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Biomedical subjects

P Brachet

Publications and source records attributed to P Brachet.

At least 109 records · Page 6Linked to original sources
PubMed1981

The hydrophobic character of the membrane-bound phosphodiesterase from Dictyostelium discoideum.

A phosphodiesterase activity is shown to copurify with the plasma membrane fraction prepared by the two-phase partition method. The enrichment in phosphodiesterase parallels that of alkaline phosphatase, which is thought to be a typical membranous enzyme. Up to 66% of the phosphodiesterase activity can be solubilized by a treatment with 0.2% Triton X-100. Higher doses were ineffective in solubilizing more activity. Analysis by native gel electrophoresis showed that an activity extracted by 2 M NaCl migrated at the same position as 'soluble' phosphodiesterase of cytosolic or extracellular origin. In contrast, the Triton-solubilized enzyme had an apparently higher molecular weight. When subjected to charge shift electrophoresis on agarose gels in the presence of an ionic detergent, the Triton-solubilized phosphodiesterase displayed a hydrophobic character. This behaviour contrasts with that of 'soluble' phosphodiesterases, the electrophoretic mobility of which is unaffected by the presence of an anionic detergent. The hydrophobic character of the membranous enzyme was lost after gentle hydrolysis by papain.

Cell Membrane↗
PubMed1980

Non-michaelian kinetics of adenylate cyclase in Dictyostelium discoideum.

Dependance of adenylate cyclase activity from "aggregation-component" Dictyostelium discoideum cells on ATP concentration has been examined. The enzyme exhibits on-Michaelian kinetics towards its substrate, from which two apparent Km values of 17 micro M and 0.4 mM can be calculated. Several experiments were performed in order to test for a difference between adenylate cyclase activities measured at ATP concentrations corresponding to each Km. Activities were associated with the particulate fraction of the extracts; however attempts have failed to recover them from purified plasma membranes. Loss of activity in cellular extracts, its stimulation by Mn2+ ions, and the increase in basal specific activity during differentiation were identical whether the enzyme was tested at either of the ATP concentrations. Although these experiments do not exclude the possible existence of two distinct molecular entities, they rather favour the hypothesis of an adenylate cyclase exhibiting anti-cooperative kinetics towards ATP.

Adenosine Triphosphate↗
PubMed1980

A separate phosphodiesterase for the hydrolysis of cyclic guanosine 3',5'-monophosphate in growing Dictyostelium discoideum amoebae.

The presence of a cyclic guanosine 3',5'-monophosphate (cGMP) phosphodiesterase in mutant Dictyostelium discoideum amoebae, deficient in cyclic adenosine 3',5'-monophosphate (cAMP) phosphodiesterase is demonstrated. This enzyme shows a high affinity for cGMP and is not affected by cAMP at concentrations of up to 0.3 mM. It is activated by Mg2+ and Mn2+, and displays no apparent heterogeneity. Biochemical analysis of wild-type amoebae revealed the presence of a cGMP-specific phosphodiesterase sharing several common characteristics with the enzyme of the mutant amoebae. From the evidence presented in this report, it is concluded that the cGMP and cAMP phosphodiesterases of D. discoideum are under separate genetic control.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1979

Multiple forms of an extracellular cyclic-AMP phosphodiesterase from Dictyostelium discoideum.

The extracellular cyclic-AMP phosphodiesterase of a mutant of Dictyostelium discoideum which accumulates this enzyme was found to exist in multiple forms. Using the isoelectric focusing technique the phosphodiesterase activity was distributed into three peaks with isoelectric points of 4.6, 6.5 and 8.3, designated as p4, p6 and p8. Gel filtration and sucrose gradient analysis showed that the p4 activity consisted of two forms of different sedimentation coefficients. At high enzyme concentrations, the heavy form was favored. Dilution of enzyme activity shifted the equilibrium toward the light form. Direct analysis by sucrose gradient sedimentation of all isoelectric forms demonstrated that besides p4, p6 activity also existed as a mixture of the heavy (9.7 S) and the light (5.4 S) components. In contrast, the p8 activity displayed only the light form. The heterogeneity of the p4 and p6 isoelectric forms was also observed by polyacrylamide gel electrophoresis. A procedure for a partial purification of the extracellular enzyme to about 70-fold is presented.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1979

Cyclic GMP and cyclic AMP changes in response to folic acid pulses during cell development of Dictyostelium discoideum.

Folic acid pulses induced developmental processes in agip 71, a morphogenetic mutant of Dictyostelium discoideum, strain Ax-2. Cells that had received folic acid pulses were able to form EDTA-stable cell aggregates and to complete full differentiation to fruiting bodies. In these cells no autonomous periodic activities were observed by light scattering. Folic acid pulses elicited increases in the concentrations of cyclic GMP and cyclic AMP. In undifferentiated cells, folic acid caused a rapid increase in the level of cyclic GMP without a significant change in the level of cyclic AMP. In an advanced developmental state folic acid caused an increase in cyclic AMP in addition to two successsive peaks of cyclic GMP. Experiments performed with the parent strain, Ax-2, also showed that during the development towards aggregation competence, cells acquired the ability to produce a cyclic AMP peak in response to folic acid.

Cell Adhesion↗
PubMed1978

The effect of divalent cations on aggregation of Dictyostelium discoideum.

Following the initiation of development, amoebae of Dictyostelium discoideum aggregate chemotactically toward cyclic AMP (cAMP). Adenyl cyclase, cAMP phosphodiesterase, and cAMP binding sites all increase 20--40 fold during the first few hours of development. It has been shown that addition of 1 mM EDTA and 5 mM MgCl2 accelerates the aggregation process. Likewise, the calcium ionophore, A23187, leads to precocious aggregation while 4 X 10(-5) M progesterone considerably delays it. These treatments have now been shown to result in increased accumulation of adenyl cyclase in the case of EDTA and Mg2+ or the ionophore and greatly decreased accumulation in the case of the steroid. Treatment with EDTA and Mg2+ or the ionophore has been shown not only to accelerate aggregation in wild-type amoebae but to overcome complete blocks to aggregation in certain mutant strains. We have found that addition of Mn2+ will also permit aggregation of mutant cells otherwise unable to aggregate. This divalent ion, unlike EDTA and Mg2+ or the ionophore, was shown to directly stimulate adenyl cyclase. Calcium ions were also found to affect the enzyme such that at Ca2+ concentrations found within the cells the great majority of the activity is inhibited. Manganese ions can overcome the inhibition by Ca2+. These findings show that conditions which stimulate aggregation result in increased activity of adenyl cyclase either by increased accumulation of the enzyme or by increased activity of the available enzyme, and support the proposed central role of adenyl cyclase in aggregation.

Adenylyl Cyclases↗
PubMed1978

The role of phosphodiesterase in aggregation of Dictyostelium discoideum.

The role of cAMP phosphodiesterase in the cAMP-mediated aggregation of the cellular slime mould Dictyostelium discoideum was investigated with a morphogenetic mutant defective in phosphodiesterase production. Mutant cells become capable of aggregating normally when incubated in the presence of exogenous phosphodiesterase isolated from Idictyostelium or rat brain. Direct contact between enzyme and the cell membrane is not required for this phenotypic suppression. The aggregateless character of this strain presumably results from an over-accumulation of cAMP in the extracellular medium since aggregation can be induced in the absence of added phosphodiesterase under conditions facilitating diffusion of the nucleotide. This suggests that phosphodiesterase is not involved in the generation or recognition of cAMP signals, but that the enzyme is essential in the control of the cAMP signal-to-noise ratio.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1976

Specificity of polarity suppression in E. coli: correction of defects in gene N, but not in gene Q, of phage lambda.

The bacterial mutation psuA1, known as (suA) a polarity suppressor, partially relieves all N defects in bacteriophage lambda growth. No evidence is found that psuA1 relieves Q defects in lambda growth. Specific mechanisms of action by the N and Q gene products are discussed. The psuA1 mutation was also found to suppress IS1 type but not IS2 type insertion mutations in lambda.

Coliphages↗
PubMed1976

[The effect of an ionophore on the aggregation of Dictyostelium discoideum].

Treatment during starvation of D. discoideum amoebae with micromolar amounts of A23187 causes an enhanced aggregation. Cells develop the properties of differentiated, aggregation competent amoebae earlier than untreated populations. Ionophore increases the release of calcium, and prevents the excretion of the phosphodiesterase ihibitor normally released in the media. A23187 suppresses the morphogenetic block of some aggregates mutants, suggesting that the ionophore either activates cAMP synthesis and excretion, or increases the cellular sensitivity to extracellular cAMP signals. This might result from the enhanced mobilisation of intracellular calcium

Calcimycin↗