Partial purification and characterization of glycogen phosphorylase from Dictyostelium discoideum

Glycogen phosphorylase was isolated from cells of Dictyostelium discoideum in the culmination stage of development and purified 35-fold. The enzyme had a pH optimum of 6.9 and contained sulfhydryl groups essential for activity. The K(m) values for phosphate and glycogen were 3 mm and 0.06% (w/v), respectively. No dependence on, or stimulation by, any nucleotide was observed and a wide variety of nucleotides and glycolytic intermediates did not inhibit the enzyme. Nucleotide sugars competitively inhibited the enzyme. Guanosine diphosphoglucose and adenosine diphosphoglucose were the most effective, and uridine diphosphoglucose was the least effective of the nucleotide sugars tested. The specific activity of glycogen phosphorylase increased from about 0.004 unit per mg of protein in aggregating cells to about 0.024 unit per mg in culminating cells, and then decreased during sorocarp formation. This increase in enzyme specific activity during the starvation and aging of the system can account for the increased rate of glycogen degradation during this period of development. Amylase specific activity, measured at pH 4.8 and 6.9, varied between 0.005 and 0.013 unit per mg of protein during all stages of development.     

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.     

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.     

Partial purification and properties of citrate synthase from sea urchin eggs.

Citrate synthase [EC 4.1.3.7] was purified from sea urchin eggs about 14-fold with a 23% yield, based on the activity of the crude extract. The molecular weight of the enzyme was about 100,000 as determined by gel filtration. The optimum pH was about 7.8 in 100 mM Tris-HCl. The apparent Km values for acetyl-CoA and for oxaloacetate were 33 and 3.2 muM, respectively. Monovalent and divalent cations inhibited the enzyme. Iodoacetamide, pCMB, EDTA, NaF, and dithiothreitol did not affect the enzyme activity. Oxaloacetate protected the enzyme against heat denaturation. Among nucleotides, ATP was the most potent inhibitor of the enzyme. The inhibition by ATP was competitive with respect to acetyl-CoA and mixed with respect to oxaloacetate.     

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.     

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.     

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.     

Purification and characterization of tripeptidyl peptidase I from Dictyostelium discoideum.

A tripeptidyl peptidase I from Dictyostelium discoideum was purified 744-fold to near homogeneity. The enzyme is 214 kDa in size and is composed of two monomers with a M(r) of 107 kDa. It has two pH optima at pH 4.5 and 5.9 and is a serine peptidase with no aminopeptidase or dipeptidyl peptidase activity. The enzyme was relatively specific showing activity on ala-ala-phe-p-nitroaniline but also acted on substrates with proline in the P1 position in contrast to mammalian TPP I. The K(m) values of the enzyme at pH 4.5 for ala-ala-phe-, ala-phe-pro- and ala-ala-pro-p-nitroanilines were 27 microM, 437 microM and 888 microM, respectively. The enzyme is most abundant during the amoeba stage of the life cycle but is present in the early stages of development and may therefore have a dual role in the organism in mobilizing amino acids or in processing specific peptides or proteins.     

Partial purification and characterization of dolichol phosphate mannose synthase from Entamoeba histolytica

Dolichol phosphate mannose synthase, an essential enzyme in glycoprotein biosynthesis, was partially purified from E.histolytica by hydrophobic interaction and affinity chromatography with octyl Sepharose CL-4B and Affi-Gel 501, respectively. Reducing agents, particularly dithiothreitol, positively influenced enzyme activity and stability, indicating a role of sulfhydryl groups on the transferase function. Activity did not depend on phospholipids; however, it was significantly stimulated by phosphatidylethanolamine and to a lower extent by other common phospholipids. Mixtures consisting of activating phospholipids did not exert an additive effect. In vitro phosphorylation with a cAMP-dependent protein kinase resulted in enzyme activation. This alteration was not associated with a change in the K(m) for the substrate but rather with a 2.6-fold increase in V(max). Phosphorylation in the presence of [gamma-(32)P]ATP resulted in strong labeling of two polypeptides, one of which exhibited the molecular mass reported for the enzyme from other organisms. Whether phosphorylation functions in vivo as a mechanism of regulation of dolichol phosphate mannose synthesis in E.histolytica remains to be determined.     

Isolation and characterization of a cdc 2 cDNA from Dictyostelium discoideum.

A cdc2 homologous sequence was amplified from Dictyostelium discoideum by the polymerase chain reaction and used to isolate several cDNA clones. The amino acid sequence encoded by these cDNAs exhibited approx. 60% identity to the Cdc2 proteins of other species. A cDNA containing the entire coding sequence complemented the temperature sensitive cdc28 mutant of Saccharomyces cerevisiae, although growth of the transformants was slow and limited. Southern blot analysis of restriction digests under high stringency conditions provided evidence that Dictyostelium contains a single cdc2 gene, although at lower stringency multiple fragments were detected, suggesting the existence of a cdc2 gene family. Northern blot analysis of RNA from different stages of Dictyostelium development showed that cdc2 mRNA levels increased during aggregation and then decreased to low levels by the pseudoplasmodial stage of development. By contrast, cdc2 mRNA levels remained relatively constant as cells passed from exponential growth to the stationary phase.     

Spinach leaf dihydrodipicolinate synthase: Partial purification and characterization

The first enzyme unique to lysine biosynthesis in higher plants, dihydrodipicolinate synthase, has been partially purified from spinach leaves, using ion exchange chromatography, hydrophobic interaction chromatography and gel filtration. The spinach enzyme is moderately stable to short-term exposure to heat, in contrast to the pea leaf enzyme, but is unstable on storage even at ?20°. Thiol reagents interfere with the calorimetric assay used, and so cannot be routinely used to stabilize the enzyme, which has an active sulphydryl group. The MW of the enzyme is 115000 (gel filtration). Lysine is a potent inhibitor with an I_(0.5) of 2OμM, whilst the lysine analogue S-β-aminoethylcysteinc has an I_(0.5) of 400 μM. The Kt´_m for aspartic-β-semialdehyde was determined to be 1.4mM, but this compound demonstrated marked substrate inhibition at concentrations above 7 mM, increasing the apparent S_(0.5)for the second substrate, pyruvate.     

An improved procedure for the purification of plasma membranes from Dictyostelium discoideum.

A novel procedure was recently described for the purification of plasma membranes of Dictyostelium discoideum (Gilkes, N. R. & Weeks, G. (1977) Biochim. Biophys. Acta 464, 142-156). Considerable enrichment of plasma membrane marker enzymes was achieved, but since purified mitochondrial and endoplasmic reticulum fractions were unavailable, it was not possible to accurately assess the contamination level of these organelles. We have therefore slightly modified the plasma membrane preparation procedure, improving purification, and have prepared partially purified mitochondrial and endoplasmic reticulum fractions. The data suggest that the contamination of the plasma membranes by endoplasmic reticulum membranes is no greater than 10%, and probably considerably less. No mitochondrial contamination is detectable.     

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.     

Isolation and characterization of a plasma membrane calcium pump from Dictyostelium discoideum.

During the aggregation and differentiation of amoebae of Dictyostelium discoideum, changes in free cytosolic Ca2+ appear to regulate a number of physiological processes. To understand the mechanisms regulating free intracellular Ca2+ in this organism, we have isolated and characterized an ATP/Mg2+-dependent, high-affinity Ca2+ pump. When homogenates of 2 h starved cells were fractionated on Percoll/KCl gradients, one peak of high-affinity Ca2+-pumping activity was detected. This activity was resolved from enzyme markers of the mitochondrion and the rough endoplasmic reticulum but it cosedimented with the plasma membrane marker, alkaline phosphatase. Further studies suggested that the pump was associated with 'inside-out' plasma membrane vesicles. Like plasma membrane Ca2+-transport ATPases from other systems, this isolated Ca2+ pump: (1) was Mg2+-dependent, (2) displayed a high specificity for ATP as an energy source, (3) exhibited a high affinity for free Ca2+ with a Km of 0.3 microM, and (4) was very sensitive to inhibition by vanadate (IC50 2 microM) but was unaffected by mitochondrial inhibitors, ouabain and Ca2+-channel blockers. Unlike plasma membrane Ca2+ pumps from most other systems, this enzyme appeared not to be regulated by calmodulin. During development, non-mitochondrial, vanadate-sensitive, high-affinity Ca2+-pumping activity in crude lysates remained relatively constant for at least 15 h. These observations suggest that this plasma membrane Ca2+ pump probably functions in Dictyostelium to maintain Ca2+ homeostasis by extruding free cytosolic Ca2+ from the cells.     

Partial purification and properties of phosphatidylserine synthase from Clostridium perfringens.

The membrane-associated phospholipid biosynthetic enzyme cytidine 5'-diphospho-1,2-diacyl-sn-glycerol:L-serine O-phosphatidyltransferase (phosphatidylserine synthase; EC 2.7.8.8) was partially purified 337-fold from a cell-free extract of the gram-positive pathogenic anaerobe Clostridium perfringens (ATCC 3624). The purification procedure included extraction from the cell envelope with the nonionic detergent Triton X-100, followed by affinity chromatography on cytidine 5'-diphosphate-diacylglycerol-Sepharose. When the partially purified enzyme was subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, two major bands were evident with apparent minimum molecular weights of 39,000 and 31,000. Activity of phosphatidylserine synthase was dependent on the addition of manganese ions (3 mM) and Triton X-100 (2.7 mM) for maximum activity. The rate of catalysis was maximal at 40 degrees C (with rapid thermal inactivation above this temperature), and the pH optimum was 8.5. The apparent Km values for cytidine 5'-diphosphate-diacylglycerol and L-serine were 0.24 and 0.26 mM, respectively. The synthetic (forward) reaction was favored, as indicated by an equilibrium constant of 82, and the energy of activation was found to be 18 kcal/mol (75,362 J/mol).     

Purification and characterization of a novel dipeptidyl peptidase from Dictyostelium discoideum

A dipeptidyl peptidase (DPP) was purified to homogeneity using lys-ala-beta-naphthylamide, the standard substrate for DPP II. The enzyme is a monomer with a Mr of 70kDa, pl 5.2, and Km 5.0 microM. Its terminal amino acid sequence was XXLLYAIQKRLF and was not identical to that of any known protein. Although initially considered to be a DPP II, the enzyme differed in some properties from classical DPP IIs. It had a pH optimum of 7.9, was not active on X-pro-naphthylamides, the usual substrates of mammalian DPP II, but was active on arg-arg- and asp-arg-naphthylamides, substrates acted on by the DPP III class of enzymes. This enzyme therefore combines properties typical of both DPP II and III and differs from all previously described DPPs. Activity on lys-ala-beta-naphthylamide was most abundant during aggregation and its activity is consistent with processing specific peptides during development.