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Major changes in phosphorylation of chromatin-associated non-histone proteins accompany development in the slime mould Dictyostelium discoideum.

ABSTRACT: Striking changes in protein and RNA synthesis were shown to accompany development in the slime mould Dictyostelium discoideum. These changes are, at least in part, directly attributable to control at the level of transcription. Analysis of nuclear proteins and their states of phosphorylation by two-dimensional gel electrophoresis and autoradiography showed only minor changes in the species of proteins detectable by Coomassie Blue staining during the vegetative growth and development phases. Major changes, however, were detected in their patterns of phosphorylation, with major differences from the vegetative pattern occurring during both early development (0-2h) and late development (8-12h). These changes coincide with major changes in polypeptide synthesis and in nuclear and cytoplasmic RNA complexity.

SUBMITTER: Sinclair JH

PROVIDER: S-EPMC1145123 | biostudies-other | 1985 Aug

REPOSITORIES: biostudies-other

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Putrescine uptake by the cellular slime mould dictyostelium discoideum.

Project description:1. Rapid labelling occurs when myxamoebae of Dictyostelium discoideum strain AX2 are incubated with [1,4-14C]putrescine. Labelling is energy-dependent. 2. The label enters a pool from which rapid exchange with extracellular putrescine does not occur, and labelling is believed to represent uptake into the cells. 3. The concentration-dependence of putrescine uptake indicates that a number of systems are involved, at least one of which is saturable, with a Km of 9.1 micro M-putrescine. At high putrescine concentrations the overall uptake process is non-saturable. 4. Significant metabolism of putrescine and loss of intracellular putrescine to the medium only occurred when cells were incubated with millimolar concentrations of extracellular putrescine. 5. Putrescine uptake was inhibited by diamines, polyamines, bivalent metal ions and omega-aminocarboxylic acids. 6. The ability to take up putrescine at low concentrations decreased during starvation of myxamoebae. 7. The results are interpreted in terms of a model for putrescine uptake involving adsorptive pinocytosis at low concentrations and fluid-phase pinocytosis at high concentrations.

| S-EPMC1161026 | biostudies-other

A bacterial factor induces changes in cysteine proteinase forms in the cellular slime mould Dictyostelium discoideum.

Project description:The electrophoretic pattern of cysteine proteinases in axenically grown myxamoebae of Dictyostelium discoideum can be altered by the addition of either Gram-negative (Klebsiella aerogenes, Escherichia coli) or Gram-positive (Micrococcus lysodeikticus, Bacillus subtilis) bacteria to the culture. No changes occurred, however, if either yeast or latex beads were used in place of bacteria. The changes involved the simultaneous loss of proteinases characteristic of the axenic cells (the A-forms) and the acquisition of those found in cells which have been grown on bacteria (the B-forms). Using K. aerogenes the conversion was complete within 4 h. Extracellular proteinase activity was unaffected during this period. After the D. discoideum cells had been lysed, no equivalent change in proteinase band pattern could be produced either by prolonged incubation of cell extracts or by treatment with proteinases. An identical conversion could be induced in cultures of myxamoebae by a factor, cysteine proteinase converting factor (CPCF), present in the 15,000 g supernatant of a sonicated suspension of K. aerogenes. CPCF was macromolecular, as demonstrated by both ultrafiltration and gel filtration, acid-precipitable, but was soluble in ethanol or alkali. Its activity was unaffected by treatment with trypsin. The results suggested that CPCF might be a component of the bacterial cell wall, and since its activity was affected by lysozyme treatment, peptidoglycan is implicated. The results can be interpreted in terms of a novel nutrient-dependent post-translational change which affected most of the cysteine proteinases present in D. discoideum myxamoebae.

| S-EPMC1135067 | biostudies-other

Metabolism of the cellular slime mould Dictyostelium discoideum grown in axenic culture.

Project description:1. The DNA, RNA, protein and carbohydrate contents of myxamoebae of Dictyostelium discoideum strain Ax-2 were measured after growth on bacteria or in various axenic media. 2. Myxamoebae grown in the different axenic media have similar DNA, RNA and protein contents, but there are marked differences in the contents of glycogen and free sugars. The DNA and protein contents of myxamoebae grown on bacteria are different from those in myxamoebae grown axenically. 3. Approximately half the DNA found in myxamoebae grown on bacteria is of bacterial rather than of slime-mould origin. 4. The specific activities of some enzymes (including UDP-glucose pyrophosphorylase) are higher in myxamoebae grown axenically than in myxamoebae grown on bacteria. Nevertheless the characteristic increase in the specific activity of UDP-glucose pyrophosphorylase occurring during differentiation of cells of the wild-type strain NC-4 is also found in cells grown axenically. 5. The rate of amino acid oxidation during axenic growth of the myxamoebae is decreased when the cells are supplied with glucose.

| S-EPMC1179340 | biostudies-other

Growth of myxameobae of the cellular slime mould Dictyostelium discoideum in axenic culture.

Project description:1. A simple axenic medium suitable for the growth of the myxamoebae of a strain of the cellular slime mould Dictyostelium discoideum is described. 2. Procedures suitable for the growth of this strain in liquid and on solid media are described. 3. Conditions suitable for initiating the cell differentiation of myxamoebae grown axenically are described.

| S-EPMC1179339 | biostudies-other

Adenosine 3':5'-cyclic monophosphate-binding protein from the cellular slime mould Dictyostelium discoideum.

Project description:During the differentiation of myxamoebae of Dictyostelium discoideum strain Ax-2 grown in axenic medium there is a seven- to ten-fold increase in the specific activity of cyclic AMP-binding protein(s). Evidence is presented for the belief that cyclic AMP phosphodiesterase and cyclic AMP-binding protein are distinct molecular species.

| S-EPMC1177739 | biostudies-other

The purification and properties of extracellular glycosidases of the cellular slime mould Dictyostelium discoideum.

Project description:The purification of beta-N-acetylhexosaminidase, alpha-glucosidase, alpha-mannosidase and beta-glucosidase from the spent growth medium of Dictyostelium discoideum strain Ax-2 myxamoebae is described. beta-N-Acetylhexosaminidase and alpha-glucosidase were obtained in high yield and as homogeneous preparations whereas the alpha-mannosidase preparation consisted of two electrophoretically distinct isoenzymes. The physical, chemical and kinetic properties of these enzymes are described.

| S-EPMC1177668 | biostudies-other

The isolation and subfractionation of plasma membrane from the cellular slime mould Dictyostelium discoideum.

Project description:A procedure for the isolation and separation of three different subfractions of plasma membrane from the cellular slime mould Dictyostelium discoideum is described. The cells were disrupted by freeze-thawing in liquid N(2) and plasma membranes were purified by equilibrium centrifugation in a sucrose gradient. The cell surface was labelled with radioactive iodide by using the lactoperoxidase iodination method. Alkaline phosphatase was identified as a plasma-membrane marker by its co-distribution with [(125)I]iodide. 5'-Nucleotidase, which has been widely described as a plasma-membrane marker enzyme in mammalian tissues, was not localized to any marked extent in D. discoideum plasma membrane. The isolated plasma membranes showed a 24-fold enrichment of alkaline phosphatase specific activity relative to the homogenate and a yield of 50% of the total plasma membranes. Determination of succinate dehydrogenase and NADPH-cytochrome c reductase activities indicated that the preparation contained 2% of the total mitochondria and 3% of the endoplasmic reticulum. When the plasma-membrane preparation was further disrupted in a tight-fitting homogenizer, three plasma-membrane subfractions of different densities were obtained by isopycnic centrifugation. The enrichment of alkaline phosphatase was greatest in the subfraction with the lowest density. This fraction was enriched 36-fold relative to the homogenate and contained 19% of the total alkaline phosphatase activity but only 0.08% of the succinate dehydrogenase activity and 0.34% of the NADPH-cytochrome c reductase activity. Electron microscopy of this fraction showed it to consist of smooth membrane vesicles with no recognizable contaminants.

| S-EPMC1168002 | biostudies-other

Biochemical and cytological heterogeneity of the differentiating cells of the cellular slime mould Dictyostelium discoideum.

Project description:1. The slug stage of the cellular slime mould Dictyostelium discoideum has been shown to contain two types of cell, which differ in buoyant density. 2. These two cell types also differ in cytological appearance and histochemical behaviour and have very different enzymic activities. 3. Evidence is presented suggesting that the lighter of these two cell types corresponds to cells from the posterior region of the slug (pre-spore cells) and the heavier of the two to cells from the anterior region of the slug (pre-stalk cells).

| S-EPMC1184969 | biostudies-other

Incorporation of bisphosphonates into adenine nucleotides by amoebae of the cellular slime mould Dictyostelium discoideum.

Project description:Bisphosphonates are a class of synthetic pyrophosphate analogues. Some are known to be potent inhibitors of osteoclast-mediated bone resorption in vivo, but their mechanisms of action are unclear. The order of potency of bisphosphonates as inhibitors of bone resorption closely matches the order of potency as inhibitors of growth of amoebae of the slime mould Dictyostelium discoideum, indicating that bisphosphonates may have a mechanism of action that is similar in both osteoclasts and Dictyostelium. Methylenebisphosphonate and several halogenated derivatives, which have low potency as antiresorptive agents and as growth inhibitors of Dictyostelium, are metabolized intracellularly by Dictyostelium amoebae into methylene-containing adenine nucleotides. We have used a combination of n.m.r. and f.p.l.c. analysis to determine whether incorporation into nucleotides is a feature of other bisphosphonates, especially those that are potent antiresorptive agents. Only bisphosphonates with short side chains or of low potency are incorporated into adenine nucleotides, whereas those with long side chains or of high potency are not metabolized. Bisphosphonate metabolism in cell-free extracts of Dictyostelium was accompanied by inhibition of aminoacylation of tRNA by several aminoacyl-tRNA synthetases. These enzymes were barely affected by the bisphosphonates that were not metabolized. The results indicate that some bisphosphonates are not metabolically inert analogues of pyrophosphate and appear to be metabolized by aminoacyl-tRNA synthetases. The cellular effects of some bisphosphonates may be the result of their incorporation into adenine nucleotides or inhibition of aminoacyl-tRNA synthetases, although the potent bisphosphonates appear to act by a different mechanism.

| S-EPMC1137591 | biostudies-other

Enzyme synthesis in the cellular slime mould Dictyostelium discoideum during the differentiation of myxamoebae grown axenically.

Project description:1. Myxamoebae of the cellular slime mould Dictyostelium discoideum Ax-2 were grown on different media, and were harvested either in the stationary or exponential phases of the growth cycle to yield samples of myxamoebae differing in enzymic composition. 2. Morphogenesis and cell differentiation phenomena in D. discoideum appear to be similar in myxamoebae grown and harvested under different conditions. 3. The specific activity of the enzymes beta-N-acetylglucosaminidase, acid phosphatase, alpha-mannosidase, beta-glucosidase and alkaline phosphatase have been determined during cell differentiation of myxamoebae grown and harvested under different conditions. 4. The pattern of synthesis of these enzymes, all of which have been claimed to be part of the ;developmental programme', either remains unaffected despite the origin of the myxamoebae (alkaline phosphatase) or is qualitatively similar but quantitatively affected (acid phosphatase, beta-glucosidase) or is both qualitatively and quantitatively affected by changes in the myxamoebae (alpha-mannosidase, beta-N-acetylglucosaminidase). 5. The implications of these results for the concept of a ;developmental programme' are discussed.

| S-EPMC1178418 | biostudies-other

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