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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The initial step in the purification of Dictyostelium myosin II heavy chain kinase A (MHCK A) is chromatography over phosphocellulose. Fractions containing MHCK A are pooled and chromatographed over a Mono Q column (Pharmacia LKB Biotechnology) equilibrated in 0.15 M KCl. Under these conditions MHCK A and most of the contaminating proteins elute in the flowthrough. The addition of Mg2+ and ATP to the Mono Q flowthrough results in the phosphorylation, within 15 min, of MHCK A to a level of 10 mol of phosphate per mole of 130-kDa kinase subunit. The hyperphosphorylated MHCK A binds to Mono Q columns in the presence of 0.15 M KCl and can be eluted, as a single homogeneous band, by a salt gradient to 0.35 M KCl. A similar purification procedure may prove useful for other proteins which can be highly phosphorylated. Hyperphosphorylation is shown to have no effect on the position at which MHCK A elutes from gel filtration columns (apparent M(r) greater than 700,000).
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PMID:Purification of Dictyostelium myosin II heavy chain kinase A based on the increase in negative charge accompanying hyperphosphorylation. 196 23

Discoidin I is the most abundant galactose binding lectin produced by the cellular slime mold Dictyostelium discoideum and has been implicated in cell-substratum adhesion. We have developed an assay of carbohydrate binding activity utilizing binding of 125I-asialofetuin to discoidin I, or to other lectins, immobilized on nitrocellulose. Among the proteins examined, only lectins exhibited the ability to bind asialofetuin. Specificity of asialofetuin binding was demonstrated by competition with monosaccharides, which inhibited binding consistent with the known sugar specificity of the lectins examined. Experiments with fetuin and derivatives differing in their oligosaccharide structure indicated a requirement for terminal galactosyl residues for probe binding to discoidin I. We have used this assay to characterize the carbohydrate binding behavior of discoidin I. The extent of asialofetuin binding to discoidin I was dependent on the concentrations of both lectin and ligand. Interpretation of equilibrium binding data suggested that, under saturating conditions, 1 mol of oligosaccharide was bound per mole discoidin I monomer. Furthermore, discoidin I in solution and discoidin I on nitrocellulose were equally effective at competing for soluble asialofetuin, suggesting that immobilization had no effect on the carbohydrate binding behavior of discoidin I. Binding was strongly inhibited by ethylenediaminetetraacetic acid; both Ca2+ and Mn2+ could overcome that inhibition, but Mg2+ could not. Preincubation of discoidin I at 60 degrees C stimulated asialofetuin binding 2-fold by increasing the affinity, while preincubation at higher temperatures resulted in a complete loss of activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Assay and characterization of carbohydrate binding by the lectin discoidin I immobilized on nitrocellulose. 244 64

We purified to homogeneity the Dictyostelium discoideum myosin heavy chain kinase that is implicated in the heavy chain phosphorylation increases that occur during chemotaxis. The kinase is initially found in the insoluble fraction of developed cells. The major purification step was achieved by affinity chromatography using a tail fragment of Dictyostelium myosin (LMM58) expressed in Escherichia coli (De Lozanne, A., Berlot, C. H., Leinwand, L. A., and Spudich, J. A. (1988) J. Cell Biol. 105, 2990-3005). The kinase has an apparent molecular weight of 84,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent native molecular weight by gel filtration is 240,000. The kinase catalyzes phosphorylation of myosin heavy chain or LMM58 with similar kinetics, and the extent of phosphorylation for both is 4 mol of phosphate/mol. With both substrates the Vmax is about 18 mumol/min/mg and the Km is 15 microM. The myosin heavy chain kinase is specific to Dictyostelium myosin heavy chain, and the phosphorylated amino acid is threonine. The kinase undergoes autophosphorylation. Each mole of kinase subunit incorporates about 20 mol of phosphates. Phosphorylation of myosin by this kinase inhibits myosin thick filament formation, suggesting that the kinase plays a role in the regulation of myosin assembly.
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PMID:Myosin heavy chain kinase from developed Dictyostelium cells. Purification and characterization. 254 52

Homogeneous S-adenosylhomocysteinase (AdoHcyase) from rat liver is a tetrameric enzyme that contains four molecules of tightly bound NAD per mole of enzyme. We report here that incubation of the rat liver enzyme with ATP, Mg2+, and KCl leads to conversion of the active enzyme to an inactive form with release of all enzyme-bound NAD which can be recovered quantitatively by gel filtration. At various concentrations of ATP, the release of NAD corresponds closely with the degree of inactivation, suggesting that the four subunits are equivalent. Hydrolysis of ATP is not required for the inactivation process since nonhydrolyzable ATP analogues can replace ATP in the inactivation process. The ATP-dependent inactivation is fully reversible upon incubation of the inactivated enzyme with NAD. The ATP-dependent inactivation of the enzyme appears to be analogues to the cAMP-dependent inactivation of the enzyme from Dictyostelium discoideum described earlier by Hohman et al. (1985) [Hohman, R. J., Guitton, M. C., & Veron, M. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 4578-4581; Hohman, R. J., Veron, M., & Guitton, M. C. (1985) Curr. Top. Cell. Regul. 26, 233-245] but differs from the irreversible inactivation studied earlier by Abeles et al. (1982) [Abeles, R. H., Fish, S., & Lapinskas, B. (1982) Biochemistry 21, 5557-5562]. These authors have ascribed the time-dependent inactivation that results from incubation of the enzyme with 2'-deoxyadenosine at the C-3' and concluded that AdoHcyase "probably consists of two nonequivalent pairs of subunits".(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:S-adenosylhomocysteinase: mechanism of reversible and irreversible inactivation by ATP, cAMP, and 2'-deoxyadenosine. 302 76

As the cellular slime mold, Dictyostelium discoideum, undergoes development, a phospholipid fraction containing 80% N-acylethanolamine glycerophospholipids (NAEGPs) and 20% acylphosphatidylglycerol (APG) disappears during the aggregation stage. In this study, the subcellular distribution of that NAEGP phospholipid fraction and the precise time period of disappearance of the fraction were determined. The content of the NAEGP fraction was determined in aggregating cells at 2-h intervals from the beginning of the developmental phase through 14 h, when the cells were completely aggregated. The NAEGP fraction comprised about 8% of the phospholipids in amoebae just starting the development cycle and about 12% in cells between 2 and 6 h of development; then its level decreased until it could not be detected at 12 and 14 h of development. The mole percentage of the total lipid phosphate in the NAEGP fraction was determined in isolated subcellular organelles. The phagolysosomes were enriched in the NAEGP fraction 1.7-2-fold over the level found in the amoebae and about 8-fold over the level in fractions highly enriched in the plasma membrane, mitochondria or peroxisomes. The content of phagolysosomes was determined by electron microscopy of aggregating cells. The amoebae contained large amounts of phagolysosomes up to 6 h of development, and then they gradually disappeared between 6 and 12 h of development. This combination of quantitative phospholipid analysis, subcellular organelle isolation and electron microscopy has revealed that in D. discoideum amoebae, the phagolysosomes were selectively enriched in the NAEGP fraction and both the NAEGP-enriched phagolysosomes and the NAEGPs disappeared concurrently between 6 and 12 h of development.
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PMID:Concurrent disappearance of N-acylethanolamine glycerophospholipids and phagolysosomes enriched in N-acylethanolamine glycerophospholipids as Dictyostelium discoideum cells aggregate. 396 14

The assembly of highly purified actin from Dictyostelium discoideum amoebae and rabbit skeletal muscle by physiological concentrations of KCI proceeds through successive stages of (a) rapid formation of a distinct monomeric species referred to as KCI-monomer, (b) incorporation of KCI-monomers into an ATP-containing filament, and (c) ATP hydrolysis that occurs significantly after the incorporation event. KCI-monomer has a conformation which is distinct from that of either conventional G- or F-actin, as judged by UV spectroscopy at 210-220 nm and by changes in ATP affinity. ATP is not hydrolyzed during conversion of G-actin to KCI-monomer. KCI-monomer formation precedes filament formation and may be necessary for the assembly event. Although incorporation of KCI-monomers into filaments demonstrates lagphase kinetics by viscometry, both continuous absorbance monitoring at 232 nm and rapid sedimentation of filaments demonstrate hyperbolic assembly curves. ATP hydrolysis significantly lags the formation of actin filaments. When half of the actin has assembled, only 0.1 to 0.2 mole of ATP are hydrolyzed per mole of actin present as filaments.
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PMID:Mechanism of K+-induced actin assembly. 688 98

The G protein alpha-subunit G alpha 2 is essential to the developmental program of Dictyostelium. G alpha 2 is transiently phosphorylated on a serine residue(s) following stimulation with extracellular cAMP (Gundersen, R. E., and Devreotes, P.N. (1990) Science 248, 591-593). To aid in defining the function of alpha-subunit phosphorylation, we identified the site of G alpha 2 phosphorylation. Comparison of the isoelectric points (pI) of the phosphorylated and nonphosphorylated forms indicated that a single mole of phosphate is added to G alpha 2. Cleavage at tryptophan residues and immunoprecipitation with a specific peptide antibody localized the phosphorylated serine in the N-terminal 119 residues. Analysis of a series of G alpha 1 and G alpha 2 chimeras further confined the site between amino acids 33 and 215. Site-directed mutagenesis of serines between amino acids 33 and 119 produced two mutants that were not phosphorylated, S45A and S113A. Ser113 was identified as the site by sequential Edman degradation of 32P-radiolabeled G alpha 2 digested with endoproteinase Glu-C. We have expressed the G alpha 2 mutants S113A, S113I, S113T, and S113D in a G alpha 2 null cell line to examine the function of phosphorylation.
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PMID:Serine 113 is the site of receptor-mediated phosphorylation of the Dictyostelium G protein alpha-subunit G alpha 2. 806 9

The interaction of the two N-terminally myristoylated isoforms of Dictyostelium hisactophilin with lipid model membranes was investigated by means of the monolayer expansion method and high-sensitivity titration calorimetry. The two isoforms, hisactophilin I and hisactophilin II, were found to insert with their N-terminal myristoyl residue into an electrically neutral POPC monolayer corresponding in its lateral packing density to that of a lipid bilayer. The partition coefficient for this insertion process was Kp = (1.1 +/- 0.2) x 10(4) M-1. The area requirement of the protein in the lipid membrane was estimated as 44 +/- 6 A2 which corresponds to the cross sectional area of the myristoyl moiety with an additional small contribution from amino acid side chains. The interaction of hisactophilin I (hisactophilin II) with negatively charged membrane surfaces is modulated in a pH-dependent manner by charged amino acid residues clustered around the myristoyl moiety. The electrostatic binding site consists of three lysine (one arginine and two lysine), seven (nine) histidine, and four (four) glutamic acid residues and has an isoelectric point of 6.9 (7.1). For small unilamellar POPC/POPG (75/25 mole/mole) vesicles, an apparent binding constant, K(app) = (8 +/- 1) x 10(5) M-1, was measured at pH 6.0 by means of high-sensitivity titration calorimetry. Electrostatic interactions hence increase the binding constant by about 2 orders of magnitude compared to hydrophobic binding alone. With increasing pH, the electrostatic attraction decreases and turns into an electrostatic repulsion at pH > 7.0 +/- 0.1. The area occupied by the cluster of charged residues constituting the membrane binding region was 280 +/- 20 A2 as derived from monolayer measurements in close agreement with molecular modeling data derived from the NMR structure of hisactophilin I [Habazettl et al. (1992) Nature 359, 855-858].
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PMID:Binding of hisactophilin I and II to lipid membranes is controlled by a pH-dependent myristoyl-histidine switch. 878 May 5

Myosin II heavy chain (MHC)-specific protein kinase C (MHC-PKC) isolated from the ameba, Dictyostelium discoideum, regulates myosin II assembly and localization in response to the chemoattractant cAMP. cAMP stimulation of Dictyostelium cells leads to translocation of MHC-PKC from the cytosol to the membrane fraction, as well as causing an increase in both MHC-PKC phosphorylation and its kinase activity. MHC-PKC undergoes autophosphorylation with each mole of kinase incorporating about 20 mol of phosphate. The MHC-PKC autophosphorylation sites are thought to be located within a domain at the COOH-terminal region of MHC-PKC that contains a cluster of 21 serine and threonine residues. Here we report that deletion of this domain abolished the ability of the enzyme to undergo autophosphorylation in vitro. Furthermore, after this deletion, cAMP-dependent autophosphorylation of MHC-PKC as well as cAMP-dependent increases in kinase activity and subcellular localization were also abolished. These results provide evidence for the role of autophosphorylation in the regulation of MHC-PKC and indicate that this MHC-PKC autophosphorylation is required for the kinase activation in response to cAMP and for subcellular localization.
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PMID:Autophosphorylation of Dictyostelium myosin II heavy chain-specific protein kinase C is required for its activation and membrane dissociation. 899 70

Dynacortin is a novel protein that was discovered in a genetic suppressor screen of a Dictyostelium discoideum cytokinesis-deficient mutant cell line devoid of the cleavage furrow actin bundling protein, cortexillin I. While dynacortin is highly enriched in the cortex, particularly in cell-surface protrusions, it is excluded from the cleavage furrow cortex during cytokinesis. Here, we describe the biochemical characterization of this new protein. Purified dynacortin is an 80-kDa dimer with a large 5.7-nm Stokes radius. Dynacortin cross-links actin filaments into parallel arrays with a mole ratio of one dimer to 1.3 actin monomers and a 3.1 microm K(d). Using total internal reflection fluorescence microscopy, GFP-dynacortin and the actin bundling protein coronin-GFP are seen to concentrate in highly dynamic cortical structures with assembly and disassembly half-lives of about 15 s. These results indicate that cells have evolved different actin-filament cross-linking proteins with complementary cellular distributions that collaborate to orchestrate complex cell shape changes.
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PMID:Dynacortin is a novel actin bundling protein that localizes to dynamic actin structures. 1178 90


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