Synthesis and characterisation of a nucleotide based pro-drug formulated with a peptide into a nano-chemotherapy for colorectal cancer.

Recent studies in colorectal cancer patients (CRC) have shown that increased resistance to thymidylate synthase (TS) inhibitors such as 5-fluorouracil (5-FU), reduce the efficacy of standard of care (SoC) treatment regimens. The nucleotide pool cleanser dUTPase is highly expressed in CRC and is an attractive target for potentiating anticancer activity of chemotherapy. The purpose of the current work was to investigate the activity of P1, P4-di(2',5'-dideoxy-5'-selenouridinyl)-tetraphosphate (P4-SedU2), a selenium-modified symmetrically capped dinucleoside with prodrug capabilities that is specifically activated by dUTPase. Using mechanochemistry, P4-SedU2 and the corresponding selenothymidine analogue P4-SeT2 were prepared with a yield of 19% and 30% respectively. The phosphate functionality facilitated complexation with the amphipathic cell-penetrating peptide RALA to produce nanoparticles (NPs). These NPs were designed to deliver P4-SedU2 intracellularly and thereby maximise in vivo activity. The NPs demonstrated effective anti-cancer activity and selectivity in the HCT116 CRC cell line, a cell line that overexpresses dUTPase; compared to HT29 CRC cells and NCTC-929 fibroblast cells which have reduced levels of dUTPase expression. In vivo studies in BALB/c SCID mice revealed no significant toxicity with respect to weight or organ histology. Pharmacokinetic analysis of blood serum showed that RALA facilitates effective delivery and rapid internalisation into surrounding tissues with NPs eliciting lower plasma Cmax than the equivalent injection of free P4-SedU2, translating the in vitro findings. Tumour growth delay studies have demonstrated significant inhibition of growth dynamics with the tumour doubling time extended by >2weeks. These studies demonstrate the functionality and action of a new pro-drug nucleotide for CRC.

Nucleotide Prodrug Containing a Nonproteinogenic Amino Acid To Improve Oral Delivery of a Hepatitis C Virus Treatment.

Delivery of pharmacologically active nucleoside triphosphate analogs to sites of viral infection is challenging. In prior work we identified a 2'-C-methyl-1'-cyano-7-deaza-adenosine C-nucleotide analog with desirable selectivity and potency for the treatment of hepatitis C virus (HCV) infection. However, the prodrug selected for clinical development, GS-6620, required a high dose for meaningful efficacy and had unacceptable variability due to poor oral absorption as a result of suboptimal solubility, intestinal metabolism, and efflux transport. While obtaining clinical proof of concept for the nucleotide analog, a more effective prodrug strategy would be necessary for clinical utility. Here, we report an alternative prodrug of the same nucleoside analog identified to address liabilities of GS-6620. A phosphoramidate prodrug containing the nonproteinogenic amino acid methylalanine, an isopropyl ester and phenol in the (S) conformation at phosphorous, GS2, was found to have improved solubility, intestinal stability, and hepatic activation. GS2 is a more selective substrate for hepatically expressed carboxyl esterase 1 (CES1) and is resistant to hydrolysis by more widely expressed hydrolases, including cathepsin A (CatA) and CES2. Unlike GS-6620, GS2 was not cleaved by intestinally expressed CES2 and, as a result, was stable in intestinal extracts. Levels of liver triphosphate following oral administration of GS2 in animals were higher than those of GS-6620, even when administered under optimal conditions for GS-6620 absorption. Combined, these properties suggest that GS2 will have better oral absorption in the clinic when administered in a solid dosage form and the potential to extend the clinical proof of concept obtained with GS-6620.

Host genetic factors in predicting response status in chronic hepatitis B patients discontinuing nucleos(t)ide analogs.

Background/Aims: The optimal duration of nucleos(t)ide analogs (NAs) therapy in chronic hepatitis B (CHB) patients remains unsatisfactory. Previous studies have confirmed the important role of host genetic factors in determining the outcome of HBV infection. This study tries to determine the role of host genetic factors in predicting response status in CHB patients discontinuing NAs according to stringent cessation criteria. Patients and Methods: Participating patients came from a prospective NAs- discontinuation cohort since June 1999. Six single-nucleotide polymorphisms (SNPs) were selected according to previous report. SNaPshot assay was used for DNA SNPs analyses. Results: Seventy-six CHB patients were enrolled in our study, of which 61 patients were HBeAg-positive and 15 patients were HBeAg-negative. rs1883832 in the Kozak sequence of CD40 displayed an AUROC of 0.778 in predicting response status in CHB patients with HBeAg seroconversion and a genotype of CT was associated with sustained response in this subpopulation. The diagnostic performance of combinative index (rs1883832, age, and HBsAg at discontinuation) seemed to be better than that of rs1883832, but no statistical difference was observed. rs1883832 was also evaluated as an independent factor for response status by multivariate logistic regression. For HBeAg-negative CHB patients, rs9277535 at HLA-DP presents a Spearman correlation coefficient of 0.582 (P = 0.023) with virological relapse after discontinuation of NAs. Conclusions: rs1883832 serves as a valuable predictive factor for CHB patients with HBeAg seroconversion. rs9277535 at HLA-DP might also be a valuable predictive factor for CHB patients with HBeAg-negative, however, further verifications are recommended due to study limitations.

Discovery of Novel Nucleotide Prodrugs with Improved Potency Against HCV Variants Carrying NS5B S282T Mutation.

Resistant HCV variants carrying NS5B S282T mutation confer reduced sensitivity to sofosbuvir, the sole marketed NS5B polymerase inhibitor. On the basis of the finding that 2'-α-F-2'-ß-C-methylcytidine 5'-triphosphate (8) was more potent than sofosbuvir's active metabolite on inhibition of both wild-type and S282T mutant polymerase, a dual-prodrug approach has been established. Twenty-nine phosphoramidates with N4-modified cytosine were designed, synthesized, and evaluated for anti-HCV activity. The results showed that compounds 4c-4e and 4m (EC50 = 0.19-0.25 µM) exhibited comparable potency to that of sofosbuvir (EC50 = 0.15 µM) on inhibition of wild-type replicons. Notably, 4c (EC50 = 0.366 µM) was 1.5-fold more potent than sofosbuvir (EC50 = 0.589 µM) on inhibition of S282T mutant replicons. In vitro metabolic studies disclosed the possible metabolic pathways of 4c. The toxicity study results indicated a good safety profile of 4c. Together, 4c-4e and 4m hold promise for drug development for the treatment of HCV infection, especially the resistant variants with NS5B S282T mutation.

Controlled synthesis of polyethylenimine coated gold nanoparticles: Application in glutathione sensing and nucleotide delivery.

Synthesis of functional gold nanoparticles (AuNPs) justifying selectivity in biochemical interaction along with biocompatibility suited for in vivo biomedical applications has been a challenging issue. We report herein the role of polyethylenimine (PEI) in controlled synthesis of AuNPs under ambient conditions which has potentiality for sensing glutathione and selective interaction with DNA binding proteins facilitating endosomal escape for the nucleotide delivery. The choice of organic reducing agents like formaldehyde/acetaldehyde/acetyl acetone/tetrahydrofuran hydroperoxide and other similar compounds allow rapid conversion of PEI capped gold cations into AuNPs at room temperature thus controlling the functional ability of nanoparticles as a function of organic reducing agents. Both small and higher molecular weight PEI facilitates fast synthesis of AuNPs controlling cytotoxicity during in vivo biomedical applications. The AuNPs have been characterized by UV-Vis and transmission electron microscopy revealing excellent polycrystallinity and controlled nanogeometry. The cationic polymer coating enhances the electrocatalytic performances of nanoparticles. The typical biomedical application on glutathione (GSH) sensing based on peroxidase mimetic ability of as made AuNPs is studied. The as synthesized AuNPs are extreme salt and pH resistant and have potentiality for both homogeneous and heterogeneous biocatalysis. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1191-1199, 2017.

An orally administrated nucleotide-delivery vehicle targeting colonic macrophages for the treatment of inflammatory bowel disease.

Tumor necrosis factor-alpha (TNF-α) plays a central role in the pathogenesis of inflammatory bowel disease (IBD). Anti-TNF-α therapies have shown protective effects against colitis, but an efficient tool for target suppression of its secretion - ideally via oral administration - remains in urgent demand. In the colon tissue, TNF-α is mainly secreted by the colonic macrophages. Here, we report an orally-administrated microspheric vehicle that can target the colonic macrophages and suppress the local expression of TNF-α for IBD treatment. This vehicle is formed by cationic konjac glucomannan (cKGM), phytagel and an antisense oligonucleotide against TNF-α. It was given to dextran sodium sulfate (DSS) colitic mice via gastric perfusion. The unique swelling properties of cKGM enabled the spontaneous release of cKGM& antisense nucleotide (ASO) nano-complex from the phytagel scaffold into the colon lumen, where the ASO was transferred into colonic macrophages via receptor-mediated phagocytosis. The treatment significantly decreased the local level of TNF-α and alleviated the symptoms of colitis in the mice. In summary, our study demonstrates a convenient, orally-administrated drug delivery system that effectively targets colonic macrophages for suppression of TNF-α expression. It may represent a promising therapeutic approach in the treatment of IBD.

Transport of platinum bonded nucleotides into proteoliposomes, mediated by Drosophila melanogaster thiamine pyrophosphate carrier protein (DmTpc1).

The results of the present study suggest that DmTpc1 is actively implicated in the specific uptake of free cytoplasmic Pt bonded nucleotides, and therefore could be linked to the mechanism of action of some platinum-based antitumor drugs. Although DmTpc1 has a low affinity for model [Pt(dien)(N7-5'-dGTP)] and cis-[Pt(NH3)2(py)(N7-5'-dGTP)] compared to dATP it's well known that DNA platination level of few metal atoms per double-stranded molecule may account for the pharmacological activity of platinum based antitumor drugs. This is the first investigation where it has been demonstrated that a mitochondrial carrier is directly involved in the transport of metalated purines related with the cisplatin mechanism of action. Moreover it is shown as a lower hindrance of nucleotide bonded platinum complexes could strongly enhance mitochondrial uptake. Furthermore, a new application of ICP-AES addressed to measure the transport of metalated nucleobases, by using a recombinant protein reconstituted into liposomes, has been here, for the first time, developed and compared with a standard technique such as the liquid scintillation counting.

Target specific and long-acting delivery of protein, peptide, and nucleotide therapeutics using hyaluronic acid derivatives.

Hyaluronic acid (HA) is a biodegradable, biocompatible, non-toxic, non-immunogenic and non-inflammatory linear polysaccharide, which has been used for various medical applications such as arthritis treatment, ocular surgery, tissue augmentation, and so on. In this review, the effect of chemical modification of HA on its distribution throughout the body was reported for target specific and long-acting delivery applications of protein, peptide, and nucleotide therapeutics. According to the real-time bio-imaging of HA derivatives using quantum dots (QDot), HA-QDot conjugates with 35mol% HA modification maintaining enough binding sites for HA receptors were mainly accumulated in the liver, while those with 68mol% HA modification losing much of HA characteristics were evenly distributed to the tissues in the body. The results are well matched with the fact that HA receptors are abundantly present in the liver with a high specificity to HA molecules. Accordingly, slightly modified HA derivatives were used for target specific intracellular delivery of nucleotide therapeutics and highly modified HA derivatives were used for long-acting conjugation of peptide and protein therapeutics. HA has been also used as a novel depot system in the forms of physically and chemically crosslinked hydrogels for various protein drug delivery. This review will give you a peer overview on novel HA derivatives and the latest advances in HA-based drug delivery systems of various biopharmaceuticals for further clinical development.

Recent advances in cell-penetrating, non-peptide molecular carriers.

The intracellular delivery of proteins and other bioactive molecules by employing membrane-permeable carrier peptide vectors, e.g. HIV-1 Tat, Antp-HD, and related arginine-rich peptides are well known for a number of years. Because of some real and potential problems associated with these peptide carriers, such as instability due to various endogenous peptidases, uncertain in vivo delivery efficiency, potential neurotoxicity and immunogenicity, an urgent need exists for the development of efficient, non-peptide molecular carriers. This review briefly summarizes the structural characteristics and the delivery properties of the newly developed non-peptide carriers, in particular the ones developed in the author's laboratory, together with their potential as delivery vectors for poorly bioavailable drugs including small molecules, proteins, and nucleotides.

Exogenous nucleic acids and nucleotides are efficiently hydrolysed and taken up as nucleosides by intestinal explants from suckling piglets.

Human milk is a rich source of RNA, free nucleotides (NT) and nucleosides (NS). To determine the uptake of different NS sources by the intestinal epithelium, jejunal explants from suckling piglets were cultured in a medium supplemented with a mixture of NS (adenosine, cytidine, guanosine, inosine, uridine; 10 mg/l each), a mixture of five NT (AMP, CMP, GMP, IMP, UMP; 7 mg/l each) or RNA (60 mg/l), respectively. Aliquots from the media were taken at different times (0.5, 2, 5, 15, 30, 60, 180 min). NS and NT concentrations were analysed in the different supernatants at those periods using solid-phase extraction followed by HPLC. When explants were cultured in the presence of NS the concentration of these compounds, excepting cytidine, rapidly decreased, suggesting that they are efficiently taken up. When explants were incubated in the presence of NT, the total concentration of these compounds decreased while the total concentration of NS increased, suggesting that enterocytes efficiently hydrolyse NT into NS. Likewise, when explants were incubated in the presence of RNA, the total concentration of both NT and NS increased, indicating that intestinal explants are able to hydrolyse RNA to NT and then to NS in the absence of luminal enzymes. In conclusion, the jejunum of piglets at weaning is able to hydrolyse RNA and free NT to NS, and NS, excepting cytidine, are efficiently taken up by the small intestine. These results suggest that the current concentration of NT used to supplement infant formulas should be reconsidered.

The characteristics of nucleobase transport and metabolism by the perfused sheep choroid plexus.

The uptake of nucleobases was investigated across the basolateral membrane of the sheep choroid plexus perfused in situ. The maximal uptake (U(max)) for hypoxanthine and adenine, was 35.51+/-1.50% and 30.71+/-0.49% and for guanine, thymine and uracil was 12.00+/-0.53%, 13.07+/-0.48% and 12.30+/-0.55%, respectively with a negligible backflux, except for that of thymine (35.11+/-5.37% of the U(max)). HPLC analysis revealed that the purine nucleobase hypoxanthine and the pyrimidine nucleobase thymine can pass intact through the choroid plexus and enter the cerebrospinal fluid CSF so the lack of backflux for hypoxanthine was not a result of metabolic trapping in the cell. Competition studies revealed that hypoxanthine, adenine and thymine shared the same transport system, while guanine and uracil were transported by a separate mechanism and that nucleosides can partially share the same transporter. HPLC analysis of sheep CSF collected in vivo revealed only two nucleobases were present adenine and hypoxanthine; with an R(CSF/Plasma) 0.19+/-0.02 and 3.43+/-0.20, respectively. Xanthine and urate, the final products of purine catabolism, could not be detected in the CSF even in trace amounts. These results suggest that the activity of xanthine oxidase in the brain of the sheep is very low so the metabolic degradation of purines is carried out only as far as hypoxanthine which then accumulates in the CSF. In conclusion, the presence of saturable transport systems for nucleobases at the basolateral membrane of the choroidal epithelium was demonstrated, which could be important for the distribution of the salvageable nucleobases, adenine and hypoxanthine in the central nervous system.

The effect of nicotine on DNA repair in adult myocytes.

Recent findings have demonstrated that terminally differentiated adult ventricular myocytes are capable of repairing DNA that has been damaged by exposure to oxygen free radicals. Despite the potential importance of DNA repair in cells that may survive many decades after injury, little is known about the mechanisms or regulation of repair. Since tobacco use has a well-defined role in the epidemiology and pathophysiology of heart disease, we tested the effects of nicotine on repair of free radical damaged plasmids by whole-cell protein extracts from adult myocytes. Exposure to a concentration of 25 microM nicotine increased incorporation of (32P)dCTP into damaged plasmids by 16%, and 50 or 100 microM nicotine increased incorporation by 32%. Nicotine did not alter the rate or amount of poly (ADP-ribose) on the major protein acceptor of molecular weight 113-116 kDa. Inhibition of DNA polymerase activity with pyridoxal 5'-phosphate revealed greater plasmid degradation in the presence of nicotine. We conclude that nicotine enhances DNA degradation and the increased repair is a consequence of this greater degradation.

Targeting antiviral nucleotide analogues to macrophages.

Macrophages are important target cells for human immunodeficiency virus type 1 (HIV-1) infection. We have developed a drug targeting system for the selective delivery of phosphorylated nucleoside analogues to these phagocytosing cells. This system is based on the possibility of encapsulating the phosphorylated drugs into autologous erythrocytes and on the subsequent selective modification of their membranes to promote macrophage recognition and phagocytosis. Targeted delivery of phosphorylated nucleoside analogues to human, feline, and murine macrophages inhibits the infectivity of HIV-1, feline immunodeficiency virus, and LP-BM5 viruses more efficiently than the administration of the corresponding nucleoside analogues. In vivo administration of 2',3'-dideoxycytidine 5'-triphosphate (ddCTP) encapsulated into autologous erythrocytes to LP-BM5-infected mice was found to reduce infectivity and disease progression. Furthermore, the simultaneous administration of AZT or ddC produced additive antiviral effects. The possibility of using red cells as drug targeting systems was useful for the design, synthesis, and delivery of new antiviral nucleoside analogues. As a prototype of these new drugs, di-(thymidine-3'-azido-2',3'-dideoxy-D-riboside)-5'-5'-p1-p2-pyrophospha te (AZTp2AZT) was prepared. Although this drug in solution has the same antiviral activity as AZT, when administered encapsulated into erythrocytes it was several times more efficient in inhibiting the infectivity of human, feline, and murine immunodeficiency viruses. Thus, the availability of a drug targeting system for the selective delivery of antivirals to macrophages offers an additional possibility for the development of new drugs and of new combination antiviral therapies.

The well-balanced nucleoside-nucleotide mixture "OG-VI" for special medical purposes.

Nucleotides and nucleosides are essential components in all cells. However, nucleotides have not been supplied in parenteral nutrition regimens. We developed a well-balanced nucleoside-nucleotide mixture "OG-VI" and examined its effect in animals under some stressed conditions. OG-VI was composed of 30 mmol/l of inosine, 30 mmol/l of guanosine monophosphate, 30 mmol/l of cytidine, 22.5 mmol/l of uridine and 7.4 mmol/l of thymidine. The whole body protein turnover increased significantly in rats receiving total parenteral nutrition (TPN) with OG-VI solution after 70% hepatectomy, compared with rats receiving normal TPN without OG-VI (122.1 +/- 20.9 mgN.kg-1.h-1 vs. 97.4 +/- 10.1 mgN.kg-1.h-1, P < 0.01, n = 10). OG-VI significantly enhanced protein synthesis while it did not decrease protein breakdown. The effect of OG-VI on myocardium after hypoxic challenge was also examined in rats. The creatine phosphate (PCr)/inorganic phosphate (Pi) was decreased in normal rat myocardium after hypoxic challenge. However, in the rats administered OG-VI, PCr/Pi was maintained at baseline level and did not decrease after hypoxia. There was no significant change in the level of adenosine triphosphate (ATP) between before and after hypoxic challenge in myocardium of the rats administered OG-VI. In the rats receiving normal saline, instead of OG-VI, the ATP level decreased significantly after hypoxic challenge (4132 +/- 276 nmol/g tissue, n = 3, vs. 3439 +/- 465 nmol/g tissue, n = 5, P < 0.05). These data suggested that the well-balanced nucleoside-nucleotide mixture, OG-VI improved nitrogen metabolism and might stimulate synthesis of high-energy phosphate in recovery after severe surgical stress.

19F NMR of 2-deoxy-2-fluoro-D-glucose for tumor diagnosis in mice. An NDP-bound hexose analog as a new NMR target for imaging.

A well-known radiopharmaceutical 2-deoxy-2-fluoro-D-glucose widely used for positron emission tomography diagnosis in terms of glucose utilization, was re-evaluated here as a nuclear magnetic resonance pharmaceutical for cancer detection. The uptake and metabolism of FDG in the experimental tumor, MH134, transplanted to the peritoneum of C3H mice as an ascitic tumor was studied extensively by ex vivo 19F NMR. Prolonged retention of FDG and its metabolites over 2 days was confirmed in the tumor cells as well as in the heart. In these tissues, the 6-phosphate of the injected compound was converted reversibly to its epimer 2-deoxy-2-fluoro-D-mannose and further to their NDP bound forms. The metabolites were almost cleared within a day from the other healthy organs where the formation of NDP-2-deoxy-2-fluoro-D-mannose was low. Thus, the 19F NMR signal of NDP-FDM detected 1 day after the FDG injection could be used as a target signal for tumor detection. Through the use of in vivo 19F NMR spectra and 19F chemical shift images, the feasibility of this proposal was demonstrated. It was concluded that FDG-NMR has a potential for tumor diagnosis in animals.

Iontophoresis of bases, nucleosides, and nucleotides.

PURPOSE: To investigate whether transdermal iontophoresis may be potentially useful for delivery of oligonucleotide drugs, the electrotransport of representative bases (uracil and adenine), nucleosides (uridine and adenosine) and nucleotides (AMP, ATP, GTP and imido-GTP) across mammalian skin in vitro has been considered. METHODS: While the passive permeability of all compounds investigated (from 1 mM solutions at pH 7.4) was very low, the application of constant current iontophoresis (0.55 mA/cm2) significantly enhanced the transport of both charged and uncharged species. RESULTS: The efficiency of delivery depended only weakly upon lipophilicity, varied quite linearly with concentration (for AMP and ATP), was inversely sensitive to molecular weight, and was strongly influenced by charge. Neutral solutes were delivered better from the anode than the cathode, as expected; post-iontophoresis, passive permeabilities were greater than those of the untreated controls, suggesting that iontophoretically-induced changes in barrier function cannot be completely repaired in in vitro model systems. The triphosphate nucleotides, ATP and GTP, were essentially completely metabolized (presumably to their corresponding mono-phosphates) during their iontophoretic delivery, while imido-GTP was apparently resistant to enzymatic attack; however, comparison of the transport data from AMP and ATP suggested that ATP metabolism occurred primarily after the rate-limiting step of iontophoresis. CONCLUSIONS: The results obtained are consistent with the general patterns of behavior previously observed in investigations of amino acid and peptide electrotransport. It remains to be seen whether extension of the research described here to larger oligonucleotide species is a feasible long-term objective.

Nucleotide uptake and metabolism by intestinal epithelial cells.

The epithelial cells of the gastrointestinal tract are the first to encounter ingested nucleotides. Enterocytes metabolize or transport nucleotides (often partially metabolized) to other cell types. Nucleotides may also affect enterocyte gene expression. These interactions in intestinal cell lines (Caco-2 and IEC-6 cells) are described. Nucleotides and nucleosides are efficiently taken up by neoplastic cells (Caco-2) and substantially metabolized during absorption by epithelial monolayers. In nonmalignant cells (IEC-6), nucleotide pools are smaller than enterocytes of neoplastic origin (Caco-2). Consequently, cell proliferation in IEC-6 cells is more dependent on an external supply of nucleotides. Cell differentiation was examined by measuring brush border enzyme activities (sucrase, lactase and alkaline phosphatase). Nucleotides enhanced the expression of brush border enzymes in carcinoma cells only when stressed by glutamine deprivation. IEC-6 cells, which are poorly differentiated in optimal media, require basement membrane (Matrigel) for expression of brush border enzymes. Under these conditions, adding nucleotides to the culture medium enhanced enzyme activity. In addition to being substrates for intestinal absorption, nucleotides may affect enterocyte differentiation.

[Protection and latent and patent sensitization by nucleotides of radiation-induced transformations of gylcyltryptophan and serum albumin]. / Zashchita, skrytaia i iavnaia sensibilizatsiia nukleotidami radiatsionnykh prevrashchenii glitsiltriptofana i syvorotochnogo al'bumina.

A study was made of the influence of nucleotides (AMP, GMP, UMP, and CMP) on x-ray chemiluminescence of glycyltryptophan and serum albumin solutions in humans. The coefficient of modification of radiation transformations of peptide (10(-4) M) and protein (1.47.10(-4) M) was shown to be a function of nucleotide concentration representing smooth curves with a plateau at the nucleotide concentration of above 2.10(-3) M. The extreme values of the modification coefficient vary from 0.35 to 2.18 and from 1 to 2 for peptide and protein respectively. The experimental data follow the kinetic mechanism suggesting that the protective effect is implemented by the nucleotide reactions with hydroxyl radicals whereas sensitization is implemented by the reactions of free radical nucleotides with peptide and protein molecules.

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

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