calcimycin and aluminum-fluoride

The structural natures of stable analogues for the ADP-insensitive phosphoenzyme (E2P) of Ca(2+)-ATPase formed in sarcoplasmic reticulum vesicles, i.e. the enzymes with bound beryllium fluoride (BeF.E2), bound aluminum fluoride (AlF.E2), and bound magnesium fluoride (MgF.E2), were explored and compared with those of actual E2P formed from P(i) without Ca(2+). Changes in trinitrophenyl-AMP fluorescence revealed that the catalytic site is strongly hydrophobic in BeF.E2 as in E2P but hydrophilic in MgF.E2 and AlF.E2; yet, the three cytoplasmic domains are compactly organized in these states. Thapsigargin, which was shown in the crystal structure to fix the transmembrane helices and, thus, the postulated Ca(2+) release pathway to lumen in a closed state, largely reduced the tryptophan fluorescence in BeF.E2 as in E2P, but only very slightly (hence, the release pathway is likely closed without thapsigargin) in MgF.E2 and AlF.E2 as in dephosphorylated enzyme. Consistently, the completely suppressed Ca(2+)-ATPase activity in BeF-treated vesicles was rapidly restored in the presence of ionophore A23187 but not in its absence by incubation with Ca(2+) (over several millimolar concentrations) at pH 6, and, therefore, lumenal Ca(2+) is accessible to reactivate the enzyme. In contrast, no or only very slow restoration was observed with vesicles treated with MgF and AlF even with A23187. BeF.E2 thus has the features very similar to those characteristic of the E2P ground state, although AlF.E2 and MgF.E2 most likely mimic the transition or product state for the E2P hydrolysis, during which the hydrophobic nature around the phosphorylation site is lost and the Ca(2+) release pathway is closed. The change in hydrophobic nature is probably associated with the change in phosphate geometry from the covalently bound tetrahedral ground state (BeF(3)(-)) to trigonal bipyramidal transition state (AlF(3) or AlF(4)(-)) and further to tetrahedral product state (MgF(4)(2-)), and such change likely rearranges transmembrane helices to prevent access and leakage of lumenal Ca(2+).

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Aluminum Compounds; Animals; Beryllium; Biochemical Phenomena; Biochemistry; Calcimycin; Calcium; Calcium-Transporting ATPases; Catalytic Domain; Cell Membrane; Chelating Agents; Crystallography, X-Ray; Cytoplasm; Dose-Response Relationship, Drug; Fluorides; Hydrogen-Ion Concentration; Hydrolysis; Magnesium Compounds; Models, Chemical; Muscle, Skeletal; Phosphorylation; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Rabbits; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Spectrometry, Fluorescence; Thapsigargin; Time Factors; Tryptophan; Vanadates

The acrosome reaction is a specialized exocytotic process. In the mouse there is compelling evidence that receptor-mediated activation of GTP-binding proteins by factors in the zona pellucida of oocytes is a central event in the acrosome reaction. Several reagents are able to affect GTP-binding proteins directly, bypassing the receptor-ligand step for activation. We have assessed the effect of several of these compounds on human spermatozoa, monitoring cell vitality and the acrosome reaction simultaneously using the triple-stain technique. GTP gamma S and aluminium fluoride complexes promote sperm activation very efficiently; amphiphilic peptides capable of activating G(o) and Gi, also elicit the acrosome reaction. The results indicate that activation of heterotrimeric GTP-binding proteins is sufficient to trigger acrosome exocytosis in human spermatozoa.

Topics: Acrosome; Aluminum Compounds; Amino Acid Sequence; Calcimycin; Fluorides; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Intercellular Signaling Peptides and Proteins; Male; Molecular Sequence Data; Peptides; Reference Values; Spermatozoa; Wasp Venoms

Several workers have described desensitization of endothelial prostacyclin production but conflicting evidence has been published regarding the mechanism of desensitization, whether it is homologous (agonist specific) or heterologous, and whether inactivation of cyclooxygenase is involved. The purpose of the present study was to determine the relation between the intensity of a first thrombin stimulus and the subsequent response to a repeat thrombin, histamine, ionophore A23187 or aluminium fluoride (AlF4) stimulation and to determine possible targets of desensitization. Following thrombin stimulation of confluent cultured human umbilical vein endothelial cells (HUVEC) only homologous desensitization of inositol phosphate production was observed. Both homologous and heterologous desensitization of arachidonic acid release and prostacyclin production occurred, the latter towards both histamine and the ionophore A23187. For any given dose of the first stimulant there was a much greater effect on the homologous response than on the heterologous response. These differences suggest different mechanisms. The homologous desensitization probably involves the receptor whereas the present results suggest that the target of heterologous desensitization is distal to calcium mobilization in the signal transduction pathway. The possibilities include decreased activity of phospholipase A2 or a decreased pool of accessible arachidonic acid.

Topics: Aluminum; Aluminum Compounds; Arachidonic Acid; Calcimycin; Cells, Cultured; Endothelium, Vascular; Epoprostenol; Fluorides; Histamine; Humans; Inositol Phosphates; Ionophores; Stimulation, Chemical; Thrombin

Pretreatment of bovine aortic endothelial cells with pertussis toxin inhibited partially the accumulation of inositol phosphates in response to ATP, whereas cholera toxin had no effect. Both pertussis and cholera toxins enhanced the stimulatory effect of ATP on prostacyclin release from the same cells. This action of cholera toxin was mimicked neither by an increase of cyclic AMP nor by the dissociated subunits of the toxin. Cholera and pertussis toxins, as well as aluminum fluoride, also potentiated the release of prostacyclin induced by ionophore A23187. These results suggest that a pertussis toxin-sensitive GTP-binding protein is involved in the coupling between P2-purinergic receptors and phospholipase C. In addition, another GTP-binding protein would play a crucial role at a further step in the control of PGI2 biosynthesis.

Topics: Adenosine Triphosphate; Aluminum; Aluminum Compounds; Calcimycin; Cholera Toxin; Endothelium, Vascular; Epoprostenol; Fluorides; GTP-Binding Proteins; Inositol Phosphates; Pertussis Toxin; Sugar Phosphates; Virulence Factors, Bordetella