Opioid Signal Transduction in Intact and Fragmented SH-SY5Y Neural Cells

Parameters of ligand binding, stimulation of low-Km GTPase, and inhibition of adenylate cyclase were determined in intact human neuroblastoma SH-SY5Y cells and in their isolated membranes, both suspended in identical physiological buffer medium. In cells, the mu-selective opioid agonist [3H]Tyr-D-Ala-Gly(Me)Phe-Gly-ol ([3H]DAMGO) bound to two populations of sites with KD values of 3.9 and 160 nM, with less than 10% of the sites in the high-affinity state. Both sites were also detected at 4 degrees C and were displaced by various opioids, including quaternary naltrexone. The opioid antagonist [3H]naltrexone bound to a single population of sites, and in cells treated with pertussis toxin the biphasic displacement of [3H]naltrexone by DAMGO became monophasic with only low-affinity binding present. The toxin specifically reduced high-affinity agonist binding but had no effect on the binding of [3H]naltrexone. In isolated membranes, both agonist and antagonist bound to a single population of receptor sites with affinities similar to that of the high-affinity binding component in cells. Addition of GTP to membranes reduced the Bmax for [3H]DAMGO by 87% and induced a linear ligand binding component; a low-affinity binding site, however, could not be saturated. Compared with results obtained with membranes suspended in Tris buffer, agonist binding, including both receptor density and affinity, in the physiological medium was attenuated. The results suggest that high-affinity opioid agonist binding represents the ligand-receptor-guanine nucleotide binding protein (G protein) complex present in cells at low density due to modulation by endogenous GTP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Long-lived primary radical pair state detected by time-resolved fluorescence and absorption spectroscopy in an isolated Photosystem two core

A Photosystem two (PS II) core preparation containing the chlorophyll a binding proteins CP 47, CP 43, D1 and D2, and the non-chlorophyll binding cytochrome-b559 and 33 kDA polypeptides, has been isolated from PS II-enriched membranes of peas using the non-ionic detergent heptylthioglucopyranoside and elevated ionic strengths. The primary radical pair state, P680⁺Pheo^-, was studied by time-resolved absorption and fluorescence spectroscopy, under conditions where quinone reduction and water-splitting activities were inhibited. Charge recombination of the primary radical pair in PS II cores was found to have lifetimes of 17.5 ns measured by fluorescence and 21 ns measured by transient decay kinetics under anaerobic conditions. Transient absorption spectroscopy demonstrated that the activity of the particles, based on primary radical pair formation, was in excess of 70% (depending on the choice of kinetic model), while time-resolved fluorescence spectroscopy indicated that the particles were 91% active. These estimates of activity were further supported by steady-state measurements which quantified the amount of photoreducible pheophytin. It is concluded that the PS II core preparation we have isolated is ideal for studying primary radical pair formation and recombination as demonstrated by the correlation of our absorption and fluorescence transient data, which is the first of its kind to be reported in the literature for isolated PS II core complexes from higher plants.Abbreviations CP 43 and CP 47 chlorophyll binding proteins of PS II having apparent molecular weights on SDS-PAGE of 43 kDa and 47 kDa, respectively - D1 and D2 polypeptides PS II reaction centre polypeptides encoded by the psbA and psbD genes, respectively - HPLC high performance liquid chromatography - PS II Photosystem two - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - P680 primary electron donor of PS II - Pheo phenophytin a - SPC single photon counting - PBQ phenyl-p-benzoquinone - DPC 1,5-diphenylcarbazide AFRC Photosynthesis Research Group, Department of Biochemistry     

Racemization of the aqua-{N-salicylidene-(S)-alaninato}copper(II) by reaction with potassium cyanate

Copper(II) N-salicylidene-(S)-alaninate trihydrate reacting as the S-enantiomeric parent compound with KOCN in hot diluted methanol yielded by slow crystallisation from the cooled reaction mixture (in the course of 1 day) the racemic product K[Cu{sal-(RS)-ala}(NCO)]. The parameters of the axial type EPR spectrum in X-band region and the LF band position in the electronic spectrum are typical of an axially distorted square pyramidal coordination of the Cu(II) atom in this complex. The spectral properties of the complex cuprate prepared and its basal crystallographic data are consistent with those of the earlier studied¹⁵ K₂[Cu₂{sal-(RS)-ala}₂(μ-NCO)₂] synthetized by using [Cu{sal-(RS)-ala}(H₂O)].H₂O as the racemic parent complex in the reaction mixture with KOCN.     

Investigation of double turnovers in Photosystem II charge separation and oxygen evolution with excitation flashes of different duration

The characteristics of double hitting in Photosystem II charge separation and oxygen evolution in algae and chloroplasts were investigated with saturating excitation flashes of 3 μs, 300 ns and 5 ns duration. Two types of double hitting or advancement in S-states were found to occur in oxygen evolution: a non-photochemical type found even with 5 ns flashes and a photochemical type seen only with microsecond-long flashes, which have extensive tails. The non-photochemical type, occurring with a probability of about 3%, is sensitive to the physiological condition of the sample, and is only present in algae or chloroplast samples that have been freshly prepared. In chloroplasts incubated with ferricyanide, a 3-fold increase in double advancement of S-states is observed with xenon-flash illumination but not with 300 ns or 5 ns laser illumination. However, double turnovers in Photosystem II reaction center charge separation are large with xenon flash or 300 ns laser illumination but not with 5 ns laser illumination. This indicates that quite different kinetic processes are involved in double advancement in S-states for oxygen evolution and double turnovers in charge separation. Various models of the Photosystem II reaction center are discussed. Also, based on experiments with chloroplasts incubated with ferricyanide, an unique solution to the oxygen S-state distribution in the dark suggested by Thibault (Thibault, P. (1978) C.R. Acad. Sci. Paris 287, 725–728) can be rejected.     

On the origin of mitochondria: a reexamination of the molecular structure and kinetic properties of pyruvate dehydrogenase complex from brewer''s yeast

45Ca2+ incorporated in response to glucose was selectively mobilized from the beta-cell-rich pancreatic islets of ob/ob-mice after raising the intracellular Na+ by removal of K+ or addition of ouabain or veratridine. Also studies of insulin release indicated opposite effects of glucose and Na+ on the intracellular sequestration of calcium. The fact that glucose inhibits insulin release induced by raised intracellular Na+ indicates that this sugar can lower the cytoplasmic [Ca2+]. The concept of a dual action of glucose on the cytoplasmic [Ca2+]. The concept of a dual action of glucose on the cytoplasmic [Ca2+] might well explain previous observations of an inhibitory component in the glucose action on the 45Ca2+ efflux.     

Toxic ligand conjugates as tools in the study of receptor-ligand interactions

We have constructed hybrid proteins in which the toxic A chains of ricin or diptheria toxin have been linked to either asialofetuin, fetuin, or epidermal growth factor (EGF). Both ASF-RTA and ASF-DTA are potent toxins on cultured rat hepatocytes, cells that display the asialoglycoprotein receptor. Toxicity of these two compounds is restricted to hepatocytes and can be blocked by asialoglycoproteins but not the native glycoproteins or asialoagalactoglycoprotein derivatives, indicating that the toxicity of the conjugates is mediated by the hepatic asialoglycoprotein receptor. The EGF-RTA conjugate is an extremely potent toxin on cells that can bind the hormone, but is only poorly effective on cells that are unable to bind EGF. The EGF-DTA conjugate, in contrast, is unable to kill 3T3 cells and is at least two orders of magnitude less effective than EGF-RTA on A431 cells, a cell line with 1-2 X 10(6) EGF receptors per cell. However, when EGF-RTA and EGF-DTA were tested on primary liver hepatocyte cultures, which were susceptible to both ASF-RTA and ASF-DTA, both EGF conjugates were potent toxins. Sensitivity of the hepatocyte cultures to ricin toxicity increases slightly during a 52-hr culture period. In contrast, sensitivity to EGF-RTA and ASF-RTA decline dramatically during this period. Receptors for both ligands remain plentiful on the cell surface during this time.     

The Benzodiazepine/GABA Receptor Complex: Molecular Size in Brain Synaptic Membranes and in Solution

Abstract: The molecular size of the benzodiazepine (BZ) receptor in the synaptic membrane of brain cortex (bovine or rat) was determined by an improved version of the radiation inactivation method to be 220,000. An identical size was found simultaneously for the associated γ-aminobutyric acid (GABA) receptor and for the component binding β-carboline esters. It is proposed that all three activities reside in a single protein or protein complex in the membrane. The size in solution, after extraction into Triton X-100 medium from exhaustively washed membranes, was estimated by sedimentation constant (9.4S) and by gel filtration (∼230,000 apparent MW), again with the BZ and GABA binding activities behaving identically. This size applies to the component that undergoes photoaffinity labelling by [³H]flunitrazepam in the membrane, and contains a 51,000 M_r polypeptide as the BZ-binding subunit. It is concluded that a protein complex or oligomer of 200,000–220,000 MW carries a class of BZ-binding sites and an associated class of GABA_A sites.     

Multiple Molecular Forms of Acetylcholine Receptors in Cultured Skeletal Muscle Cells: Subcellular Localization and Characterization

Abstract: Skeletal muscle cells of newborn rats, cultured in the absence of neuronal influence, were found to contain two types of cell surface acetylcholine receptors as demonstrated by isoelectric focusing. The isoelectric points of the two types of receptors were indistinguishable from those of junctional and extrajunctional types of receptors in mature animals. The cultured cells had two classes of intracellular α-bungarotoxin (αBT) binding components; one had the same sedimentation coefficient as that of surface receptors (9S), and the other had much smaller apparent molecular weights. Only a single major component was detected by isoelectric focusing analysis of the 9s intracellular aBT binding component, with a PI value close to that of the extra junctional receptor. These results suggest that the junctional and extrajunctional types of receptors may be synthesized through a common precursor.     

Steroid hormone receptor studies in melanoma model systems

The Transplantable B-16 melanotic melanoma carried in syngeneic C57B1/6J female mice and the Syrian hamster melanoma cell line, RPMI 3460, were utilized to determine whether steroid-hormone receptors are present in animal melanomas. In the B-16 melanoma, a cytoplasmic-estrogen receptor is detectable, but there is no evidence for androgen or progestin receptors. Some tumors contain a glucocorticoid-binding macromolecule. Sucrosedensity gradient centrifugation of cytosol after incubation with [³H]-estradiol revealed an 8S peak that was suppressed by excess radioinert diethylstilbesterol. Binding varied from 5–35 fmoles per mg cytosol protein. Scatchard analysis of [³H]-estradiol binding in cytosol yielded a single class of high-affinity binding sites; the dissociation constant is 6 × 10^?10 M. The receptor molecule is shown to be estrogen-specific by ligand competition assays. In contrast to B-16 melanoma, no estrogen, androgen, or progestin receptor can be found in the Syrian hamster melanoma cell line. However, a substantial level of specific binding is observed using [³H]-dexamethasone. Sucrose-gradient centrifugation of cytosol from this cell line after incubation with [³H]-dexamethasone revealed a 7S peak that was suppressed by excess radioinert dexamethasone. Scatchard analysis indicated a single class of high affinity sites with a dissociation constant of 2 × 10^?9 M. Binding levels from 70–610 fmoles per mg cytosol protein were observed. The Syrian hamster melanoma cells also exhibit a biological response to glucocorticoids: Dexamethasone causes both an inhibition of growth and a decrease in final-cell density in these cells.