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Chorismate synthase was purified 1200-fold from Euglena gracilis. The molecular mass of the native enzyme is in the range of 110 to 138 kilodaltons as judged by gel filtration. The molecular mass of the subunit was determined to be 41.7 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified chorismate synthase is associated with an NADPH-dependent flavin mononucleotide reductase that provides in vivo the reduced flavin necessary for catalytic activity. In vitro, flavin reduction can be mediated by either dithionite or light. The enzyme obtained from E. gracilis was compared with chorismate synthases purified from a higher plant (Corydalis sempervirens), a bacterium (Escherichia co/i), and a fungus (Neurospora crassa).These four chorismate synthases were found to be very similar in terms of cofactor specificity, kinetic properties, isoelectric points, and pH optima. All four enzymes react with polyclonal antisera directed against chorismate synthases from C. sempervirens and E. coli. The closely associated flavin mononucleotide reductase that is present in chorismate synthase preparations from E. gracilis and N. crassa is the main difference between those synthases and the monofunctional enzymes from C. sempervirens and E. coli.The three aromatic amino acids are synthesized via the shikimate pathway in microorganisms and plants (Fig. 1). Although the reaction sequence in the prechorismate pathway is identical in all organisms investigated so far, there are considerable differences in enzyme organization, as well as regulation, between organisms of different taxonomic groups. In Escherichia coli, the seven enzymatic activities of the prechorismate pathway are associated with monofunctional polypeptides. In contrast, in (reviewed in ref. 4). In Euglena gracilis, the organization of the prechorismate pathway appears to resemble that found in fungi. The first and the last steps are catalyzed by single enzymes (DAHP-synthase and chorismate synthase, respectively), whereas activities 2 to 6 form a large complex resembling the fungal arom complex (27). Euglena and fungi are strikingly similar in other biochemical pathways as well. Thus, both groups of organisms use the L-a-aminoadipate pathway for lysine biosynthesis, whereas bacteria, algae, and plants follow the diaminopimelate route (30). On the basis of such findings, a close evolutionary relationship was proposed for euglenoids and fungi. But aromatic biosynthesis shows several features that, among all organisms studied so far, are unique for E. gracilis. Anthranilate synthase, the first enzyme in the tryptophan branch ( Fig. 1), is monomeric in Euglena, whereas in all other anthranilate synthases characterized so far the chorismate and glutamine binding sites reside on distinct polypeptide chains (16). The synthesis of tryptophan from anthranilate is catalyzed by a single protein in Euglena (17), but in all other known examples tryptophan synthase is separable from the other activities of the tryptophan branch (17). Unlike ot...

Phosphoinositide specific phospholipase C from rabbit fast skeletal muscle has been enriched ca. 1,000‐fold with a specific activity of 40 μmol × min−1 × mg−1. Following SDS‐PAGE, renaturation of the enzyme protein in the presence of deoxycholate allowed the determination of an apparent molecular weight of 110 kDa. Gel‐filtration of the native enzyme resulted in a very similar apparent molecular weight of 115 kDa, however, associated proteins of higher molecular weight were also found. Free Ca2+ concentrations needed for half‐maximal activation of PtdIns(4,5)P2, PtdIns4P and PtdIns hydrolysis are 6.3 μM, 85 μM and 1.8 mM, and the Km values for these substrates 102, 340 and 937 μM, respectively.