Membrane chloroplast , ATPase

Kometani etal.(93) used a theory for energy transfer from a donor to acceptors in a plane to determine the location of the retinal chromophore relative to the membrane surface. Another similar study on the location of the active site of chloroplast ATPase relative to the membrane surface has also been carried out.(94)... 

Transporting ATP synthase [EC 3.6.1.34] in plants, also referred to as chloroplast ATPase and CFiCFo-ATPase, catalyzes the hydrolysis of ATP to produce ADP and orthophosphate. When coupled with proton transport the reverse reaction results in the synthesis of ATP by this multisubunit complex. CFi, isolated from the rest of the membrane-bound complex, retains the ATPase activity but not the proton-translocating activity. 

F0Fi ATPase/ATP synthase of mitochondrial inner 19-58 membrane, chloroplast thylakoid, and bacterial plasma membrane... 

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. 

Membrane ATPases have also been inhibited by carbodiimides. This reaction is associated with the membrane lipoprotein. Carbodiimide binding proteins have been isolated from bacterial membranes, chloroplasts, animal liver mitochondria, bovine heart mitochondria,molds and yeasts. The site of carbodiimide attack in the protein is probably in the hydrophobic region because only lipophilic carbodiimides are effective inhibitors. The addition of methyl glycinate protects erythrocyte membrane ATPase against carbodiimide inhibition. The inhibition reaction of carbodiimides may involve an O N acyl shift in the initially formed O-acylurea. 

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]. 

Use of a high field to activate a membrane enzyme was first reported by Witt et al. (25) in 1976. They used dc pulses of approximately 1 kV/cm and of 1-ms duration to induce ATP synthesis by the chloroplast ATPase. Following this initial work, there have been many reports on 1-kV/ cm dc field-induced ATP synthesis in different ATP synthetic systems (see the literature cited in references 13 and 14). The main conclusion from these studies is that an applied field-induced transmembrane potential can facilitate ATP release from the enzyme whether a PEF can affect enzyme turnover is not clear. Because 1-kV/ cm dc fields also cause severe Joule heating of a sample suspension, thermal effects cannot be easily avoided except when very short electric pulses (microseconds) are used. Thus, the method has limited utility for electroactivation experiments. The PEF method is, however, quite popular for the study of electroporation and electrofusion of cell membranes (see the chapter by J. Weaver in this volume), electroinsertion of membrane proteins (26), and electrotransfection of cells (27). 

The observed single channel currents were carried by H through the isolated and reconstituted chloroplast ATPase. We demonstrate that it is the intact enzyme complex CFqCF] and not the membrane sector CFq alone that constitutes a voltagegated, proton selective channel with a hi unit conductance of 1-5 pS at pH 53-pH 8.0. The open probability P of the CFqCFj channel increased considerably with increasing membrane voltage (from Pq < 1% (V < 120 mV) to Pq < 30% (120 mV < V 200 mV)). In the presence of ADP (3 jxM) and Pj (5 jiM), wich specifically bind to CF the open probability decreased and venturiddin (1 i,M), a specific inhibitor of H flow through GFq in thylakoid membranes, blocked the chaimel almost completely. 

FIGURE 1 Reaction scheme for ATP synthesis and hydrolysis by membrane- bound chloroplast ATPase... 

Bar-Zvi D and Shavit N (1982) Modulation of the chloroplast ATPase by tight binding of nucleotides. Biochim. Biophys. Acta 681, 451-458. Bickel-Sandkotter S (1983) Loose and tight binding of adenine nucleotides by membrane-associated chloroplast ATPase. Biochim. Bioophys. Acta 723, 71-77. 

LOOSE AND TIGHT BINDING OF ADENINE NUCLEOTIDES BY MEMBRANE ASSOCIATED CHLOROPLAST ATPase... 

Roos P, Berzborn RJ (1983) Electroimmunodiffusion- a-powerful tool for quantitative determinations of both soluble and membrane bound chloroplast ATPase CF. Z.f. Naturforsch. in press... 

A well-known example of active transport is the sodium-potassium pump that maintains the imbalance of Na and ions across cytoplasmic membranes. Flere, the movement of ions is coupled to the hydrolysis of ATP to ADP and phosphate by the ATPase enzyme, liberating three Na+ out of the cell and pumping in two K [21-23]. Bacteria, mitochondria, and chloroplasts have a similar ion-driven uptake mechanism, but it works in reverse. Instead of ATP hydrolysis driving ion transport, H gradients across the membranes generate the synthesis of ATP from ADP and phosphate [24-27]. 

The marker enzymes used in this experiment are as follows vanadate-sensitive H+-ATPase (plasma membrane), nitrate-sensitive H+-ATPase or pyrophosphatase (tonoplast), TritonX-100 stimulated-UDPase or IDPase (Golgi complex), antimycin A-insensitive NADPH cytochrome c reductase (ER), and cytochrome c oxidase (mitochondria inner membrane). NADH cytochrome c reductase activity is found to be 10 times higher than NADPH cytochrome c reductase activity. Chlorophyll content can be measured as the chloroplast marker. The chlorophyll content is calculated by the following equation. Before measurement, auto zero is performed at 750 ran. 

F-ATPases (including the H+- or Na+-translocating subfamilies F-type, V-type and A-type ATPase) are found in eukaryotic mitochondria and chloroplasts, in bacteria and in Archaea. As multi-subunit complexes with three to 13 dissimilar subunits, they are embedded in the membrane and involved in primary energy conversion. Although extensively studied at the molecular level, the F-ATPases will not be discussed here in detail, since their main function is not the uptake of nutrients but the synthesis of ATP ( ATP synthase ) [127-130]. For example, synthesis of ATP is mediated by bacterial F-type ATPases when protons flow through the complex down the proton electrochemical gradient. Operating in the opposite direction, the ATPases pump 3 4 H+ and/or 3Na+ out of the cell per ATP hydrolysed. 

A crucially important finding is that submitochon-drial particles or vesicles from broken chloroplasts will synthesize ATP from ADP and P , when an artificial pH gradient is imposed.172186 Isolated purified FjF0 ATPase from a thermophilic Bacillus has been coreconstituted into liposomes with the light-driven proton pump bacteiiorhodopsin (Chapter 23). Illumination induced ATP synthesis.187 These observations support Mitchell s proposal that the ATP synthase is both spatially separate from the electron carriers in the membrane and utilizes the protonmotive force to make ATP. Thus, the passage of protons from the outside of the mitochondria back in through the ATP synthase induces the formation of ATP. What is the stoichiometry of this process ... 

Extensive studies have been carried out on the proton-translocating ATPase of mitochondrial, bacterial and chloroplast membranes. This enzyme can also function in reverse to exploit the electrochemical potential of protons built up by respiration for the synthesis of ATP from ADP and P .298 The synthesis of ATP can be effected by the application of external electrical pulses to the ATPase vesicles in suspension with submitochondrial particles, showing that the diffusion potential of the protons (ApH) is not used. The yield of ATP was linearly dependent on the number of pulses.299... 

An H+ electrochemical gradient (ApH+) provides the energy required for active transport of all classical neurotransmitters into synaptic vesicles. The Mg2+-dependent vacuolar-type H+-ATPase (V-ATPase) that produces this gradient resides on internal membranes of the secretory pathway, in particular endosomes and lysosomes (vacuole in yeast) as well as secretory vesicles (Figure 3). In terms of both structure and function, this pump resembles the F-type ATPases of bacteria, mitochondria and chloroplasts, and differs from the P-type ATPases expressed at the plasma membrane of mammalian cells (e.g., the Na+/K+-, gastric H+/K+-and muscle Ca2+-ATPases) (Forgac, 1989 Nelson, 1992). The vacuolar and F0F1... 

H+-ATPase in membranes of mitochondria, chloroplasts and bacteria catalyzes ATP synthesis coupled with electron transport.7) The catalytic portion of the enzyme Fi is composed of five different subunits, denoted a through e in order of decreasing molecular weight. The ATPase activity can be reconstituted from isolated a, fi and y subunits, each of which is devoid of catalytic activity in the separated state.7) Interestingly, the y subunit is indispensable for the functional reconstitution, although the catalytic site is suggested to be located on the fi subunit or at the interface of the a and fi subunits.7 ... 

The main argument in favour of this hypothesis is the presence in chloroplasts and mitochondria of DNA, which is different from the nuclear DNA and similar to the DNA of prokaryotes, as well as the similarity of chloroplast, mitochondrial and bacterial ribosomes and their significant difference from cytoplasmic ribosomes of eukaryotic cells. The chloroplasts and mitochondria were found to be close to bacterial cells in additional other biochemical features the presence in their membranes of phospholipid cardiolipin, which is absent in the plasma membrane of eukaryotes, and ATPases of one and the same type FiF0. 

Tricolorin A (46) and G (47) are prototype members of this class of amphiphilic glycoconjugates (24). They constitute the allelochemical principles of Ipomoea tricolor Cav., a plant used in traditional agriculture in Mexico as a cover crop to protect sugar cane against invasive weeds. Their molecular mechanism of action likely involves the inhibition of the FT-ATPase of the plasma membrane, an enzyme that plays a crucial role in plant cell physiology. Moreover, 46 acts as a natural uncoupler of photophosphorylation in spinach chloroplasts. This compound also displays general cytotoxicity against several... 

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