4] Preparation and characterization of procollagens and procollagen-collagen intermediates

Abstract

This chapter describes specific methodology for the purification of the five different procollagen types both from tissues and from sources that synthesize these proteins in vitro. Smooth muscle cell cultures are chosen to isolate procollagen because the cells are easily propagated in tissue culture, they produce relatively large amounts of types I and III procollagen, and they alsosynthesize small but detectable amounts of types IV and V procollagen. Types I and III procollagen purification involves: isolation of smooth muscle cells; metabolic labeling; and ion-exchange chromatography. Because fractionation on DEAE-cellulose is performed under conditions in which procollagen retains its native conformation, it is critical, after removal of the labeled culture medium from the cells, to perform all procedures at 0–4 ° to prevent denaturation of the protein. Type II pro T collagen is prepared from chick sternal cartilages or from matrix-free cartilage cells. After removal of sterna the cells were filtered through lens paper and centrifuged. The culture medium was processed and chromatographed on DEAE-cellulose. Type II procollagen is migrated on SDS-PAGE similarly to type I procollagen before reduction and to the pros α(I) chain after reduction. Type IV procollagen can be prepared and characterized predominantly from three biosynthetic systems: the EHS sarcoma, human amniotic fluid cells, and rat parietal yolk sac. Procedures for isolation of type IV procollagen are the same as those just described for types I and III. In type V(AB) rocollagen purification are excised from fifty 19-day-old chick embryos and incubated in DMEM. Labeling is performed using 50 μCi of [3H]proline. To remove any precipitated material and was chromatographed on a DEAE-cellulose column.