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 mitochondrial energy-linked nicotinamide nucleotide transhydrogenase is a homodimer of monomer Mr = 109,228. Hydropathy analysis of its cDNA-deduced amino acid sequence (1043 residues) has indicated that the molecule is composed of 3 domains: a 430-residue-long hydrophilic N-terminal domain which binds NAD(H), a 200-residue-long hydrophilic C-terminal domain which binds NADP(H), and a 400-residue-long hydrophobic central domain which appears to be made up mainly of about 14 hydrophobic clusters of approximately 20 residues each. In this study, antibodies were raised to the hydrophilic N- and C-terminal domains cleaved from the isolated transhydrogenase by proteolytic digestion, and to a synthetic, hydrophilic pentadecapeptide, which corresponded to position 540-554 within the central hydrophobic domain. Immunochemical experiments with mitoplasts (mitochondria denuded of outer membrane) and submitochondrial particles (inside-out inner membrane vesicles) as sources of antigens showed that essentially the entire N- and C-terminal hydrophilic domains of the transhydrogenase, as well as epitopes from the central pentadecapeptide, protrude from the inner membrane into the mitochondrial matrix, where the N- and C-terminal domains would be expected to come together to form the enzyme's catalytic site. Treatment of mitoplasts with several proteolytic enzymes indicated that large protease-sensitive masses of the transhydrogenase are not exposed on the cytosolic side of the inner membrane, which agreed with the exception that the central highly hydrophobic domain of the molecule should be largely membrane-intercalated. Trypsin, alpha-chymotrypsin, and papain had little or no effect on the mitoplast-embedded transhydrogenase. Proteinase K, subtilisin (Nagarse), thermolysin, and pronase E each split the mitoplast-embedded enzyme into two fragments only, a fragment of approximately 70 kDa containing the N-terminal hydrophilic domain, and one of approximately 40 kDa bearing the C-terminal hydrophilic domain. The cleavage site of proteinase K was determined to be A690 -A691, which is located in a small hydrophilic segment within the central hydrophobic domain. This protease-sensitive loop appears to be exposed on the cytosolic side of the inner membrane. The proteinase K-nicked enzyme containing two peptides of 71 and 39 kDa was isolated from mitoplasts and shown to have high transhydrogenase activity.
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PMID:Mitochondrial energy-linked nicotinamide nucleotide transhydrogenase. Membrane topography of the bovine enzyme. 200 10

Light-mediated conformational changes in highly purified 124-kDa phytochrome preparations from etiolated oat seedlings have been identified by steric exclusion high performance liquid chromatography and limited proteolytic studies. Steric exclusion high performance liquid chromatography studies of oat and rye phytochromes show photoreversible changes in retention times, with the red absorbing form of phytochrome (Pr form) eluting later than the far red absorbing form of phytochrome produced by saturating red light illumination of Pr (Pfr form) in a variety of different mobile phase buffers. Molecular mass calibration with globular protein standards in Tris-glycol buffers provides estimates of 318-349 and 363-366 kDa for the molecular sizes of the Pr and Pfr forms, respectively. These analyses support earlier studies that phytochrome is a nonglobular homodimer of 124-kDa subunits in vitro. Limited proteolytic dissection of phytochrome in nondenaturing buffers with seven different endoproteases provides evidence for two "operational" domains within the 124-kDa subunit with molecular mass values of 69-72 and 52-55 kDa. The larger 69-72-kDa domain contains the site for the chromophore attachment as shown by gel electrophoresis derived enzyme-linked immunosorbent assay utilizing site-directed rabbit antiserum to a synthetic undecapeptide which is homologous with the chromophore binding site on oat phytochrome. This chromophore domain exhibits a compact structure, resistant to further proteolysis except near its N terminus. By contrast, the 52-55-kDa nonchromophore domain contains multiple sites for further proteolytic cleavage as revealed by rapid cleavage to smaller polypeptide fragments. Detailed kinetic analyses of the limited proteolytic cleavage of phytochrome with four endoproteases, subtilisin BPN', thermolysin, trypsin, and clostripain, has mapped specific regions within the 124-kDa subunit that participate in light-induced conformational changes. These include a 4-10-kDa region near the N terminus of the chromophore binding domain and at least two regions within the nonchromophore domain. A comprehensive peptide map of the oat phytochrome subunit is presented, which incorporates the results of these proteolytic studies with the recent, yet unpublished sequence analyses of Avena phytochrome cDNA clones which show the N-terminal localization of the chromophore binding site (Hershey, H. P., Colbert, J. T., Lissemore, J. L., Barker, R. F., and Quail, P. H. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 2332-2336).
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PMID:Structure function studies on phytochrome. Identification of light-induced conformational changes in 124-kDa Avena phytochrome in vitro. 388 93

A protein capable of inhibiting trypsin and other pancreatic proteases has been purified to homogeneity from Escherichia coli by conventional procedures and affinity chromatography. It is stable for at least 30 min at 100 degrees C and pH 1.0, but it is inactivated by digestion with pepsin. The inhibitor has an apparent molecular weight of 38,000 as determined by gel filtration and must be a homodimer since it contains a single 18,000-dalton subunit upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The inhibitor has an isoelectric point of 6.1. One dimeric molecule of the inhibitor can bind two trypsin molecules to form a mixed tetrameric complex, in which trypsin molecules are completely inhibited. The inhibitor is not digested by the trypsin. When N-benzoyl-DL-arginine-p-nitroanilide was used as a trypsin substrate, half-maximal inhibition was observed at 22 nM. This protein also inhibits chymotrypsin, pancreatic elastase, rat mast cell chymase, and human serosal urokinase, but it does not inhibit human pulmonary tryptase, kallikrein, papain, pepsin, Staphylococcus aureus V8 protease, subtilisin, and thermolysin. Surprisingly, it did not inhibit any of the eight soluble endoproteases recently isolated from E. coli (i.e. proteases Do, Re, Mi, Fa, So, La, Ci, and Pi) nor the chymotrypsin-like (protease I) and trypsin-like (protease II) esterases in E. coli. The inhibitor is localized to the periplasmic space and its level did not change with different growth media or stages of cell growth. The physiological function of this E. coli trypsin inhibitor is unknown. We suggest that E. coli trypsin inhibitor be named "Ecotin."
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PMID:Purification from Escherichia coli of a periplasmic protein that is a potent inhibitor of pancreatic proteases. 641 24

Functional analysis of hepatitis B virus (HBV) core particles has associated a number of biological roles with the C terminus of the capsid protein. One set of functions require the C terminus to be on the exterior of the capsid, while others place this domain on the interior. According to the crystal structure of the capsid, this segment is strictly internal to the capsid shell and buried at a protein-protein interface. Using kinetic hydrolysis, a form of protease digestion assayed by SDS-PAGE and mass spectrometry, the structurally and biologically important C-terminal region of HBV capsid protein assembly domain (Cp149, residues 1-149) has been shown to be dynamic in both dimer and capsid forms. HBV is an enveloped virus with a T=4 icosahedral core that is composed of 120 copies of a homodimer capsid protein. Free dimer and assembled capsid forms of the protein are readily hydrolyzed by trypsin and thermolysin, around residues 127-128, indicating that this region is dynamic and exposed to the capsid surface. The measured conformational equilibria have an opposite temperature dependence between free dimer and assembled capsid. This work helps to explain the previously described allosteric regulation of assembly and functional properties of a buried domain. These observations make a critical connection between structure, dynamics, and function: made possible by the first quantitative measurements of conformational equilibria and rates of conversion between protein conformers for a megaDalton complex.
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PMID:Conformational equilibria and rates of localized motion within hepatitis B virus capsids. 1802 40