Partial purification and characterization of citrate synthase from Dictyostelium discoideum.

The effect of thyroidectomy on oxidative metabolism of rat liver, kidney, and brain mitochondria has been examined. The respiration in liver, kidney, and brain mitochondria was affected differentially after thyroidectomy, the common effect in all the tissues being the impairment in state 3 as well as state 4 rates of succinate oxidation. Thyroidectomy did not have any effect on $\text{ADP}\text{O}$ ratios; however, compared to normal, respiratory control indexes were, in general, somewhat higher. Thyroidectomy also did not alter total ATPase activity of liver, kidney, and brain mitochondria, although the basal ATPase activity had decreased significantly under these conditions. The cytochrome content of the mitochondria also showed tissue-specific changes after thyroidectomy; however, no significant changes in the absorption characteristics of the cytochromes were seen. The succinate and glutamate dehydrogenase activities of mitochondria from liver, kidney, and brain were not affected by thyroidectomy, thereby ruling out the possibility that the decrease in substrate oxidation may be due to alterations in the primary dehydrogenase levels. It is concluded that thyroid hormone(s) may have a tissue-specific role in regulating the metabolic functions of mitochondria.     

Partial purification and characterization of trehalase from axenically grown myxamoebae of Dictyostelium discoideum

Lysosomal trehalase from the myxamoebae of Dictyostelium discoideum has been partially purified. The behavior of the enzyme under different chromatographic and electrophoretic conditions reveals its close similarities to other lysosomal enzymes that have been studied earlier. The cellular trehalase, which is electrophoretically homogeneous, appears as two peaks of activity when subjected to hydroxyapatite and gel filtration chromatography. The enzyme has isoelectric points of 4.0 and less than 2.5. Among natural disaccharides tested, the purified trehalase showed absolute specificity for trehalose with an apparent Km of 1.15 mM. However, the enzyme efficiently utilized the synthetic sugar alpha-D-glucosyl fluoride as a substrate. Various methods were employed to estimate the apparent molecular weight, which was found to lie in the range of 30-162 kDa.     

Purification and kinetic characterization of uridine phosphorylase from Dictyostelium discoideum

The purification and kinetic characterization of uridine phosphorylase from Dictyostelium discoideum are described. Matrex Green A, a dye-affinity chromatography gel, was used for the purification. The enzyme was specifically eluted from the dye bead matrix with the use of its substrate, uridine, resulting in a purification of 70- to 2000-fold. The enzyme preparation exhibited stoichiometry. For nucleoside phosphorolysis, the K_m values for phosphate and uridine were 0.42 and 0.24 mm, respectively, and the K_i for phosphate was 3.0 mm. For nucleoside synthesis, the K_m values for uracil and ribose 1-phosphate were 0.06 and 0.14 mm, respectively, and the K_i for ribose 1-phosphate was 0.05 mm. An ordered sequential bi:bi mechanism is proposed based on product inhibition studies.     

Isolation and characterization of glycogen synthase in Dictyostelium discoideum

We have partially purified the protein and isolated the glcS gene for glycogen synthase in Dictyostelium. glcS mRNA is present throughout development and is the product of a single gene coding for 775 amino acids, with a predicted molecular mass of 87 kD. The sequence is highly similar to glycogen synthase from human muscle, yeast, and rat liver, diverging significantly only at the amino and carboxy termini. Phosphorylation and UDPG binding sites are conserved, with K_m values for UDPG being comparable to those determined for other organisms, but in vitro phosphorylation failing to convert between the G6P-dependent (D) and -independent (I) forms. Enzyme activity is relatively constant throughout the life cycle: the I form of the enzyme isolates with the soluble fraction in amoebae, switches to the D form, becomes pellet-associated during early development, and finally reverts during late development to the I form, which again localizes to the soluble fraction. Deletion analysis of the promoter reveals a GC-rich element which, when deleted, abolishes expression of glcS. © 1996 Wiley-Liss, Inc.     

The relationship between the two forms of glycogen phosphorylase in Dictyostelium discoideum

The cellular slime mold, Dictyostelium disoideum, provides an ideal model system to study eukaryotic cell differentiation. In D. discoideum, glycogen degradation provides precursors for the synthesis of developmentally regulated structural products. The enzyme responsible for glycogen degradation, glycogen phosphorylase, exists in active and inactive forms. The active, or 'a' form, is independent of 5'adenosine monophosphate (5'AMP) while the inactive, or 'b' form, is 5'AMP-dependent. The activity of the 'b' form predominates early in development, while the activity of the 'a' form peaks in mid-late development; their combined specific activities remain constant at any point. Polyclonal antibodies raised to the purified forms of this enzyme showed low cross-reactivity. The anti-'a' serum reacted with a 104-kDa protein that was associated with phosphorylase 'a' activity; the anti-'b' serum reacted with a 92-kDa protein that was associated with phosphorylase 'b' activity and weakly cross-reacted with the 104-kDa protein. Immunoblots of peptide maps of the purified enzyme forms showed that each antibody was specific for the proteolytic fragments of its respective antigen. We also demonstrated in vitro phosphorylation of the 'b' form by an endogenous protein kinase. Cyclic AMP perturbation of intact cells caused induction of both phosphorylase-'a' activity and the 104-kDa protein. Immunotitration data suggested that the 'a' form accumulates due to de novo protein synthesis, although this result must be interpreted with caution.     

The purification and characterization of Dictyostelium discoideum plasma membranes.

A new procedure for the purification of plasma membranes of Dictyostelium discoideum is described. Cells are broken by vigorously stirring in the presence of glass beads, and plasma membranes are isolated by equilibrium sucrose density centrifugation. The purified membranes are considerably enriched in alkaline phosphatase and 5'-nucleotidase and contain very low levels of succinate dehydrogenase and NADPH-cytochrome c reductase. The purified membranes contain relatively high levels of phospholipid, sterol and carbohydrate. They appear as a relatively homogeneous population of membrane vesicles in the electron microscope. This new method of purification is compared to previously published procedures which have been found to be unsuitable for our purposes.     

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.     

Partial purification and kinetic characterization of transaldolase fromDictyostelium discoideum

Transaldolase was purified 42-fold fromDictyostelium discoideum and the resulting preparation exhibited stoichiometry. Kinetic analyses consisted of initial velocity and product inhibition studies in both the forward and the reverse directions. The enzyme exhibited ping-pong kinetics with sedoheptulose 7-phosphate adding first and erythrose 4-phosphate releasing first. TheK_m values for sedoheptulose 7-phosphate, glyceraldehyde 3-phosphate, erythrose 4-phosphate, and fructose 6-phosphate were 0.46, 0.072, 0.10, and 1.6 mM, respectively. TheK_i values for sedoheptulose 7-phosphate and erythrose 4-phosphate were 3.6 and 0.062 mM, respectively. Inorganic phosphate inhibited enzymatic activity and showed mixed-type inhibition when fructose 6-phosphate was varied. AK_i value of 35.2 mM was determined for inorganic phosphate.     

Regulation of the two forms of glycogen phosphorylase by cAMP and its analogs in Dictyostelium discoideum

Summary We have recently reported the existence of two forms of glycogen phosphorylase (1,4--D-glucan: orthophosphate--glucosyltransferase; EC 2.4.1.1) in Dictyostelium discoideum. During development the activity of the glycogen phosphorylase b form decreased as the activity of the a form increased. The total phosphorylase activity remained constant. The physical and kinetic properties of the Dictyostelium enzyme were similar to those of the mammalian enzyme. In mammals, cAMP regulates the conversion of the two forms by a cAMP dependent protein kinase (cAMPdPK). We report here that if cAMP is added to a single cell suspension, the Dictyostelium phosphorylase activity becomes independent of 5AMP and a 104kd peptide appears. We also show the effect of several cAMP analogs on the phosphorylase activity in these single-cell suspensions. The cAMP analogs were selected on the basis of their affinities for the membrane-bound cAMP receptor or the cytoplasmic cAMPdPK. We found that relatively low levels, 100 M, of cAMP or 2'd-cAMP added to aggregation-competent cells in shaking culture caused a loss of phosphorylase b activity and the appearance of phosphorylase a activity. The analog, 2'd-cAMP, has a high affinity for the cAMP receptor but a low affinity for the cAMPdPK. Two other analogs, Bt₂-cAMP and 8-Br-cAMP, which have low affinities for the cAMP receptor but high affinities for the cAMPdPK, required high levels (500 M) for b to a conversion. cDNAs to three cAMP-regulated genes-PL3, Dll, and D3-were used as controls in the above experiments. In order to determine if intracellular levels of cAMP were involved in the regulation of phosphorylase activity, both the phosphorylase and the PL3, D11 and D3 mRNA levels were examined in cells suspended in a glucose/albumin mixture - a medium in which adenylate cyclase is inhibited. Under these conditions, neither gene regulation nor a change in the phosphorylase b to a activity occurred in response to added extra cellular cAMP. The results suggest that an intracellular increase in cAMP is involved in the regulation of the two forms of glycogen phosphorylase in Dictyostelium.Abbreviations EGTA Ethyleneglycol-bis-(-aminoethyl ether) - N,N,N N-tetra acetic acid - SDS Sodium Dodecyl Sulfate - PAGE Polyacrylamide Gel Electrophoresis     

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.     

The NAD-dependent glutamate dehydrogenase from Dictyostelium discoideum: purification and properties

The NAD-dependent glutamate dehydrogenase (GDH) from Dictyostelium discoideum was purified 1101-fold with a yield of 23.4%. The enzyme has an apparent Mr of 356 kDa, determined using Sephacryl S400, and a subunit molecular weight of 54 kDa on SDS-polyacrylamide gel electrophoresis. The Kms for alpha-ketoglutarate, NADH, and NH4+ are 0.36 +/- 0.03 mM, 16.0 +/- 0.1 microM, and 34.5 +/- 2.7 mM, respectively. The purified enzyme has a pH optimum of pH 7.25-7.5. At 0.1 mM, ADP and AMP stimulate GDH activity 25 and 102%, respectively. Half-maximal activity in the presence of 0.1 mM AMP for alpha-ketoglutarate, NADH, and NH4+ is reached at 2.3 +/- 0.1 mM, 71.4 +/- 5.5 microM, and 27.9 +/- 3.6 mM, respectively.     

Purification and characterization of NAD-dependent isocitrate dehydrogenase from Dictyostelium discoideum

Isocitrate dehydrogenase (threo-d_s-isocitrate: NAD oxidoreductase (decar☐ylating) EC 1.1.1.41) from Dictyostelium dicoideum was purified 161-fold. The purified enzyme was NAD specific and required Mn²⁺ for activity. Isocitrate consumption and 2-oxoglutarate and NADH production were stoichiometric; no NADH oxidase or glutamate dehydrogenase activities were detected. The pH optimum range for activity was pH 7.5–8.5. Reductive car☐ylation of 2-oxoglutarate with NADH could not be demonstrated. Lineweaver - Burk plots of data from initial velocity studies were linear. There was no evidence of allosteric control by reported effectors (AMP, ADP, citrate) of isocitrate dehydrogenase activity. The reaction was inhibited by NADH. The inhibition by NADH was competitive when either isocitrate or NAD was the variable substrate. 2-Oxoglutarate was not inhibitory at concentrations below 4 mm. The Michaelis constant (K_m) and dissociation constant (K_ib) for isocitrate were 0.16 mm; and K_m and dissociation constant (K_ia) for NAD were 0.34 mm. The inhibition constant for NADH was 0.02 mm. The data are consistent with a rapid equilibrium random bi-bi reaction mechanism (Cleland nomenclature). The NAD-linked isocitrate dehydrogenase activity was also demonstrated in crude extracts of isolated mitochondria.     

Biochemical and structural characterization of actin from Dictyostelium discoideum.

Actin has been purified from amoebae of Dictyostelium discoideum by a procedure which is notable in that proteolysis has been diminished to undetectable levels and "selective" purification steps have been avoided. The overall yield of this procedure is 5- to 10- fold greater than that of a previous report (Spudich, J. A. (1974) J. Biol. Chem. 249, 6013-6020). The detailed biochemical and structural properties of this new preparation (preparation B) have been compared to those of Dictyostelium actin prepared by the previous procedure (preparation A) as well as to rabbit skeletal muscle actin. Preparation B actin is similar to muscle actin in its molecular weight, ability to activate myosin, filament structure, and polymerization properties. Preparation B actin has the same molecular weight and isoelectric point as preparation A actin, which is more acidic than that of skeletal muscle actin. However, preparation B actin and muscle actin form longer filaments than preparation A actin, as judged by viscometry and electron microscopy.     

Identification of two forms of glycogen phosphorylase in Dictyostelium

It has been known for 20 years that during cellular differentiation of Dictyostelium discoideum, glycogen is degraded to provide the glucose precursors that are required for the synthesis of the end-products of development. Because this pathway provided a distinct developmentally regulated event, a number of laboratories have investigated the regulation of the first step in glycogen degradation, glycogen phosphorylase. Of particular interest was the possible regulation of this enzyme by cAMP. Cyclic AMP is know to act as a signal in this organism for both chemotaxis and cell differentiation. The phosphorylase activity was found to increase during development and, therefore, it has been used in many studies as a marker for late stage development. However, only one form of the phosphorylase was found, and therefore it was concluded that cAMP was not involved in regulation of this key step in the developmental pathway. Here we report the discovery of a second form of the enzyme. This form is completely dependent on AMP for activity and is found only in the undifferentiated stage. This second form contains several of the properties of the nonphosphorylated enzyme that occurs in systems that are regulated by cAMP. This result and the recent discovery of a cAMP-dependent protein kinase has rekindled the possibility that at least one of the effects of cAMP in this organism occurs via a cAMP-dependent cascade of phosphorylation; that is, the activation of glycogen phosphorylase and subsequent production of the precursors for the end-products of development.     

Trehalose 6-phosphate synthase from Dictyostelium discoideum: partial purification and characterization of the enzyme from young sorocarps.

Trehalose 6-phosphate synthase was solubilized from young sorocarps of the cellular slime mold, Dictyostelium discoideum, by a freeze-thaw cycle and was subsequently purified about 160-fold using streptomycin sulfate precipitation, (NH₄)₂SO₄ fractionation, DEAE-cellulose chromatography, heat treatment in the presence of heparin, and molecular sieve chromatography on columns of Bio-Gel A-1.5m. The purified enzyme was maximally active at pH 6.5, showed an absolute specificity for glucose 6-phosphate as glucosyl acceptor and a relative specificity for the glucosyl donor in the order: UDP-glucose, GDP-glucose, and ADP-glucose. Although heparin and chondroitin sulfate activated the synthase, the order of glucosyl donor specificity was not affected. Other activators of trehalose 6-phosphate synthase were KCL, Mg²⁺, and EDTA, while detergents had little effect. Although synthase activity was reduced 60 to 80% upon the omission of Mg²⁺ from the assay mixture, an absolute dependency for Mg²⁺ could not be demonstrated. Evaluation of the apparent K_m values for partially purified synthase preparations demonstrated that for each of the synthase substrates, the Line weaver-Burk plots displayed complex bimodal kinetics. Estimation of the Michaelis constants after extrapolation of the straight line portions of these plots yielded values of (a) 0.2 and 3.2 mm glucose 6-phosphate and (b) 0.5 and 2.2 mm UDP-glucose. Comparison of the latter parameters with the cellular levels of UDP-glucose and glucose 6-phosphate in Dictyostelium suggests that if the observed bimodal kinetics are the consequence of multiple kinetically distinct forms of the synthase, the activation of trehalose synthesis during slime mold culmination could provide a rationale for the presence of these isozymes.