Studies on ATP Deaminase

Water insoluble ATP deaminase was prepared by binding native ATP deaminase to DEAE-cellulose and several enzymatic properties of bound ATP deaminase were compared with those of free deaminase. The optimum pH of the former was 3.0 and shifted toward acid side by about 2 pH units compared with that of the latter. The activity of bound deaminase to ATP corresponded to about one fourth of free deaminase at pH 3.0. The possibility of continuous preparation of IMP from AMP by use of bound deaminase was suggested.     

Studies on Enzymatic Production of ATP

A study of the microflora of radurized Vienna sausages which did not contain preservatives revealed gram-negative diplococci and paired short rods. These radio-resistant isolates were the main residual flora in radurized viennas. The cultures were aerobic, oxidase positive and non-motile, and sugars were oxidized or not attacked at all. The population at the low temperature incubation from 0 to 10°C was abundant and these isolates may be psychrophilic bacteria. The cultures are also capable of growing in media supplemented with 7.5% sodium chloride. The present study has demonstrated that all of these organisms should be tentatively classified as an Intermediate type of Moraxella and Acinetobacter.

D₁₀ values of typical isolates ranged from 44 to 54 krad when irradiated in 0.067 m phosphate buffer, pH 7.0, and were about 4 times larger than that of Acinetobacter calcoaceticus.     

Studies on photophosphorylation utilizing methylene diphosphonate analogs of ADP and ATP

Spinach chloroplasts were able to photophosphorylate the ADP analog α,β-methylene adenosine 5′-diphosphate (AOPCP). Phosphorylation of AOPCP was catalyzed by chloroplasts that were washed or dialyzed to remove free endogenous nucleotides. In the presence of glucose, hexokinase, AOPCP and ³²P_i, the ³²P label was incorporated into α,β-methylene adenosine 5′-triphosphate (AOPCPOP).In contrast to photophosphorylation of AOPCP, the ATP analog AOPCPOP was a poor substrate for the ATP-P_i exchange reaction and its hydrolysis was neither stimulated by light and dithiothreitol nor inhibited by Dio-9.Photophosphorylation of AOPCP was inhibited by the α,β- and β,γ-substituted methylene analogs of ATP, while phosphorylation of ADP was unaffected by them. The ATP-P_i exchange was also unaffected by both ATP analogs, while the weak AOPCPOP-P_i exchange was inhibited by the β,γ-methylene analog of ATP.Direct interaction of methylene analogs with the chloroplast coupling factor ATPase was indicated by the enzymatic hydrolysis of AOPCPOP on polyacrylamide gels.     

Studies on ATP hydrolysis in medium for histochemical demonstration of ATPase activity

Summary Nonenzymatic ATP hydrolysis in medium of Wachstein and Meisel for histochemical demonstration of ATPase activity was investigated. In this medium considerable amounts of phosphorus are released without the participation of the enzyme. ATP hydrolysis in Wachstein-Meisel's medium increase with the concentration of Pb⁺⁺ and decrease at its small concentrations. The degree of ATP hydrolysis appeared to increase with increase both temperature and pH. At high concentration of ATP (5.76 mM) the degree of ATP hydrolysis in Wachstein-Meisel's medium is lower than at 1.44 mM ATP. 10.0 mM Ca⁺⁺ or 3.6 mM Fe⁺⁺ speed up ATP hydrolysis after 30- and 60-minute incubation. In the presence of 3.6 mM Co⁺⁺ or 2.6 mM Cu⁺⁺ ATP hydrolysis in Wachstein-Meisel's medium increased throughout the whole period examined. On the contrary, 3.6 mM Fe⁺⁺⁺ decreases ATP hydrolysis in this medium.10.0 mM F^– raises the degree of ATP hydrolysis which is, however, lowered in the presence of 2.5 mM pCMB or 3.6 mM KCN. 2.0 mM cysteine highly inhibits the process of nonenzymatic ATP hydrolysis in Wachstein-Meisel's medium.These data show that the histochemical reaction for ATPase activity in Wachstein-Meisel's medium does not originate exclusively from the hydrolysis of ATP in the presence of Pb⁺⁺, but take rise, above all, as a result of an enzymatic reaction.     

Studies on photophosphorylation utilizing methylene diphosphonate analogs of ADP and ATP.

Spinach chloroplasts were able to photophosphorylate the ADP analog alpha,beta-methylene adenosine 5'-diphosphate (AOPCP). Phosphorylation of AOPCP was catalyzed by chloroplasts that were washed or dialyzed to remove free endogenous nucleotides. In the presence of glucose, hexokinase, AOPCP and 32Pi, the 32P label was incorporated into alpha,beta-methylene adenosine 5'-triphosphate (AOPCPOP). In contrast to photophosphorylation of AOPCP, the ATP analog AOPCPOP was a poor substrate for the ATP-Pi exchange reaction and its hydrolysis was neither stimulated by light and dithiothreitol nor inhibited by Dio-9. Photophosphorylation of AOPCP was inhibited by the alpha,beta- and beta,gamma-substituted methylene analogs of ATP, while phosphorylation of ADP was unaffected by them. The ATP-Pi exchange was also unaffected by both ATP analogs, while the weak AOPCPOP-Pi exchange was inhibited by the beta,gamma-methylene analog of ATP. Direct interaction of methylene analogs with the chloroplast coupling factor ATPase was indicated by the enzymatic hydrolysis of AOPCPOP on polyacrylamide gels.     

Studies on Hydrogenases in Legume Root Nodule Bacteroids: Effect of ATP

Hydrogenase activity (Tritium uptake activity) in cell-free extracts of bacteroids from soybean and lupin nodules was separated into two parts, whose activities were different from each other in responsiveness to ATP, by differential centrifugation, Sephadex gel filtration or DEAE-cellulose column chromatography. One was stimulated and the other was either inhibited or not affected by ATP.     

Studies on the mechanism of oxidative phosphorylation. Positive cooperativity in ATP synthesis

Kinetic and nucleotide binding studies have shown that submitochondrial particles from bovine heart possess three exchangeable binding sites for ADP or GDP. In order of decreasing affinity at neutral pH, these sites will be referred to as sites I, II, and III, and their respective dissociation constants as KI, KII, and KIII. In oxidative phosphorylation experiments in the presence of saturating amounts of inorganic phosphate, rapid ATP (or GTP) synthesis occurred only upon ADP (or GDP) binding to site III. The Eadie-Hofstee plots (v/[S] on the ordinate versus v on the abscissa) of the kinetics of ATP (or GTP) synthesis at variable ADP (or GDP) were, therefore, composed of an initial upward phase, indicating positive cooperativity with respect to substrate concentration, followed by a downward phase where rapid product formation took place. These data allowed calculation of KII from the upward phase and KIII (equivalent to apparent Km) from the downward phase. KI was estimated from Scatchard plots of binding data with radiolabeled ADP or GDP. Thus, together with our previous results, these findings have allowed characterization of the process of ATP or GTP synthesis by bovine-heart submitochondrial particles in terms of KI, KII, KIII, and kcat.     

Location of the ATP gamma-phosphate-binding sites on rat liver carbamoyl-phosphate synthetase I. Studies with the ATP analog 5'-p-fluorosulfonylbenzoyladenosine.

The gamma-phosphate subsites of the MgATP sites of rat liver carbamoyl-phosphate synthetase I have been defined by use of the ATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA). The synthetase utilizes two molecules of MgATP, apparently in mechanistically discrete steps and at separate MgATP sites. Sequence analysis has revealed internal duplication within the synthetase molecule (Nyunoya, H., Broglie, K.E., Widgren, E.E., and Lusty, C.J. (1985) J. Biol. Chem. 260, 9346-9356) and, based on sequence similarity with other nucleotide-binding proteins, potential ATP sites have been predicted for each of the duplicated sequences. The present FSBA studies have identified four peptides within carbamoyl-phosphate synthetase I that are involved in binding MgATP. Differential effects of N-acetylglutamate, a required allosteric activator, on the interaction of FSBA with the peptides were utilized to develop the following model for two distinct MgATP sites. Peptides 631-638 and 1327-1348 (with Cys1327 and/or Cys1337 modified by FSBA) apparently form part of the binding site for the MgATP involved in bicarbonate activation. Peptides 1310-1317 and 1445-1454 (with Tyr1450 modified by FSBA) apparently form part of the binding site for the MgATP involved in phosphorylation of enzyme-bound carbamate. Each of these MgATP sites contains a peptide from one of the internal duplicated regions of the enzyme molecule, which have previously been suggested as containing MgATP sites (Nyunoya, H., Broglie, K. E., Widgren, E. E., and Lusty, C. J. (1985) J. Biol. Chem. 260, 9346-9356; Powers-Lee, S. G., and Corina, K. (1987) J. Biol. Chem. 262, 9052-9056), as well as a peptide from the flexible C-terminal region.     

Studies on the immunological properties of complex V (mitochondrial ATP synthetase complex)

Antibody raised in rabbits against Complex V (miochondrial ATP synthetase complex) purified from beef heart mitochondria cross-reacted with Complex V and submitochondrial particles from beef heart, beef adrenals, and rat liver as shown by double-diffusion and rocket immunoelectrophoresis analysis. Of the various isolated and purified components of Complex V, only the oligomycin sensitivity-conferring protein showed strong reactivity with the anti-Complex V antibody, soluble F₁-ATPase reacted very faintly, while F₆ and ATPase inhibitor protein showed no precipitin lines. Crossed immunoelectrophoresis indicated that antigenic determinants recognized by the antibody were present on OSCP and possibly on the dicyclohexylcarbodiimide-binding protein. The components of Complex V could be precipitated from beef heart submitochondrial particles dissolved in Triton X-100 and pretreated with control IgG. When the composition of the immunoprecipitate was compared to that of purified Complex V, all the constituent polypeptides of the latter were present in the immunoprecipitate, except for one polypeptide in the low-molecular-weight region. Incubation of Complex V or submitochondrial particles with the anti-Complex V antibody in the absence of Triton X-100 caused inhibition of ATP-P_i exchange but not of ATPase activity. In the presence of Triton X-100, oligomycin sensitivity of Complex V was lost and the antibody was able to inhibit also the ATPase activity. The enzymic activity of soluble F₁-ATPase was unaffected by the antibody in the absence or presence of Triton X-100. These results suggest that the anti-Complex V antibody might be a useful tool for identifying and probing the role of Complex V components involved in energy transduction.     

Studies on the mechanism of oxidative phosphorylation. Catalytic site cooperativity in ATP synthesis

Oxidative phosphorylation catalyzed by bovine heart submitochondrial particles appears to exhibit negative cooperativity with respect to [ADP] and positive cooperativity in catalysis. Eadie-Hofstee plots (v/[S]versus v) of the kinetics of oxidative phosphorylation at the variable ADP concentration range of 1-1200 microM were curvilinear and could be analyzed for two apparent KmADP values differing by one order of magnitude, and two apparent Vmax values. The KmADP values with either NADH or succinate as the respiratory substrate were in the ranges of 10 and 100 microM, and the Vmax values in nmol of ATP formed X min-1 (mg of protein)-1 were, respectively, 500 and 1840 when NADH was the oxidizable substrate, and 550 and 100 when succinate was the energy source. Site-site cooperativity of the ATP synthase, which is a central feature of current theories for the mechanism of oxidative phosphorylation, has been well-documented for ATP hydrolysis by isolated F1-ATPase, but never before demonstrated for mitochondrial ATP synthesis.