Stability in vitro of uridine diphosphoglucose pyrophosphorylase in Dictyostelium discoideum

The stability of uridine diphosphoglucose pyrophosphorylase was examined in extracts prepared at different stages of development in Dictyostelium discoideum. In the early stages, the kinetics of inactivation were nonlinear, and, therefore, it was not possible to determine the specific enzyme activity. In the later stages of development, the enzyme was stable, but it could be rapidly inactivated by a heat-labile inhibitor present in extracts prepared at an early stage.     

Accumulation of uridine diphosphoglucose pyrophosphorylase in Dictyostelium discoideum via preferential synthesis

At the end of the exponential growth phase, the enzyme UDP-glueose pyrophosphorylase is present in the vegetative cells of Dictyostelium discoideum NC4 (haploid) at a low level (about 0.05% of total protein). During the initial stages of fruiting body construction, while the cells are entering into multicellular aggregates, the enzyme level remains constant, but increases dramatically thereafter reaching a peak (about 0.5% of total protein) at the end of fruiting body construction, and then partially decreasing. Previous studies have shown that both the accumulation and disappearance are keyed to the flow of morphogenetic events.In this study, cells were labeled with amino acids for different periods throughout the sequence. The enzyme was quantitatively immune-precipitated from crude cell extracts, the precipitate was washed and redissolved, and the enzyme protein separated by acrylamide gel electrophoresis in order to estimate the differential incorporation ratio, i.e. $\text{disints}\text{min in enzyme protein per 10}{}^8\text{cells}\text{disints}\text{min in total protein per 10}{}^8\text{cells}\text{× 100}$ for each labeling period. During the initial stages, when the enzyme level remained relatively constant, this ratio was about 0.03 to 0.04%. As the enzyme began to accumulate it rose progressively, attaining levels of 0.6 to 0.8% toward the end of fruiting body construction before declining. The data are not consistent with the theory of Gustafson and Wright (1973) that differential turnover controls the level of this enzyme during the development of D. discoideum. They are consistent with the conclusion that directed changes in the differential rate of synthesis of UDP-glucose pyrophosphorylase is the controlling element.The estimates of enzyme content are based on a value for the specific enzyme activity of 100,000 units/mg enzyme, which had been determined previously using samples of the enzyme purified to apparent physical homogeneity. This figure has been confirmed in the present study by quantitative immuneprecipitation of the enzyme from crude extracts of homogeneously labeled cells. The method can be generally used to determine if a specific biological activity estimate obtained with a purified protein is consistent with its activity when measured before or during purification.     

Uridine diphosphoglucose pyrophosphorylase activity and differentiation in the cellular slime mold Physarum polycephaluno

The specific activity of uridine 5'-triphosphate:alpha-d-glucose 1-phosphate uridyltransferase (EC 2.7.7.9) (also called uridine 5'-diphosphate [UDP]-glucose pyrophosphorylase) has been found to increase up to eightfold during spherule formation by the slime mold Physarum polycephalum. The enzyme accumulates during the first 8 to 9 h after initiation of spherule formation, declines to basal levels found in vegetative microplasmodia by 15 h, and is undetectable in completed spherules. Specific activities for UDP-glucose pyrophosphorylase in vegetative microplasmodia range from 15 to 30 nmol of UDP-glucose formed per min per mg of protein, whereas accumulated levels during spherule formation can attain a specific activity as high as 125 nmol of UDP-glucose formed per min per mg of protein. The scheduling and extent of accumulation are critically dependent on an early log-phase age of microplasmodia originally induced to form spherules. Spherule induction by 0.2 M or 0.5 M mannitol delays this schedule in a variable and unpredictable manner. Spherule-forming microplasmodia which have accumulated high levels of UDP-glucose pyrophosphorylase spontaneously excrete the enzyme when transferred to salts medium containing 0.2 M or 0.5 M mannitol. The excreted enzyme is subsequently destroyed or inactivated. Studies with preferential inhibitors of macromolecular synthesis indicate that accumulation of UDP-glucose pyrophosphorylase requires concomitant protein synthesis and prior ribonucleic acid synthesis.     

Cytokinin oxidase activity in the cellular slime mold, Dictyostelium discoideum

The cytokinin N6-(delta 2-isopentenyl)adenine (i6Ade) is produced during the development of the cellular slime mold, Dictyostelium discoideum, and functions in this organism as the immediate precursor of the spore germination inhibitor, discadenine. The metabolism of i6Ade in axenic cultures of D. discoideum Ax-3 amoebae has been investigated in the present study. An enzyme activity that specifically catalyzes the degradation of i6Ade has been detected in Ax-3 amoebae. This enzyme is similar to the cytokinin oxidases present in higher plant systems and cleaves the N6-side chain of i6Ade to form adenine. Discadenine synthase activity was also detected in axenically cultured Ax-3 amoebae. The cytokinin oxidase activity detected in Dictyostelium decreased during aggregation and development of Ax-3 amoebae and in starving Ax-3 amoebae maintained under either fast-shake (230 rpm) or slow-shake (70 rpm) conditions. In the latter case, the fall in enzyme activity was accelerated by treatment with cyclic AMP. In contrast to these results, discadenine synthase activity in Ax-3 amoebae rose sharply during the culmination phase of development, exhibited little change in starving Ax-3 amoebae maintained under fast-shake conditions, and fell under slow-shake conditions unless the amoebae were treated with cyclic AMP. Possible functions of the Dictyostelium cytokinin oxidase and the significance of the i6Ade metabolism observed in vegetative Dictyostelium amoebae are discussed.     

6-Phosphogluconate dehydrogenase and the assay of uridine diphosphate glucose pyrophosphorylase in the cellular slime mould Dictyostelium discoideum

1. 6-Phosphogluconate dehydrogenase activity is present in all morphogenetic stages during cell differentiation in the cellular slime mould. 2. The different ratios of 6-phosphogluconate dehydrogenase/UDP-glucose pyrophosphorylase observed during this process can render spectrophotometric assays of UDP-glucose pyrophosphorylase inaccurate. 3. The disputed occurrence of increases in specific activity of UDP-glucose pyrophosphorylase during cell differentiation in the cellular slime mould is discussed in the light of these observations.     

Signal transduction pathways involved in the expression of the uridine diphosphoglucose pyrophosphorylase gene of Dictyostelium discoideum

The uridine diphosphoglucose pyrophosphorylase (UDPGP1) gene of Dictyostelium discoideum is an excellent marker to study the pathways that control the expression of genes during development. We have previously shown that the UDPGP1 gene is regulated by exogenous cAMP acting on cell-surface cAMP receptors. Various steps in the signal transduction pathway between receptor stimulation and the induction of the gene can now be studied. Induction does not require the synthesis of intracellular cAMP, but does require new protein synthesis. By deletion and transformation with altered genes, two cis-acting sequences that are required for UDPGP1 expression have been identified. A GC-rich palindromic sequence located between -410 and -374 is essential for induction of the gene by extracellular cAMP, but not for its basal expression. A sequence element located between -374 and -337 is required for any basal expression of this gene. When the polarity of the palindromic sequence was reversed such that it resembled the H2K enhancer element, the gene could still be induced by exogenous cAMP. Two DNA binding activities were detected in gel mobility shift assays using a fragment containing both of the regulatory sequence elements of UDPGP1 gene. Transformation with a vector that resulted in the synthesis of anti-sense UDPGP1 RNA led to almost total elimination of the enzyme antigen and no detectable enzyme activity. However, these transformants developed normally, indicating that either UDPGP is not required for development or residual synthesis of UDPGP may be sufficient for normal development.     

Nuclear ribonucleoprotein particles in the cellular slime mold Dictyostelium discoideum

The nuclear ribonucleoprotein (RNP) particles containing rapidly labeled RNA were isolated from interphase cells of the cellular slime mold Dictyostelium discoideum and characterized. The size of the isolated RNP particles was small (10S to 50S) in comparison with that of nuclear RNP particles found in higher eukaryotes. These small RNP particles do not seem to be artifacts due to degradation during the preparation of nuclear extracts. The rapidly labeled RNA of the nuclear RNP particles was heterogeneous in size and a considerable amount contained polyadenylic acid sequences. Synthesis of RNA in the nuclear RNP particles was resistant to a relatively high concentration of actinomycin D. The protein component of the RNP particle consists of at least four proteins with molecular weights of 80,000, 66,000, 60,000, and 42,000. Thus it is suggested that almost all of the nuclear RNP particles containing rapidly labeled RNA in interphase cells are RNP complexes consisting of Heterogeneous nuclear RNA and several protein species.     

Cyclic AMP inhibits dedifferentiation in the cellular slime mold Dictyostelium discoideum

When aggregating amoebas of the cellular slime mold Dictyostelium discoideum are disaggregated and morphogenesis is reinitiated, the amoebas will reaggregate in less than $\text{1}\text{10}\text{th}$ the original time. When aggregating amoebas are disaggregated and resuspended either in full nutrient medium or in buffered salts solution containing dextrose, they retain this developmentally acquired capacity to rapidly reaggregate for approximately 1 hr and then lose it completely in a synchronous and discrete step which we have referred to as the “erasure event.” In this report, it is demonstrated that micromolar concentrations of cAMP completely block this transition from the developmental to vegetative state, and that other cyclic nucleotides also inhibit it, but they do so at 20-fold higher concentrations. Neither the hydrolysis products of cAMP nor the vegetative chemoattractant folic acid inhibit dedifferentiation at concentrations as high as 10^?3M, demonstrating a specificity for cyclic nucleotides and cAMP in particular. The addition of cAMP at any time during the lag period preceding the erasure event inhibits it and addition immediately after the erasure event reverses it. Since cAMP may inhibit the transition from the developmental to vegetative state intracellularly or extracellularly, we have also examined the intracellular concentration of cAMP and the levels of cAMP binding sites on the cell surface during the erasure process. Evidence is presented that the majority of cAMP binding sites on the cell surface are not necessary for the inhibition of erasure by cAMP. The results of these latter studies are discussed in terms of alternative models for the involvement of cAMP in the transition from the developing to vegetative state.     

Characterization of pyrimidine metabolism in the cellular slime mold, Dictyostelium discoideum

The arginine-independent, de novo biosynthetic pathway of pyrimidines in Dictyostelium discoideum is initiated by a class II carbamoyl-phosphate synthetase (EC 6.3.5.5) specific for pyrimidine biosynthesis which utilized L-glutamine as its N donor and was partially inhibited by both UTP and CTP. The second step in the de novo pathway was provided by an unregulated aspartate transcarbamoylase (EC 2.1.3.2) which primarily appeared as a multimeric enzyme of 105 kilodaltons. The next enzyme, dihydroorotase (EC 3.5.2.3), was approximately 90-100 kilodaltons. Although the early enzymatic activities of the pyrimidine pathway appeared to reside in independent protein complexes, various unstable molecular species were observed. These structural variants may represent proteolytic fragments of a multienzyme complex. In addition to de novo synthesis, the amoeba demonstrated the capacity for salvage utilization of uracil, uridine, and cytidine. Upon starvation on a solid substratum, axenically grown amoebas began a concerted developmental program accompanied by a restructuring of nucleotide metabolism. The absolute levels of the ribonucleotide pools droppedby 98% within 30 h; however, both the adenylate energy charge and the GTP/ATP ratios were maintained for 50 h after the initiation of development. The maintenance of these metabolic energy parameters required the tight cell-cell contact necessary for development, and the capacity for pyrimidine metabolism was maintained throughout developmental morphogenesis.     

Inhibition of aggregation by light in the cellular slime mold Dictyostelium discoideum

Aggregation of Dictyostelium amoebae is inhibited by light. White light intensities 10² W · cm^-2 cause an inhibition which reaches a saturation at 2 · 10³ W · cm^-2. The action spectrum, based on photon fluence-response curves, shows a major peak around 405 nm and extends through most of the visible spectrum with a secondary maximum at about 530 nm. The action spectrum of the inhibition of aggregation resembles the action spectrum of accumulations of amoebae in light traps and the action spectrum of photodispersal from light traps; it does not resemble the action spectrum of phototaxis in pseudoplasmodia.     

EDTA treatment alters protein glycosylation in the cellular slime mold Dictyostelium discoideum

We have found that treatment of cells with EDTA resulted in the accumulation of lower molecular weight forms of two cell-type-specific glycoproteins. These new glycoproteins lacked a developmentally regulated glycoantigen defined by monoclonal antibody 54.2. Since EDTA dissociated the cells, the possible involvement of cell separation was tested by immobilizing cells in soft agarose. Glycoantigen expression on these proteins was found to be dependent on cAMP and high oxygen tension but not on cell contact, and was reversibly sensitive to EDTA regardless of the state of cell association. The EDTA effect was mimicked by other soluble, but not particulate, membrane impermeable chelators, could be competed by Zn2+ better than Mg2+, and appeared to involve an intracellular mechanism. Studies with [14C]EDTA showed that EDTA equilibrated with a cellular compartment in a temperature-dependent, Zn2+-insensitive fashion with half-time kinetics of loading and unloading of 30-40 min. If the compartment was assumed to be labeled with the same concentration of EDTA as was present extracellularly, calculations showed that its volume was circa 2% of the total cell volume. This compartment probably consists of intracellular vesicles based on the similar labeling of this compartment with a bulk fluid phase marker, inulin. The data suggest that this step in glycosylation, which was found to be delayed 1 or more hours subsequent to protein synthesis, involves an intracellular, transition metal ion-dependent process which can be modulated by chelators entering the cell through the endocytic pathway.