Protein fluorescence changes associated with ATP and adenosine 5'-[gamma-thio]triphosphate binding to skeletal muscle myosin subfragment 1 and actomyosin subfragment 1.
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ABSTRACT:
SUBMITTER: Millar NC
PROVIDER: S-EPMC1148768 | biostudies-other | 1988 Feb
REPOSITORIES: biostudies-other
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Project description:Electrically permeabilized human neutrophils were used to study the mechanism of activation of the NADPH oxidase by chemotactic factors. The respiratory burst elicited by formyl-methionyl-leucyl-phenylalanine (fMLP) was strictly dependent on the addition of ATP. The response was also supported by adenosine 5'-[gamma-thio]triphosphate (ATP[S]), but not by the non-hydrolysable analogue (p[NH]ppA). In the presence of ATP, displacement of fMLP from its receptor by antagonist peptides resulted in the abrupt termination of the O2-consumption burst. In contrast, the response persisted after displacement of fMLP when ATP[S] was present. This finding is consistent with the formation of biologically active thiophosphoproteins which are resistant to cleavage by cellular phosphatases. Accordingly, lower concentrations of ATP[S], as compared with ATP, were required to support the fMLP response. The data indicate that protein phosphatases control the extent and duration of the response in cells stimulated with chemoattractants. Unlike ATP, sub-millimolar concentrations of ATP[S] elicited a spontaneous respiratory burst in the absence of fMLP or other stimuli. This effect was inhibited by p[NH]ppA and was not observed in intact (non-permeabilized) cells, indicating interaction of ATP[S] with an intracellular adenine-nucleotide-binding site, possibly a protein kinase. These results suggest that protein kinases are active in neutrophils in the absence of exogenous stimuli, but that accumulation of the essential phosphoprotein(s) is normally prevented by the ongoing vigorous phosphatase activity. It is conceivable that control of the respiratory burst is exerted by inhibition of phosphatase activity, instead of or in addition to the more commonly postulated activation of protein kinases.
| S-EPMC1138896 | biostudies-other
Project description:BackgroundTimely detection of cleaning failure is critical for quality assurance within Sterilising Service Units (SSUs). Rapid Adenosine Triphosphate (ATP) testing provides a real time and quantitative indication of cellular contaminants, when used to measure surface or device cleanliness. The aim of this study was to investigate the use of an ATP algorithm and to whether it could be used as a routine quality assurance step, to monitor surgical instruments cleanliness in SSUs prior to sterilisation.MethodsCleanliness monitoring using rapid ATP testing was undertaken in the SSUs of four hospitals located in the western (Amazonia) region of Brazil. ATP testing was conducted (Clean Trace, 3M) on 163 surgical instruments, following manual cleaning. A sampling algorithm using a duplicate swab approach was applied to indicate surgical instruments as (i) very clean, (ii) clean, (iii) equivocal or (iv) fail, based around a 'clean' cut-off of 250 Relative Light Units (RLU) and a 'very clean' <100 RLU.ResultsThe four cleanliness categories were significantly differentiated (P≤0.001). The worst performing locations (hospitals A & C) had failure rates of 39.2% and 32.4%, respectively, and were distinctly different from hospitals B & D (P≤0.001). The best performing hospitals (B & D) had failure rates of 7.7% and 2.8%, respectively.ConclusionThe ATP testing algorithm provides a simple to use method within SSUs. The measurements are in real time, quantitative and useful for risk-based quality assurance monitoring, and the tool can be used for staff training. The four-tiered approach to the grading of surgical instrument cleanliness provides a nuanced approach for continuous quality improvement within SSU than does a simple pass/fail methodology.
| S-EPMC10426997 | biostudies-literature
Project description:BackgroundEx vivo and in vivo detection and imaging of adenosine triphosphate (ATP) is critically important for the diagnosis and treatment of diseases, which still remains challenges up to present.ResultsWe herein demonstrate that ATP could be fluorescently detected and imaged ex vivo and in vivo. In particular, we fabricate a kind of fluorescent ATP probes, which are made of titanium carbide (TC) nanosheets modified with the ROX-tagged ATP-aptamer (TC/Apt). In the constructed TC/Apt, TC shows superior quenching efficiency against ROX (e.g., ~ 97%). While in the presence of ATP, ROX-tagged aptamer is released from TC surface, leading to the recovery of fluorescence of ROX under the 545-nm excitation. Consequently, a wide dynamic range from 1 μM to 1.5 mM ATP and a high sensitivity with a limit of detection (LOD) down to 0.2 μM ATP can be readily achieved by the prepared TC/Apt. We further demonstrate that the as-prepared TC/Apt probe is feasible for accurate discrimination of ATP in different samples including living cells, body fluids (e.g., mouse serum, mouse urine and human serum) and mouse tumor models.ConclusionsFluorescence detection and imaging of ATP could be readily achieved in living cells, body fluids (e.g., urine and serum), as well as mouse tumor model through a new kind of fluorescent ATP nanoprobes, offering new powerful tools for the treatment of diseases related to abnormal fluctuation of ATP concentration.
| S-EPMC8220756 | biostudies-literature
Project description:For the simplest kinetic model the reverse rate constants (k(-1) and k(-2)) associated with ATP binding and cleavage on purified heavy meromyosin and heavy meromyosin subfragment 1 from rabbit skeletal muscle in the presence of 5mm-MgCl(2), 50mm-KCl and 20mm-Tris-HCl buffer at pH8.0 and 22 degrees C are: k(-1)<0.02s(-1) and k(-1)=16s(-1). Apparently, higher values of k(-1) and k(-2) are found with less-purified protein preparations. The values of k(-1) and k(-2) satisfy conditions required by previous (18)O-incorporation studies of H(2) (18)O into the P(i) moiety on ATP hydrolysis and suggest that the cleavage step does involve hydrolysis of ATP or formation of an adduct between ATP and water. The equilibrium constant for the cleavage step at the myosin active site is 9. If the cycle of events during muscle contraction is described by the model proposed by Lymn & Taylor (1971), the fact that there is only a small negative standard free-energy change for the cleavage step is advantageous for efficient chemical to mechanical energy exchange during muscle contraction.
| S-EPMC1177701 | biostudies-other
Project description:Benefits of single molecule studies of biomolecules include the need for minimal amounts of material and the potential to reveal phenomena hidden in ensembles. However, results from recent single molecule studies of fluorescent ATP turnover by myosin are difficult to reconcile with ensemble studies. We found that key reasons are complexities due to dye photophysics and fluorescent contaminants. After eliminating these, through surface cleaning and use of triple state quenchers and redox agents, the distributions of ATP binding dwell times on myosin are best described by 2 to 3 exponential processes, with and without actin, and with and without the inhibitor para-aminoblebbistatin. Two processes are attributable to ATP turnover by myosin and actomyosin respectively, whereas the remaining process (rate constant 0.2-0.5 s-1) is consistent with non-specific ATP binding to myosin, possibly accelerating ATP transport to the active site. Finally, our study of actin-activated myosin ATP turnover without sliding between actin and myosin reveals heterogeneity in the ATP turnover kinetics consistent with models of isometric contraction.
| S-EPMC7806905 | biostudies-literature
Project description:The tryptophan fluorescence of unmodified myosin subfragment 1 (S1) from rabbit and chicken skeletal muscle with various nucleotides and phosphate analogues bound was measured after rapid temperature jumps. The fluorescence decreased during the temperature rise. Under some conditions, this decrease was followed by an increase, reflecting structural transitions within the protein. With adenosine 5'-[beta,gamma-imido]triphosphate (p[NH]ppA) or with ADP and BeF(x) bound, this rise was very rapid (reciprocal time constant approx. 2000 s(-1)) and varied only slightly with starting temperature, suggesting that, with these ligands, two different protein conformations were present in rapid equilibrium over a large temperature range. In the presence of ATP, the transient included several relaxation processes. Overall, the results suggest that complexes of S1 with ATP or with a number of other ligands exist as a mixture of two forms in temperature-dependent equilibrium. The results throw light on the finding of different forms of S1 in recent crystallographic studies and indicate a surprising lack of strong coupling between myosin's structural state and the nature of the nucleotide bound.
| S-EPMC1222044 | biostudies-other
Project description:The addition of Ca2+, adenosine 5'-[gamma-thio]triphosphate (ATP[S]) or guanosine 5'-[gamma-thio]triphosphate (GTP[S]) to digitonin-permeabilized PC12 cells stimulates noradrenaline secretion. Both ATP[S] and GTP[S] stimulate release in the absence of Ca2+. Whereas ADP and adenosine 5'-[beta gamma-imido]triphosphate inhibited ATP[S]-stimulated release, they did not inhibit Ca(2+)-stimulated release even in the absence of added ATP. This suggests that the kinase which uses ATP[S] to induce secretion may not play an essential role in Ca(2+)-stimulated release. As GTP[S]-stimulated and ATP[S]-stimulated secretions were not additive, it seemed possible that stimulation by ATP[S] might in part result from the thiophosphorylation of GDP by nucleoside-diphosphate (NDP) kinase to form GTP[S]. The following results are consistent with this possibility. (1) A low concentration of GDP increased ATP[S]-stimulated secretion, but not GTP[S]-stimulated or Ca(2+)-stimulated secretion. (2) A variety of ribo- and deoxyribo-nucleoside di- and tri-phosphates inhibited ATP[S]-stimulated secretion, but not GTP[S]-stimulated or Ca(2+)-stimulated secretion. Thus, like NDP kinase, the kinase which uses ATP[S] to cause noradrenaline release appears to have a very low specificity for ATP. (3) Incubation of permeabilized cells in a sucrose-containing buffer resulted in the preferential loss of ATP[S]-stimulated secretion and a decrease in the level of NDP kinase. The addition of rat liver NDP kinase to those depleted cells partially restored ATP[S]-stimulated secretion.
| S-EPMC1137670 | biostudies-other
Project description:The data presented here confirm and provide further experimental evidence that rabbit skeletal-muscle myosin subfragment-1 (S-1) binds to the postulated actin-(338-348) hydrophobic segment [Kabsch, Mannherz, Suck, Pai and Holmes (1990) Nature (London) 347, 37-44] with high affinity in the absence and presence of MgATP. The apparent dissociation constant of the S-1 interaction (5.5 x 10(-7) M) with the actin-(338-348) peptide was of the same order of magnitude as that of the actin-(18-28) binding site (2 x 10(-6) M). In similar conditions, fragmented (27 kDa-50 kDa-20 kDa) S-1 also bound to the peptide. Antibodies directed to the vicinal sequence 348-358 were rapidly eliminated from actin by S-1 interaction and weakened S-1 binding to monomeric or filamentous actin. The antigenic site (348-358) is located very close to the C-terminal S-1-binding site (360-369) and encompasses some residues (Leu-349 and Phe-352) included in the hydrophobic S-1-binding region [Schröder, Manstein, Jahn, Holden, Rayment, Holmes and Spudich (1993) Nature (London) 364, 171-174]. It was observed that anti-[actin-(348-358)] antibodies were also unable to decrease actomyosin ATPase activity, in contrast with previous results obtained with anti-[actin-(18-28)] antibodies [Adams and Reisler (1993) Biochemistry 32, 5051-5056]. The hydrophobic actin-(338-348) peptide used in considerable excess was unable to perturb acto-S-1 and S-1 activities in contrast with results obtained with the N-terminal actin peptide [Kôgler, Moir, Trayer and Ruegg (1991) FEBS Lett. 294, 31-34].
| S-EPMC1138102 | biostudies-other
Project description:Cell-cell adhesions are important sites through which cells experience and resist forces. In endothelial cells, these forces regulate junction dynamics and determine endothelial barrier strength. We identify the Ig superfamily member EMMPRIN (also known as basigin) as a coordinator of forces at endothelial junctions. EMMPRIN localization at junctions correlates with endothelial junction strength in different mouse vascular beds. Accordingly, EMMPRIN-deficient mice show altered junctions and increased junction permeability. Lack of EMMPRIN alters the localization and function of VE-cadherin (also known as cadherin-5) by decreasing both actomyosin contractility and tugging forces at endothelial cell junctions. EMMPRIN ensures proper actomyosin-driven maturation of competent endothelial junctions by forming a molecular complex with γ-catenin (also known as junction plakoglobin) and Nm23 (also known as NME1), a nucleoside diphosphate kinase, thereby locally providing ATP to fuel the actomyosin machinery. These results provide a novel mechanism for the regulation of actomyosin contractility at endothelial junctions and might have broader implications in biological contexts such as angiogenesis, collective migration and tissue morphogenesis by coupling compartmentalized energy production to junction assembly.
| S-EPMC4150062 | biostudies-literature
Project description:Polymorphonuclear neutrophils release ATP in response to stimulation by chemoattractants, such as the peptide N-formyl-methionyl-leucyl-phenylalanine. Released ATP and the hydrolytic product adenosine regulate chemotaxis of neutrophils by sequentially activating purinergic nucleotide and adenosine receptors, respectively. Here we show that that ecto-nucleoside triphosphate diphosphohydrolase 1 (E-NTPDase1, CD39) is a critical enzyme for hydrolysis of released ATP by neutrophils and for cell migration in response to multiple agonists (N-formyl-methionyl-leucyl-phenylalanine, interleukin-8, and C5a). Upon stimulation of human neutrophils or differentiated HL-60 cells in a chemotactic gradient, E-NTPDase1 tightly associates with the leading edge of polarized cells during chemotaxis. Inhibition of E-NTPDase1 reduces the migration speed of neutrophils but not their ability to detect the orientation of the gradient field. Studies of neutrophils from E-NTPDase1 knock-out mice reveal similar impairments of chemotaxis in vitro and in vivo. Thus, E-NTPDase1 plays an important role in regulating neutrophil chemotaxis by facilitating the hydrolysis of extracellular ATP.
| S-EPMC2568933 | biostudies-literature