ATPase activity assay

P-gp-ATPase Activation Assays H-Bonding Determines Activation Rate... 

Fig. 9. (Panel A) ATPase activity assay in the rat homogenate expressed as pMol min-1 mg-1 protein, either in the controls or in the presence of different concentrations of Al(III). Bars SD P< 0.01. (Panel B) ATPase activity assay in rat liver mitochondria expressed as pMol min-1 mg-1 protein, in the controls or in presence of 9 pM Al(III). Bars SD. P<0.01. Data are from Zatta et al. (1995) [34]...

Evaluation of the brewery CIP system and its effectiveness is usually determined using off-line laboratory analysis or portable measuring devices. This typically involves dyes such as riboflavin or ATPase activity assays used to determine the presence of hving microorganisms. 

Assays. Protein concentrations were measured by the method of Bradford (18) and the various contractile protein ATPase activities by tRe method of Martin and Doty (19). Gel electrophoresis was carried out by the method of Ames (20) on 1.5 ran polyacrylamide slabs using the discontinuous SDS buffer system of Laemnli (21). Dried gels were scanned at 550 nm for densiometry measurements. 

I. H+-ATPase, H+-PPase, and UDPase. Enzyme activities of vanadate- or nitrate-sensitive H+-ATPase, H+-PPase, and UDPase are measured by the quantitation of released inorganic phosphate from the substrate by enzymatic hydrolyzation. When H+-ATPase activities are assayed, at least three substrate mixtures should be prepared (1) a substrate mixture containing nitrate, (2) a substrate mixture containing vanadate, and... 

A variety of in vitro assays are available to identify compounds as substrates and inhibitors of P-gp. These assays, which have been reviewed elsewhere in great detail [20-24], can be classified into three general categories (1) transport, (2) accumulation/ efflux and (3) ATPase activity [20-28]. It is important to note that these in vitro model systems can be adapted for measuring the interaction of dmgs with other important drug transporter systems [22]. 

Effects on Mg -ATPase activity were determined with mitochondria that were ruptured by freezing (-20°C) and subsequent thawing (room temperature). The assay medium and procedure was essentially that of Blackmon and Moreland (18) except that DNP was... 

ATPase activity was also studied by Friebe et al. in 1997.17 They correlated the BOA and DIBOA effects on radicle elongation of Avena sativa seedlings with their effects on the activity of plasma membrane H+-ATPase from roots of Avena sativa cv. Jumbo and from Vida faba cv. Alfred. They hypothesized that an alteration in the plasma membrane ATPase activity could be the reason for an abnormal nutrient absorption in plants exposed to hydroxamic acids, because of the role that this enzyme plays in the ion gradient and, therefore, in the ionic transport through plasma membrane. The results of this experiment showed a strong inhibition in the activity of this enzyme in the plasma membrane of chloroplast and mitochondria when it was exposed to BOA and DIMBOA. This alteration implies early interactions with the assayed hydroxamic acids. 

Kashem, M.A. et al. 2007. Three mechanistically distinct kinase assays compared measurement of intrinsic ATPase activity identified the most comprehensive set of ITK inhibitors. J. Biomol. Screen. 12, 70-83. 

Rowlands, M.G. et al. 2004. High-throughput screening assay for inhibitors of heat-shock protein 90 ATPase activity. Anal. Biochem. 327, 176-183. 

A variety of ATPase activities are present in biological systems. This assay is novel in that it uses hydrophilic interaction chromatography to measure release of labeled 32P from [y-32P ATP. 

The assay was used in inhibition studies of Fi-ATPase partially purified from rat liver mitochondria, and the ATPase activity of plasma membranes. 

A substantial number of observations have demonstrated that the stress-70 proteins bind denatured proteins, as well as some short peptides, and that ATP hydrolysis (possibly with concomitant release of Pj or ADP) results in the release of bound peptides (de Silva et ai, 1990 Hendershot, 1990 Hurtley 1989 Kassenbrock 1988 Palleros etai, 1991). Conversely, bindingof peptides to stress-70 proteins induces ATPase activity above basal levels peptide-induced ATPase activity is often used as a facile in vitro assay for stress-70 protein activity. Schematically, this activity can be subdivided into (1) ATP binding and hydrolysis, followed by product release (P and ADP), (2) peptide binding and release, and (3) a mechanism of coupling peptide binding/release and nucleotide hydrolysis/product release. 

Hence, although the specific numerical values reported for ATPase rates of stress-70 proteins show some variation, possibly attributable to differences in specific protein preparation and assay procedures utilized by different individuals, the consensus scheme that these data show is that in the absence of substrate peptide, the stress-70 proteins have a low basal ATPase rate, typically found to be 0.01-0.03 (mol ATP/mol stress-70 protein min). This can be enhanced severalfold either by binding of peptides or denatured proteins, or (as demonstrated by the effect of grpE and dnaJ proteins on dnaK) by the action of ancillary proteins. To the extent that the observations on HSC70 can be generalized to other members of the stress-70 protein family, peptide binding appears to relieve the attenuation of ATPase activity and allow it to proceed at the rate characteristic of the ATPase fragment of the protein alone. 

An ATPase isolated from H. salinarium (halobium) superficially resembles the enzyme from H. saccharovorum. The purified enzyme (Mr 320 000) is composed of two subunits (Mr 86 000 and 64 000) but there is no evidence that other subunits are associated with the enzyme [25]. The purified enzyme is most active when assayed in the presence of 2M (NH4)2S04 and is about twice as active when assayed in the presence of 1.5 M Na2S03. Little if any activity is detected when the enzyme is assayed in the presence of 4M NaCl or KCl. ATPase activity is not affected by azide (1 mM) but is inhibited by NEM (1 mM), DCCD (300 pM), and NBD-Cl (500 p,M). The inhibition by NBD-Cl is antagonized by ADP. The enzyme from H. salinarium (halobium) rapidly loses activity when incubated at room temperature in absence of NaCl and slowly... 

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