Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.27 (thermolysin)
 1,894 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The number of free cysteines in each polypeptide of acetylcholine receptor from the electric organ of Torpedo californica has been assessed by alkylating the native protein with N-ethylmaleimide and iodoacetamide during homogenization of the tissue and alkylating the polypeptides with N-ethylmaleimide as they were unfolded in solutions of dodecyl sulfate. The cysteines unavailable for alkylation could be accounted for as specific cystines, connecting positions in the amino acid sequences of the individual polypeptides. Unreduced, alkylated polypeptides of acetylcholine receptor were digested with thermolysin or trypsin. Cystine-containing peptides in the chromatograms of the digests were identified electrochemically by the use of a dual gold/mercury electrode. Three thermolytic peptides and three tryptic peptides have been isolated from these digests and shown to contain intact cystines that were originally present in the native protein. The majority of these peptides contained an intact, intramolecular cystine connecting two cysteines in locations homologous to cysteines 128 and 142 from the alpha polypeptide. Each of these cystines from each of the polypeptides of acetylcholine receptor was isolated in at least one peptide, respectively. Each of these cystine-containing peptides also contained glucosamine. It can be concluded that each asparagine in the sequence Asn-Cys-Thr/Ser, which occurs in the respective, homologous location in every polypeptide, is glycosylated even though a cystine sits between the asparagine and the threonine or serine. In addition, the existence of the cystine connecting the adjacent cysteines, alpha 192 and alpha 193, in the alpha subunit of acetylcholine receptor [Kao, P. N., & Karlin, A. (1986) J. Biol. Chem. 261, 8085-8088] has been confirmed.
 ...
PMID:Assessment of the number of free cysteines and isolation and identification of cystine-containing peptides from acetylcholine receptor. 274 50

We have developed a new method to assess the binding site on alpha-bungarotoxin (alpha-BGT) for the acetylcholine receptor. It involves the covalent attachment of a palmitic acid chain to the toxin molecule, generating monopalmitoyl-alpha-bungarotoxin (PBGT) which is then immobilized on the surface of a lipid vesicle by a process of spontaneous insertion via the acyl chain into preformed unilamellar vesicles (approximately 800 A in diameter). PBGT itself is able to bind specifically to Triton X-100 solubilized acetylcholine receptors with an association constant, KA, of 5.56 X 10(6) M-1 which is approximately 20-fold lower in affinity than native alpha-BGT. Vesicle-associated PBGT binds to acetylcholine receptor enriched microsac membrane vesicles in aqueous buffer with a KA for both lipid and protein of 4.26 X 10(7) M-1. The putative site of acylation on the PBGT molecule is determined by extensive cleavage of a reduced and carboxymethylated PBGT with thermolysin. An acylated fragment is purified by hydrophobic column chromatography and identified by high-pressure liquid chromatography methods from the known primary sequence of the native toxin as a decapeptide including residues Thr47-Glu56 [C. Y. Lee convention used; see Mebs, D., Narita, K., Iwanaga, S., Samejuma, Y., & Lee, C. Y. (1971) Biochem. Biophys. Res. Commun. 44, 711-716]. Sequential hydrolysis of the fragment from the carboxy terminus with carboxypeptidase Y indicates that Lys51 is the sole site of acylation.(ABSTRACT TRUNCATED AT 250 WORDS)
 ...
PMID:alpha-Bungarotoxin immobilized and oriented on a lipid bilayer vesicle surface. 399 62

The transmembrane topology of acetylcholine receptor (AChR) delta subunit, synthesized in vitro and co-translationally integrated into dog pancreas rough microsomal membranes, was studied using limited proteolysis and domain-specific immunoprecipitation. Forty-four kilodaltons (kd) of the 65-kd delta subunit comprise a single fragment that is inaccessible to exhaustive proteolytic digestion from the cytoplasmic surface of the membrane by trypsin, chymotrypsin, thermolysin, and pronase. Previously, we have shown that this 44-kd "protected" fragment contains the amino terminus of the intact molecule and all of the core oligosaccharides (Anderson, D.J., P. Walter, and G. Blobel (1982) J. Cell Biol. 93: 501-506). Here we demonstrate that this domain can be further dissected into a 26-kd fragment, together with low molecular weight material, when the membranes are rendered permeable to trypsin by low concentrations of deoxycholate (Kreibich, G., P. Debey, and D. D. Sabatini (1973) J. Cell Biol. 58: 436-462). This 26-kd fragment contains all of the core oligosaccharides present on the intact subunit and therefore constitutes at least part, if not all, of the extracellular domain. The remaining low molecular weight material may derive from the membrane-embedded domain; our data imply that as much as 18 kd may be internal to the lipid bilayer. On the other hand, part of the cytoplasmic pole of AChR-delta can be recovered as a discrete, 12-kd fragment upon mild trypsinization of intact vesicles. We have used this 12-kd fragment to identify anti-AChR-delta monoclonal antibodies (mAbs) that react with the cytoplasmic domain of this subunit. Partial proteolytic fragmentation of the AChR in vitro translation products, in topologically well defined rough microsomes, may be used as a general assay to characterize the domain specificity of anti-AChR mAbs. For example, in the case of AChR-beta, we were able to identify two mAbs that recognize extracellular and cytoplasmic fragments, respectively.
 ...
PMID:Transmembrane orientation of an early biosynthetic form of acetylcholine receptor delta subunit determined by proteolytic dissection in conjunction with monoclonal antibodies. 619 54

Studies of the regulation of tyrosine phosphorylation at the neuromuscular junction during development and following denervation suggest that tyrosine phosphorylation of the nicotinic acetylcholine receptor is regulated by neuronal innervation of muscle. The finding that agrin, a neuronally derived extracellular matrix protein also induces tyrosine phosphorylation of the nicotinic receptor, suggests that nerve-induced tyrosine phosphorylation may be mediated by agrin. To study this further, we have examined the regulation of tyrosine phosphorylation of the nicotinic receptor by innervation in vitro using muscle-neuron cocultures. Innervation of chick myotubes by chick ciliary ganglia neurons induced tyrosine phosphorylation of the nicotinic receptor with the same subunit specificity seen with bath applied purified agrin. Both innervation and agrin-induced phosphorylation of the nicotinic receptor resulted in an increase in tyrosine and serine phosphorylation. In addition, thermolysin phosphopeptide maps of the subunits after innervation or agrin-treatment were identical. The similarity in the agrin- and nerve-induced phosphorylation of the acetylcholine receptor suggests that agrin mediates the nerve-induced phosphorylation during development in vivo and that phosphorylation of the acetylcholine receptor may play an important role in the development of the neuromuscular junction.
 ...
PMID:Comparison of innervation and agrin-induced tyrosine phosphorylation of the nicotinic acetylcholine receptor. 796 81

The location, with respect to the membrane, of Lys 165 in the folded beta polypeptide of native nicotinic acetylcholine receptor has been determined by site-directed immunochemistry. Sealed, right-side-out vesicles rich in acetylcholine receptor were modified with pyridoxal phosphate and sodium [3H]-borohydride. Saponin was added to one portion of the vesicles to make them permeable to the pyridoxal phosphate and sodium borohydride; the other portion was modified in the absence of saponin. Both samples were then exhaustively succinylated and digested with trypsin and thermolysin to produce the peptide LDAKGER, which contains Lys beta 165. The digests were passed over an immunoadsorbent specific for peptides with the sequence LDAXGER, where X represents any modified or unmodified amino acid, and specifically bound peptides were eluted with 0.1 M sodium phosphate, pH 2.5. The eluates were submitted to high-pressure liquid chromatography, and two peptides, N epsilon-phospho[3H]pyridoxalLDAKGER and N epsilon-succinylLDAKGER, modified at the epsilon amino group of lysine with pyridoxal phosphate and sodium [3H]-borohydride or succinic anhydride, respectively, were identified by comparison to standards. The relative specific radioactivity of N epsilon-phospho[3H]pyridoxalLDAKGER modified in the presence or absence of saponin, respectively, was 0.9 +/- 0.4. The incorporation of phospho[3H]pyridoxyl groups into Lys alpha 380, a residue located on the cytoplasmic surface of acetylcholine receptor, was also monitored. The relative specific radioactivity of the peptide that contains the modified Lys alpha 380, N epsilon-phospho[3H]pyridoxalGVKYIAE, increased 3.6-fold when the modification was performed in the presence of saponin. This result verifies that the vesicles used in these experiments were sealed and right-side-out. Because the incorporation of [3H]pyridoxyl groups into Lys beta 165 is the same in the presence or absence of saponin, Lys beta 165 must have been located on the outside surface of the sealed, right-side-out vesicles, and therefore on the extracytoplasmic surface of native acetylcholine receptor.
 ...
PMID:Extracytoplasmic disposition of lysine beta 165 of acetylcholine receptor. 817 83