Membrane biochemistry最新文献

Proteoliposomes containing adenylate cyclase (AC) from rabbit heart ventricles were obtained using a novel reconstitution procedure from solubilized state. The enzyme preparation can be stimulated by 5'guanylylimidodiphosphate (GppNHp) and NaF, but not by isoproterenol. Hormonal responsiveness of AC is restored by either isoproterenol trapped by the proteoliposomes during the reconstitution or pretreatment of proteoliposomes with alamethicin. GTP in the presence of alamethicin decreases the affinity of beta-adrenoceptors to the agonist, thus confirming that the properties of reconstituted AC system do not differ from the native one. It is demonstrated that the degree of AC activation by isoproterenol depends strongly on the beta-adrenoceptors content in the proteoliposomes, which in turn can be changed artificially in the process of reconstitution. The described reconstitution technique might be a useful tool for investigating the role of component stoichiometry in the functioning of hormone-regulated AC-system.

Calmodulin has been shown to stimulate the initial rates of Ca2+-uptake and Ca2+-ATPase in cardiac sarcoplasmic reticulum, when it is present in the reaction assay media for these activities. To determine whether the stimulatory effect of calmodulin is mediated directly through its interaction with the Ca2+-ATPase, or indirectly through phosphorylation of phospholamban by an endogenous protein kinase, two approaches were taken in the present study. In the first approach, the effects of calmodulin were studied on a Ca2+-ATPase preparation, isolated from cardiac sarcoplasmic reticulum, which was essentially free of phospholamban. The enzyme was preincubated with various concentrations of calmodulin at 0 degrees C and 37 degrees C, but there was no effect on the Ca2+-ATPase activity assayed over a wide range of [Ca2+] (0.1-10 microM). In the second approach, cardiac sarcoplasmic reticulum vesicles were prephosphorylated by an endogenous protein kinase in the presence of calmodulin. Phosphorylation occurred predominantly on phospholamban, an oligomeric proteolipid. The sarcoplasmic reticulum vesicles were washed prior to assaying for Ca2+ uptake and Ca2+-ATPase activity in order to remove the added calmodulin. Phosphorylation of phospholamban enhanced the initial rates of Ca2+-uptake and Ca2+-ATPase, and this stimulation was associated with an increase in the affinity of the Ca2+-pump for calcium. The EC50 values for calcium activation of Ca2+-uptake and Ca2+-ATPase were 0.96 +/- 0.03 microM and 0.96 +/- 0.1 microM calcium by control vesicles, respectively. Phosphorylation decreased these values to 0.64 +/- 0.12 microM calcium for Ca2+-uptake and 0.62 +/- 0.11 microM calcium for Ca2+-ATPase. The stimulatory effect was associated with increases in the apparent initial rates of formation and decomposition of the phosphorylated intermediate of the Ca2+-ATPase. These findings suggest that calmodulin regulates cardiac sarcoplasmic reticulum function by protein kinase-mediated phosphorylation of phospholamban.

The transport of ascorbate into cultured bovine retinal pigment epithelial (RPE) cells is reported. Primary or subcultured RPE cells were incubated in the presence of 10-500 microM L-[carboxyl-14C]-ascorbate for various periods of time. Accumulation of ascorbate into RPE cells followed a saturable active transport with a Km of 125 microM and a Vmax of 28 pmole/micrograms DNA/min. RPE intracellular water was calculated to be 0.8 pL/cell, and the transported cellular ascorbate concentration was 7.5 +/- 0.8 mM. Replacement of 150 mM NaCl in the incubation media with choline-Cl strongly inhibited (80 +/- 8%) ascorbate uptake into cultured RPE cells. Although the depletion of cellular ATP by 2,4-dinitrophenol and the inhibition of Na+-K+-ATPase by ouabain reduced ascorbate transport into RPE significantly, active transport of ascorbate was not entirely inhibited by these metabolic inhibitors. The ascorbate analogue, D-isoascorbate, competitively inhibited ascorbate transport into cultured RPE with a Ki of 12.5 mM. Cells grown in the presence of 5 to 50 mM alpha-D-glucose in the growth media did not differ in their ability to transport ascorbate. In contrast, the presence of alpha-D-glucose or its nonmetabolizable analogues, 3-0-methyl-glucose, alpha-methyl-glucose, and 2-deoxy-glucose, but not L-glucose or beta-D-fructose, in the incubation media inhibited ascorbate transport. myo-Inositol (10 or 20 mM) also inhibited ascorbate transport into RPE cells. The active uptake of ascorbate into cultured RPE cells was primarily coupled to the movement of sodium ion down its electrochemical gradient. A bifunctional, cotransport carrier possessing an ascorbate-binding site and a sodium-binding site may be involved in the ascorbate uptake system. The inhibition of ascorbate uptake by sugars appeared to be heterologous in nature, occurring between two distinct carrier systems, both of which were dependent on the sodium ions.

The addition of the protein kinase C activator phorbol esters to cell suspension a few minutes prior to stimulation inhibits the agonists-induced biochemical changes and cell responses. This inhibition is prevented by protein kinase C inhibitors. Activation of protein kinase C down regulates the stimulated responses by affecting one or more of the steps in the exitation-response coupling. This includes the receptors, the quanine-nucleotide-binding protein, the activity or distribution of phospholipase C, and other steps.

This investigation shows the effect of a Ca2+ addition on the structural and physiochemical properties of microvillus plasma membranes obtained from human placenta. Ca2+ addition induces an increase in microviscosity, as shown by the increase of order parameter and rotational correlation time of 5- and 16-doxylsterate derivatives and by the increase of fluorescence polarization of diphenylhexatriene. All the effects were obtained in a wide temperature range. The morphometric analysis of the ultrastructural images shows that the vesicle profiles of syncytiotrophoblast membranes decrease both area and form factor (FF) in the presence of Ca2+ with respect to the controls. The freeze-fracture results also show that Ca2+ induces an enhanced tendency to IMP clusterization. The Ca2+-induced changes were observed in both E and P faces. Our results underline the important role of Ca2+ in the cell membrane structure per se and in modulating interactions between cytoplasmic and extracellular microenvironments. The results of morphometric analysis of the ultrastructural images agree with biochemical data showing an increased stability induced by calcium on plasma membranes.

Fusion between membranes of Sendai virus and liposomes or human erythrocytes ghosts was studied using an assay for lipid mixing based on the relief of self-quenching of octadecylrhodamine (R18) fluorescence. We considered only viral fusion that reflects the biological activity of the viral spike glycoproteins. The liposomes were made of phosphatidylcholine, and the effects of including cholesterol, the sialoglycolipid GD1a, and/or the sialoglycoprotein glycophorin as receptors were tested. Binding of Sendai virus to those liposomes at 37 degrees C was very weak. Fusion with the erythrocyte membranes occurred at a 30-fold faster rate than with the liposomes. Experiments with biological and liposomal targets of different size indicated that size did not account for differences in fusion efficiency.

Competitive binding and fluorescence energy transfer experiments were used to examine the binding of 2'[3']-O-(2,4,6-trinitrophenyl) adenosine-5'-diphosphate (TNP-ADP) to the catalytic site of Ca ATPase. Fluorescein isothiocyanate (FITC), which is known to covalently bind near the catalytic site (13), was shown to exclude all TNP-ADP binding. TNP-ADP, in turn, will protect against FITC labeling of the Ca ATPase in its native state and in both phosphoenzyme forms. The competitive nature of these probes indicates that TNP-ADP binds to the catalytic site exclusively. Fluorescence energy transfer studies using TNP-ADP as an energy acceptor and 1,N6-ethenoadenosine-5'-diphosphate (epsilon-ADP) as an energy donor were used to estimate the distance between nucleotide binding sites in the enzyme complex. A lower limit for the distance measured was 44 A. This is interpreted to be the distance between catalytic sites on adjacent monomers of a dimer unit. The results of this work are consistent with a single nucleotide site per ATPase monomer.

Rat brain proteins presenting high-affinity binding of S-adenosyl-L-homocysteine were solubilized and purified. Extraction of binding protein was carried out in the presence of Triton X-100 and 1 M NaCl; this solubilized fraction exhibits similar kinetic properties than the membrane proteins. Purification was performed using affinity chromatography on S-adenosyl-L-homocysteine carboxyhexyl Sepharose 48 conjugate. The analysis of the affinity gel eluate by SDS-PAGE showed high purification ratios for two proteins exhibiting 54 and 68 kDa. Three activity peaks were separated when solubilized membrane proteins were submitted to isoelectric focusing; the activity peaks corresponded to proteins of pH1 6.0, 6.5, and 7.2. SDS-PAGE separation of proteins contained in each peak showed protein aggregation; a 54-kDa subunit was present in each aggregate. Solubilized membrane proteins were labeled by photoaffinity labeling with tritiated S-adenosyl-L-homocysteine; the 54- and 68-kDa proteins were found among the specifically labeled proteins. Finally, according to the previous data from the literature, the purified S-adenosyl-L-homocysteine binding proteins do not seem to be the same as adenosine receptors or phosphatidylethanolamine-N-methyltransferase.

Studies on the high-affinity receptor for IgE from rat basophilic leukemia cells (RBL-2H3) have shown that the phospholipid sphingomyelin remains attached to the protein complex during washing of the affinity immobilized complex under solubilizing conditions. Here we extended these findings and compared the species distribution patterns in sphingomyelin and phosphatidylcholine of the receptor-bound lipids to those of the plasma membrane lipids. FC epsilon-receptor-bound sphingomyelin but not phosphatidylcholine was enriched in long-chain fatty acids. We then examined other membrane proteins with respect to sphingomyelin enrichment. RBL-2H3 cell surface proteins, immobilized on concanavalin A-Sepharose and washed under solubilizing conditions, also showed a two- to six-fold enrichment in the associated sphingomyelin. Similar observations were also derived from other cell types, such as the mouse fibroblast cell line A-9 and the pig kidney epithelial cell line PK-1. Since this has been observed in all the three cell sources, it was suggested that sphingomyelin enrichment in FC epsilon-receptor preparations, although reproducible, was not specific for this protein. That this phenomenon was not specific for a particular protein might also be concluded from experiments that have shown nonhomogenous distribution of sphingomyelin in protein-free lipid-detergent mixtures. These results are compatible with a model whereby the interaction between sphingomyelin and soluble membrane proteins results from preference to nonmicellar phases or to structures with extended hydrophobic domains, probably due to the imperfect fitness of the detergent micelles to properly accomodate these lipids. This feature makes long-chain sphingomyelin a plausible candidate for the lipid responsible for the stabilizing effect that crude lipid preparations exert on the structural and functional properties of some membrane protein, e.g., FC epsilon R.