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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The structural features of the active site of human monoamine oxidase B (MAO-B) were investigated by affinity labeling and site-directed mutagenesis. The pseudosubstrate inhibitor N-[2-aminoethyl]-5-chloro-2-pyridine carboxamide HCl (lazabemide) can be irreversibly linked to MAO-B by reduction of the enzyme-inhibitor complex with NaBH(3)CN. Analysis of the flavin spectrum of [(3)H]lazabemide-labeled human MAO-B indicated that insertion of the inhibitor did not occur into the isoalloxazine ring of FAD. After trypsin digestion and HPLC peptide mapping of the radiolabeled enzyme, two labeled peptides were observed. Sequence analysis showed that both peptides started at Val371 of human MAO-B. These results indicate that [(3)H]lazabemide is incorporated into the MAO-B peptide stretch containing the FAD-modified Cys397. The function of putative active-site residues contained in this region was investigated by site-directed mutagenesis and expression of the mutant proteins in HEK-293 cells. Substitution of His382 of MAO-B with an Arg greatly reduced the enzymic activity, suggesting that this residue may represent a nucleophile relevant for the MAO-B catalytic mechanism. Whereas it has been shown that mutation of Cys389 with a Ser residue does not markedly affect the activity of the enzyme [Wu, H.-F., Chen, K. and Shih, J.C. (1993) Mol. Pharmacol. 43, 888-893] the mutant carrying an Ala at this position was virtually inactive. Conversely, substitution of Lys386 (to Met) and Ser394 (to Ala) did not markedly modify the kinetic properties of the enzyme. We also report that mutation of MAO-B Thr158 (to Ala) resulted in a dramatic loss of enzymic activity.
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PMID:Investigation on the structure of the active site of monoamine oxidase-B by affinity labeling with the selective inhibitor lazabemide and by site-directed mutagenesis. 866 24

Through expression of the cloned mouse (mSlo) or human (hSlo) large-conductance (BK) Ca(2+)-activated K+ channel in Xenopus laevis oocytes and HEK 293 cells, we characterized the effects of reported blockers and openers of BK channels to initiate the study of the molecular determinants of BK channel modulation. In oocytes, iberiotoxin and charybdotoxin, peptidyl scorpion toxins, were both equally effective blockers of BK current, although iberiotoxin was significantly more potent than charybdotoxin. The structurally related peptide kaliotoxin was not a potent blocker of BK current. Paxilline, a fungal tremorgenic alkaloid, was an effective but complex blocker of BK current. Tetrandrine, a putative blocker of type II BK channels, and ketamine were relatively ineffective. The putative BK openers NS004 and NS1619, phloretin, niflumic acid, flufenamic acid, and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) increased BK current in oocytes at microM concentrations; many of these produced biphasic concentration-response relationships. Coapplication of representative blockers and openers revealed several patterns of interaction, including competitive and noncompetitive antagonism. NS1619, niflumic acid, and phloretin were tested by using excised inside-out membrane patches from HEK 293 cells and were found to increase the activity of hSlo BK channels and produce a leftward shift in the G/Gmax-versus-voltage relationship of these channels. These results represent the first comprehensive examination of the molecular pharmacology of BK channels.
Mol Pharmacol 1996 Jul
PMID:Effects of channel modulators on cloned large-conductance calcium-activated potassium channels. 870 Jan 14

The introduction of D1A dopamine receptors and mu-opioid receptors into HEK 293 cells that were also transiently transfected with adenylyl cyclase cDNA imparted to dopamine and to mu-opioid receptor agonists the ability to modulate the activity of the expressed adenylyl cyclase. Dopamine added to cells expressing D1A receptors and type V adenylyl cyclase significantly stimulated type V enzyme activity. The concomitant addition of morphine produced a dose-dependent inhibition of dopamine-stimulated type V adenylyl cyclase activity. On the other hand, if the HEK 293 cells were transfected with cDNA for type VII adenylyl cyclase instead of the type V isoform, morphine stimulated this adenylyl cyclase activity beyond the stimulation produced by dopamine. Both the inhibitory and stimulatory effects of morphine were blocked by naloxone or pretreatment of the transfected HEK 293 cells with pertussis toxin. When expressed in the HEK 293 cells, the alpha subunit of transducin, which is considered to be the putative scavenger of the beta gamma subunits of G proteins, suppressed the stimulatory effect of morphine on type VII adenylyl cyclase. We also expressed the adenylyl cyclases in cells that were transfected with D1A receptor and G beta 1 and G gamma 2 cDNAs. Dopamine was more efficacious in stimulating type VII adenylyl cyclase activity in cells concomitantly transfected with the beta gamma subunit cDNAs than in cells not transfected with these G protein subunits. Transfection with beta gamma subunit cDNAs did not affect dopamine stimulation of type V adenylyl cyclase activity, and morphine-induced inhibition of type V adenylyl cyclase activity was still evident in cells cotransfected with the alpha subunit of transducin. These data support the contention that the effects on type VII adenylyl cyclase activity mediated through the G1/G(o) proteins may depend on the actions of the beta gamma subunits. The same is not the case for type V adenylyl cyclase. Our data demonstrate that both qualitative and quantitative responses to mu-opioid receptor stimulation depend on the isoform of adenylyl cyclase expressed in neurons or other cells of the body.
Mol Pharmacol 1996 Jul
PMID:mu-Opioid receptors inhibit dopamine-stimulated activity of type V adenylyl cyclase but enhance dopamine-stimulated activity of type VII adenylyl cyclase. 870 Jan 17

Excessive activation of glutamate receptors is thought to play a critical role in neuronal excitotoxicity. To compare the cytotoxic potential of different glutamate receptor subtypes and correlate receptor biophysical properties with cytotoxicity, we have expressed recombinant receptors in human embryonic kidney 293 (HEK-293) cells. Survival of transfected cells was analyzed under conditions of defined agonist concentration and exposure time. For HEK-293 cells transfected with N-methyl-D-aspartate (NMDA) receptors, the EC50 for NMDA-induced cytotoxicity was 300 microM. Experiments using ion substitution, or cells expressing mutant NMDA receptors with low calcium permeability, suggested that both calcium and sodium influx through NMDA receptors contributed to cytotoxicity. In contrast, cytotoxicity was not observed in cells transfected with calcium permeable alpha-amino 3-hydroxy-5-methyl-4-isoxazole propionate- or kainate-type glutamate receptors even at saturating agonist concentrations, unless inhibitors of agonist-dependent desensitization were included. These results directly demonstrate that calcium permeability and desensitization kinetics play important roles in determining the excitotoxic potential of different glutamate receptor subtypes.
Mol Cell Neurosci 1996 Feb
PMID:Glutamate receptor ion channel properties predict vulnerability to cytotoxicity in a transfected nonneuronal cell line. 873 79

The human neuropeptide Y Y1 receptor cDNA was transfected into human embryonic kidney 293 (HEK 293) cells and used to determine the selectivity of newly developed Y1 and Y2 radioligands in a model which expresses a single NPY receptor subtype. The Y1 receptor probe, [125I][Leu31,Pro34]PYY, binds with high afinity (KD of 0.4-0.6 nM) to Y1-transfected HEK 293 cells whereas the Y2 radioligand, [125I]PPY3-36 failed to demonstrate any significant labelling. Only non-selective (PYY) or selective Y1 receptor agonists behaved as potent competitors for [125I][Leu31,Pro34]PYY binding in transfected cells. Additionally, the efficacy of the transfection method used was evaluated at both the transcriptional and translational levels. In situ hybridization revealed the heterogeneous distribution of the NPY Y1 receptor mRNA expressed in transfected HEK 293 cells. Similarly, the levels of NPY Y1 binding sites per transfected cell varied as shown using [125I][Leu31,Pro34]PYY receptor autoradiography. Taken together, these results demonstrate that HEK 293 cells transfected with the NPY Y1 receptor cDNA expressed both the related receptor mRNA and protein albeit at different levels depending upon each transfected cell. Additionally, these data further establish the selectivity of the newly developed Y1 and Y2 radioligands.
Brain Res Mol Brain Res 1995 Dec 28
PMID:Expression of the neuropeptide Y Y1 receptor in human embryonic kidney 293 cells: ligand binding characteristics, in situ hybridization and receptor autoradiography. 875 Aug 33

The physiological and pharmacological properties of GABAA receptors have been studied extensively after the expression of subunits in non-neural cells. Many of these studies have used the human embryonic kidney cell line HEK 293. We examined the properties of subunits that result in the expression of low levels of functional receptors and found that the properties of the gamma-aminobutyric acid (GABA)-elicited responses in transfected HEK 293 cells differ from expectations based on previous work and are consistent with the idea that the expressed receptors do not necessarily contain the expected subunits. In particular, expression of a mutated beta 2 subunit [beta 2(Y205S)] in combination with alpha 1 and gamma 2L results in cells that have large responses to pentobarbital (as expected) but also show appreciable responses to GABA (contrary to expectation). Furthermore, transfection of HEK 293 cells with alpha 1 plus gamma 2L subunits results in responses to GABA that are potentiated by the drug loreclezole, suggesting that a subunit resembling the beta 2 or beta 3 subunit had assembled with the alpha 1 gamma 2L subunits. In addition, some nontransfected HEK 293 cells respond to applications of GABA, and transfection of cells with alpha 1, beta 1, or gamma 2L subunits alone can result in the expression of GABA-elicited currents. In comparison, when QT6 quail fibroblasts are used as the expression system, no responses were seen in untransfected cells or in cells transfected with alpha 1, beta 1, or gamma 2L subunits alone or alpha 1 gamma 2L subunits. Furthermore, no response to GABA was seen in QT6 cells transfected with alpha 1 beta 2(Y205S) gamma 2L subunits, although cells gave strong responses to pentobarbital. These observations indicate that caution must be taken in interpreting the results of studies of the properties of GABAA receptors expressed in HEK 293 cells if the exogenous subunits result in the expression of low levels of functional GABAA receptors.
Mol Pharmacol 1996 Oct
PMID:Endogenous subunits can cause ambiguities in the pharmacology of exogenous gamma-aminobutyric acidA receptors expressed in human embryonic kidney 293 cells. 886 39

Mutations in genes encoding the epsilon, delta, beta and alpha subunits of the end plate acetylcholine (ACh) receptor (AChR) are described and functionally characterized in three slow-channel congenital myasthenic syndrome patients. All three had prolonged end plate currents and AChR channel opening episodes and an end plate myopathy with loss of AChR from degenerating junctional folds. Genetic analysis revealed heterozygous mutations: epsilon L269F and delta Q267E in Patient 1, beta V266M in Patient 2, and alpha N217K in Patient 3 that were not detected in 100 normal controls. Patients 1 and 2 have no similarly affected relatives; in Patient 3, the mutation cosegregates with the disease in three generations. epsilon L269F, delta Q267E and beta V266M occur in the second and alpha N217K in the first transmembrane domain of AChR subunits; all have been postulated to contribute to the lining of the upper half of the channel lumen and all but delta Q267E are positioned toward the channel lumen, and introduce an enlarged side chain. Expression studies in HEK cells indicate that all of the mutations express normal amounts of AChR. epsilon L269F, beta V266M, and alpha N217K slow the rate of channel closure in the presence of ACh and increase apparent affinity for ACh; epsilon L269F and alpha N217K enhance desensitization, and epsilon L269F and beta V266M cause pathologic channel openings in the absence of ACh, rendering the channel leaky, delta Q267E has none of these effects and is therefore a rare polymorphism or a benign mutation. The end plate myopathy stems from cationic overloading of the postsynaptic region. The safety margin of neuromuscular transmission is compromised by AChR loss from the junctional folds and by a depolarization block owing to temporal summation of prolonged end plate potentials at physiologic rates of stimulation.
Hum Mol Genet 1996 Sep
PMID:New mutations in acetylcholine receptor subunit genes reveal heterogeneity in the slow-channel congenital myasthenic syndrome. 887 60

A novel Cre-lox system was used to construct an adenovirus encoding kappa-bungarotoxin (kappa-Bgt), modified to be secreted by attachment of a bovine prolactin signal sequence at the N-terminus of the toxin. Western blot of medium from HEK-293 cells infected with the virus demonstrated that recombinant kappa-Bgt (R-kappa-Bgt) was secreted. The biological activity of the secreted R-kappa-Bgt was investigated in Xenopus oocytes that expressed neuronal nicotinic acetylcholine receptor (nAChR) subtypes alpha3beta2 and alpha2beta2. The recombinant toxin inhibited the response of alpha3beta2 type AChRs to ACh, but did not inhibit the response of alpha2beta2 type AChRs. These data demonstrated that the recombinant adenovirus directs the secretion of biologically active kappa-Bgt from a mammalian cell line. Because adenovirus can be used to infect post-mitotic cells, recombinant adenoviruses encoding biologically active peptides may be of use as delivery vehicles for in vivo experiments where repeated application of the purified peptide is unfeasible.
Brain Res Mol Brain Res 1997 Feb
PMID:A recombinant adenovirus that directs secretion of biologically active kappa-bungarotoxin from mammalian cells. 903 Jul 9

GH release is thought to occur under the reciprocal regulation of two hypothalamic peptides, GH releasing hormone (GHRH) and somatostatin, via their engagement with specific cell surface receptors on the anterior pituitary somatotroph. In addition, GH-releasing peptides, such as GHRP-6 and the nonpeptide mimetics, L-692,429 and MK-0677, stimulate GH release through their activation of a distinct receptor, the GH secretagogue receptor (GHS-R). The recent cloning of the GHS-R from human and swine pituitary gland identifies yet a third G protein-coupled receptor (GPC-R) involved in the control of GH release and further supports the existence of an undiscovered hormone that may activate this receptor. Using the human GHS-R as a probe, we report the isolation of a rat pituitary GHS-R cDNA derived from an unspliced, precursor mRNA. The rat cDNA encodes a protein of 364 amino acids containing seven transmembrane domains (7-TM) with >90% sequence identity to both the human and swine GHS-Rs. A single intron of approximately 2 kb divides the open reading frame into two exons encoding TM 1-5 and TM 6-7, thus placing the GHS-R into the intron-containing class of GPC-Rs. The intron maps to the site of sequence divergence between the human and swine type 1a and 1b GHS-R mRNAs. In addition, determination of the nucleotide sequence for the human GHS-R gene confirmed the position of an intron in the human GHS-R gene at this position. A full-length contiguous cDNA from rat hypothalamus was isolated and shown to be identical in its nucleotide and deduced amino acid sequence to the rat pituitary GHS-R. The cloned rat GHS-R binds [35S]MK-0677 with high affinity [dissociation constant (K(D)) = 0.7 nM] and is functionally active when expressed in HEK-293 cells. Expression of the rat GHS-R was observed specifically in the pituitary and hypothalamus when compared with control tissues.
Mol Endocrinol 1997 Apr
PMID:Molecular analysis of rat pituitary and hypothalamic growth hormone secretagogue receptors. 909 93

Rat recombinant (rec) luteinizing hormone (LH) was produced in Chinese hamster ovary (CHO) cells, to enable studies on LH physiology in this species with homologous hormone. The synthesized hormone was purified, and characterized physico-chemically and biologically in comparison with highly purified preparations of rat pituitary (pit) LH (NIDDK-rLH-I-7 and I-9) and to highly purified urinary (NIH, CR-121) and rec forms of human chorionic gonadotropin (hCG). The 33 kD molecular mass of rat recLH, as determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, was comparable with the 32 kD size of pitLH. In chromatofocusing, the isoforms of rat recLH distributed in the pI range 6.5-7.8, similar to rat pitLH. In receptor binding assays using rat testicular membranes, and physiologic salt concentration, rat recLH displayed a 5-10-fold higher affinity than rat pitLH, but about 100-fold lower affinity than hCG. In contrast, in low salt concentrations the affinities of rat recLH and rechCG to rat LH receptor were rather similar. The differences in potency in the mouse Leydig cell in vitro bioassay were in agreement with the receptor binding data at physiologic salt concentration. Neither rat recLH nor pitLH stimulated cAMP production or bound specifically to HEK 293 cells expressing the rec human LH receptor. When injected subcutaneously on four consecutive days to male rats (8.4-33.7 microg/rat/day) rat recLH did not induce seminal vesicle growth in comparison with a significant effect of human menopausal gonadotropin (hMG; 12.5-50 IU/rat/day). In contrast, ovulation was induced in 5/6 and 6/6 female rats following single injections of 3.75 and 7.5 microg of rat recLH, respectively, after pretreatment with 10 microg/kg of a GnRH-antagonist (Org 30850). In conclusion, rat recLH displays clearly lower in vivo and in vitro bioactivity than hCG. Nevertheless, it binds effectively to the rat LH receptor (with affinity dependent on salt concentration) and is bioactive in the mouse Leydig cell bioassay. This newly synthesized recombinant hormone provides a useful tool for further studies on the physiology of LH action in the rat, the most common animal model in reproduction research.
Mol Cell Endocrinol 1997 Apr 04
PMID:Recombinant rat luteinizing hormone; production by Chinese hamster ovary cells, purification and functional characterization. 914 75


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