Ultracentrifugal studies of the effect of molecular crowding by trimethylamine N-oxide on the self-association of muscle glycogen phosphorylase b.
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The suitability of sedimentation equilibrium for characterizing the self-association of muscle glycogen phosphorylase b has been reappraised. Whereas sedimentation equilibrium distributions for phosphorylase b in 40 mM Hepes buffer (pH 6.8) supplemented with 1 mM AMP signify a lack of chemical equilibrium attainment, those in buffer supplemented additionally with potassium sulfate conform with the requirements of a dimerizing system in chemical as well as sedimentation equilibrium. Because the rate of attainment of chemical equilibrium under the former conditions is sufficiently slow to allow resolution of the dimeric and tetrameric enzyme species by sedimentation velocity, this procedure has been used to examine the effects of thermodynamic nonideality arising from molecular crowding by trimethylamine N-oxide on the self-association behaviour of phosphorylase b. In those terms the marginally enhanced extent of phosphorylase b self-association observed in the presence of high concentrations of the cosolute is taken to imply that the effects of thermodynamic nonideality on the dimer-tetramer equilibrium are being countered by those displacing the T<==>R isomerization equilibrium for dimer towards the smaller, nonassociating T state. Because the R state is the enzymically active form, an inhibitory effect is the predicted consequence of molecular crowding by high concentrations of unrelated solutes. Thermodynamic nonideality thus provides an alternative explanation for the inhibitory effects of high concentrations of glycerol, sucrose and ethylene glycol on phosphorylase b activity, phenomena that have been attributed to extremely weak interaction of these cryoprotectants with the T state of the enzyme. (+info)
Cleavage of SNAP-25 by botulinum toxin type A requires receptor-mediated endocytosis, pH-dependent translocation, and zinc.
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Previously we reported that SNAP-25, synaptobrevin II, and syntaxin I, the intracellular substrates of botulinum toxin originally identified in nontarget tissues, were present in a recognized mammalian target tissue, the mouse hemidiaphragm. Furthermore, we reported that SNAP-25, syntaxin I, and synaptobrevin II were cleaved by incubation of the intact hemidiaphragm in botulinum serotypes A, C, and D, respectively. The objective of the current study was to use the mouse phrenic nerve-hemidiaphragm preparation and botulinum serotype A to investigate 1) the relationship of substrate cleavage to toxin-induced paralysis, and 2) the relevance of substrate cleavage to the mechanism of toxin action. Immunoblot examination of tissues paralyzed by botulinum toxin type A (10(-8) M) revealed < or =10% loss of SNAP-25 immunoreactivity at 1 h postparalysis, and > or =75% loss at 5 h postparalysis. Triticum vulgaris lectin, an agent that competitively antagonizes toxin binding, antagonized toxin-induced paralysis as well as SNAP-25 cleavage. Methylamine hydrochloride, an agent that prevents pH-dependent translocation, also antagonized toxin-induced paralysis and SNAP-25 cleavage. Furthermore, zinc chelation antagonized toxin-induced paralysis and SNAP-25 cleavage. These results demonstrate that cleavage of SNAP-25 by botulinum serotype A fulfills the requirements of the multistep model of botulinum toxin action that includes receptor-mediated endocytosis, pH-dependent translocation, and zinc-dependent proteolysis. Furthermore, the minimal amount of SNAP-25 cleavage at 1 h postparalysis suggests that inactivation of only a small but functionally important pool of SNAP-25 is necessary for paralysis. (+info)
Chaperonin-assisted folding of glutamine synthetase under nonpermissive conditions: off-pathway aggregation propensity does not determine the co-chaperonin requirement.
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One of the proposed roles of the GroEL-GroES cavity is to provide an "infinite dilution" folding chamber where protein substrate can fold avoiding deleterious off-pathway aggregation. Support for this hypothesis has been strengthened by a number of studies that demonstrated a mandatory GroES requirement under nonpermissive solution conditions, i.e., the conditions where proteins cannot spontaneously fold. We have found that the refolding of glutamine synthetase (GS) does not follow this pattern. In the presence of natural osmolytes trimethylamine N-oxide (TMAO) or potassium glutamate, refolding GS monomers readily aggregate into very large inactive complexes and fail to reactivate even at low protein concentration. Surprisingly, under these "nonpermissive" folding conditions, GS can reactivate with GroEL and ATP alone and does not require the encapsulation by GroES. In contrast, the chaperonin dependent reactivation of GS under another nonpermissive condition of low Mg2+ (<2 mM MgCl2) shows an absolute requirement of GroES. High-performance liquid chromatography gel filtration analysis and irreversible misfolding kinetics show that a major species of the GS folding intermediates, generated under these "low Mg2+" conditions exist as long-lived metastable monomers that can be reactivated after a significantly delayed addition of the GroEL. Our results indicate that the GroES requirement for refolding of GS is not simply dictated by the aggregation propensity of this protein substrate. Our data also suggest that the GroEL-GroES encapsulated environment is not required under all nonpermissive folding conditions. (+info)
Trimethylaminuria: the fish malodor syndrome.
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The fish malodor syndrome (also known as the fish odor syndrome and trimethylaminuria) is a metabolic disorder characterized by the presence of abnormal amounts of the dietary-derived tertiary amine, trimethylamine, in the urine, sweat, expired air, and other bodily secretions. Trimethylamine itself has the powerful aroma of rotting fish, and this confers upon the sufferer a highly objectionable body odor, which can be destructive to the personal, social, and work life of the affected individual. In recent years, much progress has been made at all levels-clinical, epidemiological, biochemical, and genetic-in our understanding of this unfortunate condition. The present article summarizes this progress, draws attention to the different types of fish malodor syndrome, and highlights the current needs in the treatment of such patients. (+info)
N-Methylglutamate synthetase. Substrate-flavin hydrogen transfer reactions probed with deazaflavin mononucleotide.
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N-Methylglutamate synthetase, reconstituted from apo-protein with 5-deazaFMN, catalyzes the reversible formation of N-methylglutamate via the same two-step mechanism previously elucidated for native enzyme (Reactions 1 and 2).(See article). This conclusion is based on the observation that: 1. Enzyme-bound deazaFMN (gamma-max equals 410, 338, epsilon410 equals 10,400 m-minus 1 cm-minus 1) is reduced by L-glutamate, N-methyl-L-glutamate but not D-glutamate. At saturating concentrations of L-glutamate Reaction 1 proceeds at 1% of the rate observed with FMN-reconstituted enzyme. 2. Substrate-reduced deazaFMN enzyme is reoxidized by methylamine or ammonia. 3. A glutaryl enzyme intermediate, isolated by Sephadex G-25 chromatography, contains radioactivity when prepared from [U-14C]glutamate, [alpha-3H]glutamate, or N-[glutaryl U-14C]methylglutamate; however, this intermediate is not labeled from N[methyl 14C]methylglutamate. 4. The amount of radioactivity incorporated into the intermediate is stoichiometric with the amount of deazaFMN reduced during its formation. 5. Intermediate prepared with [U-14C]glutamate yields alpha-[14C]ketoglutarate when denatured with acid and N-[glutaryl-U-14C]methylglutamate when incubated with methylamine. In the absence of methylamine deazaFMN enzyme intermediate slowly decays to yield alpha-hydroxyglutarate. 6. The rate of deazaFMN glutaryl enzyme intermediate formation at a fixed glutamate concentration is equal to the rate of the over-all reaction while the rate of intermediate reaction with methylamine is approximately 50 times greater than the over-all reaction. DeazaFMN enzyme intermediate prepared with [alpha-3H]-glutamate yields [3H]deazaFMNH2 when denatured with acid or phenol and N-[3H]methylglutamate when incubated with methylamine. These results show that the alpha-hydrogen of glutamate is transferred to deazaFMNH2, presumably at the 5 position, during Reaction 1 and that the same hydrogen is utilized for the reformation of the alpha C-H bond during Reaction 2. These results provide the first direct evidence for enzymic hydrogen transfer from substrate to flavin. (+info)
Rapid dephosphorylation of the TorR response regulator by the TorS unorthodox sensor in Escherichia coli.
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Induction of the torCAD operon, encoding the trimethylamine N-oxide (TMAO) respiratory system, is tightly controlled by the TorS-TorR phosphorelay system in response to TMAO availability. TorS is an unorthodox sensor that contains three phosphorylation sites and transphosphorylates TorR via a four-step phosphorelay, His443-->Asp723-->His850-->Asp(TorR). In this study, we provide genetic evidence that TorS can dephosphorylate phospho-TorR when TMAO is removed. Dephosphorylation probably occurs by a reverse phosphorelay, Asp(TorR)-->His850-->Asp723, since His850 and Asp723 are both essential in this process. By using reverse transcriptase PCR, we also show that TMAO removal results in shutoff of tor operon transcription in less than 2 min. Based on our results and on analogy to other phosphorelay signal transduction systems, we propose that reverse phosphotransfer could be a rapid and efficient mechanism to inactivate response regulators. (+info)
The conformation of the glucocorticoid receptor af1/tau1 domain induced by osmolyte binds co-regulatory proteins.
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The activation domains of many transcription factors appear to exist naturally in an unfolded or only partially folded state. This seems to be the case for AF1/tau1, the major transactivation domain of the human glucocorticoid receptor. We show here that in buffers containing the natural osmolyte trimethylamine N-oxide (TMAO), recombinant AF1 folds into more a compact structure, as evidenced by altered fluorescence emission, circular dichroism spectra, and ultracentrifugal analysis. This conformational transition is cooperative, a characteristic of proteins folding to natural structures. The structure resulting from incubation in TMAO causes the peptide to resist proteolysis by trypsin, chymotrypsin, endoproteinase Arg-C and endoproteinase Gluc-C. Ultracentrifugation studies indicate that AF1/tau1 exists as a monomer in aqueous solution and that the presence of TMAO does not lead to oligomerization or aggregation. It has been suggested that recombinant AF1 binds both the ubiquitous coactivator CBP and the TATA box-binding protein, TBP. Interactions with both of these are greatly enhanced in the presence of TMAO. Co-immunoadsorption experiments indicate that in TMAO each of these and the coactivator SRC-1 are found complexed with AF1. These data indicate that TMAO induces a conformation in AF1/tau1 that is important for its interaction with certain co-regulatory proteins. (+info)
Breast cancer metastatic potential correlates with a breakdown in homospecific and heterospecific gap junctional intercellular communication.
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Breast cancer progresses toward increasingly malignant behavior in tumorigenic and metastatic stages. In the series of events in the metastatic stage, tumor cells leave the primary tumor in breast and travel to distant sites where they establish secondary tumors, or metastases. In this report, we demonstrate that cell-cell communication via gap junctions is restored in the metastatic human breast carcinoma cell line MDA-MB-435 when it is transfected with breast metastasis suppressor 1 (BRMS1) cDNA. Furthermore, the expression profile of connexins (Cxs), the protein subunits of gap junctions, changes. Specifically, the expression of BRMS1 in MDA-MB-435 cells increases Cx43 expression and reduces Cx32 expression, resulting in a gap junction phenotype more similar to normal breast tissue. Taken together, these results suggest that gap junctional communication and the Cx expression profile may contribute to the metastatic potential of these breast cancer cells. (+info)