Six distinct collagenases present in the culture filtrate of Clostridium histolyticum have been purified to homogeneity and characterized in detail. To accomplish this end, an accurate and convenient assay for collagenase activity based on the hydrolysis of soluble {('14)CH(,3)}collagen has been developed. This assay, which can detect nanogram quantities of collagenase, has greatly facilitated the purification. The key step in the isolation of the individual collagenases, which have very similar physicochemical properties, is chromatography over a red dye-ligand column. The purified enzymes are homogeneous by sodium dodecyl sulfate gel electrophoresis and are devoid of any contaminating enzymatic activities. / The six collagenases, designated (alpha), (beta), (gamma), (delta), (epsilon), and (zeta), have been characterized as to their molecular weights, isoelectric points, carbohydrate content and amino acid compositions. Atomic absorption spectroscopy shows that all of the enzymes contain one gram atom of zinc per mole of protein, and variable amounts of calcium. The activities of the enzymes towards collagen, gelatin, and several synthetic peptides have been determined. These activities, taken together with other data, strongly support the assignment of the collagenases into two distinct classes. / The relationship between the individual collagenases has also been examined. Chemical modification reactions establish that all six enzymes contain essential lysine, tyrosine, and carboxyl residues. Moreover, all six enzymes cross react with antiserum prepared against (beta)-collagenase. Information about the sequence relationships between the enzymes has been obtained by comparison of their tryptic digestion and of their cyanogen bromide reaction products. The results of these experiments indicate that the collagenases within each class have extensive sequence homology with each other, but that there is little sequence homology between the two classes. In addition, the data show that (beta)-collagenase probably consists of domains that have homologous amino acid sequences. Gene duplication is implicated as a possible evolutionary mechanism for the development both of two classes of enzymes and of homologous domains in (beta)-collagenase. / Source: Dissertation Abstracts International, Volume: 44-11, Section: B, page: 3384. / Thesis (Ph.D.)--The Florida State University, 1983.
| Identifer | oai:union.ndltd.org:fsu.edu/oai:fsu.digital.flvc.org:fsu_75229 |
| Contributors | BOND, MICHAEL DOUGLAS., Florida State University |
| Source Sets | Florida State University |
| Detected Language | English |
| Type | Text |
| Format | 193 p. |
| Rights | On campus use only. |
| Relation | Dissertation Abstracts International |
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