Pierre Volfin

Effect of an anti-tumor platinum complex, Pt(II)diaminotoluene, on mitochondrial membrane properties

The effects of platinum complexes, selected for their potent anti-tumor activities, have been studied on rat liver mitochondria. Among the mitochondrial properties which have been studied, the most marked effects of platinum complexes were obtained on functions linked to the inner membrane. cis-Pt(II)(3,4-diaminotoluene) dichloride is shown to stimulate state 4 respiration. It inhibits the phosphate transport into mitochondria, decreases the accumulation of Ca2+, and induces a more rapid release of the accumulated Ca2+. A release of Mg2+ from mitochondria incubated in the absence of added divalent cations, and an efflux of divalent cations from mitochondrial membranes are also observed. All these results indicate a profound modification of the of the permeability of mitochondrial membrane.

Surface charge of purple membranes measured by laser Doppler velocimetry.

Laser Doppler velocimetry measurements on purple membrane suspensions from Halobacterium halobium showed a linear correlation between electrophoretic mobility and applied electric field, electrokinetic responses could be rapidly monitored. Native membranes are less charged than white membrane preparations (from the R1mW mutant). Chemical modification of carboxyl residues reduces surface charge, and nitrotyrosine modified membranes are more or less charged than native membranes at pH greater than or less than 6.5, respectively. Changes in surface charge are found upon actinic illumination and are greatest (Ca 5 X 10(-4)/A2) under conditions where decay of the M412 intermediate of the photoreaction cycle is inhibited, such as at high pH or after chemical modification.

Design of a chemiluminescent and bioluminescent photometer.

An apparatus for chemi- and bioluminescence measurements is described. The main features are in high sensitivity: for example, 5 10(-12) M adenosine triphosphate, 5 10(-7) M glucose, 2 10(-9) M perhydrol and 4 10(-13) M peroxidase are easily detected; its fast mixing time and the possibility of injecting a reactant, at any time, enabling kinetic studies. Moreover, its compactness and 12 V battery supply allow measurements outside of laboratories.

Influence of oestradiol and progesterone injections on the acid-soluble phosphate fractions of the rat uterus

Abstract Various fractions of acid-soluble phosphates in the rat uterus were investigated. Oestradiol was found to increase greatly the high-energy phosphate content of the immature rats uterus. Progesterone provoked a further significant increase of the phosphocreatine-like fraction, as compared with the rat treated with oestradiol only. The influence of ovarietomy on the acid-soluble phosphate fractions of the adult rat was likewise studied. The physiological significance of the results is discussed.

Aggregation and proton release of purple and white membranes following cleavage of the carboxyl-terminal tail of bacteriorhodopsin.

Our results indicate that the previously reported decrease in proton release by proteolyzed purple membrane sheets was due merely to the aggregation state of these preparations and not to the loss of the carboxyl-terminal tail. Changes in H+/M412 ratios obtained for purple and white membrane preparations correlate with the measured aggregation. White membrane preparations consistently exhibit H+/M412 ratios more than twice those measured for native purple membranes under the same conditions. Quasi-elastic light scattering was used to characterize the size of isolated purple and white membrane sheets before and after proteolysis. The results clearly show that native purple membrane preparations are larger in size than would be expected and that, following trypsin treatment, they are on average more than an order of magnitude larger. Negative staining electron microscopy showed that the purple membrane became aggregated in stacked arrays. Bleaching and reconstitution with retinal also affect aggregation, but iodination or nitration of purple membrane does not affect the measured size. The average size of white membranes is smaller; this is consistent with results of electron microscopy and the size increase is much less than that of purple membranes following trypsin treatment. No size change occurs with retinal reconstitution. In aggregated purple membrane preparations, protons and other cations are unable to exchange freely with the aqueous medium, explaining why proteolysis lowers the proton release from purple membrane sheets in suspension.

Dinoflagellate luciferases: Purification of luciferases from Gonyaulax polyedra, Pyrocystis lunula, and Pyrocystis fusiformis

Abstract The isolation and purification of three luciferases from Pyrocystis lunula, Pyrocystis fusiformis , and Gonyaulax polyedra are described in this paper. The three luciferases have low molecular weights, 30,000 for G. polyedra and 40,000 for P. lunala and P. fusiformis , and each is composed of a single polypeptidic chain. The molecular weight of these luciferases is independent of both the period of the circadian rhythm and the pH of the extraction medium between pH 5.4 and pH 8. These enzymes are probably metalloproteins. Indeed, chelating agents such as EDTA, EGTA, and chlorotetracycline and also sodium azide and potassium cyanide inhibit the light emission. Three cations (Mn 2+ , Mg 2+ , and Ca 2+ ) increase the flash height and the total light emitted, whereas other cations (Fe 2+ , Fe 3+ , Cu 2+ , Ni 2+ , and Zn 2+ ) inhibit the light emission. The three luciferases cannot be replaced by peridoxases or oxidases as in the Balanoglossus and the Pholas systems. The pH dependence of the luciferase activities is represented by a symmetrical function with optimum near pH 7. Thus, the flashing mechanism cannot be explained by means of a switch mechanism controlled by the pH. The presence of a specific luciferin-binding protein has not been observed in the three extracts of dinoflagellates. The difference between our observations and those described in the literature may be explained by the difference of the degree of purification of these enzymes.

Importance of Mn++ in the regulation of gluconeogenesis in cellular and acellular kidney cortex systems: new data on mechanism of action of biguanides

Summary The study of the mechanism of action of the hypoglycemic biguanides (dimethyl and phenethyl biguanides) known as neoglycogenesis inhibitors, lead us to investigate this metabolic pathway on various cellular and acellular kidney cortex systems where, in the absence of glycogen, free glucose is obtained. o 1 On cellular systems: cortex slices, whatever substrates were employed, biguanide inhibition of neoglycogenesis has been observed. A selective stimulation by Mn++ at the level of reactions from pyruvate to triose-phosphates has been demonstrated. 2 With regard to acellular systems, studies of cortex homogenate have shown the absolute Mn++ requirement for glucose formation from substrates below the triose phosphate level. A relation between Mn++ and stimulation of neoglycogenesis occuring during fasting is demonstrated and a biguanide Mn++ interaction (as one explanation for there inhibitory action) is postulated. An action of biguanides on glucose-6-phosphatase activity is also indicated. 3 Experiments on kidney cortex cytosol concerning the reaction from malate to phosphoenol pyruvate (in presence of fluoride, an inhibitor of enolase) or to glucose-6-phosphate (in the absence of fluoride) confirm the importance of an Mn++-PEP carboxykinase regulation and inhibition by biguanides at this level.

Does a proton-pumping ATPase exist in the tonoplast?

In order to account for the accumulation of metabolites in plant vacuoles, the existence of a proton-pumping ATPase has been widely suggested in the literature. The demonstration of such a tonoplast-bound ATPase was merely based on the characterization of a nitrate-sensitive microsomal fraction. In some examples, this ATPase activity has been evidenced on vacuole preparations obtained under conditions which were criticized by Boller. The application of the reverse phase high-performance liquid chromatography method (RP-HPLC) to the simultaneous separation of adenine nucleotides, in the presence of tonoplast vesicles isolated from Catharanthus roseus, showed results not necessarily correlated with the ATPase hypothesis. Moreover, in light of the H+-quenching of quinacrine fluorescence observed during ATP hydrolysis by vacuoles or tonoplast vesicles, the existence of a proton-pumping ATPase may be questioned.

Dinoflagellate luminescence: Purification of a NAD(P)H-dependent reductase and of its substrate

The soluble enzymatic luminescent system of the dinoflagellate Pyrocystis lunula (luciferase-luciferin) is coupled with an enzymatic NAD(P)H-dependent reaction. The enzyme is a soluble reductase (Mr 47,000) which catalyzes, in the presence of NAD(P)H, the reduction of a molecule called P630. Reduced P630 has the same spectral characteristics as the purified luciferin. The luciferase can oxidize this reduced molecule with a light emission at 480 nm. These observations suggest that reduced P630 is a luciferin molecule. The oxidized form seems, in these conditions, to be the precursor of luciferin.

Influence of adrenaline and hypophyseal growth hormone on the acid-soluble phosphate fractions of the rat uterus

Abstract Adrenaline injections are followed by an increase of “true” inorganic phosphate and a decrease of high-energy phosphate fractions in the rat uterus. Growth hormone injections provoke an increase of high-energy phosphates in the same organ. The relationship between these changes and oxidative phosphorylation phenomena is discussed with regard to the growth hormone- and adrenaline-promoted inhibitions of the uterine metabolism, which have been previously reported.