling enzyme as in Tube E through another affinity absorption procedure and a third DEAE chromatography (Table I).

f a

a 3000 I 0 % ,’ 4 Y

2000 I’

2

“I

k z”

1000 %/ 0 0 2 4

TIME (hours)

FIG. 5. Effect of collagen substrates from different tissues on apparent lysyl oxidase activity. Samples of either bone (Panel a) or skin collagen (Panel b) 500,000 ‘H cpm/incubation tube were incu- bated with highly purified lysyl oxidase after preincubation in phos- phate/saline buffer (0-A) or the same buffer with either 0.05 M aspartic acid (A---A) or 0.05 M arginine (04) added before incubation. See text for details of assay.

from the gel and recover enzyme activity were unsuccessful. However, it was possible to show that the protein eluted from the gel was immunologically similar to highly purified lysyl oxidase. There was significantly less inhibition by crude antisera directed against highly purified lysyl oxidase when the protein eluted from the gel was added to an incubation mixture containing highly purified lysyl oxidase (Table III).

Comparison of Lysyl Oxidase Activity with Different Sub- strates-since the previous study (10) showed that lysyl oxidase

TABLE III

Immunologic cross-reaction of highly purified lysyl oxidase with SDS-acrylamide gel protein band”

Rabbit antisera’

Net $H Per cent CPm

released’ inhibition

Phosphate/saline

Net *H Per cent EleaSe inhibition

Lysyl oxidased Lysyl oxidase” +

SDS’-acrylamide gel protein band elute’

Lysyl oxidased + SDS-acrylamide gel eluateg

307 91.8 3750 0 3190 15.0 3800 -1.3

320 91.3 3800 -1.3

a All incubations for 2 h at 37”. * 50 pl per incubation tube plus 0.1 mg of bovine serum albumin. ’ 5 x lo5 cpm substrate per assay tube. dOne microgram of highly purified lysyl oxidase per incubation

tube. e SDS, sodium dodecyl sulfate. ’ Extracted in PBS and dialyzed versus PBS. g Gel slice, from area without protein, extracted and dialyzed uersus

PBS.

TABLE IV

Lysyl oxidase activity with uarious collagen substrates

Net 3H cpm re- Per cent of re- leased”/5 x 10’ lease compared cpm substrate to reconsti-

tuted fibril

Native collagen fibrils Intact chick calvaria’ 0.02 M phosphate extract’

Pellet Supernatant

Nonhydroxylated collagerid Reconstituted bone collagen fi-

brils Reconstituted bone collagen

fibrils + 0.05 M arginine’ Reconstituted bone collagen

fibrils + 0.05 M aspartic acid

54.0 2.2

74.0 3.0 23.0 0.92

120.0 4.80

2490 100

125.0 5.0

2590 104

n Assays for 2 h at 37O. All substrates preincubated for 1 h at 37” prior to addition of enzyme.

b Unhomogenized chick calvaria suspended in PBS. c Prepared by homogenizing [6-‘Hllysine-labeled chick calvaria in

0.02 M NaH2P0,, pH 7.4, and dialyzing against 0.02 M NaH*PO, for 48 h.

d Prepared from chick calvaria by labeling with [6-3H]lysine in the presence of 2.5 x lo-’ M a+--dipyridyl and lo-’ M 8-aminopropioni- trile.

e Added to substrate prior to initial incubation at 37 “.

has high activity with reconstituted native fibrils, it was of interest to study the nature of the substrate specificity further with highly purified lysyl oxidase. As illustrated in Table IV, there was little activity with native, presumably tightly packed fibrils. This was true whether the fibrils were present in tissue such as whole calvaria labeled with [6-3H]lysine in the pres- ence of &aminopropionitrile or when calvarial homogenates prepared in 0.02 M Na2HP0,, pH 7.4, to prevent dissolution of native fibrils were used as substrates. There was also low activity with nonhydroxylated collagen prepared from chick calvaria. As previously noted (lo), there was little enzyme activity when 0.05 M arginine, an inhibitor of fibril formation,

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Substrate Specificity of Lysyl Oxidase 5783

FIG. F. Electron micrographs of re- constituted lathyritic chick bone colla- gen fibrils. Samples were heated to 37” for 1 h and then stained with 2% sodium phosphotungstate. The bar is 100 nm. Magnification is x 66,150.

was added before fibril formation. However, with 0.05 M

aspartic acid, heat precipitation of the bone collagen substrate from solution increased from 75 to 81% and the apparent enzyme activity also increased slightly. The effect of aspartic acid on fibril formation and apparent enzyme activity was much greater with chick skin than chick bone collagen. After incubation at 37” for 1 h, 3.3% of the radioactivity in the skin collagen substrate precipitated. With arginine this fell to zero and with aspartic acid, this increased to 35.3%. As illustrated in Fig. 5, the apparent enzyme activity reflects the increased radioactive precipitation and presumed increase in fibril for- mation. With bone collagen, the activity was low with arginine and high with either aspartic acid or the control phosphate/ saline solution. With skin collagen on the other hand, the activity was low with either arginine or the control phosphate/

saline solution but increased significantly with aspartic acid. Since the bone collagen substrate is pure collagen (Table I), but the skin collagen substrate is only one-third collagen by amino acid analysis, the lower activity observed with aspartic acid in skin collagen as compared to bone collagen is probably due to the presence of less collagen. The skin collagen substrate is largely type I collagen and similar to the bone collagen substrate since it has 2/l al/a2 ratio on carboxymethyl- cellulose chromatography and the ratio of hydroxylysine to lysine is 0.42. In addition to characterizing the collagen substrate by amino acid analysis (Table I), the morphology was evaluated by electron microscopy. As illustrated in Fig. 6, native collagen fibrils were present after heat precipitation. However, much of the collagen was present as loosely packed fibrils with 640 A repeats or overlapping aggregates of mole-

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5784 Substrate Specificity of Lysyl Oxidase

cules that seem to align with the larger fibrils at some points (Fig. 65). The same morphology was found with aspartic acid present. However, with arginine, neither native fibrils nor the overlapping aggregates of molecules were seen.

To study the activity of lysyl oxidase with reconstituted fibrils in more detail, a time curve of enzyme activity was run and additional substrate and additional enzyme added to some tubes as the reaction was slowing at 6 h (Fig. 7). In this experiment, the rate of tritium release during the early part of the incubation was approximately 800 cpm/h. At 7 h this had slowed to 140 cpm/h. With addition of enzyme equal to the initial enzyme the rate increased to 529 cpm/h or 66% of the early velocity. With additional substrate equal to the initial substrate, there was a short lag and then the rate increased to 730 cpm/h or 91% of the early velocity. Without additional substrate or enzyme the curve leveled off after approximately 1.2% of the tritium had been released from the fibrils. Since there are 95 lysyl and hydroxylsyl residues in type I chick collagen, this amount of tritium release indicates that less than two aldehyde cross-link intermediates formed per molecule before the reaction stopped.

To determine whether this aldehyde and cross-link concen- tration was sufficient to affect collagen solubility, the solubility of collagen was monitored during incubation by determining the radioactivity in the supernatant after incubation and dialysis. As illustrated in Fig. 8, soluble radioactivity decreased linearly during the first few hours of incubation and then remained fairly constant. Forty-two per cent of the total radioactivity was insoluble at 6 h. In this experiment, the total radioactivity per tube was 695,000 cpm and 6,403 cpm were released at 6 h. If one assumes that each lysyl residue has similar specific activity and that all the tritium was released from the 42% of the collagen that became insoluble, a maximum of 2.08 tritium atoms were released per collagen molecule.

DISCUSSION

Since Narayanan et al. (3) found that lysyl oxidase is stable in 6 M urea, workers from several laboratories (3-5, 18) have been able to purify the enzyme extensively from chick aorta

5ccc-

T Jj 4000-

0 I 2 3 4 5 6 7 8

INCUBATION TIME (hours)

FIG. 7. Effect of additional substrate and enzyme on lysyl oxidase activity when the enzymatic reaction was slowing. Pure lysyl oxidase (400 na) was added to tubes with 500,000 corn of 16-3Hllysine collagen substrate precipitated as reconstituted fibrils. At 6 h (1) either an additional 400 ng of pure lysyl oxidase (O----O), or an additional 500,006 cpm of substrate (0~~~~[7) was added. Final volume of all tubes was 0.9 ml. Aliquots (0.6 ml) of each tube were analyzed for tritium water after distillation.

and epiphyseal cartilage and bovine aorta. In this study lysyl oxidase from embryonic chick cartilage was purified to homogeneity on sodium dodecyl sulfate acrylamide gel electro- phoresis and to constant specific activity. This preparation differs from others since the bulk of the enzyme activity was extracted with urea after removal of a significant fraction of other soluble proteins by extraction with phosphate/saline. The urea extract was purified by sequential chromatography on DEAE, collagen-Sepharose 4B “affinity” resin, and DEAE. This procedure is relatively rapid, requires few intervening steps except dialysis and yields enzyme that remains active for at least 1 year. Since recycling the highly purified enzyme does not increase the specific activity significantly and the single protein band on sodium dodecyl sulfate acrylamide gels cross-reacts with inhibiting antisera developed against highly purified lysyl oxidase, this preparation seems free of obvious protein contaminants. On the other hand, the per cent of native enzyme molecules present cannot be estimated since the gels must be run under denaturing conditions. Many inactive enzyme molecules may be present as a result of using urea with partially purified enzyme. The increase in specific activity observed after the second DEAE-procedure may in fact be due to removal of inactive enzyme molecules rather than other proteins since the gels after the affinity step and second DEAE-step are quite similar.

In a recent report, Shieh et al. (5) found that urea extracts of bovine aorta contained two peaks of lysyl oxidase activity on DEAE that were not immunologically identical. Stassen (18) found that urea extracts of embryonic chick cartilage gave four activity peaks on DEAE, each with 28,000 molecular weight. In this study, two major activity peaks with molecular weight of 62,000 were found. As in Stassen’s paper (18), each peak has similar activity profiles with collagen and elastin substrates. Although the reasons for these differences are unknown, it is possible that they are due to differences in preparation. In this study, enzyme was always prepared from fresh tissue. If the dissected cartilage was frozen and then extracted the following day, the first activity peak in the initial DEAE chromatogram

was no longer present. The molecular weight of the purified second activity peak was unchanged. The other workers used tissue that had been frozen and used different extraction

UJ 2 60 3

51 6s

40

t \

l - m-0

\ 0

I I I I 0 2 4 6 8

TIME (hrs)

FIG. 8. Solubility of collagen incubated with lysyl oxidase. [6-3H]Lysine chick calvarium collagen (695,000 cpm) was incubated with 806 ng of lysyl oxidase for varying incubation times. Following incubation, 5 mg of carrier collagen was added and the samples suspended in 5 ml of 6 M urea, 0.05 M Tris, pH 7.5 at 25”, and then dialvzed against 0.06 M sodium acetate for 24 h at 4”. Soluble radioactivity after filtration through glass wool was compared to samples incubated with fl-aminopropionitrile at each point.

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Substrate Specificity of Lysyl Oxidase 5785

procedures. Degradation during freezing, thawing, and extrac- tion may have caused chromatographic or immunologic hetero- geneity.

The principal reason for the rigorous purification done here was to study the substrate specificity of highly purified enzyme. This study demonstrates that highly purified lysyl oxidase is active on elastin as well as type I collagen substrates from both bone and skin. The critical determinant of apparent enzyme activity seems to be the physical state of the substrate rather than the enzyme concentration or tissue of origin of the substrate. As previously noted (lo), there is higher activity with reconstituted fibrils than monomeric collagen. In this study, ‘the enzyme initially seemed to have greater activity with bone collagen than skin collagen. However, this was due to decreased fibril formation with skin collagen. When aspartic acid was added to accelerate fibril formation, the apparent enzyme activity with the skin substrate increased markedly. There was little effect with the bone substrate. If fihril formation was inhibited by compounds such as arginine, there was an apparent decrease in enzyme activity. From these findings, it seems likely that the concentration of lysyl oxidase is only one of the factors regulating the rate of collagen cross-linking in vim.

The experiments described in this paper indicate that lysyl oxidase has maximum activity on ordered aggregates that may be only a few collagen molecules across or reconstituted microfibrils rather than tightly packed native fibrils. Electron microscopy of the collagen substrate confirmed that fibrils with native banding patterns (19, 20) were present. However, most of the collagen was present as loosely packed, sideways associations a few molecules across. The addition of arginine inhibited formation of these aggregates and decreased appar- ent enzyme activity. Aspartic acid stimulated aggregation and increased enzyme activity. There was little activity with nonhydroxylated collagen, which does not form fibrils, or with tightly packed native fibrils present either in intact calvaria or in solution.

Additional information as to the nature of the enzyme-sub- strate interaction was provided by the experiment in which additional enzyme and substrate were added after enzymatic activity had decreased. With additional substrate, reaction velocity increased to 91% of the initial rate. Besides suggesting lack of product inhibition, this implies that most of the enzyme is bound to the exterior of collagen aggregates or growing fibrils rather than trapped in the interior of fibrils. It is unlikely that the enzyme is free in solution rather than bound to collagen since the reaction velocity also increased when additional enzyme was added to the solution. Furthermore, very little enzyme activity is present in the supernatant 1 h after the start of an incubation if the collagen fibrils are removed by centrifugation. In vivo, bound lysyl oxidase may stabilize the orientation of nascent collagen molecules to the ordered

molecular arrangement of existing fibrils (19) by rapid enzymatic aldehyde formation and cross-linking.

These considerations suggest that lysyl oxidase may act on a molecular aggregate that is an intermediate in fibril biosynthe- sis. Current theories suggest that such intermediates in vivo may be either four-stranded head-to-tail aggregates of mole- cules (20) or a five-stranded microfibril (21). The evidence presented here indicates that collagen molecules become insoluble and presumably incorporated into fibrils after a maximum of two aldehydes are formed per molecule. Possibly, the growing fibril is formed from four-stranded head-to-tail aggregates that are stabilized by cross-links at each end of the molecule. Further study of collagen cross-linking in vitro with purified lysyl oxidase should be useful in investigating this possibility and further defining the biosynthesis of the mature collagen fibril.

Acknowledgments-We wish to thank Dr. Karl Piez and Dr. John Daniels for encouragement and helpful suggestions dur- ing preparation of this manuscript. We wish to express our appreciation to Mrs. Pat Miller for typing this manuscript and to Ms. Jan Parker for taking the electron micrographs.

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R C Siegel and J C FuCollagen cross-linking. Purification and substrate specificity of lysyl oxidase.

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