Biosynthesis of Arabinogalactan-Protein in Lolium multiflorum (Ryegrass) Endosperm Cells : III. Subcellular Distribution of Prolyl Hydroxylase.

The peptidyl prolyl hydroxylase responsible for the formation of hydroxyproline during arabinogalactan-protein biosynthesis in Lolium multiflorum (ryegrass) endosperm cells is a membrane-associated enzyme which will catalyze the hydroxylation of poly(l-proline) in the presence of oxygen, alpha-ketoglutarate, ferrous ion, and ascorbate. The K(m) for poly(l-proline) (8000 molecular weight) is 40 micromolar. The enzyme will also hydroxylate the protocollagen analog (Pro-Pro-Gly)(5).4H(2)O.Fractionation of membranes from protoplast lysates on a discontinuous sucrose/sorbitol density gradient, followed by centrifugation on a linear sucrose gradient in the presence of Mg(2+), leads to a clear separation of a number of membrane components. The membrane components have been tentatively identified using marker enzymes and assayed for peptidyl prolyl hydroxylase. It is concluded that the ryegrass prolyl hydroxylase is enriched in Golgi-derived membranes, but that significant amounts are also located in other subcellular fractions, including the rough endoplasmic reticulum.