Ionic Strength Of Incubation Medium Research Articles

Competitive interactions of amphipathic polycationic peptides and cationic fluorescent probes with lipid membrane: Experimental approaches and computational model

The electrostatic interaction of polycationic peptides with negatively charged biomembranes has been recognized as the first and very important step of their selective binding to many bacteria and transformed cells. In this work we demonstrated the phenomenon of competition of some earlier designed polycationic peptides and fluorescent probes for their binding to the negatively charged inner membrane of mitochondria and to the PC/PG (9:1) liposomes. Rat liver mitochondria swelling induced by the antimicrobial polycationic peptide BTM-P1 (VAPIAKYLATALAKWALKQGFAKLKS) and by the retro-BTM-P1 was significantly diminished in the presence of 10μM fluorescent probe safranin O. In experiments with liposomes, the polycationic peptides BTM-P1 and P7-5 (IYLATALAKWALKQGF-GG-RRRRRRR) at the concentrations of 2–3μM completely displaced the membrane-bound fluorescent probe DiSC3(5) in a low ionic strength medium. The developed computational model allowed a mathematical description of such interactions, predicting membrane surface concentrations of bound peptides as the function of the membrane surface charge and lipid quantity in the sample, the peptide charge, hydrophobicity and concentration, the ionic strength of incubation medium and of the presence of a charged fluorescent probe used for monitoring the membrane surface potential under real-time peptide–membrane interactions.

Computational Model for Monitoring of the Membrane Potential with the Fluorescent Probe DiSC3(5)

The optical properties of cationic dicarbocyanide dyes have been shown to depend on the transmembrane electrical potential of liposomes or living cells. In this work, we developed the most complete computational model allowing optimization of experimental conditions to monitor the membrane potential of membrane vesicles composed of neutral and charged phospholipids at fixed ionic strength of incubation media in a desirable voltage range. The model allows predicting potential‐dependent changes of the fluorescent properties of DiSC3(5) on concentration of the dye and on the lipid composition of the liposomes of certain sizes. It also takes into account the values of the surface potentials of the inner and outer leaflets of the lipid bilayer, as well as their changes due to the DiSC3(5) incorporation into the lipid phase. The probe dimerization in the aqueous and lipid phases, and the possibilities of the voltage‐dependent transitions of the dimers between two leaflets of the lipid bilayer and between the aqueous and lipid phases were also included in the model, in addition to the monomer transitions. The model was adjusted to several literature data obtained with DiSC3(5) probe as an indicator of the membrane potential in experiments with liposomes and biomembrane vesicles. Colciencias (Colombia) research grants #111840820380 and #111852128625.

A possible restriction of ferro- and ferricyanide oxidoreductase activities of rat liver mitochondria by the outer membrane

In this work, various ferro-ferricyanide oxidoreductase activities of rat liver mitochondria were studied to find conditions under which the outer membrane might restrict the flux of these highly charged non-biological anions. When the isotonic low ionic strength medium was supplemented with 25 mM KCl, a several-fold increase in the succinate-ferricyanide reductase activity of mitochondria and in the rate of external NADH oxidation in the presence of ferrocyanide was observed. Mitochondrial respiration with 5 mM ferrocyanide was almost completely inhibited after consumption of 3.8–18.5% of the dissolved oxygen, depending on the medium and the presence of 2,4-dinitrophenol. These and other experimental data together with mathematical modeling of the redox-state equilibrium suggest that the measured activities might be restricted by two factors: first, the permeability of the outer mitochondrial membrane and second, a strong influence of the ionic strength of incubation media on the intermembrane space redox reactions.

Characteristics of receptors for VIP in rat peritoneal macrophage membranes

Vasoactive intestinal peptide (VIP) receptors were investigated in rat peritoneal macrophage membranes (RPMM) using [ 125I]VIP as ligand. The receptor binding was rapid, reversible, saturable, specific, and dependent on time, temperature, and membrane concentration. The Scatchard analysis of binding data was consistent with the existence of two classes of VIP binding sites with K d values of 0.60 ± 0.08 and 275 ± 39 n M and binding capacities of 580 ± 71 and 72,500 ± 810 fmol VIP/mg protein, respectively. The interaction showed a high degree of specificity, as suggested by competitive displacement experiments with several peptides structurally or not structurally related to VIP. These pharmacological studies showed the following order of potency: VIP (IC 50 = 1 n M) > rGRF (IC 50 = 13 rmn M) > PHI (IC 50 = 421 n M) ⪢ secretin. Glucagon, somatostatin, insulin, octapeptide of cholecystokinin [CCK(26–33)], and pancreastatin were ineffective at concentrations up to 1 μ M. Binding of [ 125I]VIP to membranes is markedly reduced by increasing the ionic strength of incubation medium. Treatment of membranes with dithiothreitol, trypsin, and phospholipases A 2 and C resulted in a loss of the ability of these membranes to bind VIP. However, treatment with phospholipase D did not affect binding of VIP by membranes. The molecular characterization of VIP receptors in RPMM was performed after [ 125I]VIP cross-linking to membranes using the cross-linker dithiobis (succinimidyl propionate). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane proteins revealed specific [ 125I]VIP-protein complexes of M r 55,000 ± 1700, 35,000 ± 900, and 22,000 ± 500.

In Vitro Effect of Bean Amylase Inhibitor on Insect Amylases

The activity of a bean amylase inhibitor against amylases extracted from several insects was tested. Amylases extracted from Mediterranean flour moth larvae (Anagasta kuhniella), red flour beetle adults (Tribolium castaneum), both adults and larvae of Tribolium confusum (confused flour beetle) and yellow mealworm larvae (Tenebrio molitor) were inhibited while adult granary weevil (Sitophilus granarius) amylase was not inhibited by the bean inhibitor. The T. molitor amylase interaction with the bean inhibitor was studied further. Inhibition of the Tenebrio enzyme is expressed slowly at pH 5.4, but lowering the pH or raising the ionic strength of incubation media caused a marked increase in rate of expression of the inhibition.

RNA synthesis in maturing avian erythrocyte nuclei

The rate of RNA synthesis and its inhibition by α-amanitin in the nuclei of mature and immature avian erythrocytes are increased with the increase in ionic strength of incubation medium. Polyacrylamide gel electrophoresis indicates that heterogeneous species of RNAs are synthesised in the mature and immature erythrocyte nuclei. However, a large number of high molecular weight RNAs are synthesised in the nuclei of immature erythrocytes. Elution profiles on poly(U)-sepharose chromatography indicate that the RNAs synthesised in the nuclei of two types of cell contain poly(A) segments. Sixteen per cent of mature erythrocyte nuclear RNA syntbesised are polyadenylated, while it is 13% in immature erythrocyte nuclei. However, the total RNA synthesised is 2–3 fold higher in immature erythrocyte nuclei than that in mature erythrocyte nuclei.

Influence of Ionic Strength of Incubation Medium on the Release of Some Enzymes from Rat Liver Mitochondria

Influence of ionic strength of incubation medium on the release of adenylate kinase (AK), isocitrate dehydrogenase (IDH) and glutamate dehydrogenase (GDH) from intact and injured rat liver mitochondria has been studied. It has been found that raised ionic strength (mu equal 0.16-0.32) has increased the release of GDH and AK activities - IDH to a lesser degree - for mitochondrial preparations only with damaged inner membrane. Treatment of mitochondria with potassium chloride solution of definite ionic strength has permitted to detect the alteration of these organelles occurring in vivo and in vitro.