Extração de Colágeno

Extração de Colágeno

(Parte 1 de 2)

Volume 85, number 2 FEBS LETTERS January 1978

David SCHWARTZ and Arthur VEIS+ Northwestern University Dental and Medical Schools, 303 E. Chicago Avenue, Chicago, IL 60611, USA

Received 6 November 1977

1. Introduction

Basement membranes (BM) contain a class or classes of collagen distinct from the Type I, I and II interstitial collagens. The differences can be grouped under two general categories: first, the peptide chain sequences of the collagen, and their heightened content of 3-hydroxyproline and glycosylated hydroxylysine; and second, the presence of covalently attached non- collagenous protein moieties [ 1,2] . The molecular weights of individual o-chains of BM collagen are greater, even after pepsin digestion, than the cr-chains of Type I collagen [2,3], although the presence of a second species of BM collagen with a mol. wt 5.5 0 has been proposed [3],

The essential question to us is structural. BM collagens exist in the form of non-ftlamentous sheets rather than in striated D-periodic fibrils as in the interstitial collagens. Which features of the BM collagen prevent the formation of periodic fibrils, the non-collagenous moieties, or the distribution of inter- active groups in the collagen helical region? Electron microscopy of the interstitial collagens has provided many insights into their molecular structure and inter- molecular interactions, but little data of this nature has been available [4,5] relative to collagens of BM Origin.

We have succeeded in producing segment-long- spacing precipitates (SLS) from bovine anterior lens capsule BM collagen [6]. This technique enables us to examine BM collagen at several stages of enzymic

+ TO whom correspondence should be addressed 326 pretreatment and compare the band pattern of the lens capsule collagen SLS directly with the pattern of Type I interstitial collagen. In addition, we find that BM collagen is susceptible to specific cleavage by pepsin, giving rise to the appearance of pepsin resistant half-molecules.

2. Materials and methods

2.1. Preparation of BM collagen

Anterior lens capsules were dissected from 200 fresh bovine eyes and placed in 0.15 M sodium chlo- ride. The dissected capsules were sonicated briefly

(<I min) and then collected by low speed centrifuga- tion. The capsules were suspended in 50 ml 0.075 M sodium citrate, pH 3.7, containing 0.001 M phenyl- methylsulfonyl fluoride (PMSF). After stirring for 48 h at 10°C the residual capsular material was again collected by low speed centrifugation.

The supernatant of the centrifugation was made 4.0 M in sodium chloride. The precipitate which formed was collected and redissolved in the pH 3.7 buffer, reprecipitated once again with sodium chloride, and f%urlly dissolved in 0.5 N acetic acid. This solu- tion was desalted by dialysis and lyophilized.

The residual capsular material was suspended in 50 ml 0.5 M acetic acid, pH 2.5 at 4°C and, after dialysis against more 0.5 M acetic acid to remove the PMSF, 0.1 mg pepsin (Worthington) was added. After 24 h the supematant was decanted and dialyzed against 0.9 M sodium chloride [7]. The precipitate was discarded and the remaining solution taken as the pepsin solubilized BM collagen fraction (P-I).

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Volume 85, number 2 FEBS LETTERS January 1978

2.2. Second pepsin digestion

P-I was reduced and alkylated following the procedure in [5] , but in the absence of urea. The

reduced and alkylated collagen was then digested a second time with pepsin (1:50, pepsin:collagen) at

pH 2.5 for 16 h at lO”C, following the general scheme in [7]. The collagen produced in this treatment is

designated P-I.

2.3. Collagen digestion To determine the collagenous character of certain

fractions and components, samples were digested with bacterial collagenase (Worthington) purified further by the method in [8] . Digestion was carried out for 4 h at pH 7.5 in Tris buffer containing N-ethyl-male- imide to inhibit non-specific proteolysis.

2.4. Electrophoresis and amino acid analysis

Polyacrylamide gel electrophoresis in sodium- dodecylsulfate followed the procedure in [9] . Buffer solutions were used both with and without mercapto- ethanol.

Samples for amino acid analysis were hydrolyzed at 105°C for 2 h in 6 N HCl in sealed, nitrogen- flushed tubes. Analyses were carried out in a JEOL 6AH analyzer with a single column program.

2.5. Electron microscopy and SLS formation Collagen solutions, at 0.1 mg/ml in 0.5% acetic

acid, were dialyzed against 0.4% adenosine triphos- phoric acid (ATP) (Aldrich) in the cold, from 24-48 h. Precipitates did not develop rapidly, as with Type I collagen, but appeared only after many hours. Droplets of precipitate suspension were placed on carbon/ formvar coated grids and negatively stained with 2% phosphotungstic acid containing 100 pg bacitracin,

adjusted to pH 7.5 with NaOH. Grids were viewed on an Hitachi I-ID-1 1 A microscope.

.3. Results

Little protein was recovered from the initial citrate buffer extraction of the lens capsules. Although a collagen was isolated from a similar preparation [ 51, amino acid analyses indicated that very little of the citrate extracted protein was collagenous. However, fraction P-I was collagenous. Its amino acid compo- sition, including 4 residues hydroxylysine, 10.7 residues 3-hydroxyproline, 134 residues 4-hydroxy- proline and 296 residues glycine per 1000 amino acids, was virtually identical with that of a standard BM collagen preparation from bovine lens capsule base- ment membrane, generously supplied by Dr N. A. Kefalides. All further work began with the P-I preparation.

Acrylamide gel electrophoresis; in SDS showed P-I contains a mixture of r-like high molecular weight

1.0 -

E c 0.75- g .

8 s 0.5- n

3 0.25 - b 2 4 6 6 IO

Migration (m)

Fig.la. Polyacrylamide gel electrophoresis in SDS of the collagenous material derived from the first pepsin digest (P-I) of bovine anterior lens capsule. F&lb. P-I, with 1% mercapto- ethanol in the running buffer.

Volume 85, number 2 FEBS LETTERS January 1978

components, some material with weights between o(I) and p(I) chain weights, and a small amount of lower molecular weight material, fig.la. Electropho- resis carried out in the presence of 1% mercapto- ethanol, figlb, showed that most of the y compo- nents were converted to a single chain type migrating between al(I) and &(I) positions at app. mol. wt 160 0 and three lower molecular weight fractions with app. mol. wt 115 0,85 0 and 50 0. All of the major bands are removed upon digestion with bacterial collagenase. Hence these components are BM collagen chains or chain fragments.

SLS precipitates develop from P-I slowly and with difficulty, however, copious amounts of loosely- ordered SLS can eventually be seen, iig.2a. In some isolated SLS spools the band pattern is occasionally very distinct, fig.2b. From the asymmetry of the banding it is evident that each P-I molecule is also asymmetric and that each is aligned and pointing in the same direction within the SLS spool. The BM

Fig.2a. SLS crystallites of basement membrane collagen after a single pepsin digestion (P-I) X 100 0,2% PTA, pH 7. Fig.Zb. Enlargement of a SLS spool from a similar preparation. Note the different character to each end of the crystallite and the asym-

metric banding, X 200 0,2% PTA, pH 7.

Volume 85, number 2 FEBS LETTERS January 1978

Fig.3a. P-I mixed with salt extracted type I collagen and dialysed against ATP. Upper right is P-I (BM) SLS, lower left is type I SLS, x 100 0,2% PTA, pH 7. Fig.3b. Comparison of SLS, type I (left) and P-I, basement membrane collagen (right), in the relative orientation of ends which provide the most number of matched bands. The N-terminus of type I is at the bottom of the

fgure, x 200 0,2% PTA, pH 7.

Fig.4. Composite of type I (left), P-I (middle) and P-I, resulting from a second pepsin digestion of P-I after reduction and alkyla- tion (right), x 170 0, 2% PTA, pH 7.

Fig.5 330

Volume 85, number 2 FEBS LETTERS January 1978

SLS stop abruptly in register at one end but charac- teristically have a bush-like appendage at the other end. The appendages appear to inhibit close packing

(Parte 1 de 2)

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