A new method for measuring the rate of enzymatic excision of the carboxy-terminal, nonhelical fragment from type I procollagen is presented. Human procollagen containing [3H]tryptophan-labeled carboxy-terminal extensions was used as the substrate, and the enzyme was derived from the culture medium of mouse 3T6 fibroblasts. Incubation mixtures of substrate with enzyme were made 25% in ethanol which left the excised radiolabeled carboxy-terminal fragment in solution, whereas all other radiolabeled components were precipitated. Enzymatic activity was measured by radioactive counting of the ethanol supernatant. The assay is simple, rapid, sensitive and generates valid kinetic data.
Soluble secreted precursors (procollagens) have been demonstrated for each of the four major genetic types of collagen (1,2 for general reviews). To date, the conversion of type I procollagen to collagen has been studied in greatest detail and has been shown to require the enzymatic excision of nonhelical peptides from both ends of the precursor molecule. Available data indicate that at least some of these excisions involve endopeptidases; cleavages at the aminoterminus release peptide chains of about 11,500 and 17,500 daltons (3), whereas, the cleavages at the carboxy-terminus generate a three-chain, disulfide-linked fragment of approximately 75,000 daltons (45).
Purification of the enzymes involved in the conversion of type I procollagen to collagen has been hampered by lack of practical assays for these activities. The gel electrophoresis (3,4) ion-exchange, and immunoprecipitation techniques applied to date are laborious or require complete conversion of the precursor substrate (6). In this paper we describe a simple and rapid assay for the endopeptidase(s) which releases the three-chain fragment from the carboxy-terminal end of type I procollagen. The method is based on the observation that the excised fragment is soluble in 25% ethanol, whereas the undigested substrate, digestion intermediates, and collagen are insoluble in this concentration of ethanol.