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91.
92.
Socrates J. Tzartos Heleni V. Loutrari Fen Tang Anna Kokla Susan L. Walgrave Robert P. Milius† Bianca M. Conti-Tronconi 《Journal of neurochemistry》1990,54(1):51-61
Most anti-nicotinic acetylcholine receptor (AChR) antibodies in myasthenia gravis are directed against an immunodominant epitope or epitopes [main immunogenic region (MIR)] on the AChR alpha-subunit. Thirty-two synthetic peptides, corresponding to the complete Torpedo alpha-subunit sequence and to a segment of human muscle alpha-subunit, were used to map the epitopes for 11 monoclonal antibodies (mAbs) directed against the Torpedo and/or the human MIR and for a panel of anti-AChR mAbs directed against epitopes on the alpha-subunit other than the MIR. A main constituent loop of the MIR was localized within residues alpha 67-76. Residues 70 and 75, which are different in the Torpedo and human alpha-subunits, seem to be crucial in determining the binding profile for several mAbs whose binding to the peptides correlated very well with their binding pattern to native Torpedo and human AChRs. This strongly supports the identification of the peptide loop alpha 67-76 as the actual location of the MIR on the intact AChR molecule. Residues 75 and 76 were necessary for binding of some mAbs and irrelevant for others, in agreement with earlier suggestions that the MIR comprises overlapping epitopes. Structural predictions for the sequence segment alpha 67-76 indicate that this segment has a relatively high segmental mobility and a very strong turning potential centered around residues 68-71. The most stable structure predicted for this segment, in both the Torpedo and human alpha-subunits, is a hairpin loop, whose apex is a type I beta-turn and whose arms are beta-strands. This loop is highly hydrophilic, and its apex is negatively charged. All these structural properties have been proposed as characteristic of antibody binding sites. We also localized the epitopes for mAbs against non-MIR regions. Among these, the epitope for a monoclonal antibody (mAb 13) that noncompetitively inhibits channel function was localized within residues alpha 331-351. 相似文献
93.
Endogenous Benzodiazepine Receptor Ligands in Human and Animal Hepatic Encephalopathy 总被引:6,自引:1,他引:5
Marjut Olasmaa Jeffrey D. Rothstein Alessandro Guidotti Richard J. Weber† Steven M. Paul‡ Sydney Spector§ Maria L. Zeneroli Mario Baraldi Erminio Costa 《Journal of neurochemistry》1990,55(6):2015-2023
The role of endogenous benzodiazepine receptor ligands in the pathogenesis of hepatic encephalopathy was studied in humans and in rat models of hepatic encephalopathy. Endogenous benzodiazepine ligands were extracted from rat brain and human CSF by acid treatment and purification by HPLC. Detection and partial characterization of these endogenous benzodiazepine ligands were carried out using both radioreceptor binding assays and radioimmunoassays with anti-benzodiazepine antibodies. Four different benzodiazepine receptor ligands were identified in human and rat tissue, two of which may be diazepam and desmethyldiazepam, based on elution profiles and anti-benzo-diazepine antibody reactivity. Human CSF and serum from patients with hepatic encephalopathy contained approximately 10 times more endogenous benzodiazepine receptor ligand than CSF from controls or nonencephalopathic patients with liver disease. The levels of brain benzodiazepine receptor ligand compounds were also increased approximately 10-fold in rats suffering from fulminant hepatic failure, but not in rats with portacaval shunts, a model of chronic hepatic disease. The increased concentrations of these substances could be behaviorally significant and may contribute to the pathogenesis of hepatic encephalopathy. 相似文献
94.
Hydrolysis of bis(5''-nucleosidyl) polyphosphates by Escherichia coli 5''-nucleotidase. 总被引:1,自引:1,他引:0
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A Ruiz C Hurtado J Meireles Ribeiro A Sillero M A Günther Sillero 《Journal of bacteriology》1989,171(12):6703-6709
Two enzymatic activities that split diadenosine triphosphate have been reported in Escherichia coli: a specific Mg-dependent bis(5'-adenosyl) triphosphatase (EC 3.6.1.29) and the bis(5'-adenosyl) tetraphosphatase (EC 3.6.1.41). In addition to the activities of these two enzymes, a different enzyme activity that hydrolyzes dinucleoside polyphosphates is described. After purification and study of its molecular and kinetic properties, we concluded that it corresponded to the 5'-nucleotidase (EC 3.1.3.5) that has been described in E. coli. The enzyme was purified from sonic extracts and osmotic shock fluid. From sonic extracts, two isoforms were isolated by chromatography on ion-exchange Mono Q columns; they had a molecular mass of about 100 kilodaltons (kDa). From the osmotic shock fluid, a unique form of 52 kDa was recovered. Mild heating transformed the 100-kDa isoform to a 52-kDa form, with an increase in activity of about threefold. The existence of a 5'-nucleotidase inhibitor described previously, which associates with the enzyme and is not liberated in the osmotic shock fluid, may have been responsible for these results. The kinetic properties and substrate specificities of both forms (52 and 100 kDa) were almost identical. The enzyme, which is known to hydrolyze AMP and uridine-(5')-diphospho-(1)-alpha-D-glucose, but not adenosine-(5')-diphospho-(1)-alpha-D-glucose, was also able to split adenosine-(5')-diphospho-(5)-beta-D-ribose, ribose-5-phosphate, and dinucleoside polyphosphates [diadenosine 5',5'-P1,P2-diphosphate,diadenosine 5',5'-P1,P3-triphosphate, diadenosine 5',5'-P1,P4-tetraphosphate, and bis(5'-guanosyl) triphosphate]. The effects of divalent cations and pH on the rate of the reaction with different substrates were studied. 相似文献
95.
ATP hydrolysis by ischemic mitochondria 总被引:5,自引:0,他引:5
Cellular ATP levels are determined by the rates of ATP production and ATP hydrolysis. Both phenomena are affected by ischemia. Mitochondrial enzymes are damaged, inhibiting this organelle's ability to make ATP. Mitochondria are also uncoupled by ischemia and have the ability to hydrolyze ATP. We designed a series of experiments to determine whether decreased production or increased hydrolysis of ATP was the primary effect of mitochondrial damage. Rat hearts were subjected to 45 min of warm ischemia in order to induce irreversible cell damage. ATP or ADP was injected into cuvettes containing mitochondria isolated from normal myocardium or myocardium damaged by ischemia. Luciferin-luciferase, which fluoresces in the presence of ATP, was also added to the tubes as an indicator of ATP levels. Mixtures of uncoupled and coupled mitochondria were made and compared with the mitochondria damaged by ischemia. The results showed that mitochondria damaged by prolonged ischemia hydrolyze ATP more rapidly than normal mitochondria; however, normal mitochondria can easily compensate for increased ATP hydrolysis when in mixture with equal amounts of uncoupled mitochondria. These data suggests that the low cellular levels of ATP following irreversible ischemia are primarily due to decreased ATP synthesis and not to increased hydrolysis. 相似文献
96.
The aim of the present study was to investigate the existence of aging- and sex-related alterations in the permeability of the blood-brain barrier (BBB) in the rat, by calculating a unidirectional blood-to-brain transfer constant (Ki) for the circulating tracer [14C]-alpha-aminoisobutyric acid. We observed that: a) the permeability of the BBB significantly increased within the frontal and temporo-parietal cortex, hypothalamus and cerebellum in 28-30 week old rats, in comparison with younger animals; b) in several brain areas of female intact rats higher Ki values (even though not significantly different) were calculated at oestrus than at proestrus; c) in 1-week ovariectomized rats there was a marked increase of Ki values at the level of the frontal, temporo-parietal and occipital cortex, cerebellum and brain-stem. One can speculate that aging- and sex-related alterations in the permeability of the BBB reflect respectively changes in brain neurochemical system activity and in plasma steroid hormone levels. 相似文献
97.
gamma-Aminobutyric acidB (GABAB) receptor recognition sites that inhibit cyclic AMP formation, open potassium channels, and close calcium channels are coupled to these effector systems by guanine nucleotide binding proteins (G proteins). These G proteins are ADP-ribosylated by islet-activating protein (IAP), also known as pertussis toxin. This process prevents receptor coupling to these G proteins. In slices of cerebral cortex and hippocampus from rat, stimulation of GABAB receptors with baclofen, a receptor agonist, also potentiates the accumulation of cyclic AMP stimulated by beta-adrenergic agonists. It was unknown whether those GABAB receptors that potentiate the beta-adrenergic response were also sensitive to IAP. IAP was injected intracerebroventricularly into rats to ADP-ribosylate IAP-sensitive G proteins. Four days after the IAP injection, 38% and 52% of these G proteins from cerebral cortex and hippocampus, respectively, were ADP-ribosylated by the IAP injection. In slices of both structures prepared from IAP-treated rats, the GABAB receptor-mediated potentiation of the beta-adrenergic receptor response was attenuated. Thus, many GABAB receptor-mediated responses are coupled to IAP-sensitive G proteins. 相似文献
98.
E. Slobodyansky A. Guidotti C. Wambebe A. Berkovich E. Costa 《Journal of neurochemistry》1989,53(4):1276-1284
This report describes the purification and characterization from rat brain of triakontatetraneuropeptide (TTN, DBI 17-50), a major biologically active processing product of diazepam binding inhibitor (DBI). Brain TTN was purified by immunoaffinity chromatography with polyclonal octadecaneuropeptide, DBI 33-50) antibodies coupled to CNBr-Sepharose 4B followed by two reverse-phase HPLC steps. The amino acid sequence of the purified peptide is: Thr-Gln-Pro-Thr-Asp-Glu-Glu-Met-Leu-Phe-Ile-Tyr-Ser-His-Phe-Lys-Gln-Ala-Thr-Val - Gly-Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly-Leu-Leu-Asp-Leu-Lys. Synthetic TTN injected intracerebroventricularly into rats induces a proconflict activity (IC50 0.8 nmol/rat) that is prevented by the specific "peripheral" benzodiazepine (BZ) receptor antagonist isoquinoline carboxamide, PK 11195, but not by the "central" BZ receptor antagonist imidazobenzodiazepine, flumazenil. TTN displaces [3H]Ro 5-4864 from synaptic membranes of olfactory bulb with a Ki of approximately 5 microM. TTN also enhances picrotoxinin inhibition of gamma-aminobutyric acid (GABA)-stimulated [3H]flunitrazepam binding. These data suggest that TTN, a natural DBI processing product acting at "Ro 5-4864 preferring" BZ binding site subtypes, might function as a putative neuromodulator of specific GABAA receptor-mediated effects. 相似文献
99.
Opioid Receptors of Neuroblastoma Cells Are in Two Domains of the Plasma Membrane that Differ in Content of G Proteins 总被引:1,自引:0,他引:1
Opioid receptors of NG 108-15 cell membranes are distributed in two membrane fractions sedimenting at 20,000 g (P2) and 200,000 g(P3). The number of receptors is identical in P2 and P3, but in P2 all sites are present in one high-affinity state (2 nM), whereas in P3 60% of these receptors display lower affinity (150 nM). Upon addition of GTP or pretreatment with pertussis toxin, 80% of the sites exist in low affinity in both P2 and P3. Therefore, the effect of GTP and pertussis toxin on agonist binding appears to be smaller in P2 than in P3. In contrast, sodium inhibits agonist binding in P2 and P3 to the same extent and with identical potency. Opioid-mediated stimulation of GTPase is much greater in P2 than in P3, whereas inhibition of adenylate cyclase does not differ in the two fractions. Using site-specific antibodies and pertussis toxin-catalyzed ADP-ribosylation, we found that the amount of G proteins in P3 is only 30-50% of that in P2. Treatment of intact cells with the hydrophilic protein-modifying agent sulfosuccinimido-biotin results in biotinylation of proteins from both fractions and in a similar reduction of opioid binding in P2 and P3. Likewise, exposure of intact cells to the alkylating opioid antagonist, chlornaltrexamine, produces identical degrees of receptor inactivation in P2 and P3. The rate of in vivo pertussis toxin-mediated modification of G proteins is not different in the two fractions.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
100.
Chronic Exposure of NG 108-15 Cells to Opiate Agonists Does Not Alter the Amount of the Guanine Nucleotide-Binding Proteins Gi and GO 总被引:1,自引:1,他引:0
We have characterized the pertussis toxin substrate in NG 108-15 cell membranes using site-specific antisera and ADP-ribosylation. Cell membranes contain two pertussis toxin-sensitive guanine nucleotide-binding protein alpha-subunits (G alpha) whose Rf values in gel electrophoresis coincide with those of G alpha o and G alpha i2. The total quantity of Gi and Go immunoreactivity amounted to 24.3 +/- 2.8 pmol/mg, whereas only 1.5 +/- 0.2 pmol/mg are capable of undergoing ADP-ribosylation catalyzed by pertussis toxin. Pretreatment of cells with the agonist [D-Ala2,D-Leu2]-enkephalin (DADLE) for 24 h and DADLE or morphine for 72 h did not alter the incorporation of ADP-ribose or the immunoreactive amount of Gi and Go subunits. However, pretreatment for 72 h with naloxone increased the incorporation of ADP-ribose without an apparent change in affinity or in the immunochemically determined protein levels of Gi and Go. This indicates that the process of down-regulation and desensitization of the delta-opioid receptor neither requires quantitative alterations in the levels of Gi and Go nor changes in the degree of coupling among their subunits. In contrast, chronic exposure to antagonists seems to alter the degree of precoupling between alpha- and beta-subunits of Gi and/or Go. 相似文献