Of membrane-bound ATPases

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

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. 

Biochemical Implications for the Reversible Function of Membrane-Bound Atpases... 

There has been considerable discussion regarding the mode of action of the sea cucumber and starfish saponins. Both the triterpene and steroidal glycosides inhibit both Na/K ATPase and Ca/Mg ATPase 06) possibly as a result of their aglycone structures. However, their detergent properties cause membrane disruption which will influence the activity of membrane-bound enzymes such as the ATPases. In investigating the actions of saponins on multilamellar liposomes, it was found that cholesterol serves as the binding site for such saponins and that cholesterol-free lip-somes are not lysed by saponins 107). 

Fig. 3. (A) Disposition of afi unit in the membrane, based on sequence information [14,15], selective proteolytic digestion of the a subunit [5,6] and hydrophobic labelling (Table 1). The model for the (S subunit is based on sequencing of surface peptides and identification of S-S bridges [64,65]. T, T2 and C3 show location of proteolytic splits. CHO are glycosylated asparagines in the P subunit. (B) Peptide fragments remaining in the membrane after extensive tryptic digestion of membrane-bound Na,K-ATPase from outer medulla of pig kidney as described by Karlish et al. [7,58].

Until recently, the possibility that H,K-ATPase consists not only of a catalytic a subunit but also of other subunits was not examined. This was mainly due to the fact that SDS-PAGE of purified gastric H,K-ATPase preparations principally gave one protein band with an apparent molecular mass of about 100 kDa, which was reported to comprise 75% or more of the total amount of protein [6,66,67]. This mass is lower than the mass deduced from its cloned cDNA [40], but may be due to the higher electrophoretic mobility of membrane-bound proteins, as consequence of having relatively high contents of hydrophobic amino acid residues [68]. 

An important question arises about the effects of phospholipid composition and the function of membrane-bound enzymes. The phospholipid composition and cholesterol content in cell membranes of cultured cells can be modified, either by supplementing the medium with specific lipids or by incubation with different types of liposomes. Direct effects of phospholipid structure have been observed on the activity of the Ca2+-ATPase (due to changes in the phosphorylation and nucleotide binding domains) [37]. Evidence of a relationship between lipid structure and membrane functions also comes from studies with the insulin receptor [38]. Lipid alteration had no influence on insulin binding, but modified the kinetics of receptor autophosphorylation. 

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 ... 

Impairment of membrane-bound Na,K-ATPase, which is responsible for maintaining and restoring membrane potential, and an increased level of malondialdehyde (MDA), which is a known as an index of lipid peroxidation, are seen after unilateral focal cerebral ischemia in the mouse. Pretreatment with EGb (100 mg/kg/day, p.o. for 10 days) preserves the Na,K-ATPase activity during cerebral ischemia and prevents the increased MDA levels caused by cerebral... 

Matthyssee and Phillips (20) isolated two nuclear proteins, from tobacco cells, that bound specifically to 2,4-D. Receptor proteins for auxins, kinetins, and GA have been found (21). Sub-cellular fractions from bean leaves were recently shown to bind abscisic acid (22). Preliminary experiments (22) indicated that maximum ABA binding activity coincides with the activities of membrane-bound Mg -dependent, K+-stimulated ATPase and glucan synthetase. Table I of Biswas and Roy (21) lists hormone receptor proteins reported in plant tissue. For a protein to qualify as a receptor molecule, it should have a high stereo-specific binding capacity (Kd 10 6 to 10 SM) for its particular hormone. In com coleoptiles, both IAA and NAA are equally effective in inducing cell elongations fractions of plasma membrane and endoplasmic reticular membrane contain receptor proteins with Kd values of 10 M to 10 M for auxins (5, 18). When one considers procedural... 

The modulation of synaptosomal plasma membranes (SPMs) by adriamycin and the resultant effects on the activity of membrane-bound enzymes have been reported [58]. Again DPH was used as fluorescence probe. Adriamycin increased the lipid fluidity of the membrane labeled with DPH, as indicated by the steady-state fluorescence anisotropy. The lipid-phase separation of the membrane at 23.3 °C was perturbed by adriamycin so that the transition temperature was reduced to 16.2 °C. At the same time it was found that the Na+,K+-stimulated ATPase activity exhibits a break point at 22.8 °C in control SPMs. This was reduced to 15.8 °C in adriamydn-treated SPMs. It was proposed that adriamycin achieves this effect through asymmetric perturbation of the lipid membrane structure and that this change in the membrane fluidity may be an early key event in adriamycin-induced neurotoxicity. 

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]. 

Further sequelae to lipid peroxidation include inhibition of membrane-bound, phospholipid-dependent sodium-potassium ATPase and calcium ATPase with changes in the electrolyte milieu, especially calcium overloading of the cell with subsequent further cell damage and cell death as well as inhibition of adenylate cyclase. This results in loss of function in the mitochondria and microsomes. Damage to the DNA leads to enzyme defects or impaired enzyme synthesis, which triggers further metabolic changes. This also causes a cellular overload with calcium and subsequent activation of... 

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