Zinc transport by fibroblasts from patients with acrodermatitis enteropathica

Acrodermatitis enteropathica (AE) is a zinc deficiency disease. To date, the only defect has been demonstrated in the gut. We have investigated zinc uptake in fibroblasts established from four unrelated patients with AE using normal skin fibroblasts as controls. Zinc content of AE and control cells was similar (0.3 fmol/cell). Zinc accumulation over 24 h from a complete culture medium was similar in both normal controls and mutant cells. The fraction of zinc removed by Pronase treatment remained constant at 50 pmol/micrograms DNA, whereas the zinc remaining after Pronase treatment accumulated rapidly for 8 h, then more slowly. Analysis of binding data showed no significant difference between AE and control cells, with apparent Ka values of 4-6 X 10(6) M-1 and between 1 and 2 X 10(8) receptors/cell. Analysis of Pronase resistant data showed no difference between the control and the mutant cells with apparent Km values of 0.2-0.3 microM and Vmax values of 17-19 pmol/micrograms DNA/h. No difference in zinc efflux rates was detected. We conclude that the defect that underlies acrodermatitis enteropathica is either not expressed in fibroblasts or cannot be detected under these experimental conditions.     

Initial steps in Haemophilus influenzae transformation. Donor DNA binding in the com10 mutant

The com10 mutant of Haemophilus influenzae binds donor DNA reversibly, but is deficient in uptake. The DNA binding has all the characteristics of interaction with a protein receptor; it is saturable, reversible, and specific. However, binding specificity is 6-fold weaker in com10 than is uptake specificity in wild-type. The binding of small (120 base pairs) and large (14,400 base pairs) DNA molecules were compared. For small molecules, binding data fitted a straight line by Scatchard analysis (Bmax = 4.8 DNA molecules/cell, Kd = 0.5 X 10(-9) M). In contrast, for large DNA molecules, the Scatchard plot was not linear. A high affinity binding (Kd = 0.4 X 10(-12) M) and a lower affinity binding (Kd = 1.2 X 10(-11) M) were found with a total number of 3 molecules bound per cell. In wild-type cells, 3.2 large molecules were taken up per cell, whereas up to 40 small 120-base pair DNA fragments were taken up per cell. Uptake of small DNA molecules followed a Michaelis-Menten function with a Km of 0.5 X 10(-9) M and a maximal initial velocity of 1.5 molecules/cell/min at room temperature. For large DNA molecules, maximal initial velocity was approximately 2 molecules/cell/min at room temperature. The analysis of the binding and uptake data suggest to us that a receptor or a receptor complex is responsible for the uptake of either a single large DNA molecule or, successively, a number of small DNA molecules.     

Zinc biosorption from aqueous solution by a halotolerant cyanobacterium Aphanothece halophytica

We have investigated the characteristics of zinc biosorption by Aphanothece halophytica. Zinc could be rapidly taken up from aqueous solution by the cells with an equilibrium being reached within 15 min of incubation with 100 mg L⁻¹ ZnCl₂. The adsorbed zinc was desorbed by treatment with 10 mM EDTA. The presence of glucose, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and N,N′-dicyclohexylcarbodiimide (DCCD) did not affect the uptake of zinc. The specific uptake of zinc increased at low cell concentration and decreased when cell concentration exceeded 0.2 g L⁻¹. The binding of zinc followed Langmuir isotherm kinetics with a maximum zinc binding capacity of 133 mg g⁻¹ and an apparent zinc binding constant of 28 mg L⁻¹. The presence of an equimolar concentration of Mn²⁺, Mg²⁺, Co²⁺, K⁺, or Na⁺ had no effect on zinc biosorption, whereas Ca²⁺, Hg²⁺, and Pb²⁺ showed an inhibitory effect. The biosorption of zinc was low at a pH range from 4 to 6, but increased progressively at pH 6.5 and 7. Received: 12 December 2001 / Accepted: 11 January 2002     

Zinc exchange by blood cells in nearly physiologic standard conditions

Determination of zinc concentrations in white blood cells has been used to establish zinc deficiency. During pathological conditions changes in zinc concentrations in these blood cells were observed. However, these investigations were hampered by the low amount of zinc in this form per mL blood. Earlier we demonstrated that, in the case of zinc deficiency, the uptake of zinc was increased, using the in vitro exchange of zinc by the various blood cells with extracellular zinc labeled with⁶⁵Zn in fairly physiologic conditions. In case of inflammation, no increase in zinc uptake by erythrocytes was seen, indicating that this method probably can be used to differentiate real from apparent zinc deficiency. Only during the first days of the inflammatory process, probably representing the redistribution phase during which zinc moves from the serum to the liver, a small increase in in vitro zinc uptake was seen in mononuclear cells (MNC) and polymorphonuclear cells (PMNC). Earlier papers raised some questions; e.g., is the uptake part of an exchange process and can the efflux of zinc by the cells be measured by the same method; what is the influence of time on the process of zinc uptake; what is the magnitude of the uptake of zinc by the cells compared to the zinc concentration in the cells; and, what is the influence of temperature on the uptake of zinc? In the present study, the influence of incubation time and temperature on the uptake of zinc by human and rat blood cells and on the release of zinc by rat blood cells was studied. At least three phases of uptake of zinc in the various cells were found by varying the incubation time—a fast phase during the first half hour, probably caused by an aspecific binding of zinc on or in the cell membrane; a second fast uptake between 60–330 min, probably caused by an influx of zinc in the cell as part of the exchange process of zinc; and a slow third phase after 5.5 h, in which probably the in- and efflux of the rapidly exchangeable intracellular pool is more or less equilibrated. For mononuclear cells, polymorphonuclear cells, and erythrocytes of rats, the rapidly exchangeable intracellular pool is 40%, 53%, and 10%, respectively, of the total zinc content of the cells. This study is also performed in human cells; in human cells the exchangeable pool of mononuclear cells and erythrocytes is 17 and 3.5% of the total zinc content of the cells, respectively. The efflux of zinc by blood cells can be measured by the same method. Both the uptake and the loss of zinc by blood cells of rats were compared and are of the same magnitude, indicating that the in vitro uptake of zinc described elsewhere is part of an exchange process. Increasing temperature during incubation procedures results in an increase of zinc uptake by human blood cells, even at high temperatures of 41°C, although there are gradual differences between the various blood cells. Both the in- and efflux of zinc by blood cells are very small at 4°C.     

Mechanism of Zinc Uptake in Bean (Phaseolus vulgaris) Tissues

Uptake of micronutrient zinc by intact leaves, enzymically isolated leaf cells, leaf disks, excised roots, and stem-callus tissue of two field bean cultivars 'Saginaw’ and ‘Sanilac’) was studied using radio-isotope tracer technique. Radio-phosphorus absorption by these tissues was also followed under comparable experimental conditions. A rapid absorption of the micronutrient and strong dependency on external zinc concentration and pH were revealed. Absorption of zinc was not inhibited by respiratory inhibitors (dinitrophenol, azide, cyanide, and amytal), and was not light or temperature dependent. Q₁₀ values for zinc uptake ranged between 1 and 1.2. Uptake of phosphate, on the other hand, was temperature and light dependent and drastically reduced by the presence of metabolic inhibitors. Differences in responses to respiratory inhibitors, temperature, pH, light and darkness, and kinetic data, strongly suggest that zinc uptake in bean tissues occurs primarily by a passive mechanism, involving possibly a physical or physiochemical binding of the micronutrient ions to the cell wall and free space components, and a passive diffusion into the interior of the cell.     

Pathways involved in fluid phase and adsorptive endocytosis in neuroblastoma

The endocytosis of ricin, horseradish peroxidase (HRP), and a conjugate of ricin-HRP by monolayer cultures of murine neuroblastoma was studied using morphological and biochemical techniques. The binding of (125)I-ricin and (125)I-ricin-HRP to cells at 4 degrees C, as a function of ligand concentration, was a saturable process. The apparent affinity constants, determined at equilibrium, were 2.8 X 10(6) M(-1) for ricin and 1 x 10(6) M(-1) for ricin-HRP. The number of binding sites per cell was 8 x 10(7) and 3 x 10(7) for the lectin and the conjugate, respectively. The binding of (125)I-ricin to monolayers as not proportional to cell density. We found reduced binding at higher cell concentrations, suggesting a decrease in the accessibility of the ligand for the receptor site or fewer sites with increasing cell population. Neuroblastoma cells have an acid-phosphatase-positive network of cisternae and vesicles near the Golgi apparatus (GERL). Ricin-HRP undergoes endocytosis in vesicles and cisternae corresponding to GERL, and in residual bodies (dense bodies). The cellular uptake of ricin-HRP was 100-200 times greater than free HRP and there was no stimulation of fluid phase endocytosis by ricin. When monolayers were exposed to concentrations of native HRP 100-fold that of the conjugate, cellular uptake of peroxidase was comparable, but HRP was localized only in residual bodies and never in elements of GERL. These results support the conclusion that GERL is involved in the adsorptive endocytosis of ricin-HRP, while residual bodies are involved in the bulk uptake of HRP. In addition, the binding, uptake, and possible recycling of (125)I- subunit B (the binding subunit) of ricin and of (125)I-ricin was examined by quantitative electron microscope autoradiography. Both ricin and its binding subunit displayed similar autoradiographic grain distributions at 4 degrees C, and there was no evidence of their breakdown or recycling to the plasma membrane during endocytosis for 2 h.     

Quantitative requirements for exponential growth of Alcaligenes eutrophus.

Quantitative nutrient requirements for unrestricted autotrophic growth of Alcaligenes eutrophus were determined. Minimum saturating concentrations of Mg2+, SO42-, PO43-, Fe3+, and Na2+ for an optical density increase of 2 were 10(-4) M 8 X10(-5) M, 5 X 10(-4) to 6 X 10(-4) M, 10(-5) M, and 10(-7) to 2 X 10(-7) M, respectively. Trace metal requirements for cobalt, chromium, and copper were also demonstrated, but minimum concentrations could not be determined because other reagents contributed a high background of these metals. Under certain conditions an apparent response to zinc was observed, although other experiments suggest the zinc salt contained another metal that was required for growth. Poly-beta-hydroxybutyrate biosynthesis was shown to be initiated by a magnesium or sulfate deficiency as well as by a nitrogen or phosphate deficiency.     

Alpha-fetoprotein receptors in a human breast cancer cell line

Evidence is presented for the existence of specific receptors for alpha-fetoprotein on the surface of MCF-7 human breast cancer cells. At 4 degrees C, the binding of alpha-fetoprotein to these cells displayed a biphasic saturation curve. Scatchard analysis revealed the presence of at least two binding sites with dissociation constants of 4.5 X 10(-9) M (2,000 sites/cell) and 1.3 X 10(-8) M (135,000 sites/cell), respectively. Binding was inhibited by 85% in the presence of a 5,000-fold excess of unlabeled alpha-fetoprotein and by 50% with the same excess of serum albumin. Competition by other serum proteins was not significant. At 37 degrees C, alpha-fetoprotein was endocytosed and the uptake curve reached a plateau after 3-4 hours of incubation.     

A variant of human transferrin with abnormal properties.

Normal human skin fibroblasts cultured in vitro exhibit specific binding sites for 125I-labelled transferrin. Kinetic studies revealed a rate constant for association (Kon) at 37 degrees C of 1.03 X 10(7) M-1 X min-1. The rate constant for dissociation (Koff) at 37 degrees C was 7.9 X 10(-2) X min-1. The dissociation constant (KD) was 5.1 X 10(-9) M as determined by Scatchard analysis of binding and analysis of rate constants. Fibroblasts were capable of binding 3.9 X 10(5) molecules of transferrin per cell. Binding of 125I-labelled diferric transferrin to cells was inhibited equally by either apo-transferrin or diferric transferrin, but no inhibition was evident with apo-lactoferrin, iron-saturated lactoferrin, or albumin. Preincubation of cells with saturating levels of diferric transferrin or apo-transferrin produced no significant change in receptor number or affinity. Preincubation of cells with ferric ammonium citrate caused a time- and dose-dependent decrease in transferrin binding. After preincubation with ferric ammonium citrate for 72 h, diferric transferrin binding was 37.7% of control, but no change in receptor affinity was apparent by Scatchard analysis. These results suggest that fibroblast transferrin receptor number is modulated by intracellular iron content and not by ligand-receptor binding.     

Pathways in the binding and uptake of ferritin by hepatocytes

The binding and uptake of rat liver ferritin by primary cultures of rat liver hepatocytes was studied in order to assess the relative importance of saturable, high-affinity pathways and nonspecific processes in the incorporation of the protein by the cells. To minimize artifacts, ferritin not subjected to heat treatment and labeled in vivo with 59Fe was used. Binding to cell membranes was estimated from incubations performed at 4 degrees C. After 2 h, when a steady state in cell-associated ferritin had been achieved, approx. 4-10(4) binding sites per cell were observed, with an affinity constant for ferritin of 1 x 10(9) M-1. At 37 degrees C, the maximal uptake from these sites was 1.3 x 10(5) ferritin molecules/cell per h. For ferritin molecules bearing an average of 2400 iron atoms, this uptake amounts to 5 x 10(6) iron atoms/cell per min. Half-maximal uptake was achieved at a ferritin concentration, or KM1, of 3 x 10(-9) M. Although uptake rates at least a thousand times greater could be achieved by binding to the much larger number of low-affinity sites, the apparent KM2 for such 'nonspecific' uptake was 4 x 10(-7) M. At ferritin concentrations up to 2 nM, at least 90% of ferritin bound and taken up by hepatocytes involves saturable, high-affinity sites, presumably true ferritin receptors.     

Localization of vitamin B12 binding in Euglena gracilis

Different fractionation procedures were used to determine the location of vitamin B12 binding sites in Euglena gracilis. Using uptake measurements, cell fractionation, and light and electron microscopy, the cuticle of the cell was found to be the fraction containing the majority of B12 binding sites. The apparent distribution of vitamin binding sites differed according to the cell lysis method used. The cuticle fraction was responsible for the binding of 80% of the vitamin taken up by the cell during both the rapid and the slow phase of uptake. These results suggest that vitamin B12 binding is regulated, in part, at the cuticle level, and support our previous conclusion that the secondary phase of uptake represents the synthesis of new receptor sites and not the unloading of vitamin inside the cell.     

Stabilizing influence of calcium and magnesium ions on the decameric structure of chiton hemocyanins

The stabilizing effects of Ca2+ and Mg2+ ions on the decameric structure of hemocyanins from two representative chitons, Stenoplax conspicua and Mopalia muscosa were investigated by light-scattering molecular weight measurements, ultracentrifugation, absorbance, and circular dichroism methods. The dissociation profiles at any given pH resulting from the decrease in divalent ion concentration, investigated at a fixed protein concentration of 0.1 g.liter-1, could be fitted by a decamer-to-dimer-to monomer scheme of subunit dissociation. The initial decline in the light-scattering molecular weight curves required one or two apparent binding sites per hemocyanin dimer formed as intermediate dissociation product, with apparent dissociation constants (kD,2) for Ca2+ ions of 0.7 to 7 X 10(-4) M, not very different from the value of 2.5 X 10(-4) M obtained by Makino by equilibrium dialysis for the hemocyanin of the opistobranch, Dolabella auricularia. The binding of Mg2+ ion to S. conspicua and M. muscosa hemocyanins appears to be both weaker than the binding of Ca2+ and more pH dependent, with kD,2 values ranging from the 3 X 10(-4) to 4 X 10(-2) M at pH 8.5 to 9.5. The dissociation the decameric hemocyanin species (sedimentation coefficient ca. 60 S) is also observed in the ultracentrifugation with the initial appearance of 18-20 S dimers, followed by a shift in equilibrium to monomeric species of lower sedimentation rates of 11-12 S as the divalent ion concentration is reduced below 1 X 10(-4) M Ca2+ and Mg2+. The dissociation of dimers to monomers in the second step of the reaction is characterized by one or two binding sites per subunit and a somewhat stronger affinity for divalent ions, indicated by apparent dissociation constants (kD,1) of 0.7 X 10(-4) to 3 X 10(-3) M. Circular dichroism and absorbance measurements at 222 and 346 nm suggest no significant changes in the conformation of the hemocyanin subunits produced by the different stages of subunit dissociation.     

Interaction of zinc and hemoglobin: binding of zinc and the oxygen affinity.

Stripped human hemoglobin was shown to have a high apparent zinc association constant of 1.3 X 10(7) M-1 with a stoichiometry of one zinc for every two hemes. The saturation of this site produces a dramatic 3.7-fold increase in the oxygen affinity. The effect of zinc on the oxygen affinity is interrelated with the interaction of 2,3-diphosphoglyceric acid (2,3-DPG) and hemoglobin. Thus, a smaller zinc effect is observed in the presence of added 2,3-DPG. Information about the location of the zinc-binding site responsible for the increased oxygen affinity has been obtained by comparing the binding of zinc to various hemoglobins. Blocking the beta93 sulfhydryl group decreases the apparent zinc association constant by an order of magnitude. The substitution of histidine-beta143 in hemoglobin Abruzzo [beta143 (H21) His leads to Arg] and hemoglobin Little Rock [beta143 (H21) His leads to Gln] decreases the apparent zinc association constant by two orders of magnitude. The substitution of histidine-beta143 by other amino acids and the reaction of the beta93 sulfhydryl group are known to produce dramatic increases in the oxygen affinity. The binding of zinc to one or both of these amino acids can, therefore, explain the zinc-induced increase in the oxygen affinity.     

The effect of zinc and other divalent cations on the structure and function of human alpha 2-macroglobulin

Zinc binding to human alpha 2-macroglobulin was studied to assess its involvement in the structure and function alpha 2-macroglobulin. Equilibrium dialysis experiments indicated multiple classes of zinc-binding sites, the one of highest affinity having a site number of 20 and a Kd value of 8 X 10(-7) M. Native alpha 2-macroglobulin and alpha 2-macroglobulin-trypsin complexes bound comparable amount of zinc. The proteinase inhibitory activity of alpha 2-macroglobulin was not affected by zinc binding at physiological concentrations nor by the removal of zinc by EDTA. Above 25 microM zinc, alpha 2-macroglobulin activity decreased, although binding of [125I]trypsin was not affected. When nondenaturing gel electrophoresis was performed, the preparation of alpha 2-macroglobulin migrated as half-molecules at increasing zinc concentration. Experiments with other divalent cations correlated decreases in alpha 2-macroglobulin activity with apparent dissociation of the alpha 2-macroglobulin tetramer in the presence of copper and mercury, but not barium, cadmium or nickel. While zinc binding to alpha 2-macroglobulin does not function in proteinase inhibition, it might be involved in zinc transport in vivo. At nonphysiological concentrations, zinc and other divalent cations are useful as probes of protein quaternary structure.     

65Zn2+ transport by isolated gill epithelial cells of the American lobster, Homarus americanus

Gills are the first site of impact by metal ions in contaminated waters. Work on whole gill cells and metal uptake has not been reported before in crustaceans. In this study, gill filaments of the American lobster, Homarus americanus, were dissociated in physiological saline and separated into several cell types on a 30, 40, 50, and 80% sucrose gradient. Cells from each sucrose solution were separately resuspended in physiological saline and incubated in ⁶⁵Zn²⁺ in order to assess the nature of metal uptake by each cell type. Characteristics of zinc accumulation by each kind of cell were investigated in the presence and absence of 10 mM calcium, variable NaCl concentrations and pH values, and 100 μM verapamil, nifedipine, and the calcium ionophore A23187. ⁶⁵Zn²⁺ influxes were hyperbolic functions of zinc concentration (1–1,000 μM) and followed Michaelis–Menten kinetics. Calcium reduced both apparent zinc binding affinity (K _m) and maximal transport velocity (J _max) for 30% sucrose cells, but doubled the apparent maximal transport velocity for 80% sucrose cells. Results suggest that calcium, sodium, and protons enter gill epithelial cells by an endogenous broad-specificity cation channel and trans-stimulate metal uptake by a plasma membrane carrier system. Differences in zinc transport observed between gill epithelial cell types appear related to apparent affinity differences of the transporters in each kind of cell. Low affinity cells from 30% sucrose were inhibited by calcium, while high affinity cells from 80% sucrose were stimulated. ⁶⁵Zn²⁺ transport was also studied by isolated, intact, gill filament tips. These intact gill fragments generally displayed the same transport properties as did cells from 80% sucrose and provided support for metal uptake processes being an apical phenomenon. A working model for zinc transport by lobster gill cells is presented.     

Reconstitution of high cell binding affinity of a marine sponge aggregation factor by cross-linking of small low affinity fragments into a large polyvalent polymer

Species-specific reaggregation of cells from the marine sponge Microciona prolifera is mediated by a proteoglycan-like aggregation factor (MAF) of Mr = 2 X 10(7) which has two functional domains, a cell binding domain and an aggregation factor interaction domain. After extensive trypsin digestion, over 60% of the MAF mass was converted into a glycopeptide fragment of Mr = 10,000 (T-10) which is therefore a representative part of the major portion, but not of the entire MAF molecule. The T-10 fragment has a similar amino acid and carbohydrate composition as the intact MAF and displays species-specific binding. Although T-10 also inhibited MAF association with homotypic cells, its apparent affinity is 3 X 10(6) M-1, i.e. 13,000 times lower than that of native MAF. Reconstitution of binding affinity in the same order of magnitude as native MAF (Ka = 10(10) M-1) was obtained by cross-linking the glycopeptide fragment into polymers of the approximate size of MAF (Mr greater than 1.5 X 10(7) using diepoxybutane and glutaraldehyde, or periodate oxidation and glutaraldehyde. The apparent association constants of intermediate polymers with Mr = 1 X 10(5), 6 X 10(5), 9 X 10(5), 2 X 10(6) and above 1.5 X 10(7) increased proportionally to their size and were in line with association constants of MAF degradation fragments. Since the binding affinity of the T-10 glycopeptide fragment could be reconstituted by cross-linking, and since this fragment accounts for over 60% of MAF, we propose that the specificity and high affinity of the MAF-cell association is based on a highly polyvalent interaction of low affinity cell-binding sites. Such a polyvalency of the cell binding domain is advantageous for efficient cell-cell interactions and thus differs from most known interaction molecules and receptors characterized.     

Receptor binding of pancreatic spasmolytic polypeptide (PSP) in rat intestinal mucosal cell membranes inhibits the adenylate cyclase activity

The recently isolated pancreatic spasmolytic polypeptide, PSP, interacted with specific binding sites in the gastrointestinal tract and inhibited the adenylate cyclase activity in rat intestinal mucosal cell membranes. The binding sites appeared to be heterogeneous and Scatchard analysis of the binding data indicated the presence of at least two classes of sites. The high-affinity low-capacity binding sites and the low-affinity high-capacity binding sites had apparent dissociation constants of 1.3 X 10(-7) mol/l and 4.2 X 10(-6) mol/l, respectively. The PSP induced inhibition of the adenylate cyclase activity was independent of the stimulatory state of the enzyme. The basal activity as well as that stimulated by VIP and secretin was half maximally inhibited at approximately 3 X 10(-5) mol/l of PSP. The inhibitory effect of PSP was independent of the agonist concentration employed. PSP did not affect the receptor binding of VIP nor did VIP affect the receptor binding of PSP.     

A murine IgM monoclonal antibody binds an antigenic determinant in outer surface protein A, an immunodominant basic protein of the Lyme disease spirochete

A hybridoma cell line formed by the fusion of the P3x63-Ag8.653 myeloma cell line with splenocytes from BALB/c mice immunized with Borrelia burgdorferi produced an IgM monoclonal antibody (mAb-11G1) with kappa-light chains which detected an antigenic determinant in a major spirochetal protein of m.w. approximately 31,000, also known as outer surface protein A (OSP-A). Apparent saturation was reached in approximately 35 min with 34 ng of mAb-11G1 binding to 5 X 10(7) spirochetes giving an estimated 4.8 X 10(2) IgM molecules per spirochete and thus a minimum of 480 binding sites per organism. Enzymatic digestion studies suggest that the antigenic determinant to mAb-11G1 is contained within the peptide chain of OSP-A as binding could be eliminated by treatment of the spirochetes with proteinase K, Pronase and pepsin (100 to 200 micrograms/ml of enzyme) but not by trypsin or bromelain treatment. Periodate oxidation as well as mixed and endoglycosidase treatment of the spirochetes did not alter the binding of mAb-11G1. Two-dimensional gel electrophoresis of whole spirochetal cell lysates disclosed that OSP-A is a heterogeneously charged basic protein with an apparent isoelectric point range from 8.5 to 9.0. Amino acid analysis of OSP-A showed a 10% lysine component which could provide the basic nature to the protein. OSP-A with the intact antigenic determinant for mAb-11G1 can be found in the urine of hamsters experimentally infected with B. burgdorferi.     

Attractant-induced changes and oscillations of the extracellular Ca++ concentration in suspensions of differentiating Dictyostelium cells

We used a Ca++-sensitive electrode to measure changes in extracellular Ca++ concentration in cell suspensions of Dictyostelium discoideum during differentiation and attractant stimulation. The cells maintained an external level of 3-8 microM Ca++ until the beginning of aggregation and then started to take up Ca++. The attractants, folic acid, cyclic AMP, and cyclic GMP, induced a transient uptake of Ca++ by the cells. The response was detectable within 6 s and peaked at 30 s. Half-maximal uptake occurred at 5 nM cyclic AMP or 0.2 microM folic acid, respectively. The apparent rate of uptake amounted to 2 X 10(7) Ca++ per cell per min. Following uptake, Ca++ was released by the cells with a rate of 5 X 10(6) ions per cell per min. Specificity studies indicated that the induced uptake of Ca++ was mediated by cell surface receptors. The amount of accumulated Ca++ remained constant as long as a constant stimulus was provided. No apparent adaptation occurred. The cyclic AMP-induced uptake of Ca++ increased during differentiation and was dependent on the external Ca++ concentration. Saturation was found above 10 microM external Ca++. The time course and magnitude of the attractant-induced uptake of external Ca++ agree with a role of Ca++ during contraction. During development the extracellular Ca++ level oscillated with a period of 6-11 min. The change of the extracellular Ca++ concentration during one cycle would correspond to a 30-fold change of the cellular free Ca++ concentration.     

Studies of the cell surface of Dictyostelium discoideum during differentiation. The binding of 125I-concanavalin A to the cell surface.

125I-concanavalin A (125I-Con A) was found to be equally effective as native Con A in binding to and agglutinating cells of Dictyostelium discoideum, suggesting that iodination of the molecule had no effect on the interaction of the protein with the cell surface. Almost all of the 125I-Con A binding to the cells was inhibited by alpha-methyl glucoside. The binding of 125I-Con A to the cells was extremely rapid, and once bound, the molecule was not readily displaced by prolonged incubation or by the addition of excess native concanavalin A (Con A). In contrast, the 125I-Con A was displaced rapidly from the cell surface by alpha-methyl glucoside. The binding of 125I-Con A to D. discoideum was identical at 22 degrees and 4 degrees, and was unaffected by metabolic inhibitors, suggesting that the protein was not subject to endocytosis. The cell surface Con A binding sites became saturated at high 125I-Con A concentrations. Scatchard plots of the data indicated that growing cells possessed 4 X 10(7) sites/cell, all of equal affinity. Similar plots for "aggregation phase" cells indicated at least two classes of binding sites. A small proportion of the sites had an affinity close to that for the sites on growing cells, but the majority of the sites had a markedly decreased affinity. The total number of binding sites increased only slightly during aggregation to 5.6 X 10(7) sites/cell.