Inactivation of the chemoattractant folic acid by cellular slime molds and identification of the reaction product.

Enzymes inactivating the chemoattractants folic acid and pterin were detected in extracellular, intracellular, and particulate fractions obtained from Dictyostelium discoideum strains NC4 and AX-2 and Polysphondylium violaceum. The products of the inactivation reaction were analyzed by thin-layer chromatography and UV spectroscopy. Results obtained indicate that folic acid and pterin were deaminated to 2-deamino folic acid and lumazine, respectively.     

Molecular probe for selective detection of thiols in water of neutral pH

The ternary complex, [Zn(cyclen)(lumazine)](+) that can be readily obtained by mixing cyclen, zinc perchlorate, and lumazine in water in a 1:1:1 molar ratio serves as a molecular probe that detects selectively thiols in an aqueous solution of neutral pH. Thiols successfully displace the lumazine in the molecular probe, which is accompanied by a decrease of the fluorescence. The molecular probe is useful for identification of cysteine among essential amino acids and detection of glutathione in aqueous solution of neutral pH.     

Purification of lumazine proteins from Photobacterium leiognathi and Photobacterium phosphoreum: bioluminescence properties

Bright strains of the marine bioluminescent bacterium Photobacterium leiognathi produce a "lumazine protein" in amounts comparable to that previously found in Photobacterium phosphoreum. New protocols are developed for the purification to homogeneity of the proteins from both species in yields up to 60%. In dimmer strains the amounts of lumazine protein in extracts are less, and also there is an accompanying shift of the bioluminescence spectral maximum to longer wavelength, 492 nm. Both types of lumazine proteins have identical fluorescence spectra, with maxima at 475 nm, so it is suggested that, whereas lumazine protein is the major emitter in bright strains, there is a second emitter also present with a fluorescence maximum at longer wavelength. The two species of lumazine protein have the same 276 nm/visible absorbance ratio, 2.2, but differ in visible maxima: P. phosphoreum, 417 nm; P. leiognathi, 420 nm. For the latter the bound lumazine has epsilon 420 = 10 100 M-1 cm-1, practically the same as in free solution. The two lumazine proteins also differ quantitatively in their effect on the in vitro bioluminescence reaction, i.e., at blue shifting the bioluminescence spectrum or altering the kinetics. The P. phosphoreum lumazine protein is more effective with its homologous luciferase or with P. leiognathi luciferase than is the lumazine protein from P. leiognathi. These differences may have an electrostatic origin.     

Electronic excitation transfer in the complex of lumazine protein with bacterial bioluminescence intermediates

Fluorescence dynamics measurements have been made on the bioluminescence reaction intermediates using Photobacterium leiognathi, Vibrio fischeri, and Vibrio harveyi luciferases, both alone and in mixtures with Photobacterium phosphoreum lumazine protein. Each luciferase produces a "fluorescent transient" intermediate on reaction with the bioluminescence substrates, FMNH2, tetradecanal, and O2, and all have a fluorescence quantum yield about 0.3, with a predominant lifetime around 10 ns. The P. leiognathi luciferase fluorescent transient has a rotational correlation time of 79 ns at 2 degrees C, as expected for the rotational diffusion of a 77-kDa macromolecule. In the presence of lumazine protein however a faster correlation time of about 3 ns predominates. This rapid channel of anisotropy loss is attributed to energy transfer from the flavin intermediate bound on the luciferase to the lumazine ligand, reflects the presence of protein-protein complexation, and is greatest in the case of P. leiognathi, but not at all for V. fischeri. This fact is consistent with the strong influence of lumazine protein on the bioluminescence reaction of P. leiognathi, and not at all with V. fischeri. The rate of energy transfer is of order 10(9) s-1, much greater than the 10(8) s-1 fluorescence rate of the donor. Thus the bioluminescence excitation of lumazine protein could occur by a similar photophysical mechanism of interprotein energy transfer from a chemically excited fluorescent transient donor to the lumazine acceptor.     

Fluorescence study of the ligand stereospecificity for binding to lumazine protein.

6,7-Dimethyllumazine derivatives, substituted at the 8-position with aldityls or monohydroxyalkyl groups, have been examined for their binding ability to lumazine apo-protein from two strains of Photobacterium phosphoreum using fluorescence dynamics techniques. On the protein the lumazine has a nearly monoexponential decay of fluorescence with lifetime 13.8 ns (20 degrees C). In free solution the lifetime is 9.6 ns. The concentration of free and bound lumazine in an equilibrium mixture can be recovered readily by analysis of the fluorescence decay. Only the aldityl derivatives D-xylityl and 3'-deoxy-D-ribityl, having stereoconfigurations at the 2' and 4' positions identical to the natural ligand, 8-(1'-D-ribityl), show comparable dissociation constants (0.3 microM, 20 degrees C, pH 7.0). D-Erythrityl and L-arabityl have dissociation constants of 1-2 microM. All other ligands show no interaction at all or have dissociation constants in the range 6-80 microM, which can still be determined semi-quantitatively using the fluorescence decay technique. In the case of these very weakly bound ligands, unambiguous detection of bound ligand can be shown by a long correlation time (23 ns, 2 degrees C) for the fluorescence anisotropy decay. Examination of the bound D-xylityl compound's fluorescence anisotropy decay at high time resolution (< 100 ps) shows rigid association, i.e. no mobility independent of the macromolecule. All bound ligands appear to be similarly positioned in the binding site. The influence of the stereoconfiguration at the 8-position found for lumazine protein parallels that previously observed for the enzyme riboflavin synthase, where the lumazines are substrates or inhibitors. This is consistent with the finding of significant sequence similarity between these proteins. The binding rigidity may have implications for the mechanism of the enzyme.     

Determination of rotational correlation times from deconvoluted fluorescence anisotropy decay curves. Demonstration with 6,7-dimethyl-8-ribityllumazine and lumazine protein from Photobacterium leiognathi as fluorescent indicators

The experimental and analytical protocols required for obtaining rotational correlation times of biological macromolecules from fluorescence anisotropy decay measurements are described. As an example, the lumazine protein from Photobacterium leiognathi was used. This stable protein (Mr 21 200) contains the noncovalently bound, natural fluorescent marker 6,7-dimethyl-8-ribityllumazine, which has in the bound state a long fluorescence lifetime (tau = 14 ns). Shortening of the fluorescence lifetime to 2.6 ns at room temperature was achieved by addition of the collisional fluorescence quencher potassium iodide. The shortening of tau had virtually no effect on the rotational correlation time of the lumazine protein (phi = 9.4 ns, 19 degrees C). The ability to measure biexponential anisotropy decay was tested by the addition of Photobacterium luciferase (Mr 80 000), which forms an equilibrium complex with lumazine protein. Under the experimental conditions used (2 degrees C) the biexponential anisotropy decay can best be described with correlation times of 20 and 60 ns, representing the uncomplexed and luciferase-associated lumazine proteins, respectively. The unbound 6,7-dimethyl-8-ribityllumazine itself (tau = 9 ns) was used as a model compound for determining correlation times in the picosecond time range. In the latter case rigorous deconvolution from the excitation profile was required to recover the correlation time, which was shorter (100-200 ps) than the measured laser excitation pulse width (500 ps).     

The membrane potential of the cellular slime mold Dictyostelium discoideum is mainly generated by an electrogenic proton pump

Trans membrane potential or ionic current changes may play a role in signal transduction and differentiation in the cellular slime mold dictyostelium discoideum. Therefore, the contribution of electrogenic ion pumps to the membrane potential of D. discoideum cells was investigated. the (negative) peak-value of the rapid potential transient, seen upon microelectrode impalement, was used to detect membrane potential changes upon changes in the external pH in the range of 5.5 to 8.0. The membrane potential was close to the Nernstian potential for protons over the pH range 5.5 to 7.5. The acid-induced changes in membrane potential were consistent with outward-proton pumping. The maximal membrane potential was at pH 7.5. Furthermore, the proton pump inhibitors diethylstilbestrol, miconazole and zearalenone directly depolarize the membrane. Cyanide and temperature decrease cause membrane depolarization as well. During recovery from cyanide poisoning a H+ efflux is present. From these measurements we conclude that the membrane potential of d. discoideum cells is mainly generated by an electrogenic proton pump. Measurements in cells with different extracellular potassium and H+ concentrations suggest a role for potassium in the function of the electrogenic proton pump. These results provide a framework for future research towards a possible role for the proton pump in signal transduction and differentiation.     

Bioluminescence spectral and fluorescence dynamics study of the interaction of lumazine protein with the intermediates of bacterial luciferase bioluminescence

The mechanism of the shifting of the bioluminescence spectrum from the reaction of bacterial luciferase by lumazine protein is investigated by methods of fluorescence dynamics. A metastable intermediate is produced on reaction of Vibrio harveyi luciferase with FMNH2 and O2. It has an absorption maximum at 374 nm and a rotational correlation time (phi) derived from the decay of its fluorescence (maximum 500 nm) anisotropy of 90 ns (2 degrees C). Lumazine protein from Photobacterium phosphoreum has an absorption maximum at 417 nm and a fluorescence maximum at 475 nm. Lumazine protein forms a protein-protein complex with luciferase, and the complex has a phi of approximately 100 ns. A mixture of lumazine protein and the intermediate would be expected to have an average correlation time (phi av) around 100 ns, but instead, the result is anomalous. The phi av is much lower and is also wavelength dependent. For excitation at 375 nm, which is mainly absorbed in the flavin chromophore of the intermediate, phi av = 25 ns, but at 415 nm, mainly absorbed by the lumazine derivative ligand of lumazine protein, phi av approximately 50 ns. It is proposed that protein-protein complexation occurs between lumazine protein and the luciferase intermediate and that in this complex energy transfer from the flavin to the lumazine is the predominant channel of anisotropy loss. A distance of 20 A between the donor and acceptor is calculated. In the bioluminescence reaction of intermediate with tetradecanal, a fluorescent transient species is produced which is the bioluminescence emitter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Time-resolved fluorescence spectroscopy of lumazine protein from Photobacterium phosphoreum using synchrotron radiation

Time-resolved fluorescence on lumazine protein from Photobacterium phosphoreum was performed with synchrotron radiation as a source of continuously tunable excitation. The experiments yielded structural and dynamic details from which two aspects became apparent. From fluorescence anisotropy decay monitoring of lumazine fluorescence with different excitation wavelengths, the average correlation times were shown to change, which must indicate the presence of anisotropic motion of the protein. A similar study with 7-oxolumazine as the fluorescent ligand led to comparable results. The other remarkable observation dealt with the buildup of acceptor fluorescence, also observed with 7-oxolumazine although much less pronounced, which is caused by the finite energy transfer process between the single donor tryptophan and the energy accepting lumazine derivatives. Global analytical approaches in data analysis were used to yield realistic correlation times and reciprocal transfer rate constants. It was found that the tryptophan residue has a large motional freedom as also reported previously for this protein and for the related protein from P. leiognathi (Lee et al. 1985; Kulinski et al. 1987). The average distance between the tryptophan residue and the ligand donor-acceptor couple has been determined to be 2.7 nm for the same donor and two different acceptors.     

Folic acid deaminase activity during development in Dictyostelium discoideum.

Folic acid attracts vegetative amoebae of Dictyostelium discoideum. Secreted by bacteria, it may act as a food-seeking device. The inactivation of this attractant is catalyzed by a deaminase. As assay has been developed to measure the folic acid deaminase activity. In addition to cell-surface an intracellular deaminase, the amoebae of D. discoideum release the enzyme into the medium. The pH optimum of the extracellular enzyme was 6.0, and higher for the cell-associated deaminases. The extracellular enzyme was secreted maximally by vegetative amoebae, and its activity diminished during cell differentiation. The cell-surface bound enzyme was less active than the extracellular enzyme, and its activity decreased twofold during a 6-h starvation period. The enzyme activity of homogenates and 48,000 x g pellets diminished during this period 35 to 40%. The supernatant of a homogenate had a higher deaminase activity than the homogenate itself or its pellet; this suggests the presence of an inhibitor in the particulate fraction. The underlying mechanism for inactivation of folic acid has similar characteristics as that for inactivation of cyclic adenosine monophosphate.     

Chemical characterization of lumazine protein from Photobacterium leiognathi: comparison with lumazine protein from Photobacterium phosphoreum

The properties of lumazine proteins purified from the marine bioluminescent bacteria Photobacterium phosphoreum, a psychrophile, and Photobacterium leiognathi, a relatively thermophilic species, are compared. An accurate 1:1 stoichiometry of binding of the ligand 6,7-dimethyl-8-ribityllumazine to each lumazine protein is established by back-titration of the apoprotein with the authentic ligand, using both fluorescence and absorption measurements. Neither protein contains metal cofactors, organic phosphorus, or carbohydrate. Both proteins are anionic and hydrophilic. They each contain a single Trp residue and have blocked amino terminals but otherwise differ in amino acid composition and other properties (P. phosphoreum and P. leiognathi, respectively): Met (internal), 1, 2; Cys, 2, 1; Arg, 4, 7; pI, 4.78 and 4.83, 4.38 and 4.45; Mr, 19 750, 21 300. In the P. phosphoreum protein both Cys residues are accessible, but in the P. leiognathi protein the single Cys is "buried". Modification of this buried Cys and at least one Cys in the P. phosphoreum protein prevents binding of the ligand. The UV and visible absorption spectra of both lumazine proteins denatured in 6 M guanidine hydrochloride can be accurately modeled by using the number of equivalents of the lumazine derivative and blocked aromatic amino acid model compounds determined by chemical and spectrophotometric analyses for Trp, Tyr, and Phe.     

Dynamic fluorescence study of the interaction of lumazine protein with bacterial luciferases

The equilibrium association of lumazine protein from Photobacterium phosphoreum with luciferases from either P. phosphoreum or an aldehyde-requiring dark mutant of Vibrio harveyi is measured from changes of the rotational correlation time which is derived from the decay of the lumazine ligand's fluorescence anisotropy. The rotational correlation time of lumazine protein is 23 ns (2 degrees C, 0.25 M Pi) and is increased on addition of luciferase due to the formation of a higher molecular weight complex. The V. harveyi luciferase exhibits full competence for the association and a 1:1 stoichiometry with a Kd in the range 40-90 microM. At lower ionic strength (0.05 M Pi), the Kd increases but is reduced again by the addition of dodecanol or dimyristoyllecithin. In contrast, tetradecanal, a substrate for the bioluminescence reaction, exerts no influence on the association. The equilibration rate is found to be too slow and for both luciferases the Kd values are too high for the interaction of the native proteins to account quantitatively for the spectral shifting of the bioluminescence by lumazine protein.     

Purification and properties of a secreted and developmentally regulated alpha-L-fucosidase from Dictyostelium discoideum.

During its development the eukaryotic microorganisms Dictyostelium discoideum secretes an alpha-L-fucosidase (EC 3.2.1.51). In cells of the growth phase almost no alpha-L-fucosidase activity is detectable. The activity increases steadily up to the aggregation stage and accumulates also in the extracellular medium. The developmental regulation is mediated by pulsatile cAMP signals. The alpha-L-fucosidase was purified from extracellular medium. The isolation procedure started with concentration of the enzyme by batchwise anion-exchange chromatography and ammonium sulfate precipitation, followed by Sephacryl S-300 gel filtration and further purification by fast protein liquid chromatography on Mono Q, phenyl-Superose, and finally Superose 12. The purified preparation was found to be essentially free of activities of six other glycosidases also secreted by D. discoideum. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme showed one major band with an apparent molecular mass of 62 kilodalton. Gel filtration of the enzyme on a Superose 12 column was consistent with an active monomer. A monoclonal antibody was produced, which recognizes a carbohydrate epitope shared by all lysosomal enzymes in D. discoideum. The pH optimum of the alpha-L-fucosidase is at 3.7. The apparent Michaelis constant for p-nitrophenyl alpha-L-fucoside as substrate is 1.2 mM. The enzyme catalyzes preferentially the hydrolysis of alpha 1----6GlcNAc but also of alpha 1----2Gal and alpha 1----3Glc fucosyl linkages.     

Measurement of cytoplasmic pH in Dictyostelium discoideum by using a new method for introducing macromolecules into living cells

We have developed a novel method for introducing exogenous macromolecules from solution into the cytoplasm of living amoebae of the cellular slime mold Dictyostelium discoideum and have used it to measure the cytoplasmic pH of these cells. Amoebae (strain NC-4) were loaded with fluorescein-labelled dextran by sonication in a solution containing 17 mM phosphate buffer, 1 mM CaCl2, and 10 mg/ml of fluorescein-labelled dextran, pH 6.1. The recovery of living cells was approximately 40% after sonication and washing. A significant fraction (10%) of the recovered cells were loaded and contained 10(5) to 10(7) molecules of fluorescein-labelled dextran per cell as assessed by flow cytometry. The cells loaded by sonication appeared both viable and healthy, since they exhibited normal morphology and locomotion, could differentiate to form mature fruiting bodies, could chemotax in a gradient of extracellular cAMP, and could endocytose latex microspheres. The pH of single cells was estimated by using flow cytometry to measure the fluorescence ratio (fluorescein/rhodamine) in cells loaded with a mixture of the two fluorochrome-labelled dextrans. The fluorescence ratios were calibrated in situ with the flow cytometer after treatment of the cells with either weak acid or weak base to clamp the internal pH at known values. The intracellular pH measured in cells loaded with dextran in a simple salt solution was 5.9. The intracellular pH measured in cells loaded with dextran in the same solution supplemented with amino acids and glucose was 6.7. The novel sonication loading technique described may have general utility for loading diverse types of macromolecules into suspensions of living cells.     

Lysophosphatidic acid- and Gbeta-dependent activation of Dictyostelium MAP kinase ERK2

Exogenous lysophosphatidic acid (LPA) has been shown to evoke a chemotactic response in aggregative cells of the social amoeba Dictyostelium discoideum. In this paper, we demonstrate that extracellular LPA is also able to induce activation of mitogen-activated protein (MAP) kinase DdERK2 (extracellular signal regulated kinase 2) in these cells. This activation is independent of cyclic AMP receptors, yet fully dependent on the single Gbeta subunit, hinting to the presence of functional heptahelical LPA receptors in a primitive eukaryote. We did not observe LPA-dependent cyclic GMP accumulation, which suggests that the pathways for LPA-induced and "classical" chemotaxis of D. discoideum cells are substantially different.     

Multiple assembly states of lumazine synthase: a model relating catalytic function and molecular assembly

Lumazine synthases have been observed in the form of pentamers, dimers of pentamers, icosahedral capsids consisting of 60 subunits and larger capsids with unknown molecular structure. Here we describe the analysis of the assembly of native and mutant forms of lumazine synthases from Bacillus subtilis and Aquifex aeolicus at various pH values and in the presence of different buffers using small angle X-ray scattering and electron microscopy. Both wild-type lumazine synthases are able to form capsids with a diameter of roughly 160 A and larger capsids with diameters of around 300 A. The relative abundance of smaller and larger capsids is strongly dependent on buffer and pH. Both forms can co-exist and are in some cases accompanied by other incomplete or deformed capsids. Several mutants of the B. subtilis lumazine synthase, in which residues in or close to the active site were replaced, as well as an insertion mutant of A. aeolicus lumazine synthase form partially or exclusively larger capsids with a diameter of about 300 A. The mutations also reduce or inhibit enzymatic activity, suggesting that the catalytic function of the enzyme is tightly correlated with its quaternary structure. The data show that multiple assembly forms are a general feature of lumazine synthases.     

Cellulose-binding modules from extracellular matrix proteins of Dictyostelium discoideum stalk and sheath.

Cellulose-binding modules (CBMs) of two extracellular matrix proteins, St15 and ShD, from the slime mold Dictyostelium discoideum were expressed in Escherichia coli. The expressed proteins were purified to > 98% purity by extracting inclusion bodies at pH 11.5 and refolding proteins at pH 7.5. The two refolded CBMs bound tightly to amorphous phosphoric acid swollen cellulose (PASC), but had a low affinity toward xylan. Neither protein exhibited cellulase activity. St15, the stalk-specific protein, had fourfold higher binding affinity toward microcrystalline cellulose (Avicel) than the sheath-specific ShD CBM. St15 is unusual in that it consists of a solitary CBM homologous to family IIa CBMs. Sequence analysis of ShD reveals three putative domains containing: (a) a C-terminal CBM homologous to family IIb CBMs; (b) a Pro/Thr-rich linker domain; and (c) a N-terminal Cys-rich domain. The biological functions and potential role of St15 and ShD in building extracellular matrices during D. discoideum development are discussed.     

Spectral properties and function of two lumazine proteins from Photobacterium

The spectral properties are compared for two 6,7-dimethyl-8-ribityllumazine proteins from marine bioluminescent bacteria, one from a psychrophile, Photobacterium phosphoreum, and the other from a thermophile, Photobacterium leiognathi. The visible spectral properties, which are the ones by which the protein performs its biological function of bioluminescence emission, are almost the same for the two proteins: at 2 degrees C and 50 mM Pi, pH 7, fluorescence quantum yield phi F = 0.59 and 0.54, respectively; fluorescence lifetime tau = 14.4 and 14.8 ns, respectively; fluorescence maxima, both 475 nm; absorption maximum, 417 and 420 nm, respectively; circular dichroism minima at around 420 nm, both -41 X 10(3) deg cm2 dmol-1. The ligand binding sites therefore must provide very similar environments, and arguments are presented that the bound ligand is relatively exposed to solvent. The dissociation equilibrium was studied by steady-state fluorescence polarization. The thermophilic protein binds the ligand with Kd (20 degrees C) = 0.016 microM, 10 times more tightly than the other protein [Kd (20 degrees C) = 0.16 microM]. The origin of the binding difference probably resides in differences in secondary structure. The tryptophan fluorescence spectra of the two proteins are different, but more significant is an observation of the decay of the tryptophan emission anisotropy. For the psychrophilic lumazine protein this anisotropy decays to zero in 1 ns, implying that its single tryptophan residue lies in a very "floppy" region of the protein. For the other protein, the anisotropy exhibits both a fast component and a slow one corresponding to rotation of the protein as a whole.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measurement of the cytoplasmic pH of Dictyostelium discoideum using a low light level microspectrofluorometer

Pyranine was employed as a sensitive pH indicator in a low light level microspectrofluorometer. The in vivo and in vitro standard curves of the 460/410-nm fluorescence excitation ratio of pyranine as a function of pH are identical. Therefore, pyranine is specifically sensitive to cytoplasmic pH in Dictyostelium. The cytoplasmic pH of single cells in a population of Dictyostelium discoideum amoebae was obtained for the first time. The median cytoplasmic pH of vegetative amoebae was 7.19. Carbonyl cyanide m-chlorophenylhydrazone, a mitochondrial uncoupler and a protonophore, lowered the median cytoplasmic pH to 6.12 when the extracellular pH was 6.1. This result is in accord with the protonophore activity of carbonyl cyanide m-chlorophenylhydrazone. Interest in the cytoplasmic pH of Dictyostelium has been greatly stimulated by the theory that cytoplasmic acidification promotes development of pre-stalk cells, while cytoplasmic alkalinization favors the pre-spore pathway (Gross, J. D., J. Bradbury, R. R. Kay, M. J. Peacey. 1983. Nature (Lond.). 303:244-245). The theory postulates that diethylstilbestrol (DES), an inducer of stalk cell differentiation and a plasma membrane proton translocating ATPase inhibitor, should cause acidification of the cytosol. Previous measurements of the effects of stalk cell inducers including DES on intracellular pH using 31P nuclear magnetic resonance measurements have failed to confirm the predictions of the theory, and have suggested that significant modification of the model may be required. Using pyranine as the pH indicator, we find that the median cytoplasmic pH in cells treated with 10 microM DES dropped from 7.19 to pH 6.02. This effect is consistent with the pharmacological action of DES and with the proposal that DES, a stalk cell inducer, should acidify the cytosol. These results provide direct support for the theory that cytoplasmic pH is an essential regulator of the developmental pathway in Dictyostelium.     

Differentiation of Dictyostelium discoideum in monolayer cultures and its modification by ionic conditions

We have compared the pattern of enzyme expression in cyclic AMP-induced monolayer cultures of Dictyostelium discoideum with that found during normal development. We find that both the temporal and quantitative pattern of enzyme expression are initially similar in the two situations, although the developmental sequence is more protracted and terminal cell differentiation is delayed in the monolayer situation. We describe differentiation conditions that permit the expression of only one terminal phenotype, which may be useful for further biochemical studies. Enzyme accumulation patterns under these conditions indicate that UDP gal transferase is not required for stalk cell differentiation (i.e., it is a prespore enzyme). We have shown that, when cell monolayers are incubated with cAMP, the presence of a weak acid at low extracellular pH favors stalk-cell differentiation, while a weak base at high extracellular pH favors spore differentiation. Finally, we show that variations in the monovalent cation content of the buffer, or the addition of an ion transport inhibitor (scillaren), or an ionophore (valinomycin) all affect the ratio of stalk cells to spores. Taken together, these results suggest that intracellular H+ and/or other cations may play an important role in regulating differentiation of specific cell types in D. discoideum.