scholarly journals Structural and biochemical aspects of cell motility in amebas of Dictyostelium discoideum.

1977 ◽  
Vol 72 (2) ◽  
pp. 339-350 ◽  
Author(s):  
B S Eckert ◽  
R H Warren ◽  
R W Rubin
Keyword(s):  
Life Cycle ◽  
Cyclic Amp ◽  
Cell Motility ◽  
Dictyostelium Discoideum ◽  
Cell Shape ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Saltatory Movement ◽  
Filament Network ◽  
Motility Rate

Amebas of Dictyostelium discoideum contain both microfilaments and microtubules. Microfilaments, found primarily in a cortical filament network, aggregate into bundles when glycerinated cells contract in response to Mg-ATP. These cortical filaments bind heavy meromyosin. Microtubules are sparse in amebas before aggregation. Colchicine, griseofulvin, or cold treatments do not affect cell motility or cell shape. Saltatory movement of cytoplasmic particles is inhibited by these treatments and the particles subsequently accumulate in the posterior of the cell. Cell motility rate changes as Dicytostelium amebas go through different stages of the life cycle. Quantitation of cellular actin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the quantity of cellular actin changes during the life cycle. These changes in actin are directly correlated with changes in motility rate. Addition of cyclic AMP to Dictyostelium cultures at the end of the feeding stage prevents a decline in motility rate during the preaggregation stage. Cyclic AMP also modifies the change in actin content of the cells during preaggregation.

scholarly journals Phosphorylation of synaptic-membrane proteins from ox cerebral cortex in vitro. Preparation of fractions enriched in phosphorylated proteins by using extraction with detergents and urea, and gel filtration

1977 ◽  
Vol 163 (2) ◽  
pp. 369-378 ◽  
Author(s):  
P R Dunkley ◽  
H Holmes ◽  
R Rodnight
Keyword(s):  
Cerebral Cortex ◽  
Cyclic Amp ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Synaptic Membrane ◽  
Hydroxyl Groups ◽  
Phosphorylated Proteins

Synaptic-membrane fragments from ox cerebral cortex contain basal and cyclic AMP-stimulated protein kinase(s) that transfer 32P from [gamma-32P]ATP to hydroxyl groups of serine and threonine residues in membrane-protein substrates. In the present work, labelled membrane fragments were partitioned into soluble and insoluble fractions with Triton X-100, Nonidet P. 40, sodium deoxycholate and urea, and the distribution of 32P-labelled protein in the fractions was determined by polyacrylamide-gel electrophoresis and radioautography. A high percentage of phosphorylated protein sustrates remained insoluble, including those whose phosphorylation was most highly stimulated by cyclic AMP. Whole membrane fragments and samples prepared by detergent extraction were fractionated on Sepharose 6B columns in the presence of low concentrations of sodium dodecyl sulphate and pooled fractions were analysed by polyacrylamide-gel electrophoresis and radioautography. Phosphorylated proteins were fractionated on the basis of their molecular weight, but homogeneous protein was not obtained. The results are discussed in relation to the techniques used and the results obtained in other laboratories.

Effects of calcium antagonists on cyclic AMP phosphodiesterase induction in Dictyostelium discoideum

1987 ◽  
Vol 88 (3) ◽  
pp. 379-388
Author(s):  
M.B. Coukell ◽  
A.M. Cameron
Keyword(s):  
Cyclic Amp ◽  
Cell Viability ◽  
Dictyostelium Discoideum ◽  
Cyclic Gmp ◽  
Cell Shape ◽  
Second Messengers ◽  
Channel Blockers ◽  
Cyclic Amp Phosphodiesterase ◽  
Dose Dependent Fashion ◽  
Altered Cell

Previous studies have suggested that cyclic GMP and/or Ca2+ might function as second messengers in the induction by exogenous cyclic AMP of the cyclic AMP phosphodiesterase (PD) in Dictyostelium discoideum. To assess further the role of Ca2+ in PD induction we examined the effect on this process of a number of putative Ca2+-channel blockers. At relatively low micromolar concentrations, TMB-8, nicardipine, nifedipine, diltiazem and verapamil all altered cell shape and inhibited PD induction in a similar dose-dependent fashion. Concentrations of these drugs that abolished PD induction had no effect on cell viability; however, higher concentrations reduced viability and caused cell lysis. All effects of these compounds on the cells were antagonized at least partially by 5–10 mM-Ca2+. Other cations tested were considerably less effective. Like the organic inhibitors, La3+ also altered cell shape, inhibited PD induction and reduced cell viability at elevated concentrations, but its effect on the cells appeared to be more complex. Inhibition of PD induction by the organic antagonists could not be attributed solely to an impaired uptake of extracellular Ca2+, a reduction of ATP pools in the cells or a direct effect on calmodulin. Concentrations of TMB-8 that inhibited PD induction had little effect on the cyclic GMP response. Therefore, this compound did not inhibit PD induction indirectly by blocking cyclic GMP synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Effects of collagen, ionophore A23187 and prostaglandin E1 on the phosphorylation of specific proteins in blood platelets

1979 ◽  
Vol 178 (2) ◽  
pp. 397-406 ◽  
Author(s):  
Richard J. Haslam ◽  
James A. Lynham ◽  
Joan E. B. Fox
Keyword(s):  
Cyclic Amp ◽  
Prostaglandin E1 ◽  
Blood Platelets ◽  
Short Circuit ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Platelets ◽  
Ionophore A23187 ◽  
Release Reaction ◽  
Specific Proteins

Human platelets that had been preincubated with 5-hydroxy[3H]tryptamine and [32P]Pi were stirred with various agents; the secretion of 5-hydroxy[3H]tryptamine from platelet granules and the radioactivity of platelet [32P]phosphopolypeptides separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis were then measured. Exposure of the platelets to collagen fibres or ionophore A23187 selectively increased the phosphorylation of polypeptides with apparent mol.wts. of 47000 (P47) and 20000 (P20) by approx. 3-fold, in association with the release of 5-hydroxy[3H]tryptamine. The 47000-mol.wt. phosphopolypeptide (P47) was clearly separated from platelet actin by the electrophoresis system used. Prostaglandin E1, which inhibits platelet function by increasing platelet cyclic AMP, decreased the phosphorylation of polypeptides caused by collagen as well as the release of 5-hydroxy[3H]tryptamine. Prostaglandin E1 also selectively increased the phosphorylation of distinct polypeptides with apparent mol.wts. of 24000 (P24) and 22000 (P22) by approx. 2-fold. As the phosphorylation reactions caused by collagen are probably mediated by an increase in Ca2+ concentration in the platelet cytosol and may have a role in the release reaction [Haslam & Lynham (1977) Biochem. Biophys. Res. Commun.77, 714–722; (1978) Thromb. Res.12, 619–628], we suggest that a cyclic AMP-dependent phosphorylation of the 24000- and/or 22000-mol.wt. polypeptides caused by prostaglandin E1 may initiate processes that decrease the Ca2+ concentration in the cytosol, so inhibiting both the Ca2+-dependent phosphorylation reactions and the release reaction. Treatment of platelets with prostaglandin E1 did not inhibit the increased phosphorylation of polypeptides with apparent mol.wts. of 47000 and 20000 (P47 and P20) caused by ionophore A23187, which may therefore short-circuit cyclic AMP-dependent mechanisms that decrease the Ca2+ concentration in the platelet cytosol. As prostaglandin E1 did inhibit the release of 5-hydroxy[3H]tryptamine by ionophore A23187, cyclic AMP may also inhibit the release reaction by additional mechanisms.

scholarly journals Human platelets are defective in processing of cholera toxin

1983 ◽  
Vol 212 (3) ◽  
pp. 669-678 ◽  
Author(s):  
R J Hughes ◽  
P A Insel
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Mammalian Cells ◽  
Time Course ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Platelets ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity

Cholera toxin is unable to elevate cyclic AMP levels in intact human platelets despite being very efficacious in this respect in other mammalian cells; in the presence of 0.5 mM-isobutylmethylxanthine, we found that 3-6nM-cholera toxin over 3h at 37 degrees C elevated platelet cyclic AMP from 33 +/- 13 to 39 +/- 12pmol/mg of protein (means +/- S.D.; n = 12). We have investigated the basis for this lack of response. 125I-labelled cholera toxin bound to platelets both saturably and with high affinity (Kd congruent to 60pM; Bmax. congruent to 50fmol/mg of protein). Incubation of platelets with the putative cholera toxin receptor monosialoganglioside GM1 enhanced 125I-labelled cholera toxin binding at least 40-fold but facilitated only a minimal (less than or equal to 3-fold) elevation of platelet cyclic AMP levels. In contrast, dithiothreitol-activated cholera toxin markedly stimulated adenylate cyclase activity in platelet membranes. Platelet cytosol both enhanced stimulation of adenylate cyclase activity by activated cholera toxin (A1 subunit) and supported stimulation by the A1-A2 subunit of cholera toxin. Neither GTP nor NAD+, both necessary for response to cholera toxin, was lacking in intact platelets. However, we found that platelets were unable to cleave cholera toxin to the active A1 subunit (as assessed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis). By contrast, murine S49 lymphoma cells were able to generate the A1 subunit with a time course that closely resembled the kinetics of toxin-mediated cyclic AMP accumulation in these cells. Thus we conclude that human platelets are defective in their ability to process surface-bound cholera toxin. These results indicate that binding of cholera toxin to surface receptors is necessary, but not sufficient, for expression of the toxin effect and the generation of the A1 subunit of the toxin may be rate-limiting for expression of cholera toxin response.

scholarly journals Accumulation of UDP-glucose pyrophosphorylase by axenically grown amoebae of Dictyostelium discoideum

1979 ◽  
Vol 177 (1) ◽  
pp. 21-28 ◽  
Author(s):  
B D Hames ◽  
B A Hodson
Keyword(s):  
Affinity Chromatography ◽  
Dictyostelium Discoideum ◽  
Sodium Dodecyl Sulphate ◽  
Vegetative Growth ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Klebsiella Aerogenes ◽  
Axenic Medium ◽  
Axenic Growth

Amoebae of the slime mould Dictyostelium discoideum AX2 possess only low UDP-glucose pyrophosphorylase activity when grown on autoclaved Klebsiella aerogenes (approx. 30 units/mg of protein), but accumulate the enzyme to approx. 150-200 units/mg of protein during vegetative growth in axenic medium. The vegetative accumulation of UDP-glucose pyrophosphorylase by axenically grown cells is prevented if autoclaved K. aerogenes are included in the axenic medium, suggesting the absence of a specific inducer. Affinity chromatography using anti-(UDP-glucose pyrophosphorylase) antibody and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicate that the enzyme accumulated during axenic growth and that normally accumulated during development are immunologically cross-reactive and that both are composed of two subunits with mol.wts. 55,600 and 57,500 present in approximately equal amounts in the active enzyme.

The microcyst wall of Didymium iridis: chemical analyses

1984 ◽  
Vol 30 (2) ◽  
pp. 162-170
Author(s):  
Thomas J. Raub
Keyword(s):  
Gel Electrophoresis ◽  
Cell Shape ◽  
Physarum Polycephalum ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Insoluble Fraction ◽  
Chemical Analyses ◽  
Neutral Sugars ◽  
Didymium Iridis ◽  
Sulfur Containing

Chemical analyses of the microcyst cell wall of Didymium iridis were done to compare it with other well-studied organisms, Physarum polycephalum and Physarum flavicomum. Large wall fragments were obtained by breakage in a Braun homogenizer. Chemical analyses of purified walls identified neutral sugars, protein, and hexosamine as the major components. Wall polysaccharides were mostly composed of galactosamine with smaller amounts of glucose and galactose. The protein component consisted of large quantities of threonine and aspartate–asparagine with trace amounts of the sulfur-containing amino acids. Most of the wall protein was soluble in alkaline urea. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis was used to identify seven major bands, at least four of which are acidic glycoproteins. Most of the galactosamine was associated with the urea – hot alkali insoluble fraction comprised mostly of glucose. This galactosaminoglucan was partially sulfated and acetylated and arranged as microfibrils that maintain cell shape.

scholarly journals Transit of alpha-mannosidase during its maturation in Dictyostelium discoideum.

1985 ◽  
Vol 101 (6) ◽  
pp. 2063-2069 ◽  
Author(s):  
L Wood ◽  
A Kaplan
Keyword(s):  
Plasma Membrane ◽  
Dictyostelium Discoideum ◽  
Gel Electrophoresis ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Percoll Gradient ◽  
Density Fractions ◽  
Physical Evidence ◽  
Immune Precipitation

We proposed that Dictyostelium discoideum contains two linked pools of mature alpha-mannosidase (Wood, L., R. N. Pannell, and A. Kaplan, 1983, J. Biol. Chem., 258:9426-9430). To obtain physical evidence for these pools, cells were pulse-labeled with [35S]methionine, homogenized, and subjected to Percoll gradient centrifugation. After immune precipitation of alpha-mannosidase, its polypeptides were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and detected by fluorography. After a 30-min pulse with [35S]methionine, the precursor and small amounts of cleaved enzyme were detected in a low density fraction (1.04 g/ml). Subsequently, cleaved enzyme was transferred to higher density fractions (1.05 and 1.07 g/ml) that were enriched in lysosomal enzymes. The half time for formation of the 1.07 g/ml pool was approximately 45 min, whereas formation of the 1.05 g/ml pool was not detected until 1.5 h after the pulse. The transfer of mature forms out of the 1.04 g/ml pool was inhibited by monensin (3.5 microM). Thus, alpha-mannosidase precursor appears to be cleaved in a prelysosomal organelle. The data also indicate that starving cells secrete precursor directly from this organelle to the extracellular space, whereas cleaved forms are first transferred into lysosomes before they are secreted. Furthermore, 2 h after starvation, the secretion of mature forms ceases even though both transit of mature forms between the two pools and secretion of precursor continues. From this we inferred that the cessation of secretion of mature forms is due to a halt in fusion of lysosomes with the plasma membrane and that precursor follows a different route to the plasma membrane.

scholarly journals The biosynthesis of plasmodial myosin during starvation of Physarum polycephalum

1973 ◽  
Vol 135 (4) ◽  
pp. 639-647 ◽  
Author(s):  
F. Harry White ◽  
June Lascelles
Keyword(s):  
Life Cycle ◽  
Physarum Polycephalum ◽  
Adenosine Triphosphatase ◽  
De Novo ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Low Molecular Weight ◽  
Time Intervals

The actomyosin protein complex of Physarum polycephalum was prepared from vegetative and starved plasmodia. The yield of actomyosin per unit wet wt. was the same from both types of plasmodia. Myosin was resolved from the complex by gel filtration and purified by ion-exchange chromatography. The Ca2+-stimulated adenosine triphosphatase activities of myosin preparations from vegetative and starved plasmodia were not appreciably different. Synthesis of myosin de novo was shown to occur during the starvation phase of the life-cycle by the isolation of labelled myosin preparations from plasmodia starved in the presence of [2-14C]glycine. Fractionation of polyacrylamide gels after gel filtration of labelled myosin confirmed the presence of label in the adenosine triphosphatase-active myosin band. It is concluded that during starvation myosin synthesis continues although there is a net loss of approx. 50% of the total protein. Sodium dodecyl sulphate–polyacrylamide-gel electrophoresis of Physarum myosin showed the presence of low-molecular-weight components of the molecule, similar to those of muscle myosins. The content and composition of the free amino acid pool of Physarum was measured at various time-intervals during the vegetative and starvation phases of the life-cycle.

scholarly journals Rates of accumulation of glycosidase activities during growth and differentiation of Dictyostelium discoideum

1975 ◽  
Vol 148 (2) ◽  
pp. 161-167 ◽  
Author(s):  
D Every ◽  
J M Ashworth
Keyword(s):  
Life Cycle ◽  
Cyclic Amp ◽  
Dictyostelium Discoideum ◽  
Growth Medium ◽  
Specific Activity ◽  
Growth Conditions ◽  
Cellular Protein ◽  
Total Cellular Protein ◽  
Early Differentiation ◽  
Rate Of Accumulation

1. The rates of accumulation (enzyme units/h per 10(8) cells) of a number of glycosidase activities were studied in Dictyostelium discoideum cells during the growth and differentiation phases of this organism's life cycle. 2. The rates of accumulation of the enzymes β-N-acetylglucosaminidase, α-glucosidase and β-galactosidase remain unchanged during the growth and early differentiation phases. 3. The considerable changes in specific activity of the enzymes which occur in the early differentiation phase are due to the massive loss of total cellular protein which occurs at this time. 4. Significant alterations can occur in the rates of accumulation of α-mannosidase during both the growth and differentiation phases, and since, on the onset of differentiation, β-glucosidase activity is excreted and degraded, the rate of accumulation of this enzyme differs in the growth and differentiation phases. 5. The characteristic rates of accumulation of all these glycosidases change markedly with changes in the growth conditions of the myxamoebae, and thus these rates of synthesis must be regulated independently; however, addition of cyclic AMP to the growth medium has no effect on them.

A stalk-specific wheat germ agglutinin binding protein, wst34, in Dictyostelium discoideum can be detected with antiserum raised against Dictyostelium mucoroides stalk

1990 ◽  
Vol 68 (4) ◽  
pp. 699-704 ◽  
Author(s):  
Yuzuru Kubohara ◽  
Koji Okamoto
Keyword(s):  
Dictyostelium Discoideum ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyclonal Antiserum ◽  
Stalk Cell ◽  
Affinity Column ◽  
Western Blots

A new stalk-specific wheat germ agglutinin (WGA) binding protein, wst34, has been identified in Dictyostelium discoideum and purified by the use of preparative sodium dodecyl sulfate - polyacrylamide gel electrophoresis and a WGA-affinity column. In normal development, wst34 appears during culmination and is maintained in stalk cells. It has a molecular mass of 34 kilodaltons and a pI value of 5.5–6.5. A polyclonal antiserum raised against stalk cell proteins of Dictyostelium mucoroides recognizes wst34 in western blots of D. discoideum proteins.Key words: Dictyostelium discoideum, Dictyostelium mucoroides, wheat germ agglutinin.

Sign in / Sign up

Export Citation Format

Share Document