ISOLATION AND CHARACTERIZATION OF PARAMYLON SYNTHASE FROM EUGLENA GRACILIS (EUGLENOPHYCEAE)^1,

Abstract

The aim of this study was to isolate and characterize the paramylon synthesizing enzyme from Euglena gracilis Klebs. A method for enzyme solubilization with high synthase activity using the zwitterionic detergent 3‐[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propane sulfonate is presented. Fractionated purification showed that the main enzyme activity was associated with the paramylon granula fraction, isolated from heterotrophically grown cells of E. gracilis. Further purification by sucrose density centrifugation resulted in a large enzyme complex with an apparent molar mass of 670 kDa (native). The complex remained active throughout the isolation procedures and produced beta‐1,3‐glucan in vitro. Two polypeptides of 37 and 54 kDa could be identified by photoaffinity labeling with [32P]‐UDP‐glucose as substrate after SDS‐PAGE.

The Transition from Glycogen to Starch Metabolism in Cyanobacteria and Eukaryotes

“…Strikingly, storage "-glucans in eukaryotes can thus be found, as is the case for '-glucans, in either watersoluble or solid crystalline form. The very few studies performed on paramylon metabolism suggest that such storage polysaccharides are synthesized from UDPGlc by a glycosyltransferase (Bäumer et al 2001). However, there is not one single report of a mutant, or antisense RNA or RNAi with decreased biochemical activity, associated with an alteration in "-glucan metabolism to confirm this reasonable hypothesis.…”

“…It must be stressed that "-1,3-linked storage "-glucans are widely distributed in the bikont branch of the eukaryotes (Fig. 4.1, see legend for definition of bikont) and that very few reports address the biochemistry of their synthesis or mobilization (Bäumer et al 2001;Goldemberg and Marechal 1963;Tomos and Northcote 1978;Vogel and Barber 1968). Most soluble "-glucan forms, such as laminarin, chrysolaminarin, and mycolaminarin, contain, in addition, "-1,6 branches, while the very highly crystalline paramylon found in Euglena (an excavate unicellular green alga) and Traditionally the eukaryotes are divided into the unikonts and bikonts which were previously thought to contain only uniflagellated cells or biflagellated cells.…”

Structural and physiological studies on the storage β-polyglucan of haptophyte Pleurochrysis haptonemofera

“…Concerning the enzymes involved in -polyglucan synthesis, paramylon synthase has been isolated from the paramylon granule membranes in the euglenophyte Euglena gracilis (Bäumer et al 2001). In the Bacillariophyceae, chrysolaminaran synthesis is suggested to be mediated through a pathway including the reactions of UDP-glucose pyrophosphorylase and glucosyl transferase, as in other organisms performing UDP-glucose-utilizing polyglucan synthesis (Roessler 1987(Roessler , 1988.…”

Biology of (1,3)-β-Glucans and Related Glucans in Protozoans and Chromistans

“…Biosynthesis of paramylon in E. gracilis involves a membrane-bound (1,3)--glucan synthase using UDP-glucose as substrate (Marechal and Goldemberg, 1964;Tomos and Northcote, 1978;Bäumer et al, 2001; see also Chapter 3.3.2). During photoheterotrophic growth, Calvayrac et al (1981) observed that mitochondria and chloroplasts develop a common network of vesicles in which paramylon granules are formed.…”