Identification of the acrasin of Dictyostelium minutum as a derivative of folic acid

The acrasin of the slime mold Dictyostelium minutum was isolated from aggregating cells and purified. The compound was species specific and more active in the aggregative than in the vegetative stage. Three observations strongly suggest a structural relationship between the acrasin and folic acid. (1) Folic acid inhibited acrasin degradation by D. minutum. (2) Methotrexate, an antagonist of chemotaxis towards folic acid, also inhibited the response to the acrasin. (3) The chemotactic response to an excess of folic acid was delayed. The response was also delayed to simultaneously tested low amounts of a related compound, but not to unrelated compounds (Van Haastert, 1982). The response to the acrasin was observed to be delayed by excess of folic acid. The acrasinase was identified as a folic acid C9-N10 splitting enzyme. Based on chromatographic properties and biological activity of the acrasin and folate derivatives, the chemical structure of the acrasin is discussed.     

Determination of the active portion of the folic acid molecule in cellular slime mold chemotaxis.

From earlier work it is known that folic acid attracts the amoebae of various species of cellular slime molds (11). Here we have tested a wide variety of pteridines, pyrimidines, and pyrazines to determine what part of the folic acid molecule is chemotactically active. It was shown that the activity lies in the pteridine ring itself. Furthermore, the cell-free supernatants of slime mold amoebae contain an enzyme that renders pterin and folic acid chemotactically inactive, which apparently increases the chemotactic sensitivity of the amoebae to those compounds. Despite the fact that slime mold amoebae secrete small amounts of folic acid-related compounds, there is no evidence that folates are acrasins; rather it is postulated that attraction to folates may be a food-seeking device for the amoebae which prey on folate-secreting bacteria in the soil.     

Substrate specificity of cyclic nucleotide phosphodiesterase from beef heart and from Dictyostelium discoideum

The substrate specificity of beef heart phosphodiesterase activity and of the phosphodiesterase activity at the cell surface of the cellular slime mold Dictyostelium discoideum has been investigated by measuring the apparent Km and maximal velocity (V) of 24 derivatives of adenosine 3',5'-monophosphate (cAMP). Several analogs have increased Km values, but unaltered V values if compared to cAMP; also the contrary (unaltered Km and reduced V) has been observed, indicating that binding of the substrate to the enzyme and ring opening are two separate steps in the hydrolysis of cAMP. cAMP is bound to the beef heart phosphodiesterase by dipole-induced dipole interactions between the adenine moiety and an aromatic amino acid, and possibly by a hydrogen bond between the enzyme and one of the exocyclic oxygen atoms; a cyclic phosphate ring is not required to obtain binding. cAMP is bound to the slime mold enzyme via a hydrogen bond at the 3'-oxygen atom, and probably via a hydrogen bond with one of the exocyclic oxygen atoms. A cyclic phosphate ring is necessary to obtain binding to the enzyme. A specific interaction (polar or hydrophobic) between the base moiety and the enzyme has not been demonstrated. A negative charge on the phosphate moiety is not required for binding of cAMP to either enzyme. The catalytic reaction in both enzymes is restricted to the phosphorus atom and to the exocyclic oxygen atoms. Substitution of the negatively charged oxygen atom by an uncharged dimethylamino group in axial or equatorial position renders the compound non-hydrolyzable. Substitution of an exocyclic oxygen by a sulphur atom reduces the rate of the catalytic reaction about 100-fold if sulphur is placed in axial position and more than 10000-fold if sulphur is placed in equatorial position. A reaction mechanism for the enzymatic hydrolysis of cAMP is proposed.     

Evidence for a second chemotactic system in the cellular slime mold, Polysphondylium.

An unidentified substance(s) in a commercial guanosine 3′,5′-cyclic monophosphoric acid (cyclic 3′,5′-GMP) preparation is effective in attracting the aggregating amoebae of the cellular slime mold, Polysphondylium pallidum. Bacterial extracts (Escherichia coli) and amoeba extracts (P. pallidum) attract both vegetative and aggregating amoebae. A crude enzyme preparation from amoebae is capable of reducing the chemotactic activity of the extracts on aggregating amoebae and eliminating the activity of the unknown substance in the commercial cyclic 3′,5′-GMP preparation. As only the extracts were shown to contain folic acid, and since the enzyme does not reduce folic acid activity, it is suggested that the extracts contain a factor (possibly folic acid) primarily active on vegetative amoebae and an acrasin. The commercial cyclic 3′,5′-GMP preparation contains only an acrasin. The acrasin is heat stable and nondialyzable.     

Folic acid and pterin deaminases in Dictyostelium discoideum: kinetic properties and regulation by folic acid, pterin, and adenosine 3'',5''-phosphate.

Kinetic data obtained for deamination of pterin by the extracellular fraction from Dictyostelium discoideum yielded apparently linear Lineweaver-Burk plots for pterin. The Michaelis constant for pterin was 30 microM. The data for folic acid deamination yielded convex Lineweaver-Burk plots. Convex Lineweaver-Burk plots could result from the presence of two types of enzymes with different affinities. The data for folic acid deamination were analyzed mathematically for two types of enzymes. This analysis produced Michaelis constants for folic acid of 1.8 and 23 microM competition studies suggested that an enzyme with low affinity nonspecifically catalyzed the deamination of folic acid and pterin, whereas an enzyme with high affinity was a specific folic acid deaminase. A specific folic acid deaminase with high affinity appeared to be present on the surface of D. discoideum cells. The Michaelis constant for this enzyme was 2.6 microM. Cells growing in nutrient broth and cells starved in phosphate buffer released folic acid and pterin deaminases. The quantity of deaminase activities released by the cells appeared to be controlled by chemoattractants. Starving cells that were supplied with folic acid, pterin, or adenosine 3',5'-phosphate increased their extracellular folic acid and pterin deaminase activities to a larger extent than did cell suspensions to which no chemoattractants were added. Administration of folic acid or pterin to starving cells caused increases of the activity of extracellular adenosine 3',5'-phosphate phosphodiesterase and repressed increases of the activity of phosphodiesterase inhibitor.     

A cell surface-localized acetylcholinesterase in the cellular slime mold Polysphondylium violaceum

Abstract The cellular slime mold Polysphondylium violaceum was found to synthesize a plasma membrane-bound acetylcholinesterase that is located on the surface of growing amoebae. Enzyme activity declines as cells approach stationary phase and is undetectable 2h after asexual differentiation has begun. The enzyme has a K _m value of approx. 46 μM and a V _max of 3 pmol · min⁻¹ (10⁷ cells)⁻¹. It shows substrate inhibition by acetylcholine and is inhibited by the true cholinesterase inhibitor BW248c51 but is weakly inhibited by N, N '-diisopropylphosphorodiamidic anhydride ( iso -OMPA), a pseudocholinesterase inhibitor. These results expand upon an earlier study which suggested that P. violaceum amoebae are able to respond to acetylcholine and other pharmacologically related compounds. Both studies suggest that an acetylcholine-based sensory system might operate during growth and early stages of P. violaceum differentiation.     

α-Mannosidase and Microcyst Differentiation in the Cellular Slime Mold Polysphondylium pallidum

The intracellular and extracellular pattern of α-mannosidase (EC 3.2.1.24) activity was studied during microcyst differentiation in the cellular slime mold, Polysphondylium pallidum. The evidence suggests that microcyst differentiation requires continuous protein synthesis. α-Mannosidase activity is present in amoebae and increases with differentiation, and the data indicate that this increase in activity requires concurrent protein synthesis. The enzyme is excreted during the differentiation process, and the release of the enzyme is not stopped by cycloheximide. A cystless mutant does not show the normal intracellular pattern of α-mannosidase but does excrete the enzyme. Microcyst differentiation is proposed as an alternative system to multicellular slime mold development for the biochemical analysis of certain aspects of cellular differentiation.     

Purification and characterization of a neutral ceramidase from mouse liver. A single protein catalyzes the reversible reaction in which ceramide is both hydrolyzed and synthesized

We report here a novel ceramidase that was purified more than 150, 000-fold from the membrane fraction of mouse liver. The enzyme was a monomeric polypeptide having a molecular mass of 94 kDa and was highly glycosylated with N-glycans. The amino acid sequence of a fragment obtained from the purified enzyme was homologous to those deduced from the genes encoding an alkaline ceramidase of Pseudomonas aeruginosa and a hypotheical protein of the slime mold Dictyostelium discoideum. However, no significant sequence similarities were found in other known functional proteins including acid ceramidases of humans and mice. The enzyme hydrolyzed various N-acylsphingosines but not galactosylceramide, sulfatide, GM1a, or sphingomyelin. The enzyme exhibited the highest activity around pH 7.5 and was thus identified as a type of neutral ceramidase. The apparent K(m) and V(max) values for C12-4-nitrobenzo-2-oxa-1, 3-diazole-ceramide and C16-(14)C-ceramide were 22.3 microM and 29.1 micromol/min/mg and 72.4 microM and 3.6 micromol/min/mg, respectively. This study also clearly demonstrated that the purified 94-kDa ceramidase catalyzed the condensation of fatty acid to sphingosine to generate ceramide, but did not catalyze acyl-CoA-dependent acyl-transfer reaction.     

Developmental Regulation of α-Mannosidase in Dictyostelium discoideum

The specific activity of alpha-mannosidase (EC 3.2.1.24) has been found to increase more than a thousandfold during development of the cellular slime mold, Dictyostelium discoideum. The enzyme accumulates in both spore and stalk cells. Studies with preferential inhibitors of macromolecular synthesis indicate that accumulation of alpha-mannosidase requires concomitant protein synthesis and prior ribonucleic acid synthesis. Control of the period of synthesis by the overall developmental program is demonstrated in two temporally deranged morphological mutants. alpha-Mannosidase is found in lysosomes of D. discoideum in association with other acid hydrolases which may be involved in metabolism of extracellular polysaccharide.     

Chemotaxis in Dictyostelium discoideum: effect of concanavalin A on chemoattractant mediated cyclic gmp accumulation anlight scattering decrease.

In cells of the cellular slime mold Dictyostelium discoideum concanavalin A (Con A), at a concentration of 100 microgram per ml, inhibits folic acid and cyclic AMP induced decrease in light scattering. Con A has no effect on folic acid mediated cyclic GMP accumulation and increases cyclic AMP mediated cyclic GMP accumulation two-fold. At a lower Con A concentration, 10 microgram per ml, changes in light scattering induced by folic acid are normal and cyclic AMP induces a monophasic instead of a biphasic response. The stimulatory effect of Con A on cyclic AMP mediated cyclic GMP accumulation is still observable at 10 microgram Con A per ml. When cells are repeatedly stimulated with cyclic AMP, a decrease in light scattering without being accompanied by changes in cyclic GMP concentration is observed. Based on these results a model for chemotaxis is proposed.     

Folic acid deaminase activity during development in Dictyostelium discoideum.

Folic acid attracts vegetative amoebae of Dictyostelium discoideum. Secreted by bacteria, it may act as a food-seeking device. The inactivation of this attractant is catalyzed by a deaminase. As assay has been developed to measure the folic acid deaminase activity. In addition to cell-surface an intracellular deaminase, the amoebae of D. discoideum release the enzyme into the medium. The pH optimum of the extracellular enzyme was 6.0, and higher for the cell-associated deaminases. The extracellular enzyme was secreted maximally by vegetative amoebae, and its activity diminished during cell differentiation. The cell-surface bound enzyme was less active than the extracellular enzyme, and its activity decreased twofold during a 6-h starvation period. The enzyme activity of homogenates and 48,000 x g pellets diminished during this period 35 to 40%. The supernatant of a homogenate had a higher deaminase activity than the homogenate itself or its pellet; this suggests the presence of an inhibitor in the particulate fraction. The underlying mechanism for inactivation of folic acid has similar characteristics as that for inactivation of cyclic adenosine monophosphate.     

Participation of calcium in the induction of phosphodiesterase by cyclic adenosine 3',5'-monophosphate in Dictyostelium discoideum

The effects of divalent cations on the induction of phosphodiesterase [EC 3.1.4.17] by cyclic adenosine 3',5'-monophosphate (cyclic AMP) were studied in Dictyostelium discoideum. When cells were incubated with 1 mM ethylene glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) in 20 mM Tris-HCl buffer, pH 7.5, for 2 h, the induction of cellular phosphodiesterase was inhibited by about 80%, and that of extracellular phosphodiesterase by about 65%. When cells were incubated with 1 mM EGTA for 1 h, 2 mM CaCl2 was added and the cells were further incubated for 1 h, the activities of cellular and extracellular phosphodiesterases were increased about 5 and 2.5 times, respectively, compared with those in the EGTA-inhibited cells. Although various other kinds of divalent cations were also studied, Ca2+ had the greatest effect on the induction. These results suggest that Ca2+ may participate in the induction of phosphodiesterase, and thus in the regulation of the development of the cellular slime mold.     

Inhibition of mammalian xanthine oxidase by folate compounds and amethopterin

We have examined the effects of folate compounds and the folate analog amethopterin (methotrexate) as inhibitors of mammalian xanthine oxidase and have found that they offer potent inhibition of the enzyme. We have compared the inhibitory potency of folic acid and its coenzyme derivative tetrahydrofolic acid to that of allopurinol, a known inhibitor of xanthine oxidase, and have demonstrated that folic acid and tetrahydrofolic acid are severalfold more potent than allopurinol as inhibitors of xanthine oxidase. Comparative inhibition constants calculated were 5.0 X 10(-7) M for folic acid. 1.25 X 10(-6) M for tetrahydrofolic acid, and 4.88 X 10(-6) M for allopurinol. Incubation of xanthine oxidase with folic acid at a concentration of 10(-6) M abolished 94% of the enzymic activity within 1 min of incubation with the enzyme. At the same concentration, allopurinol was almost ineffective as an inhibitor of xanthine oxidase. The substrate xanthine protected the enzyme against total inhibition by folic acid. Reversibility of the enzymic inhibition by folic acid was demonstrated. Folic acid-inactivated enzyme was totally regenerated either by filtration through Sephadex G-200 or by precipitation with ammonium sulfate. 2-Amino-4-hydroxypteridine was a poor substrate for the enzyme but a potent inhibitor for the oxidation of xanthine by the enzyme. The inhibition constant calculated was 1.50 X 10(-6) M. In the presence of an excess of xanthine oxidase, neither folic acid nor tetrahydrofolic acid and allopurinol exhibited any change in intensity of their absorbance or in the wavelength of their maximal absorbance that might have been suggestive of substrate utility. The folate analog amethopterin was also determined a potent inhibitor of mammalian xanthine oxidase. The inhibition constant calculated was 3.0 X 10(-5) M.     

Change in ATP-pyrophosphohydrolase activity during spherule formation of Physarum polycephalum.

The activity of Ca2+-dependent ATP pyrophosphohydrolase was found to fluctuate during spherule formation of the acellular slime mold Physarum polycephalum under starving incubation. The enzyme activity increased up to 16-fold at the 3rd day of the starvation, then decreased drastically to less than its original level. Column chromatography of the enzyme preparation suggested that the increase in the activity was due to de novo synthesis of a new isozyme. Cycloheximide inhibited the synthesis. The two isozymes were different in their Ca2+ sensitivity, the new one being less sensitive.     

The intracellular location of adenylyl cyclase in the cellular slime molds Dictyostelium discoideum and Polysphondylium pallidum

Adenylyl cyclase activity was low or not detectable on intact cells and in isolated plasma membranes, phagocytic vacuoles and nuclei of the two slime mold species examined. The entire activity of homogenates was sedimentable and concentrated in a light membrane fraction. When this fraction was centrifugated through sucrose density gradients the adenylyl cyclase activity sedimented differently from all other enzymes measured. The gradient fractions with the highest specific activity of adenylyl cyclase consisted mainly of small vesicles. No changes in adenylyl cyclase distribution were associated with development. The possibility that cellular slime mold adenylyl cyclase activity is associated with vesicles in vivo, as already suggested by Maeda & Gerisch [10], is discussed.     

Characterization of a cDNA encoding subunit VI of cytochrome c oxidase from the slime mold Dictyostelium discoideum.

The primary structure of subunit VI of cytochrome c oxidase from the slime mold Dictyostelium discoideum has been determined by sequencing cDNA and N-terminus of the protein. The 92 amino acid residues long polypeptide (Mr = 10,535) shows homology with subunit IV of mammalian and subunit V of yeast cytochrome c oxidase. Though smaller and synthesized without a cleavable presequence, the slime mold oxidase subunit maintains the presence of a putative membrane spanning region.     

A protein kinase assayable with intact cells of the cellular slime mold Dictyostelium discoideum

A protein kinase activity assayable with whole cells of the slime mold is described. This activity is largely lost if the cells are disrupted by sonication or freeze-thawing. The cation and pH requirements of the enzyme are described. Neither cAMP nor dibutyryl cAMP stimulate the activity under a variety of conditions. In addition, cells harvested from different periods in the growth cycle or from different stages in development yielded the same activity per cell and showed no cAMP or dibutyryl cAMP effect. With histone type II as the substrate, the product of the reaction was judged to be a phosphorylated serine or threonine by its lability in hot alkali and its stability to hot acid.     

Antagonists of chemoattractants reveal separate receptors for cAMP, folk acid and pterin in Dictyostelium

Adenosine 3′,5′-monophosphate (cAMP), folic acid and pterin are chemoattractants in the cellular slime molds. The cAMP analog, 3′-amino-cAMP, inhibits a chemotactic reaction to cAMP at a concentration at which the analog is chemotactically inactive. The antagonistic effect of 3′-amino-cAMP on the chemotactic activity of cAMP is competitive, which suggests that 3′-amino-cAMP antagonizes cAMP via the chemotactic receptor for cAMP. 3′-Amino-cAMP does not antagonize folic acid or pterin. The binding of folic acid to post-vegetative Dictyostelium discoideum cells is inhibited by low concentrations of 2-deamino-2-hydro folic acid (DAFA [7]). DAFA is neither chemotactically active, nor does it inhibit a chemotactic reaction to folic acid. This questions the involvement of the main folic acid cell surface-binding sites in the chemotactic response to folic acid. The pterin analog, 6-aminopterin, is an antagonist of pterin, but not of cAMP or folic acid. Our results show that cAMP, folic acid and pterin are detected by different receptors. Furthermore, they suggest that the antagonistic action of 3′-amino-cAMP and 6-aminopterin is localized in the signal transduction pathway at a step before the signals from the separate receptors have arrived at a single pathway.     

Acetylglucosaminidase, an Early Enzyme in the Development of Dictyostelium discoideum

The specific activity of acetylglucosaminidase has been found to increase more than 10-fold during the first 10 hr of development in the cellular slime mold Dictyostelium discoideum. The specific activity then remained essentially constant until after germination. The activity was purified 36-fold and found to behave as a single protein species. The increase in specific activity required concomitant protein synthesis. If ribonucleic acid synthesis was preferentially inhibited during the period of synthesis of acetylglucosaminidase, further increase in enzymatic activity stopped after 2 hr. The increase in activity did not occur in a mutant strain which did not undergo the first step in morphogenesis. Mutant strains, blocked slightly later in morphogenesis, synthesized the enzyme at the normal rate but for an extended period. It was concluded that the initiation and termination of synthesis of acetylglucosaminidase are controlled by the developmental program.