Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.5.1.52 (PNGase F)
 1,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Brain membrane preparations of different vertebrates were photoaffinity labeled with [3H]flunitrazepam and subsequently deglycosylated with endoglycosidase F and peptide N-glycopeptidase. SDS-polyacrylamide gel electrophoresis followed by fluorography revealed that each benzodiazepine-binding protein is deglycosylated in two steps, indicating that each protein has two glycosylation sites. Species variation of the apparent molecular masses of the benzodiazepine-binding proteins and regional heterogeneity in avians persist after deglycosylation. These results indicate that the alpha-subunit(s) of the GABA/benzodiazepine receptor has undergone electrophoretically detectable changes in its amino acid composition during vertebrate evolution. The existence of at least two different alpha-subunits in avians is further substantiated.
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PMID:Persistence of species variation and regional heterogeneity of the apparent molecular masses of benzodiazepine-binding proteins after deglycosylation. 284 88

Various alpha and beta 3 subunit-specific antibodies were used to characterize some of the heterogeneous ligand-binding properties of gamma-aminobutyric acidA receptors. Polyclonal antibodies that were raised against the cytoplasmic amino acid sequence (380-392) of the rat beta 3 subunit recognized a single polypeptide of molecular mass of 58 kDa in Western blots with Ro7-1986 affinity-purified GABAA receptors from the rat brain, and a doublet of molecular mass of 54 kDa and 56 kDa in receptors from the bovine cortex, hippocampus, and cerebellum. Deglycosylation of purified receptors from the bovine cortex with N-glycanase resulted in a single band immunostained at molecular mass of 52 kDa. These anti-beta 3 subunit antibodies immunoprecipitated approximately 50% of [3H]flunitrazepam binding sites from soluble extracts of bovine cortex, whereas beta cyto antibodies, which probably recognize all beta subunit isoforms, precipitated almost 100% of benzodiazepine binding sites. These results indicate heterogeneity of GABAA receptor subunit composition with respect to the nature of beta subunits. The GABA analogue 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), like GABA, shows heterogeneous binding affinities in brain homogenates. The higher affinity sites were previously suggested as corresponding to a 58-kDa polypeptide in rat that is photoaffinity-labeled with [3H]muscimol, a band that comigrates with the one stained by anti-beta 3 antibodies. However, THIP affinity was not significantly different between receptors containing beta 3 subunits and those lacking beta 3, as demonstrated by similar affinities in receptors that ere immunoprecipitated by anti-beta 3 antibodies and those that were not. Also, THIP displaced [3H]muscimol binding with similar multiple affinities across brain regions where different beta subunit variants are expressed with varying abundances. These observations suggest that the property of high affinity THIP binding cannot be explained solely by beta 3 subunits. The coupling efficiency between GABA and benzodiazepine binding sites appears to be determined by the nature of alpha subunits rather than of beta subunits. GABA enhanced [3H]flunitrazepam binding with different efficacies and potencies in receptors immunoprecipitated by anti-alpha 1, -alpha 2, and -alpha 3 subunit antibodies. In contrast, beta 3 subunit-enriched and disenriched receptors did not differ in this property. [3H]Flunitrazepam binding in GABAA receptors containing alpha 2 and alpha 3 subunits was enhanced to a significantly greater extent than were those with alpha 1. In addition, receptors containing alpha 1 and alpha 3 subunits had higher potencies of enhancement than did those with alpha 2 subunits.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Pharmacological subtypes of the gamma-aminobutyric acidA receptors defined by a gamma-aminobutyric acid analogue 4,5,6,7-tetrahydroisoxazolo[5,4-c] pyridin-3-ol and allosteric coupling: characterization using subunit-specific antibodies. 747 92

The alpha subunit of the gamma-aminobutyric acid type A (GABA(A)) receptor is known to be photoaffinity labeled by the classical benzodiazepine agonist, [3H]flunitrazepam. To identify the specific site for [3H]flunitrazepam photoincorporation in the receptor subunit, we have subjected photoaffinity labeled GABA(A) receptors from bovine cerebral cortex to specific cleavage with cyanogen bromide and purified the resulting photolabeled peptides by immunoprecipitation with an anti-flunitrazepam polyclonal serum. A major photolabeled peptide component from reversed-phase high performance liquid chromatography of the immunopurified peptides was resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The radioactivity profile indicated that the [3H]flunitrazepam photoaffinity label is covalently associated with a 5.4-kDa peptide. This peptide is glycosylated because treatment with the enzyme, peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase, reduced the molecular mass of the peptide to 3.2 kDa. Direct sequencing of the photolabeled peptide by automated Edman degradation showed that the radioactivity is released in the twelfth cycle. Based on the molecular mass of the peptides that can be generated by cyanogen bromide cleavage of the GABA(A) receptor alpha subunit and the potential sites for asparagine-linked glycosylation, the pattern of release of radioactivity during Edman degradation of the photolabeled peptide was mapped to the known amino acid sequence of the receptor subunit. The major site of photoincorporation by [3H]flunitrazepam on the GABA(A) receptor is shown to be alpha subunit residue His102 (numbering based on bovine alpha 1 sequence).
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PMID:The major site of photoaffinity labeling of the gamma-aminobutyric acid type A receptor by [3H]flunitrazepam is histidine 102 of the alpha subunit. 862 79