Membrane bound ATPase

What is the principal difference between the ATPase activity of F, in solution and the membrane-bound ATPase activity apart from the pro-tontransfer function ... 

Manganese(II) and Gadolinium(III) EPR Studies of Membrane-Bound ATPases... 

The activation and inactivation of the membrane-bound ATPase occur also in vivo and can be demonstrated in intact chloroplasts. Here, a thiol reductant need not be added, since the photochemically reduced protein, thioredoxin, seems to fulfill this function [36]. 

The activated membrane-bound ATPase is functionally coupled to proton movements. Thus, a transmembrane pH gradient (acid inside) of a magnitude similar to that observed during light-induced coupled electron flow is developed during ATP hydrolysis. ATP hydrolysis is stimulated, while the coupled proton transport is inhibited, by the addition of uncouplers, indicating that the rate of ATP hydrolysis is also partially limited by the electrochemical gradient which it creates. Nevertheless, attempts to measure H /ATP ratios in this system yielded numbers much below the expected ratio of 3. 

Figure 6.3 (Right) Schematic depiction of anammox cell showing the anammoxozome and nucleoid. (Left) Postulated pathway of anaerobic ammonium oxidation coupled to the ana-mmoxosome membrane resulting in a proton motive force and ATP synthesis via membrane-bound ATPases. HH, hydrazine hydrolase HZO, hydrazine oxidizing enzyme NIR, nitrite reductase. (Redrawn from van Niftrik etal., 2004 and Kuypers et al., 2006).

P-type ATPase. Methanococcus voltae, a marine organism growing on H2/CO2, contains high activities of a membrane-bound ATPase, which was sensitive towards vanadate rather than to DCCD [147], suggesting the presence of a P-type ATPase (see ref [141]). Accordingly the purified enzyme, composed of one 74 kD subunit, could be phosphorylated in a vanadate-sensitive fashion [148], a characteristic property for P-type ATPases, which involve a phosphoprotein as intermediate in the catalytic cycle. 

The ATPase from H. halobium is more firmly membrane-bound than is the enzyme from H. saccharovorum, and solubilization of the former requires treating membranes with EDTA before sonication [25]. EDTA by itself is effective for solubilizing the ATPases from Methanosarcina barkeri [26], Methanolobus tindarius [27], and S. acidocaldarius [2%]. On the other hand, the membrane-bound ATPase from Methanobacterium thermoauto trophicum requires extraction with EDTA followed by... 

The membrane-bound archaeal ATPases have attracted considerable interest since patterns of inhibitor sensitivity, subunit structure, and amino-acid sequence homologies suggest a close relationship to the V-type ATPases [3,41]. In addition, the membrane-bound ATPases from S. acidocaldarius, H. salinarium (halobium), Methanosarcina barken, and the V-type ATPase from Saccharomyces cerevisiae are immunologically related [42]. 

The membrane-bound ATPase from M barkeri is inhibited by DCCD and the inhibitor is bound to a small hydrophobic peptide (Mr 6000). A DCCD-sensitive ATPase, solubilized by octylglucoside, has been purified in the presence of the detergent. This form of the ATPase contains six subunits (Mr 62000, 49000, 40000, 27000, 23000, and 6000). When incubated with C-DCCD, all the radioactivity is associated with the Mr 6000 subunit. [56]. This subunit is smaller than the DCCD-binding peptides from S. acidocaldarius [57] and coated vesicles [58]. The function of the Mr 6000 peptide has yet to be establishe4 although its properties suggest that it may be equivalent to the subunit c, the DCCD-binding peptide of F-type ATPases. 

Several membrane-bound ATPases occur in the genus Sulfolobus. There are two ATP-hydrolyzing activities in S. acidocaldarius strain 7. One has a pH optimum at 6.5 in the absence of sulfate, and the presence of that anion activates the enzyme and shifts the pH optimum to 5.0. ATP hydrolysis is unaffected by DCCD, azide, NEM, /7-hydroxymercuribenzoate, or vanadate [59]. The other ATPase is most active at pH 2.5, is inhibited by sulfate, and appears to be a pyrophosphatase [16]. The purified sulfate-activated ATPase (M, 360000) is composed of three subunits (Mr 69000, 54000, and 28000). It is most active at 85 C, stimulated some three-fold by sulfate, sulfite, and bicarbonate, but is unaffected by chloride. There are two pH optima. One is located at pH 5 and the other at pH 8.5 and neither is affected by sulfite. ATPase activity is inhibited by nitrate (63% at 20 mM) and NBD-Cl (90% at 1 mM) but is not significantly affected by azide (5mM), vanadate (100 pM), and NEM (100pM)[28]. 

The membrane-bound ATPase from H. saccharovorum exhibits different properties when solubilized with Triton X-100 and subsequently purified in the... 

N,N -Dicyclohexylcarbodiimide (DCCD), an inhibitor of membrane bound ATPase, has been shown to strongly inhibit IAA-induced elongation of cucumber hypocotyl sections, while it has no effect on GA-induced elongation (36). DCCD markedly inhibits BR-induced elongation (8), suggesting that BR acts differently from GA, but similarly to IAA in this particular case. 

Inatomi K-J (1986) Characterization and purification of the membrane-bound ATPase of the archaebacterium Methanosarcina barkeri. J Bacteriol 167 837-841 Ingledew WJ (1982) Thiobacillus ferrooxidans. The bioenergetics of an acidophilic chemolitho-autotroph. Biochim Biophys Acta 683 89-117... 

Tsong, T. Y. Astumian, R. D. Electroconformational coupling An efficient mechanism for energy transfection by membrane-bound ATPases. Ann. Rev. Physiol. 50, 273-290. 

ATPases participate directly in various transport and motile functions. Of significance for this review is a manganese-specific ATPase found in rat brain [146], and Mn(II) which interacts specifically with other membrane-bound ATPases from brain and heart cells, including phospholipid-dependent ATPase activity exhibited by protein kinase C [147-149]. Interestingly hydrolysis of the cholinesterase-inhibitor Soman (isopropyl methyl-phosphonyl fluoride) is catalyzed by a manganese-dependent enzyme isolated from clonal neuroblastoma cells [150]. 

Sakamoto, K., van Veen, H. W., Saito, H., Kobayashi, H., Konings, W. N. (2002). Membrane-bound ATPase contributes to hop resistance of Lactobacillus brevis. Applied and Environmental Microbiology, 68, 5374—5378. 

Secretion of certain types of compounds into nonplasmic compartments may be followed by the passive penetration of other substances. Basic compounds may accumulate in ion traps, as do epinephrine and dopamine (D 22.1.1) in the chromaffin granules of adrenal gland cells. The driving force of the accumulation of these amines in the nonplasmic lumen of chromaffin granules is a proton gradient dependent on ATP and most probably generated by a membrane-bound ATPase. Epinephrine and dopamine in their unprotonated form easily penetrate the membranes of the chromaffin granules and are trapped in the vesicles in their protonated form (Fig. 4). Probably the same mechanism is involved in accumulation of certain alkaloids in plant cells. Nicotine, for instance, in Nicotiana rustica is synthesized mainly in the roots. It is released from the root cells, transported to the aerial parts in the stream of water driven by transpiration, and accumulated, probably in the protonated form, in several nonplasmic compartments of the leaf cells. 

Several established protocols have been adapted for 96-well plate readers including catalase, hyaluronidase, acetylcholinesterase, protein phosphatases and membrane-bound ATPases (22-26). In several instances these have involved novel protocols that are well suited to the ELISA format. For example, a sensitive, rapid microtitre-based assay for hyaluronidase activity was described by Frost and Stem (23). The free carboxyl groups of hyaluronan are biotinylated in a one-step reaction using biotin-hydrazide. This substrate is then covalently coupled to a 96-well microtitre plate. At the completion of the enzyme reaction, residual substrate is detected with an avidin-peroxidase reaction that can be read in a standard ELISA plate reader. Because the substrate is covalently bound to the microtitre plate, artefacts such as pH-dependent displacement of the biotinylated substrate do not occur. The sensitivity permits rapid measurement of hyaluronidase activity from cultured cells and biological samples, with an interassay variation of less than 5%. 

---