Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.3.1 (Mg2+-ATPase)
1,484 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The specific activity of the Mg2+-ATPase and the (Ca2+ + Mg2+)-ATPase has been measured in a microsomal fraction from pig antral smooth muscle with the phosphate-release assay and the NADH-coupled enzyme assay, and the release of inorganic phosphate as a function of time is compared with the concomitant production of ADP. Both assays are found to overestimate the true Mg2+-ATPase activity. The adenylate kinase inhibitor P1,P5-di(adenosine-5'-)pentaphosphate (Ap5A) reduces the specific activity of the Mg2+-ATPase measured in the NADH-coupled enzyme assay to about half of its original value; however, it does not affect the specific activity of the Mg2+-ATPase in the Pi-release assay. The considerable overestimation of the Mg2+-ATPase activity in the NADH-coupled enzyme assay results from a combined action of an ATP pyrophosphatase (ATP in equilibrium AMP + PPi) and adenylate kinase activity contaminating the microsomes. The adenylate kinase activity in the microsomes catalyses the conversion of AMP formed by the ATP pyrophosphatase together with ATP into two ADP's. Also the phosphate-release assay is prone to an overestimation artefact because an inorganic pyrophosphatase will degrade the pyrophosphate and thus lead to additional Pi-production. Measurements of AMP and NAD+ production by HPLC confirmed our proposed reaction scheme. The same (Ca2+ + Mg2+)-ATPase activity is found in both assays, because the (Ca2+ + Mg2+)-ATPase activity is calculated from the difference in ATPase activity in the presence and absence of Ca2+, so that as a consequence the interfering activities are automatically subtracted.
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PMID:Measurement of microsomal ATPase activities: a comparison between the inorganic phosphate-release assay and the NADH-coupled enzyme assay. 253 60

The apparent Mg2+-activated ATPase activity measured by the continuous NADH-coupled enzyme assay was studied in a number of microsomal preparations obtained from smooth muscle of the myometrium from pregnant or 17 beta-oestradiol-pretreated rats, the bovine aorta, the guinea-pig taenia coli, the rabbit ear artery and pig antrum. It was shown that this ATPase assay is prone to the effects of a number of artefacts that are tissue-dependent. The apparent Mg2+-ATPase activity in microsomes (microsomal fractions) from myometrium, aorta and taenia coli declines non-linearly during the assay. Its initial high rate gradually diminishes over 15-60 min, depending on the type of smooth muscle, to a constant value. This decline depends on the presence of ATP and can be partially prevented by concanavalin A. The non-linearity is limited in microsomes from rabbit ear artery. In microsomes from antrum the apparent Mg2+-ATPase activity actually increases with time, albeit gradually. Storage on ice of the microsomes of the aorta, and especially of myometrium of pregnant rats and of taenia coli, is accompanied over a few hours after their preparation by a gradual suppression of the component of the Mg2+-ATPase activity that is inhibited by ATP. The Mg2+-ATPase activity in microsomes from antrum remains constant. NADH oxidase activity accounts for 10% of the Mg2+-ATPase activity in microsomes from stomach smooth muscle. The apparent initial non-linearity of the Mg2+-ATPase activity in that tissue is due to a time-dependent decrease of a rotenone-sensitive NADH oxidase activity. The adenylate kinase activity, as deduced from the effect of the adenylate kinase inhibitor P1,P5-di(adenosine-5') pentaphosphate, could account for 45.0, 35.0 and 31.0% respectively of the Mg2+-ATPase activity in microsomes from stomach, myometrium and aorta. No adenylate kinase activity could be detected in microsomes from ear artery and taenia coli. When microsomes from stomach smooth muscle were separated on a sucrose gradient, the contribution of adenylate kinase and NADH oxidase to the Mg2+-ATPase activity was most pronounced in the higher-density fractions. Part of the NADH oxidase activity and of the Mg2+-ATPase activity, and most of the adenylate kinase activity, are not sedimented at 224000 gmax. for 30 min and may therefore be present as soluble enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Characterization of the Mg2+-activated ATPase activity in smooth-muscle membranes. NADH oxidase and adenylate kinase interfere with the NADH-coupled enzyme assay. 283 48

After 10 wash cycles, 0.8 u.e. of creatine kinase activity remained bound per mg of chicken pectoralis myofibrils which had been freed of soluble creatine kinase, mitochondria, and membranes. The bound creatine kinase is located at the M-band and contributes to the electron density of this sarcomeric structure (Wallimann, T., Pelloni, G.W., Turner, D.C., and Eppenberger, H. M. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 4296-4300). By measuring the combined actin-activated Mg2+-ATPase and creatine kinase reactions of myofibrils by pH-stat, it was shown that the amount of M-line-bound creatine kinase activity was sufficient to rephosphorylate the ATP hydrolyzed in vitro by the actin-activated Mg2+-ATPase. The amount of M-line-bound creatine kinase and thus the ATP regeneration potential depended on the muscle type. It was higher in fast muscles and lower in slow muscles. Inhibition of myofibrillar creatine kinase or extraction of the M-line-bound enzyme abolished the ATP regeneration potential without affecting ATPase activity. Inhibitors of myokinase, mitochondrial ADP/ATP translocase, and respiration did not affect the ATP regeneration potential or the ATPase. M-line-bound creatine kinase, sufficient to support an ATP turnover rate of 6s-1 per myosin head, seems to have the capacity for the intramyofibrillar regeneration of most or all of the ATP hydrolyzed by the myofibrillar ATPase during muscle contraction. Thus, M-line-bound creatine kinase at the myofibrillar receiving end of the phosphorylcreatine shuttle is of physiological significance.
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PMID:Function of M-line-bound creatine kinase as intramyofibrillar ATP regenerator at the receiving end of the phosphorylcreatine shuttle in muscle. 614 55

ATP-binding cassette (ABC) transporters, a superfamily of integral membrane proteins, catalyse the translocation of substrates across the cellular membrane by ATP hydrolysis. Here we demonstrate by nucleotide turnover and binding studies based on ³¹P solid-state NMR spectroscopy that the ABC exporter and lipid A flippase MsbA can couple ATP hydrolysis to an adenylate kinase activity, where ADP is converted into AMP and ATP. Single-point mutations reveal that both ATPase and adenylate kinase mechanisms are associated with the same conserved motifs of the nucleotide-binding domain. Based on these results, we propose a model for the coupled ATPase-adenylate kinase mechanism, involving the canonical and an additional nucleotide-binding site. We extend these findings to other prokaryotic ABC exporters, namely LmrA and TmrAB, suggesting that the coupled activities are a general feature of ABC exporters.
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PMID:Coupled ATPase-adenylate kinase activity in ABC transporters. 2800 95

The ATP-binding cassette (ABC) transporter MsbA is an ATP-driven lipid-A flippase. It belongs to the ABC protein superfamily whose members are characterized by conserved motifs in their nucleotide binding domains (NBDs), which are responsible for ATP hydrolysis. Recently, it was found that MsbA could catalyze a reverse adenylate kinase (rAK)-like reaction in addition to ATP hydrolysis. Both reactions are connected and mediated by the same conserved NBD domains. Here, the structural foundations underlying the nucleotide binding to MsbA were therefore explored using a concerted approach based on conventional- and DNP-enhanced solid-state NMR, pulsed-EPR, and MD simulations. MsbA reconstituted into lipid bilayers was trapped in various catalytic states corresponding to intermediates of the coupled ATPase-rAK mechanism. The analysis of nucleotide-binding dependent chemical shift changes, and the detection of through-space contacts between bound nucleotides and MsbA within these states provides evidence for an additional nucleotide-binding site in close proximity to the Q-loop and the His-Switch. By replacing Mg²⁺ with Mn²⁺ and employing pulsed EPR spectroscopy, evidence is provided that this newly found nucleotide binding site does not interfere with the coordination of the required metal ion. Molecular dynamic (MD) simulations of nucleotide and metal binding required for the coupled ATPase-rAK mechanism have been used to corroborate these experimental findings and provide additional insight into nucleotide location, orientation, and possible binding modes.
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PMID:Unexplored Nucleotide Binding Modes for the ABC Exporter MsbA. 3028 53

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