Recommendations concerning the use of bacterial collagenase for isolating collagen-associated molecules from tissues

Benya, Paul D.; Berger, K.; Golditch, M.; Schneir, Michael

doi:10.1016/0003-2697(73)90438-7

Cited by 39 publications

(8 citation statements)

References 4 publications

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“…Some of these limitations can be overcome by the use of bacterial collagenases which have a high degree of specificity for proteolysis of either native or denatured collagen ). Proteolysis of tissue preparations or extracts with partially purified collagenases from Clostridium histolyticum in the presence of N-ethylmaleimide, dithiothreitol, and a serine protease inhibitor permits relatively specific digestion of collagen (Peterkofsky and Diegelmann, 1971;Benya et al, 1973). The utility of the method depends on the completeness of the digestion of the collagen, the lack of precipitability of the digestion products with reagents such as trichloroacetic acid-tannic acid, and the absence of nonspecific proteolytic activity.…”

Section: Assaysmentioning

confidence: 98%

The Chemistry and Biology of Collagen

Börnstein¹,

Traub²

1979

The Proteins

183

119

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Section: Assaysmentioning

confidence: 98%

The Chemistry and Biology of Collagen

Börnstein¹,

Traub²

1979

The Proteins

183

119

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“…Worthington CLSPA collagenase was purified (13,14) and incubated with samples in 1 M NaCl/50 mM Tris/5 mM CaC12/0.1% Triton X-100,' pH 7.5, for 3 hr at 350C. Controls were incubated identically but without enzyme.…”

mentioning

confidence: 99%

Biosynthesis of A,B procollagen.

Kumamoto¹,

Fessler²

1980

Proc. Natl. Acad. Sci. U.S.A.

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Interest in cell-associated collagens led others to isolate A and B collagen chains, also known as type V collagen, from many tissues, but only after pepsin cleavage. Soluble precursors of these chains are synthesized in vitro by crop, a chicken embryo muscle tissue, and are converted by at least two processing steps from procollagens via intermediates to final forms which are larger than the pepsin-derived A and B chains. Heterotrimeric, disulfide-bridged procollagen molecules corresponding to B2A exist. Components were separated by ion exchange chromatography, velocity sedimentation, and electrophoresis, and the relationships between them were establis ed by sequential radioactive labeling and comparison of peptides generated by protease cleavage. Collagens form a family of related gene products composed of two general groups: the interstitial and the basement membrane collagens. Two (4,5) reported different ratios of these chains and also pepsin-resistant B chains, indicating 13 trimers. We find proaA chains disulfide-linked to proaB chains, which establishes the heterotrimer [(proaB)2proaA]. However, an excess of B chains suggests that homotrimer B3 also exists. Because type V collagens have been located immunologically in muscle tissue (6, 7), have been found in chicken embryo tissues (7-9), and have been shown to be made by cultured smooth muscle (10) and chicken cells (7, 11), we chose a rapidly growing muscle tissue of the chicken embryo, the crop. Because this thin-walled diverticulum of the esophagus does not produce digestive enzymes, accidental proteolysis was minimized; furthermore, other studies support our conclusion of stepwise processing.t MATERIALS AND METHODS Typically, crops from 50 19-day chicken embryos were incubated in 15 ml of Dulbecco's modified Eagle's medium (DME medium) by described procedures (12) with the modifications given below. The medium lacked lactalbumin hydrolysate and the amino acids used for labeling; it contained 2-aminopro- 40C. Crops were homogenized in 1 M NaCl/50 mM Tris/10 mM EDTA, pH 7.5 (buffer H), and centrifuged (40,000 rpm, 15 min). The pellet was rehomogenized in 0.5 M acetic acid and the suspension was digested with pepsin (100 gg/ml; Worthington) for 18 hr at 40C. The supernatant was diluted with 1 vol of 4 M urea/0.2% Triton X-100/50 mM Tris, pH 7.5, and, after dialysis against 0.5 M acetic acid/0.2 M NaCl/0.1% Triton X-100 (Buffer P), it was digested with pepsin as above. Further purification of A and B chains was as described (2, 7) except that NaCl was added to 5% by dialysis and type I and III carrier collagens (0.1 mg/ml) were added to help precipitate material from the buffer H extract. CNBr cleavage and peptide separation were as described (2).Extraction of Type V Precursors, DEAE-Cellulose Chromatography, and Velocity Sedimentation. Tissue was homogenized in buffer I [buffer H plus 1250 Ag N-ethylmaleimide (MaINEt) and 150 ,g of phenylmethylsulfonyl fluoride (PheMeSO2F) per ml] and centrifuged (40,000, rpm, 15 min). The supernatant was ...

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“…Control = Not treated; +R = reduction with 2-mercaptoethanol (0.7%); +P = pepsin treatment, 100 pg/ml for 2 h at 4'C ; P+R = pepsin and reduction; +C = collagenase digestion. 5 pg, 2 h at 37"C, general protease-free [20]; C+R = colla genase and reduction.…”

Section: Resultsmentioning

confidence: 99%

EC Collagen: Biosynthesis by Corneal Endothelial Cells and Separation from Type IV without Pepsin Treatment or Denaturation

Benya¹

1980

Kidney Blood Press Res

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Primary cultures of rabbit corneal endothelial cells were labeled with proline and the medium proteins were fractionated by sedimentation through sucrose density gradients. A new collagen, endothelial cell (EC) collagen, was completely separated from type IV collagen without denaturation by this technique. Sodium dodecyl sulfate electrophoresis demonstrated EC collagen fragments of 180K, 120K, and 60K daltons. Prior pepsin treatment produced only the 60K fragment after electrophoresis. The cyanogen bromide peptide pattern of the 180K EC peptide demonstrated that EC was different from type IV. A preliminary structural model for the largest helical form (∼ 540K daltons) of EC collagen is presented.

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Recommendations concerning the use of bacterial collagenase for isolating collagen-associated molecules from tissues

Cited by 39 publications

References 4 publications

The Chemistry and Biology of Collagen

The Chemistry and Biology of Collagen

Biosynthesis of A,B procollagen.

EC Collagen: Biosynthesis by Corneal Endothelial Cells and Separation from Type IV without Pepsin Treatment or Denaturation

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