Overexpression of recombinant proteins with a C-terminal thiocarboxylate: implications for protein semisynthesis and thiamin biosynthesis.
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A facile and rapid method for the production of protein C-terminal thiocarboxylates on DNA-encoded polypeptides is described. This method, which relies on the mechanism of the cleavage reaction of intein-containing fusion proteins, can produce multi-milligram quantities of protein C-terminal thiocarboxylate quickly and inexpensively. The utility of this method for protein semisynthesis and implications for studies on the biosynthesis of thiamin are discussed. (+info)
The effect of muscle contraction on the regulation of adenosine formation in rat skeletal muscle cells.
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1. The present study examined the effect of muscle contraction on the rate of extracellular adenosine formation and on the distribution of 5' nucleotidase in primary rat skeletal muscle cells in culture. Experiments were also performed to determine whether the muscle cells release a metabolite upon contraction which may influence the extracellular production of adenosine. 2. Muscle contraction, induced by electrical stimulation, increased (P < 0.05) the rate of adenosine formation in the presence of physiological concentrations (2 and 5 microM) of adenosine monophosphate (AMP). Muscle contraction also led to an increase (P < 0.05) in the maximal rate of extracellular adenosine formation from 4.09 +/- 0.19 to 7.04 +/- 0.27 micromol (g protein)-1 min-1. Similarly, homogenates of contracted muscle cells had a higher (by 19.5 +/- 10.5 %; P < 0.05) AMP 5' nucleotidase activity than homogenates of control cells. 3. Addition of buffer from contracted cells to control cells induced an elevation (18.4 +/- 5.3 %; P < 0.05) in the rate of adenosine formation. The rate of adenosine formation was also increased with decreased intracellular adenylate charge (P < 0.05). 4. Cell homogenates treated with detergent had a higher (by 58.0 +/- 16.3 %; P < 0.05) AMP 5' nucleotidase activity than untreated homogenates, suggesting the existence of an enclosed pool of 5' nucleotidase within the muscle cells. The rate of adenosine formation in the detergent-treated homogenates was similar for electrically stimulated and non-electrically stimulated cells. 5. The present data show that muscle contraction induces an enhanced extracellular adenosine production via an increase in the activity of ecto AMP 5' nucleotidase. The activity of 5' nucleotidase can be elevated via a compound released by muscle cells during contraction and by alteration in intracellular adenylate charge. It is furthermore proposed that the extracellular adenosine formation is increased by translocation of 5' nucleotidase from an enclosed intracellular pool to the muscle membrane. (+info)
Hypothalamic glucose sensor: similarities to and differences from pancreatic beta-cell mechanisms.
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Glucose-responsive neurons in the ventromedial hypothalamus (VMH) are stimulated when glucose increases from 5 to 20 mmol/l and are thought to play an essential role in regulating metabolism. The present studies examined the role of glucose metabolism in the mechanism by which glucose-responsive neurons sense glucose. The pancreatic, but not hepatic, form of glucokinase was expressed in the VMH, but not cerebral cortex, of adult rats. In brain slice preparations, the transition from 5 to 20 mmol/l glucose stimulated approximately 17% of the neurons (as determined by single-cell extracellular recording) from VMH but none in cortex. In contrast, most cells in both VMH and cortex were silent below 1 mmol/l and active at 5 mmol/l glucose. Glucosamine, 2-deoxyglucose, phloridzin, and iodoacetic acid blocked the activation of glucose-responsive neurons by the transition from 5 to 20 mmol/l glucose. Adding 15 mmol/l mannose, galactose, glyceraldehyde, glycerol, and lactate to 5 mmol/l glucose stimulated glucose-responsive neurons. In contrast, adding 15 mmol/l pyruvate to 5 mmol/l glucose failed to activate glucose-responsive neurons, although pyruvate added to 0 mmol/l glucose permitted neurons to maintain activity. Tolbutamide activated glucose-responsive neurons; however, diazoxide only blocked the effect of glucose in a minority of neurons. These data suggest that glucose-responsive neurons sense glucose through glycolysis using a mechanism similar to the mechanism of pancreatic beta-cells, except that glucose-responsive neurons are stimulated by glycerol and lactate, and diazoxide does not generally block the effect of glucose. (+info)
Site-directed mutagenesis establishes cysteine-110 as essential for enzyme activity in human gamma-glutamyl hydrolase.
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Gamma-glutamyl hydrolase (GH), which hydrolyses the gamma-glutamyl conjugates of folic acid, is a key enzyme in the maintenance of cellular folylpolyglutamate concentrations. The catalytic mechanism of GH is not known. Consistent with earlier reports that GH is sulphydryl-sensitive, we found that recombinant human GH is inhibited by iodoacetic acid, suggesting that at least one cysteine is important for activity [Rhee, Lindau-Shepard, Chave, Galivan and Ryan (1998) Mol. Pharmacol. 53, 1040-1046]. Using site-directed mutagenesis, the cDNA for human GH was altered to encode four different proteins each with one of four cysteine residues changed to alanine. Three of the mutant proteins had activities similar to wild-type GH and were inhibited by iodoacetic acid, whereas the C110A mutant had no activity. Cys-110 is conserved among the human, rat and mouse GH amino acid sequences. The wild-type protein and all four mutants had similar intrinsic fluorescence spectra, indicating no major structural changes had been introduced. These results indicate that Cys-110 is essential for enzyme activity and suggest that GH is a cysteine peptidase. These studies represent the first identification of the essential Cys residue in this enzyme and provide the beginning of a framework to determine the catalytic mechanism, important in defining GH as a therapeutic target. (+info)
Purification of collagenase and specificity of its related enzyme from Bacillus subtilis FS-2.
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A collagenase in the culture supernatant of B. subtilis FS-2, isolated from traditional fish sauce, was purified. The enzyme had a molecular mass of about 125 kDa. It degraded gelatin with maximum activity at pH 9 and a temperature of 50 degrees C. The purified enzyme was stable over a wide range of pH (5-10) and lost only 15% and 35% activity after incubation at 60 degrees C and 65 degrees C for 30 min, respectively. Slightly inhibited by EDTA, soybean tripsin inhibitor, iodoacetamide, and iodoacetic acid, the enzyme was severely inhibited by 2-beta-mercaptoethanol and DFP. The protease from B. subtilis FS-2 culture digested acid casein into fragments with hydrophilic and hydrophobic amino acids as C-terminals, in particular Asn, Gly, Val, and Ile. (+info)
Energy dependence of restitution in the gastric mucosa.
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Rapid epithelial repair (restitution) after injury is required to maintain barrier function of the gastrointestinal mucosa and skin and is thought to be a highly ATP-dependent process that would be inhibited under hypoxic conditions. However, little is known about the metabolic pathways required for restitution. Thus, this study was undertaken to evaluate, in vitro, the role of oxidative respiration and glycolysis in restitution after injury. To this end, restitution of the bullfrog gastric mucosa was evaluated under the following conditions: 1) blockade of mitochondrial respiration; 2) blockade of glycolysis; or 3) absence of glucose. The extent of mucosal repair after injury was evaluated by electrophysiology and morphology. Cell migration, repolarization, and the formation of tight junctions after injury occurred during blockade of mitochondrial respiration, whereas the recovery of mucosal barrier function did not. In contrast, glycolytic inhibition completely blocked all aspects of restitution by inhibiting the migration of surface epithelial cells. Restitution occurred in tissues incubated with glucose-free solutions, suggesting that cells contain sufficient glucose (glycogen) to drive glycolysis for many hours. Our results demonstrate that the glycolytic pathway is essential for restitution after injury in the bullfrog gastric mucosa and that all but complete repair of barrier function occurs in the absence of mitochondrial respiration. (+info)
Alternative metabolic pathways for energy supply and resistance to apoptosis in Fanconi anaemia.
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Deregulation of control of the apoptotic process in Fanconi anaemia (FA) appears to be one of the main features of this disease at the cellular level. We show here that FA cells are resistant to treatments with rhodamine-1,2,3 and doxycycline, which both interfere with mitochondrial functionality by different mechanisms. In contrast, normal lymphoblastoid cells are severely affected by these treatments, which result in acute ATP depletion and a significant enhancement of the fraction of cells undergoing apoptotic cell death. FA cells are very sensitive to the action of 2-deoxy-D-glucose (2dG) and iodoacetic acid (IAA), two inhibitors of glycolytic metabolism. The ability of FA cells to sustain metabolic insults interfering with energy production and balance may be linked with the pathological manifestations of the disease, including susceptibility to acute myeloid leukemia. These findings suggest that FA genes may be involved in a pathway that mediates a protective response to stress. We suggest that a peculiar metabolic regulation in FA cells could explain both defective apoptosis and susceptibility to oxidative stress. (+info)
Purification and properties of membrane-bound D-sorbitol dehydrogenase from Gluconobacter suboxydans IFO 3255.
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D-Sorbitol dehydrogenase was solubilized from the membrane fraction of Gluconobacter suboxydans IFO 3255 with Triton X-100 in the presence of D-sorbitol. Purification of the enzyme was done by fractionation with column chromatographies of DEAE-Cellulose, DEAE-Sepharose, hydroxylapatite, and Sephacryl HR300 in the presence of Triton X-100. The molecular mass of the enzyme was 800 kDa, consisting of homologous subunits of 80 kDa. The optimum pH of the enzyme activity was 6.0, and the optimum temperature was 30 degrees C. Western blot analysis suggested the occurrence of the enzyme in all the Gluconobacter strains tested. (+info)