Partial purification and characterization of prolyl hydroxylase from the free-living nematode Panagrellus silusiae

Abstract

Prolyl hydroxylase was isolated from the free-living nematode Panagrellus silusiae by ammonium sulfate precipitation and calcium phosphate gel ion exchange from Triton X-100 treated homogenates. The enzyme preparation was highly purified but not to homogeneity as judged by agarose gel filtration and sodium dodecyl sulfate (SDS) – polyacrylamide gel electrophoresis. Gel filtration indicated that the molecular weight of the enzyme approximated 285 000. Analysis by SDS–acrylamide electrophoresis revealed subunits of ~67 000. Complete enzymic activity required α-ketoglutarate, Fe2+, ascorbate, catalase, atmospheric oxygen, and dithiothreitol. This activity was dramatically inhibited by α, α-dipyridyl, phenanthroline, and polyproline II. The purified enzyme preparation synthesized 50 to 100 μg hydroxyproline per milligram chick protocollagen per hour at 30 °C with an estimated Km for the substrate of 80 μg. Thus, a purified prolyl hydroxylase from a simple invertebrate possesses many of the properties of the prolyl hydroxylases of various vertebrates.