Characterization, Distribution, and Protein Kinase C-Mediated Regulation of [35S]Glutathione Binding Sites in Mouse and Human Spinal Cord

Abstract: We have characterized a high-affinity [³⁵S]-glutathione ([³⁵S]GSH) binding site in mouse and human spinal cord. [³⁵S]GSH binding sites in mouse and human spinal cord were observed largely within the gray matter in both the dorsal and ventral horns of spinal cord at cervical, thoracic, and lumbosacral segments. High-affinity [³⁵S]GSH binding was saturable, showing a B _max of 72 fmol/mg of protein and a K _D of 3.0 n M for mouse spinal cord and a B _max of 52 fmol/mg of protein and a K _D of 1.6 n M for human spinal cord. [³⁵S]GSH binding was displaceable by GSH, l -cysteine, and S -hexyl-GSH, but not by glutamate, glycine, or NMDA. These [³⁵S]GSH binding sites exhibited kinetic and saturation characteristics similar to GSH binding sites in rat brain astrocytes. To determine whether [³⁵S]GSH binding sites could be regulated by protein kinase C, we exposed human spinal cord sections to phorbol 12,13-diacetate for 1 h before ligand binding. Phorbol ester treatment increased [³⁵S]GSH binding by ∼60%, an effect that could be blocked by exposure of spinal cord sections to 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a general protein kinase inhibitor. [³⁵S]GSH binding sites in the spinal cord of both species exhibited many of the characteristics of a receptor including saturable binding, high affinity, ligand specificity, and modulation by kinase activity. These data suggest that GSH is a neurotransmitter in the CNS.     

High light fluence impairs light-harvesting Chl a/b complex apoprotein syntheses and/or membrane uptake in the CD3 mutant of wheat

The CD3 mutant of wheat is a chlorophyll(Chlo-deficient mutant the phenotype of which depends upon the accumulation of the light-harvesting Chl a/b protein complex in leaves in response to the intensity of illumination. In the present studies, the rates of synthesis and/or uptake, and degradation of the light-harvesting Chl apoprotein in chloroplasts of wild-type wheat ( Triticum aestivum L. selection ND 496) and CD3 wheat leaf segments were examined in response to two different intensities of illumination. We were interested particularly in the 21. 23 kDa proteins of the light-harvesting Chl a/b complex of photosystem I (LHCI) and the 25. 27. 29 kDa proteins of the light-harvesting Chl a/b complex of photosystem II (LHCII). The accumulation of [³⁵S]-Met into the light-harvesting Chl protein of CD3 wheat chloroplasts was impaired by a high but not by a low light fluence. The levels of radiolabel in the supernatant fractions of leaf tissue homogenates from the wild-type and CD3 wheats were not significantly different over time, suggesting that the cellular uptake of [³⁵S]-Met was not limiting in the mutant. The high fluence did not enhance the degradation of light-harvesting Chl protein from CD3 wheat thylakoids. Our data indicate an impairment in the light-harvesting Chl protein synthesis/membrane uptake system in CD3 wheat leaves under high fluence. A recovery in levels of the inner LHCPII, but not of LHCPI, was observed in the Chl-deficient wheat mutant after a prolonged (4 days) exposure to high fluence. Under low fluence, LHCP was added to both photosystem II (PSH) and photosystem I (PSI) but only that added to PSI remained in thylakoids after seedlings were switched to high fluence.     

COMPARATIVE STUDIES ON THE DEGRADATION OF GABA and TAURINE IN THE BRAIN

Abstract— The degradation of taurine and GABA in mammalian brain was studied in vivo and in vitro. Small amounts of [³⁵S]isethionate (10–20 pmol/g brain wet weight) and [³⁵S]sulphate (about 2 pmol/g) were detected in mouse brain after intramuscular injection of [³⁵S]taurine. Taurine also produced isethionate in rat brain homogenates (about 20 nmol/h/g protein) and subcellular fractions (about 40 nmol/h/g protein in synaptosomes and about 300 nmol/h/g in mitochondria), but the reaction was not stimulated either by external electrical pulses or by the addition of various cofactors (NAD and NADP in both oxidized and reduced forms, riboflavin, glutathione. pyridoxal-5'-phosphate, ATP) to the incubation medium. [¹⁴C]GABA was readily metabolized to [¹⁴C]succinate both in vivo and in vitro. Isethionate formation activity was concentrated in the mitochondrial fraction, as was also GABA-T activity. Partially purified GABA-T from calf brain also slightly catalysed the formation of [³⁵S]isethionate (about 1.3 μmol/min/g protein) from [³⁵S]taurine. It appears that the slight formation of isethionate from taurine is coupled to GABA-T activity. The formation of isethionate from taurine is so small, that it apparently has no role in the control of the brain taurine pool.     

Formation and Utilization of the Active Sulfate Donor [35S]3'-Phosphoadenosine 5'-Phosphosulfate in Brain Slices: Effects of Depolarizing Agents

Abstract: The accumulation and utilization of [³⁵S]3'-phos-phoadenosine 5'-phosphosulfate (PAPS) were studied in slices from rat cerebral cortex incubated in the presence of inorganic [³⁵S]sulfate. [³⁵S]PAPS levels were directly evaluated after either isolation by ion-exchange chromatography or quantitative enzymatic transfer of its active [³⁵S]sulfate group to an acceptor phenol under the action of added phenolsulfotransferase activity. [³⁵S]PAPS formation was also indirectly followed by incubating slices in the presence of β-naphthol and measuring the levels of [³⁵S]β-naphthyl sulfate ([³⁵S]β-NS). Whereas [³⁵S]PAPS levels rapidly reached a plateau, [³⁵S]β-NS formation proceeded linearly with time for at least 1h, an observation indicating that the nucleotide was continuously synthesized and utilized for endogenous sulfation reactions. [³⁵S]PAPS formation in ices was completely and rather potently blocked by 2,6-dichloro-4-nitrophenol (IC₅₀= .10 μM), an inhibitor of the PAPS-synthesizing enzyme system in a cytosolic preparation. [³⁵S]PAPS accumulation and [³⁵S]β-NS'formation were strongly reduced by depolarizing agents such as potassium or veratridine. At millimolar concentrations, various excitatory amino acids (glutamate, aspartate, cysteate, quisqualate, and homocysteate) also elicited similar effects, whereas kainate and N -methyl-D-aspartate were inactive. This suggests that PAPS synthesis is turned off when cerebral cells are strongly depolarized.     

Methionine Recycling in Brain: A Role for Folates and Vitamin B-12

Abstract: The recycling of methionine via homocysteine was measured in vivo in brain. After constant intravenous infusions (5 h) of both [³H-methyl] methionine and [³⁵S]methionine into rats, the ratios of [³H-methyl]methionine to [³⁵S]methionine in liver, brain and plasma were determined, Similar experiments were performed in rabbits, except that the [³H-methyl]- and [³S]methionine were injected intraventricularly. If the methyl group of methionine was removed with the formation of homocysteine and then replaced by another (unlabeled) methyl group, the specific activity of the [³H-methyl]methionine would decrease more than that of [³⁵S]methionine; i.e., the ratio of [³H-methyl]- to [³⁵S]methionine in the tissue would decline. The results showed that the ratios of [³H-methyl]- to [³⁵S]methionine in liver and brain were less than the same ratio in plasma in the rats. The comparable ratios in the brain and CSF of rabbits were less than the ratio in the injectate. Since brain contains only one enzyme capable of remethylating homocysteine to methionine, the vitamin B-12–dependent methyltetrahydrofolate-homocysteine methyltransferase (EC 2.1.1.13), our results for methionine recycling via homocysteine in brain strongly support the activity of this enzyme in brain in vivo.     

ELECTROPHORETIC ANALYSES OF PROTEINS TRANSPORTED TO THE RAT POSTERIOR PITUITARY

Abstract— [³⁵S]cysteine, [³H]methionine, or [³H]fucose were injected into the supraoptic nuclei (SON) of rats, and the labelled proteins that were transported to and accumulated in the posterior pituitary 24h post-injection were analyzed electrophoretically. The transported, labelled proteins which were soluble in 0.1 m -HCl were primarily of low molecular weight (about 12,000 on SDS gels). However, the selectivity of labelling of these proteins by the three different labelled precursors could be revealed by isoelectric focusing. The 0.1 m -HCl insoluble labelled proteins, presumably reflecting membrane proteins transported from the SON to the pituitary, were more diverse and generally of higher molecular weight (> 43,000 on SDS gels).     

Time-dependent changes in polypeptide and translatable mRNA levels caused by NaCl in barley roots

The effect of salt stress on polypeptide and translatable mRNA levels was examined in roots of barley ( Hordeum vulgare L. cv. CM 72). A salt-shock time course (200 m M NaCl added to the nutrient solution of 5-day old seedlings for up to 24 h) and a 6-day salt treatment (seedlings grown in nutrient solution containing 200 m M NaCl for 6 days) were compared. Roots of intact seedlings were labeled in vivo with [³⁵S]-methionine. Poly(A)⁺ RNA was isolated and assayed in an in vitro translation system. The changes in the levels of the labeled polypeptides and translation products were analyzed following separation on two-dimensional polyacrylamide gels. Over the salt-shock time course, the majority of the changes in polypeptides and translation products were quantitative, as were the changes at 6 days. Qualitative changes occurred during the salt-shock time course, but were not observed at 6 days. The levels of polypeptides and translation products differed at each point of the salt-shock time course and at 6 days. In addition, changes were observed at the shortest time points examined, indicating a rapid response to salt.     

Endomorphin-Stimulated [35S]GTPγS Binding in Rat Brain: Evidence for Partial Agonist Activity at μ-Opioid Receptors

Abstract: Endomorphin-1 is a peptide whose binding selectivity suggests a role as an endogenous ligand at μ-opioid receptors. In the present study, the effect of endomorphin-1 on μ receptor-coupled G proteins was compared with that of the μ agonist DAMGO by using agonist-stimulated [³⁵S]GTPγS binding in rat brain. [³⁵S]GTPγS autoradiography revealed a similar localization of endomorphin-1 and DAMGO-stimulated [³⁵S]GTPγS binding in areas including thalamus, caudate-putamen, amygdala, periaqueductal gray, parabrachial nucleus, and nucleus tractus solitarius. Naloxone blocked endomorphin-1-stimulated labeling in all regions examined. Although the distribution of endomorphin-1-stimulated [³⁵S]GTPγS binding resembled that of DAMGO, the magnitude of endomorphin-1-stimulated binding was significantly lower than that produced by DAMGO. Concentration-effect curves of endomorphin-1 and DAMGO in thalamic membranes confirmed that endomorphin-1 produced only 70% of DAMGO-stimulated [³⁵S]GTPγS binding. Differences in maximal stimulation of [³⁵S]GTPγS binding between DAMGO and endomorphin-1 were magnified by increasing GDP concentrations, and saturation analysis of net endomorphin-1-stimulated [³⁵S]GTPγS binding revealed a lower apparent B _max value than that obtained with DAMGO. Endomorphin-1 also partially antagonized DAMGO stimulation of [³⁵S]GTPγS binding. These results demonstrate that endomorphin-1 is a partial agonist for G protein activation at the μ-opioid receptor in brain.     

Micromere Differentiation in the Sea Urchin Embryo: Two-Dimensional Gel Electrophoretic Analysis of Newly Synthesized Proteins

A method for large-scale culture of isolated blastomeres of sea urchin embryos in spinner flasks was developed. Micromeres and meso-, macromeres isolated from sea urchin embryos at the 16-cell stage were cultured by this method and the patterns of protein synthesis by their descendants were examined by two-dimensional gel electrophoresis of [³⁵S] methionine-labeled proteins. Six distinct proteins with molecular weights of 140–kDa, 105–kDa, 43–kDa, 32–kDa, and 28–kDa (two components) were specifically synthesized by differentiating micromeres. Quantitative analysis of the two-dimensional gel patterns demonstrated that all these proteins, except the 32–kDa protein, appeared at the time of ingression of primary mesenchyme cells (PMC's) in vivo , several hours earlier than the onset of spicule formation. The synthesis of 32–kDa protein was paralleled to active spicule formation and the uptake of Ca²⁺. Cell-free translation products directed by poly (A)⁺ RNAs isolated from descendant cells of micromeres and meso-, macromeres were compared by two-dimensional gel electrophoresis. Several spots specific to the micromere lineage were detected. However, none of them comigrated with the proteins synthesized specifically by the cultured micromeres. The results suggest that the expression of these proteins specific to differentiating micromeres may involve post-translational modification.     

TAURINE IN DEVELOPING RAT BRAIN: CHANGES IN BLOOD-BRAIN BARRIER

Abstract— The fate of [³⁵S]taurine injected intraperitoneally or intracranially has been compared in rats throughout neonatal development. The amount of [³⁵S]taurine present in the whole rat pup 24 h after intraperitoneal injection increases during development to a maximum 15 days after birth, and declines thereafter, whereas the amount of [³⁵S]taurine reaching the brain 24 h after intraperitoneal injection was greatest in the first 5 days after birth. The amount of [³⁵S]taurine remaining in the brain 24 h after intracranial injection does not vary throughout the period of neonatal development. These results suggest that the 'blood-brain' barrier is more accessible to taurine in the first few days after birth than later in neonatal development, and that factors other than simple exchange are involved.     

Outer membrane complex proteins of Chlamydia pneumoniae

Abstract The protein composition of the outer membrane complex (OMC) of Chlamydia pneumoniae strain AR-39 was analyzed by metabolic labeling with [³⁵S]methionine and [³⁵S]cysteine. Cysteine-rich proteins with molecular masses of 98, 60 doublet, 39.5 (MOMP) and 15.5 kDa were found in the OMC of C. pneumoniae . The cysteine-rich proteins of the OMCs of the threee Chlamydia species showed specific reaction patterns by immunoassay and autoradiography to rabbit or turkey immune sera. Recognition of the MOMP and 60-kDa proteins of the three species was cross-reactive. However, the C. pneumoniae 98-kDa protein was recognized by anti- C. pneumoniae (AR-39) and anti- C. psittaci (TT3) immune sera. None of the immunee sera recognized the 12-kDa cysteine-rich complex.     

Culture of cotyledons of Douglas-fir on a medium for the induction of adventitious shoots induces rapid changes in polypeptide profiles and mRNA populations

Cotyledons of 3- to 4-week-old seedlings of Douglas-fir [ Pseudotsuga menziesii (Mirb). Franco] were treated with shoot induction medium (SIM) containing 5 μ M 6N-benzylaminopurine (BAP) and 5 n M naphthaleneacetic acid (NAA). Fresh weight, dry weight and soluble protein levels were not altered within the first 48 h of SIM treatment. SIM-treated cotyledons were labelled in vivo with ³⁵SO₄^2-, and TCA-insoluble proteins were analyzed electrophoretically by a 2-dimensional system consisting of non-equilibrium electrophoresis (NEPHGE) followed by sodium dode-cylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). A basic polypeptide with a relative molecular weight of 14.5 kDa was detected 32 h after induction and after 48 h a number of polypeptides in the 14 to 35 kDa range were induced. Translation products of poly-A⁺ RNA isolated from cotyledons treated with SIM for 4, 16, 32 and 48 h were analyzed by using 2-dimensional NEPHGE-SDS-PAGE. Both qualitative and quantitative differences in the translation products were observed at all time points investigated. Expression of a specific RNA coding for a 30 kDa polypeptide was demonstrated as early as 4 h after culture on SIM. This RNA was also present at 16 h, but decreased with longer SIM treatments. Thus, culture of excised cotyledons on a medium inducing the formation of adventitious shoots results in rapid quantitative and qualitative changes in the polypeptide composition and translatable RNA population prior to morphological evidence of shoot induction.     

Immunocytochemical Localization and Biological Activity of Hydroxysteroid Sulfotransferase in the Frog Brain

Abstract : Biosynthesis of the neuroactive steroids pregnenolone sulfate (▵⁵PS) and dehydroepiandrosterone sulfate (DHEAS) is catalyzed by the enzyme hydroxysteroid sulfotransferase (HST), which transfers the sulfonate moiety from 3'-phosphoadenosine 5' -phosphosulfate (PAPS) on thye 3-hydroxy site of steroids. Although high concentrations of ▵⁵PS and DHEAS have been detected in the rat brain, the anatomical localization of HST in the CNS has never been determined. Using an antiserum against rat liver HST, we have investigated the distribution of HST-like Immunoreactivity in the CNS of the frog Rana ridibunda. Two populations of HST-immunoreactive neurons were observed in the hypothalamus, and several bundless of positive nerves fibers were visualized in the telencephalon and diencephalon. lncubation of frog brain homogenates with [³⁵S]PAPS and [³H] pregnenolone yielded the formation of several ³H, ³⁵S-labeled compounds, including ▵⁵PS and testosterone sulfate. When [³] dehydroepiandrosterone and [³⁵S]PAPS were used as precursors, one of the ³H, ³⁵S-labeled metabolities coeluted with DHEAS. Neosynthesis of [³H]▵⁵PS and [³H]DHEAS was reduced significantly by 2,4-dichloro-6-nitrophenol, a specific inhibitor of sulfotransferases. The present study provides the first immunocytochemical mapping of HSt in the brain. Our data also demonstrate for the first time that biopsynthesis of the highly poten neuroactive steroids ▵⁵PS and DHEAS occurs in the CNS of nonmammalian vertebrates.     

Amyloid Precursor Protein Is Synthesized by Retinal Ganglion Cells, Rapidly Transported to the Optic Nerve Plasma Membrane and Nerve Terminals, and Metabolized

Abstract: We have investigated the synthesis, axonal transport, and processing of the β-amyloid precursor protein (APP) in in vivo rabbit retinal ganglion cells. These CNS neurons connect the retina to the brain via axons that comprise the optic nerve. APP is synthesized in retinal ganglion cells and is rapidly transported into the optic nerve in small transport vesicles. It is then transferred to the axonal plasma membrane, as well as to the nerve terminals and metabolized with a f_1/2 of less than 5 h. A significant accumulation of C-terminal amyloidogenic or nonamyloidogenic fragments is seen in the optic nerve 5 h after [³⁵S]- methionine, [³⁵S]cysteine injection, which disappears by 24 h. The major molecular mass species of APP in the optic nerve is ∼110 kDa, and is an APP isoform that does not contain a Kunitz protease inhibitor domain. Higher molecular mass species containing this sequence are seen mostly in the retina. A protease(s) that can potentially cleave APP to generate an amyloidogenic fragment is present in the same optic nerve membrane compartment as APP.     

STUDIES ON THE POLYSACCHARIDE-SULFATING SYSTEM IN SEA URCHIN EMBRYOS

The sulfating system in sea urchin embryos was examined, using the labeled precursor inorganic [³⁵S]sulfate in vivo and [³⁵S]3'-phosphoadenosine 5'-phosphosulfate ([³⁵S]PAPS) in a cell-free system. In vivo incorporation of [³⁵pS]sulfate into the trichloroacetic acid (TCA)-insolubte fraction increased gradually during sea urchin development, whereas radioactivity of [³⁵S]sulfate contained in the TCA-soluble fraction showed a conspicuous peak at the late gastrula stage.
In a cell-free system, the particulate fraction showed marked incorporation of [³⁵pS]JPAPS. This sulfating activity was highest at pH 6.4 to 7.2 and at 27°C, and it was strongly inhibited by Hg ²⁺and p-chloromercuribenzoic acid.
The sulfating activity was quite low in fertilized eggs, but then increased rapidly up to the swimming blastula stage. The activity in the particulate fraction precipitated at 10,000 xg increased gradually and that in the particulate fraction precipitated at 100,000 xg was almost constant from the swimming blastula stage to the pluteus stage.     

INCORPORATION OF RADIOACTIVE SULPHATE INTO SULPHATIDE DURING MYELINATION IN CULTURES OF RAT CEREBELLUM

Abstract— Explants of rat cerebellum obtained 12-24 h after birth were maintained in culture in Maximow assemblies. About 90 per cent of the cultures myelinated after 10–12 days in vitro . Cultures maintained for varying periods of time were exposed to [³⁵S]Na₂SO₄; labelled sulphatide was recovered from the total homogenate. Preparation of a subcellular fraction with density properties corresponding to those of myelin indicated that labelled sulphatide appeared in this fraction. Cultures which were poorly-myelinated always exhibited a lower rate of inccrpomtion than well-myelmated cultures from littermate animals, but the distribution of labelled sulphatide into the 'myelin' fraction was similar in the two groups. The rate of incorporation of [³⁵S]Na₂SO₄ into total sulphatide increased with the duration of the culture, with a low level of incorporation until the seventh day in vitro , followed by a sharp increase in rate up to the 21st day. This pattern resembles that observed for rat cerebellum in vivo .     

N-Linked Glycosylation of the α-Amino-3-Hydroxy-5-Methylisoxazole-4-Propionate(AMPA)-Selective Glutamate Receptor Channel α2 Subunit Is Essential for the Acquisition of Ligand-Binding Activity

Abstract: The N-linked glycosylation of the α2 subunit of the mouse α-amino-3-hydroxy-5-methylisoxazole-4-propionate(AMPA)-selective glutamate receptor (GluR) channel was characterized. The receptor subunit protein has five putative N -glycosylation sites. The recombinant receptor proteins were identified by [³⁵S]methionine/[³⁵S]cysteine metabolic labeling, western blot analysis, immunocytochemical detection, and [³H]AMPA binding experiments when expressed in insect Spodoptera frugiperda cells using a baculovirus system. The effect of tunicamycin on the metabolic labeling and immunoblots suggested that the two products, a major protein species of ∼102 kDa and a minor species of ∼98 kDa, correspond to glycosylated and unglycosylated forms, respectively, which was also supported by the enzymic deglycosylation experiments. Immunofluorescence staining of tunicamycin-treated cells expressing only the unglycosylated form differed little from that of tunicamycin-nontreated cells expressing both glycosylated and unglycosylated forms. The lack of AMPA-binding activity of the unglycosylated form expressed in the presence of tunicamycin suggested that N -glycosylation is required, directly or indirectly, for functional expression in insect cells for ligand binding. These results demonstrate that occupancy of at least one N -glycosylation site is required for the formation and maintenance of the GluRα2 subunit protein in an active conformation for ligand binding. Possible roles of N -glycosylation of GluRα2 subunit protein are discussed.     

Lindane Administration to the Rat Induces Modifications in the Regional Cerebral Binding of [3H]Muscimol, [3H]-Flunitrazepam, and t-[35S]Butylbicyclophosphorothionate: An Autoradiographic Study

Abstract: The effect of lindane administration on the specific binding of ligands to different sites on the GABA_A receptor-ionophore complex was studied in the rat brain by receptor mapping autoradiography. [³H]Muscimol (Mus), [³H]flunitrazepam (Flu), and t -[³⁵S]butylbicyclophosphorothionate (TBPS) were used as specific ligands of GABA, benzodiazepine, and picrotoxinin binding sites, respectively. Rats received a single oral dose of 30 mg/kg lindane and they were classified into two groups according to the absence or presence of convulsions. Vehicle-treated groups acted as controls. The effect of the xenobiotic on ligand binding was measured in different brain areas and nuclei 12 min or 5 h after its administration. Lindane induced a generalized decrease in [³⁵S]TBPS binding, which was present shortly after dosing. In addition, [³H]Flu binding was increased in lindane-treated animals, this modification also appearing shortly after administration but diminishing during the studied time. Finally, lindane induced a decrease in [³H]Mus binding, which became more evident over time. These modifications were observed both in the presence and in the absence of convulsions. However, an increase in [³H]-Mus binding was detected shortly after lindane-induced convulsions. The observed decrease in [³⁵S]TBPS binding is in agreement with the postulated action of lindane at the picrotoxinin binding site of the GABA_A receptor chloride channel. The effects observed on the binding of [³H]Flu and [³H]Mus may be secondary to the action of lindane as an allosteric antagonist of the GABA_A receptor.