An acrasin-like attractant from yeast extract specific for Dictyostelium lacteum.

The transition of the unicellular to the multicellular stage in Dictyostelium lacteum is not mediated by cyclic AMP. The attractant for aggregative amoebae of this cellular slime mold species was isolated from yeast extract and purified more than 1000-fold without a significant loss of activity. Several characteristics of the chemotactic molecule specific for D. lacteum are reported, and the presence of an inactivating enzyme has been demonstrated.     

Isolation and regulatory properties of two glutamate dehydrogenases from the cellular slime mold Dictyostelium discoideum.

Two distinct glutamate dehydrogenases are present in amoebae of the cellular slime mold Dictyostelium discoideum. One enzyme has been extracted from a crude mitochondrial fraction, and the other from an extramitochondrial cytoplasmic fraction. Both enzymes have been partially purified and characterized. The mitochondrial enzyme can utilize both NAD⁺ and NADP⁺ as coenzyme, while the extramitochondrial is NAD⁺ specific. When the mitondrial enzyme is assayed in the presence of either a rate-limiting or saturating concentration of glutamate, its activity is stimulated by both AMP and ADP and is inhibited by ATP. When the extramitochondrial enzyme is assayed in the presence of a rate-limiting concentration of glutamate, its activity is sensitive to modulation by a number of intermediates in carbohydrate metabolism and is inhibited by ADP, ATP, GTP, and CTP.     

Isolation and Characterization of an Extracellular Polysaccharide from Physarum polycephalum

The myxomycetes are called slime molds because of the synthesis of copious amounts of extracellular material (slime) during parts of the life cycle. In Physarum polycephalum, small amounts of slime are produced during exponential growth of microplasmodia in shake flasks, but the amount of this slime increased 10- to 20-fold at 16 to 34 hr after microplasmodia were induced to form spherules by transferring them to salt solution. The slime obtained during both periods is the same; an acidic polysaccharide consisting of galactose, sulfate, and trace amounts of rhamnose. Analysis of the galactose-to-sulfate ratio gave a value of about 4 to 1. Infrared spectroscopy showed increased absorbance at 820 cm⁻¹ characteristic of C-O-S vibrations. Electrophoresis on polyacrylamide gel revealed that the material moved as a single band which stained with Alcian Blue and periodic acid Shiff reagent. However, fractionation of identical material on Dowex columns and electrophoresis on cellulose acetate showed the slime to be made up of three major fractions. The polysaccharide appeared as an extracellular capsule closely adhering to the walls of the spherules. It could be separated from the wall by vigorous shaking. The increased synthesis of slime during spherulation was not blocked by cycloheximide, suggesting that new enzyme synthesis was not necessary for its formation.     

Poly(adenosine diphosphate ribose) glycohydrolase in Physarum polycephalum.

Poly(adenosine diphosphate ribose) glycohydrolase, which has thus far only been found in mammalian tissues, was found for the first time in the primitive eukaryotic slime mold Physarum polycephalum. The hydrolytic product of poly(adenosine diphosphate ribose) with this enzyme was identified as adenosine diphosphate ribose by paper and thin-layer chromatography. It is likely that the enzyme caused exoglycosidic hydrolysis. The optimal pH of this enzyme was 6.0, and the K_m value was 4.3 μm, as adenosine diphosphate ribose residues of polymer. Adenosine diphosphate ribose, ADP and ATP at a concentration of 0.1mm strongly inhibited the enzyme activity. 3′,5′-Cyclic AMP was inhibitory at a concentration of 1mm. The molecular weight of this enzyme was estimated to be 57,000.     

Chemical Characterization of Polysaccharide from the Slime Layer of the Cyanobacterium Microcystis flos-aquae C3-40

Macromolecular material from the slime layer of the cyanobacterium Microcystis flos-aquae C3-40 was defined as material that adhered to cells during centrifugation in growth medium but was dislodged by washing with deionized water and retained within dialysis tubing with a molecular-weight cutoff of 3,500. At each step of this isolation procedure, the slime was observed microscopically. Cells in the centrifugal pellet were surrounded by large amounts of slime that excluded negative stain, whereas cells that had been washed with water lacked visible slime. Two independently isolated lots of slime contained no detectable protein (<1%, wt/wt) and consisted predominantly of anthrone-reacting polysaccharide. Sugars in a hydrolysate of slime polysaccharide were derivatized with trimethylsilylimidazole and examined by gas chromatography-mass spectrometry. The composition of the slime polysaccharide was 1.5% (wt/wt) galactose, 2.0% glucose, 3.0% xylose, 5.0% mannose, 5.5% rhamnose, and 83% galacturonic acid. This composition resembles that of the plant polysaccharide pectin, which was treated in parallel as a control. Consistent with earlier indications that M. flos-aquae slime preferentially binds certain cations, the ratio of Fe to Na in the dialyzed slime was 10⁴ times that in the growth medium. The composition of the slime is discussed with respect to possible mechanisms of cation binding in comparison with other cyanobacterial exopolysaccharides and pectin.     

Intracellular and extracellular beta-N-acetylhexosaminidases from Physarum polycephalum. Comparison of vegetative and spherulation enzymes.

Vegetative microplasmodia of the slime mold, Physarum polycephalum, produce an intracellular β-N-acetylhexosaminidase enzyme when grown on a medium containing 1% glucose, 0.15% yeast extract, and 1% peptone. When early log-phase microplasmodia are induced to differentiate to spherules by starvation in a salts medium, they excrete an extracellular β-N-acetylhexosaminidase. Both of these enzymes have been purified to apparent homogeneity. Characterization studies showed that the extracellular enzyme was nonidentical to the preexisting, vegetative enzyme and the enzyme in completed spherules. Evidence demonstrating dissimilarities between the two proteins included marked differences in (i) specificities for several natural and synthetic substrates, (ii) various kinetic parameters, (iii) relative net charges as evidenced by different elution behavior from similar DE-52 cellulose chromatography columns, (iv) carbohydrate contents, and (v) subunit polypeptide molecular weights. Conclusive evidence for their nonidentity was shown in their respective amino acid compositions and divergent immunological properties. The extracellular β-N-acetylhexosaminidase demonstrated a subunit molecular weight of 25,300; the intracellular enzyme subunit molecular weight was 40,500. The extracellular enzyme, with the smaller polypeptide subunit, contained 1.79 times as many aromatic amino acid residues in tyrosine, phenylalanine, and tryptophan as the intracellular enzyme. Thus, the extracellular enzyme could not have been comprised of subunits derived from limited proteolytic hydrolysis of the larger subunits of the intracellular enzyme. Rabbit antisera prepared against each purified β-N-acetylhexosaminidase failed to yield precipitin bands with the heterologous antigen in immunodiffusion tests. Thus, apparently distinct structural genes code for these two enzymes and they may serve different, but unidentified, physiological functions.     

(1,3)-β-D-Glucan Synthase Activity in Mycelial and Cell Wall-less Phenotypes of the fz,sg, os-1 ("Slime") Mutant Strain of Neurospora crassa

Polizeli, M. L. T. M., Noventa-Jordāo, M. A., Marques da Silva, M., Jorge, J. A., and Terenzi, H. F. 1995. (1,3)-β-D-Glucan synthase activity in mycelial and cell wall-less phenotypes of the fz, sg, os-1 ("slime") mutant strain of Neurospora crassa. Experimental Mycology 19, 35-47. The cell wall-less fz, sg, os-1 ("slime") triple mutant of Neurospora crassa lacks (1,3)-βD-glucan synthase activity. fz, sg, os-1 segregants from slime × wild-type crosses initially germinate as a plasmodium (slime-like), but develop hyphae in a few hours and acquire a stable mycelial phenotype (mycelial intermediate). The cell wall-less phenotype (stable slime) can be reisolated from mycelial intermediates by filtration-enrichment selection in medium of high osmolarity. Pairs of mycelial intermediate and stable slime obtained from a single slime-like segregant were comparatively studied. Mycelial intermediate strains synthesize a cell wall with normal amounts of (1,3)-β-glucan, chitin, and other polysaccharides and possess (1,3)-β-glucan synthase activity with apparently normal properties (i.e., association with membranes, stability, K_m app, V_max, stimulation by GTP). The enzyme was dissociated by treatment with Tergitol NP-40 and NaCl into a membrane-bound catalytic center and a soluble factor which activates the enzyme in the presence of GTP. Heterologous reconstitution assays demonstrated that stable slime spheroplasts had normal activity of the soluble activating factor, but were severely deficient in membrane-bound activity. The genetic composition of the viable progeny of stable slime or mycelial intermediate × wild-type crosses failed to show differences between the two extreme phenotypes of slime. However, the analysis of heterokaryons demonstrated that the stable slime homokaryotic progeny of stable slime/wild-type heterokaryons were not viable. In contrast, the behavior of mycelial intermediate/wild-type heterokaryons was normal. Apparently, stable slime strains differed from the original mycelial intermediate in a mutation(s) which arose spontaneously during the filtration-enrichment selection applied to mycelial intermediates in order to obtain the cell wall-less phenotype. This new trait impaired conidial germination and might be the actual cause of the loss of (1,3)-β-glucan synthase activity and cell wall.     

Isolation and partial characterization of a cyclic GMP-dependent cyclic GMP-specific phosphodiesterase from Dictyostelium discoideum

The cellular slime mold, Dictyostelium discoideum, contains at least two classes of phosphodiesterase activity. One class of enzymes hydrolyses cyclic AMP (cAMP) and cyclic GMP (cGMP) with approximately equal rates. Another enzyme, which is less than 5% of the total activity, specifically hydrolyses cGMP. The cGMP-specific enzyme does not bind to a Con A-Sepharose column, while all the cAMP-hydrolyzing activities are retarded by this column. The cGMP-specific enzyme is activated by low cGMP concentrations (10^?8-10^?6 M); the enzyme has normal Michaelis-Menten kinetics at high substrate concentrations with a K_m of about 3–6 μM. The cGMP-binding sites for activation and for catalysis show different cyclic nucleotide specificity, but they are probably located on one protein with a molecular weight of about 70 000. The enzyme is stable only under specific conditions, and the activation property of the enzyme is lost relatively easy. Irreversible modifications occur at temperatures below 0° and above 30°C, and at pH below 6.0. Several other conditions such as high ion concentrations, temperatures just above 0°C and pH above 8.0 lead to reversibel modifications of enzyme activity.     

Regulation of Polyglutamic Acid Synthesis by Glutamate in Bacillus licheniformis and Bacillus subtilis

The synthesis of polyglutamic acid (PGA) was repressed by exogenous glutamate in strains of Bacillus licheniformis but not in strains of Bacillus subtilis, indicating a clear difference in the regulation of synthesis of capsular slime in these two species. Although extracellular γ-glutamyltranspeptidase (GGT) activity was always present in PGA-producing cultures of B. licheniformis under various growth conditions, there was no correlation between the quantity of PGA and enzyme activity. Moreover, the synthesis of PGA in the absence of detectable GGT activity in B. subtilis S317 indicated that this enzyme was not involved in PGA biosynthesis in this bacterium. Glutamate repression of PGA biosynthesis may offer a simple means of preventing unwanted slime production in industrial fermentations using B. licheniformis.     

Evidence for the Heterolobosea from Phylogenetic Analysis of Genes Encoding Glyceraldehyde-3-Phosphate Dehydrogenase

ABSTRACT The phylogenetic relationships between major slime mould groups and the identification of their unicellular relatives has been a subject of controversy for many years. Traditionally, it has been assumed that two slime mould groups, the acrasids and the dictyostelids were related by virtue of their cellular slime mould habit; a view still endorsed by at least one current classification scheme, However, a decade ago, on the basis of detailed ultrastructural resemblances, it was proposed that acrasids of the family Acrasidae were not relatives of other slime moulds but instead related to a group of mostly free-living unicellular amoebae, the Schizopyrenida. The class Heterolobosea was created to contain these organisms and has since figured in many discussions of protist evolution. We sought to test the validity of Heterolobosea by characterizing homologs of the highly conserved glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from an acrasid, Acrasis rosea ; a dictyostelid, Dictyostelium discoideum ; and the schizopyrenid Naegleria andersoni. Phylogenetic analysis of these and other GAPDH sequences, using maximum parsimony, neighbour-joining distance and maximum likelihood methods strongly supports the Heterolobosea hypothesis and discredits the concept of a cellular slime mould grouping. Moreover, all of our analyses place Dictyostelium discoideum as a relatively recently originating lineage, most closely related to the Metazoa, similar to other recently published phylogenies of protein-coding genes. However, GAPDH phylogenies do not show robust branching orders for most of the relationships between major groups. We propose that several of the incongruencies observed between GAPDH and other molecular phylogenies are artifacts resulting from substitutional saturation of this enzyme.     

Characterization of GDP-Fucose: Polysaccharide Fucosyl Transferase in Corn Roots (Zea mays L.)

The peripheral root cap cells of corn (cv. SX-17A) secrete a fucose-rich, high molecular weight, polysaccharide slime via the dictyosome pathway. To study the synthesis of this polysaccharide, a technique for isolating and assaying GDP-fucose:polysaccharide fucosyl transferase activity was developed. Corn roots were excised from germinated seeds, incubated 12 hours at 10 C in water, and ground in 100 millimolar Tris or Pipes buffer (pH 7.0) with or without 0.5 molar sucrose. The membrane-bound enzyme was solubilized by sonication in the presence of 2 molar urea and 1.5% (v/v) Triton X-100 and assayed by monitoring the incorporation of GDP-[¹⁴C]fucose into endogenous acceptors. Optimum enzyme activity is expressed at pH 7.0 and 30 C in the presence of 0.8% (v/v) Triton X-100. The enzyme does not require divalent cations for activation and is inhibited by concentrations of MnCl₂ or MgCl₂ greater than 1 millimolar. Corn root cap slime will serve as an exogenous acceptor for the enzyme if it is first hydrolyzed in 5 millimolar trifluoroacetic acid for 60 minutes at 18 pounds per square inch, 121 C. This procedure prepares the acceptor by removing terminal fucose residues from the slime molecule. Kinetics of fucose release during hydrolysis of native slime and in vitro synthesized product suggests that the two polymers possess similar linkages to fucose.     

Characteristics of Gloeophyllum trabeum Alcohol Oxidase, an Extracellular Source of H2O2 in Brown Rot Decay of Wood

A novel alcohol oxidase (AOX) has been purified from mycelial pellets of the wood-degrading basidiomycete Gloeophyllum trabeum and characterized as a homooctameric nonglycosylated protein with native and subunit molecular masses of 628 and 72.4 kDa, containing noncovalently bonded flavin adenine dinucleotide. The isolated AOX cDNA contained an open reading frame of 1,953 bp translating into a polypeptide of 651 amino acids displaying 51 to 53% identity with other published fungal AOX amino acid sequences. The enzyme catalyzed the oxidation of short-chain primary aliphatic alcohols with a preference for methanol (K_m = 2.3 mM, k_cat = 15.6 s⁻¹). Using polyclonal antibodies and immunofluorescence staining, AOX was localized on liquid culture hyphae and extracellular slime in sections from degraded wood and on cotton fibers. Transmission electron microscopy immunogold labeling localized the enzyme in the hyphal periplasmic space and wall and on extracellular tripartite membranes and slime, while there was no labeling of hyphal peroxisomes. AOX was further shown to be associated with membranous or slime structures secreted by hyphae in wood fiber lumina and within the secondary cell walls of degraded wood fibers. The differences in AOX targeting compared to the known yeast peroxisomal localization were traced to a unique C-terminal sequence of the G. trabeum oxidase, which is apparently responsible for the protein's different translocation. The extracellular distribution and the enzyme's abundance and preference for methanol, potentially available from the demethylation of lignin, all point to a possible role for AOX as a major source of H₂O₂, a component of Fenton's reagent implicated in the generally accepted mechanisms for brown rot through the production of highly destructive hydroxyl radicals.     

Purification and some properties of discadenine synthase from Dictyostelium discoideum

The enzyme discadenine synthase, which transfers the 3-amino-3-carboxypropyl residue of S-adenosylmethionine to an acceptor, N⁶-isopentenyladenine, from the fruiting body of the cellular slime mold Dictyostelium discoideum, was purified 420-fold. Its apparent molecular mass was 82 000 Da and the isoelectric point was pH 5.8. The K_m value for S-adenosylmethionine was 1.85 · 10⁻⁵ M and for benzyladenine was 7.0 · 10⁻⁷ M. In contrast to theenzyme which catalyzes the formation of 3-(3-amino-3-carboxypropyl)uridine in tRNAs, the present enzyme did not require Mg²⁺ and was not stimulated by ATP. Some other metal ions (Zn²⁺, Mn²⁺, Ca²⁺) showed inhibition at 10–100 mM.     

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.     

Amino Acid sequence of a novel calmodulin from the unicellular alga chlamydomonas

An amino acid sequence for a Chlamydomonas calmodulin has been elucidated with emphasis on the characterization of differences that are unique to Chlamydomonas and Dictyostelium calmodulin. While the concentration of calmodulin required for half-maximal activation of plant NAD kinase varies among vertebrate, higher plant, algal, and slime mold calmodulins, only calmodulins from the unicellular alga Chlamydomonas and the slime mold Dictyostelium show increased maximal activation of NAD kinase (Roberts, Burgess, Watterson 1984 Plant Physiol 75: 796-798; Marshak, Clarke, Roberts, Watterson 1984 Biochemistry 23: 2891-2899). The same preparations of calmodulin do not show major differences in phosphodiesterase or myosin light chain kinase activator activity.

We report here that a Chlamydomonas calmodulin has four primary structural features similar to Dictyostelium that are not found in other calmodulins characterized to date: an altered carboxy terminus including a novel 11-residue extension for Chlamydomonas calmodulin, unique residues at positions 81 and 118, and an unmethylated lysine at position 115. The only amino acid sequence identity unique to Chlamydomonas and Dictyostelium calmodulin is the presence of a lysine at position 115 instead of a trimethyllysine. These studies indicate that the methylation state of lysine 115 may be important in the maximal NAD kinase activator activity of calmodulin and support the concept that calmodulin has multiple functional domains in addition to multiple structural domains.

     

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.     

Partial purification and characterization of dipeptidyl-aminopeptidase III fromDictyostelium discoideum

Dipeptidyl-aminopeptidase III was isolated from cells of the cellular slime moldDictyostelium discoideum in the culmination stage of development. The enzyme was purified 18-fold by precipitation with ammonium sulfate and gel filtration chromatography and was shown to have a molecular weight of 158,000 and a sharp pH optimum at pH 10.2 and to be inhibited by sulfhydryl reagents. The enzyme acted upon the artificial substratearginyl-arginyl-β-naphthylamide, producing arginyl-arginine andβ-naphthylamine but notarginyl-β-naphthylamide. Activity towardarginyl-arginyl-β-naphthylamide was strongly inhibited by physiological concentrations of angiotensin III and, to a lesser extent, by angiotensins I and II and other angiotensin-related peptides but not by enkephalin peptides. Several dipeptides known to inhibit mammalian dipeptidyl-aminopeptidase III also inhibited theDictyostelium enzyme. Incubation of the enzyme preparation with angiotensins resulted in their conversion into a complex mixture of products. Thus dipeptidyl-aminopeptidase III fromDictyostelium closely resembles the mammalian enzyme in many of its characteristics.     

'Ghosts' formation and their isolation from the cellular slime mold Dictyostelium discoideum.

Conditions for isolating ghosts from the cellular slime mold Dictyostelium discoideum are described. The cells were washed with a 20 mM KCl, 2.5 mM MgCl₂ solution and homogenized vigorously in 15 mM lactate buffer, pH 4.8, using a tight-fitting Dounce homogenizer. The resultant spherical ghosts were purified by the dextran-polyethylene glycol aqueous two-phase system described by Brunette &; Till [1], The proportion of ghosts which are finally purified by 3rd partition in the aqueous two-phase system is 5.6% of those present in the homogenate. As shown by phase-contrast and scanning electron microscopy, the plasma membrane fractions are almost completely uncontaminated by other identifiable subcellular components. On the basis of enzyme assays the ghosts isolated showed a 9- to 11-fold enrichment of alkaline phosphatase relative to the homogenate. They are free of succinic dehydrogenase, glucose 6-phosphatase but do contain some acid phosphatase and N-acetylglucosaminidase activity. Further purification using a sucrose-density gradient removes the residual lysosomal enzyme activities.     

Novel chlorinated dibenzofurans isolated from the cellular slime mold,Polysphondylium filamentosum,and their biological activities

Cellular slime molds are expected to have the huge potential for producing secondary metabolites including polyketides, and we have studied the diversity of secondary metabolites of cellular slime molds for their potential utilization as new biological resources for natural product chemistry. From the methanol extract of fruiting bodies of Polysphondylium filamentosum, we obtained new chlorinated benzofurans Pf-1 (4) and Pf-2 (5) which display multiple biological activities; these include stalk cell differentiation-inducing activity in the well-studied cellular slime mold, Dictyostelium discoideum, and inhibitory activities on cell proliferation in mammalian cells and gene expression in Drosophila melanogaster.     

Isolation and characterization of mutations affecting UDPG pyrophosphorylase activity in Dictyostelium discoideum

A procedure for screening large numbers of clones for an enzyme activity was used to isolate mutations which affect UDPG pyrophosphorylase activity (EC 2.7.7.9) in the cellular slime mold Dictyostelium discoideum. Five strains were recovered which have little or no UDPG pyrophosphorylase activity. Ten other strains were found which have significant activity in vivo which is rapidly inactivated upon cell lysis. These strains have permitted us to evaluate the role of UDPG pyrophosphorylase during growth and development. The enzyme affects the growth rate of the cells but is not essential for growth. However, during development the lack of enzyme activity leads to cell death and lysis. Strains which lack UDPG pyrophosphorylase accomplish early developmental events but are unable to culminate. However, certain biochemical and cytological differentiations associated with late stages were observed.