Guanosine 5′-(γ-[35S]Thio)triphosphate Autoradiography Allows Selective Detection of Histamine H3 Receptor-Dependent G Protein Activation in Rat Brain Tissue Sections

Abstract: Histamine elicits its biological effects via three distinct G protein-coupled receptors, termed H₁, H₂, and H₃. We have used guanosine 5′-(γ-[³⁵S]thio)triphosphate (GTPγ[³⁵S]) autoradiography to localize histamine receptor-dependent G protein activation in rat brain tissue sections. Initial studies revealed that in basal conditions, adenosine was present in tissue sections in sufficient concentrations to generate an adenosine A₁ receptor-dependent GTPγ[³⁵S] signal in several brain regions. All further incubations therefore contained 8-cyclopentyl-1,3-dipropylxanthine (10 µM), a selective A₁ receptor antagonist. Histamine elicited dose-dependent increments in GTPγ[³⁵S] binding to discrete anatomical structures, most notably the caudate putamen, cerebral cortex, and substantia nigra. The overall anatomical pattern of the histamine-evoked binding response closely reflects the known distribution of H₃ binding sites and was faithfully mimicked by N^α-methylhistamine, (R)-α-methylhistamine, and immepip, three H₃-selective agonists. In all regions examined, the GTPγ[³⁵S] signal was reversed with thioperamide and clobenpropit, two potent H₃-selective antagonists, whereas mepyramine, a specific H₁ antagonist, and cimetidine, a prototypic H₂ antagonist, proved ineffective. These data indicate that in rat brain tissue sections, GTPγ[³⁵S] autoradiography selectively detects H₃ receptor-dependent signaling in response to histamine stimulation. As the existing evidence suggests that GTPγ[³⁵S] autoradiography preferentially reveals responses to G_i/o-coupled receptors, our data indicate that most, if not all, central H₃ binding sites represent functional receptors coupling to G_i/o, the inhibitory class of G proteins. Besides allowing more detailed studies on H₃ receptor signaling within anatomically restricted regions of the CNS, GTPγ[³⁵S] autoradiography offers a novel approach for functional in vitro screening of H₃ ligands.     

The heavy form of elongation factor 1 in Artemia salina embryos is functionally analogous to a complex of bacterial factors EF-Tu and EF-Ts

The purified heavy form of elongation factor 1 (EF-1) from cysts of $\text{Artemia salina}$ was found to catalyze the exchange of free GTP with a complex of EF-1_L (EF-1_α) and GDP. Furthermore, after heat treatment of EF-1_H in the presence of GTP, the factor, while inactive by itself, stimulated aminoacyl-tRNA binding to ribosomes as well as polyphenylalanine synthesis when combined with EF-1_α. These functional properties are similar to those reported recently for purified EF-1_β from pig liver [ Nagata,S., Motoyoshi,K., and Iwasaki,K. (1976) Biochem. Biophys. Res. Comm. $\text{71}$, 933–938 ]. We suggest that Artemia EF-1_H consists of a EF-1_α. EF-1_β complex which is functionally analogous to bacterial EF-Tu · EF-Ts.     

Autoradiography of Serotonin 5-HT1A Receptor-Activated G Proteins in Guinea Pig Brain Sections by Agonist-Stimulated [35S]GTPγS Binding

Abstract: G protein activation mediated by serotonin 5-HT_1A and 5-HT_1B/D receptors in guinea pig brain was investigated by using quantitative autoradiography of agonist-stimulated [³⁵S]GTPγS binding to brain sections. [³⁵S]GTPγS binding was stimulated by the mixed 5-HT_1A/5-HT_1B/D agonist L694247 in brain structures enriched in 5-HT_1A binding sites, i.e., hippocampus (+140 ± 14%), dorsal raphe (+70 ± 8%), lateral septum (+52 ± 12%), cingulate (+36 ± 8%), and entorhinal cortex (+34 ± 5%). L694247 caused little or no stimulation of [³⁵S]GTPγS binding in brain regions with high densities of 5-HT_1B/D binding sites (e.g., substantia nigra, striatum, central gray, and dorsal subiculum). The [³⁵S]GTPγS binding response was antagonized by WAY100635 (10 µM) and methiothepin (10 µM). In contrast, the 5-HT_1B inverse agonist SB224289 (10 µM) did not affect the L694247-mediated [³⁵S]GTPγS binding response, and the mixed 5-HT_1B/D antagonist GR127935 (10 µM) yielded a partial blockade. The distribution pattern of the [³⁵S]GTPγS binding response and the antagonist profile suggest the L694247-mediated response in guinea pig brain to be mediated by 5-HT_1A receptors. In addition to L694247, 8-hydroxy-2-(di-n-propylamino)tetralin, and flesinoxan also stimulated [³⁵S]GTPγS binding; their maximal responses varied between 46 and 52% compared with L694247, irrespective of the brain structure being considered. Sumatriptan, rizatriptan, and zolmitriptan (10 µM) stimulated [³⁵S]GTPγS binding in the hippocampus by 20–50%. Naratriptan, CP122638, and dihydroergotamine stimulated [³⁵S]GTPγS binding to a similar level as L694247 in hippocampus, lateral septum, and dorsal raphe. It appears that under the present experimental conditions, G protein activation through 5-HT_1A but not 5-HT_1B/D receptors can be measured in guinea pig brain sections.     

Selective Increases in Phosphoinositide Signaling Activity and G Protein Levels in Postmortem Brain from Subjects with Schizophrenia or Alcohol Dependence

Abstract: Comparisons of the activity of the G protein-mediated phosphoinositide signal transduction system and of G protein levels were made in two regions of frontal cortex from eight schizophrenic, alcohol-dependent, and control subjects. G protein-mediated phosphoinositide hydrolysis was measured by stimulating cortical membranes incubated with [³H]phosphatidylinositol with 0.3–10 µM guanosine 5′-O-(3-thio)triphosphate (GTPγS). In frontal cortex areas 8/9, GTPγS-induced phosphoinositide hydrolysis was 50% greater in schizophrenic than control or alcohol-dependent subjects, whereas there were no differences among these groups of subjects in the response to GTPγS in frontal cortex area 10. Agonists for dopaminergic, cholinergic, purinergic, serotonergic, histaminergic, and glutamatergic receptors coupled to the phosphoinositide signaling system increased [³H]phosphatidylinositol hydrolysis in a GTPγS-dependent manner. Responses to most agonists were similar in all three subject groups in both cortical regions, with the largest difference being a 40% greater response to dopaminergic receptor stimulation in frontal cortex 8/9 from schizophrenic subjects. Measurements of the levels of phospholipase C-β, and of α-subunits of G_q, G_o, G_i1, G_i2, and G_s, made by immunoblot analyses revealed no differences among the groups of subjects except for increased Gα_o in schizophrenic subjects and increased Gα_o and Gα_i1 in alcohol-dependent subjects. These results demonstrate that schizophrenia is associated with increased activity of the phosphoinositide signal transduction system and increased levels of Gα_o, whereas the phosphoinositide system was unaltered in alcohol dependence, but Gα_o and Gα_i1 were increased.     

GPCR-induced dissociation of G-protein subunits in early stage signal transduction

G-protein coupled receptors (GPCRs) form a ternary complex of agonist, receptor and G-proteins during primary signal transduction at the cell membrane. Downstream signalling is thought to be preceded by the process of dissociation of Gα and Gβγ subunits, thus exposing new surfaces to interact with downstream effectors. We demonstrate here for the first time, the dissociation of heterotrimeric G-protein subunits (i.e., Gα and Gβγ) following agonist-induced GPCR (α_2A-adrenergic receptor; α_2A-AR) activation in a cell-free assay system. α_2A-AR membranes were reconstituted with the G-proteins (±hexahistidine-tagged) Gα_i1 and Gβ₁γ₂ and functional signalling was determined following activation of the reconstituted receptor:G-protein complex with the potent agonist UK-14304, and [³⁵S]GTPγS. In the presence of Ni²⁺-coated agarose beads, the activated his-tagged Gα_i1his-[³⁵S]GTPγS complex was captured on the Ni²⁺-presenting surface. When his-tagged Gβ₁γ₂ (Gβ₁γ₂his) was used with Gα_i1, the [³⁵S]GTPγS-bound Gα_i1 was not present on the Ni²⁺-coated beads, but rather, it was separated from the β₁γ₂(his)-beads, demonstrating receptor-induced dissociation of Gα and Gβγ subunits. Treatment of the reconstituted α_2A-AR membranes containing Gβ₁γ₂his:Gα_i1 with imidazole confirmed the specificity for the Ni²⁺:G-protein surface dissociation of Gα_i1 from Gβ₁γ₂his. These data demonstrate for the first time, the complete dissociation of the G-protein subunits and extend observations on the role of G-proteins in the assembly and disassembly of the ternary complex in the primary events of GPCR signalling.     

Exchange of free GTP with EF-1α·GDP complex promoted by a factor EF-1β from pig liver

Eukaryotic polypeptide chain elongation factor 1 (EF-1) has been resolved into two complementary factors, EF-1α and EF-1β, both of which were purified. Recently, we find that [³H] GDP bound to purified EF-1α is replaced by exogenous GTP rather slowly when the reaction is carried out at ionic strength optimal for polyphenylalanine synthesis. EF-1β stimulates the exchange of free GTP with EF-1α·GDP, indicating that the function of EF-1β is, at least in part, similar to that of bacterial EF-Ts.     

Molecular Cloning of cDNA for BRab from the Brain of Bombyx mori and Biochemical Properties of BRab Expressed in Escherichia coli

From a brain cDNA library of Bombyx mori, we cloned cDNA for BRab, which encoded a 202-amino-acid polypeptide sharing 60-80% similarity with rab1 family members. To characterize its biochemical properties, cDNA for BRab was inserted into an expression vector (pGEX2T) and expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. The recombinant protein was purified to homogeneity with glutathione S-Sepharose. The purified GST-BRab bound [³⁵S]-GTPγS and [³H]-GDP with association constants of 1.5×10⁶ M^-1 and 0.58×10⁶ M^-1, respectively. The binding of [³⁵S]-GTPγS was inhibited with GTP and GDP, but with no other nucleotides. The GTP-hydrolysis activity was evaluated to be 5 m mole/min/mole of BRab. In the presence of 6 mM MgCl₂, bound [³⁵S]-GTPγS and [³H]-GDP were exchanged with GTPγS most efficiently. These results suggest that BRab, having a higher affinity for GTP than GDP, converts from the GTP-bound state into the GDP-bound state by intrinsic GTP hydrolysis activity and returns to the GTP-bound state with the exchange of GDP with GTP.     

Unsaturated Free Fatty Acids Increase Benzodiazepine Receptor Agonist Binding Depending on the Subunit Composition of the GABAA Receptor Complex

Abstract: It has been shown previously that unsaturated free fatty acids (FFAs) strongly enhance the binding of agonist benzodiazepine receptor ligands and GABA_A receptor ligands in the CNS in vitro. To investigate the selectivity of this effect, recombinant human GABA_A/benzodiazepine receptor complexes formed by different subunit compositions (α_xβ_yγ₂, x = 1, 2, 3, and 5; y = 1, 2, and 3) were expressed using the baculovirus-transfected Sf9 insect cell system. At 10^?4M, unsaturated FFAs, particularly arachidonic (20:4) and docosahexaenoic (22:6) acids, strongly stimulated (>200% of control values) the binding of [³H]flunitrazepam ([³H]FNM) to the α₃β₂γ₂ receptor combination in whole cell preparations. No effect or small increases in levels of unsaturated FFAs on [³H]FNM binding to α₁β_xγ₂ and α₂β_xγ₂ receptor combinations were observed, and weak effects (130% of control values) were detected using the α₅β₂γ₂ receptor combination. The saturated FFAs, stearic and palmitic acids, were without effect on [³H]FNM binding to any combination of receptor complexes. The hydroxylated unsaturated FFAs, ricinoleic and ricinelaidic acids, were shown to decrease the binding of [³H]FNM only if an α₁β₂γ₂ receptor combination was used. Given the heterogeneity of the GABA_A/benzodiazepine receptor subunit distribution in the CNS, the effects of FFAs on the benzodiazepine receptor can be assumed to vary at both cellular and regional levels.     

Receptor-Mediated Activation of G Proteins Is Increased in Postmortem Brains of Bipolar Affective Disorder Subjects

Abstract: Guanine nucleotide binding proteins (G proteins) have been implicated in the pathophysiology of bipolar affective disorder. In the present investigation receptor-mediated G protein activation and changes in G protein trimeric state were examined in frontal cortical membranes obtained from postmortem brains of bipolar affective disorder subjects and from age-, sex-, and postmortem interval-matched controls. Stimulation of cortical membranes with serotonin, isoproterenol, or carbachol increased guanosine 5′-O-(3-[³⁵S]thiophosphate) ([³⁵S]GTPγS) binding to specific G_α proteins in a receptor-selective manner. The abilities of these receptor agonists to stimulate the binding of [³⁵S]GTPγS to the G_α proteins was enhanced in membranes from bipolar brains. Immunoblot analyses showed increases in the levels of membrane 45- and 52-kDa G_αs proteins but no changes in the amounts of G_αi, G_αo, G_αz, G_αq/11, or G_β proteins in membrane or cytosol fractions of bipolar brain homogenates. Pertussis toxin (PTX)-activated ADP-ribosylations of G_αi and G_αo were enhanced by ～80% in membranes from bipolar compared with control brains, suggesting an increase in the levels of the trimeric state of these G proteins in bipolar disorder. Serotonin-induced, magnesium-dependent reduction in PTX-mediated ADP-ribosylation of G_αi/G_αo in cortical membranes from bipolar brains was greater than that observed in controls, providing further evidence for enhanced receptor-G protein coupling in bipolar brain membranes. In addition, the amounts of G_β proteins that coimmunoprecipitated with the G_α proteins were also elevated in bipolar brains. The data show that in bipolar brain membrane there is enhanced receptor-G protein coupling and an increase in the trimeric state of the G proteins. These changes may contribute to produce exaggerated transmembrane signaling and to the alterations in affect that characterize bipolar affective disorder.     

Characterization of (2S,2'R,3'R)-2-(2',3'-[3H]-Dicarboxycyclopropyl)glycine Binding in Rat Brain

Abstract: [(2S,2′R,3′R)-2-(2′,3′-[³H]Dicarboxycyclopropyl)glycine ([³H]DCG IV) binding was characterized in vitro in rat brain cortex homogenates and rat brain sections. In cortex homogenates, the binding was saturable and the saturation isotherm indicated the presence of a single binding site with a K_D value of 180 ± 33 nM and a B_max of 780 ± 70 fmol/mg of protein. The nonspecific binding, measured using 100 µM LY354740, was <30%. NMDA, AMPA, kainate, l (?)-threo-3-hydroxyaspartic acid, and (S)-3,5-dihydroxyphenylglycine were all inactive in [³H]DCG IV binding up to 1 mM. However, several compounds inhibited [³H]DCG IV binding in a concentration-dependent manner with the following rank order of potency: LY341495 = LY354740 > DCG IV = (2S,1′S,2′S)-2-(2-carboxycyclopropyl)glycine > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid > (2S,1′S,2′S)-2-methyl-2-(2-carboxycyclopropyl)glycine > l -glutamate = ibotenate > quisqualate > (RS)-α-methyl-4-phosphonophenylglycine = l (+)-2-amino-3-phosphonopropionic acid > (S)-α-methyl-4-carboxyphenylglycine > (2S)-α-ethylglutamic acid > l (+)-2-amino-4-phosphonobutyric acid. N-Acetyl-l -aspartyl-l -glutamic acid inhibited the binding in a biphasic manner with an IC₅₀ of 0.2 µM for the high-affinity component. The binding was also affected by GTPγS, reducing agents, and CdCl₂. In parasagittal sections of rat brain, a high density of specific binding was observed in the accessory olfactory bulb, cortical regions (layers 1, 3, and 4 > 2, 5, and 6), caudate putamen, molecular layers of the hippocampus and dentate gyrus, subiculum, presubiculum, retrosplenial cortex, anteroventral thalamic nuclei, and cerebellar granular layer, reflecting its preferential (perhaps not exclusive) affinity for pre- and postsynaptic metabotropic glutamate mGlu2 receptors. Thus, the pharmacology, tissue distribution, and sensitivity to GTPγS show that [³H]DCG IV binding is probably to group II metabotropic glutamate receptors in rat brain.     

Biological characterization of various forms of elongation factor 1 from rabbit reticulocytes

Two forms of elongation factor 1 (EF-1) have been tested for a variety of biological functions. One form, EF-1H, is a high-molecular-weight aggregate (M_r > 500,000) containing four distinct polypeptides (α, β, γ, δ). The other form, EF-1α, consists of a single polypeptide which is the same as the α subunit of EF-1H. Both EF-1α and EF-1H function catalytically in binding Phe-tRNA to ribosomes, and in poly(U)-directed polyphenylalanine synthesis. The activity of EF-1α is enhanced in polyphenylalanine synthesis by a complementary component, EF-1βδ. It is also shown that EF-1βδ can facilitate an exchange of EF-1α-bound GDP for GTP. The EF-1α dissociation constants for GDP and GTP were 0.47 and 0.55 μm respectively, while the EF-1H dissociation constants for GDP and GTP were 2.0 and 1.6 μm, respectively. Thus, while EF-1α and EF-1H had approximately the same affinities for GDP and GTP, the EF-1α dissociation constants were about fourfold lower than the EF-1H dissociation constants. Attempts to isolate complexes of EF-1α or EF-1H with GTP and Phe-tRNA or with GTP, Phe-tRNA, and ribosomes were unsuccessful using either Millipore filters, gel filtration, or sucrose density gradients. The results presented in this report, along with studies from other laboratories, strengthen the hypothesis that the general mechanism of the elongation cycle is similar in eucaryotes and procaryotes.     

Regulation of Na+-coupled glucose carrier SGLT1 by AMP-activated protein kinase

Abstract

AMP-activated protein kinase (AMPK), a serine/threonine kinase activated upon energy depletion, stimulates energy production and limits energy utilization. It has previously been shown to enhance cellular glucose uptake through the GLUT family of facilitative glucose transporters. The present study explored the possibility that AMPK may regulate Na⁺-coupled glucose transport through SGLT1 (SLC5A1). To this end, SGLT1 was expressed in Xenopus oocytes with and without AMPK and electrogenic glucose transport determined by dual electrode voltage clamping experiments. In SGLT1-expressing oocytes but not in oocytes injected with water or expressing constitutively active ^γR70QAMPK (α1β1γ1(R70Q)) alone, the addition of glucose to the extracellular bath generated a current (I_g), which was half maximal (K_M) at ≈ 650 μM glucose concentration. Coexpression of ^γR70QAMPK did not affect K_M but significantly enhanced the maximal current (≈ 1.7 fold). Coexpression of wild type AMPK or the kinase dead ^αK45RAMPK mutant (α1(K45R)β1γ1) did not appreciably affect I_g. According to confocal microscopy and Western Blotting, AICAR (1 mM), phenformin (1 mM) and A-769662 (10 μM) enhanced the SGLT1 protein abundance in the cell membrane of Caco2 cells suggesting that AMPK activity may increase membrane translocation of SGLT1. These observations support a role for AMPK in the regulation of Na⁺-coupled glucose transport.     

Purification of various forms of elongation factor 1 from rabbit reticulocytes

Previous studies have indicated that the high-molecular-weight form of elongation factor 1 (EF-1H) contained four subunits (α, β, γ, and δ). Using the conventional methods of gel-filtration and ion-exchange chromatography, various forms of elongation factor 1 (EF-1α, EF-βδ, EF-1βγδ) have been purified from rabbit reticulocyte lysate. The procedure described allows one to purify these factors from a single batch of lysate in sufficient amounts for physical and biochemical studies. EF-1α is a single polypeptide of M_r 52,000, and has an isoelectric point of 9.1. EF-1βδ and EF-1βγδ are composed of two and three nonidentical polypeptides, respectively, as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Both proteins can form stable aggregates in native conditions that can reach more than 2,000,000 Da. The isoelectric point for each polypeptide was determined; 5.8 for EF-1β, 5.5 for EF-1γ, and 4.8 for EF-1δ. The activity of both proteins was compared on a molecular basis by their ability to stimulate EF-1α in the poly(U)-directed synthesis of polyphenylalanine. On the basis of this assay EF-1βγδ is slightly more active than EF-1βδ. The similarity of the amino acid composition of EF-1γ and EF-1δ and the molar ratio of α:β:γ:δ in EF-1H of approximately 1:1:0.5:0.5 have led to the conclusion that EF-1δ is probably a breakdown product of EF-1γ, and that the native form of EF-1H probably contains only the α, β, and γ subunits.     

Adrenocorticotropin/α-Melanocyte-Stimulating Hormone (ACTH/MSH)-Like Peptides Modulate Adenylate Cyclase Activity in Rat Brain Slices: Evidence for an ACTH/MSH Receptor-Coupled Mechanism

Abstract: The regulation of adenylate cyclase activity by adrenocorticotropin/α-melanocyte–stimulating hormone (ACTH/MSH)-like peptides was investigated in rat brain slices using a superfusion method. Adenylate cyclase activity was concentration-dependently increased by ACTH-(1–24), α-MSH (EC₅₀ values 16 and 6 nM, respectively), and [Nle⁴,D-Phe⁷]α-MSH (EC₅₀ value 1.6 nM), in the presence of forskolin (1 μM, optimal concentration). 1-9-Dideoxy-forskolin did not augment the response of adenylate cyclase to ACTH-(1–24). Various peptide fragments were tested for their ability to enhance [³H]cyclic AMP production. [Nle⁴,D-Phe⁷]α-MSH increased [³H]cyclic AMP formation with a maximal effect of 30% and was more potent than ACTH-(1–24), ACTH-(1–16)-NH₂, α-MSH, ACTH-(1–13)-NH₂, [MetO⁴]α-MSH, [MetO₂⁴,D-Lys⁸,Phe⁹]ACTH-(4–9), ACTH-(7–16)-NH₂, ACTH-(1–10), and ACTH-(11–24), in order of potency. This structure–activity relationship resembles that found for the previously described peptide-induced display of excessive grooming. ACTH-(1–24) stimulated adenylate cyclase activity in both striatal (maximal effect, ?20%) and septal slices (maximal effect, ?40%), but not in hippocampal or cortical slices. Lesioning of the dopaminergic projections to the striatum did not result in a diminished effect of [Nle⁴,D-Phe⁷]α-MSH on [³H]cyclic AMP accumulation, which indicates that the ACTH/MSH receptor–stimulated adenylate cyclase is not located on striatal dopaminergic terminals. ACTH-(1–24) did not affect the dopamine D₁ or D₂ receptor–mediated modulation of adenylate cyclase activity. Based on the present data, we suggest that the binding of endogenous ACTH or α-MSH to a putative ACTH/MSH receptor in certain brain regions leads to the activation of a signal transduction pathway using cyclic AMP as a second messenger.     

Contribution of G Protein Activation to Fluoride Stimulation of Phosphoinositide Hydrolysis in Human Neuroblastoma Cells

Abstract: To examine the possibility that NaF enhances phosphoinositide-specific phospholipase C (PIC) activity in neural tissues by a mechanism independent of a guanine nucleotide binding protein (G_p), we have evaluated the contribution of G_p activation to NaF-stimulated phosphoinositide hydrolysis in human SK-N-SH neuroblastoma cells. Addition of NaF to intact cells resulted in an increase in the release of inositol phosphates (450% of control values; EC₅₀ of ～ 8 mM). Inclusion of U-73122, an aminosteroid inhibitor of guanine nucleotide-regulated PIC activity in these cells, resulted in a dose-dependent inhibition of NaF-stimulated inositol lipid hydrolysis (IC₅₀ of ～ 3.5 μM). When added to digitonin-permeabilized cells, NaF or guanosine-5′-O-thiotriphosphate (GTPγS) resulted in a three- and sevenfold enhancement, respectively, of inositol phosphate release. In the combined presence of optimal concentrations of NaF and GTPγS, inositol phosphate release was less than additive, indicative of a common site of action. Inclusion of 2–5 mM concentrations of guanosine-5′-O-(2-thiodiphosphate) (GDPβS) fully blocked phosphoinositide hydrolysis elicited by GTPγS, whereas that induced by NaF was partially inhibited (65%). However, preincubation of the cells with GDPβS resulted in a greater reduction in the ability of NaF to stimulate inositol phosphate release (87% inhibition). Both GTPγS and NaF-stimulated inositol phosphate release were inhibited by inclusion of 10 μM U-73122 (54–71%). The presence of either NaF or GTPγS also resulted in a marked lowering of the Ca²⁺ requirement for activation of PIC in permeabilized cells. These results indicate that in SK-N-SH cells, little evidence exists for direct stimulation of PIC by NaF and that the majority of inositol phosphate release that occurs in the presence of NaF can be attributed to activation of G_p.     

Ca2+-Guanine Nucleotide Interactions in Brain Membranes. II. Characteristics of [3H]Guanosine Triphosphate and [3H]β, γ-Imidoguanosine 5'-Triphosphate Binding and Catabolism in the Rat Hippocampus and Striatum

Abstract: With [³H]guanosine triphosphate ([³H]GTP) and [³H]β, γ -imidoguanosine 5′-triphosphate ([³H]GppNHp) as the labelled substrates, both the binding and the catabolism of guanine nucleotides have been studied in various brain membrane preparations. Both labelled nucleotides bound to a single class of noninteracting sites (K_D= 0.1-0.5 μm ) in membranes from various brain regions (hippocampus, striatum, cerebral cortex). Unlabelled GTP, GppNHp, and guanosine diphosphate (GDP) but not guanosine monophosphate (GMP) and guanosine competitively inhibited the specific binding of [³H]guanine nucleotides. Calcium (0.1–5 mm ) partially prevented the binding of [³H]GTP and [³H]GppNHp to hippocampal and striatal membranes. This resulted from both an increased catabolism of [³H]GTP (into [³H]guanosine) and the likely formation of Ca-guanine nucleotide^2- complexes. The blockade of guanine nucleotide catabolism was responsible for the enhanced binding of [³H]GTP to hippocampal membranes in the presence of 0.1 mm -ATP or 0.1 mm -GMP. Striatal lesions with kainic acid produced both a 50% reduction of the number of specific guanine nucleotide binding sites and an acceleration of [³H]GTP and [³H]GppNHp catabolism (into [³H]guanosine) in membranes from the lesioned striatum. This suggests that guanine nucleotide binding sites were associated (at least in part) with intrinsic neurones whereas the catabolising enzyme(s) would be (mainly) located to glial cells (which proliferate after kainic acid lesion). The characteristics of the [³H]guanine nucleotide binding sites strongly suggest that they may correspond to the GTP subunits regulating neurotransmitter receptors including those labelled with [³H]5-hydroxytryptamine ([³H]5-HT) in the rat brain.     

Chemical synthesis of the 17-propanamide derivatives of stereoisomeric Δ14-17α- and 17β-estradiols: potential 17β-hydroxysteroid dehydrogenase inhibitors

The 17-propanamide derivatives of diastereomeric Δ¹⁴-17α- and 17β-estradiols, the potential candidates of a 17β-hydroxysteroid dehydrogenase (17β-HSD) inhibitor, were synthesized in 11 steps from estrone. The principal reactions employed involved in (1) conversion of estrone to the corresponding Δ¹⁴-estrone, (2) Grignard reaction of Δ¹⁴-estrone with allylmagnesium bromide followed by regioselective hydroboration of the resulting stereoisomeric 17ξ-allyl-Δ¹⁴-17ξ-ols with 9-borabicyclo[3.3.1]nonane (9-BBN), and (3) direct amidation of the 17ξ-O-/17ξ-C-spiro-γ-lactones with NH₃ under positive pressure of H₂.     

Polypeptide Elongation Factors of the Developing Chick Brain

Abstract: The polypeptide elongation factors (EF-1_L, EF-1_H, and EF-2) of the developing chick brain were separated and purified by means of a combination of gel chromatographic methods. The molecular weight of EF-1_H of the chick brain ranged from 5 to 10 × 10⁵, and was different from that of the chick liver (about 7 × 10⁵). The molecular weight of other purified factors was about 5 × 10⁴ for EF-1_L. and 9.4 × 10⁴ for EF-2. High activities of polyphenylalanine (poly-Phe) synthesis per mg protein in the developing chick brain were observed between the 3rd embryonic week and the 1st post-hatch week and declined afterwards. On the other hand, the levels of both EF-1 and EF-2 per mg protein in the brain were observed to be high in an early embryonic stage, gradually declining afterwards to the adult level. The brain EF-1_L was a major component of EF-1 in an early embryonic stage, while EF-1_H became recognizable in the 3rd embryonic week. Moreover, the EF-1_H activities were found to be more than double with regard to the binding reaction and to be more than 10-fold as active in respect to poly-Phe synthesis in comparison with the activities of EF-1_L. It is proposed that the brain EF-1_H could be due to aggregates consisting of EF-1_L, a stimulatory factor, and other components.     

Purification and Characterization of Lactobacillus bulgaricus Diaphorase

γ-Isomer of 1,2,3,4,5,6-hexachlorocyclohexane (BHC) showed greater decomposition on γ or UV irradiation of five isomers of BHC in crystalline state or in 2-propanol solution. The α- and δ-isomer of BHC and known la, 2a, 3e, 4e, 5e-pentachlorocyclohexane were separated from the irradiation product of crystalline γ-BHC. Four compounds were isolated from the irradiation product of γ-BHC in 2-propanol. Two compounds were tetrachloro-cyclohexenes (C₆H₆C1₄): γ-isomer (mp 86 ~87°C) and ?-isomer (mp 99 ~ 100°C). The other two were isomers of pentachlorocyclohexane (C₆H₇C1₅). One of them (mp 78 ~ 8.5°C) was consistent with known meso-1e,2a,3a,4a,5e isomer. The molecular structure of the other (mp 75°C) established by X-ray crystal structure analysis was 1α, 2α, 3α, 4β, 5α configuration or le 2a 3e 4e 5e conformation of CI atoms. A reaction mechanism was proposed that included a radical chain reaction and chlorine atom migration.