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

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.
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PMID:Extracytoplasmic disposition of lysine beta 165 of acetylcholine receptor. 817 83

Purified phosphofructokinase 1 from baker's yeast (Saccharomyces cerevisiae) was subjected to proteolysis by thermolysin, endoproteinase lys-C, trypsin and chymotrypsin under defined solvent conditions. In the absence of substrates and allosteric effectors, the catalytic activity of phosphofructokinase rapidly disappeared in the presence of each proteolytic enzyme. The presence of a saturating concentration of ATP protected phosphofructokinase activity from proteolytic inactivation while the collective presence of fructose 6-phosphate, AMP and fructose 2,6-bisphosphate provided transient activation during proteolysis. Changes in the quaternary structure of phosphofructokinase resulting from proteolysis were estimated by high performance size exclusion chromatography while changes in the primary sequence of the individual alpha and beta polypeptide chains were estimated by polyacrylamide-gel electrophoresis in sodium dodecylsulfate. The site(s) of proteolytic cleavage were identified by N-terminal sequence analysis of resolved electrophoretic components. The presence of ATP protects phosphofructokinase from thermolysin proteolysis, while the collective presence of fructose 6-phosphate, AMP and fructose 2,6-bisphosphate restricts proteolysis to one site in each polypeptide chain involving the peptide bonds preceding Leu199 in the alpha chain and Leu192 in the beta chain. The truncated phosphofructokinase retains its octameric structure. The presence of ATP largely restricts endoproteinase lys-C proteolysis to a single site in the alpha chain involving the peptide bond preceding Val914. This cleavage results in the dissociation of the octameric form of phosphofructokinase into two tetramers. The presence of ATP restricts both trypsin and chymotrypsin proteolysis to the N-terminal and C-terminal regions described above, resulting in the preferential stabilization of the tetrameric form of phosphofructokinase. It would appear that the first 200 and last 80 residues which are unique to the sequence of the yeast phosphofructokinase are not directly involved in catalysis or its allosteric regulation. However, the last 80 residues of the alpha polypeptide chain do appear to stabilize an octameric structure which is unique to yeast phosphofructokinase.
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PMID:Limited proteolysis of yeast phosphofructokinase. Sequence locations of cleavage sites created by the actions of different proteinases. 822 96