11] Posttranslational enzymes in the biosynthesis of collagen: Extracellular enzymes

Abstract

This chapter describes the assay, purification, and properties of the enzymes catalyzing the unique extratracellular modifications— procollagen N-, C-proteinases, and lysyl oxidase. Two general methods are used to assay the procollagen N- and C-proteinases. One is to use polyacrylamide gel electrophoresis for examining the cleavage products obtained with the enzymes. The other is to precipitate the reaction products under conditions in which uncleaved procollagen, partially cleaved procollagen, and collagen are recovered in the pellet, but the released N- or C-propeptides are in the supernatant. Procollagen N-proteinase can be purified from leg tendons of chick embryos. Partial purification has been carried on what appears to be the same enzyme from calf tendons and the medium of cultured chick embryo tendon fibroblasts. The steps involves are: tendon extraction; ammonium sulfate precipitation; Con A-Sepharose ; and Heparin-Sepharose column chromatography. For purification of procollagen C-proteinase, one laboratory has taken the medium from cultured fibroblasts of chick embryo tendons, precipitated the proteins with 50% saturated ammonium sulfate, and chromatographed the protein in DEAE-cellulose. However, there no successful procedure for purifying the C-proteinase has been developed. Purification of lysyl oxidase include following steps: extraction of femoral and tibial epiphysial cartilages from 17-day-old chick embryos: chromatography on DEAE-cohmn I and DEAE-column II.