Affinity For ADP Research Articles

The effect of phosphate on light-induced exchange of ADP at the tight nucleotide binding site of CF1.

The effects of phosphate on the energy-dependent exchange of the tightly bound adenine nucleotides in chloroplast thylakoids has been studied. 1. Phosphate was found to have a dual effect on ADP binding; at low light intensities Pi enhanced the rate of binding and the total amount of tightly bound ADP, while at saturating light intensities, it decreased the level of bound nucleotides. Half-maximal stimulation or inhibition of ADP binding occurred at about 30 microM phosphate. 2. Phosphate inhibition of ADP binding at saturating light intensities is prevented in the presence of an ADP-regenerating system. Pi had no effect on the amount of ATP bound to chloroplast coupling factor 1 (CF1), which was lower than the amount of ADP bound. 3. Arsenate acted similarly to phosphate at low light intensities but not at saturating light intensities. 4. At low light intensities the interaction of phosphate with the membrane-bound CF1 increased the binding affinity for ADP about 8-fold. 5. Kinetic analysis of the ADP binding showed that phosphate increased the rate constant for ADP binding to the adenine nucleotide-depleted form of CF1.

Concomitance of an Active and an Inactive Mutant of Red Cell Pyruvate Kinase (PK)

A new mutant red cell PK associated with mild chronic haemolytic anaemia is described. The propositus, double heterozygous for a maternal gene coding for a structural abnormal enzyme and a paternal gene coding for a catalitically inactive enzyme, was suitable for an accurate functional characterization of the PK variant since his erythrocytes contained only one active mutant form of this enzyme. The active isoenzyme was characterized by low activity, decreased affinity for phosphoenolpyruvate, incomplete fructose-1,6-diphosphate activation, increased 'zero-time transition temperature', increased stability to guanidine-HCl and storage at +4 degrees C, increased guanosine-5'-diphosphate and cytidine-5'-diphosphate utilization, altered electrophoretic pattern with a single slow-moving component and abnormal isoelectric point. Affinity for ADP, ATP inhibition, optimum pH, molecular weight of the subunits, antigen concentration and immunological properties were in the normal range.

Morphological, histochemical and biochemical studies on germ cell mitochondria of normal rats

Morphological changes in rat germ cell mitochondria are described. In diplotene and secondary spermatocytes and in the spermatids of the Golgi, cap and acrosomal phases, the mitochondria take on a rounded appearance with the inner space containing the matrix flattened against the outer membrane and the intracristal spaces considerably swollen ("condensed" mitochondria). Functional studies on "condensed" mitochondria isolated from the germ cells of normal rats have been performed. The following parameters have been evaluated: ADP/O ratio, respiratory control ratio (RCR) and ADP affinity. The ADP/O values found in the presence of various substrates are in agreement with the theoretical figures. The RCR is remarkably high. Moreover, the ADP affinity of these mitochondria is very high, as demonstrated by the low values of the "apparent Km". These biochemical findings, which demonstrate a high oxidative capacity coupled with a marked phosphorylation, suggest that the "condensed" appearance of germ cell mitochondria is the expression of an active functional state.

Effects of palmityl coenzyme A and palmitylcarnitine on phosphorylating respiration in heart mitochondria

Glutamate-supported respiration in mitochondria is inhibited by palmityl-CoA in the presence of carnitine. Palmityl-CoA-induced lag phase and depressed state 3 rates increase with increasing ADP. Palmityl-CoA inhibition of state 3 respiration with glutamate shows an increased I 50 for palmityl-CoA (three to fourfold) when ADP increases and carnitine is present. ADP alone has a small effect. Glutamate-supported respiration is more profoundly inhibited by palmityl-CoA (+carnitine) than palmityl-CoA oxidation. With palmityl-CoA (+ carnitine) alone, the I 50 for palmityl-CoA is two-to threefold greater than when glutamate is also present. Active respiration with palmityl-CoA as substrate demonstrates a 2.5-fold greater apparent affinity for ADP than when glutamate is also present. The kinetics are competitive in both cases. Palmitylcarnitine, above 30 μ m, produces inhibition of glutamate-supported respiration, concomitant with mitochondrial swelling and eventual lysis. At 15 μ m palmitylcarnitine (minimal swelling), succinate (+ rotenone)-supported respiration decreases with a decrease in K app for ADP; no effect of 15–20 μ m palmitylcarnitine on glutamate-supported respiration is observed. However, palmityl-CoA (+ carnitine)-inhibited respiration with glutamate is further decreased with 15 and 20 μ m palmitylcarnitine, i.e., by 13 and 29%, respectively. Inhibition is competitive with ADP. With 3 μ m palmitylCoA and 20 μ m palmitylcarnitine, a decrease in carnitine (1.5 to 0.25 m m) decreases the apparent K i for palmityl-CoA from 2.6 to 1.8 μ m. The results suggest that glutamate increases the palmityl-CoA available to inhibit adenine nucleotide transport. Inhibition may take place external to the inner membrane. Competition of carnitine and palmitylcarnitine for substrate sites may explain the decreased apparent K i for palmityl-CoA as carnitine decreases.

Reconstitution of inhibitor binding properties of the isolated adenosine 5'-diphosphate,adenosine 5'-triphosphate carrier-linked binding protein.

We studied the binding of carboxyatractylate (CAT) and bongkrekate (BKA) to the solubilized ATP,ADP carrier-linked binding protein, which had been incorporated into liposomes. After solubilization with 3-lauramido-N,N-dimethylpropylamine oxide the binding protein had largely lost it affinity and binding capacity for both CAT and BKA. On incorporation into phospholipid vesicles, CAT binding was restored to a considerable extent (3.5 mumol of CAT/g of protein), reaching the original affinity as observed in mitochondria (Kd = 10(-8) M). With high amounts of CAT and under the influence of ADP the binding can be increased to 6.8 mumol of CAT/g of protein, indicating a movement of binding sites in the liposomal membrane. The binding of BKA was also reconstituted with high affinity (Kd = 8 X 10(-8) M) and to the same extent (6.4 mumol of BKA/g of protein). As in the case of intact mitochondria, this reconstituted binding depends on the presence of ADP. This dependence on ADP has an apparent Km = 7 muM, similar to the carrier affinity for ADP in intact mitochondria. The reorientation model of Klingenberg for the ADP,ATP carrier implicating an ADP-catalyzed transition between the CAT binding form (c state) and BKA binding form (m state) in the inner mitochondrial membrane has been confirmed in this reconstituted system.

A deficient pyruvate kinase with an electrophoretically slow-moving component.

A deficient erythrocyte pyruvate kinase observed in a patient with congenital non-spherocytic anaemia was characterized by the following properties: very low activity in haemolysates, decreased thermal stability, slightly increased urea denaturation, high affinity for PEP, poor FDP activation, normal ATP inhibition, decreased affinity for ADP, normal pH of optimal activity, and presence of an abnormal slow-moving component in this layer polyacrylamide gel electrophoresis. The patient was probably double heterozygous for two different deficient mutants of erythrocyte pyruvate kinase.

Availability of GTP-Sepharose for the purification of glutamate dehydrogenase from eel liver.

Glutamate dehydrogenase from eel liver was purified 75.5 fold in 33% yield by ammonium sulfate fractionation, protamine and heat treatment, gel filtration and affinity chromatography on GTP-Sepharose. When the partially purified enzyme from gel filtration was applied to a GTP-Sepharose column, it was retained with very few other proteins and recovered in 80 to 90% yield. The high affinity for ADP of glutamate dehydrogenase from eel liver was applied to the purification of this enzyme by affinity chromatography. Althouth this enzyme was retained on the ADP-Sepharose column, it could not be cluted either by a KCl gradient or by a pulse of NADH.

Studies on the kinetic effects of adenosine-3':5'-monophosphate-dependent phosphorylation of purified pig-liver pyruvate kinase type L.

The effect of cyclic-AMP-dependent phosphorylation on the activity of isolated pig liver pyruvate kinase was studied. It was found that the major kinetic effect of the phosphorylation was to reduce the affinity for the substrate phosphoenolpyruvate, K0.5 for this substrate increasing from 0.3 to 0.9 mM upon phosphorylation. The cooperative effect with phosphoenolpyruvate was enhanced, the Hill constant nH increasing concomitantly from 1.1 to 1.5. V was unaltered. The change in activity occurred in parallel with the phosphate incorporation, except during the initial part of the reaction, when inactivation was correspondingly slower. The affinity for the second substrate ADP was unchanged, with an apparent Km of 0.3 mM at saturating concentration of phosphoenolpyruvate. Likewise, the requirement for potassium was unaffected, whereas the phosphoenzyme required a higher concentration of magnesium ions for maximal activity, compared with the control enzyme. The inhibitory effect of the phosphorylation was counteracted by positive effectors, fructose 1,6-biphosphate in micromolar concentrations completely activated the phosphoenzyme, resulting in an enzyme with properties similar to the fructose 1,6-biphosphate-activated unphosphorylated enzyme, with K0.5 for phosphoenolpyruvate about 0.025 mM and with a Hill constant of 1.1. Hydrogen ions were also effective in activating the phosphoenzyme. Thus, when pH was lowered from 8 to 6.5 the inhibition due to phosphorylation was abolished. The phosphoenzyme was sensitive to further inhibition by negative effectors such as ATP and alanine. 2 mM ATP increased K0.5 for phosphoenolpyruvate to 1.5 mM and nH to 2.3. The corresponding values with alanine were 1.3 mM and 1.9. Phosphorylation is thought to be an additional mechanism of inhibition of the enzyme under gluconeogenetic conditions.

Effect of F-actin upon the binding of ADP to myosin and its fragments.

The effect of F-actin upon the binding of ADP to rabbit skeletal muscle myosin, heavy meromyosin, and subfragment 1 was studied by equilibrium dialysis, ultracentrifuge transport, and light scattering techniques. Both myosin and H-meromyosin (HMM) bind a maximum of approximately 1.6 mol of ADP/mol of protein, while S-1 binds approximately 0.9 mol of ADP/mol of protein. The affinity for ADP of all three preparations was similar at a given ionic strength (approximately 10(6) M-1 at 0.05 M KCl) and decreased with increasing ionic strength. Under conditions similar to those used for the measurement of ADP binding, the binding sites of myosin, HMM, and subfragment 1 (S-1) are saturated with actin at molar ratios of 2, 2, and 1 mol of actin monomer/mol of protein, respectively, as determined by light scattering, ultracentrifuge transport, and in the case of myosin by ATPase measurements. F-actin was found to inhibit ADP binding, but even at an actin concentration at least twice that required for saturation of myosin, HMM, or S-1, significant ADP binding remained. This ADP binding was inhibited by 10(-4) M pyrophosphate. The observations are consistent with the formation of an actomyosin-ADP complex in which actin and ADP are bound to myosin at distinct but interacting sites.

Some kinetic properties of M 2-type pyruvate kinase from rat liver at physiological Mg 2+ concentration

1. 1.|The influence of the free Mg 2+ concentration on the kinetic parameters of M 2-type pyruvate kinase (ATP; pyruvate phosphotransferase, EC 2.7.1.40) from rat liver, isolated in the presence of the physiological free Mg 2+ concentration, is investigated. 2. 2.|At 1 mM Mg free 2+ and pH 7.5 the affinity of the enzyme for the substrate phosphoenolpyruvate is decreased as compared to 10 mM Mg free 2+, whereas the affinity for the second substrate ADP does not change. The decrease in affinity for phosphoenolpyruvate at low free Mg 2+ concentrations appears to be the consequence of the presence of a low affinity form of the enzyme. 3. 3.|It is shown that the ATP inhibition is mainly due to Mg 2+ binding and that it is probably not of physiological importance. 4. 4.|The enzyme possesses a high affinity for Fru-1,6- P 2 (half maximal activation at 1 μM) which is decreased by the addition of alanine. The affinity of the enzyme for Fru-1,6- P 2 at 0.1 mM phosphoenolpyruvate is not influenced by the free Mg 2+ concentration. In the presence of alanine the affinity for Fru-1,6- P 2 is increased by increasing the phosphoenolpyruvate concentration. 5. 5.|It is concluded that at physiological concentrations of alanine, Mg 2+, ADP and phosphoenolpyruvate the M 2-type pyruvate kinase activity is mainly dependent upon the Fru-1,6- P 2 concentration.

The value of Dowex-50 in fractionation of nucleotides from acid soluble pool. A caution

Problems were encountered in separating acid soluble nucleotides into four main groups by chromatography on Dowex-50(H +) columns. These difficulties derived from the low affinity of ATP, ADP, GDP, GTP, CDP, and CTP for Dowex-50 (H +) ion exchanger under mild acid conditions. Thus, under the conditions described, these compounds travel together with uridine compounds.

Study of the differential modifications of ( [formula omitted])-ATPase and its partial reactions by dimethylsulfoxide

1. 1. Dimethylsulfoxide inhibits ( Na + + K +)-activated ATPase but increases [ 14C]ADP binding to the enzyme and stimulates the associated K +-dependent p- nitrophenylphosphatase . 2. 2. Enhancement of [ 14C]ADP binding occurs by a decrease of the dissociation constant from K D = 0.95 μ M to 0.09 μM in the presence of 30% dimethylsulfoxide. 5 mM Na + enhances ADP affinity in the presence and absence of dimethylsulfoxide about 3 fold. However, the decrease of the ADP affinity in the presence of K + is abolished by increasing concentrations of dimethylsulfoxide. Two ADP-binding sites, a high-affinity ( K D = 0.17 μ M ) and a low-affinity site ( K D = 0.40 μ M , appeared in the presence of 30% dimethylsulfoxide and 0.2 mM Mg 2+ or Ca 2+. Mg 2+ and Ca 2+ decreased the affinity of the enzyme for ADP in the presence and absence of dimethylsulfoxide. 3. 3. Dimethylsulfoxide decreased the rate of Na + -dependent phosphorylation of an EDTA-washed enzyme (E 1 conformation) and reduced the hydrolysis of the [ 32P]phosphoprotein. However, dimethylsulfoxide enhanced the Na +-dependent phosphorylation of the Rb +-bearing dephosphoenzyme ( Rb + · E 2 ). 4. 4. Dimethylsulfoxide increased the dissociation constant of the ouabain-receptor complex when ouabain binding was studied via the Na +-dependent pathway. 5. 5. The results suggest that dimethylsulfoxide acts on several sites: It favors the E 1 conformational state of the enzyme by an increased affinity for nucleotide triphosphates and diphosphates, by reducing the affinity of K + for the enzyme in the presence of nucleotides and possibly by an expulsion of K + from the K +-dephospho-enzyme. Inhibition of ( Na + + K +)-ATPase may occur by a decreased interaction of the phosphokinase and phosphatase sites with the phosphorylacceptor site.

Differential Effects of Adenylyl Imidodiphosphate on Adenosine Triphosphate Synthesis and the Partial Reactions of Oxidative Phosphorylation

Abstract A study of the interaction of the ATP analog, adenylyl imidodiphosphate (AMP-PNP), with mitochondrial ATPase revealed that the analog was a strong competitive inhibitor of both the soluble, purified enzyme, Ki = 0.33 µm, and of the membrane-bound enzyme, Ki = 0.16 µm. AMP-PNP also was a strong inhibitor of the partial reactions of oxidative phosphorylation catalyzed by phosphorylating submitochondrial particles. Thus, the ATP-dependent reduction of NAD by succinate was inhibited competitively with Ki = 13 µm and the ATP-dependent enhancement of fluorescence of 1,8-anilinonaphthalene sulfonate and the exchange between 32Pi and ATP also were inhibited. However, AMP-PNP did not affect the forward reactions of oxidative phosphorylation, that is, the synthesis of ATP from ADP and Pi linked to the oxidation of succinate. It is proposed that the failure of AMP-PNP to inhibit oxidative phosphorylation may be traceable to a high affinity for ADP of the phosphorylating sites on F1 or may reflect two catalytic sites on the enzyme which are specialized, respectively, for ATP synthesis and ATP utilization.

Adenine nucleotide translocation in yeast mitochondria. Effect of inhibitors of mitochondrial biogenesis on the ADP translocase

1. Optimal test conditions for adenine nucleotide translocation in Candida utilis mitochondria are a standard medium, consisting of 0.63 M mannitol, 2 mM EDTA (or ethylene glycol tetraacetic acid, EGTA), 10 mM morpholinopropane sulfonic acid (pH 6.8), and a temperature of 0 °C. 2. Adenine nucleotide translocation in C. utilis mitochondria is an exchange-diffusion process. The whole pool of internal adenine nucleotides is exchangeable, ADP being the most readily exchangeable nucleotide. The rate of mitochondrial ADP exchange, but not the K m value, depends on growth conditions. At 0 °C, the rate is about 3 to 4 nmoles ADP/min per mg protein for mitochondria obtained from yeast grown in the presence of 1.5% glucose; it rises to 11.5 nmoles when glucose is replaced by 3% ethanol in the growth medium. The K m value for ADP is 2 μM. The Q 10 is about 2 between 0 and 20 °C. Among other exchangeable adenine nucleotides are ATP, dADP and the methylene and the hypophosphate analogues of ADP. Unlike mammalian mitochondria, C. utilis mitochondria are able to transport external UDP by a carboxyatractyloside-sensitive process. 3. Under conditions of oxidative phosphorylation (phosphate and substrate present in an aerated medium), added ADP is exchanged with internal ATP. A higher ATP/ADP ratio was found in the extramitochondrial space than in the intramito-chondrial space. The difference between the calculated phosphate potentials in the two spaces was 0.9–1.7 kcal/mole. 4. Atractyloside, carboxyatractyloside, bongkrekic acid and palmityl-CoA inhibit mitochondrial adenine nucleotide translocation in C. utilis as they do in mammalian mitochondria, but 2 to 4 times less efficiently. The inhibition due to atractyloside or palmityl-CoA is competitive with respect to ADP whereas that due to bongkrekic acid and carboxyatractyloside is non-competitive. Carboxyatractyloside and atractyloside inhibitions are additive. The apparent K d for the binding of [ 35S]-carboxyatractyloside and [ 14C]bongkrekic acid is 10–15 nM and the concentration of sites 0.4–0.6 nmole/mg protein in both cases. [ 35S]Carboxyatractyloside binding is competitively displaced by atractyloside and vice versa. 5. Binding of [ 14C]ADP has been carried out with mitochondria depleted of their endogenous adenine nucleotides by incubation with phosphate and Mg 2+ at 20 °C. The amount of bound [ 14C]ADP which is atractyloside removable is 0.08–0.16 nmole/mg protein. 6. The rate of ADP transport is quite different in mitochondria isolated from C. utilis, according to whether it is grown on glucose, or on ethanol or in the presence of chloramphenicol; for instance, it decreases by 10 times when 3% ethanol in the growth medium is replaced by 10% glucose and by 5 times when chloramphenicol is added to the medium. These variations are accompanied by parallel variations in cytochrome aa 3. The number of atractyloside-sensitive ADP binding sites is not modified by the above conditions of culture, nor the number of [ 35S]carboxyatractyloside binding sites. The affinity for ADP is apparently not significantly modified, nor the size of the endogenous adenine nucleotide pool. In contrast to glucose repression or chloramphenicol inhibition, semi-anaerobiosis in C. utilis lowers significantly the mitochondrial binding capacity for carboxyatractyloside. Strict anaerobiosis in S. cerevisiae results in a practical loss of the cytochrome oxidase activity, and also of the carboxyatractyloside and ADP binding capacity. Transition from anaerobiosis to aerobiosis restores the cytochrome oxidase activity and the ADP and carboxyatractyloside binding capacities.

Bohr effect, 2,3-DPG and ATP concentrations in the blood of premature infants with RDS.

In children with RDS, the concomitant respiratory-metabolic acidosis leads with the Bohr effect to a lowering of oxygen affinity (increases of P<sub>50</sub>).However, pH reduction and resultant lowering of 2,3-DPG concentration in the erythrocytes make one expect an oxygen affinity increase. The question is thus to what extent the two influences overlap and neutralize each other. We wanted to take a look at this problem as part of a more extensive study, and so we measured the acid-base status, the oxygen affinity of hemoglobin, the 2,3-DPG, ATP, ADP and AMP contents in the blood of premature infants with RDS and in some term infants with RDS; then we compared these measurements with those of premature infants without RDS.

Respiration and phosphorylation by mitochondria from the hepatopancreas of the blue crab ( Callinectes sapidus)

Mitochondria isolated from the hepatopancreas of the blue crab Callinectes sapidus show high rates of oxidation of pyruvate + proline and of various intermediates of the tricarboxylic acid cycle in a 280- to 380-mOsm sucrose-mannitol medium supplemented with bovine serum albumin. The respiratory control ratio ranged from 6 to 10. Respiration was accompanied by phosphorylation of ADP, with the expected ADP:O ratio for all substrates tested except α-ketoglutarate, indicating that all three energy-conserving sites were functional. Fatty acids were also oxidized, but no oxidation of β-hydroxybutyrate, glycerol 3-phosphate, or NADH was observed. The crab mitochondria showed a relatively low affinity for phosphate, but a normal affinity for ADP. Respiration and phosphorylation gave the normal responses to respiratory chain inhibitors, uncoupling agents, oligomycin, and ionophores. Crab mitochondria have an exceptionally high content of phosphate, exceeding 1000 nmoles per mg protein, but a normal energy charge of the adenylic system. An unusual feature is the presence of considerable arginine kinase activity, which is usually thought to be restricted to muscle and nerve tissue in anthropods. This enzyme allows arginine to act as secondary phosphate acceptor. The arginine kinase is located on the cytosol side of the atractyloside-sensitive barrier and is thus unable to transfer the terminal phosphate group of matrix ATP directly to arginine.

Some principle effects of bongkrekic acid on the binding of adenine nucleotides to mitochondrial membranes.

The effect of bongkrekic acid on the binding of ADP to mitochondrial membranes is characterized and studied in relation to a number of parameters. Bongkrekic acid increases the binding of ADP to mitochondrial membranes (from beef heart, rat heart and rat liver) in contrast to atractyloside. The “specific” ADP binding (i.e. binding at the carrier sites), under the influence of bongkrekic acid, is differentiated by the combined application of bongkrekic acid and atractyloside so that the unspecific, non‐carrier‐linked binding portions are substracted. The binding increase by bongkrekic acid is specific both for ADP and ATP and excludes AMP and other nucleotides such as IDP etc. The dependence of the binding on the concentration of ADP shows that under the influence of bongkrekic acid the affinity for ADP is increased by about 25‐ to 100‐fold, yielding a dissociation constant KD of 0.2 to 0.02 μM. The dissociation rate of bound ADP in the presence of bongkrekic acid as measured by the exchangeability of bound [14C]ADP against ADP decreases more than 200‐fold. The removal of [14C]ADP by atractyloside is even fully inhibited by bongkrekic acid. The inhibition by bongkrekic acid of the ADP dissociation from the carrier explains the observed inhibition by bongkrekic acid of the overall ADP translocation. The specific ADP binding is smaller when bongkrekic acid is added before than after ADP. The binding sites, differentiated by the sequence of bongkrekic acid addition, are interpreted to reflect inner binding sites which can be reached by bongkrekic acid due to its lipophilic nature. In certain cases this difference is of equal size to the portion of high affinity sites for ADP without bongkrekic acid which previously also had been explained to represent the inner sites exposed to endogenous adenine nucleotides. With double labelling the binding of endogenous adenine nucleotides can be directly shown to be increased during preincubation with bongkrekic acid causing an apparent decrease in binding of exogenous ADP. Thus at high concentration of bongkrekic acid all sites can be blocked by endogenous adenine nucleotides. The additional binding of ADP by bongkrekic acid is remarkably slow depending on several parameters (e.g. half‐time, t1/2, ranging from 0.5 to 5 min at pH 7.0, 20°C). Preincubation with bongkrekic acid only partially eliminates the slow time course, indicating that transition of sites to high affinity requires simultaneous presence of bongkrekic acid and ADP. The influence of bongkrekic acid on the binding of ADP is strongly temperature‐dependent. The temperature effect is of kinetic nature since once applied at increased temperature, bongkrekic acid remains effective in the ADP binding at lower temperature. The temperature effect is diminished at lower pH and at high concentration of bongkrekic acid. The bongkrekic acid effect on ADP binding is strongly pH‐dependent. It is effective only at pH < 7.2 and completely abolished at pH > 7.6. The pK of the bongkrekic acid effect is independent of the concentration of ADP but is increased with the concentration of bongkrekic acid. Two theories are discussed: (a) a cooperative increase of the binding for ADP by bongkrekic acid, i.e., existence of substrate and regulatory sites, or (b) binding of bongkrekic acid to the substrate site of the carrier, however, preferentially when located on the inner membrane surface, resulting in a redistribution of all carrier sites to the inner surface. Thus the function of a mobile carrier would be demonstrated on the molecular level.

On the mechanism of carbamoylphosphate synthetase.

Studies were undertaken to investigate whether 2‐acetamido glutaric anhydride is an intermediate in the synthesis of carbamoylphosphate. Experiments were carried out with pools of 2‐acetamido‐glutaric anhydride, its hydroxamate and NH2OH to trap the anhydride. The anhydride was also tested as an inhibitor. The experiments show no evidence for the participation of free 2‐acetamido‐glutaric anhydride as an intermediate.The effect of NH2OH on the synthesis of ATP from carbamoylphosphate and ADP was investigated in order to clarify whether this reaction is an integral part of the overall reaction catalyzed by carbamoylphosphate synthetase and whether there is a common intermediate which could react with NH2OH. The unexpected observation was made that NH2OH is an inhibitor of ATP synthesis at low levels of ADP, and that the enzyme has a high affinity for ADP. These results together with other experiments on carbamoylphosphate binding by the enzyme point to a competition between ADP site and NH2OH. No increase in the release of Pi from carbamoylphosphate in the presence of both acetylglutamate and NH2OH was observed; therefore the intermediate responsible for the acetylglutamate NH2OH‐dependent ATPase is not formed extensively from carbamoylphosphate.The activation of carbamoylphosphate synthetase by acetylglutamate for both the forward and back reactions was studied. The enzyme was activated with acetylglutamate and rapidly separated from the cofactor on Sephadex. There was no evidence for retention of activation, indicating a fast return to the non‐activated state. Therefore, enzyme samples were activated, diluted and assayed immediately and after time intervals. A time‐dependent reversibility of the activated enzyme for both the forward and back reactions is illustrated.

A survey of the interaction of calcium ions with mitochondria from different tissues and species.

A survey was made of the capacity of mitochondria isolated from a number of different tissues and species to accumulate Ca(2+) from the suspending medium during electron transport. The species examined included the rat, mouse, rabbit, hamster, guinea pig, cow, chicken, turtle, blowfly, yeast and Neurospora crassa. The tissues examined included vertebrate liver, kidney, brain, heart, spleen, thyroid and adrenal cortex, and the flight muscle of the blowfly. The mitochondria from all vertebrate tissues examined showed: (a) stimulation of State 4 respiration by added Ca(2+) (Ca(2+)/~ activation ratio about 2.0), accompanied by accumulation of Ca(2+) and ejection of H(+), with a H(+)/Ca(2+) ratio about 1.0; (b) a requirement of phosphate for accumulation of large amounts of Ca(2+); (c) respiration-independent high-affinity binding sites for Ca(2+); (d) endogenous Ca(2+), which is largely released by uncoupling agents. However, mitochondria from yeast and blowfly flight muscle are unable to accumulate Ca(2+) in a respiration-dependent process and possess no high-affinity Ca(2+)-binding sites. These findings support the view that the high-affinity sites represent the ligand-binding sites of a specific Ca(2+) ;permease' or transport system in the membrane. The relatively high affinity for Ca(2+), which equals or exceeds the affinity for ADP, and the generally uniform characteristics of Ca(2+) transport in all the vertebrate mitochondria tested strongly suggest that respiration-linked Ca(2+) accumulation plays a general and fundamental role in vertebrate cell physiology.

Biochemical and ultrastructural properties of a mitochondrial inner membrane fraction deficient in outer membrane and matrix activities.

Treatment of the inner membrane matrix fraction of rat liver mitochondria with the nonionic detergent Lubrol WX solubilized about 70% of the total protein and 90% or more of the following matrix activities: malate dehydrogenase, glutamate dehydrogenase, and isocitrate dehydrogenase (NADP). The Lubrol-insoluble fraction was enriched in cytochromes, phospholipids, and a Mg(++)-stimulated ATPase activity. Less than 2% of the total mitochondrial activity of monoamine oxidase, an outer membrane marker, or adenylate kinase, an intracristal space marker could be detected in this inner membrane fraction. Electron micrographs of negatively stained preparations showed vesicles (</=0.4 micro diameter) literally saturated on the periphery with the 90 A ATPase particles. These inner membrane vesicles, which appeared for the most part to be inverted with respect to the normal inner membrane configuration in intact mitochondria, retained the succinicoxidase portion of the electron-transport chain, an intact phosphorylation site II with a high affinity for ADP, and the capacity to accumulate Ca(++). A number of biochemical properties characteristic of intact mitochondria and the inner membrane matrix fraction, however, were either absent or markedly deficient in the inner membrane vesicles. These included stimulation of respiration by either ADP or 2,4-dinitrophenol, oligomycin-sensitive ADP-ATP exchange activity, atractyloside sensitivity of adenine nucleotide requiring reactions, and a stimulation of the Mg(++)-ATPase by 2,4-dinitrophenol.