Apurinic endonuclease from Saccharomyces cerevisiae.

An endonuclease cleaving depurinated and alkylated double-stranded DNA has been purified 500-fold from Saccharomyces cerevisiae, strain MB 1052. The enzyme has an Mr of 31 000 +/- 2000, a sedimentation value of 3.2S and a diffusion coefficient of 9.5 X 10-7 cm2/s. The enzyme was active only at apurinic/apyridiminic sites, regardless of whether they were produced by heating the DNA at acidic pH or by alkylation with the ultimate carcinogen methyl methanesulphonate. Native DNA was not acted upon. U.v.-irradiated DNA and DNA treated with the ultimate carcinogen N-acetoxy-2-acetylaminofluorene were cleaved to an extent related to the extent of apurinic/apyridiminic sites. Enzymic activity was not dependent upon Mg2+, but was stimulated approx. 3-fold by 4mM-Mg2+. The enzyme did not bind to DEAE-cellulose or CM-cellulose at KCl concentrations greater than 160 mM. The endonuclease was obtained free of exonuclease and 3-methyladenine-DNA glycosylase activity in five chromatographic steps.     

Apurinic DNA endonuclease activities in repair-deficient human cell lines.

Several autosomal recessive diseases are associated with apparent DNA repair defects in cell culture. It seemed likely that a defect in excision repair reported for ataxia telangiectasia cells might reflect a lack of apurinic endonuclease activity. We report here normal levels of apurinic endonuclease activity in extracts of cell lines derived from patients with ataxia telangiectasia, xeroderma pigmentosum (complementation group D), Cockayne dwarfism, Fanconi anemia and Bloom syndrome.     

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.     

Apurinic acid endonuclease activity from mouse epidermal cells.

An endonuclease activity making single-strand breaks into depurinated and alkylated DNA has been purified 500-fold from carcinogen-transformed mouse epidermal cells. The enzyme was active only at apurinic/apyrimidinic sites, regardless of whether they were produced by heating at an acidic pH or by alkylation with the ultimate carcinogen MeSO2OMe. The enzyme did not act on native DNA nor on ultraviolet-induced pyrimidine-dimers nor on steric distortions caused by modification of DNA with the carcinogen (Ac)2ONFln. The enzyme was active in the presence of 1 mM EDTA; however, at pH 7.4 optimal conditions were: 6mM MgCl2 and 40--120 mM KCl or 10--40 mM potassium phosphate. The enzyme eluted from hydroxyapatite, phosphocellulose and heparin-cellulose between 100--250 mM potassium phosphate but did not bind to DEAE-cellulose. Using four chromatographic steps the endonuclease was obtained free of exonuclease, demethylase and DNA glycosylase activity specific for DNA bases methylated with MeSO2OMe or MeNOUr. The molecular weight was 31 000 +/- 3000 as calculated from the diffusion coefficient (8.2 x 10-7 cm2/s) and the sedimentation value (2.7 S).     

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.     

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.     

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.     

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.     

Motile activities of Dictyostelium discoideum differ from those in Protista or vertebrate animal cells

Cell movement in the amoebae Dictyostelium discoideum has been examined in media differing in monovalent cation concentration (i.e. Na+ and K+). Under isotonic or even slightly hypertonic conditions, the cells move equally well in solutions in which either potassium or sodium ions dominate. However, in strongly hypertonic solutions the amoebae showed motility in a 2% potassium chloride solution, but remained motionless in a hypertonic 2% sodium chloride solution. This inhibition of D. discoideum amoebae movement in a hypertonic sodium chloride solution was fully reversible. Such behaviour corresponds to that of plant, fungi, and some invertebrate animal cells rather than protozoan or vertebrate cells. These observations suggest that studies using D. discoideum as a model for cell motility in vertebrate animal tissue cells should be considered with caution, and would seem to confirm the classification of cellular slime moulds as related rather to Fungi than to Protista. This also shows that the cell membrane models should consider the asymmetry in sodium/potassium ion concentrations found in vertebrate animal cells as one of various possibilities.     

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.     

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).     

Expression of glycosidase activities during germination of Dictyostelium discoideum spores.

Several lysosomal glycosidase activities were examined in vitro during heat-induced germination of Dictyostelium discoideum spores and were found not to be coordinately controlled. The level of beta-glucosidase activity increased significantly during the emergence stage of germination. Both alpha-glucosidase and N-acetyl-beta-glucosaminidase activities remained relatively constant until postemergence, when they increased slightly; alpha-mannosidase activity decreased during all stages of germination. The activity of beta-galactosidase increased slightly during spore swelling, fell below the level initially found in spores at zero time, and increased slightly during postemergence. The expression of all of these enzyme activities, except the increase in beta-galactosidase, appeared to require protein synthesis. Spores in the lag phase of germination which were exposed to severe environmental stress were deactivated and exhibited reduced levels of alpha-glucosidase, beta-glucosidase, and N-acetyl-beta-glucosaminidase activities. Prolonged heat activation treatment reduced the levels of lysosomal glycosidase activities in postactivated spores but did not change the subsequent enzyme patterns during the spore-swelling and emergence stages of germination.