The nature of inactivation of rat muscle 5'-adenylate aminohydrolase by fluorodinitrobenzene.
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ABSTRACT:
SUBMITTER: Raggi A
PROVIDER: S-EPMC1165202 | biostudies-other | 1975 Feb
REPOSITORIES: biostudies-other
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Project description:Reaction of rat muscle AMP deaminase with low molar excess of tetranitromethane results in a rapid loss of free thiol groups and a concomitant decrease in enzyme activity at high, but not at low, AMP concentration. This modification appears to be limited to the same non-essential thiol groups reactive towards specific reagents in non-denaturing conditions. On incubation with higher molar excess of tetranitromethane, a loss of enzyme activity is observed, which correlates with nitration of tyrosine residues. By amino acid analysis, approximately there tyrosine residues per subunit are estimated to be nitrated in the completely inactivated enzyme. The kinetic properties of the partially inactivated AMP deaminase reveal a negative co-operatively behaviour at approximately half saturation. This suggests that modification of tyrosine residues is also responsible for alteration of the binding properties of the hypothesized activating site of AMP deaminase.
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Project description:The direct effects of pituitary adenylate cyclase-activating polypeptides (PACAP) on sympathetic neurons were investigated using rat superior cervical ganglion neurons. Electrophysiological and pharmacological analyses were used to evaluate PACAP modulation of sympathetic neuron membrane potentials and to investigate potential ionic and intracellular signaling mechanisms mediating the responses. More than 90% of the sympathetic neurons were depolarized by the PACAP peptides even when stimulated release was blocked, indicating that the PACAP peptides elicited primary responses in the postganglionic neurons. The response profile was consistent for activation of PACAP-selective PAC(1) receptors: nanomolar concentrations of PACAP27 and PACAP38 were required to stimulate depolarization, whereas vasoactive intestinal peptide failed to evoke any response. Furthermore, depolarizations elicited by PACAP27 were reduced by the PAC(1) receptor antagonist PACAP(6-38). Both sodium influx and inhibition of a potassium current contributed to the peptide-induced depolarizations. Activation of neither pertussis toxin- nor cholera toxin-sensitive G-proteins was required for generation of the depolarizations. cAMP and diacylglycerol production and activation of protein kinase A or protein kinase C also were not requisite for the responses. By contrast, phospholipase C (PLC)-dependent inositol 1,4,5-triphosphate (IP(3)) synthesis was crucial to the PACAP-mediated depolarizations. Although calcium release from IP(3)-sensitive stores was not required for the PACAP-induced responses, inhibition of IP(3) receptors reduced the depolarizations. Thus, among the many signal transduction pathways coupled to the PAC(1) receptor, the PACAP-induced depolarization of sympathetic neurons appears to require activation of PLC and subsequent generation of IP(3).
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Project description:Site I inactivation of calmodulin (CaM) was used to examine the importance of aspartic acid 22 at position 3 in CaM calcium binding, protein folding, and activation of the Bordetella pertussis adenylate cyclase toxin domain (CyaA-ACD). NMR calcium titration experiments showed that site I in the CaM mutant (D22A) remained largely unperturbed, while sites II, III, and IV exhibited calcium-induced conformational changes similar to wild-type CaM (CaMWt). Circular dichroism analyses revealed that D22A had comparable ?-helical content to CaMWt, and only modest differences in ?-helical composition were detected between CaMWt-CyaA-ACD and D22A-CyaA-ACD complexes. However, the thermal stability of the D22A-CyaA-ACD complex was reduced, as compared to the CaMWt-CyaA-ACD complex. Moreover, CaM-dependent activity of CyaA-ACD decreased 87% in the presence of D22A. Taken together, our findings provide evidence that D22A engages CyaA-ACD, likely through C-terminal mediated binding, and that site I inactivation exerts functional effects through the modification of stabilizing interactions that occur between N-terminal CaM and CyaA-ACD.
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Project description:In response to the enormous threat to human survival and development caused by the large number of viruses, it is necessary to strengthen the defense against and elimination of viruses. Metallic materials have been used against viruses for thousands of years due to their broad-spectrum antiviral properties, wide sources and excellent physicochemical properties; in particular, metal nanoparticles have advanced biomedical research. However, researchers in different fields hold dissimilar views on the antiviral mechanisms, which has slowed down the antiviral application of metal nanoparticles. As such, this review begins with an exhaustive compilation of previously published work on the antiviral capacity of metal nanoparticles and other materials. Afterwards, the discussion is centered on the antiviral mechanisms of metal nanoparticles at the biological and physicochemical levels. Emphasis is placed on the fact that the strong reducibility of metal nanoparticles may be the main reason for their efficient inactivation of viruses. We hope that this review will benefit the promotion of metal nanoparticles in the antiviral field and expedite the construction of a barrier between humans and viruses.
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Project description:A unique sequence within the giant titin molecule, the PEVK domain, has been suggested to greatly contribute to passive force development of relaxed skeletal muscle during stretch. To explore the nature of PEVK elasticity, we used titin-specific antibodies to stain both ends of the PEVK region in rat psoas myofibrils and determined the region's force-extension relation by combining immunofluorescence and immunoelectron microscopy with isolated myofibril mechanics. We then tried to fit the results with recent models of polymer elasticity. The PEVK segment elongated substantially at sarcomere lengths above 2.4 micro(m) and reached its estimated contour length at approximately 3.5 micro(m). In immunofluorescently labeled sarcomeres stretched and released repeatedly above 3 micro(m), reversible PEVK lengthening could be readily visualized. At extensions near the contour length, the average force per titin molecule was calculated to be approximately 45 pN. Attempts to fit the force-extension curve of the PEVK segment with a standard wormlike chain model of entropic elasticity were successful only for low to moderate extensions. In contrast, the experimental data also could be correctly fitted at high extensions with a modified wormlike chain model that incorporates enthalpic elasticity. Enthalpic contributions are likely to arise from electrostatic stiffening, as evidenced by the ionic-strength dependency of titin-based myofibril stiffness; at high stretch, hydrophobic effects also might become relevant. Thus, at physiological muscle lengths, the PEVK region does not function as a pure entropic spring. Rather, PEVK elasticity may have both entropic and enthalpic origins characterizable by a polymer persistence length and a stretch modulus.
| S-EPMC20927 | biostudies-literature
Project description:Bordetella pertussis produces a calmodulin-dependent adenylate cyclase (AC) which acts as a toxin capable of penetrating eukaryotic cells and generating high levels of intracellular cyclic AMP. Transfer of target cells into B. pertussis AC-free medium leads to a rapid decay in the intracellular AC activity, implying that the invasive enzyme is unstable in the host cytoplasm. We report here that treatment of human lymphocytes with a glycolysis inhibitor and an uncoupler of oxidative phosphorylation completely blocked the intracellular inactivation of B. pertussis AC. Lymphocyte lysates inactivated all forms of B. pertussis AC in the presence of exogenous ATP. This inactivation was associated with degradation of an 125I-labelled 200 kDa form of B. pertussis AC. It appears that ATP is required for the proteolytic pathway, but not as an energy source, since non-hydrolysable ATP analogues supported inactivation and complete degradation of the enzyme. The possibility that binding of ATP to B. pertussis AC renders it susceptible to degradation by the host cell protease is discussed.
| S-EPMC1133224 | biostudies-other
Project description:Oxidative stress and adenine nucleotide catabolism occur concomitantly in several disease states, such as cardiac ischaemia-reperfusion, and may act as synergistic determinants of tissue injury. However, the mechanisms underlying this potential interaction remain ill-defined. We examined the influence of oxidative stress on the molecular, kinetic and regulatory properties of a ubiquitous AMP-catabolizing enzyme, adenylate deaminase (AMPD) (EC 3.5.4.6). To this intent, rabbit heart AMPD and an H2O2/ascorbate/iron oxidation system were employed. Enzyme exposure to the complete oxidation system acutely impaired its catalytic activity, lowered the Vmax. by 7-fold within 5 min, and rendered the enzyme unresponsive to nucleotide effectors. Irreversible AMPD inactivation resulted within about 15 min of oxidative insult and was not prevented by free-radical scavengers. Oxidative stress did not affect the molecular mass, tetrameric nature, Km, immunoreactivity or trypsinolytic pattern of the enzyme; nor did it induce carbonyl formation, Zn2+ release from the holoenzyme or net AMPD S-thiolation. This injury pattern is inconsistent with a radical-fragmentation mechanism as the basis for the oxidative AMPD inactivation observed. Rather, the sensitivity of the enzyme to both S-thiolation and thiol alkylation and the significant (3 of 9/mol of denatured enzyme) net loss of DTNB-reactive thiols on exposure to oxidant strongly implicate the conversion of essential thiol moieties into stable higher-oxidation states in the oxidative inactivation of cardiac AMPD. The altered thiol status of the enzyme on oxidative insult may prohibit a catalytically permissible conformation and, in so doing, increase AMP availability to 5'-nucleotidase in vivo.
| S-EPMC1136536 | biostudies-other
Project description:Skeletal muscle accounts for the largest proportion of human body mass, on average, and is a key tissue in complex diseases and mobility. It is composed of several different cell and muscle fiber types. Here, we optimize single-nucleus ATAC-seq (snATAC-seq) to map skeletal muscle cell-specific chromatin accessibility landscapes in frozen human and rat samples, and single-nucleus RNA-seq (snRNA-seq) to map cell-specific transcriptomes in human. We additionally perform multi-omics profiling (gene expression and chromatin accessibility) on human and rat muscle samples.
| EGAS00001005730 | EGA
Project description:Binding assays and assays of activation of adenylate cyclase with the agonists 5'-N-ethylcarboxyamidoadenosine (NECA) and CGS21680 have been used to compare adenosine receptors in rat pheochromocytoma PC12 cells and in rat striatum. The [3H]NECA binding showed two components, whereas [3H]CGS21680 bound to one component in both tissues. The Kd value for the high affinity site labeled with [3H]NECA in PC12 cell membranes (2.3 nM) was lower than that in striatum (6.5 nM). The [3H]CGS21680 binding site showed a Kd value of 6.7 nM and 11.3 nM in PC12 cells and striatum, respectively. In the presence of GTP the KD values of [3H]NECA and [3H]CGS21680 for the high affinity site were increased severalfold, whereas the low affinity sites for [3H]NECA were no longer detected with filtration assays. A comparison of the ability of a series of agonists and antagonists to inhibit high affinity binding of [3H]NECA to A2 receptors in PC12 cell and striatal membranes indicated that agonists had higher affinities and antagonists had lower affinities in PC12 cells, compared with affinities in striatal membranes. Analysis of activation of adenylate cyclase in PC12 cell membranes suggested that the dose-dependent stimulation by NECA involved two components, whereas CGS21680 stimulated via one component. The maximal stimulation by NECA significantly exceeded that caused by CGS21680. In intact PC12 cells, NECA caused a greater accumulation of AMP than did CGS21680, as was the case in membranes. In striatal membranes, NECA and CGS21680 showed similar maximal stimulations of adenylate cyclase. Both NECA and CGS21680 were more potent in PC12 cell membranes than in striatal membranes, in agreement with binding data. However, in contrast to binding data, antagonists were not less potent versus stimulation of adenylate cyclase by NECA or CGS21680 in PC12 cell membranes, compared with striatal membranes. In toto, the results suggest that A2A receptors in striatum are virtually identical to the A2A receptors in PC12 cells. But, in addition to an A2A receptor, it appears that a lower affinity functional receptor, probably an A2B receptor, is present in PC12 cells and PC12 cell membranes, whereas such a functional low affinity receptor is not detectable in striatal membrane.
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Project description:1. The solubilization and partial purification of a proteinase from the intestinal smooth muscle of rats fed on protein-free diets are described. 2. It has a mol.wt. of about 33000 and it is stable over a narrow pH range. 3. From its susceptibility to known modifers of proteolytic enzymes, it appears to be a serine proteinase of a trypsin-like nature. Active-site titration with soya-bean trypsin inhibitor shows that the concentration of proteinase was about 3 microgram/g wet wt. of intestinal smooth muscle. However, the muscle proteinase demonstrates a marked ability for inactivating enzymes in their native conformation at neutral pH. It is about 100 times more efficient than pancreatic trypsin when the inactivating activities are compared on an approximately equimolar basis. 4. Inactivation of the substrate enzymes is accompanied by limited proteolysis, as demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 5. An endogenous inhibitor was separated from the proteinase by fractionation with (NH4)2SO4. 6. Contamination of the muscle tissue by lumen, mucosal or blood proteinases and inhibitors is shown to be unlikely. 7. A role for the neutral trypsin-like proteinase in initiating the degradation of intracellular enzymes is considered.
| S-EPMC1185773 | biostudies-other