Inhibition in vitro of lipogenic enzymes from bovine (Bos taurus) mammary tissue by methylmalonyl-coenzyme A and coenzyme A

Bovine mammary fatty acid synthetase was inhibited by approximately 50% by 40 microM methylmalonyl-CoA; this inhibition was competitive with respect to malonyl-CoA (apparent Ki = 11 microM). Similarly, 6.25 microM coenzyme A inhibited the synthetase by 35% and this inhibition was again competitive (apparent Ki = 1.7 microM). Apparent Km for malonyl-CoA was 29 microM. The short-chain dicarboxylic acids malonic, methylmalonic and ethylmalonic at high concentrations (160-320 microM) and ATP (5 mM) enhanced the synthetase activity by about 50% respectively; the activating effects of methylmalonic acid and ATP on the synthetase were additive. Methylmalonyl-CoA at 50 microM concentration inhibited the partially purified acetyl-CoA carboxylase uncompetitively by 10% and the propionyl-CoA carboxylase activity of the enzyme preparation competitively (apparent Ki = 21 microM) by 40%. Malonyl-CoA also inhibited the acetyl-CoA carboxylase activity competitively (apparent Ki = 7 microM) by 35% and the propionyl-CoA carboxylating activity of the preparation competitively (apparent Ki = 4 microM) by 82%. The possibility that methylmalonyl-CoA may be a causal factor in the aetiology of the low milk-fat syndrome in high yielding dairy cows is discussed.     

Evidence that F-actin can hydrolyze ATP independent of monomer-polymer end interactions

The rate of ATP hydrolysis in solutions of F-actin at steady state in 50 mM KC1, 0.1 mM CaC12 was inhibited by AMP and ADP. The inhibition was competitive with ATP (Km of about 600 microM) with Ki values of 9 microM for AMP and 44 microM for ADP. ATP hydrolysis was inhibited greater than 95% by 1 mM AMP. AMP had no effect on the time course of actin polymerization, ATP hydrolysis during polymerization, or the critical actin concentration. Simultaneous measurements of G-actin/F-actin subunit exchange and nucleotide exchange showed that nucleotide exchange occurred much more rapidly than subunit exchange; during the experiment over 50% of the F-actin-bound nucleotide was replaced when less than 1% of the F-actin subunits had exchanged. When AMP was present it was incorporated into the polymer, preventing incorporation of ADP from ATP in solution. F-actin with bound Mg2+ was much less sensitive to AMP than F-actin with bound Ca2+. These data provide evidence for an ATP hydrolysis cycle associated with direct exchange of F-actin-bound ADP for ATP free in solution independent of monomer-polymer end interactions. This exchange and hydrolysis of nucleotide may be enhanced when Ca2+ is bound to the F-actin protomers.     

Inhibition of lipid peroxidation by a novel compound (CV-2619) in brain mitochondria and mode of action of the inhibition

Lipid peroxidation in rat brain mitochondria was induced by NADH in the presence of ADP and FeCl3. CV-2619 inhibited the lipid peroxidation in a concentration-dependent manner; the concentration giving 50% inhibition (IC50) was 84 microM. In addition, the inhibitory effect of CV-2619 was strongly enhanced by adding substrates of mitochondrial respiration; when succinate, glutamate, or succinate plus glutamate was added, the IC50 of CV-2619 was changed to 1.1, 10, or 0.5 microM, respectively. Metabolites of CV-2619 also inhibited the lipid peroxidation. The inhibitory effect of CV-2619 on mitochondrial lipid peroxidation disappeared when TTFA, an inhibitor of complex II in mitochondrial respiratory chain, was added. The results indicate that in mitochondria CV-2619 is changed to its reduced form which inhibits lipid peroxidation.     

Phloretin - an uncoupler and an inhibitor of mitochondrial oxidative phosphorylation

The effect of phloretin on respiration by isolated mitochondria and submitochondrial particles was studied. In submitochondrial particles, both NADH- and succinate-dependent respiration was inhibited by phloretin. 50% maximum inhibition was reached at phloretin concentrations of 0.1 mM (NADH oxidation) and 0.7 mM (succinate oxidation). In isolated mitochondria, phloretin inhibited glutamate oxidation in both State 3 and State 4; 50% maximum inhibition occurred at about 30 microM. Succinate oxidation is inhibited in State 3 by phloretin, inhibition being half its maximum value at 0.5 mM, but in State 4 it is stimulated about 2-fold by phloretin at a concentration of 0.6 mM. Ascorbate oxidation is stimulated in both State 3 and State 4, maximum stimulation being equal to that obtained with an uncoupler of oxidative phosphorylation. Under all circumstances, phloretin lowered the transmembrane electrical potential difference in isolated mitochondria. These results are discussed in terms of mosaic non-equilibrium thermodynamics. We conclude that phloretin is both an uncoupler and an inhibitor of oxidative phosphorylation.     

Effects of suramin, an anti-human immunodeficiency virus reverse transcriptase agent, on protein kinase C. Differential activation and inhibition of protein kinase C isozymes.

Suramin inhibited protein kinase C (PKC) type I-III activity in a concentration-dependent manner. Similar inhibitory effects were observed with M-kinase, the constitutively active catalytic fragment of PKC, and autophosphorylation of PKC types I-III. Kinetic experiments indicated that suramin competitively inhibits activity with respect to ATP (Ki = 17, 27, and 31 microM, respectively) and that it can also inhibit by interaction with the substrate histone III-S. With protamine as the Pi acceptor, suramin inhibition was dependent on lipid, being approximately 4-fold less sensitive to inhibition in the absence of phosphatidylserine and diacylglycerol than in their presence. Suramin at low concentrations (10-40 microM), in the presence of Ca2+ and absence of lipid, was able to stimulate kinase activity (approximately 200-400%) in a type-dependent manner and at higher concentrations inhibited activity with histone III-S as substrate. These results indicate that suramin, a hexa-anionic hydrophobic compound, can act as a negatively charged phospholipid analog in activating PKC in the presence of Ca2+ and absence of lipid and can inhibit Ca2+/phosphatidylserine/diacylglycerol-stimulated kinase activity at higher concentrations by competing with ATP or by interaction with the exogenous substrate. Suramin inhibited cAMP-dependent protein kinase much less potently (IC50 = 656 microM) than PKC. The ability of suramin to inhibit PKC-mediated processes in intact cells was tested using the phorbol ester-stimulated respiratory burst of neutrophils as a model system. The respiratory burst of human neutrophils, when preincubated with suramin and then stimulated with phorbol ester, was inhibited in a concentration-dependent manner, suggesting that suramin may also be able to inhibit PKC-mediated processes in intact cells.     

Magnesium-dependent ecto-ATP diphosphohydrolase activity in Herpetomonas muscarum muscarum

In the present work we characterized the ecto-ATP diphosphohydrolase activity of the trypanosomatid parasite Herpetomonas muscarum muscarum. This parasite hydrolyzed ATP at a rate of 15.52 nmol Pi/mg protein/min and this activity reached a maximum at pH 7.5. Classical inhibitors of acid phosphatases, such as sodium orthovanadate (NaVO3), sodium fluoride (NaF), and ammonium molybdate presented no effect on this activity. MgCl2, ZnCl2, and MnCl2 stimulated the ATP hydrolysis by H. m. muscarum. The ecto-ATPase activity was insensitive to oligomycin and sodium azide, two inhibitors of mitochondrial Mg-ATPase, bafilomycin A1, a V-ATPase inhibitor, ouabain, a Na(+)+K+-ATPase inhibitor and to levamizole, an inhibitor of alkaline phosphatase. An extracellular impermeant inhibitor 4,4'-diisothiocyanostylbene 2',2'-disulfonic acid (DIDS) and a inhibitor of some ecto-ATPases, suramin, which is also a competitive antagonist of P2-purinergic receptors, promoted a great inhibition on the ATP hydrolysis. This enzyme is able to hydrolysis ATP, ADP, UTP, and UDP, but not GTP, GDP, CTP, or CDP. ADP inhibited the enzymatic activity in a concentration dependent manner, reaching 70% inhibition.     

Decavanadate induces mitochondrial membrane depolarization and inhibits oxygen consumption

Decavanadate induced rat liver mitochondrial depolarization at very low concentrations, half-depolarization with 39 nM decavanadate, while it was needed a 130-fold higher concentration of monomeric vanadate (5 microM) to induce the same effect. Decavanadate also inhibits mitochondrial repolarization induced by reduced glutathione in vitro, with an inhibition constant of 1 microM, whereas no effect was observed up to 100 microM of monomeric vanadate. The oxygen consumption by mitochondria is also inhibited by lower decavanadate than monomeric vanadate concentrations, i.e. 50% inhibition is attained with 99 M decavanadate and 10 microM monomeric vanadate. Thus, decavanadate is stronger as mitochondrial depolarization agent than as inhibitor of mitochondrial oxygen consumption. Up to 5 microM, decavanadate does not alter mitochondrial NADH levels nor inhibit neither F(O)F(1)-ATPase nor cytochrome c oxidase activity, but it induces changes in the redox steady-state of mitochondrial b-type cytochromes (complex III). NMR spectra showed that decameric vanadate is the predominant vanadate species in decavanadate solutions. It is concluded that decavanadate is much more potent mitochondrial depolarization agent and a more potent inhibitor of mitochondrial oxygen consumption than monomeric vanadate, pointing out the importance to take into account the contribution of higher oligomeric species of vanadium for the biological effects of vanadate solutions.     

Inhibition of acetyl-carnitine oxidation in rat brown-adipose-tissue mitochondria by erucoyl-carnitine is due to sequestration of CoA

The cause underlying the inhibitory effect of erucoyl-carnitine on acetyl-carnitine oxidation in rat brown-adipose-tissue mitochondria was investigated. The inhibition was shown to be of a noncompetitive nature, with an I50 of about 10 microM erucoyl-carnitine. Erucoyl-carnitine did not inhibit the respiratory chain or the citric acid cycle to a significant degree. Incubation of mitochondria with erucoyl-carnitine led to about 2/3 of all CoA being sequestered in the form of acid-insoluble esters (probably erucoyl-CoA). This sequestration had an apparent Km of about 10 microM. Erucoyl-carnitine also inhibited pyruvate oxidation with an I50 of about 10 microM. When added at this concentration, it also inhibited the oxidation of a wide variety of acyl-carnitines by about 50%, despite very different oxidation rates of these different acyl-carnitines. It was concluded that the inhibitory effect of erucoyl-carnitine on all CoA-dependent substrates could be adequately explained by the suggestion that erucoyl sequesters a significant fraction of mitochondrial matrix CoA as slowly metabolizable erucoyl-CoA esters. Possible physiological effects of this sequestration for brown adipose tissue thermogenesis are discussed.     

Plant succinic semialdehyde dehydrogenase. Cloning, purification, localization in mitochondria, and regulation by adenine nucleotides.

Succinic semialdehyde dehydrogenase (SSADH) is one of three enzymes constituting the gamma-aminobutyric acid shunt. We have cloned the cDNA for SSADH from Arabidopsis, which we designated SSADH1. SSADH1 cDNA encodes a protein of 528 amino acids (56 kD) with high similarity to SSADH from Escherichia coli and human (>59% identity). A sequence similar to a mitochondrial protease cleavage site is present 33 amino acids from the N terminus, indicating that the mature mitochondrial protein may contain 495 amino acids (53 kD). The native recombinant enzyme and the plant mitochondrial protein have a tetrameric molecular mass of 197 kD. Fractionation of plant mitochondria revealed its localization in the matrix. The purified recombinant enzyme showed maximal activity at pH 9.0 to 9.5, was specific for succinic semialdehyde (K(0.5) = 15 microM), and exclusively used NAD+ as a cofactor (Km = 130 +/- 77 microM). NADH was a competitive inhibitor with respect to NAD+ (Ki = 122 +/- 86 microM). AMP, ADP, and ATP inhibited the activity of SSADH (Ki = 2.5-8 mM). The mechanism of inhibition was competitive for AMP, noncompetitive for ATP, and mixed competitive for ADP with respect to NAD+. Plant SSADH may be responsive to mitochondrial energy charge and reducing potential in controlling metabolism of gamma-aminobutyric acid.     

Transport and metabolism of adenosine in human erythrocytes: effect of transport inhibitors and regulation by phosphate

Rapid kinetic techniques were applied to determine the effect of transport inhibitors on the transport and metabolism of adenosine in human red cells. Dipyridamole inhibited the equilibrium exchange of 500 microM adenosine by deoxycoformycin-treated cells in a similar concentration dependent manner as the equilibrium exchange and zero-trans influx of uridine with 50% inhibition being observed at about 20 nM. Intracellular phosphorylation of adenosine at an extracellular concentration of 5 microM was inhibited only by dipyridamole concentrations greater than or equal to 100 nM, which inhibited transport about 95%. Lower concentrations of dipyridamole actually stimulated adenosine phosphorylation, because the reduced influx of adenosine lessened substrate inhibition of adenosine kinase. When the cells were not treated with deoxycoformycin, greater than 95% of the adenosine entering the cells at a concentration of 100 microM became deaminated. A 95-98% inhibition of adenosine transport by treatment with dipyridamole, dilazep, or nitrobenzylthioinosine inhibited its deamination practically completely, whereas adenosine phosphorylation was inhibited only 50-85%. Whether adenosine entering the cells is phosphorylated or deaminated is strictly based on the kinetic properties of the responsible enzymes, substrate inhibition of adenosine kinase, and the absolute intracellular steady state concentration of adenosine attained. The latter approaches the extracellular concentration of adenosine, since transport is not rate limiting, except when modulated by transport inhibitors. In spite of the extensive adenosine deamination in cells incubated with 100 microM adenosine, little IMP accumulated intracellularly when the medium phosphate concentration was 1 mM, but IMP formation increased progressively with increase in phosphate concentration to 80 mM. The intracellular phosphoribosylation of adenine and hypoxanthine were similarly dependent on phosphate concentration. The results indicate that adenosine is the main purine source for erythrocytes and is very efficiently taken up and converted to nucleotides under physiological conditions, whereas hypoxanthine and adenine are not significantly salvaged. Hypoxanthine resulting from nucleotide turnover in these cells is expected to be primarily released from the cells. Adenosine was also dephosphorylated in human red cells presumably by 5'-methylthioadenosine phosphorylase, but this reaction seems without physiological significance as it occurs only at high adenosine and phosphate concentrations and if deamination is inhibited.     

Inhibition of bovine heart NAD-specific isocitrate dehydrogenase by reduced pyridine nucleotides: modulation of inhibition by ADP, NAD+, Ca2+, citrate, and isocitrate

The activity of NAD-dependent isocitrate dehydrogenase from bovine heart was inhibited by NADH (apparent Ki about 4.3 microM) and NADPH (Ki about 9.8 microM) at subsaturating substrate concentrations at pH 7.4. The inhibition by NADH or NADPH was reversed competitively by magnesium isocitrate in the presence of ADP, but not without ADP. Reversal of inhibition by NADH or NADPH with respect to NAD+ was competitive or of the linear mixed type depending on whether ADP was absent or present. ADP3- (0.2 mM) increased the Ki(app) for NADPH from 9.8 to 27.1 microM; further addition of Ca2+ (0.2 mM) raised the Ki(app) to 127 microM. For the modification of NADPH inhibition by ADP, S0.5 for Ca2+ was approximately 48 microM. This compares to the Km for Ca2+ of 0.3-1 microM for the activation of the enzyme without NADPH [Denton, R. M., Richards, D. A., & Chin, J. G. (1978) Biochem. J. 176, 899-906; Aogaichi, T., Evans, J., Gabriel, J., & Plaut, G. W. E. (1980) Arch. Biochem. Biophys. 204, 350-360]. ADP did not affect the Ki for NADH. Magnesium citrate, which was about 100-fold more effective as a positive modifier of the enzyme with ADP than without ADP [Gabriel, J. L., & Plaut, G. W. E. (1983) Fed. Proc., Fed. Am. Soc. Exp. Biol. 42, 2082], reversed competitively the inhibition by NADPH in the presence of ADP, but not without ADP. Magnesium citrate did not reverse NADH inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative effects of three naturally occurring furanocoumarins on mitochondrial bioenergetics

Oxygraphic measurements of the rates of mitochondrial respiration in the presence of varying amounts of chalepin, imperatorin and marmesin, three naturally occurring furanocoumarins, revealed that the oxidation of NAD(+)-linked substrates was inhibited by chalepin and imperatorin and less significantly by marmesin. The order of potency being rotenone much greater than chalepin imperatorin greater than marmesin. There was no effect whatsoever on succinate oxidation by the furanocoumarins tested (up to 60 microM). State 3 respiration was also inhibited by these furanocoumarins; by at least 80% by 10 microM chalepin and by 48 and 29% with 60 microM imperatorin and 60 microM marmesin, respectively. Consequently, ADP control of respiration was diminished by those concentrations of furanocoumarins that inhibited respiration. At 60 microM, respiratory control ratio was reduced by about 88, 49 and 28% with chalepin, imperatorin and marmesin, respectively. A measurement of the rate of proton and Ca2(+)-movements across the mitochondrial coupling membrane demonstrated that succinate-supported transport was not affected by these furanocoumarins. On the other hand, pyruvate/malate-supported proton ejection was significantly inhibited by chalepin, imperatorin and marmesin. The order of the degree of inhibition of proton flux is rotenone much greater than chalepin greater than imperatorin greater than marmesin. The pattern of the inhibition of pyruvate/malate-supported Ca2(+)-transport was identical to that seen during proton transport. A comparison of the effects of chalepin to that of rotenone suggests that chalepin might be about 10 times less potent than rotenone.     

Duramycin effects on the structure and function of heart mitochondria. II. Energy conversion reactions.

The polypeptide antibiotic duramycin has been reported to interact selectively with phosphatidylethanolamine (PE) and monogalactosyldiacylglycerol (Navarro et al., 1985, Biochemistry 24, 4645-4650). PE is a major component of mitochondrial membranes. Duramycin was used to probe the role of PE in mitochondrial energy conversion reactions with the following results: (i) Duramycin uncoupled mitochondrial respiration, decreasing the respiratory control ratio to 1 at 5 microM. At concentrations of duramycin in excess of 10 microM, ADP addition inhibited electron transport. (ii) Duramycin inhibited oxidative phosphorylation (C50 less than 2 microM). (iii) Duramycin stimulated mitochondrial ATP hydrolysis modestly. The antibiotic was 7- to 16-fold less effective in this regard than concentrations of carbonylcyanide p-trifluoromethoxyphenylhydrazone (F-CCP) which produced comparable uncoupling. (iv) Duramycin inhibited uncoupled ATPase activity (C50 = 8 microM). Inhibition of the ATPase activity of intact mitochondria was blocked by 1 mM MgCl2 and 5 mM CaCl2; inhibition persisted in sub-mitochondrial particles assayed in the presence of 3 mM MgCl2. The effects on mitochondrial function of free fatty acids (FFA) and duramycin are similar in many respects. It is suggested that duramycin, like FFA, uncouples via a nonclassical mechanism, possibly by disrupting intramembrane H+ transfer between redox and ATPase complexes. In addition, interaction of duramycin, either direct or indirect, with the F0 moiety of the mitochondrial ATPase and with one or more components of the respiratory electron transport chain is proposed.     

Dicyclohexylcarbodiimide-sensitive ATPase in Halobacterium saccharovorum

Membranes from Halobacterium saccharovorum contained a cryptic ATPase which required Mg2+ or Mn2+ and was activated by Triton X-100. The optimal pH for ATP hydrolysis was 9-10. ATP or GTP were hydrolyzed at the same rate while ITP, CTP, and UTP were hydrolyzed at about half that rate. The products of ATP hydrolysis were ADP and phosphate. The ATPase required high concentrations (3.5 M) of NaCl for maximum activity. ADP was a competitive inhibitor of the activity, with an apparent Ki of 50 microM. Dicyclohexylcarbodiimide (DCCD) inhibited ATP hydrolysis. The inhibition was marginal at the optimum pH of the enzyme. When the ATPase was preincubated with DCCD at varying pH values, but assayed at the optimal pH for activity, DCCD inhibition was observed to increase with increasing acidity of the preincubation medium. DCCD inhibition was also dependent on time of preincubation, and protein and DCCD concentrations. When preincubated at pH 6.0 for 4 h at a protein:DCCD ratio of 40 (w/w), ATPase activity was inhibited 90%.     

Characterization of mitochondrial monoamine oxidase of Ascaridia galli

Oxidative deamination of various biogenic monoamines by Ascaridia galli monoamine oxidase (MAO) was blocked by different mammalian MAO inhibitors, namely, iproniazid, trans-PcP, nialamide and pargyline and the blockade was observed to be time as well as concentration dependent. The binding of inhibitors with chick ascarid MAO was of the irreversible type and the nature of the inhibition was competitive. Pargyline showed lowest I50 (8 microM) and Ki (12 microM) values. Chlorgyline and deprenyl at 100 microM concentration inhibited MAO by about 60 and 40% respectively, indicating the presence of both type A and type B MAO in A. galli.     

Characterization of triacsin C inhibition of short-, medium-, and long-chain fatty acid: CoA ligases of human liver

Short-, medium-, and long-chain fatty acid:CoA ligases from human liver were tested for their sensitivity to inhibition by triacsin C. The short-chain fatty acid:CoA ligase was inhibited less than 10% by concentrations of triacsin C as high as 80 microM. The two mitochondrial xenobiotic/medium-chain fatty acid:CoA ligases (XM-ligases), HXM-A and HXM-B, were partially inhibited by triacsin C, and the inhibitions were characterized by low affinity for triacsin C (K(I) values > 100 microM). These inhibitions were found to be the result of triacsin C competing with medium-chain fatty acid for binding at the active site. The microsomal and mitochondrial forms of long-chain fatty acid:CoA ligase (also termed long-chain fatty acyl-CoA synthetase, or long-chain acyl-CoA synthetase LACS) were potently inhibited by triacsin C, and the inhibition had identical characteristics for both LACS forms. Dixon plots of this inhibition were biphasic. There is a high-affinity site with a K(I) of 0.1 microM that accounts for a maximum of 70% of the inhibition. There is also a low affinity site with a K(I) of 6 microM that accounts for a maximum of 30% inhibition. Kinetic analysis revealed that the high-affinity inhibition of the mitochondrial and microsomal LACS forms is the result of triacsin C binding at the palmitate substrate site.The high-affinity triacsin C inhibition of both the mitochondrial and microsomal LACS forms was found to require a high concentration of free Mg(2+), with the EC(50) for inhibition being 3 mM free Mg(2+). The low affinity triacsin C inhibition was also enhanced by Mg(2+). The data suggests that Mg(2+) promotes triacsin C inhibition of LACS by enhancing binding at the palmitate binding site. In contrast, the partial inhibition of the XM-ligases by triacsin C, which showed only a low-affinity component, did not require Mg(2+).     

Inhibition of growth and of thymidine incorporation into DNA in Tetrahymena by chlorpromazine, pimozide and penfluridol

The antipsychotic drugs chlorpromazine, pimozide, and penfluridol caused a 50% inhibition of growth of Tetrahymena at concentrations of 4.5, 5.5, and 1.5 microM, respectively. The degree of growth inhibition was dependent on the concentration of cells; higher drug concentrations were needed to produce inhibition of denser cell cultures. Binding studies with penfluridol showed that 50% growth inhibition resulted when approximately 50 mumoles of drug were bound per 10(6) cells. A 20-min preincubation of cells with chlorpromazine (14.7 microM) inhibited DNA synthesis by 46%, and with penfluridol (4 microM) DNA synthesis was inhibited by 27%. The incorporation of labeled thymidine into the thymidine triphosphate pool was inhibited by chlorpromazine but not by penfluridol, indicating that the drugs produce their growth inhibitory effects by different mechanisms. TDP kinase activity was demonstrated in a particle-free fraction of the cells. Its enzymatic activity was not affected by added chlorpromazine, penfluridol, or calmodulin, suggesting that inhibition of DNA synthesis by these drugs may be a consequence of growth inhibition.     

Effects of extramitochondrial ADP on permeability transition of mouse liver mitochondria

Carboxyatractylate (CAT) and atractylate inhibit the mitochondrial adenine nucleotide translocator (ANT) and stimulate the opening of permeability transition pore (PTP). Following pretreatment of mouse liver mitochondria with 5 microM CAT and 75 microM Ca2+, the activity of PTP increased, but addition of 2 mM ADP inhibited the swelling of mitochondria. Extramitochondrial Ca2+ concentration measured with Calcium-Green 5N evidenced that 2 mM ADP did not remarkably decrease the free Ca2+ but the release of Ca2+ from loaded mitochondria was stopped effectively after addition of 2 mM ADP. CAT caused a remarkable decrease of the maximum amount of calcium ions, which can be accumulated by mitochondria. Addition of 2 mM ADP after 5 microM CAT did not change the respiration, but increased the mitochondrial capacity for Ca2+ at more than five times. Bongkrekic acid (BA) had a biphasic effect on PT. In the first minutes 5 microM BA increased the stability of mitochondrial membrane followed by a pronounced opening of PTP too. BA abolished the action about of 1 mM ADP, but was not able to induce swelling of mitochondria in the presence of 2 mM ADP. We conclude that the outer side of inner mitochondrial membrane has a low affinity sensor for ADP, modifying the activity of PTP. The pathophysiological importance of this process could be an endogenous prevention of PT at conditions of energetic depression.     

Regulatory properties of carbamoyl-phosphate synthetase II from the parasitic protozoan Crithidia fasciculata

1. At the lowered concentrations of 0.5 mM ATP and 1.5 mM MgCl2, 2.0 mM UTP, UDP and UMP inhibited the activity of Crithidia fasciculata carbamoyl-phosphate synthetase II by about 65, 80 and 40% respectively. 2. The result suggests that feedback inhibition of the activity by uridine nucleotides is a mechanism of regulation of the de novo pyrimidine biosynthetic pathway in C. fasciculata. 3. ADP, AMP and CDP inhibited the activity (about 70, 40 and 40%). 4. Excess Mg2+ at around 1 mM, relative to the ATP concentration, was required for the maximum activity. 5. 5-Phosphoribosyl 1-pyrophosphate had no significant effect on the activity under various conditions examined.