This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Dabbous, M. K. Articles by Brinkley, S. B. Search for Related Content PubMed PubMed Citation Articles by Dabbous, M. K. Articles by Brinkley, S. B.

Collagenase and Neutral Protease Activities in Cultures of Rabbit VX-2 Carcinoma¹

Departments of Biochemistry [M. K. D., B. B.] and Microbiology [A. N. R.], College of Basic Medical Sciences, University of Tennessee Center for Health Sciences, Memphis, Tennessee 38163

Primary cultures of minced rabbit VX-2 carcinoma tissue were shown to release collagenolytic activity in serum-free medium. This activity appeared to be a multienzyme system composed of specific collagenase and neutral protease. The collagenase was released at an increasing rate for 4 days and then at a relatively lower rate for 3 to 4 more days. Collagenase release was subject to inhibition by actinomycin D and by cyclohexamide. The collagenase (M.W. approximately 34,000) was purified 7,000-fold by ultrafiltration, (NH₄)₂SO₄ precipitation, column chromatography, and acrylamide gel electrophoresis. The purified enzyme solubilized radioactive peptides from ¹⁴C-labeled collagen fibrils at a linear rate as a function of both enzyme concentration and incubation time at 36°, decreased the specific viscosity of acid-soluble collagen solutions to a limit value of 40% of the original viscosity, and cleaved tropocollagen at one site one-fourth of the length from the C-terminal end of the molecule producing TC^a (75%) and TC^b (25%) fragments as evidenced by disc electrophoresis and electron microscopy of segment-long-spacing aggregates of the products. The purified collagenase had a pH optimum of 7 to 8.5 and was inhibited by sera from normal and tumor-bearing rabbits, ethylenediaminetetraacetic acid, and cysteine. The neutral protease activity released radioactive trichloroacetic acid-soluble peptides from ¹⁴C-labeled gelatin. At temperatures below collagen denaturation, the protease released telopeptides from native tropocollagen without attacking the main triple helical body of the tropocollagen macromolecule. It is suggested that both the collagenase and neutral protease activities may play cooperative roles during tumor invasion of the surrounding tissues.

¹ This paper is one of a series on collagenolytic activity in invasive tumors. This work was supported by USPHS Grant CA 15677 from the National Cancer Institute.

Received 11/17/76. Accepted 6/30/77.