[35] Lysyl oxidase: Preparation and role in elastin biosynthesis

[35]

LYSYL OXIDASE IN ELASTIN BIOSYNTHESIS

637

more mild treatments involving collagenase and denaturants, 2r may be removing what might be considered as partially insoluble elastin. Thus newly incorporated tropoelastin molecules, not yet fully cross-linked, may be released by such treatments. Therefore one must fully describe the system being studied and define quite clearly the insoluble elastin obtained under specified conditions. A t best, all definitions o f insoluble elastin are strictly operational.

An interesting approach in this regard, which should be useful in the cell culture systems as well, is that described by Sykes and Hawber. 2s They studied the formation of elastin biosynthesis using an immunoprecipitant procedure. After obtaining a sheep anti-chick elastin serum they examined the incorporation of tritiated valine and/or proline into both the soluble and the insoluble elastin produced by organ cultures of chick embryo aortas. After appropriate incubation times of up to 3 hr they extracted the aortas with 0.5 M acetic acid or neutral salt solutions with and without protease inhibitors. Significant amounts of immunoprecipitated radioactivity were detected in the extracts, suggesting partial solubilization of previously insolubilized elastin. It would be of interest to evaluate the cross-link content in these solubilized materials. Such observations tend to reinforce the suggestion that well defined operational procedures must be employed in all experimental approaches. As one becomes more sophisticated in the definition of insoluble elastin, the experimental protocols and interpretation of data from organ and cell cultures will have to be reevaluated. Acknowledgments The authorswishto thank Diane Dunnfor the pulmonaryartery smoothmusclecell data; Dr. Paul Toselli, who performedthe electron microscopy;and Dr. Lily L. Salcedofor her many contributionsin the preparation of this manuscript. 2r L. B. Sandberg,Int. Rev. Connect. Tissue Res. 7, 159 (1976). 2s B. Sykes and S. Hawber,Adv. Exp. Med. Biol. 79, 453 (1977).

[35] L y s y l O x i d a s e : P r e p a r a t i o n a n d R o l e in Elastin Biosynthesis By HERBERT M. KAGAN and KATHLEEN A. SULLIVAN

Lysyl oxidase is the enzyme that initiates the formation of covalent cross-linkages in elastin and collagen by oxidatively deaminating certain endopeptidyl lysine residues in these proteins to peptidyl a-aminoadipic-

METHODS IN ENZYMOLOGY, VOL. 82

Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181982-5

638

ELASTIN

NH2 I CH2 I (CHz)3 - H N-C H - C O PEPTIDYL LYSINE

+

02 + H20

~

[35]

CliO I (CH~)3

+

NH 3

+ H202

-HN-C H --COPEPTIDYL AMINOADIPIC SEMIALDEHYDE

FIG. 1. Reaction catalyzed by lysyl oxidase.

6-semialdehyde. Once generated, these peptidyl aldehydes can undergo a series of spontaneous condensation reactions with e-amino groups or other aldehyde residues to yield the inter- and intramolecular crosslinkages in these connective tissue proteins. The chemistry of the reaction catalyzed by lysyl oxidase is presented in Fig. 1. The chemistry of the cross-linkages and proposed biosynthetic routes for their formation are reviewed in this volume. 1 A review of the properties and biological role of lysyl oxidase has appeared. 2 Clearly, lysyl oxidase plays an essential role in elastin biosynthesis by converting soluble elastin precursors to the insoluble cross-linked elastic fibers, which are essential to the normal function of the connective tissue matrix. Lysyl oxidase has been demonstrated in several connective tissues that vary in their relative contents of elastin and collagen. Further, substrates utilized for its assay have included various forms of elastin, collagen, mixtures of both, and chemically undefined protein preparations which presumably contain forms of collagen and/or elastin. Studies of this enzyme are rendered still more complex by the finding in each of several connective tissues of multiple, catalytically functional enzyme forms. In all cases, however, enzyme activity oxidizing peptidyl lysine in elastin or collagen substrates should be 50% inhibited by less than 10 /zM /3-aminopropionitrile (BAPN), an irreversible naturally occurring inhibitor of this enzyme, 3-5 to confirm such amine oxidase activity as deriving from lysyl oxidase. The present review describes purification and assay methods utilized in our laboratory for the study of aortic lysyl oxidase and its activity toward elastin substrates. 1 M. A. Paz, D. A. Keith, and P. M. Gallop, this volume [31]. R. C. Siegel, Int. Rev. Connect. Tissue Res. 8, 73 (1979). 3 S. R. Pinnell and G. R. Martin, Proc. Natl. Acad. Sci. U.S.A. 61, 708 (1968). 4 A. S. Narayanan, R. C. Siegel, and G. R. Martin, Biochem. Biophys. Res. C o m m u n . 46, 745 (1972). 5 p. C. Trackman and H. M. Kagan, J. Biol. Chem. 254, 7831 (1979).

[35]

LYSYL OXIDASE IN ELASTIN BIOSYNTHESIS

639

Assay Methods Tritium Release Assay Principle. The original report of Pinnell and Martin z identifying lysyl oxidase activity in an extract of chick cartilage also first defined the tritium release assay, which remains a principal method of assay for lysyl oxidase, with some modification. Chick embryo aortas are incubated in organ culture with [6-3H]lysine in the presence of BAPN. The salineinsoluble, elastin-rich fraction is isolated from the pulsed aortas after incubation in organ culture. Oxidative deamination of the E-carbon of radioactive peptidyl lysine in this insoluble substrate releases a tritium ion that forms tritiated water by exchange during the assay. Tritiated water is distilled in vacuo from assay mixtures and quantified by liquid scintillation spectrometry. Substrates can also be prepared by pulsing aortic tissue with [4,53H]lysine, which is available in higher specific activity than is [63H]lysine. Release of tritium from C-5 accompanies keto-enol tautomerism of the enzyme-produced aldehyde. 6 Reagents e-[4,5-ZH]Lysine, 60-80 Ci-mmol BAPN fumarate (Aldrich Chemical Company) Sodium ascorbate Sodium bicarbonate Penicillin (10,000 units/ml)-streptomycin (10,000/xg/ml) e-Valine Chick embryos, 16 days old Dulbecco's modified Eagle medium (dry concentrate) without lysine, containing glucose (4.5 mg/liter) and L-glutamine NaC1, 0.15 M Sodium borate, 0.1 M, pH 8.0, containing NaCI, 0.15 M Purified bacterial collagenase (Biofactures; 2500 units/ml) Tris, 0.05 M, pH 7.4, containing CaCI2, 1 mM, and BAPN, 0.1 mM Pulsing of Tissue. NaHCOz (3.7 g) and L-valine (97 mg) are added to a liter equivalent of medium concentrate dissolved in 500 ml of distilled, deionized water. The pH is adjusted to 7.35, the volume brought to 1 liter with water, and the solution is sterilized by filtration through a 0.2 tzm filter assembly (Millipore). The medium is warmed to 37° and supplemented with sterile precautions with BAPN (50/zg/ml), sodium ascorbate (50/zg/ml), and with the antibiotic solution (10 ml/liter). The supplemented 6 R. C. Siegel, S. R. Pinnell, and G. R. Martin,

Biochemistry 9, 4486 (1970).

640

ELASTIN

[35]

medium is dispensed in 15-ml aliquots into sterile, plastic petri dishes (100 × 15 mm) to serve as a preincubation medium into which freshly excised aortas are placed (60 aortas/dish). Chick embryos, 16 days postfertilization, are r e m o v e d from the eggs and approximately 0.5 to 1 cm lengths o f the ascending thoracic aortas are excised. In practice, the aortic segments are r e m o v e d along with corresponding lengths o f the pulmonary artery and pulmonary vein. The vascular tissue is blotted free o f excess blood and placed into the preincubation medium for 30 rain at 37° to deplete the issue o f endogenous lysine. The aortas are then transferred to sterile 125-ml E d e n m e y e r flasks containing fresh 25-ml aliquots o f medium further supplemented with [4,5-3H]lysine (20/zCi per milliliter of medium). Each flask contains 60 aortas in 25 ml of radioactive medium. The flasks are incubated with gentle shaking at 37 ° for 22 hr in a 95% 0 2 - 5 % COa atmosphere. The aortas are then r e m o v e d and rinsed several times in distilled water, lyophilized, and stored at - 20°. It should be noted that the decanted medium contains tritium-labeled soluble proteins that may be prepared for use as a soluble substrate for lysyl oxidase, as described by Harris et al. 7,8 Substrate may also be prepared using aortic tissue o f 1-day-old hatchling chicks, thus obtaining more tissue from each animal. The procedure is the same as outlined above, with the exceptions that the aortas are cut with a razor blade to 1-mm 3 pieces and the tissue is incubated at the equivalent o f 20 aortas/50 ml o f radioactive medium. Extraction o f Pulsed Tissue. Pulsed, lyophilized aortas are extracted in cold 0.15 M NaCl (5 aortas/ml) in a conical, ground-glass homogenizer until an even suspension is obtained. The homogenate is sedimented at 10,000g for 15 min, the supernatant is removed, and the pellet is extracted and sedimented again in this manner. The resulting pellet is homogenized in 1 N HC1 to inactivate endogenous lysyl oxidase a and centrifuged as above. Two additional extractions in 0.1 M sodium borate, 0.15 M NaCl, p H 8.0, neutralize the remaining acid. The pellet is finally suspended in this b o r a t e - N a C l buffer at 5 aortas per milliliter, and the radioactivity in an aliquot of the evenly stirred suspension is quantified by liquid scintillation spectrometry. Typically, one aorta equivalent contains 6 × 105 cpm of tritium, counted at 30% efficiency. The insoluble substrate has been found to contain approximately 88% elastin and 12% collagen? ° To remove the collagen contaminant, the inTE. D. Harris, W. A. Gonnerman, J. E. Savage, and B. L. O'Dell, Biochim. Biophys. Acta 341, 332 (1974). 8 E. D. Harris and M. C. Garcia-de-Quevedo,Arch. Biochem. Biophys. 190, 227 (1978). 9 H. M. Kagan, N. A. Hewitt, L. L. Saleedo, and C. Franzblau, Biochim. Biophys. Acta 365, 223 (1974). lo A. S. Narayanan, R. C. Page, and G. R. Martin, Biochim. Biophys. Acta 351, 126 (1974).

[35]

LYSYL OXIDASE IN ELASTIN BIOSYNTHESIS

641

soluble substrate pellet equivalent to 20 mg of lyophilized aortas is incubated with 600 units of bacterial collagenase in 50 mM Tris, 1 mM CaCl2, 0.1 mM BAPN, pH 7.4, for 3 hr at 37°. The suspension is centrifuged; the isolated pellet is extracted three times with 0.15 M NaCl and finally suspended in borate-NaC1 buffer, and the radioactivity of the pellet is quantified as above. Assay Procedure. Aliquots of the evenly suspended substrate containing 125,000 cpm are transferred to 13 × 100 mm glass tubes on ice containing borate-NaC1 buffer. Lysyl oxidase is added to the assays in 0.1-ml aliquots or less to bring the final volume of each assay to 0.75 ml. Control assays are incubated without added enzyme or with enzyme and BAPN (50/zg/ml). The assay tubes are incubated at 37° with shaking for 2 hr. The reaction is terminated by chilling the tubes to 0° on ice. Tritiated water is distilled from the reaction mixtures under vacuum; the distillates are collected at - 5 0 °. Aliquots (0.5 ml) of the distillates are counted by liquid scintillation spectrometry. A microdistillation apparatus suitable for this procedure has been described. 1~ Assay Precautions. The assay is linear over a range of 600 cpm of tritium released in the 2-hr incubation. Lysyl oxidase is inhibited by urea carried over from stock solutions of enzyme, although inhibition plots can be used to correct for this effect.~z Collagen, elastin, or nonspecific proteins contaminating enzyme extracts interfere with the assay. Multiple dilutions of enzyme samples should be assayed to ensure linearity in each case.

Specific Activity. A convenient enzyme unit is defined as 1 cpm released in 2 hr per milliliter of assay mixture under the assay conditions described. There is no uniformly accepted enzyme unit for lysyl oxidase owing to the different substrates and assay conditions employed by investigators thus far. Specific activity is expressed as units per milligram of enzyme protein, the latter determined by the Lowry procedure. 13 Chemical Identification of Enzyme Product. It is advisable to confirm the generation of aldehyde products of lysyl oxidase activity by direct chemical analysis of forms of substrates not previously analyzed. The radioactive aldehyde produced from tritium-labeled or 14C-labeled peptidyllysine by the action of lysyl oxidase can be reduced with sodium borohydride to peptidyl-e-hydroxynorleucine, the alkali-stable alcohol derivative. The substrate is then base hydrolyzed, and the E-hydroxynorleucine product is identified by split-stream amino acid analI I R. Misiorowski, J. B. Ulreich, and M. Chvapil, Anal. Biochem. 71, 186 (1976). ~2 H. M. Kagan, K. A. Sullivan, T. A. Olsson, and A. L. Cronlund, Biochem. J: 177, 203 (1979). ~30. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).

642

ELASTIN

[35]

ysis.9'14 Alternatively, radioactive aldehyde deriving from 14C-labeled peptidyllysine can be oxidized to peptidylaminoadipic acid by treatment o f the substrate with performic acid after its incubation with lysyl oxidase. The 14C-labeled aminoadipic acid is released by acid hydrolysis and identified by split-stream amino acid analysis. 3 Enzyme-Coupled Spectrophotometric Assays Principle. Bovine aortic lysyl oxidase has been shown to oxidize a variety of nonpeptidyl amines producing the corresponding aldehydes and H202. 5 The oxidation o f n-butylamine, for example, can be followed fluorometrically by coupling this assay to the H2Os-dependent oxidation o f homovanillic acid by horseradish peroxidase. The accumulation o f the fluorescent product of homovanillate oxidation is followed at 37° at an excitation wavelength o f 320 nm and an emission wavelength o f 420 nm. Alternatively, the production o f n - b u t y r a l d e h y d e by lysyl oxidase can be coupled to the reduction of N A D ÷ by aldehyde dehydrogenase, measuring the change in absorption at 340 nm. 5 Although the oxidation o f nonpeptidyl amines is limited to 300 turnovers of lysyl oxidase, since the enzyme becomes inactivated in the process, initial rates o f oxidation are proportional to e n z y m e concentration. ~ This assay is reliable for partially or highly purified lysyl oxidase of bovine aorta, although its application to e n z y m e o f other tissues or crude extracts remains to be established.

Purification L y s y l oxidase is mostly insoluble in neutral salt buffers in a variety of connective tissues, although e n z y m e activity is present in the growth medium of fibroblast cell cultures as a soluble protein. 15 The e n z y m e can be solubilized from connective tissue sources by buffers containing 4 - 6 M urea. TM Most purification schemes that have evolved begin with the ureasolubilized fraction and generally share other purification steps as well. We shall describe the method used in our laboratory for the purification o f lysyl oxidase from bovine aorta.12 Reagents

Thoracic aortas o f 2- to 6-week-old calves, obtained at slaughter Potassium phosphate, 0.016 M, p H 7.7 14R. C. Siegel, J. Biol. Chem. 251, 5786 (1976). 15D. L. Layman, A. S. Narayanan, and G. R. Martin, Arch. Biochem. Biophys. 149, 97 (1972). 16A. S. Narayanan, R. C. Siegel, and G. R. Martin, Arch. Biochem. Biophys. 162, 231 (1974).

[35]

LYSYL OXIDASE IN ELASTIN BIOSYNTHESIS

643

Potassium phosphate, 0.016 M, pH 7.7, containing 1 M NaCI or containing urea at 1 M, 2 M, 4 M, or 6 M Urea, 8 M, freshly deionized by passage through a mixed-bed ionexchange resin (Barnstead, Ultrapure). This stock solution is used to prepare each of the buffered urea solutions. (Diethylaminoethyl) cellulose (DE-52, Whatman) Sephacryl S-200 (Pharmacia) Sepharose CL-4B (Pharmacia) Citrate-soluble calf skin collagen Cyanogen bromide Extraction. The entire purification procedure is carried out at 4° . Each liter of extracting buffer receives 0.5 ml of a 2 M solution of phenylmethanesulfonyl fluoride (PMSF; Sigma) in dimethyl sulfoxide just prior to each extraction. The calf aortas are cleaned of adhering muscle and fat and are finely ground by passage through a chilled meat grinder. The ground tissue is homogenized at high speed in a Waring blender for 90 sec in 0.016 M potassium phosphate, pH 7.7, containing 0.15 M NaC1, and 1 mM PMSF at a ratio of 2.5 ml of buffer per gram of ground tissue. This ratio is utilized for all extractions. The homogenate is centrifuged at 11,000 g for 20 min, the supernatant is decanted, and the pellet is extracted once more with this buffer. The salt extracts contain negligible lysyl oxidase activity and may be discarded. The pellet is homogenized in 0.016M potassium phosphate, pH 7.7, containing PMSF. The supernatant is discarded after centrifugation, and the pellet is homogenized in 0.016 M potassium phosphate, pH 7.7, containing 1 M urea and 1 mM PMSF, and stirred slowly at 4° for 1 hr. The pellet is isolated by centrifugation and homogenized in 4M urea in phosphate buffer and PMSF. The homogenate is stirred slowly for 18 hr and centrifuged; the supernatant is decanted and saved. The 4 M urea extraction is repeated twice more, and the three 4 M urea supernatants are pooled and saved. Affinity Chromatography. The collagen affinity column is prepared by coupling citrate-soluble calf skin collagen, prepared as described, 17 to Sepharose CL-4B, activated with CNBr. TM Lyophilized calf skin collagen is denatured and dissolved in water at 60° just prior to coupling, utilizing a ratio of 1 g of collagen per 200 g of Sepharose. One 200-g affinity column retains enzyme extracted from 160 g of aortic tissue. The pooled 4 M urea extracts are diluted with an equal volume of 0.016 M potassium phosphate, pH 7.7, and passed into the affinity column. Unbound material is removed by washing the column with 2 M urea, 0.016 M potassium phosphate, until the Azs0 falls to zero. The column is succeslr p. M. Gallop and S. Seifter, this series, Vol. 6, p. 635. is p. Cuatrecasas, J. Biol. Chem. 245, 3059 (1970).

644

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i

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FIG. 2. Chromatography o f 6 M urea effiuent o f affinity column on DEAE-cellulose. Extraction was performed in the presence o f phenylmethanesulfonyl fluoride (0.001 M). . . . . . , E n z y m e activity; , A2ao; 0 , salt gradient.

sively eluted with 600-ml quantities per 200 g of collagen-Sepharose of 0.016 M potassium phosphate; 1 M NaC1, 0.016 M potassium phosphate; 0.016 potassium phosphate; and, finally, 6 M urea, 0.016 M potassium phosphate, each adjusted to pH 7.7. The 1 M NaCI wash elutes considerable bound protein as measured at 280 nm but does not remove enzyme. Lysyl oxidase elutes with the 6 M urea buffer. DE-52 Chromatography. The conductivity of the 6 M urea effluent of the affinity column is measured to ensure that its ionic strength is not greater than that of 0.016 M potassium phosphate, 6 M urea, pH 7.7. A maximum volume of 1 liter of this effluent is then loaded onto a 2.5 × 84 cm column of DE-52 equilibrated in this buffer at 4°. The column is eluted with a linear gradient of NaCI (0 to 0.4M) in 6M urea, 0.016M potassium phosphate, pH 7.7. The total gradient volume is 2680 ml. Fractions (8.5 ml) are collected and analyzed for A2s0, enzyme activity, and conductivity. A typical elution pattern is shown in Fig. 2. Although the same multiplicity of peaks of enzyme activity results regardless of the presence or the absence of PMSF in the extraction buffers, the addition of PMSF as described yields purified enzyme species with somewhat greater stability to storage, suggestive of the effects of serine protease activity.

/

[35]

LYSYL OXIDASE IN ELASTIN BIOSYNTHESIS

645

Gel Exclusion Chromatography. The pools of the four species resolved in DE-52 (Fig. 2) are individually concentrated to approximately 15-ml volumes by ultrafiltration (Amicon type YM-10 filters at 30 psi). Each enzyme concentrate is passed through a 120 × 3 cm column of Sephacryl S-200 equilibrated and eluted with 6 M urea, 0.016 M potassium phosphate, pH 7.7. Each enzyme species elutes at or just prior to the elution position of carbonic anhydrase (Mr = 29,000) (Fig. 3). Typical purification results are summarized in Table I. Purity. Polyacrylamide gel electrophoresis in SDS resolves each purified enzyme species into a major band at a molecular weight of 32,000 --- 100012 but reveals the presence of a minor band at 24,000 molecular weight constituting <5 to 15% of the stainable protein on the gel. However, only the major band of each preparation of each enzyme species becomes labeled when the preparations are incubated with [14C]BAPN at 37° and then analyzed by SDS gel electrophoresis. Further, the peptide maps of the minor band and of the major band isolated from the gels are very similar in each case. 19We conclude that the band at 32,000 -+ I000 is the protomer of catalytically functional lysyl oxidase. The smaller band appears to be a degradation product of the enzyme. Properties

Stability. The enzyme in the final purification step as described above elutes from the Sephacryl column in 6 M urea, 0.016 M potassium phosphate, pH 7.7. Storage of the enzyme at 4° as the pooled fractions pooled from the Sephacryl column in the 6 M urea buffer results in 85-90% recovery of activity for as long as 6 weeks. Dialysis of enzyme into ureafree buffers or into water decreases activity in shorter time periods. As much as 90% of the activity is lost upon lyophilization of enzyme previously dialyzed into water. Enzyme activity is not restored by addition of copper salts, pyridoxal phosphate, or flavins to assays. Enzyme can be dialyzed out of urea into 0.1 M sodium borate, 0.15 M NaC1, pH 8.0, assay buffer and stored at 4° with nearly full retention of activity for 1 week. Freezing of such solutions, however, causes marked losses of activity. Assay Optima. The enzyme has a fairly broad pH optimum between 7.8 and 8.3, and is routinely assayed at pH 8.0. The plot of the temperature dependency of the assay using the aortic elastin substrate exhibits a sharp maximum at 50-520. 9 The enzyme is irreversibly inactivated at 85° and is, therefore, quite thermostable. The optimal assay temperature may reflect temperature-induced changes in the conformational properties of the substrate. Assays are usually conducted at 37°, however, because of the ~9K. A. Sullivanand H. M. Kagan,in preparation, 1981.

646

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FIG. 3. C h r o m a t o g r a p h y o f DEAE-cellulose peaks I - I V on Sephacryl S-200 in 6 M u r e a - 0 . 0 1 6 M p o t a s s i u m phosphate, p H 7.7. Abbreviations used to identify elution positions o f s t a n d a r d s are as follows: v, void; a, bovine s e r u m albumin; o, ovalbumin; c, carbonic a n h y d r a s e ; cy, c y t o c h r o m e c. R o m a n n u m e r a l s refer to peaks o f activity resolved on DEAE-cellulose, as labeled in Fig. 2. - . . . . , E n z y m e activity; , A2so.

[35]

LYSYL OXIDASE IN ELASTIN BIOSYNTHESIS

647

TABLE I PURIFICATION OF FOUR SPECIES OF LYSYL OXIDASE FROM BOVINE AORTA a'b

Purification step Urea extract, 4 M Affinity chromatography DEAE-cellulose Peak I Peak II Peak III Peak IV (Sum of four peaks) Sephacryl S-200 Peak I Peak II Peak III Peak IV (Sum of four peaks)

Activity (total units x 10 -b) 11.7 8.75 3.02 1.59 1.03 0.41 (6.05) 0.49 0.30 0.48 0.26 (1.54)

Total protein (mg)

Specific activity (× 10 -5)

Yield (%)

0.075 0.547

100 74.7

1 7.3

0.994 0.443 0.627 0.557

25.8 13.5 8.8 3.5 (51.6)

13.2 5.9 8.3 7.4

1554 160 30.4 35.8 16.4 7.4 (90.0) 0.54 0.39 0.67 0.37 (1.97)

11.0 6.9 12.2 6.0

4.2 2.3 4.1 2.2 (12.8)

Purification (fold)

146 92 162 80

Reproduced, with permission from the publisher, from Kagan et al. 12 b Purification began with 200 g of bovine aorta.

increased nonenzymic release of tritium from the substrate, which occurs at elevated temperatures. The aortic elastin substrate is not markedly affected by ionic strength above that of the borate-NaC1 buffer. However, significant inhibition results at unusually high (e.g., 2 M NaC1) ionic strength. Amino Acid Composition. Amino acid analyses have been reported for the four species of bovine aortic enzyme, 4 and one species of the chick cartilage enzyme 2o (Table II) with similarities but not identities in composition noted. The aortic enzymes are each apparently acidic species, although the amide contents have not been established. Preliminary isoelectric focusing experiments on the bovine aortic enzyme species reveal pI values between 4.5 and 5. Molecular Weight. The molecular weights of the bovine aortic lysyl oxidase species, determined by the SDS-gel electrophoresis technique of Laemmli, 21 each are 32,000 _+ 1000,12 consistent with their elution just 2o F. L. H. Stassen, Biochirn. Biophys. Acta 438, 49 (1976). 21 U. K. Laemmli, Nature (London) 72, 248 (1976).

648

ELASTIN

[35]

TABLE II AMINO ACID ANALYSES OF PURIFIED LYSYL OXlDASE SPECIES a'b

Content (residues/1000 residues)

Residue

Peak I

Peak II

Peak HI

Peak IV

Chick-embryo cartilage enzyme c

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Cysteine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine

123 57 104 113 60 120 71 39 27 15 30 64 25 27 31 39 56

125 55 86 136 51 87 82 42 24 16 27 86 31 30 36 25 61

122 56 104 136 50 108 83 35 18 15 31 78 21 26 36 27 56

94 48 122 124 51 177 85 32 14 17 29 65 18 25 29 23 49

136 53 82 106 58 97 66 39 30 15 40 67 65 27 31 29 59

Reproduced, with permission from the publisher, from Kagan et al. 1~ b Tryptophan was not determined. Cysteine was analyzed as cysteic acid in separate samples oxidized with performic acid. c Data were taken from Stassen. 2°

prior to a 29,000 molecular weight marker from Sephacryl S-200 in 6 M urea. In the absence of denaturants, however, lysyl oxidase polymerizes into a series of multimeric states, ranging up to 1,000,000 Mr. Iz Such enzyme polymers may exist under assay conditions, although the functional molecular weight of the enzyme has not been established. Cofactors. A number of investigations support the conclusion that lysyl oxidase is a copper metailoprotein. Nutritional deprivation of copper lowers cross-linkage content and biosynthesis of elastin and collagen, z2'2~ Enzyme activity is markedly reduced in copper-deficient chick aorta but can be restored by incubating the copper-deficient aortas with Cu 2+ in organ culture. 24 However, the stoichiometric relationship between the metal atom and enzyme protein is uncertain owing to the states of purity and/or 22 E. J. Miller, G. R. Martin, C. E. Mecca, and K. A. Piez, J. Biol. Chem. 240, 3623 (1965). 2a E. D. Harris and B. L. O'Dell, Adv. Exp. Biol. Med. 48, 267 (1974). 24 E. D. Harris, Proc. Natl. Acad. Sci. U.S.A. 73, 371 (1976).

[3S]

LYSYL OXIDASE IN ELASTIN BIOSYNTHESIS

649

to uncertainties about the subunit molecular weight of preparations in which Cu 2÷ has been directly measured. The bovine aortic enzymes are each inhibited by a,a'-dipyridyl and other copper chelators, 4 but the content of enzyme-bound copper has not been measured with this enzyme. There is indirect evidence for the presence o f a second organic cofactor in lysyl oxidase. Nutritional studies 25'26 and binding studies with tritiated pyridoxine 2~ suggest a role for pyridoxal phosphate. The purified bovine aortic enzymes as well as enzymes o f other sources are fully inhibitable by carbonyl reagents, such as isoniazid, semicarbizide, or NaCN, 7'1z consistent with such a prosthetic group. H o w e v e r , fluorescence and absorption spectrophotometric analysis o f the purified bovine aortic enzyme species pursued in the author's laboratory have not revealed the presence o f such an identifiable organic cofactor, either as an enzyme complex or as material solubilized from purified enzyme. Substrate Specificity. Insoluble elastin 9 and soluble tropoelastin 27 as well as fibrillar forms of collagen 28 are oxidized by lysyl oxidase. Although activity is expressed toward tropoelastin in solution, a coacervated aggregate of this protein is much more readily oxidized and becomes crosslinked to a preexistent insoluble elastin matrix 2r upon the addition of purified cartilage lysyl oxidase. Bovine aortic lysyl oxidase oxidizes elastin and collagen substrates as well as synthetic polypeptides modeled after valine- and proline-rich repeating sequences found in elastin. 29 The repeat polypentapeptide HCO-(VaI-Pro-GIy-X-Gly)~-Val-OMe, where X = Val or Lys at a 4 : 1 ratio and where n -> 40, is much more readily oxidized and cross-linked by the enzyme if it is coacervated prior to and during exposure to the enzyme. 29 Thus, intermolecular alignment, intramolecular conformational changes, and the increased hydrophobicity resulting from coacervation of natural or synthetic polypeptide substrates all may contribute to the susceptibility of potential substrates to oxidation by lysyl oxidase. This possibility seems to be supported upon examination of the sequences surrounding lysine residues in elastin, collagen, and synthetic polypeptide substrates known to be oxidized by the enzyme (Table 111).29-3.' The variety o f permissible sequences strongly suggests that facz5 B. C. Starcher, Proc. Soc. Exp. Biol. Med. 132, 379 (1969). 24 j. C. Murray and C. I. Levene, Biochem. J. 167, 463 (1978). 27 A. S. Narayanan, R. C. Page, F. Kuzan, and C. G. Cooper, Biochem. J. 173, 857 (1978). 28 R. C. Siegel, Proc. Natl. Acad. Sci. U.S.A. 71, 4826 (1974). 29 H. M. Kagan, L. Tseng, P. C. Trackman, K. Okamoto, R. S. Rapaka, and D. W. Urry, J. Biol. Chem. 255, 3656 (1980). ao j. A. Foster, R. Shapiro, P. Voynow, G. Crombie, B. Faris, and C. Franzblau, Biochemistry 14, 5343 (1975). 31 p. p. Fietzeck and K. Kuhn, Int. Rev. Connect. Tissue Res. 7, 1 (1976). az U. Becker, H. Furthmayr, and R. Timpl, Hoppe-Seyler's Z. Physiol. Chem. 356, 21 (1975).

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ELASTIN

[36]

TABLE III SPECIFICITY OF LYSYL OXIDASE FOR PEPTIDYLLYSINEa Peptide

Source

Referenc

-Ala-Ala-Lys-Ala-Ala-Ala-Lys-Ala-Gly-Tyr-Asp-Glu-Lys-Ser-Aia-Giy-Gln-Glu-Glx-Ly....2s-Ala-His-Asp-GlyHCO-(X--Pro-GIy-Gly)n-Val-OMe b HCO-(VaI-Pro-GIy-X-Gly)n-Val-OMeb HCO-VaI(AIa-Pro-GIy-X-GIy-Val)n-OMeb

Elastin and tropoelastin NH2-terminal, collagen al(I) chain COOH-terminal, collagen ~1(I) chain Synthetic repeat polytetrapeptide Synthetic repeat polypentapeptide Synthetic repeat polyhexapeptide

30 31 32 29 29 29

Lys or X (as iysine) residues susceptible to oxidation by lysyl oxidase. b~= ValorLysata4:lratio;n -->40.

tors in addition to sequence are important in the expression of enzyme activity. It has been suggested, however, that tyrosine or phenylalanine peptide linked through a-amino groups to endopeptidyl lysine in elastin prevents oxidation of lysine by lysyl oxidase, a3,a4 Purified bovine aortic lysyl oxidase can oxidize nonpeptidyl amines in addition to polypeptide substrates, and, as noted, such compounds can serve as substrates in continuously monitored spectrophotometric assays. ~ Such substrates include free lysine and its a-N-acetylated and/or esterified derivatives; monoamines, including propyl-, butyl-, and octylamine; and 3-, 4-, 5-, and 6-carbon primary diamines. The biological significance of the oxidation of such compounds by lysyl oxidase has not been established. 33 j. A. Foster, L. Rubin, H. M. Kagan, C. Franzblau, E. Bruenger, and L. Sandberg, J. Biol. Chem. 249, 6191 (1974). 34 K. M. Baig, M. Vlaovic, and R. A. Anwar, Biochem. J. 185, 611 (1980).

[36] Isolation of Soluble Elastin from Copper-Deficient Chick Aorta By ROBERTB. RUCKER Lysyl oxidase activity and the rate of cross-linking must be reduced effectively to cause an increase in the net pool of soluble elastins prior to isolation. This may be achieved by rendering animals nutritionally copper-deficient or lathrytic. 1,2 The choice of experimental animals is also R. B. Rucker and D. Tinker, Int. Rev. Exp. Pathol. 17, 1 (1977). 2 L. B. Sandberg, Int. Rev. Connect. Tissue Res. 7, 159 (1976).

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