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)

5'-p-Fluorosulfonylbenzoyladenosine (5'-FSBA) is a specific affinity label for the inhibitory NADH site of bovine liver glutamate dehydrogenase. Reaction of the enzyme with 5'-FSBA results in the loss of inhibition by high concentrations of NADH with covalent attachment of 0.53 sulfonylbenozyladenosine/subunit, i.e. modification of three subunits of the hexameric enzyme. Equal amounts of N epsilon-(4-carboxybenzenesulfonyl)lysine (Lys-(CBS] and O-(4-carboxybenzenesulfonyl)tyrosine (Tyr-(CBS] are found throughout the course of the reaction (Saradambal, K. V., Bednar, R. A., and Colman, R. F. (1981) J. Biol. Chem. 256, 11866-11872). Modified enzyme, prepared by incubating 2 mg/ml glutamate dehydrogenase with 0.3 mM 3H-labeled 5'-FSBA at pH 8 for 1 h, was carboxymethylated and digested with thermolysin. Two nucleosidyl peptides were isolated by a combination of chromatography on phenyl boronate-agarose, high-performance liquid chromatography in ammonium bicarbonate and high-performance liquid chromatography in trifluoroacetic acid. By comparison of the amino acid analysis and NH2-terminal residue of each isolated peptide with the known amino acid sequence of the enzyme, the peptides were identified as Leu-Gly-Arg-Lys(CBS) and Ile-Gly-His-Tyr(CBS)-Asp. These sequences correspond to residues 417-420 and 187-191, respectively. Lys-420 and Tyr-190 of glutamate dehydrogenase react with 5'-FSBA, and both are apparently located in the NADH inhibitory site.
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PMID:Identification of the lysine and tyrosine peptides labeled by 5'-p-fluorosulfonylbenzoyladenosine in the NADH inhibitory site of glutamate dehydrogenase. 643 99

A purification procedure for guanylate kinase from pig brain has been developed consisting of ammonium sulfate precipitation and heptane extraction of the crude extract, hydrophobic-interaction chromatography, affinity chromatography and chromatofocussing. From 1.75 kg pig brain, 1.2 mg enzyme was isolated with a yield of 18% and a purity of about 90%. For sequence determination, the protein was cleaved with trypsin, cyanogen bromide and endoproteinase Glu-C. Some of the isolated peptides were subcleaved with chymotrypsin, thermolysin or trifluoroacetic acid. The blocked N-terminus was analyzed by mass spectrometry and by amino acid analysis of a tryptic peptide, while the C-terminus was found in a tryptic and a chymotryptic peptide and confirmed by a carboxypeptidase Y digestion. The sequence contains 197 amino acids with a M(r) of 21,831, one tryptophan and one cysteine residue. It has been compared to those of the homologous enzymes of yeast and Escherichia coli, as well as to proteins from sequence data banks that show similarities. The sequence is discussed in the light of the known spatial structure of yeast guanylate kinase.
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PMID:Purification and sequence determination of guanylate kinase from pig brain. 809 61