Extração de Colágeno

Extração de Colágeno

(Parte 2 de 2)

of molecules within the spools.

The band pattern of the BM collagen was com- pared with Type I SLS banding in a preparation in which P-I and Type I SLS were mixed and deposited together on the same grid. All aspects of subsequent preparation for viewing, staining, drying, magniilca- tion and so on are thus identical. It is evident, fig.3a, that the two types of collagen have distinctly different staining patterns. A direct comparison, fig3b, with the sharply defined BM SLS segment, end matched with the amino terminal end of the Type I SLS, shows that while some striations appear to match, the central regions of the two collagens, in particular, are very different. The alternate orientation of Type I provides a still greater mismatch of band pattern. The bush- like appendage makes the P-I SLS about 40 rmr longer than Type I SLS.

Reduction and alkylation of P-I, followed by a second pepsin digestion to produce P-I, brings about two modifications in the BM collagen. First, the appendage is removed and both ends of the SLS become sharply defined. As shown in the composite set of micrographs of fig.4, the clearly banded P-I SLS is almost the same length as the Type I SLS

A second, and most striking observation, figSa, is that in addition to the ‘~300 nm full length BM SLS, there are large numbers of partial segments, with lengths close to 135 nm or 45% the length of the intact P-I SLS. Almost all of the partial segments have the same band pattern. Moreover, where the partial segments coprecipitate alongside full segments, figures 5b and 5c, it is evident that the partial seg- ments correspond to the end of the BM molecule which originally had the bush-like extensions. Although one may occasionally find what looks like an isolated half-segment corresponding to the ‘NH*-terminal’ end of the BM collagen, fig.5d, more than 95% of the partial segments are from the bush-like end region and are of uniform length. In the original P-I prepara- tion both the extra-helical depleted and the 45% SLS could be found but to a much lesser extent than after the second pepsin digest. Occasionally SLS ~85% of the P-I were also seen in P-I preparations, indicating cleavage in the helical region and possibly correspond- ing to the 85 0 mol. wt peak seen in the SDS gels.

4. Discussion

Bovine anterior lens capsule basement membrane collagen released in acid-soluble form by limited pepsin digestion contains collagenous and non-colla- genous parts. The non-collagenous region can be removed from the collagenous part by a combination of disulfide bond rearrangement, accomplished by reduction and alkylation, followed by a second pepsin digestion. SLS precipitates of this collagen, P-I, fig.4, show that the triple helical region has a length virtually identical to that of intact Type I collagen although the pattern of cross-striation of the SLS, and hence the sequence in the helical region, is distinctly different from that of Type I. The non-collagenous region, seen in SLS precipi- tates of P-I collagen, that is before cleavage of the disulfide bonds and after only a single pepsin treat- ment, is located at one end of the SLS spools, fig.2 and tig.3. The presence of the pepsin-sensitive exten- sion region interferes with the close packing of molecules and obscures the band pattern at its end of the SLS spool. This is similar to the effect of the non-helical extensions in Type I procollagen SLS [ 10,l l] . At this point we cannot say whether the end- extension region is a continuation of the main pep- tide chains, as in Type I procollagen, or a separate disulfide-linked protein as sometimes suggested [l] . Removal of the appendage appears to be facilitated by reduction of disultide bonds but extensive pepsin treatment alone can accomplish the same result. Since pepsin was used in the initial stage of extraction we

FigSa. P-I, demonstrating full-length SLS (arrow) and numerous 45% length, fragment SLS, X 69 300. Fig.Sb. Fragment SLS

(arrows) along side full size SLS, X 69 300. Fig.Sc. A partial SLS (arrowhead) placed between 2 full-length SLS. Along with a and b demonstrating that most fragment SLS are derived from one end of the molecule, tentatively identified as the C-terminal

half. X 180 0. Fig.Sd. A rare N-terminal half SLS (arrow) that was likely derived from a single pepsin cleavage at the 45-5% point along the collagen molecule, X 180 0. All preparations stained with 2% PTA, pH 7.

Volume 85, number 2 FEBS LETTERS January 1978

cannot rule out either the possibilities that, in vivo, the appendage is larger than demonstrated here, or that there may be an even more labile pepsin-sensitive region at the other end of the BM-collagen molecule prior to extraction.

The appearance of a BM collagen component with mol. wt 5 0 in pepsin digested collagen from human aortic intima has indicated the presence of BM collagen in relatively short sequences joined by non- collagenous protein [3] . Our data in the lens capsule system offer at least one alternative explanation.

The collagen solubilized from the lens capsule with one pepsin treatment (P-I) is essentially all in intact a-chain length form. The SLS are predominantly in %300 nm lengths with an asymmetric band pattern throughout, fig.2 and tig.3. Upon further pepsin treatment, and after reduction and alkylation, P-I is converted to clean, full length, %300 nm asymetri- tally banded collagen molecules (P-I) plus many ~135 nm segments which in almost all cases exactly match only one end of the intact molecules in band pattern, fig.4 and fig.5. This sequence of events is consistent with the presence of a pepsin-sensitive region or regions within the triple-helical sequence of the lens capsule BM collagen, similar to the trypsin- sensitive region found in Type I collagen [ 121 and in dentin collagen [ 131.

The number of pepsin-sensitive sites in BM collagen is difficult to assess at this time. The remaining 135 nm length segments, corresponding to that end which binds the non-collagenous protein, contains no addi- tional pepsin-sensitive sites. The region which is cleaved and reduced to dialyzable peptides may be a molecular region of low stability after opening of a single pepsin-sensitive triple-helical region or may contain several such regions. Half-segments corre- sponding to the pepsin-labile helical region have been observed, although very rarely. This might indicate that one site of pepsin susceptibility is at the 5-45% point along the molecule. The presence of a few especially pepsin-sensitive regions in the collagen helical region is supported by the SDS electrophoretic pattern of reduced P-I in which collagenase susceptible components of 115 0,85 0 and 50 0 are seen.

At the same time, the lack of other collagenase sensitive fragments is consistent with the instability of the partly-cleaved molecules.


Supported by research grant AM 13921 from the

National Institute for Arthritis, Metabolic and Digestive Diseases, National Institutes of Health (to A.V.) and General Research Support Grant from Northwestern University Medical and Dental Schools (to D.S.).


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(Parte 2 de 2)