The glycoproteins of Dictyostelium discoideum. Changes during development.

The glycoproteins of D. discoideum have been analyzed by direct binding of radio-iodinated lectins to SDS gels of the successive developmental stages. Compared with the total pattern of proteins, many changes are found in the glycoproteins during development. WGA reacts with few gel bands from the vegetative cells and most of these, including a very intense band at the top of the gel, are lost during the first few hours of development. Approximately half-way through the developmental cycle at least 14 new glycoproteins reacting with WGA begin to appear and progressively accumulate. In contrast, ConA labels many glycoproteins over the complete molecular weight range and most are unaffected during development. Lectins which bind fucose label a single component at the top of the gel of vegetative cells and this decreases rapidly as development begins. No other reactive gel bands are revealed by fucose-binding lectins until the final stages of spore and stalk formation, when four high molecular weight glycoproteins are detected. Lectins specific for terminal galactose residues and for N-acetyl-galactosamine, including the intrinsic lectins produced by D. discoideum during its development, failed to reveal any reactive glycoproteins.     

Accumulation of alpha-mannosidase-1 in Dictyostelium discoideum requires many developmentally essential genes

alpha-Mannosidase-1, one of the earliest known developmentally controlled gene products in the cellular slime mold Dictyostelium discoideum, accumulates intracellularly during both axenic growth and development. The accumulation of alpha-mannosidase-1 activity prematurely ceases in all of 125 randomly isolated aggregation-deficient mutants at discrete times in development resulting in significantly reduced levels of cellular enzyme activity. This suggests that, unlike other developmentally controlled enzymes in this organism, the continued accumulation of alpha-mannosidase-1 activity is controlled by a large number of genes essential for early development. alpha-Mannosidase-1 misregulation and the aggregation-deficient phenotype are caused by the same mutation since (1) morphological revertants exhibit a coreversion to both fruiting ability and wild-type alpha-mannosidase-1 accumulation and (2) normal enzyme accumulation depends on the ability to aggregate and ultimately fruit in a conditional aggregation-deficient mutant. This type of regulation does not appear to be due to differences in enzyme secretion or changes in the overall rate of total protein synthesis. Aggregation-deficient mutants continue to synthesize protein beyond the time in development at which alpha-mannosidase-1 accumulation ceases. Our studies indicate that most of the 50-125 genes required for aggregation in Dictyostelium are also required for the normal accumulation of alpha-mannosidase-1 activity.     

Effect of stimulus strength and adaptation on the thermotactic response of Dictyostelium discoideum pseudoplasmodia

The thermotactic responses of Dictyostelium discoideum strain HL50 and mutants derived from this strain have been characterized by curves of stimulus-strength vs response. With gradient midpoint temperatures of 16 and 24 °C, these curves are typical of those of a single response, i.e., the strength of the response increases with increasing stimulus strength until at some strength the response saturates. However, with a gradient midpoint temperature close to the transition from negative to positive thermotaxis, the sign of the thermotactic response depends on gradient strength. These observations support the hypothesis that the transduction pathways for positive and negative thermotaxis act concurrently and contain separable elements. An investigation of the adaptation of thermotaxis indicated that the stimulus-strength-dependence and midpoint-temperature-dependence of both thermosensory responses was altered by shifting the growth and development temperature.     

Effect of extracellular Ca2+ on the morphogenesis of Dictyostelium discoideum.

Effect of extracellular Ca²⁺ on the morphogenesis of the cellular slime mold Dictyostelium discoideum was examined on agar plate. The concentration of Ca²⁺ in agar plate was controlled by keeping the concentration of a chelating reagent EGTA constant and varying the concentration of total calcium. From experiments in which EGTA concentration was kept at 2.0 × 10^?3 M, it was found that by decreasing Ca²⁺ concentration the morphogenesis was modified so that development of the aggregating amebae into fruiting bodies was accelerated and the period of migrating slugs was shortened. Below 1.0 × 10^?3 M of Ca²⁺ concentration, the total number of aggregates initially increased with decreasing Ca²⁺ concentration, reached a maximum at about 3.0 × 10^?7 M of Ca²⁺ concentration and hereafter decreased with decreasing Ca²⁺ concentration. The number of mature fruiting bodies obtained at 36 h period after starvation depends on Ca²⁺ concentration and the total number of aggregates. The cell aggregation initiated at the same time period after starvation even at an extreme case of 1.0 × 10^?8 M of Ca²⁺ concentration as under enough Ca²⁺ supply, while the formation of mature fruiting body was seriously inhibited. These observation suggested that the cAMP-mediated cell aggregation in D. discoideum is a Ca²⁺-independent phenomena, although extracellular Ca²⁺ is necessary for the normal development of the aggregated amebae.     

Developmental changes in messenger RNAs and protein synthesis in Dictyostelium discoideum

The pattern of proteins synthesized at different stages of differentiation of the slime mold Dictyostelium discoideum was studied by two-dimensional polyacrylamide gel electrophoresis. Of the approximately 400 proteins detected during growth and/or development, synthesis of most continued throughout differentiation. Approximately 100 proteins show changes in their relative rates of synthesis. During the transition from growth to interphase, the major change observed is reduction in the relative rate of synthesis of about 8 proteins. Few further changes are noticeable until the stage of late cell aggregation, when production of about 40 new proteins begins and synthesis of about 10 is reduced considerably. Thereafter, there are few changes in the pattern of protein synthesis. Major changes in the relative rates of synthesis of a number of proteins are found during culmination, but few culmination-specific proteins are observed. In an attempt to understand the molecular basis for these changes, mRNA was isolated from different stages of differentiation and translated in an improved wheat germ cell-free system; the products were resolved on two-dimensional gels. The ratio of total translatable mRNA to total cellular RNA is constant throughout growth and differentiation. Messenger RNAs for many, but not all, developmentally regulated proteins can be identified by translation in cell-free systems. Actin is the major protein synthesized by vegetative cells and by early differentiating cells. The threefold increase in the relative rate of synthesis of actin during the first 2 hr of differentiation and the decrease which occurs thereafter can be accounted for by parallel changes in the amount of translatable actin mRNA. Most of the changes in the pattern of protein synthesis which occur during the late aggregation and culmination stages can also be accounted for by parallel increases or decreases in the amounts of translatable mRNAs encoding these proteins. It is concluded that mRNAs do not appear in a translatable form before synthesis of the homologous protein begins, and that regulation of protein synthesis during development is primarily at the levels of production or destruction of mRNA.     

On the mechanism of erythroid cell sensitivity to chloramphenicol: Studies on mitochondria isolated from erythroid and myeloid tumors

Erythroleukemia mitochondria (E. Mito) and chloroma mitochondria (C. Mito) were isolated from tumors grown in their hosts, DBA/2J mice and Long-Evans rats, respectively. Oxypolarographic tests showed respiratory control and ADP/O ratios typical for well-coupled mitochondria. Therapeutic concentration of chloramphenicol (CAP) had no effect on the energy transfer of those mitochondria. l-[¹⁴C]leucine incorporation into protein was comparable in both types of mitochondria. Although the incorporation at 15 min appeared higher in C. Mito, at 60 min it became similar to that in E. Mito. When CAP was used at the therapeutic concentration of 20 μg/ml about 80% inhibition was observed in both mitochondria. The exogenous amino acid mixture added to the medium was an important determinant in both the rate of leucine incorporation as well as the sensitivity to CAP. Thus, if no amino acids were added the incorporation was reduced to 18–25%. Under these conditions, however E. Mito were significantly more sensitive to the same concentration of CAP than C. Mito. The results suggest that mitochondrial amino acid pool may be involved in the greater sensitivity of erythroid precursors to CAP.     

Endogenous substrates for in vitro phosphorylation by cyclic AMP-dependent protein kinase from Dictyostelium discoideum

Endogenous proteins which could serve as substrates for cyclic AMP-dependent protein kinase in vitro were measured in cytosolic fractions at four stages of development. A peak of cyclic AMP-dependent phosphorylation occurred at the slug stage, coincident with the appearance of cyclic AMP-dependent protein kinase. After partial purification of the slug-stage extracts by DE-52 cellulose and Sephacryl S-300 chromatography, cyclic AMP dependency of six proteins was observed. The apparent subunit molecular weights of the proteins were greater than 200,000, 110,000, 107,000, 91,000, 75,000 and 69,000. Upon further purification of the cyclic AMP-dependent protein kinase by chromatofocusing, the endogenous substrates were separated from the enzyme. In addition, the enzyme separated into catalytic and regulatory subunits. If the purified catalytic subunit was added to heated S300 fractions, proteins with apparent molecular weights of 91,000 and 107,000 were specificity phosphorylated. The results show the stage-dependent appearance of a cyclic AMP-dependent protein kinase and point out several in vitro substrates for the enzyme.     

Isolation and regulatory properties of two glutamate dehydrogenases from the cellular slime mold Dictyostelium discoideum.

Two distinct glutamate dehydrogenases are present in amoebae of the cellular slime mold Dictyostelium discoideum. One enzyme has been extracted from a crude mitochondrial fraction, and the other from an extramitochondrial cytoplasmic fraction. Both enzymes have been partially purified and characterized. The mitochondrial enzyme can utilize both NAD⁺ and NADP⁺ as coenzyme, while the extramitochondrial is NAD⁺ specific. When the mitondrial enzyme is assayed in the presence of either a rate-limiting or saturating concentration of glutamate, its activity is stimulated by both AMP and ADP and is inhibited by ATP. When the extramitochondrial enzyme is assayed in the presence of a rate-limiting concentration of glutamate, its activity is sensitive to modulation by a number of intermediates in carbohydrate metabolism and is inhibited by ADP, ATP, GTP, and CTP.     

Half-lives of messenger RNA species during growth and differentiation of Dictyostelium discoideum

The half-lives of functional messenger RNAs were determined by a method employing the drugs actinomycin D and daunomycin for the inhibition of mRNA synthesis; the activity of extracted mRNAs was determined by an in vitro translation assay. Several controls indicated that this method yielded reliable values for mRNA half-lives; in particular, the declining rate of protein synthesis in the presence of the drugs is due predominantly to the decay of translatable mRNA. This method was used to determine the half-lives of two specific mRNAs—encoding actin and a protein of MW 51,000—as well as that of total cytoplasmic mRNA activity during growth and at several times in differentiation. The half-lives of at least these two mRNAs were shown to be distinctly different from that of the total mRNA population—about 4 hr. However, no significant change in any of these half-lives was observed between growing and developing cells. Therefore wholesale alterations in the degradation rates of total and at least specific messages do not appear to play a role in the regulation of gene expression during Dictyostelium development.     

X-ray diffraction from isolated metaphase chromosomes

Metaphase chromosomes were isolated from Chinese hamster fibroblasts. They were found to give the same characteristic diffraction pattern in the wet state as isolated nucleohistone but when dry did not give the reflections normally associated with dry nucleohistone. No higher structural order than that associated with the nucleohistone supercoil was detected.     

Similarity of the primary sequences of ribosomal RNAs isolated from vegetative and developing cells of Dictyostelium discoideum

Differentiating cell aggregates of Dictyostelium discoideum exhibit a pattern of rRNA metabolism quite different from that observed in the single-celled vegetative amoebas of this organism. We have examined whether the differences are related to a requirement for the production of new types of ribosomal RNA during development. Oligonucleotide maps and supplementary sequence data for 25 S, 17 S, and 5 S rRNAs from vegetative and developing cells have revealed no detectable alterations in primary sequence distinguishing any species of rRNA in developing cells from its vegetative cell counterpart.     

Regulation of pyruvate oxidation in blowfly flight muscle mitochondria: Requirement for ADP

Blowfly (Phormia regina) flight muscle mitochondria oxidized pyruvate (+ proline) in the presence of either ADP (coupled respiration) or carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP-uncoupled respiration). There was an absolute requirement for ADP (K_m = 8.0 μm) when pyruvate oxidation was stimulated by FCCP in the presence of oligomycin. This requirement for ADP was limited to the oxidation of pyruvate; uncoupled α-glycerolphosphate oxidation proceeded maximally even in the absence of added ADP. Atractylate inhibited uncoupled pyruvate oxidation whether added before (>99%) or after (95%) initiation of respiration with FCCP. In the presence of FCCP, oligomycin, and limiting concentrations of ADP (less than 110 μm), there was a shutoff in the uptake of oxygen. This inhibition of respiration was completely reversed by the addition of more ADP. Plots of net oxygen uptake as a function of the limiting ADP concentration were linear; the observed ADP/O ratio was 0.22 ± 0.025. An ADP/O ratio of 0.2 was predicted if phosphorylation occurred only at the succinyl-CoA synthetase step of the tricarboxylate cycle. Experiments performed in the presence of limiting concentrations of ADP, and designed to monitor changes in the mitochondrial content of ADP and ATP, demonstrated that the shutoff in oxygen uptake was not due to the presence of a high intramitochondrial concentration of ATP. Indeed, ATP, added to the medium prior to the addition of FCCP, inhibited uncoupled pyruvate oxidation; the apparent K_I was 0.8 mm. These results are consistent with the hypothesis that it is the intramitochondrial ATP/ADP ratio that is one of the controlling factors in determining the rate of flux through the tricarboxylate cycle. Changes in the mitochondrial content of citrate, isocitrate, α-ketoglutarate, and malate during uncoupled pyruvate oxidation in the presence of a limiting concentration of ADP were consistent with the hypothesis that the mitochondrial NAD⁺-linked isocitric dehydrogenase is a major site for such control through the tricarboxylate cycle.     

Synthesis of developmentally regulated proteins in Dictyostelium discoideum which are dependent on continued cell-cell interaction

In the previous paper we showed that the major changes in the pattern of protein synthesis during differentiation of Dictyostelium discoideum occur during the 4-hr period when the cells are forming tight, visible aggregates. During this time, synthesis of 10 discrete polypeptides made by preaggregation cells ceases or is reduced considerably, and synthesis of 40 new proteins is induced. Induction or cessation of synthesis of these proteins was parallelled by the appearance or disappearance of the corresponding messenger RNAs. In this paper we show that many of these changes are induced by continued cell-cell contact. None of these occurs in aggregation-competent cells kept in suspension culture, but changes do take place when such cells are allowed to form tight aggregates. Disaggregation of cells causes cessation of synthesis of “aggregation-stage” proteins and reinduction of synthesis of polypeptides characteristic of preaggregation cells.     

Demonstration, by renaturation in O'Farrell gels, of heterogeneity in Dictyostelium uridine diphosphoglucose pyrophosphorylase

A new procedure has been devised for the rapid isolation of yeast hexokinase isoenzymes PI and PII, giving specific activities comparable to those obtained after conventional purification. Hexokinases were bound to d-glucosamine, which had been coupled to CH Sepharose 4B using 6-aminohexanoic acid as a spacer. An ATP/d-glucose/MgCl₂ solution was used for elution. After concentration with DEAE-Sephacel, isoenzymes were separated by chromatofocusing. Hexokinase PI gave a single band on polyacrylamide gel electrophoresis, whereas one minor foreign band was seen for hexokinase PII.     

Biochemical identification of a putative glutamate receptor in housefly thoracic membranes

Specific stereoselective binding of [3H]L-glutamate was detected to membranes prepared from housefly thorax to which were added several antiproteases. A single high affinity binding site was detected (KD 0.5 +/- 0.04 microM), but total binding varied from preparation to preparation (5-60 pmoles/mg protein). Specific binding was inhibited by preincubation of the membranes with trypsin, chymotrypsin or protease, or by exposure to 70 degrees C for 5 min. It was also inhibited by several compounds, the most potent being L-glutamate and L-aspartate, followed by L-glutamate diethylester, then D-glutamate, N-methyl-D-aspartate and ibotenate. Quisqualate had little effect, while kainate, proctolin and D-aspartate had none. d-Tubocurarine stimulated [3H]L-glutamate binding. The data suggest that [3H]L-glutamate is binding to an L-glutamate receptor in housefly thoracic muscle membranes.     

Mitochondrial matrix granules in soft tissues. II. Isolation and initial characterization of a calcium-precipitable, soluble lipoprotein subfraction from brown fat and liver mitochondria

Degradation of ¹²⁵I-labelled HDL ([¹²⁵I]HDL) was measured in isolated rat hepatocytes that had been preincubated with [¹²⁵I]HDL and then reincubated in fresh medium without [¹²⁵I]HDL. About 5 % of the [¹²⁵I]HDL associated with the cells in advance were degraded per hour at 37 °C. This in vitro degradation was inhibited about 50% by lysosomal inhibitors such as chloroquine, ammonia and leupeptin. Depolymerization of microtubuli by colchicine inhibited the degradation of [¹²⁵I]HDL to about 65–75 % of the control cells. Cytochalasin B (CB), a destabilizer of microfilaments, had a less marked effect on the degradation in vitro. Degradation of [¹²⁵I]HDL associated with cells in vivo after intravenous injection was also studied in isolated cells. About 8.5% of the [¹²⁵I]HDL associated with the cells in vivo were degraded per hour in the isolated cells. The effects of ammonia, chloroquine, leupeptin and colchicine on HDL degradation were similar for [¹²⁵I]HDL taken up in vivo and in vitro. Subcellular fractionation by centrifugation in sucrose gradients indicated that [¹²⁵I]HDL associated with hepatocytes in vivo are primarily accumulated in lysosomes. [¹²⁵I]HDL associated with the cells in vitro are located in organelles whose distribution coincides with that of 5′-nucleotidase. These organelles may be endocytic vesicles. It is concluded that the internalization of [¹²⁵I]HDL in rat hepatocytes is relatively slow. The intracellular degradation of the apoproteins of HDL is at least partly lysosomal.     

Monoclonal antibodies: use to detect developmentally regulated antigens on D. discoideum amebae

We have used monoclonal antibodies to detect developmentally regulated cell surface antigens on D. discoideum amebae. A study of an antigen detected using an antibody produced by a hybridoma line implicates a previously undescribed component in the process of cell aggregation. This antigen (consisting of a doublet of 69,000 and 73,000 molecular weight) is first detected during the early hours of cell starvation and is present until cells begin slug formation. The developmental appearance of the antigen is not controlled by cAMP pulses and is distinct from that of Contact A sites. Fab fragments directed against the antigen are potent inhibitors of aggregation but do not inhibit the differentiation of cells to aggregation competence.     

Purine metabolism and urate biosynthesis in isolated chicken hepatocytes

The metabolism of some purine compounds to urate and their effects on de novo urate synthesis in chicken hepatocytes were investigated. The purines, listed in descending order of rates of catabolism to urate, were hypoxanthine, xanthine, inosine, guanosine, guanine, IMP, GMP, adenosine, AMP, and adenine. During a 1-h incubation period, conversion to urate accounted for more than 80% of the total quantities of guanine, guanosine, and inosine metabolized, but only 42% of the adenosine and 23% of the adenine metabolism. Adenine, adenosine, and AMP inhibited de novo urate synthesis [( 14C]formate incorporation into urate), whereas the other purines, especially guanine, guanosine, and GMP, stimulated de novo urate synthesis. When hepatocytes were incubated with glutamine and adenosine, AMP, guanine, guanosine, or GMP, the rates of de novo urate synthesis were lower than the additive effects of glutamine and the purine in separate incubations. Increasing phosphate concentrations had no effect on urate synthesis in the absence of added purines but, in combination with adenosine, AMP, guanosine, or GMP, increased urate synthesis. These results indicate that the ratio of adenine to guanine nucleotides and the interaction between substrates and purine nucleotides are involved in the regulation of urate biosynthesis in chicken liver.