Intact Membrane Preparations Research Articles

Diphenylene iodonium as an inhibitor for the hydrogenase complex of Rhodobacter capsulatus. Evidence for two distinct electron donor sites

The photosynthetic bacterium Rhodobacter capsulatus synthesises a membrane-bound [NiFe] hydrogenase encoded by the H 2 uptake hydrogenase (hup)SLC structural operon. The hupS and hupL genes encode the small and large subunits of hydrogenase, respectively; hupC encodes a membrane electron carrier protein which may be considered as the third subunit of the uptake hydrogenase. In Wolinella succinogenes, the hydC gene, homologous to hupC, has been shown to encode a low potential cytochrome b which mediates electron transfer from H 2 to the quinone pool of the bacterial membrane. In whole cells of R. capsulatus or intact membrane preparation of the wild type strain B10, methylene blue but not benzyl viologen can be used as acceptor of the electrons donated by H 2 to hydrogenase; on the other hand, membranes of B10 treated with Triton X-100 or whole cells of a HupC − mutant exhibit both benzyl viologen and methylene blue reductase activities. We report the effect of diphenylene iodonium (Ph 2I), a known inhibitor of mitochondrial complex I and of various monooxygenases on R. capsulatus hydrogenase activity. With H 2 as electron donor, Ph 2I inhibited partially the methylene blue reductase activity in an uncompetitive manner, and totally benzyl viologen reductase activity in a competitive manner. Furthermore, with benzyl viologen as electron acceptor, Ph 2I increased dramatically the observed lagtime for dye reduction. These results suggest that two different sites exist on the electron donor side of the membrane-bound [NiFe] hydrogenase of R. capsulatus, both located on the small subunit. A low redox potential site which reduces benzyl viologen, binds Ph 2I and could be located on the distal [Fe 4S 4] cluster. A higher redox potential site which can reduce methylene blue in vitro could be connected to the high potential [Fe 3S 4] cluster and freely accessible from the periplasm.

Energy transfer and trapping in photosystem I reaction centers from cyanobacteria.

A mutant strain of the cyanobacterium Synechocystis 6803, TolE4B, was constructed by genetic deletion of the protein that links phycobilisomes to thylakoid membranes and of the CP43 and CP47 proteins of photosystem II (PSII), leaving the photosystem I (PSI) center as the sole chromophore in the photosynthetic membranes. Both intact membrane and detergent-isolated samples of PSI were characterized by time-resolved and steady-state fluorescence methods. A decay component of approximately 25 ps dominates (99% of the amplitude) the fluorescence of the membrane sample. This result indicates that an intermediate lifetime is not associated with the intact membrane preparation and the charge separation in PSI is irreversible. The decay time of the detergent-isolated sample is similar. The 600-nm excited steady-state fluorescence spectrum displays a red fluorescence peak at approximately 703 nm at room temperature. The 450-nm excited steady-state fluorescence spectrum is dominated by a single peak around 700 nm without 680-nm "bulk" fluorescence. The experimental results were compared with several computer simulations. Assuming an antenna size of 130 chlorophyll molecules, an apparent charge separation time of approximately 1 ps is estimated. Alternatively, the kinetics could be modeled on the basis of a two-domain antenna for PSI, consistent with the available structural data, each containing approximately 65 chlorophyll a molecules. If excitation can migrate freely within each domain and communication between domains occurs only close to the reaction center, a charge separation time of 3-4 ps is obtained instead.

Comparative study of the binding of prolactin and growth hormone by rabbit and human lung cell membrane fractions.

The specific binding capacities for human prolactin (hPRL) and human growth hormone (hGH) were examined in human lung membrane preparations at different developmental stages. A parallel study was carried out on rabbit lung preparations to compare binding parameters. Lung tissues were obtained from 15 fetuses of 16-38 weeks after spontaneous or therapeutic abortion and from 7 adults (lobar resection surgery). A histological study was systematically performed with a radial alveolar count in the fetuses with suspected hypoplasia. Binding analysis was performed on both intact membrane preparations and MgCl2-treated membranes, using [125I]hGH and [125I]hPRL as tracers. In the rabbit lung, specific [125I]hGH binding was found. Scatchard analysis revealed a single class of binding sites (affinity constant: 2.6 +/- 0.8 x 10(9) l/nmol and number of binding sites: 9.5 +/- 4.3 fmol/mg protein for adult rabbit; 1.85 +/- 0.5 x 10(9) l/nmol and 27.6 +/- 3.0 fmol/mg protein for 25-day-old rabbit fetuses, respectively). In contrast, [125I]hPRL did not specifically bind to lung membrane preparations. In the human lung, no consistent specific binding sites for [125I]hPRL or [125I]hGH (less than 0.5%/mg protein) were detected in adults and in 11 of the 15 fetuses. In 4 fetuses, little specific binding was observed (0.59-1.9%/mg protein) for [125I]hGH and (1.5%/mg protein) for [125I]hPRL. There was no correlation with histological lung structure. Our findings confirm the presence of specific binding sites for GH in the adult rabbit lung and demonstrate such binding in the fetal rabbit lung. In contrast, our results showed no significant binding for PRL and GH in the human lung, suggesting that these hormones do not play a direct physiological role in human lung growth and maturation.

Small angle X-ray diffraction studies on the topography of cannabinoids in synaptic plasma membranes

In a previous publication, we have described in detail how we used small angle x-ray diffraction to determine the topography of (−)- Δ 8-tetrahydrocannabinol ( Δ 8-THC) in dimyristoylphophatidylcholine (DMPC) bilayers, and to deduce the conformation of the THC side chain by using the iodo-analog (5′-I- Δ 8-THC) in the model membrane. We have now extended our studies to synaptic plasma membrane systems where the cannabinoids are believed to exert part of their pharmacological effects. Synaptic plasma membranes (SPM) were isolated from fresh bovine brains and Δ 8-THC was incorporated into the membranes. By comparing the electron density profiles of drug free and drug-containing SPM preparations, we observed an electron density increase due to the presence of Δ 8-THC in a region centered at 9.2 Å from the terminal methyl groups of the membrane bilayer. In an attempt to dissect the effects of different membrane components on the topography of Δ 8-THC, we carried out parallel experiments using membrane preparations from the synaptosomal membrane total lipid extract (TLX) as well as from bovine brain phosphatidyl choline extract (PCX) containing 30 mole percent cholesterol (Chol). Our results regarding the topography of Δ 8-THC and 5′-I- Δ 8-THC in these lipid membranes show that the TLX bilayer simulates the natural membrane environment very closely whereas in the PCX/Chol bilayer Δ 8-THC resides at a location approximately 4 Å closer to the membrane interface, similar to that found in our previous study using DMPC model membrane. These x-ray diffraction results provide insights regarding the location of the binding sites on the cannabinoid receptor and indicate that preparations of the total lipid extract from synaptosomal membranes duplicate very well the properties of the intact membrane preparation.

Characterization of binding of [ 3H]GBR 12935 (1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)-piperazine) to membranes and to solubilized membrane extracts from terminal field regions of mesolimbic, mesocortical and nigrostriatal dopamine pathways

The binding characteristics of [ 3H]GBR 12935 (1-[2-(diphenylmethoxy)ethyl]-4-(3-phenyl-propyl)piperazine), a selective dopmaine uptake inhibitor, were examined in intact membrane preparations and solubilized extracts of terminal field regions of dopamine pathways in the brain of the rats. There were many similarities in the properties of binding sites for [ 3H]GBR 12935 in the striatum, nucleus accumbens and olfactory tubercle. The binding of [ 3H]GBR 12935 was saturable and the affinity constants were not significantly different between regions of the brain. The binding of [ 3h]GBR 12935 was inhibited by amfonelic acid, GBR 12909, mazindol, methylphenidate and cocaine, with comparable affinities in each region of the brain and with the same order of potency in both preparations. Furthermore, the rank order of potencies for inhibiting the binding of [ 3H]GBR 12935 was the same as for inhibiting the uptake of [ 3H]dopamine in these regions of the brain. There did appear to be some degree of heterogeneity of binding sites for [ 3H]GBR 12935 in each of these regions of the brain, as both amfonelic acid and mazindol were best fitted by two-site models. Whether this apparent heterogeneity was due to the existence of two distinct binding sites or to two components of a single site is unclear. It did not, however, appear to be due to binding to uptake sites for norepinephrine or serotonin, as neither nisoxetine nor fluoxetine, selective inhibitors of the uptake of norepinephrine and serotonin, respectively, inhibited the binding of [ 3H]GBR 12935, at concentrations which inhibit the uptake of norepinephrine or serotonin.

Carbamylcholine inhibits β-adrenergic receptor-coupled G s protein function proximal to adenylate cyclase

The specific mechanism by which the inhibitory guanine nucleotide binding protein (G i) mediates the inhibition of adenylate cyclase activity is still unclear. The subunit dissociation model, based on studies in purified or reconstituted systems, suggests that the βγ subunit, which is dissociated with activation of G i, inhibits the function of the stimulatory guanine nucleotide binding protein (G s) by reducing the concentration of the free α s subunit. In the present study, G s, protein function is determined by measuring cholera toxin-blockable, isoproterenol-induced increases in guanosine triphosphate (GTP) binding capacity to rat cardiac ventricle membrane preparations. Carbamylcholine totally inhibited this β-adrenergic receptor-coupled G s protein function. Pretreatment of the cardiac ventricle membrane preparation with pertussis toxin prevented this muscarinic agonist effect. These results confirm the possibility of an inhibitory agonist-receptor coupled effect through G i on G s protein function proximal to the catalytic unit of adenylate cyclase in an intact membrane preparation.

High- and low-affinity [ 3H]imipramine binding sites on human platelets: separate determination and involvement of sulphur-containing bonds

We have confirmed the presence of two different classes of [ 3H]imipramine ([ 3H]IMI) binding sites on human platelets: high-affinity (K d = 0.52 nM, B max = 1670 fmol/mg protein) and low-affinity (K d = 101 nM, B max = 8 000 fmol/mg protein) binding sites. The high-affinity component of [ 3H]IMI binding can also be obtained separately as the difference between specific [ 3H]IMI binding in Na-containing and Li-containing incubation buffer. The low-affinity component can be obtained as the difference between [ 3H]IMI binding in 50 mM Tris-HCl, 5 mM KCl, 120 mM LiCl, (pH 7.5) in the absence and presence of 0.1 mM IMI. The chemical modification of SH groups was performed with Ellman's reagent (10 mM, 40 min at 23° C). The high-affinity component of the binding was totally inhibited while the low-affinity component only decreased by 39%. No decrease in [ 3H]IMI specific binding was observed when the modification of SH groups was carried out in the presence of 1 μM IMI. The inhibition of high- and low-affinity [ 3H]IMI binding was reversible since it was completely restored by incubation of modified membranes with 1,4-dithioerythritol (DTE). The reduction of SS groups by DTE (10 mM, 1 h at 23° C) in the intact membrane preparation produced an increase in total number of binding sites of the high-affinity component of [ 3H]IMI binding by 50%.

Solubilization and properties of oxytocin receptors from rat mammary gland.

Oxytocin (OT)-binding activity was extracted with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]2-hydroxy-1-propanesulfonate (CHAPSO) from rat involuted mammary glands with about 20% yield. The binding in detergent extracts was characterized and shown to be similar or identical to that of OT receptors on intact plasma membranes. Solubilized receptors had a high affinity site (Kd approximately 2 nM) and a lower affinity component, whereas the membrane receptor has only the high affinity site. Several synthetic OT analogs inhibited [3H]OT binding in the same rank order in both solubilized and intact membrane preparations. Both solubilized and membrane-associated receptor required Mn2+ for [3H]OT binding. The concentration of OT-binding sites in solubilized extracts of uterine myometrium from rats in late pregnancy was substantially greater in uteri from rats in labor than in that from rats 2 days before labor, as we have seen previously with receptors on intact membranes. The affinity of the solubilized myometrial receptor (Kd approximately 5 nM) was comparable to that of the membrane-associated receptor. Binding of [3H]OT to solubilized extracts of intestinal smooth muscle, which is not a target for OT, was negligible. Gel filtration analysis on columns of Sepharose 6B indicated that the solubilized [3H]OT-binding component from mammary gland was present in multiple mol wt forms, but the smallest major form eluted with an average apparent mol wt of about 40,000. These studies indicate that CHAPSO-solubilized binding sites for [3H]OT are the same as those in intact membranes and, therefore, are components of the OT receptor.

Calorimetric and fluorescence characterization of interactions between enkephalins and liposomal and synaptic plasma membranes containing gangliosides.

The interactions of the opioid peptide [D-Ala2]methionine-enkephalinamide with dipalmitoylphosphatidylcholine (DPPC) large unilamellar vesicles containing gangliosides GM1, Gd1a, and Gt1b and synaptic plasma membranes selectively enriched with dimyristoylphosphatidylcholine (DMPC) and ganglioside Gd1a have been investigated by using high-sensitivity differential scanning calorimetry. In the absence of gangliosides, the addition of enkephalinamide in concentrations of up to 10(-3) M does not induce any appreciable change in the heat capacity function of DPPC. In the presence of gangliosides, however, changes in the heat capacity function were observed with as little as micromolar concentrations of the enkephalinamide; the same is true for DMPC-Gd1a-enriched synaptic membranes. The magnitude and the nature of the enkephalinamide effect depend on the type of ganglioside studied. For DPPC vesicles containing ganglioside GM1 only a slight broadening in the heat capacity function and a small upward shift in the transition temperature were observed. For DPPC vesicles containing ganglioside Gd1a the effect was more dramatic; enkephalinamide concentrations as low as 10(-5) M caused the appearance of two well-defined peaks in the heat capacity function in contrast to the one peak observed in the absence of enkephalinamide. In the case of DPPC vesicles containing ganglioside Gt1b the enkephalinamide effect was seen at concentrations of 10(-4) M or higher. Synaptic plasma membranes were isolated from bovine brain, selectively enriched with exogenous lipid, and their thermotropic behavior was characterized by steady-state fluorescence spectroscopy and differential scanning calorimetry. This lipid enrichment results in the appearance of a membrane phase transition otherwise absent in the intact membrane preparation.(ABSTRACT TRUNCATED AT 250 WORDS)

Characteristics of the guanine nucleotide regulatory component of adenylate cyclase in human erythrocyte membranes

This study probes the structure and mutual interactions of the components of adenylate cyclase. We use a complementation assay which involves the addition of an adenylate cyclase-related guanine nucleotide-binding protein component to a membrane lacking this component to measure guanine nucleotide-stimulated-adenylate cyclase. Instead of using detergent extracts we were able to achieve full complementation by mixing intact membrane preparations in the presence of the nucleotide component. Of particular interest was the human erythrocyte membrane which contains very low amounts of catalytic activity and no measurable β-adrenergic receptor but has normal amounts of the nucleotide component. This component appears to be the same, by several criteria, as components found in pigeon and turkey erythrocytes and in rat liver plasma membrane. The component confers Gpp(NH)p, fluoride, and GTP stimulation of adenylate cyclase along a single reconstitution curve. It is labeled with NAD by cholera toxin, and has an apparent molecular weight of 39 000 upon sodium dodecyl sulfate gel electrophoresis. The presence of the nucleotide unit in the virtual absence of the active catalytic unit allowed us to determine those properties intrinsic to each unit and those conferred by the association of the units. The nucleotide component binds guanine nucleotides weakly in the human erythrocyte membrane, yet produces persistent activation of adenylate cyclase and tight binding (of Gpp(NH)p) upon combination with the catalytic unit. Treatment of the human erythrocyte membrane with N-ethylmaleimide causes a simultaneous diminution in both Gpp(NH)p and fluoride stimulation in reconstituted activities, suggesting that both activities are conferred by the same component.

Action of intrinsic sialidase of rat brain synaptic membranes on membrane sialolipid and sialoprotein components in situ.

The sialo compounds in the synaptosomal membranes of young rat brain were specifically labeled in vivo by the intracranial injection of radioactive N-acetylmannosamine. More than 95% of the incorporated label was found in glycosidically bound sialic acid. Specific activities of sialic acid in the synaptic membrane gangliosides G71 (monosialo), GD1a (disialo), and GT1 (trisialo) were similar; labeling in GD1b (disialo) was consistently somewhat higher. The highest specific activity of rat brain sialidase was evenly distributed between "small myelin fragment" and synaptosomal membrane fractions, and ouabain-sensitive (Na+, K+)-ATPase also was concentrated in the latter fraction. The greatest amount of bound sialic acid was found in these subcellular fractions having the highest sialidase activity. A microsomal fraction was discovered to contain a small amount of bound sialic acid with a very high degree of radioactive labeling, but no sialidase. Release of sialic acid from the relatively intact membrane preparations by intrinsic membrane-bound sialidase occurred in two recognizable stages. There was a rapid initial release, complete within 30 min, of approximately equal amounts of lipid- and protein-bound sialic acid, corresponding to roughly half of the enzymatically releasable protein-boudn, and somewhat less than one-third of the lipid-bound, sialic acid. The remainder of the membrane sialidase-susceptible sialic acid was released in a second, slower stage. The intrinsic sialidase released 16 +/- 1% of the total sialoprotein and 31 +/- 1% of the total sialolipid sialic acid. Approximately the same amount of sialic acid is releasable from membrane sialolipid by the action of exogenous Vibrio sialidase; almost twice as much is releasable from sialoglycoprotein by this enzyme as compared with the intrinsic membrane sialidase. Each of the various membrane gangliosides appeared to be equally available to the action of the membrane sialidase. The results of this study indicate that both glycolipid- and glycoprotein-bound sialic acid in the synaptic membrane are releasable in situ by the action of the intrinsic synaptic membrane sialidase, and they suggest that this enzyme may act to modulate the physical properties of the membrane. In addition to influencing the rate of hydrolysis of endogenous membrane sialo compounds by intrinsic sialidase, pH had an effect on availability of protein-bound sialic acid. At acid pH, lipid- and protein-bound sialic acid were similarly available, but near neutral pH, gangliosides appeared to be attacked preferentially.

Significance of multiple forms of brain monoamine oxidase in situ as probed by electron spin resonance.

Spin-labeled hydroxyamphetamine, a competitive reversible inhibitor of brain monoamine oxidase, has been shown to be useful as an electron spin resonance (ESR) probe of the microenvironment of the active sites of the possible monoamine oxidase multiple forms. The ESR spectrum of spin-labeled hydroxyamphetamine was strongly quenched upon binding to the enzyme. The conformation of the active site of rat brain monoamine oxidase existing in various physical states, i.e. monoamine oxidase in situ (intact brain mitochondria), crude solubilized monoamine oxidase (MAOS) and isolated monoamine oxidase fractions (MAOa and MAOb) were critically and systematically examined. Nonlinear least squares regression analyses have been used to fit the binding data (obtained at room temperature with varying spin-labeled hydroxyamphetamine concentrations) to three groups of independent noninteracting ligand-binding models. A Gibbs-Helmholtz relationship was applied to the interpretation of the measured apparent association constant K as a function of temperature ranging from 4-50 degrees with increments of 2 degreesmfrom the extracted intensive parameters, k (intrinsic association constant) and deltaF (intrinsic free energy), as well as the apparent heat, deltaH, it was clear that the microenvironment of the binding sites existing in the more purified enzyme fractions MAOa and MAOb were similar to those found in the crude solubilized enzyme. More importantly, they correlated well with the conformation of the sites characterized in situ. The data suggested that the microenvironment of this multienzyme system was unperturbed in spite of the treatment due to the isolation process. In terms of the composition of binding sites, MAOa appeared to be heterogeneous while MAOb appeared to be more homogeneous. Since the isolated fractions MAOa and MAOb possessed marked different substrate specificities, these observations directly implied that monoamine oxidase multiple forms do exist in situ. The extracted extensive parameters, n (specific binding activity, nanomoles/mg of protein), as well as the measured characteristic transition temperatures, indicated that the relative abundance of the sites which directly affected substrate specificities was indeed altered. The consistency of the characteristic transition temperatures of 21 degrees and 38 degrees for the case of intact membrane preparations was particularly significant. A tenable hypothesis is that the manipulation in the composition of the monoamine oxidase binding forms through intimate lipid-protein interactions, which has been amply demonstrated in many biomembrane systems to be functionally important might be the underlying regulatory mechanism in vivo.

Properties of membrane-associated sialyltransferase of Escherichia coli.

Membrane-associated sialyltransferase complexes in Escherichia coli K-235 can be dissociated by lipid deletion and reassembled by the addition of undecaprenyl phosphate, a unique membrane-bound lipid coenzyme. Following disruption of the cells by pressure disintegration and centrifugal fractionation, the sialyltransferase activity is assocatied with both a "particulate" and "soluble" complex. Kinetic studies as well as sugar nucleotide, metal ion, pH, ammonium sulfate, and thiol reagent requirements showed these two complexes contained functionally identical enzymatic activities. Isopycnic sucrose density gradient centrifugation studies carried out on unfractionated total membranes established that these sialytransferase activities were associated with membrane hybrids composed of different relative amounts of inner and outer membranes. Enzyme localization studies employing DPNH oxidase, a marker for the inner membrane, and relative phospholipid to protein composition determinations in the two complexes, provided added support for this conclusion. Sialyl polymer synthesis was not dependent on the incorporation of other monosaccharides and had no demonstrable metal ion requirement. Kinetic studies showed that the Km for cytidine 5-monophospho-N-acetylneuraminic acid in intact soluble and particulate enzyme preparations was 8.1 times 10-5M and 9.2 times 105M, respectively. Similarly, both enzyme complexes had nearly identical Vmax values. Following reassembly of delipidated enzyme preparations, however, there was a 10-fold increase in the Km value for the particulate enzyme and a 3-fold increase for the soluble enzyme. This increase was accompanied by an increase of approximately the same magnitude in the Vmax values. Since the lipid coenzyme was limiting in intact enzyme preparations, the increase in Vmax reflected an increase in the concentration of the active lipid in reconstituted complexes. Sialyl polymer synthesis in intact membrane preparations was stimulated by the exogenous addition of lipid. Insertion of the carrier lipid was dependent on temperature. At 37 degrees, a 120% increase in sialytransferase activity was observed while only a 35% increase was observed at 30 percent. At 20 degrees, no stimulation occurred. Fluidity of the lipid phase is apparently required for proper function of this membraneassociated enzyme complex. Thus, at 20 degrees, a temperature below the membrane lipid transition temperature, the lipids are relatively immobile.

Orientation of the Cell Membrane in Ghosts and Electron Transport Particles of Mycobacterium phlei

Abstract The chemical and enzymatic composition and the respiratory components of an intact membrane preparation, ghosts from Mycobacterium phlei, were compared to those of the electron transport particles. The ghosts were found to contain 5 to 7% of some of the soluble enzymes associated with the cytoplasm. In contrast, 97% of the total content of cytochromes b, c1 + c, and a + a3 was found in the ghost preparation. The content of cytochromes, menaquinone, latent ATPase, and phospholipid was similar in both ghost and electron transport particle preparations. The oxidative activities of the ghosts were similar to or slightly lower than those of the electron transport particles with all substrates tested. Coupled phosphorylation, however, was cryptic with the ghost preparations. Phosphorylation was demonstrable following brief sonication or preincubation of the ghosts with substrates and other components of the phosphate acceptor system. The synthesis of ATP was found to occur within the ghosts. Evidence is presented which suggests that the cell membrane of the ghost is oriented as in the intact cell, whereas the orientation of the membrane in the electron transport particles appears to be inside out.