ABSTRACT
cDNA corresponding to human IgE heavy (epsilon) chain mRNA was cloned from human IgE-secreting myeloma U266 cells. Partial nucleotide sequence analysis demonstrated that the cloned cDNA contained the coding region for about two-thirds of the CH2 and all of the CH3 and CH4 domains as well as the 3'-untranslated region. This epsilon cDNA was inserted into expression vector pUC7 and expression of an epsilon-chain fragment in Escherichia coli was demonstrated by protein blot analysis using 125I-labeled goat anti-human IgE as probe. The expression product was purified on a column of goat anti-human IgE-conjugated Sepharose 4B and the polypeptide was found to retain binding activity to human basophils.
Subject(s)
Cloning, Molecular , DNA/metabolism , Escherichia coli/genetics , Immunoglobulin E/genetics , Immunoglobulin Heavy Chains/genetics , Immunoglobulin epsilon-Chains/genetics , Amino Acid Sequence , Base Sequence , Cell Line , DNA Restriction Enzymes , Humans , Immunoglobulin Fc Fragments/genetics , Multiple Myeloma/immunologySubject(s)
Calcium-Binding Proteins/metabolism , Calmodulin/metabolism , Islets of Langerhans/enzymology , Protein Kinases/metabolism , Animals , Cell Membrane/enzymology , Electrophoresis, Polyacrylamide Gel , Enzyme Activation , Male , Molecular Weight , Rats , Rats, Inbred Strains , Subcellular Fractions/enzymology , Trifluoperazine/pharmacologyABSTRACT
Calcium is known to play an essential part in the regulation of insulin secretion in the pancreatic beta cell. Calcium influx/efflux studies indicate that glucose promotes an accumulation of calcium by the beta cell. However, interpretation of such data is particularly difficult due to the complex compartmentalization of calcium within the cell. Although indirect evidence using chlorotetracycline suggests that control of calcium homeostasis at the plasma membrane may be central to insulin secretion, the mechanism by which secretagogues influence the handling of calcium remains unknown. Despite its continuous diffusive entry, intracellular calcium is maintained in the submicromolar range by energy-dependent mechanisms. One such process which has been well characterized in erythrocytes is a plasma membrane calcium extrusion pump whose enzymatic basis is a high affinity (Ca+2 + Mg+2)ATPase. A similar mechanism regulated by insulin has recently been identified in adipocyte plasma membranes. We report here the presence of a high affinity (Ca+2 + Mg+2)ATPase and ATP-dependent calmodulin-stimulated calcium transport system in rat pancreatic islet cell plasma membranes.
Subject(s)
Calcium-Binding Proteins/metabolism , Calcium-Transporting ATPases/metabolism , Calcium/metabolism , Calmodulin/metabolism , Cell Membrane/metabolism , Islets of Langerhans/metabolism , Adenosine Triphosphate/metabolism , Animals , Biological Transport, Active , Kinetics , Magnesium/metabolism , Male , RatsABSTRACT
In the present study, a colchicine binding assay was used to measure changes in islet polymerized and depolymerized tubulin at intervals characterizing the biphasic pattern of glucose-induced insulin release i.e., 2.5, 5.5, 10.5, and 30.5 min. At 2.5 min during the rapid onset of insulin release, a significant increase from 24% to 33% in polymerized tubulin content was observed. This increase in polymerized tubulin was followed by a reduction that temporally correlated with the disappearance of first phase release. Second phase release was also associated with a shift in equilibrium favoring tubulin polymerization at longer incubation periods. The effect of cytochalasin B on the equilibrium between polymerized and depolymerized tubulin was evaluated at a submaximal glucose concentration of 16.5 mM. Under these conditions, cytochalasin B increased polymerized tubulin content in a manner that parallels its enhancing effect on insulin release. These results indicate that the rapid events associated with the biphasic pattern of insulin release are temporally correlated with changes in polymerized tubulin.
