Morphology and structural integrity of fungal cells depend on cell wall polysaccharides. The chemical structure and biosynthesis of two types of these polysaccharides, chitin and (1-->3)-beta-glucan, have been studied extensively, whereas little is known about alpha-glucan. Here we describe the chemical structure of alpha-glucan isolated from wild-type and mutant cell walls of the fission yeast Schizosaccharomyces pombe. Wild-type alpha-glucan was found to consist of a single population of linear glucose polymers, approximately 260 residues in length. These glucose polymers were composed of two interconnected linear chains, each consisting of approximately 120 (1-->3)-linked alpha-d-glucose residues and some (1-->4)-linked alpha-D-glucose residues at the reducing end. By contrast, alpha-glucan of an alpha-glucan synthase mutant with an aberrant cell morphology and reduced alpha-glucan levels consisted of a single chain only. We propose that alpha-glucan biosynthesis involves an ordered series of events, whereby two alpha-glucan chains are coupled to create mature cell wall alpha-glucan. This mature form of cell wall alpha-glucan is essential for fission-yeast morphogenesis.
Lipoxygenases are key enzymes in the metabolism of unsaturated fatty acids. Soybean lipoxygenase-1 (LOX-1), a paradigm for lipoxygenases isolated from different sources, is composed of two domains: a ∼30 kDa N-terminal domain and a ∼60 kDa C-terminal domain. We used limited proteolysis and gel-filtration chromatography to generate and isolate a ∼60 kDa fragment of LOX-1 ("mini-LOX"), produced by trypsin cleavage between lysine 277 and serine 278. Mini-LOX was subjected to N-terminal sequencing and to electrophoretic, chromatographic, and spectroscopic analysis. Mini-LOX was found to be more acidic and more hydrophobic than LOX-1, and with a higher content of R-helix. Kinetic analysis showed that mini-LOX dioxygenates linoleic acid with a catalytic efficiency approximately 3-fold higher than that of LOX-1 (33.3 × 10 6 and 10.9 × 10 6 M -1 ‚s -1 , respectively), the activation energy of the reaction being 4.5 ( 0.5 and 8.3 ( 0.9 kJ‚mol -1 for mini-LOX and LOX-1, respectively. Substrate preference, tested with linoleic, R-linolenic, and arachidonic acids, and with linoleate methyl ester, was the same for LOX-1 and mini-LOX, and also identical was the regio-and stereospecificity of the products generated thereof, analyzed by reversed-phase and chiral high-performance liquid chromatography, and by gas chromatography/mass spectrometry. Mini-LOX was able to bind artificial vesicles with higher affinity than LOX-1, but the binding was less affected by calcium ions than was that of LOX-1. Taken together, these results suggest that the N-terminal domain of soybean lipoxygenase-1 might be a built-in inihibitor of catalytic activity and membrane binding ability of the enzyme, with a possible role in physio-(patho)logical conditions.Lipoxygenases are a family of monomeric non-heme, nonsulfur iron dioxygenases which catalyze the conversion of unsaturated fatty acids into conjugated hydroperoxides. Mammalian lipoxygenases have been implicated in the pathogenesis of several inflammatory conditions such as arthritis, psoriasis, and bronchial asthma (1). They have been also implicated in atherosclerosis (2), brain aging (3), HIV infection (4), and kidney disease (5, 6). In plants, lipoxygenases favor germination and participate in the synthesis of traumatin and jasmonic acid and in the response to abiotic stress (7). Recently, they have been also shown to initiate the programmed death (apoptosis) of plant cells (8). Soybean lipoxygenase-1 (LOX-1) 1 is widely used as a prototype for studying the homologous family of lipoxygenases from tissues of different species, both in structural (9-13) and in kinetic (14-18) investigations. The amino acid sequence (19) and three-dimensional structure (9, 10) of LOX-1 have been determined. The overall 839 amino acid residues of LOX-1, with a molecular mass of 93 840 Da, show 2 domains. The first 146 N-terminal amino acid residues form an 8-stranded -barrel, and the remaining 693 residues of the C-terminal domain are organized into 23 helices and 8 -strands. Mammalian lipoxygenases do ...
HPLC analysis of sialic acid released from recombinant variants of human tissue plasminogen activator, human chimeric plasminogen activator, human erythropoietin, and human follitropin, expressed in Chinese hamster ovary cells, demonstrates for each glycoprotein the presence of N‐acetylneuraminic and N‐glycolylneuraminic acid in a ratio of 97:3. Structural analysis by 500 MHz1H‐NMR spectroscopy, of the enzymatically released N‐linked carbohydrate chains of chimeric plasminogen activator and of erythropoietin, showed that α2‐3 linked N‐glycolylneuraminic acid can occur in different N‐acetyllactosamine type antennary structures.
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