Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.3.3.1 (citrate synthase)
 4,488 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The isolation of cell organelles from Dictyostelium discoideum was attempted using a variety of techniques. Cell homogenization (e.g. Potter-Elvehjem, glass beads) gave poor yields of organelles which were, in addition, exceptionally fragile and unstable in density gradients. An isolation method was developed using Triton X-100 in buffered sorbitol/Ficoll solutions at concentrations optimal for plasma membrane rupture. Immediately following cell lysis the solutions were diluted to sub-optimal Triton X-100 concentrations. Sedimentabilities of malate dehydrogenase, citrate synthetase, urate oxidase and catalase of around 55%, 40%, 35% and 55% respectively could be demonstrated using this method. The organelles were more resistant to breakage during resuspension following differential centrifugation and remained largely intact during density gradient centrifugation. The distribution of adenylate kinase activity in gradients showed that at least half the mitochondria retained an intact outer membrane. The mitochondria and peroxisomes could not be clearly separated using conventional sucrose-Ficoll density gradients. Separation was achieved by incubating the cell homogenate with succinate and a tetrazolium dye (2-p-iodophenyl-3-p-nitrophenyl-5-phenyl monotetrazolium chloride). Succinate dehydrogenase activity of mitochondria reduced the tetrazolium dye and the product (formazan) was deposited on the mitochondrial membranes ("heavy-labelling"). The mitochondria then sedimented to denser regions of the gradient while catalase distribution remained unchanged. The treatment left both organelles intact. The mitochondria (1.21 g/ml) were slightly denser than the peroxisomes (1.19 g/ml). The peroxisomes contained catalase and urate oxidase; no other hydrogen-peroxide-producing oxidases were detected. The slime mould urate oxidase resembled the mammalian enzyme. It had an apparent Km value of 12.5 muM, an optimum of activity at pH 8.5 in borate buffer and was competitively inhibited by trichloropurine.
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PMID:Mitochondria and peroxisomes from the cellular slime mould Dictyostelium discoideum. Isolation techniques and urate oxidase association with peroxisomes. 24 46

Non-mitochondrial citrate synthase catalyses citrate synthesis in the glyoxylate cycle in gluconeogenesis. Screening Dictyostelium discoideum mutants generated by insertional mutagenesis isolated a poor-growing mutant that displayed aberrant developmental morphology on bacterial lawns. Axenically grown mutants developed normally and formed mature fruiting bodies on buffered agar. The affected locus encoded a novel protein (CshA) that was homologous to glyoxysomal citrate synthase. cshA was expressed maximally during vegetative growth and gradually decreased through subsequent developmental stages. An in vitro citrate synthase assay revealed that cshA disruption resulted in a 50% reduction in enzyme activity, implicating CshA as an active citrate synthase. The amino-terminus of CshA was found to have an atypical mitochondrial targeting signal, instead containing a unique nonapeptide sequence (RINILANHL) that was homologous to the conserved peroxisomal targeting signal 2 (PTS2). CshA protein was shown to be localized in the peroxisomes, and the RINILANHL sequence only efficiently targeted the peroxisomal green fluorescent protein. The growth defect of cshA(-) cells was associated with the impairment of phagocytosis and fluid-phase endocytosis, independent from cytokinesis. Disrupted multicellular development on bacterial lawns resulted from the abnormal susceptibility to the environmental conditions, perhaps because of citrate insufficiency. Taken together, these results provide new insights into the function of peroxisomal citrate synthase in cell growth and multicellular development.
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PMID:Disruption of the peroxisomal citrate synthase CshA affects cell growth and multicellular development in Dictyostelium discoideum. 1522 5

In this study, a quantitative comparative proteomics approach has been used to analyze the Dictyostelium discoideum mitochondrial proteome variations during vegetative growth, starvation and the early stages of development. Application of 2-D DIGE technology allowed the detection of around 2000 protein spots on each 2-D gel with 180 proteins exhibiting significant changes in their expression level. In total, 96 proteins (51 unique and 45 redundant) were unambiguously identified. We show that the D. discoideum mitochondrial proteome adaptations mainly affect energy metabolism enzymes (the Krebs cycle, anaplerotic pathways, the oxidative phosphorylation system and energy dissipation), proteins involved in developmental and signaling processes as well as in protein biosynthesis and fate. The most striking observations were the opposite regulation of expression of citrate synthase and aconitase and the very large variation in the expression of the alternative oxidase that highlighted the importance of citrate and alternative oxidase in the physiology of the development of D. discoideum. Mitochondrial energy states measured in vivo with MitoTracker Orange CM Ros showed an increase in mitochondrial membrane polarization during D. discoideum starvation and starvation-induced development.
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PMID:Dynamics of the Dictyostelium discoideum mitochondrial proteome during vegetative growth, starvation and early stages of development. 2001 82