ATPase formate labeling

NCD-4 is a nonfluorescent carbodiimide derivative that forms a fluorescent adduct with the Ca -ATPase, accompanied by inhibition of ATPase activity and phos-phoenzyme formation [376-378]. Ca protected the enzyme against the inhibition by NCD-4 and reduced the extent of labeling, suggesting that the reaction may involve the Ca " " binding site. The stoichiometry of the Ca -protected labeling was i 2mole/mol ATPase. The fluorescence emission of the modified Ca -ATPase is consistent with the formation of a protein bound A-acylurea adduct in a relatively hydrophobic environment. After tryptic proteolysis of the NCD-4 labeled ATPase the fluorescence was associated with the A2 band of 24 kDa [376,379]. 

Na+, K+-ATPase was inactivated by FSB A in the presence of Mg23-. Although Na+ and K+ are not essential for inactivation, they accelerate the inactivation rate. In the absence of Mg2+, the enzyme was not inactivated by FSB A even in the presence of both Na+ and K+. Sequence study on the FSBA-modified dog kidney enzyme revealed that one of the major labeled sites is a lysyl residue equivalent to Lys-725 of the torpedo enzyme. The simultaneous replacement of both Lys-712 and Lys-713 by Met of SR-ATPase (The two lysyl residues are equivalent to Lys-725 and Lys-726 of the torpedo Na+, K+-ATPase) did not change the Ca2+-transporting activity or the formation of phosphoenzyme intermediate,781 indicating that these lysyl residues are not essential for enzyme activity. 

The mechanics of converting proton flow back into mitochondria into high-energy phosphate bonds is performed by the FoFj ATPase. This complex enzyme system spans the inner mitochondrial membrane and appears as particles labeled EP in Figure 17.3. Structural details are given in Figure 17.7. The enzyme consists of two sections the F0 section is embedded in the membrane and forms a channel through which protons are permitted to enter the F, section. The latter is located on the matrix side of the membrane and is attached to the F0 section. The ATPase catalyzes the formation of ATP from ADP and P,. The reverse reaction, ATP —> ADP + P, is carried out when Fj is separated from F0 hence the term ATPase. 

Coupling between the H+ movements across the thylakoid membranes associated with electron flow and ATP formation occurs via a coupling factor known as an ATP synthetase, which is usually referred to as ATP synthase but also as an ATPase (because it can catalyze the reverse reaction leading to ATP hydrolysis). As illustrated in Figure 6-5, the ATP synthase has two components (1) a five-protein factor that occurs on the stromal side of a thylakoid, which can bind ADP, Pj, and ATP (labeled CFX in Fig. 6-5) and (2) a four-protein factor that is hydrophobic and hence occurs in the thylakoid membrane, through which H+ can pass (labeled CF0).5... 

Luedi, H. Hasselbach, W. Excimer formation of ATPase from sarcoplasmic reticulum labeled with N-(3-pyrene)maleinimide. Fur. J. Biochem. 1983, 130,5-8. 

Fig. 2. Identification of fMet on subunit 9 of oligomycin-sensitive ATPase. Sac-charomyces cerevisiae, strain 4D, was grown on SSA 3% lactate, labeled with PH]formate for 6 h, and mitochondria prepared as previously described. Preparation of chloroform-methanol (CM) fractions was done as described by Sierra and Tzagoloff. Sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis was done by the method of Weber and Osborn, with 8% acrylamide for 11 h at 5 mA per gel. The 12-cm gels were cut into 1-mm slices and counted. Different patterns were normalized thru mobility of protein standards and tracking dye. (A) Final purified CM fraction after several ether...

In the experiments to be discussed here we introduced into this side-chain a thiolcapturing moiety as shown in fig. 4, which was reacted with cysteine 374 of monomeric actin. When this actin conjugate was subjected to polymerization conditions we observed that the phalloidin convalently attached to actin in this position had almost the same stabilization effect as free phalloidin (9). The parameter for filament stability that we followed in these experiments was the so-called steady state ATPase activity, which grossly reflects the number of breaks in the filaments repaired at the expense of ATP. In contrast to phalloidin linked to cysteine 374, phalloidin linked to cysteine 10, which is located in the same subdomain of actin as cysteine 374 but on the opposite side, showed no stabilization effect at all. Moreover, phalloidin fixed to this position badly disturbed the formation of actin filaments. Our finding that the binding site of phalloidin must be located close to the C-terminus of actin confirms previous results by Wieland and coworkers, who found that a photoaffinity-labeled phallotoxin reacted with methionine 355, an amino acid likewise positioned in close vicinity to the C-terminus. 

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