On the accumulation of d -aspartate in elastin and other proteins of the ageing aorta

4 therosclerosis, 91 (1992) 20 l-208 6 1992 Elsevier Scientific Publishers Ireland, Ltd. All rights reserved. 0021-9150/92/$05.00 Printed and Published in Ireland

201

ATHERO 04932

On the accumulation

of D-aspartate in elastin and other proteins of the ageing aorta

Janet T. Powell, Nicholas Vine and Margot Crossman Department

of Biochemistry.

Charing Cross and Westminster

Medical School, Fulham Palace Road, London W6 8RF (UK)

(Received 6 February, 1992) (Revised, received 10 August, 1992) (Accepted 11 August, 1992)

Summary Ageing and degenerative changes of the human aorta are associated with medial thinning and a reduced dry weight content of elastin. The metabolic stability of cross-linked elastin was investigated by measuring the accumulation of D-aspartate with ageing in insoluble elastin isolated from human aorta. D-Aspartate accumulation in elastin was compared with D-aspartate accumulation in aortic collagen and an elastin bound glycoprotein fraction. The D-aspartate content of elastin, purified from infrarenal aorta; increased linearly with age from 3% of the total aspartate in youth to 13% in the mid 80s. In contrast the D-aspartate content of aortic collagen remained invariant (3-5% of the total aspartate) from youth to old age. The apparent first order rate constant for the racemization of L-aspartate in elastin was 1.14 x 10T3. The D-aspartate content of the elastin bound glycoproteins increased by only a small amount, from 3% in the mid 30s to 6% in the mid 80s. These results argue for the metabolic stability of aortic elastin as compared with the tibrillar collagens of the human aorta. Both the rate of racemization and the specific accumulation of D-aspartate in elastin, but not collagen, indicates that mature cross-linked elastin is not synthesized in the adult aorta.

Key words: D-aspartate; Elastin; Collagen; Aorta; Ageing

Introduction The ageing of the human aorta is accompanied by increasing calcification and reduced elasticity and (in early pathological studies) medial thinning Correspondence to: Janet T. Powell, Department of Biochemistry, Charing Cross and Westminster Medical School, Fulham Palace Road, London W6 8RF, UK. Tel.: 081-8467044; Fax: 08 l-846-7099.

[l-3]. The great elastic recoil of the young aorta is lost in old age and this is accompanied by biochemical changes including the accumulation of glycoproteins and a decreased amount of elastin and elastic connective tissue [4,5]. Elastin appears to be a very stable protein, traditionally isolated after autoclaving tissues and/or exposing them to 0.1 M NaOH at 98°C [6]. There has been no convincing evidence to demonstrate that elastin is synthesized in the adult aorta. Here we have

202

investigated the accumulation of D-Uptrtak! in insoluble elastin isolated from aortas of adults, aged 18-85 years. Whilst this work was in progress we have learnt that the elastic fibres of the lung have a marked longevity, the amount of Daspartate in lung elastin increasing linearly with age 171. Newly synthesized mammalian proteins contain exclusively L-amino acids. Racemization to Damino acids occurs very slowly, the rate depending principally on the temperature and the particular amino acid. The racemization of L-aspartate to Daspartate has been used to date long lived proteins including tooth dentine and lens crystallins [g-12]. Aspartic acid is the most useful amino acid for these purposes, the first order rate constant for racemization of metabolically stable proteins at body temperature ranging from 0.794 x 10e3/year (tooth dentine) to 1.50 x 10e3/year (myelinated white matter) [lo- 121. The percentage of Daspartate in such metabolically stable proteins increases by about 1% for each decade. Other studies have indicated that the accumulation of Daspartate may derive selectively from aspartate residues where the side chain carboxyl is ionized, from deamidated asparagine residues or particular amino acid sequences [8,13]. Since purified elastin is characterised by its amino acid composition, which shows only 5-9 aspartate residues/lOOO, to determine their rate of racemization presented a challenge [14]. The challenge was made more profound when inspection of the elastin gene sequence indicated the presence of only 3 aspartate residues amongst nearly 800 and no asparagine [ 151.

Fig. I. Aortic media. The media from the aorta of a 57-year-old man has been dissected free of intima and adventitia. The section has been stained to show the elastic tissue (Van Gieson), the final magnification is x 63.

Isolation of fibrillar collagens

Materials and Methods Aortas

These were collected at post-mortem and a piece approximately 3 x 3 cm from the level of the renal arteries was dissected free of adherent clot, endothelium and adventitia and and the adequate preparation of media checked by histology (Fig. 1). Aortic aneurysms were not included. The media was stored at -20°C over a period of up to 1 year prior to the preparation of collagen, elastin and other proteins, based on the procedure of Spina et al. [14].

The frozen aortic media was finely diced at 0°C and suspended in 0.9% saline (25 ml) and agitated with end-over-end mixing for 4-6 h at 4°C. The diced aortic media was extracted for 24 h at 4°C firstly in acetone, then in chloroform-methanol (2:l v/v) and finally in chloroform-methanol (3:l v/v). The tissue was blotted dry after each extraction. The tissue was then homogenized using a Polytron (5 x 30 s) in 0.4 M Tris/Cl- containing 0.2 M EDTA (20 ml/g tissue) and left to mix end over end for a further 24 h at 4°C. This was followed by four more successive 24-h extractions at 4°C in (i) 5 M guanidinium chloride containing 0.2 M

203

EDTA, 0.4 M Tris/Cl-, pH 7.4, (ii) 5 M guanidinium thiocyanate containing 0.2 M EDTA, 1 mM dithiothreitol, 0.4 M Tris/Cl, pH 7.4, (iii) as in (ii) but increasing the concentration of dithiothreitol to 5 mM and (iv) phosphate buffered saline. After each extraction the insoluble material was collected by centrifugation at 6000 x g for 20 min and the supernatants discarded. Finally the samples were washed well with water and dried in vacua over P205 at 4°C. The dried material was resuspended in a solution of pepsin (Sigma P 7012) in 0.4 M acetic acid at 1 mg/ml. The pepsin digestion was continued at 18°C for 72 h with end-over-end mixing. All further steps were performed at 4°C. The pepsin digest was centrifuged for 1 h at 16 000 x g and the supernatant dialysed against 0.02 M Na2HP04 overnight. Further purification of collagen was as previously described [ 161. Isolation of elastin

The insoluble material remaining after the pepsin digest was washed with ice cold water and dried in vacua over P205 at 4°C. The dried precipitate was weighed (lo-30 mg) and suspended in formic acid (5 ml/l0 mg). Cyanogen bromide (3 x the weight of precipitate) was added, the tubes flushed with N2, closed and incubated at 25°C for 48 h. Finally the temperature was raised to 37°C for 1 h prior to centrifugation at 4°C (6000 x g, 20 min). The precipitate was washed at 4°C firstly with 10 mM EDTA and then water until the pH of the wash reached 6. The precipitate was then dried in vacua at 4°C over P205. The precipitate was suspended in 1% solution of TPCK treated trypsin (Sigma T8264) in 0.1 M NH4HC03 at 18°C for 48 h. The insoluble material was washed twice with 5 M guanidinium chloride containing 25 mM Tris/Cl, pH 7.4 followed by 2 M NaCl containing 25 mM Tris/Cl and water, dried and weighed, giving a final yield of l-4 mg. The elastin content of aortic media was determined by the yield of elastin prepared from defatted, dried aortic media according to the method of Lansing [6]. Elastin bound glycoproteins

The material solubilized and separated from

elastin by trypsin treatment was chromatographed over a column of Biogel P-100 (1 x 20 cm) prepared and run in 0.25 M NaCl containing 25 mM Tris/Cl, pH 7.4 at 30 ml/h and the material eluting in the void volume was dialysed against 50 mM N&HCOs and freeze-dried. Amino acid analysis

Samples were hydrolysed in 0.5 ml 6 M HCI at 110°C under Nz. Norleucine was added as a reference standard. Aliquots (0.2 ml) were removed after 6 h and the remainder left for 24 h. Samples were analysed on an LKB Biochrom 415 1 alpha plus analyser, using the Na+ column. Detection of D- and L-aspartate

The 6-h amino acid hydrolysates were derivatized as previously described with acidic isopropanol/trifluoroacetic anhydride [ 171. The derivatives were extracted into dichloromethane (2 x 0.1 ml) and transferred to an acid washed vial. The sample was evaporated to dryness and stored at -70°C if not chromatographed immediately (rapid racemization of L-aspartate derivatives occurs in samples left in solvent). The gas liquid chromatography was performed as previously described on a chiral column, XE-60-S-valine-S-a-phenylethylamide (Chrompack) on a Varian 3300 gas chromatograph [ 181. Samples were dissolved in dichloromethane (10 ~1) and 0.5 ~1 (5%) injected onto the column. The column temperature was held at 100°C for 10 mm and then a temperature gradient of 0.2Wmin applied to bring the temperature to 110°C. Control samples of D/Laspartate mixtures demonstrated a clear separation of 1.O- 1.2 min. Each sample was run 3 times and the mean % of D-aspartate calculated with the aid of a Varian 4290 integrator. Racemization kinetics

Racemization is a reversible first-order reaction [8]. When the rate of racemization is small, D/L < 0.15, it can be considered as an irreversible first order reaction and the rate constant calculated from the equation: kit = In (1 + D/L) - In (1 + D/L), = 0.

204

Results

TABLE 1 RATIO OF ASPARTATE RELEASED AFTER 6 AND 24 h OF ACID HYDROLYSIS A single protein hydrolysate was established and samples withdrawn after 6 h and 24 h. Equivalent amounts of protein were calculated from the norleucine standard added before hydrolysis. The 25-year aorta did not yield sufficient elastin bound glycoprotein fraction for analysis. Age of aorta (years)

25 42 57 70 78

6 h aspartate/24 h aspartate Collagen

Elastin

Elastin bound glycoprotein

0.87 0.87 0.83 0.90 0.88

0.92 0.89 0.83 0.99 0.85

nd 0.76 0.80 0.72 0.74

The purification of proteins from the aortic media was adapted to minimize exposure to higher temperatures. Amino acid analyses of the collagen preparations showed that they contained about 38% glycine, 10% proline, 12% hydroxyproline and 4-5% aspartate. Assuming complete release of aspartate after 24 h hydrolysis, 80-900/o of the aspartic acid had been released from collagen after 6 h of hydrolysis (Table 1). After the cyanogen bromide extraction procedure at low temperatures, elastin preparations still contained some methionine and excessive amounts of polar amino acids; even in preparations from younger aortas, the amino acid compostion of the material obtained after cyanogen bromide digestion varied considerably (Table 2). The introduction of a trypsin

TABLE 2 THE AMINO ACID COMPOSITIONS OF PROTEIN FRACTIONS PURIFIED

FROM AORTAS OF DIFFERENT AGE

CNBr, protein after cyanogen bromide extraction; elastin, after further trypsin digestion of the CNBr protein; EBG, elastin bound glycoprotein fraction. There was insuflicient elastic tibril glycoprotein fraction for analysis from the 25-year aorta. All values were obtained after 24 h acid hydrolysis. The final column shows the mean composition of elastin purified, using collagenase and trypsin, from different age thoracic aortas, and is taken from Ref. 14. Residues/1000 residues Age (years) 25

Cysteic acid Asx Thr Ser Glx Pro HYP GUY Ala Val Met Ile JAI

Tyr Phe His Lys Arg Des/ides Aminosugars

57

85

CNBr

Elastin

CNBr

Elastin

EBG

CNBr

Elastin

EBG

15.2 64.2 25.0 51.8 88.2 99.3 5.0 348.7 107.4 27.6 7.2 17.4 27.8 8.1 15.8 10.9 21.1 48.5 5.9 +

0 4.1 12.0 8.3 14.5 128.2 3.0 291.8 237.4 153.0 0 22.6 58.0 23.1 20.8 0 3.5 6.8 12.9

18.9 60.3 24.3 45.5 82.6 97.4 4.9 365.5 113.1 23.9 5.5 13.8 24.6 4.3 13.2 12.2 22.4 52.7 7.3 +

0 4.4 11.9 8.2 16.0 127.0‘ 6.1 285.6 236.7 155.2 0 23.6 62.3 19.2 21.3 0 3.8 6.5 11.3

34.2 75.3 58.1 48.7 177.0 88.4 0.0 113.8 38.9 57.8 1.7 33.0 71.5 46.3 29.6 42.5 26.6 58.2 0 ++

22.6 61.3 20.5 33.7 87.5 93.1 2.8 332.9 108.8 37.8 4.5 17.4 22.0 12.0 15.8 14.1 26.6 47.1 5.8 +

0 4.6 13.2 9.8 15.6 128.8 7.0 284.5 229.3 159.2 0 22.9 60.8 19.0 22.9 0 4.0 7.3 10.6

35.1 87.8 62.3 45.1 135.9 92.1 0.0 99.0 43.7 43.3 1.1 43.6 80.0 38.2 30.5 47.1 49.8 63.7 0 ++

Ref. 14 aortic elastin

6.3 13.7 9.3 19.5 129.4 6.5 292. I 228.5 143.5 0 23.5 56.1 23.0 20.5 0 3.9 7.2 18.2

205

digestion produced white elastin preparations with amino acid compositions which were similar irrespective of the age of the aorta from which the elastin derived (Table 2) and also similar to those previously published for aortic elastin [14]. A 6-h acid hydrolysis released 90% of the aspartate from purified elastin (Table 1). The amino acid composition of the material solubilized after trypsin digestion is also given in Table 2. This material was rich in hydrophilic amino acids and amino sugars, a composition resembling microfibrillar glycoproteins [19]. We have called this material elastin bound glycoprotein and it was obtained from aortas of all ages above 25 years. A 6-h acid hydrolysis of the elastic fibril glycoprotein released 70-75% of the total aspartate released after longer (24 h) hydrolysis (Table 1). The separation of D- and L-aspartate by gas liquid chromatography is shown in Fig. 2. The Denantiomer elutes 1.0-1.2 min ahead of the Lenantiomer. This separation was maintained for

D

D-aspartate contents of 3- 15%. Day-to-day reproducibility was ensured by interbatch, running a previous sample (stored dry at -70°C) in each run. An estimate of procedural racemization was obtained by quantitating the amount of Daspartate after subjecting L-aspartate to 6 M HCl for 6 h at llO”C, 2.9% f 0.8%: after 24 h of acid hydrolysis the amount of D-aspartate had increased to 13.2% f 2.3%. The ratio of D-aspartate to total aspartate, D/(D + L), in each of the three proteins, collagen, elastin and elastin bound glycoprotein is shown in Fig. 3. Only in elastin is there a steady increase in the D-aspartate content with age, from 3.9% f 0.3% at the age of 18 to 12.9% f 0.5% at the age of 85 (Table 3). The apparent first order rate constant for the racemization of L-aspartic acid in aortic elastin is calculated as 1.14 x 10e3/year. The variation of the elastin content of infrarenal aortic media with age is shown in Fig. 4. There is a slow decline in the dry weight elastin content

L

Fig. 2. The separation of D- and L-aspartate. D-aspartate elutes after 17 min, 1.0-1.2 min ahead of L-aspartate. The resolution of a standard 1:l mixture is shown on the left and the resolution of a sample with only a small percentage component of D-aspartate is shown on the right.

206 In (1+DL)

e!astin (%dty wt)

0.18

40

0.16

elastln 0 collagen A elastinbound glycoprote~n ??

0.14 !

1

1

t--

010 i

0.08 i

0.00

1 0

al

40

80

60

100

age(~ears)

Fig. 3. The accumulation of D-aspartate in proteins of the ageing aorta. The filled squares represent elastin, the open squares tibrillar collagen, and the triangles elastic tibril glycoproteins: error bars are standard deviations of repeat chromatography of the same sample.

with age, from over 30% in younger aortas to only 20% in older aortas. Discussion The turnover rate of proteins in the human body has a spectrum from minutes to years. Some of the TABLE 3 D-ASPARTATE IN ELASTIN AND ISOLATED FROM AGEING AORTA Age (years)

18 25 30 42 49 53 51 63 IO 16 78 85

COLLAGEN

% D-aspartate/(total aspartate) mean f SD. Elastin

Collagen

3.9 ?? 0.3 5.4 f 0.3 5.0 * 0.1 6.8 zt 0.1 6.3 zt 0.2 7.9 ?? 0.3 8.5 * 0.5 9.4 ?? 0.2 10.5 ?? 0.0 10.8 f 0.3 8.9 LIZ0.2 12.9 f 0.5

3.3 3.2 3.5 3.2 3.6 nd 3.8 3.6 3.8 nd 4.6 4.1

* 0.2 zt 0.3 ?? 0.3 -f 0.1 zt 0.2 zt 0.1 zt 0.3 f 0.1 * 0.5 f 0.2

Fig. 4. Variation in the elastin content of infrarenal aorta with age. Points represent single estimates from different aortas.

most metabolically stable proteins are also chemically stable and found in relatively avascular environments, e.g. tooth dentine, lens crystallin, aortic elastin. In this study of aortic proteins we sampled the infrarenal aorta, where in contrast to the thoracic aorta, there are very few vasa vasorum in the media. If vascularity and nutrient supply influence protein turnover we should be cautious about extrapolating our results from this study of infrarenal aorta to other regions of the aorta or other elastic arteries. The principal proteins of the aortic media are mature, cross-linked elastin and collagen, accounting for 30% and 60%, respectively, of the dry weight of abdominal aorta. The collagen fraction comprises mainly type I and type III collagen in 2:l ratio [20]. We prepared three different matrix protein fractions (elastin, fibrillar collagen and elastin bound glycoproteins) from aortic media of aortas from age 18-85 years. Avoidance of elevated temperatures dictated the purification methods and resulted in a protracted purification procedure for elastin. The elastin still contained substantial impurities after cyanogen bromide digestion since amino acid analysis showed an excess of polar amino acids. The variability of the amino acid compositions after this step may reflect a differential accumulation of glycoproteins with ageing. The final preparations of elastin did not

207

demonstrate a significant increase in polar amino acids with the increasing age of the aorta. The amount of D-aspartate in COkigen preparations (3.2-4.6%) was almost constant through this age range and this extent of racemization probably reflects procedural racemization, in large part attributed to the acid hydrolysis performed at 110°C [8, 211. Aspartate racemization accelerates at high temperatures, with 4% of the aspartates of serum albumin being racemized during a 24-h hydrolysis at 110°C [21]. An enzymic method of protein hydrolysis has been developed to avoid these problems but this would not be suitable for elastin which remains inert to proteases other than elastases [2 11. Accordingly acid hydrolysis times were limited to 6 h for the analysis of aspartate enantiomers. This short hydrolysis accomplished release of nearly all the aspartate from elastin and collagen but incomplete release from the elastin bound glycoprotein fraction. It remains possible that those aspartates/asparagines not released after 6 h of acid hydrolysis are those most susceptible to racemization. Nevertheless we have chosen to use the values obtained for collagen, thought to have a turnover time in the order of 50-300 days, to provide a better estimate of procedural racemization than that provided by the racemization of L-aspartate [22]. The preparations of elastin bound glycoprotein contained a fairly constant amount of D-aspartate, with some small increase above the age of 70 years. We have not established what this elastin bound protein fraction contains, but this protein(s) remains firmly attached to the elastin after cyanogen bromide digestion. Previous studies have indicated the efficacy and safety of introducing a trypsin digestion into the purification of elastin from ageing aorta [ 141. In our hands this removed glycoproteins and left material whose amino acid composition was identical with authentic elastin. Even though this elastin contained only 4-5 aspartate residues/1000, by modification of sample preparation conditions, we were able to provide sufficient aspartate for detection after chromatography. For elastin there was a steady increase in the Daspartate content with age of the aorta. The rate constant for racemization, apparent 1.14 x 10V3/year is considerably lower than that found for lung elastin, 1.76 x 10e3/year, but

similar to that found for lens crystallin [7,11]. The preparations of lung elastin contained twice as much aspartate as our preparations of aortic elastin and this may be one factor contributing to the different rate constants for racemization. Further, the amount of D-aspartate present in aortic elastin increases linearly with age, irrespective of the decrease in dry weight content of elastin with age. After exhaustive purification the amino acid composition of the elastin was very similar, with only 4-5 aspartates/lOOO residues, irrespective of the age of the aorta from which the elastin derived. Since the elastin from ageing aortas did not appear to be contaminated by other proteins, the accumulation of aspartate with ageing indicates a lifetime residence for cross-linked elastin in the infrarenal aortic media, with little new synthesis and deposition of mature elastin in this region of the aorta during adult life. The strongly dissociative steps early in the purification procedure would remove uncross-linked, or immature, elastin and collagen. Therefore it remains possible that elastin is synthesized in the ageing aorta, but not deposited into mature elastic fibrils. Propagation of the arterial pulse down the aorta is dependent on the elastic properties of the aorta. Fortunately elastin is very resistant to degradation: the slow decline in the dry weight content of elastin in infrarenal aorta with ageing could result from either loss of elastin or the accumulation of other components in the ageing aorta. Calcification of the ageing aorta might result in the increased ionization of aspartate side chain carboxyl in elastin which would accelerate the racemization of D-aspartate in this protein. Since mammalian cells contain a protein carboxymethyltransferase which specifically methylates only D-aspartate residues, the accumulation of D-aspartate residues could target a protein for specific proteolysis [ 131 (methylated D-aspartate is labile to acid hydrolysis and any present in our elastin preparations would have been estimated as D-aspartate). Therefore it is possible that the sequential processes of calcification, aspartate racemization and proteolysis could explain the decreased elastin content of the ageing aorta and the susceptibility of the ageing aorta to aneurysmal dilatation. In contrast there is no accumulation of D-aspartate in the fibrillar collagens from infrarenal aorta, and

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turnover of fibrillar collagen appears to continue through life. With ageing there is fibrous replacement of the smooth muscle cells of the aortic media: the replacement fibroblasts appear to maintain collagen synthesis. Alternatively our data could indicate that no aspartate residues in collagen are prone to racemization. How the primary and secondary structure of proteins influences the rate of amino acid racemization and how the metabolic stability of elastin is related to its chemical stability remain intriguing questions.

8 9

10

11

12

Acknowledgements We thank Professor M. Spina for advice on the preparation of elastin and Dr A.K. Allen for help with the gas liquid chromatography. This work was supported by a grant from the Medical Research Council.

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References Learoyd, B.M. and Taylor, M.G., Alterations with age in the visco-elastic properties of human arterial walls, Circ. Res., 18 (1966) 278. Mohiaddin, R.H., Underwood, S.H., Bogren, H.G., Firmin, D.N., Khpstein, R.H., Rees, R.S.O. and Longmore, D.B., Regional aortic compliance studied by magnetic resonance imaging: the effects of age, training and coronary artery disease, Br. Heart J., 62 (1989) 90. Crawford, T. and Levine, C.J., Medial thinning in atheroma, J. Pathol. Bacterial., 66 (1953) 19. Nejjar, I., Pieraggi, M.-T., Thiers, J.C. and Bouisson, H., Age related changes in the elastic tissue of the human aorta, Atherosclerosis, 80 (1990) 199. Powell, J.T., Dilatation through loss of elastin. In: Greenhalgh, R.M and Mannick, J.A. (Eds.), The Cause and Management of Aneurysms, WB Saunders, London, 1990, pp. 89-96. Lansing, A.I., Rosenthal, T.B., Alex, M. and Dempsey, E.W., The structure and chemical characterization of elastic tibres as revealed by elastase and electron microscopy, Anat. Rec., 114 (1952) 555. Shapiro, S.D., Endicott, S.K., Province, M.A., Pierce, J.A. and Campbell, E.J., Marked longevity of human lung parenchymal elastic fibers deduced from prevalence of D-

16 17

18

19

20

21

22

aspartate and nuclear weapons-related radiocarbon, J. Clin. Invest., 87 (1991) 1828. Bada, J.L., In vivo racemization in mammalian proteins, Methods Enzymol., 106 (1984) 98. Bada, J.L. and Pro&h, R., Racemization reaction of aspartic acid and its use in dating fossil bones, Proc. Natl. Acad. Sci. USA, 70 (1973) 1331. Helfman, P.M. and Bada, J.L., Aspartate racemization in tooth enamel from living humans, Proc. Natl. Acad. Sci. USA, 72 (1975) 2891. Masters, P.M., Bada, J.L. and Zigler, J.S., Aspartic acid racemisation in the human lens during ageing and in cataract formation, Nature, 268 (1977) 71. Man, E.N., Sandhouse, M.E., Burg, J. and Fisher, G.H., Accumulation of D-aspartic acid with age in the human brain, Science, 220 (1983) 1407. Geiger, T. and Clarke, S., Dearidation, isomerization and racemization at asparaginyl and aspartyl residues in peptides, J. Biol. Chem., 262 (1987) 785. Spina, M., Garbisa, S., Hinnie, J., Hunter, J.C. and Serafini-Fracassini, A., Age related changes in composition and mechanical properties of the tunica media of the upper thoracic human aorta, Arteriosclerosis, 3 (1983) 64. Indik, Z., Yeh, H., Ornstein-Goldstein, N., Sheppard, P., Anderson, N., Rosenbloom, J.C., Peltonen, L. and Rosenbloom, J., Alternate splicing of human elastin mRNA indicated by sequence analysis of cloned genomic and complementary DNA, Proc. Natl. Acad. Sci. USA, 84 (1987) 5680. Epstein, E.H., [aI(III)]s Human skin collagen, J. Biol. Chem., 249 (1974) 3225. van den Oetelaar, P.J.M., van Beijsterveldt, L.E.C., van Beckhoven, J.R.C.M. and Hoenders, H.J., Detection of aspartic acid enantioners by chiral capillary gas chromatography, J. Chromatogr., 368 (1986) 135. van den Oetelaar, P.J.M., van Beckoven, J.R.C.M. and Hoenders, H.J., Evaluation of a chiral capillary gas chromatographic and a diastereomeric high performance liquid chromatographic method, J. Chromatogr., 388 (1987) 441. Gibson, M.A., Kumaratilake, J.S. and Cleary, E.G., The protein components of the 12 nm microtibrils of elastic and non-elastic tissues, J. Biol. Chem., 264 (1989) 4590. Menashi, S., Campa, J.S., Greenhalgh, R.M. and Powell, J.T., Collagen in abdominal aortic aneurysms: typing, content and degradation, J. Vast. Surg., 6 (1987) 578. DAniello, A., D’Onnofrio, G. and Pischetola, M., Total hydrolysis of proteins with strongly reduced racemization of amino acids, Biochim. Biophys. Acta, 1037 (1990) 200. Murphy, G.M. and Reynolds, J.J., Collagen degradation, Bioessays, 2 (1985) 55.