Search

The most recent developments in the use of hydrolytic enzymes - proteases, lipases and nitrilases - are summarized: Sterically demanding a,a-disubstituted carboxylic esters can be enzymatically resolved using a commercially available protease derived from Aspergillus oryzae. A significant enhancement of selectivity and activity of Candida cylindracea lipase in irreversible acyl transfer reactions may be achieved by removal of by-products or via covalent modification of the enzyme. Enzymatic hydrolysis of nitriles proceeds under mild conditions but enzyme-catalysed side- reactions such as ester- or epoxide-hydrolysis may occur.

The most recent developments in the use of hydrolytic enzymes - proteases, lipases and nitrilases - are summarized: Sterically demanding a,a-disubstituted carboxylic esters can be enzymatically resolved using a commercially available protease derived from Aspergillus oryzae. A significant enhancement of selectivity and activity of Candida cylindracea lipase in irreversible acyl transfer reactions may be achieved by removal of by-products or via covalent modification of the enzyme. Enzymatic hydrolysis of nitriles proceeds under mild conditions but enzyme-catalysed side- reactions such as ester- or epoxide-hydrolysis may occur.

In yeast, as in higher eukaryotic cells, sphingolipids are essential membrane components. The yeast, Saccharomyces cerevisiae, contains three classes of sphingolipids, inositolphosphorylceramide (InsP Cer), mannosylinositolphosphorylceramide (ManInsP Cer) and mannosyldiinositolphosphorylceramide (ManP Ins2P Cer). As a prerequisite to localize these sphingolipids in subcellular membranes, authentic standards of the respective lipids were isolated and characterized using biochemical methods and electrospray ionization mass spectrometry. The complete set of yeast subcellular membranes was isolated at high purity, and sphingolipids were extracted. InsP Cer, ManInsP Cer, and ManP Ins2P Cer were separated by thin-layer chromatography, stained and densitometrically scanned along with the respective standards. These methods enable a complete overview of the subcellular distribution of yeast sphingolipids to be obtained, as far as is known, for the first time. InsP Cer was highly enriched in Golgi and vacuolar membranes, whereas the largest amounts of ManInsP Cer and ManPIns2PCer were found in the plasma membrane. The presence of inositol-containing sphingolipids in organelles of the protein-secretory pathway strongly supports the notion that protein secretion and intracellular trafficking of sphingolipids are linked processes.

In the lower eukaryote, Saccharomyces cerevisiae,the inositol-containing ceramides inositolphosphate ceramide (IPC), mannosyl inositolphosphate ceramide (MIPC) and mannosyl diinositolphosphate ceramide (M(IP)2C) were shown to be the major sphingolipids (Wagner & Zofcsik, 1966; Steiner et al., 1969; Smith & Lester, 1974).

Organelles of the early protein secretion pathway (ER, Golgi) are involved in biosynthesis and intracellular migration of the yeast sphingolipids, inositolphosphorylceramide (IPC), mannosylinositolphosphorylceramide (MIPC), and mannosyldiinositolphosphorylceramide (M(IP)2C). Cycloheximide and nocodazole neither block biosynthesis of sphingolipids, nor ER to Golgi transport of IPC. In contrast, treatment of yeast cells with brefeldin A, which affects integrity of the Golgi, decreases formation of IPC and MIPC. Interruption of late steps of protein secretion (Golgi to plasma membrane transport) in temperature-sensitive secretory mutants prevents sphingolipids from being transported to the cell periphery.