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Maltose frequently occurs as intermediate of the central carbon metabolism of prokaryotic and eukaryotic cells. Various mutants possess elevated maltose levels. Maltose exists as two anomers, (alpha- and beta-form) which are rapidly interconverted without requiring enzyme-mediated catalysis. As maltose is often abundant together with other oligoglucans, selective quantification is essential. In this communication, we present a photometric maltose assay using 4-alpha-glucanotransferase (AtDPE2) from Arabidopsis thaliana. Under in vitro conditions, AtDPE2 utilizes maltose as glucosyl donor and glycogen as acceptor releasing the other hexosyl unit as free glucose which is photometrically quantified following enzymatic phosphorylation and oxidation. Under the conditions used, DPE2 does not noticeably react with other di- or oligosaccharides. Selectivity compares favorably with that of maltase frequently used in maltose assays. Reducing end interconversion of the two maltose anomers is in rapid equilibrium and, therefore, the novel assay measures total maltose contents. Furthermore, an AtDPE2-based continuous photometric assay is presented which allows to quantify beta-amylase activity and was found to be superior to a conventional test. Finally, the AtDPE2-based maltose assay was used to quantify leaf maltose contents of both Arabidopsis wild type and AtDPE2-deficient plants throughout the light-dark cycle. These data are presented together with assimilatory starch levels. (C) 2017 Published by Elsevier Inc.
Metabolites and lipids are the final products of enzymatic processes, distinguishing the different cellular functions and activities of single cells or whole tissues. Understanding these cellular functions within a well-established model system requires a systemic collection of molecular and physiological information. In the current report, the green alga Chlamydomonas reinhardtii was selected to establish a comprehensive workflow for the detailed multi-omics analysis of a synchronously growing cell culture system. After implementation and benchmarking of the synchronous cell culture, a two-phase extraction method was adopted for the analysis of proteins, lipids, metabolites and starch from a single sample aliquot of as little as 10-15million Chlamydomonas cells. In a proof of concept study, primary metabolites and lipids were sampled throughout the diurnal cell cycle. The results of these time-resolved measurements showed that single compounds were not only coordinated with each other in different pathways, but that these complex metabolic signatures have the potential to be used as biomarkers of various cellular processes. Taken together, the developed workflow, including the synchronized growth of the photoautotrophic cell culture, in combination with comprehensive extraction methods and detailed metabolic phenotyping has the potential for use in in-depth analysis of complex cellular processes, providing essential information for the understanding of complex biological systems.
Functional interactions of plastidial phosphorylase (Phol) and starch branching enzymes (BEs) from the developing rice endosperm are the focus of this study. In the presence of both Phol and BE, the same branched primer molecule is elongated and further branched almost simultaneously even at very low glucan concentrations present in the purified enzyme preparations. By contrast, in the absence of any BE, glucans are not, to any significant extent, elongated by Phol. Based on our in vitro data, in the developing rice endosperm, Phol appears to be weakly associated with any of the BE isozymes. By using fluorophore-labeled malto-oligosaccharides, we identified maltose as the smallest possible primer for elongation by Phol. Linear dextrins act as carbohydrate substrates for BEs. By functionally interacting with a BE, Phol performs two essential functions during the initiation of starch biosynthesis in the rice endosperm: First, it elongates maltodextrins up to a degree of polymerization of at least 60. Second, by closely interacting with BEs, Phol is able to elongate branched glucans efficiently and thereby synthesizes branched carbohydrates essential for the initiation of amylopectin biosynthesis.
Lafora disease (LD, OMIM #254780) is a rare, recessively inherited neurodegenerative disease with adolescent onset, resulting in progressive myoclonus epilepsy which is fatal usually within ten years of symptom onset. The disease is caused by loss-of-function mutations in either of the two genes EPM2A (laforin) or EPM2B (malin). It characteristically involves the accumulation of insoluble glycogen-derived particles, named Lafora bodies (LBs), which are considered neurotoxic and causative of the disease. The pathogenesis of LD is therefore centred on the question of how insoluble LBs emerge from soluble glycogen. Recent data clearly show that an abnormal glycogen chain length distribution, but neither hyperphosphorylation nor impairment of general autophagy, strictly correlates with glycogen accumulation and the presence of LBs. This review summarizes results obtained with patients, mouse models, and cell lines and consolidates apparent paradoxes in the LD literature. Based on the growing body of evidence, it proposes that LD is predominantly caused by an impairment in chain-length regulation affecting only a small proportion of the cellular glycogen. A better grasp of LD pathogenesis will further develop our understanding of glycogen metabolism and structure. It will also facilitate the development of clinical interventions that appropriately target the underlying cause of LD.