Biosynthesis of Arabinogalactan-Protein in Lolium multiflorum (Ryegrass) Endosperm Cells

Abstract

The peptidyl prolyl hydroxylase responsible for the formation of hydroxyproline during arabinogalactan-protein biosynthesis in L. multiflorum (ryegrass) endosperm cells is a membrane-associated enzyme which will catalyze the hydroxylation of poly(L-proline) in the presence of O2, .alpha.-ketoglutarate, ferrous ion and ascorbate. The Km for poly(L-proline) (8000 MW) is 40 .mu.M. The enzyme will also hydroxylate the protocollagen analog (Pro-Pro-Gly)5 .cntdot. 4H2O. 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 Mg2+, leads to a clear separation of a number of membrane components. The membrane components were tentatively identified using marker enzymes and assayed for peptidyl prolyl hydroxylase. The ryegrass prolyl hydroxylase is enriched in Golgi-derived membranes, but significant amounts are also located in other subcellular fractions, including the rough endoplasmic reticulum.