Tadpole collagenase

Figure 9.22 Separation of hydrolysis products of the action of tadpole collagenase on DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Argby reversed-phase HPLC. Upper tracing shows the gradient employed. The reaction was initiated at zero time by the addition of 4 /xg of enzyme. At the indicated times, a 4.0 /xL aliquot erf the reaction mixture was injected onto the HPLC column and the peptides separated. (From Gray and Saneii, 1982.)...

D. Collagenases from Vertbbk. te Sources 1. Tadpole Collagenase... 

Experiments by Sakai and Gross (1967) provided further information on the nature of the fragments produced by tadpole collagenase. Viscosity experiments su ested persistence of a rigid rod structure for both the 3/4 piece and 1/4 piece, and optical rotatory dispersion measurements indicated preservation of the helical structure. How ever, the fragments heat denature more readily than colleen. At acid pH, Tm values were 32 C and 29"C for TC and TC , rcj peetively, as compared to 36 C for TC... 

The enzyme vertebrate collagenase was partially purified from a lyophilized tissue culture medium of back skin from tadpoles. The medium was harvested,... 

Collagenase a proteolytic enzyme, and the only enzyme capable of degrading native collagen to soluble, low M, peptides. More than 20 C. have been described from baeteiia, fungi, arthropods, amphibia and mammals, C from Clostridium histolyticum and C. from tadpole tail have been extensively studied. Clostridial C (C.A M, 105.000. CB M, 57,000) attacks mainly the peptide bond proceeding the Gly-Pro sequence (-4-Gly(16)-Pro-Ser-4—Gly-Pro-) the bonds in front of Gly-Leu and Gly-Ala are attacked to a lesser extent, e.g. in the a,-chain. 

Eisen and Gross (1965) sought to determine the tissue source of eol-lagenase and hyaluronidase as well as the role of these enzymes in tadpole tail resorption. Epithelium was separated from connective tissues of the tail fins with the aid of an elastase digestion, and the tissues were tested separately for collagenase production, Collagenase was detected only in culture fluids of epithelial cells. Hyaluronidase could be detected in either the culture fluids or in homogenates of the mesenchymal tissues (Silbert et al, 1965). 

Vertebrate tissues knowm to produce specific collagenases include tadpole skin (epithelium only), human gingivae (epithelium and connective tissues), human skin (epithelium and dermis), bone, synovium, and polymorphonuclear leukocytes from the peripheral blood. With the exception of the W hite blood cells, culture is presently required for detection of all other vertebrate collagenases. Uniformly negative results for specific collagenases have been achieved in preparations of vertebrate tissues (except for leukocytes) designed to permit detection of enzymes in lyso-somes. Present data are consistent with the view that collagenase is not stored in celis rather it is produced and released into the extracellular environment w here it may act or be inactivated. 

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