Minimum inhibitory concentration of drugs againstMycobacterium leprae as determined by anin vitro assay

The observations that liveMycobacterium leprae after entry into cultured peritoneal macrophages from mice, reduced the EA rosetting macrophages, have been exploited to determine the minimum inhibitory concentration of diamino diphenyl sulphone and rifampicin. Diamino diphenyl sulphone showed a minimum inhibitory concentration of 0.028 μg/ml and rifampicin 0.11μg/ml when given externally. However, there was accumulation of diamino diphenyl sulphone inside the macrophages. At an external concentration of 0.028 μg/ml the concentration inside the macrophage was 0.5μg/ml. The minimum inhibitory concentration for diamino diphenyl sulphone in this assay system is higher by several folds and that for rifampicin is slightly lower, than what is reported earlier with mice foot pad experiments. The minimum inhibitory concentration reported in this assay system is quite close to what is observed forin vitro inhibition ofMycobacterium lufu with both the drugs     

Analysis of the energy metabolism after incubation of Saccharomyces cerevisiae with sulfite or nitrite

After addition of 5 mM sulfite or nitrite to glucose-metabolizing cells of Saccharomyces cerevisiae a rapid decrease of the ATP content and an inversely proportional increase in the level of inorganic phosphate was observed. The concentration of ADP shows only small and transient changes. Cells of the yeast mutant pet 936, lacking mitochondrial F₁ATPase, after addition of 5 mM sulfite or nitrite exhibit changes in ATP, ADP and inorganic phosphate very similar to those observed in wild type cells. They key enzyme of glucose degradation, glyceraldehyde-3-phosphate dehydrogenase was previously shown to be the most sulfiteor nitrite-sensitive enzyme of the glycolytic pathway. This enzyme shows the same sensitivity to sulfite or nitrite in cells of the mutant pet 936 as in wild type cells. It is concluded that the effects of sulfite or nitrite on ATP, ADP and inorganic phosphate are the result of inhibition of glyceraldehyde-3-phosphate dehydrogenase and not of inhibition of phosphorylation processes in the mitochondria. Levels of GTP, UTP and CTP show parallel changes to ATP. This is explained by the presence of very active nucleoside monophosphate kinases which cause a rapid exchange between the nucleoside phosphates. The effects of the sudden inhibition of glucose degradation by sulfite or nitrite on levels of ATP, ADP and inorganic phosphate are discussed in terms of the theory of Lynen (1942) on compensating phosphorylation and dephosphorylation in steady state glucose metabolizing yeast.Abbreviations ATP adenosine triphosphate - ADP adenosine diphosphate - AMP adenosine monophosphate - P_i inorganic orthophosphate Dedicated to Prof. Dr. Hans Grisebach on the occasion of his sixtieth birthday     

Reduced pyridine nucleotides in Pseudomonas carboxydovorans are formed by reverse electron transfer linked to proton motive force

In cell suspensions of Pseudomonas carboxydovorans pulsed with lithotrophic substrates (CO or H₂) in the presence of oxygen, formation of reduced pyridine nucleotides and of ATP could be demonstrated using the bioluminescent assay. Experiments employing base-acid transition, an uncoupler and inhibitors of ATPase or electron transport enabled us to propose a model for the formation of NAD(P)H in chemolithotrophically growing P. carboxydovorans.The protonophor FCCP (carbonly-p-trifluormethoxyphenylhydrazon) inhibited both, formation of NAD(P)H and of ATP. In the absence of oxygen, a chemical potential imposed by base-acid transition resulted in the formation of NAD(P)H and ATP when electrogenic substrates (CO or H₂) were present. This suggests proton motive force-driven NAD(P)H formation. The proton motive force was generated by oxidation of substrate, and not by ATP hydrolysis, as obvious from NAD(P)H formation during inhibition of ATP synthesis by oligomycin and N,N-dicyclohexylcarbodiimide.That the CO-born electrons are transferred via the ubiquinone 10-cytochrome b region to NADH dehydrogenase functioning in the reverse direction, was indicated by inhibition of NAD(P)H formation by HQNO (2-n-heptyl-4-hydroxyquinoline-N-oxide) and rotenone, and by resistance to antimycin A.We conclude that in P. carboxydovorans, growing with CO or H₂, electrons and a proton motive force, generated by respiration, are required to drive an reverse electron transfer for the formation of reduced pyridine nucleotides.Abbreviations CODH carbon monoxide dehydrogenase - DCCD N,N-dicyclohexylcarbodiimide - FCCP carbonyl-p-trifluormethoxyphenylhydrazon - HQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - pmf proton motive force     

Evidence for the involvement of a UDP-glucose-dependent group translocator in sucrose uptake into vacuoles of storage roots of red beet

Vacuoles isolated from the storage roots of red beet (Beta vulgaris L.) accumulate sucrose via two different mechanisms. One mechanism transports sucrose directly, and its rate is increased by the addition of MgATP. The other mechanism utilizes uridine diphosphate glucose (UDP-glucose) to synthesize and simultaneously transport sucrose phosphate and sucrose into the vacuole. This group translocation mechanism has also been found in sugarcane vacuoles. As in sugarcane, the beet group translocator does not require fructose 6-phosphate, nor is the latter substance transported into the vacuole. The uptake of UDP[¹⁴C]glucose in inhibited by high concentrations of osmoticum.Abbreviations EDTA ethylenediaminetetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - UDP uridine 5-diphosphate     

The rapid polyphosphoinositide metabolism may be a triggering event for thrombin-mediated stimulation of human platelets

The metabolism of polyphosphoinositides was examined in human platelets activated by thrombin. The addition of thrombin to [3H]glycerol-labeled platelets induced an initial loss and a subsequent increase of the radioactivity in phosphatidylinositol-4,5-bisphosphate (TPI) without any significant change in phosphatidylinositol-4-phosphate (DPI). A marked enhancement of [32P]Pi incorporation into TPI occurred in parallel with an increase in this lipid content, which was accompanied with a conccurent decrease in phosphatidylinositol (PI). The rate of this subsequent increase in TPI was smaller than that observed in [3H]arachidonic acid-labeled platelets, suggesting that formed TPI in activated platelets may contain much greater amount of arachidonate than preexisting TPI in resting platelets. These data indicate that thrombin causes a rapid change in TPI metabolism (initial degradation of preexisting TPI and subsequent production of arachidonate-rich TPI), which might be a primary candidate to modulate thrombin-induced function in human platelets.     

Clathrin-associated proteins contain bound nucleotide

An alcohol dehydrogenase isolated from Zymomonas mobilis was found to be activated by ferrous ions but not by zinc, after inactivation with metal-complexing agents. Cobaltous ions also re-activated to a lesser extent. It is suggested that in this species the alcohol dehydrogenase naturally contains iron. Kinetic studies on the iron-treated enzyme indicate an 'alcohol activation' phenomenon, which may have physiological relevance in overcoming product inhibition during fermentation.     

A new system for ATP-metric immunoanalysis

Treatment of amino-group-containing antigens with adenosine-5'-trimetaphosphate results in their chemical modification by -pppA residues. An immunoanalytical system is proposed based upon competition of these ATP-labelled antigens with those of the sample for immobilized antibodies. Mild acidic treatment of complexes of ATP-labelled antigens with immobilized antibodies results in quantitative liberation of intact ATP. The latter may be determined by the ultrosenstive bioluminescent techniques based upon emission of light with firefly luciferase. The validity of the system has been studied with two clinically important antigens, thyroxine and myoglobin.     

Titration of the binding sites for the oligomycin-sensitivity conferring protein in beef heart submitochondrial particles

The binding parameters of the oligomycin-sensitivity conferring protein (OSCP) in inside-out particles from beef heart mitochondria have been tested by means of two assays, the oligomycin-sensitive ATP-Pi exchange, and the oligomycin-sensitive ATP hydrolysis. The total number of OSCP binding sites in A particles was equal to 220 pmol/mg particle protein. Each mole of ATPase active site was able to bind 1.1 +/- 0.5 mol OSCP with Kd 1.7 nM.     

Evaluation of H2O activity in the free or phosphorylated catalytic site of Ca2+-ATPase

The sarcoplasmic reticulum Ca2+-ATPase catalyses a reversible calcium transport coupled to phosphate transfer between ATP and water. It has been proposed [Biochemistry (1980) 19, 4252-4261] that the reactivity of the acyl-phosphate bond is dependent on the water activity within the catalytic site. We have tested this hypothesis and found that the polarity in the free catalytic site is lower than that of water, a further and large decrease is observed when the enzyme is phosphorylated by Pi. Phosphorylation by ATP indicates that this polarity change is specifically associated with the formation of the ADP-insensitive phosphoenzyme.     

Inorganic pyrophosphate-driven ATP-synthesis in liposomes containing membrane-bound inorganic pyrophosphatase and F0-F1 complex from Rhodospirillum rubrum

PPi driven ATP synthesis has been reconstituted in a liposomal system containing the membrane-bound energy-linked PPiase and coupling factor complex, both highly purified from Rhodospirillum rubrum. This energy converting model system was made by mixing both enzyme preparations with an aqueous suspension of sonicated soybean phospholipids and subjecting to a freeze-thaw procedure. In the presence of ADP, Mg2+, Pi and PPi the system catalyzed phosphorylation by up to 25 nmol ATP formed X mg protein-1 X min-1, at 20 degrees C, which was sensitive to uncouplers and inhibitors of phosphorylation such as oligomycin, efrapeptin and N,N'-dicyclohexylcarbodiimide.