Type I procollagen C‐proteinase from mouse fibroblasts: Purification and demonstration of a 55‐kDa enhancer glycoprotein

Efrat KESSLER, Rivka ADAR

Research output: Contribution to journalArticlepeer-review

Abstract

The enzyme procollagen C‐proteinase removes the carboxy‐terminal propeptide from procollagen. In the present study we describe an improved procedure for the purification of this enzyme. From the medium of cultured mouse fibroblasts, consisting of ammonium sulfate precipitation, gel filtration and affinity chromatography on a lysyl‐Sepharose column, followed by chromatography on a column of Sepharose coupled to the carboxy‐terminal propeptide of type I procollagen (PP‐Sepharose). This procedure yielded a practically homogeneous, 18 500‐fold‐purified enzyme preparation and the molecular mass of the purified C‐proteinase as determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis was 80 kDa. The lysyl‐Sepharose step separated the enzyme from the majority of the contaminating proteins, including a 55‐kDa protein which was further purified by PP‐Sepharose chromatography and identified as an additional form of the 36‐kDa and 34‐kDa procollagen C‐proteinase enhancer proteins described before [Adar et al. (1986) Collagen Relat. Res. 6, 267–277]. It enhanced the C‐proteinase activity, bound to the carboxyl propeptide of type I procollagen, cross‐reacted immunologically with the 36‐kDa as well as the 34‐kDa enhancer proteins, and in common with the latter proteins, it was glycosylated. In the course of PP‐Sepharose chromatography, a large proportion of the 55‐kDa protein disappeared with the concomitant appearance of the smaller enhancer proteins. All these findings suggest that the 55‐kDa protein is a precursor of the low molecular mass enhancer proteins. Also suggested from this study is that lysyl‐Sepharose chromatography is a highly beneficial purification step which may find use in the purification of the C‐proteinase from other sources as well.

Original languageEnglish
Pages (from-to)115-121
Number of pages7
JournalEuropean Journal of Biochemistry
Volume186
Issue number1-2
DOIs
StatePublished - Dec 1989

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