Metal ion dependency of serine racemase from Dictyostelium discoideum

D-Serine is known to act as an endogenous co-agonist of the N-methyl-D-aspartate receptor in the mammalian brain and is endogenously synthesized from L-serine by a pyridoxal 5'-phosphate-dependent enzyme, serine racemase. Though the soil-living mycetozoa Dictyostelium discoideum possesses no genes homologous to that of NMDA receptor, it contains genes encoding putative proteins relating to the D-serine metabolism, such as serine racemase, D-amino acid oxidase, and D-serine dehydratase. D. discoideum is an attractive target for the elucidation of the unknown functions of D-serine such as a role in cell development. As part of the elucidation of the role of D-serine in D. discoideum, we cloned, overexpressed, and examined the properties of the putative serine racemase exhibiting 46% amino acid sequence similarity with the human enzyme. The enzyme is unique in its stimulation by monovalent cations such as Na(+) in addition to Mg(2+) and Ca(2+), which are well-known activators for the mammalian serine racemase. Mg(2+) or Na(+) binding caused two- to ninefold enhancement of the rates of both racemization and dehydration. The half-maximal activation concentrations of Mg(2+) and Na(+) were determined to be 1.2?μM and 2.2?mM, respectively. In the L-serine dehydrase reaction, Mg(2+) and Na(+) enhanced the k (cat) value without changing the K (m) value. Alanine mutation of the residues E207 and D213, which correspond to the Mg(2+)-binding site of Schizosaccharomyces pombe serine racemase, abolished the Mg(2+)- and Na(+)-dependent stimulation. These results suggest that Mg(2+) and Na(+) share the common metal ion-binding site.     

Discoidin-binding polysaccharide from Dictyostelium discoideum

Extracts of Dictyostelium discoideum grown axenically in a chemically defined medium were evaluated for binding to discoidin I and discoidin II, endogenous lectins of this slime mold. Binding activity was measured by competitive inhibition of 125I-lactosyl-bovine serum albumin binding to the immobilized lectins. With the solubilization procedure used extracts of vegetative cells and of early aggregates had no significant inhibitory activity, but an abundant discoidin-binding substance was detected in late aggregates and fruiting bodies. This material was purified by ethanol and acid precipitation followed by precipitation with discoidin. It is a polysaccharide composed of 77% galactose, 15% N-acetylgalactosamine, 5% glucose, and 3% N-acetylglucosamine and may be a biologically functional ligand for the slime mold lectins, in particular discoidin II. Use of axenic cells was critical in these experiments, since extracts of Escherichia coli and Klebsiella aerogenes, commonly used as food for D. discoideum, were found to contain substances that react with discoidin. This would complicate isolation of endogenous discoidin ligands from cells raised on bacteria.     

New actin-binding proteins from Dictyostelium discoideum

Dictyostelium discoideum contains a soluble actin-binding protein that caps actin filaments at their fast growing ends. The purified protein consists of two subunits with 34 kd and 32 kd apparent mol. wts. Like similar proteins from Acanthamoeba and bovine brain the capping protein from D. discoideum acts in a Ca²⁺ -independent manner. It lacks severing activity as indicated by its inability to disrupt the stress fibers and the microfilament network in detergent-extracted cells. Two actin-binding proteins from a plasma membrane-enriched fraction were labeled with [¹²⁵I]actin using a gel overlay technique. One of these proteins, with an apparent mol. wt. of 17 kd in SDS-polyacrylamide gels, has been purified from high-salt extracts, the other protein with an apparent mol. wt. of 31 kd has been purified from Triton X-100 extracted membranes. Monoclonal antibodies were raised against D. discoideum severin, α-actinin, the larger subunit of the capping protein, and the 17-kd membrane-associated protein. Immunoblotting of proteins from whole cell lysates showed that all these actin-binding proteins were present in both growth phase and aggregation-competent cells.     

Uracil-DNA glycosylase activity from Dictyostelium discoideum

We have isolated and partially characterized a uracil-DNA glycosylase activity from the cellular slime mold, Dictyostelium discoideum. This glycosylase has a broad pH optimum (6.5-8.5) and is fully active in 10 mM EDTA or in 5 mM Mg2+. Its molecular weight by gel filtration is about 55 000. This enzyme activity may work in concert with previously described apurinic/apyrimidinic (AP) endonuclease activities in the excision repair of uracil from the DNA of this lower eukaryote.     

cAMP-dependent protein kinase from Dictyostelium discoideum

The cAMP-dependent protein kinase (cAK) from Dictyostelium discoideum is an enzyme composed of one catalytic and one regulatory subunit. Upon binding of cAMP, the holoenzyme dissociates to liberate free active catalytic subunits. The cAK is developmentally regulated, ranging from very little activity in vegetative cells to maximal expression in postaggregative cells. Although there is no immunological cross-reaction between the subunits of cAKs from Dictyostelium and from other organisms, they share several biochemical properties. A complete cDNA for the regulatory subunit has been cloned and sequenced. Only one copy of the gene for the regulatory subunit is present per haploid genome. On the basis of the comparison of the structure of the cAK from Dictyostelium with its counterparts in yeast and higher eukaryotes, we propose a model for the evolution of cyclic-nucleotide-binding proteins.     

Acid DNAase activity from Dictyostelium discoideum

We have isolated and partially characterized an acid endonuclease activity from the cellular slime mold, Dictyostelium discoideum. This activity comprises more than 90% of the nonspecific DNA-endonuclease activity of the vegetative cells. Its molecular weight is about 44 000, and its activity is enhanced 7-fold by Mg2+. The pH optimum for the nicking activity depends upon NaCl concentrations, being at pH 5.0 in 207 mM NaCl, and at pH 5.8 in 7 mM NaCl. Large quantities of this enzyme activity are released into the growth medium or buffer, with detectable amounts appearing within 15 min of incubation.     

Studies on the mechanism of action of the alkaline phosphatase from Dictyostelium discoideum

The Dictyostelium discoideum alkaline phosphatase was investigated kinetically in an attempt to elucidate its mechanism of action. Analysis of the hydrolysis of p-nitrophenyl phosphate by stopped-flow spectrophotometry revealed biphasic kinetics, suggesting a double displacement enzyme mechanism. Furthermore, Tris stimulated activity in an uncompetitive manner, a result that was consistent with this interpretation. The enzyme was inhibited reversibly by phosphate at low ionic strength, but the inhibition was irreversible at high ionic strength and the latter effect was enhanced at alkaline pH values. These results indicate that high ionic strength and alkaline pH conditions bring about a conformational change that renders the enzyme susceptible to irreversible inhibition by phosphate.     

cDNA sequence of cyclophilin from Dictyostelium discoideum

A cDNA encoding a protein homologous to cyclophilins from other species has been isolated from a Dictyostelium discoideum cDNA library. From the deduced amino acid sequence a protein with a molecular mass of 19 kD and 64% identity with human cyclophilin is predicted. Southern blot analysis indicates that there is one cyclophilin gene in the D. discoideum genome. The mRNA is present in all developmental stages.     

The mechanism of formation of Liesegang structures around Dictyostelium discoideum

A theoretical study of the phenomenon of Liesegang structure formation induced by the Dictyostelium discoideum population in a medium containing folic acid was carried out. Using a "reaction-diffusion" model proposed in this work, it was shown that the formation of Liesegang structures around the Dictyostelium discoideum population depends on two competing processes: (a) inactivation of folic acid by vegetative amoebae and (b) the chemical reaction of folic acid with the products of amoeba metabolism, which results in the formation of insoluble sediment. The dependence of the model solutions on the geometric and functional parameters was studied. The results are in good agreement with experimental data.     

Cyclic GMP-activated protein kinase from Dictyostelium discoideum

Cells of Dictyostelium discoideum respond to their chemoattractants, cAMP and folate, with a rapid increase of the cellular cGMP content. The molecular mechanisms of cGMP action are not understood. Since in many biological systems cGMP-activated protein kinase is a prominent cGMP acceptor, we searched for such an enzyme in D. discoideum. By means of affinity chromatography on cGMP-Sepharose and other chromatographic procedures (DEAE-Trisacryl, CM-Trisacryl), we separated a novel protein kinase. This preparation did not show any regulation by cGMP and may represent an enzyme modified by proteolysis. In order to establish a rapid and efficient purification step, an antiserum against the kinase preparation was raised and coupled to Sepharose. Chromatography of the supernatant from a cell homogenate on this antibody matrix yielded a protein kinase that was activated 3-fold by cGMP. Half-maximal activation occurred at about 1 nM cGMP. Cyclic AMP at a 20-fold higher concentration also activated the protein kinase. On a Superose 6HR column the cGMP-activated protein kinase eluted in the same volume as enolase (Mr = 82,000).     

Mitochondrial large-conductance potassium channel from Dictyostelium discoideum

In the present study, we describe the existence of a large-conductance calcium-activated potassium (BK_Ca) channel in the mitochondria of Dictyostelium discoideum. A single-channel current was recorded in a reconstituted system, using planar lipid bilayers. The large-conductance potassium channel activity of 258 ± 12 pS was recorded in a 50/150 mM KCl gradient solution. The probability of channel opening (the channel activity) was increased by calcium ions and NS1619 (potassium channel opener) and reduced by iberiotoxin (BK_Ca channel inhibitor). The substances known to modulate BK_Ca channel activity influenced the bioenergetics of D. discoideum mitochondria. In isolated mitochondria, NS1619 and NS11021 stimulated non-phosphorylating respiration and depolarized membrane potential, indicating the channel activation. These effects were blocked by iberiotoxin and paxilline. Moreover, the activation of the channel resulted in attenuation of superoxide formation, but its inhibition had the opposite effect. Immunological analysis with antibodies raised against mammalian BK_Ca channel subunits detected a pore-forming α subunit and auxiliary β subunits of the channel in D. discoideum mitochondria. In conclusion, we show for the first time that mitochondria of D. discoideum, a unicellular ameboid protozoon that facultatively forms multicellular structures, contain a large-conductance calcium-activated potassium channel with electrophysiological, biochemical and molecular properties similar to those of the channels previously described in mammalian and plant mitochondria.     

Multiple forms of glucokinase from Dictyostelium discoideum.

A single form of glucokinase with an apparent Km value equal to 0.12 mM glucose was detectable in extracts prepared from aggregating cells, whereas kinetic and electrophoretic evidence indicated the presence of this form as well as a second glucose-phosphorylating enzyme with a Km value of about 0.01 mM glucose in extracts from culminating cells.     

Chromosomal mapping of tRNA genes from Dictyostelium discoideum

Summary Different wild-type isolates of Dictyostelium discoideum exhibit extensive polymorphism in the length of restriction fragments carrying tRNA genes. These size differences were used to study the organisation of two tRNA gene families which encode a tRNA^Val(GUU) and a tRNA^Val(GUA) gene. The method used involved a combination of classitics. The tRNA genes were mapped to specific linkage groups (chromosomes) by correlating the presence of polymorphic DNA bands that hybridized with the tRNA gene probes with the presence of genetic markers for those linkage groups. These analyses established that both of the tRNA gene families are dispersed among sites on several of the chromosomes. Information of nine tRNA^Val(GUU) genes from the wild-type isolate NC4 was obtained: three map to linkage group I (C, E, F,), two map to linkage group II (D, I), one maps to linkage group IV (G), one, which corresponds to the cloned gene, maps to either linkage group III or VI (B), and two map to one of linkage groups III, VI or VIII (A, H). Six tRNA^Val(GUA) genes from the NC4 isolate were mapped; one to linkage group I (D), two to linkage group III, VI or VII (B, C) and three to linkage group VII or III (A, E, F).     

Cloning and characterization of beta-COP from Dictyostelium discoideum

We have isolated a cDNA coding for beta-COP from Dictyostelium discoideum by polymerase chain reaction using degenerate primers derived from rat beta-COP. The complete cDNA clone has a size of 2.8 kb and codes for a protein with a calculated molecular mass of 102 kDa. Dictyostelium beta-COP exhibits highest homology to mammalian beta-COP, but it is considerably smaller due to a shortened variable region that is thought to form a linker between the highly conserved N- and C-terminal domains. Dictyostelium beta-COP is encoded by a single gene, which is transcribed at moderate levels into two RNAs that are present throughout development. To localize the protein, full-length beta-COP was fused to GFP and expressed in Dictyostelium cells. The fusion protein was detected on vesicles distributed all over the cells and was strongly enriched in the perinuclear region. Based on coimmunofluorescence studies with antibodies directed against the Golgi marker comitin, this compartment was identified as the Golgi apparatus. Beta-COP distribution in Dictyostelium was not brefeldin A sensitive being most likely due to the presence of a brefeldin A resistance gene. However, upon DMSO treatment we observed a reversible disassembly of the Golgi apparatus. In mammalian cells DMSO treatment had a similar effect on beta-COP distribution.     

盘基网柄菌肌动蛋白的生物信息学分析

盘基网柄菌Dictyostelium discoideum是目前黏菌中研究最清楚的模式生物,其捕食过程与肌动蛋白的多聚化密切相关。为探讨盘基网柄菌肌动蛋白的序列特征,本研究利用生物信息学方法分析了盘基网柄菌32条肌动蛋白的蛋白-蛋白相互作用(protein-protein interaction)和可能含有的保守基序。结果表明:盘基网柄菌32条肌动蛋白与其他蛋白存在一组复杂相互作用关系和5组比较简单的相互作用关系;利用MEME SUITE分别分析盘基网柄菌32条肌动蛋白序列的保守基序和与actin17呈最佳匹配的21种生物肌动蛋白的保守基序,结果共获得6个保守基序,即motif1,motif2,motif3,Motif1,Motif2,Motif3。其中motif1,Motif1,Motif3为本研究新发现的保守基序,这3个保守基序可定位于Profilin-actin-VASP202–244(PDB ID:3CHW)三维结构的重要位置。以上结果表明actin3,actin10,actin14,actin15,actin17,actin31可能为盘基网柄菌比较重要的肌动蛋白;motif1,Motif1,Motif3可能是盘基网柄菌肌动蛋白在进化中重要的保守基序。