Large-scale isolation of human erythrocyte Ca2+-transport ATPase.
Ontology highlight
ABSTRACT:
SUBMITTER: Gietzen K
PROVIDER: S-EPMC1153836 | biostudies-other | 1982 Oct
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Coefficients for active transport of ions and heat in vesicles with Ca(2+)-ATPase from sarcoplasmic reticulum are defined in terms of a newly proposed thermodynamic theory and calculated using experiments reported in the literature. The coefficients characterize in a quantitative manner different performances of the enzyme isoforms. Four enzyme isoforms are examined, namely from white and red muscle tissue, from blood platelets, and from brown adipose mitochondria. The results indicate that the isoforms have a somewhat specialized function. White muscle tissue and brown adipose tissue have the same active transport coefficient ratio, but the activity level of the enzyme in white muscle is higher than in brown adipose tissue. The thermogenesis ratio is high in both white muscle and brown adipose tissue, in agreement with a specific role in nonshivering thermogenesis. Other isoforms do not have this ability to generate heat. A calcium-dependence of the coefficients is found, which can be understood as being in accordance with the role of this ion as a messenger in muscle contraction as well as in thermogenesis. The investigation points to new experiments related to structure as well as to function of the isoforms.
| S-EPMC2711487 | biostudies-literature
Project description:Background and objectivesThe human Amniotic epithelial cells (AME) derived from amniotic membrane of placenta have been considered as the potential fetal stem cell source with minimal or no ethical concerns and are important therapeutic tool for anti-fibrotic and regenerative therapies.Methods and resultsHere, we evaluated the isolation, media screening, scale-up and characterization of AME cells. The isolation, expansion of AMEs were performed by sequential passaging and growth kinetics studies. The AMEs were characterized using immunocytochemistry, immunophenotyping, In-vitro differentiation, and anti-fibrotic assays. The growth kinetics study revealed that the AME cultured in Ultraculture (UC) and DMEM knockout (DMEM-KO) have prominently higher growth rate compared to others. Overall, the AMEs cultured from 5 different media retained basic morphological characteristics and the functional characteristics.ConclusionsOur result suggests that the AMEs can be successfully cultured in UC based complete media without losing its epithelial cell characteristics even after passaging for passage 2 (P2). However, a careful and methodical pre-clinical and clinical translation studies need to be conducted to show its safety and efficacy.
| S-EPMC5984062 | biostudies-literature
Project description:Antibodies against purified Ca2+-transport ATPase from human erythrocytes were raised in rabbits. Immunodiffusion experiments revealed that precipitating antibodies had been developed. The immunoglobulin fraction inhibited solely the calmodulin-dependent fraction of erythrocyte Ca2+-transport ATPase activity, whereas the basal (in the absence of added calmodulin) activity of the enzyme was not significantly affected by the antibodies. The antibodies produced similar doseresponse curves for the calmodulin- and the oleic acid-stimulated enzyme. However, the immunoglobulin fraction was considerably less effective in inhibiting Ca2+-transport ATPase activated by limited proteolysis. The results obtained with our antibodies are compatible with the interpretation that at least one subpopulation of the antibodies attacks the enzyme at or close to the calmodulin-binding site of the ATPase. The antibodies also inhibited the calmodulin-regulated Ca2+-transport ATPase from pig smooth-muscle plasma membrane, though with lower potency. However, the immunoglobulin fraction failed to suppress pig cardiac sarcoplasmicreticulum Ca2+-transport ATPase activity in the concentration range investigated. In addition, the activity of phosphodiesterase from rat brain, another enzyme modulated by calmodulin, was not at all affected by the immunoglobulin fraction.
| S-EPMC1145006 | biostudies-other
Project description:Some microbes, among them a few species of cyanobacteria, are able to excavate carbonate minerals, from limestone to biogenic carbonates, including coral reefs, in a bioerosive activity that directly links biological and geological parts of the global carbon cycle. The physiological mechanisms that enable such endolithic cyanobacteria to bore, however, remain unknown. In fact, their boring constitutes a geochemical paradox, in that photoautotrophic metabolism will tend to precipitate carbonates, not dissolve them. We developed a stable microbe/mineral boring system based on a cyanobacterial isolate, strain BC008, with which to study the process of microbial excavation directly in the laboratory. Measurements of boring into calcite under different light regimes, and an analysis of photopigment content and photosynthetic rates along boring filaments, helped us reject mechanisms based on the spatial or temporal separation of alkali versus Acid-generating metabolism (i.e., photosynthesis and respiration). Instead, extracellular Ca(2+) imaging of boring cultures in vivo showed that BC008 was able to take up Ca(2+) at the excavation front, decreasing the local extracellular ion activity product of calcium carbonate enough to promote spontaneous dissolution there. Intracellular Ca(2+) was then transported away along the multicellular cyanobacterial trichomes and excreted at the distal borehole opening into the external medium. Inhibition assays and gene expression analyses indicate that the uptake and transport was driven by P-type Ca(2+)-ATPases. We believe such a chemically simple and biologically sophisticated mechanism for boring to be unparalleled among bacteria.
| S-EPMC3003062 | biostudies-literature
Project description:The Ca2+-pumping ATPase (Ca2+-ATPase) was purified from human and pig erythrocyte membranes by calmodulin affinity chromatography in the presence of phosphatidylcholine. The amount of enzyme present in pig erythrocytes is at least 7 times greater than that isolated from human erythrocyte ghosts. However, the properties of the enzyme from the two species are similar in many respects.
| S-EPMC1148536 | biostudies-other
Project description:A technique is described for the isolation of a plasma membrane-enriched preparation from a rat liver post-mitochondrial fraction by using discontinuous Percoll density-gradient centrifugation. The procedure is simple, of high reproducibility and yield and requires a total isolation time of only 90 min. The preparation consists almost exclusively of membrane vesicles and is enriched approx. 26-fold in plasma membrane-localized enzymes with minor contamination (less than 10%) with membranes derived mainly from the endoplasmic reticulum and Golgi apparatus. Approx. 20% of the fraction comprises tightly-sealed vesicles in the inverted orientation which are capable of accumulating calcium ions and exhibiting vanadate-insensitive Ca2+-ATPase activity. The properties of these activities, including insensitivity to vanadate, oxalate, and to p-chloromercuribenzoate as well as a lack of requirement for added Mg2+, contrast markedly with the reported properties of Ca2+ transport by the endoplasmic reticulum isolated from rat liver. The technique may have wide application in the study of plasma membrane-associated activities in rat liver, particularly in relation to sinusoidal membrane surface-related events.
| S-EPMC1144357 | biostudies-other
Project description:The lack of knowledge about the mechanism of erythrocyte biogenesis through self-replication makes the in vitro generation of large quantities of cells difficult. We show that transduction of c-MYC and BCL-XL into multipotent hematopoietic progenitor cells derived from pluripotent stem cells and gene overexpression enable sustained exponential self-replication of glycophorin A(+) erythroblasts, which we term immortalized erythrocyte progenitor cells (imERYPCs). In an inducible expression system, turning off the overexpression of c-MYC and BCL-XL enabled imERYPCs to mature with chromatin condensation and reduced cell size, hemoglobin synthesis, downregulation of GCN5, upregulation of GATA1, and endogenous BCL-XL and RAF1, all of which appeared to recapitulate normal erythropoiesis. imERYPCs mostly displayed fetal-type hemoglobin and normal oxygen dissociation in vitro and circulation in immunodeficient mice following transfusion. Using critical factors to induce imERYPCs provides a model of erythrocyte biogenesis that could potentially contribute to a stable supply of erythrocytes for donor-independent transfusion.
| S-EPMC3871399 | biostudies-literature
Project description:Treatment of extensively washed erythrocyte membranes with 0.1mm-EDTA decreased their Mg(2+)-dependent, Ca(2+)-stimulated ATPase [(Mg(2+)+Ca(2+))-ATPase] activity. An activator released by this treatment restored the (Mg(2+)+Ca(2+))-ATPase to its original value in a Ca(2+)-dependent manner. This activator was different from calmodulin, as determined by a number of criteria. It was retained on an Amicon XM-100 ultrafiltration membrane (molecular-weight cut-off 100000); it appeared in the void volume of Sephadex G-100 and G-75 columns; it was not retained on a DEAE-cellulose ion-exchange column at ionic strengths similar to those used to retain calmodulin; and it maximally activated (Mg(2+)+Ca(2+))-ATPase activity less than calmodulin and at a higher Ca(2+) concentration. Like calmodulin, the activator is heat-stable. The activator fraction isolated on a 2.5-15% sucrose gradient in 0.16m-KCl showed a single band of mol.wt. 63000 and no calmodulin on 10%-polyacrylamide/sodium dodecyl sulphate gels. A trace amount of calmodulin was detected in the activator fraction by radioimmunoassay (approx. 10pg/ml of ;ghosts'), but this amount was insufficient to account for the (Mg(2+)+Ca(2+))-ATPase activation. Furthermore, calmodulin-binding protein failed to inhibit (Mg(2+)+Ca(2+))-ATPase activity by more than 10-20% in the membrane preparations from which the activator was extracted. It was concluded that erythrocyte membranes contain a (Mg(2+)+Ca(2+))-ATPase activator that may attenuate the activation of the Ca(2+)-transport ATPase by calmodulin.
| S-EPMC1161817 | biostudies-other
Project description:The sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) is responsible for intracellular Ca(2+) homeostasis. SERCA activity in muscle can be regulated by phospholamban (PLB), an affinity modulator, and sarcolipin (SLN), an uncoupler. Although PLB gets dislodged from Ca(2+)-bound SERCA, SLN continues to bind SERCA throughout its kinetic cycle and promotes uncoupling of Ca(2+) transport from ATP hydrolysis. To determine the structural regions of SLN that mediate uncoupling of SERCA, we employed mutagenesis and generated chimeras of PLB and SLN. In this study we demonstrate that deletion of SLN N-terminal residues (2)ERSTQ leads to loss of the uncoupling function even though the truncated peptide can target and constitutively bind SERCA. Furthermore, molecular dynamics simulations of SLN and SERCA interaction showed a rearrangement of SERCA residues that is altered when the SLN N terminus is deleted. Interestingly, transfer of the PLB cytosolic domain to the SLN transmembrane (TM) and luminal tail causes the chimeric protein to lose SLN-like function. Further introduction of the PLB TM region into this chimera resulted in conversion to full PLB-like function. We also found that swapping PLB N and C termini with those from SLN caused the resulting chimera to acquire SLN-like function. Swapping the C terminus alone was not sufficient for this conversion. These results suggest that domains can be switched between SLN and PLB without losing the ability to regulate SERCA activity; however, the resulting chimeras acquire functions different from the parent molecules. Importantly, our studies highlight that the N termini of SLN and PLB influence their respective unique functions.
| S-EPMC4447977 | biostudies-literature
Project description:Extracellular vesicles (EVs) have been the focus of great interest, as they appear to be involved in numerous important cellular processes. They deliver bioactive macromolecules such as proteins, lipids, and nucleic acids, allowing intercellular communication in multicellular organisms. EVs are secreted by all cell types, including immune cells such as natural killer cells (NK), and they may play important roles in the immune system. Currently, a large-scale procedure to obtain functional NK EVs is lacking, limiting their use clinically. In this report, we present a simple, robust, and cost-effective method to isolate a large quantity of NK EVs. After propagating and activating NK cells ex vivo and then incubating them in exosome-free medium for 48 h, EVs were isolated using a polymer precipitation method. The isolated vesicles contain the tetraspanin CD63, an EV marker, and associated proteins (fibronectin), but are devoid of cytochrome C, a cytoplasmic marker. Nanoparticle tracking analysis showed a size distribution between 100 and 200 nm while transmission electron microscopy imaging displayed vesicles with an oval shape and comparable sizes, fulfilling the definition of EV. Importantly, isolated EV fractions were cytotoxic against cancer cells. Furthermore, our results demonstrate for the first time that isolated activated NK (aNK) cell EVs contain the cytotoxic proteins perforin, granulysin, and granzymes A and B, incorporated from the aNK cells. Activation of caspase -3, -7 and -9 was detected in cancer cells incubated with aNK EVs, and caspase inhibitors blocked aNK EV-induced cytotoxicity, suggesting that aNK EVs activate caspase pathways in target cells. The ability to isolate functional aNK EVs on a large scale may lead to new clinical applications. Abbreviations: NK: natural killer cells; activated NK (aNK) cells; EVs: extracellular vesicles; ALL: acute lymphoblastic leukaemia; aAPC: artificial antigen-presenting cell; TEM: transmission electron microscope; PBMC: peripheral blood mononuclear cells; FBS: foetal bovine serum.
| S-EPMC5345580 | biostudies-literature