Annexins V and XII alter the properties of planar lipid bilayers seen by conductance probes.

Annexins are proteins that bind lipids in the presence of calcium. Though multiple functions have been proposed for annexins, there is no general agreement on what annexins do or how they do it. We have used the well-studied conductance probes nonactin, alamethicin, and tetraphenylborate to investigate how annexins alter the functional properties of planar lipid bilayers. We found that annexin XII reduces the nonactin-induced conductance to approximately 30% of its original value. Both negative lipid and approximately 30 microM Ca(2+) are required for the conductance reduction. The mutant annexin XIIs, E105K and E105K/K68A, do not reduce the nonactin conductance even though both bind to the membrane just as wild-type does. Thus, subtle changes in the interaction of annexins with the membrane seem to be important. Annexin V also reduces nonactin conductance in nearly the same manner as annexin XII. Pronase in the absence of annexin had no effect on the nonactin conductance. But when added to the side of the bilayer opposite that to which annexin was added, pronase increased the nonactin-induced conductance toward its pre-annexin value. Annexins also dramatically alter the conductance induced by a radically different probe, alamethicin. When added to the same side of the bilayer as alamethicin, annexin has virtually no effect, but when added trans to the alamethicin, annexin dramatically reduces the asymmetry of the I-V curve and greatly slows the kinetics of one branch of the curve without altering those of the other. Annexin also reduces the rate at which the hydrophobic anion, tetraphenylborate, crosses the bilayer. These results suggest that annexin greatly reduces the ability of small molecules to cross the membrane without altering the surface potential and that at least some fraction of the active annexin is accessible to pronase digestion from the opposite side of the membrane.

Uptake into leishmania donovani promastigotes and antileishmanial action of an organometallic complex derived from pentamidine.

Iridium (Ir)-(COD)-pentamidine tetraphenylborate (CAS 225-75-4) was selected from a primary screening to be evaluated in vitro on three Leishmania (L.) strains comparatively to pentamidine used as reference compound. The IC50 values obtained from in vitro evaluation on promastigotes of L. major CRE 26, L. donovani DD8 and L. donovani LV9 were 3.9, 23.5, and 3.3 mumol/l for Ir-(COD)-pentamidine tetraphenylborate and 1.6, 7.7, and 3.9 mumol/l for pentamidine isethionate, respectively. Cytotoxicity on mouse peritoneal macrophages led to determine a chemotherapeutic index of 1.7 for Ir-(COD)-pentamidine tetraphenylborate and 4 for pentamidine. Considering L. donovani DD8, the uptake of iridium complex by the promastigotes was shown to be saturable with a Km value of 17.4 mumol/l and Vmax of 1.3 nmol/mg protein/2 h. After 2 and 4 h incubation of treated promastigotes in drug free medium the absence of Ir-complex efflux is in favour of intracellular drug binding. As a matter of fact iridium complex was shown to bind ribosomal subunits in vitro, with no effect on macromolecular biosynthesis.

Millimeter microwave effect on ion transport across lipid bilayer membranes.

The effects of millimeter microwaves in the frequency range of 54-76 GHz on capacitance and conductance of lipid bilayer membranes (BLM) were studied. Some of the membranes were modified by gramicidin A and amphotericin B or by tetraphenylboron anions (TPhB-). The millimeter microwaves were pulse-modulated (PW) at repetition rates ranging from 1 to 100 pps, PW at 1000 pps, or unmodulated continuous waves (CW). The maximum output power at the waveguide outlet was 20 mW. It was found that CW irradiation decreased the unmodified BLM capacitance by 1.2% +/- 0.5%. At the same time, membrane current induced by TPhB- transport increased by 5% +/- 1%. The changes in conductance of ionic channels formed by gramicidin A and amphotericin B were small (0.6% +/- 0.4%). No "resonance-like" effects of mm-wave irradiation on membrane capacitance, ionic channel currents, or TPhB- transport were detected. All changes in membrane capacitance and currents were independent of the modulation employed and were equivalent to heating by approximately 1.1 degrees C.