Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.17 (lysozyme)
 21,489 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The predicted amino acid sequence of the alpha subunit of the rat liver mitochondrial ATP synthase has been obtained by sequencing a cDNA for the alpha subunit. Analysis of the sequence shows that it contains the A and B consensus sequences found in many nucleotide-binding proteins. Twelve amino acids of the rat liver alpha subunit differ from the sequence of the bovine heart alpha subunit; four of these involve differences in charge. The rat liver alpha subunit, from arginine 15 to the C-terminal proline 510, has been overexpressed in Escherichia coli using the alkaline phosphatase promoter (phoA) and leader peptide to direct the export of the expressed protein to the bacterial periplasm. By treating the cells with lysozyme, osmotic shock, and alkaline pH washes, the alpha subunit can be extracted in high yield (greater than 25 mg/liter) and in a high state of purity. The expressed alpha subunit remains soluble at pH 9.5 or greater and precipitates when treated with Mg2+ ions at low millimolar concentration. The bacterially expressed alpha subunit interacts with 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP), resulting in a marked fluorescence enhancement upon binding. An enhancement of fluorescence is also observed upon the interaction of the alpha subunit with TNP-ADP. Preincubating the alpha subunit with 1.5 mM ATP significantly reduces the fluorescence enhancement seen with TNP-ATP. The alpha subunit binds TNP-ATP with an apparent Kd in the low micromolar range (1-5 microM) and binds TNP-ADP with an affinity at least 10-fold lower. This work shows that the rat liver alpha subunit can be overexpressed in E. coli to yield a large amount of functional protein. With the acquisition of the overexpressed alpha subunit, it is now possible to test the reconstitution of ATPase activity from a mixture of recombinant and rat liver-derived subunits and to test the formation of complexes by the overexpressed alpha and beta subunits of the rat liver F1-ATPase.
 ...
PMID:Mitochondrial ATP synthase. cDNA cloning, amino acid sequence, overexpression, and properties of the rat liver alpha subunit. 213 25

We have developed a chromatographic procedure to analyze the association of the subunits of the Escherichia coli F1Fo-ATP synthase with the cytoplasmic membrane. Minicells containing [35S]-labeled ATP synthase subunits are treated with lysozyme, solubilized, and chromatographed on a Sepharose CL-2B column in buffer containing urea and taurodeoxycholate. ATP synthase subunits are resolved into membrane intrinsic and membrane extrinsic subunits. Interestingly, a significant amount (36%) of the F1 subunit beta fractionates with the membrane intrinsic Fo subunits. About half of this amount (19%) of beta is non-specifically bound to the membrane. Interaction of beta with the membrane is not mediated by the amino terminal portion of beta.
 ...
PMID:The beta subunit of the Escherichia coli ATP synthase exhibits a tight membrane binding property. 285 19

The C-terminal two-thirds of the rat liver ATP synthase beta subunit has been overexpressed and exported to the Escherichia coli periplasm under the direction of the alkaline phosphatase (phoA) promoter and leader peptide. The processed soluble protein contains the 358 amino acids from glutamate 122 to the rat liver beta C-terminal serine 479, including all the regions that have been predicted by chemical and genetic modification studies to be involved in nucleotide, Pi, and Mg2+ binding. Through a simple sequence of Tris/EDTA/lysozyme treatment, osmotic lysis, and alkaline pH washes, the processed beta subunit fragment can be prepared in greater than 95% purity and at a yield of greater than 20 mg/liter of culture. It interacts with 2'(3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) which exhibits a strong enhancement of fluorescence upon binding. A similar enhancement is observed upon interaction with TNP-ADP. Enhancement observed with both TNP-nucleotides is markedly reduced by prior addition of either ATP or ADP and almost completely prevented by the ATP synthase inhibitor 7-chloro-4-nitrobenz-2-oxa-1,3-diazole. Both TNP-ATP and TNP-ADP bind at a stoichiometry of approximately 1 mol of nucleotide/mol of beta subunit fragment. Under the same conditions, TNP-AMP does not exhibit a fluorescence enhancement. This work demonstrates that, in the absence of interaction with other ATP synthase subunits, the rat liver beta subunit sequence from glutamate 122 to the C terminus exhibits no more than one readily detectable nucleotide binding domain. This success in producing a "functional" beta subunit fragment has significance for the pursuit of genetic and physical studies focused on the structure and function of the rat liver ATP synthase beta subunit.
 ...
PMID:Mitochondrial ATP synthase. Overexpression in Escherichia coli of a rat liver beta subunit peptide and its interaction with adenine nucleotides. 290 92

Fatty acid composition of the membrane lipids in the mesophilic cyanobacterium Synechocystis sp. PCC 6803 was altered in earlier work by targeted mutagenesis of genes for fatty acid desaturases. In this work, cells of several mutant strains, depleted in the unsaturated fatty acids in membrane lipids, were grown at 34 degrees C. Spheroplasts (permeabilized cells) were prepared by lysozyme digestion of the cell wall followed by gentle osmotic shock. The bioenergetic parameters ATP formation, electron transport, and H+ uptake were measured at various temperatures. All three bioenergetic parameters for spheroplasts from wild-type cells (which had abundant polyunsaturated fatty acids) were active down to the lowest temperatures used (1 degrees - 2 degrees C). In two strains, which lacked the capacity to desaturate fatty acids at the A 12 position and at the A 12 and A6 positions (designated as desA- and desA-/desD-, respectively), the spheroplasts lost the capacity to form ATP (measured as phenazine methosulfate cyclic phosphorylation) at about 5 degrees C but retained electron transport (water oxidation-dependent ferricyanide reduction) and H+ uptake linked to phenazine methosulfate cyclic electron transport. It appears that the absence of the unsaturation of fatty acids in the A 12 and A6 positions blocks the ability of the photosynthetic membranes to couple a bioenergetically competent proton-motive force to the ATP formation mechanism at temperatures below 5 degrees C. It remains to be determined whether the loss of ATP formation in the mutant strains is the failure of available protons to properly flow into the CF0CF1-ATP synthase or a failure in the CF1 part of the complex in coupling the dissipative H+ flow to the enzyme mechanism of the synthase.
 ...
PMID:Bioenergetic responses of Synechocystis 6803 fatty acid desaturase mutants at low temperatures. 1145 19

Clam Ruditapes philippinarum is one of the important marine aquaculture species in North China. However, pathogens can often cause diseases and lead to massive mortalities and economic losses of clam. In this work, we compared the metabolic responses induced by Vibrio anguillarum and Vibrio splendidus challenges towards hepatopancreas of clam using NMR-based metabolomics. Metabolic responses suggested that both V. anguillarum and V. splendidus induced disturbances in energy metabolism and osmotic regulation, oxidative and immune stresses with different mechanisms, as indicated by correspondingly differential metabolic biomarkers (e.g., amino acids, ATP, glucose, glycogen, taurine, betaine, choline and hypotaurine) and altered mRNA expression levels of related genes including ATP synthase, ATPase, glutathione peroxidase, heat shock protein 90, defensin and lysozyme. However, V. anguillarum caused more severe oxidative and immune stresses in clam hepatopancreas than V. splendidus. Our results indicated that metabolomics could be used to elucidate the biological effects of pathogens to the marine clam R. philippinarum.
 ...
PMID:Differential metabolic responses of clam Ruditapes philippinarum to Vibrio anguillarum and Vibrio splendidus challenges. 2405 79

Manila clam Ruditapes philippinarum is an important marine aquaculture shellfish. This species has several pedigrees including White, Zebra, Liangdao Red and Marine Red distributing in the coastal areas in North China. In this work, we studied the metabolic differences induced by Vibrio harveyi in hepatopancreas from White and Zebra clams using NMR-based metabolomics. Metabolic responses (e.g., amino acids, glucose, glycogen, ATP and succinate) and altered mRNA expression levels of related genes (ATP synthase, heat shock protein 90, defensin and lysozyme) suggested that V. harveyi induced clear disruption in energy metabolism and immune stresses in both White and Zebra clam hepatopancreas. However, V. harveyi caused obvious osmotic stress in Zebra clam hepatopancreas, which was not observed in V. harveyi-challenged White clams samples. In addition, V. harveyi challenge induced more severe disruption in energy metabolism and immune stress in White clams than in Zebra clams. Overall, our results indicated that the biological differences between different pedigrees of R. philippinarum should be considered in immunity studies.
 ...
PMID:Metabolomic analysis revealed the differential responses in two pedigrees of clam Ruditapes philippinarum towards Vibrio harveyi challenge. 2416 58