Structure elucidation of two differentiation inducing factors (DIF-2 and DIF-3) from the cellular slime mould Dictyostelium discoideum.
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ABSTRACT:
SUBMITTER: Morris HR
PROVIDER: S-EPMC1148792 | biostudies-other | 1988 Feb
REPOSITORIES: biostudies-other
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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
Project description:Stalk cell differentiation during development of the slime mould Dictyostelium is induced by a chlorinated alkyl phenone called differentiation-inducing factor-1 (DIF-1). Inactivation of DIF-1 is likely to be a key element in the DIF-1 signalling system, and we have shown previously that this is accomplished by a dedicated metabolic pathway involving up to 12 unidentified metabolites. We report here the structure of the first four metabolites produced from DIF-1, as deduced by m.s., n.m.r. and chemical synthesis. The structures of these compounds show that the first step in metabolism is a dechlorination of the phenolic ring, producing DIF metabolite 1 (DM1). DM1 is identical with the previously known minor DIF activity, DIF-3. DIF-3 is then metabolized by three successive oxidations of its aliphatic side chain: a hydroxylation at omega-2 to produce DM2, oxidation of the hydroxy group to a ketone group to produce DM3 and a further hydroxylation at omega-1 to produce DM4, a hydroxyketone of DIF-3. We have investigated the enzymology of DIF-1 metabolism. It is already known that the first step, to produce DIF-3, is catalysed by a novel dechlorinase. The enzyme activity responsible for the first side-chain oxidation (DIF-3 hydroxylase) was detected by incubating [3H]DIF-3 with cell-free extracts and resolving the reaction products by t.l.c. DIF-3 hydroxylase has many of the properties of a cytochrome P-450. It is membrane-bound and uses NADPH as co-substrate. It is also inhibited by CO, the classic cytochrome P-450 inhibitor, and by several other cytochrome P-450 inhibitors, as well as by diphenyliodonium chloride, an inhibitor of cytochrome P-450 reductase. DIF-3 hydroxylase is highly specific for DIF-3: other closely related compounds do not compete for the activity at 100-fold molar excess, with the exception of the DIF-3 analogue lacking the chlorine atom. The Km for DIF-3 of 47 nM is consistent with this enzyme being responsible for DIF-3 metabolism in vivo. The two further oxidations necessary to produce DM4 are also performed in vitro by similar enzyme activities. One of the inhibitors of DIF-3 hydroxylase, ancymidol (IC50 67 nM) is likely to be particularly suitable for probing the function of DIF metabolism during development.
| S-EPMC1136583 | biostudies-other
Project description:Previous work has led to the identification of a novel class of effector molecules [DIFs (differentiation-inducing factors) 1-3] released from the slime mould Dictyostelium discoideum. These substances induce stalk-cell differentiation in Dictyostelium discoideum and are thought to act as morphogens in the generation of the prestalk/prespore pattern during development. The DIFs are phenylalkan-1-ones, with chloro, hydroxy and methoxy substitution on the benzene ring. DIFs 1-3 and a number of their analogues have been synthesized by using a simple two-step procedure, and each analogue has been characterized by m.s., u.v. and n.m.r. spectroscopy. The crystal structure of synthetic DIF-1 [1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one, was investigated. The specific biological activity of each analogue was determined in a bioassay, where isolated Dictyostelium amoebae are induced to differentiate into stalk cells. The major biologically active substance, DIF-1, caused 50% stalk-cell differentiation at 1.8 x 10(-10) M; the C4 alkyl homologue (DIF-2) and C6 homologue possessed 40 and 16% of the activity of DIF-1 respectively. Further increase or decrease in the alkyl chain length resulted in a marked decrease in specific activity. The pattern of substitution on the benzene ring is a major determinant of bioactivity, since the specific activities of the 2,4-dihydroxy-6-methoxy and trihydroxy analogues were less than 1% of that of DIF-1. Substitution of bromine in DIF-1 had little effect on bioactivity; in contrast the activity of monochloro-DIF-1 (DIF-3) was diminished. There was no evidence for antagonism or synergy between DIF-1 and any of its analogues. This series of analogues will facilitate further studies in the biological effects and mode of action of DIF-1.
| S-EPMC1135362 | biostudies-other
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
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
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
Project description:1. The variation in cellular glycogen content of differentiating cells derived from myxamoebae that initially contained a wide range of glycogen contents (0.047-5.56mg of glycogen/10(8) myxamoebae) has been studied. 2. Myxamoebae that initially contained 0.047-3.62mg of glycogen/10(8) myxamoebae all gave rise to fruiting bodies that contained similar amounts of glycogen (0.06-0.11mg of glycogen/10(8) cells) but myxamoebae that initially contained 5.56mg of glycogen formed fruiting bodies containing 0.5mg of glycogen/10(8) cells. 3. Despite the high net rate of glycogen disappearance (during cell differentiation) from cells that contained more than 2mg of glycogen/10(8) cells initially, there were still significant variations in the rate of glycogen synthesis. The rate of glycogen synthesis reached a peak at the aggregation stage. 4. Evidence is presented showing that the rate of this synthesis of glycogen is controlled by factors other than the intracellular concentration of glycogen synthetase. 5. Our results are discussed in the context of the theory that the rates of glycogen synthesis and degradation act as a control mechanism for cell differentiation. 6. Criteria are discussed for deciding whether a biochemical event is causally or secondarily related to morphogenesis.
| S-EPMC1178420 | biostudies-other
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
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
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