Localization of the enzyme system for glycosylation of proteins via the lipid-linked pathway in rough endoplasmic reticulum.

A crude preparation of microsomal membranes (postmitochondrial supernatant fraction) from the magnum portion of the hen oviduct was further subfractionated using a discontinuous sucrose gradient. Preparations of purified smooth surfaced membranes and rough endoplasmic reticulum, characterized by electron microscopy and nucleic acid content, were isolated. The enzymes involved in formation of mannose-containing glycoproteins via the lipid-linked pathway were shown to be localized in the rough endoplasmic reticulum. In contrast, a galactosyltransferase that catalyzed transfer of galactose to asialo-agalacto-orosomucoid was localized in the smooth membrane fraction. There was no evidence for the involvement of lipid intermediates in the galactosyl transfer observed in this fraction.

Phenylalanine ammonia lyase and cinnamic acid hydroxylases as assembled consecutive enzymes on microsomal membranes of cucumber cotyledons: Cooperation and subcellular distribution.

1. Cooperation between phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and cinnamic acid hydroxylases was investigated using microsomal fractions from cotyledons of cucumber (Cucumis sativus L.). The interpretations were based on experiments which demonstrate a limited exchange between the pool of cinnamic acid formed by the membrane-bound phenylalanine ammonia-lyase and the cinnamic acid pool external to the enzyme-membrane system. 2. The extent of cooperation between the microsomal enzymes was proved to be influenced by treatment of the cotyledons with light. On exposure to UV-light, which is known to enhance greatly the soluble phenylalanine ammonia-lyase activity in cell cultures, differential effects on the levels of microsomal and soluble phenylalanine ammonia-lyase, and of cinnamic acid hydroxylases, were observed. The time course of the enzyme activities and their cooperation in vitro after treatment of the cotyledons with light were studied. 3. The extent of cooperation in vitro was found to vary depending on the concentration of L-phenylalanine. 4. Homogenates obtained from etiolated cotyledons of Cucumis sativus in the absence of Mg(2+) were fractionated by sucrose density gradient centrifugation and examined for phenylalanine ammonia-lyase, cinnamic acid o-hydroxylase, cinnamic acid o-hydroxylase, and several marker enzymes. Ammonia-lyase activity was highest in fractions with 25% sucrose, in which primarily smooth endoplasmic reticulum is localized. Hydroxylase activities co-occur with phenylalanine ammonia-lyase in these fractions (density=1.100 g/cm(3)), and also in fractions at higher densities (d=1.12-1.13 and 1.15 g/cm(3)).

A model of closely assembled consecutive enzymes on membranes: formation of hydroxycinnamic acids from L-phenylalanine on thylakoids of Dunaliella marina.

p-Hydroxycinnamic acid was found to be located within the plastids of the green alga Dunaliella marina. Thylakoid fractions desintegrated by ultrasonic treatment were capable of converting L-phenylalanine into o- and p-hydroxycinnamic acids; the hydroxylation reaction was increased by addition of NADPH. Hydroxycinnamic acids produced when [3-14C]cinnamate was incubated with varying amounts of [4'-3H]L-phenylalanine exhibited a 3H/14C ratio 10-150 times higher than that of the cinnamic acid reisolated from the incubation mixture. The lack of equilibration between cinnamate formed from L-phenylalanine and cinnamate added to the solution supports the hypothesis that cinnamate as an intermediate in hydroxycinnamate formation remains bound to the membrane enzyme complex. A model of membrane-bound multienzyme complexes is proposed for the conversion of aromatic amino acids into phenols.

Formation of p-coumaric acid and o-coumaric acid from L-phenylalanine by microsomal membrane fractions from potato: Evidence of membrane-bound enzyme complexes.

The enzymes described here are the membrane-bound L-phenylalanine ammonia-lyase and cinnamate hydroxylase. Microsomes prepared from tubers of Solanum tuberosum L. are capable of converting L-phenylalanine into both o- and p-coumaric acid. Three microsomal fractions obtained by density gradient centrifugation were characterized by their equilibrium densities. Within various subfractions we found different patterns of distribution of the two enzymes, probably because of the extent of their fixed arrangement on a membrane area. Simultaneous incubation of the microsomal fraction with L-[4-ring-(3)H]-phenylalanine and trans-[3-(14)C]cinnamate indicated the existence of two pools of substrate available to cinnamate p-hydroxylase: cinnamate formed by the L-phenylalanine-ammonialyase reaction was a more effective substrate than cinnamate added to the incubation mixture. We conclude that the coumaric acids are formed from L-phenylalanine by a mechanism by which endogenously formed cinnamate is only partially equilibrated with exogenous cinnamate supplied in the incubation medium. This effect of enzyme cooperation is dependent on the integrity of membranes. The extent of cooperation was reduced by attempts to purify the microsomal membranes and by treatment in vivo with ethylene at high concentrations.