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)

A new GTP-binding protein, which serves as a substrate for pertussis toxin, was prepared from porcine brain. The new G protein was separated from other GTP-binding proteins, Gi and Go, by an anion-exchange column chromatography. The mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the alpha subunit of the new G protein was between those of alpha subunits of Gi and Go. Evidence that the alpha subunit is not a proteolytic fragment of the alpha subunit is not a proteolytic fragment of the alpha subunit of Gi or Go was provided by experiments involving partial hydrolysis of these G proteins with thermolysin and their interaction with an antibody raised against the amino terminal peptide of the alpha subunit of Gi. In addition, the gamma subunit of the new G protein was indicated to be different from the gamma subunits of Gi and Go, because the latter were found to be phosphorylated by protein kinase C but the former was not. GTP-sensitive high affinity binding of muscarinic receptors with acetylcholine was observed when muscarinic receptors purified from porcine cerebrum were reconstituted in phospholipid vesicles with the new G protein as well as with Gi or Go. The proportion of the high affinity sites increased with the concentrations of the G proteins, the potency of the new G protein being similar to that of Gi but a little lower than that of Go. This GTP-sensitive high affinity binding was not observed when each G protein was pretreated with pertussis toxin and then reconstituted with muscarinic receptors. Acetylcholine accelerated the dissociation of [3H]GDP from the new G protein as well as from Gi and Go, which were reconstituted with muscarinic receptors. These results indicate that muscarinic receptors interact with at least the above three kinds of G proteins, in a pertussis toxin-sensitive manner.
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PMID:Cerebral muscarinic acetylcholine receptors interact with three kinds of GTP-binding proteins in a reconstitution system of purified components. 249 27

The removal of tightly bound GDP from the exchangeable nucleotide-binding site of tubulin has been performed with alkaline phosphatase under conditions which essentially retain the assembly properties of the protein. When microtubule protein is treated with alkaline phosphatase, nucleotide is selectively removed from tubulin dimer rather than from MAP (microtubule-associated protein)-containing oligomeric species. Tubulin devoid of E-site (the exchangeable nucleotide-binding site of the tubulin dimer) nucleotide shows enhanced proteolytic susceptibility of the beta-subunit to thermolysin and decreased protein stability, consistent with nucleotide removal causing changes in protein tertiary structure. Pyrophosphate ion (3 mM) is able to promote formation of normal microtubules in the complete absence of GTP by incubation at 37 degrees C either with nucleotide-depleted microtubule protein or with nucleotide-depleted tubulin dimer to which MAPs have been added. The resulting microtubules contain up to 80% of tubulin lacking E-site nucleotide. In addition to its effects on nucleation, pyrophosphate competes weakly with GDP bound at the E-site. It is deduced that binding of pyrophosphate at a vacant E-site can promote microtubule assembly. The minimum structural requirement for ligands to induce tubulin assembly apparently involves charge neutralization at the E-site by bidentate ligation, which stabilizes protein domains in a favourable orientation for promoting the supramolecular protein-protein interactions involved in microtubule formation.
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PMID:Tubulin-nucleotide interactions. Effects of removal of exchangeable guanine nucleotide on protein conformation and microtubule assembly. 303 51