Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

At least part of the gamma subunit of the catalytic portion of the chloroplast ATP synthase (CF1) is present in the middle of the alpha3beta3 heterohexamer. Interactions of the alpha/beta subunits with the gamma subunit stabilize the hexameric structure. Surprisingly, neither reduction of the gamma disulfide nor selective proteolysis of alpha, beta and gamma affects the thermal stability of EDTA-treated CF1 preparations, as determined by differential scanning calorimetry. Dissociation of the enzyme in the cold may be monitored by loss of the ATPase activity of CF1 subunit depleted of its epsilon subunit [CF1(-epsilon)]. The rate of cold inactivation of ATPase activity of reduced and alkylated CF1(-epsilon) treated with trypsin in solution was much faster than that CF1(-epsilon)(8.1 versus 38.7 min, respectively, for 50% loss of activity). The increased cold liability of the trypsin-treated enzyme was not a consequence of the cleavage of the gamma. CF1 incubated with trypsin under conditions in which gamma is not cleaved was as cold labile as CF1 with cleaved gamma. Instead, loss of the delta subunit and a few residues from the C-terminal of the beta subunits were responsible for the increased cold liability of the enzyme.
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PMID:Structural stability of chloroplast coupling factor 1 determined by differential scanning calorimetry and cold inactivation. 866 76

Scanning force microscope images of membrane-bound Escherichia coli ATP synthase F0 complexes have been obtained in aqueous solution. The images show a consistent set of internal features: a ring structure which surrounds a central dimple and contains an asymmetric lateral mass. Images of trypsin-treated F0 complexes, which have lost part of their b subunits, show a reduced asymmetric mass, while images of c-subunit oligomers, which lack both the a and b subunits, show a ring and dimple but do not have an asymmetric mass. These results support models in which the F0 complex contains a ring of 9-12 c subunits with the b subunits located outside this ring, and show that scanning force microscopy is able to provide structural information on membrane proteins of molecular mass less than 200 000 Da.
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PMID:Topographical structure of membrane-bound Escherichia coli F1F0 ATP synthase in aqueous buffer. 894 8

The delta and b subunits are both involved in binding the F1 to the F0 part in the Escherichia coli ATP synthase (ECF1F0). The interaction of the purified delta subunit and the isolated hydrophilic domain of the b subunit (bsol) has been studied here. Purified delta binds to bsol weakly in solution, as indicated by NMR studies and protease protection experiments. On F1, i.e. in the presence of ECF1-delta, delta, and bsol interact strongly, and a complex of ECF1.bsol can be isolated by native gel electrophoresis. Both delta subunit and bsol are protected from trypsin cleavage in this complex. In contrast, the delta subunit is rapidly degraded by the protease when bound to ECF1 when bsol is absent. The interaction of bsol with ECF1 involves the C-terminal domain of delta as delta(1-134) cannot replace intact delta in the binding experiments. As purified, bsol is a stable dimer with 80% alpha helix. A monomeric form of bsol can be obtained by introducing the mutation A128D (Howitt, S. M., Rodgers, A. J.,W., Jeffrey, P. D., and Cox, G. B. (1996) J. Biol. Chem. 271, 7038-7042). Monomeric bsol has less alpha helix, i.e. only 58%, is much more sensitive to trypsin cleavage than dimer, and unfolds at much lower temperatures than the dimer in circular dichroism melting studies, indicating a less stable structure. The bsol dimer, but not monomer, binds to delta in ECF1. To examine whether subunit b is a monomor or dimer in intact ECF1F0, CuCl2 was used to induce cross-link formation in the mutants bS60C, bQ104C, bA128C, bG131C, and bS146C. With the exception of bS60C, CuCl2 treatment resulted in formation of b subunit dimers in all mutants. Cross-linking yield was independent of nucleotide conditions and did not affect ATPase activity. These results show the b subunit to be dimeric for a large portion of the C terminus, with residues 124-131 likely forming a pair of parallel alpha helices.
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PMID:The subunit delta-subunit b domain of the Escherichia coli F1F0 ATPase. The B subunits interact with F1 as a dimer and through the delta subunit. 938 56

Limited trypsin digestion of isolated F1 removed 15 and 7 amino acids from the N-termini of the alpha and beta subunits respectively and left other subunits untouched as shown by electrophoresis, immunoblotting and protein sequencing. The cooperativity for ATP hydrolysis by soluble F1 was impaired by trypsin digestion. The Km2 obtained from Eadie-Hofstee plots apparently decreased in trypsin-digested F1 but the affinity for adenosine 5'-[beta,gamma-imido]triphosphate (AdoPP[NH]P) and GTP hydrolysis was not influenced. The inhibition of ATP hydrolysis by ADP was attenuated by trypsin digestion. Trypsin digestion of F1 did not affect its capacity to bind to Fo nor did it alter the sensitivity of ATP hydrolysis in the F1Fo reconstituted system to oligomycin and N,N'-dicyclohexylcarbodiimide. The cleavage of the alpha and beta subunits did, on the other hand impair: (a) the ATP-driven proton pumping in the reconstituted F1Fo complex: (b) the inhibition by F1 of passive proton conduction in Fo; (c) the inhibition of passive proton conduction in Fo by AdoPP[NH]P binding to F1. These results show that the limited cleavage of the N-termini of the alpha and beta subunits, located on the top of F1, results in decoupling of catalysis from proton transport. The possible relationship of these observations with the binding change rotatory model of the F1Fo ATP synthase is discussed.
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PMID:The N-termini of the alpha and beta subunits at the top of F1 stabilize the energy-transfer function in the mitochondrial F1Fo ATP synthase. 952 25

Amino acid substitutions at many positions in the a subunit of F1F0 ATP synthase result in impaired proton translocation and altered catalytic activity. In this work, we demonstrate that amino acid substitutions in the a subunit affect the epsilon subunit. In mutant F1F0 ATP synthases, the epsilon subunit was studied by determining its sensitivity to proteolysis and by chemical crosslinking under conditions of active turnover and in quiescent enzyme. Like native F1F0 ATP synthase, the epsilon subunit in enzymes carrying either the aarg-210-->ile or agly-218-->asp substitutions proved resistant to trypsin digestion during ATP hydrolysis. In each case, the epsilon subunit was rapidly digested in the presence of a nonhydrolyzable ligand, but this did not result in the activation of hydrolytic activity typically seen in wild-type enzyme. In enzyme carrying the aala-217-->arg substitution, the trypsin digestion of the epsilon subunit occurred regardless of ligand and was accompanied by a limited hydrolytic activation. Relative to the native F1F0 ATP synthase, the aala-217-->arg substitution resulted in reduced efficiency of crosslinking between the epsilon and beta subunits using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. These observations indicate that the structural changes resulting from amino acid substitutions in the a subunit are propagated to the epsilon subunit and are specific to the individual substitutions.
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PMID:Amino acid substitutions in the a subunit affect the epsilon subunit of F1F0 ATP synthase from Escherichia coli. 988 60

We have studied the functional effect of limited proteolysis by trypsin of the constituent subunits in the native and reconstituted F1F0 complex and isolated F1 of the bovine heart mitochondrial ATP synthase (EC 3.6.1.34). Chemical cross-linking of oligomycin-sensitivity conferring protein (OSCP) with other subunits of the ATP synthase and the consequent functional effects were also investigated. The results obtained show that the alpha subunit N-terminus is essential for the correct, functional connection of F1 to F0. The alpha-subunit N-terminus contacts OSCP which, in turn, contacts the F0I-PVP(b) and the F0-d subunits. The N-terminus of subunit alpha, OSCP, a segment of subunit d and the C-terminal and central region of F0I-PVP(b) subunits are peripherally located with respect to subunits gamma and delta which are completely shielded in the F1F0 complex against trypsin digestion. This qualifies the N-terminus of subunit alpha, OSCP, subunit d and F0I-PVP(b) as components of the lateral element of the stalk. These subunits, rather than being confined at one side of the complex which would leave most of the central part of the gamma subunit uncovered, surround the gamma and the delta subunits located in the central stalk.
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PMID:F1 and F0 connections in the bovine mitochondrial ATP synthase: the role of the of alpha subunit N-terminus, oligomycin-sensitivity conferring protein (OCSP) and subunit d. 1088 Sep 68

The antibiotic bicyclomycin inhibits rho-dependent termination processes by interfering with RNA translocation by preventing RNA binding at the translocation site or by uncoupling the translocation process from ATP hydrolysis. Previous studies have shown that bicyclomycin binds near the ATP hydrolysis pocket on rho. The hexameric structure of rho indicates that it is in a class of enzymes with strong sequence similarity to F(1)-ATP synthase. The bicyclomycin derivative 5a-formylbicyclomycin, an inhibitor comparable to bicyclomycin, was previously shown to form a stable imine with rho and when reduced to the amine with NaBH(4) to singly label five of the six rho subunits. Lysine-336 was identified by mass spectrometric analysis of trypsin-digested fragments as the site of 5a-formylbicyclomycin adduction. A model of rho was made by threading the rho sequence on the known crystal structure of the alpha and beta subunits of F(1)-ATP synthase. The model, along with information concerning the extent and site of 5a-formylbicyclomycin adduction, indicates an overall C6 symmetry for rho subunit organization. We propose that the sequence similarity between rho and F(1)-ATP synthase extends to a similar quaternary structure and an equivalent enzyme mechanism. The proposed mechanism of RNA translocation coupled with ATP hydrolysis changes the overall symmetry of rho from C6 to C6/C3.
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PMID:Rho transcription factor: symmetry and binding of bicyclomycin. 1092

We updated the two-dimensional protein database for mouse liver. Microsomal and cytosolic fractions of the liver proteins from male mice were separated by two-dimensional electrophoresis. The proteins were identified by Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) on the basis of peptide mass fingerprinting, following in-gel digestion with trypsin and matching with the theoretical peptide masses of all known proteins from all species. Approximately 5800 spots, excised from 14 two-dimensional gels, were analyzed which resulted in the identification of about 2500 proteins that were the products of 328 different genes. The database includes 112 newly identified gene products. The fractionation prior to two-dimensional electrophoresis was essential for the detection of the new proteins, 55% of which were found in the microsomal and 35% in the cytosolic fraction. The more frequently identified proteins in the various gels were heat shock proteins, house-keeping enzymes, such as ATP synthase chains, disulfide isomerase, and structural proteins, such as tropomyosin. About 45% of the identified proteins were detected 1-3 times, 45% 4-9 times, and the rest 10 or more times. Most proteins were represented by many spots. In average, about 18-20 spots were detected per gene product.
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PMID:Two-dimensional database of mouse liver proteins. An update. 1142 30

Streptococcus oralis is the predominant aciduric nonmutans streptococcus isolated from the human dentition, but the role of this organism in the initiation and progression of dental caries has yet to be established. To identify proteins that are differentially expressed by S. oralis growing under conditions of low pH, soluble cellular proteins extracted from bacteria grown in batch culture at pH 5.2 or 7.0 were analyzed by two-dimensional (2-D) gel electrophoresis. Thirty-nine proteins had altered expression at low pH; these were excised, digested with trypsin using an in-gel protocol, and further analyzed by peptide mass fingerprinting using matrix-assisted laser desorption ionization mass spectrometry. The resulting fingerprints were compared with the genomic database for Streptococcus pneumoniae, an organism that is phylogenetically closely related to S. oralis, and putative functions for the majority of these proteins were determined on the basis of functional homology. Twenty-eight proteins were up-regulated following growth at pH 5.2; these included enzymes of the glycolytic pathway (glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase), the polypeptide chains comprising ATP synthase, and proteins that are considered to play a role in the general stress response of bacteria, including the 60-kDa chaperone, Hsp33, and superoxide dismutase, and three distinct ABC transporters. These data identify, for the first time, gene products that may be important in the survival and proliferation of nonmutans aciduric S. oralis under conditions of low pH that are likely to be encountered by this organism in vivo.
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PMID:Altered protein expression of Streptococcus oralis cultured at low pH revealed by two-dimensional gel electrophoresis. 1147 10

We applied proteomic technologies to analyze the human fetal brain. Such an analysis could provide us with important information on the development of the early neuronal life in healthy and diseased states. The proteins from the cerebellum of control subjects were analyzed by two-dimensional electrophoresis and identified by matrix-assisted laser desorption/ionization-mass spectrometry on the basis of peptide mass fingerprinting, following in-gel digestion with trypsin. Approximately 3,000 spots, excised from three two-dimensional gels, were analyzed which resulted in the identification of about 1,700 proteins that were the products of 437 different genes. About half of them are enzyme subunits and are mainly localized in the cytosol and in mitochondria. The most frequently identified proteins in the various gels were heat shock proteins, house-keeping enzymes, such as ATP synthase chains, protein disulfide isomerase, and structural proteins, such as tubulin chains. Seven gene products were identified for the first time in the fetal brain. The other proteins had also been detected in other human samples which were analyzed in our laboratory. Most proteins were represented by multiple spots. In average, about 3-5 spots were detected per gene product. The fetal brain database includes proteins with important functions and also with unknown functions and represents today one of the largest two-dimensional databases for higher eukaryotic proteomes. It may be a useful tool in the investigation of protein changes in neurodegenerative diseases early in life.
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PMID:Proteomic analysis of the fetal brain. 1244 55


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