Effect of polyene antibiotics on plasma membranes of dog kidney]

Effects of amphotericine B and nistatine on plasmatic membranes of dog kidney are studied. Intravenous injection of antibiotics resulted in the change of the chemical composition of plasmatic membranes: the content of proteins, lipids and RNA. The composition of membrane proteins and the quantitative ratio of separate fatty acids in lipids of plasmatic membranes also changed in the presence of amphotericine B and nistatine. It is suggested on the basis of the data obtained that the mechanism of action of polyene antibiotics in dog kidney cells is connected with the damage of membrane structures.     

The change of template activity of dog kidney chromtin by polyene antibiotics in vivo and in vitro]

Effect of amphotericin B and nistatin on template activity of nuclear membrane-bound (DNPm) and free (DNPo) dog kidney chromatin after intravenous injections of antibiotics and after the incubation of isolated kidney cell nuclei with the antibiotics is studied. It is found that injections of amphotericin B and nistatin resulted in the increase of DNPo template activity in RNA polymerase system, the stimulating effect of nistatin being higher than that of amphotericin B. Injections of nistatine stimulated also template activity of DNPm, while amphotericin B produced no effect on DNPm. When studing the effect of polyene antibiotics on template activity of DNPo and DNPm in vitro, it is found that the intensity of RNA synthesis after incubation of isolated nuclei with antibiotics is considerably increased, and stimulating effect of nistatin is higher than of amphotericin B. Both antibiotics produced no effect on template activity of DNP in vitro. Thus, comparative analysis of changes in template activity of dog kidney chromatin under the effect of polyene antibiotics in vivo and in vitro has revealed the similarity of these drugs and draws to the conclusion that nistatin and amphotericin B produce a direct effect on template activity of chromatin.     

Effects of polyene antibiotics on the membranes of dog kidney isolated nuclei]

The effects of amphotericin B and nistatin on the membranes of dog kidney isolated nuclei after their incubation with the antibiotics in question, have been studied. It is found that the polyene antibiotics, though they are superficially-active compounds, have no solubilizing effect on nuclear membranes and do not change their chemical composition. Electrophoretic study has revealed that nuclear membrane proteins, besides high- and low-molecular protein components, also contain a large amount of histones. The incubation of the nuclei with the polyene antibiotics results in marked changes in the fractional composition of nuclear membrane proteins, the most significant changes being induced by amphotericin B. It is assumed that polyene antibiotics induce proteolytic degradation of nuclear membrane proteins.     

Action of polyene antibiotics on ribosome binding with Candida albicans membranes]

The effect of treatment of the protoplasts and cell membranes of C. albicans with polyenic entibiotics on formation of the ribosomal-membrane complex was studied in vitro. It was shown that amphotericin B and nystatin had no effect on this process. Significant suppression of the ribosome binding with the membranes was observed only in vivo in the presence of levorin. The role of the structural changes occurring in the membranes on formation of the polyen-sterol complex, as well as the role of the lipid components of the membrane in binding of the ribosomes is discussed.     

The effect of polyene antibiotics on fractions of DNP both bound and not bound to dog kidney nuclear membranes]

Effects of amphotericine B and nistatine on nuclear membrane-bound DNP (DNPm) and free DNP (DNPf) from dog kidney are studied. Intravenous injection of amphotericine B resulted in the increase of binding of DNP particles with nuclear membrane: the content of DNA in DNPm fraction was 50-fold increased. The injection of nistatine did not affect DNP binding with nuclear membrane. Amphotericine B alone increased the protein-DNA ratio and decreased the RNA/DNA ratio in DNPf fraction. Both amphotericine B and nistatine sharply increased the protein/DNA ratio and practically did not change the RNA/DNA ratio in DNPm fraction. Amphotericine B produced considerable changes in temperature denaturation of DNA in DNPo, while nistatine produced no effect. Both antibiotics considerably changed the composition of acid soluble proteins in DNPm and DNPf, non-histone proteins in DNPf, and also they caused the changed and quantitative redistribution of separate lipid components in DNPm lipids. Polyene antibiotics are suggested to effect on animal cell nuclear structures.     

Effects of polyene antibiotics and detergents on cell membranes of polyene-sensitive and polyene-resistant strains of Candida albicans]

Various detergents and EPR-probes of 4,4-dimethylspiro[5alpha-androstan-17beta-ol-3,2-(1,3-oxazolidin-3-oxyl)]2CH3OH; 2,2,6,6-tetramethylpalmitoyl-amidopiperidine-1-oxyl and 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinoxyl were used to establish structural differences in the membrane of polyene-sensitive and polyene-resistant strains of C. albicans. It was shown that the type of protein-lipid interactions is modified by the changes in the sterol component of the polyene-resistant strain membranes. This manifests itself in a decrease in sensitivity of membrane alkaline phosphatase for levorin and the detergents, as well as in the alteration of the lipid fluidity pattern of the polyene-resistant strain membranes as compared to the membranes of original culture of C. albicans. Treatment of polyene-sensitive strain membranes with amphotericine B causes more intensive protein-lipid interactions, which is not observed in case of the polyene-resistant strain. It is assumed that C. albicans resistance to polyenes is due to the existence of strong protein-lipid interactions in the membrane coupled with ergosterol substitution by other sterol components.     

Role of sterols in the interaction of polyene antibiotics with lipid membranes

The problem whether the membrane sterols are indirect acceptors of polyenic antibiotics or they play the role of substances providing conditions (at the expense of putting in order the membrane phospholipids) for formation of conductive complexes (ionic canals) from the antibiotic molecules is discussed. The comparative study on the ability of sterols of various structure (ergosterol, 7-dehydrocholesterol, cholesterol, 5 alpha-cholestan-3 beta-ol) to interact with the membrane phospholipids and to increase the sensitivity of such membranes to amphotericin B showed no correlation between the levels of these properties. The value of the changes in the cross elasticity module (E) of artificial bilayer lipid membranes from egg lecithin on introduction of the above sterols into their composition was used as the criterion for the interaction level. The absence of correlation between the above properties of the sterols indicated that the role of the sterols in interaction of polyenic antibiotics with the membranes could not be considered as the only effect of the sterols on putting in order the phospholipids, which confirmed the hypothesis on the acceptor function of the sterols with respect to polyenic antibiotics. The study of the effect of amphotericin B on the elastic properties of the cholesterol-containing bilayer membranes isolated from egg lecithin showed tha the values of the longitudinal and cross elasticity modules of the membranes did not change during introduction into the membranes of the ionic canals.     

Ionization of the acid-base groups of polyene antibiotics in aqueous solutions]

The acid-base characteristics of polyenic antibiotics, such as nystatin, mycoheptin and levorin in aqueous solutions were studied. A special procedure provided the use of potentiometric titration for investigation of ionization of the groups of vater-insoluble substances. The ionization constants of the carboxylic and amine groups of the antibiotics at several temperatures were determined. It was found that ionization of the acid group did not practically depend on the temperature. At the same time the heat effect of the amine group ionization was significant and amounted to about 10 kcal/mole. Thermodynamic analysis of the ionization process of the polyenic antibiotics in aqueous solutions was performed. Integral components defining the process energetics were calculated.     

The water and ionic permeability induced by polyene antibiotics across plasma membrane vesicles from Leishmania sp

An osmotic method has been used to study the effect of the polyene antibiotics amphotericin B, nystatin and candicidin on the water permeability of plasma membranes prepared from Leishmania sp. The effect of amphotericin B on the permeability of Leishmania membranes to a salt such as potassium nitrate was also investigated. A non-linear and saturable enhancement of water and salt permeability was measured with increasing polyene concentrations, which could be adjusted to Hill cooperativity equation. The antibiotic concentrations that induce at 30 degrees C half-maximal effects on the water permeability of Leishmania vesicles were 0.021 microM for candicidin, 0.21 microM for amphotericin B and 1.4 microM for nystatin. At 30 degrees C, the concentration of amphotericin B required to induce half of the maximal effect on the permeability of Leishmania vesicles to potassium nitrate was 1.8 microM. The temperature dependence for amphotericin B, nystatin and candicidin enhancement of the water permeability of Leishmania vesicles was determined by using Q10 data at 20 and 30 degrees C. The estimated activation energies at increasing polyene concentrations display the same general pattern for all three polyene antibiotics investigated, that is, a maximal positive value at about the polyene concentrations required for half-maximal effect. The significance of these results for understanding the mechanisms of action of polyene antibiotics on natural membranes is discussed.     

Radioprotective and antineoplastic activity of polyene antibiotics combined with dimethyl sulfoxide]

Radioprotective and antineoplastic activity of polyene, its derivatives and combinations with dimethyl sulfoxide (DMSO) was studied. The most potent radioprotective effect was demonstrated by methylated levorin, original levorin and by its isomer--isolevorin. Survival rate of the animals on 12th day after X-ray exposure was 100, 60, 60 per cent, at the control group 33.6, 20 and 0 per cent consequently. Levorin and alkyl derivatives of amphotericin B--methamphocin and buthamphocin inhibited growth of ascites and solid tumors to 46.3-79.0 per cent when compared to control group. Polyen antibiotics combined with DMSO also demonstrated antineoplastic activity at the animals treated with carcinogenic agent--diethyl nitrosoamine (DENA). 5-month survival of the animals was 76 per cent at nystatin and levorin group and 35.7 per cent at the control group (animals treated with DENA only).     

Differential effects of pectolytic enzymes on tomato polyuronides in vivo and in vitro

There are marked differences between polyuronide degradation in vivo during tomato ripening and in vitro during the autolysis of cell wall preparations. Experiments using purified enzymes and enzymically inactive wall preparations show that the combined action of polygalacturonase (E.C. 3.2.1.15) and pectinesterase(E.C. 3.1.1.11)can mimic this in vitro autolysis of cell walls. Assuming these two enzymes are also responsible for polyuronide degradation in vivo their combined action must be restricted in some way.     

Interaction of polyene antibiotics with sterols in phosphatidylcholine bilayer membranes as studied by spin probes

Interaction of filipin and amphotericin B with sterols in phosphatidylcholine membranes has been studied using various spin probes; epiandrosterone, cholestanone, phosphatidylcholine with 12-nitroxide or 5-nitroxide stearate attached to 2 position and also with tempocholine at the head group. Filipin caused increase in the fluidity of cholesterol-containing phosphatidylcholine membranes near the center, while it rather decreased the fluidity near the polar surface. On the other hand, amphotericin B did not apparently affect the fluidity. In the electron spin resonance spectrum of steroid spin probes in the antibiotic-containing membranes, both bound and free signals were observed and the association constant was calculated from the siganal intensity. In the binding of steroids with filipin, both 3 and 17 positions were involved, while the 17 position was less involved in the binding with amphotericin B. Phase change in the host membrane markedly affected the interaction of filipin with epiandrosterone probe. The bound fraction jumped from 0.4 to 0.8 on going to the crystalline state and increased further with decrease in temperature. The overall splitting of the bound signal also increased on lowering the temperature below phase transition. This change was attributed to aggregate formation of filipin-steroid complexes in the crystalline state. On the other hand, effect of phase transition was much smaller on the interaction of amphotericin B with the steroid probe.     

Effect of gamma-irradiation on some biological and physico-chemical properties of polyene antibiotics]

Deep effect of gamma-rays on polyenic antibiotics was studied. It was shown that gamma-radiation induced radiation-chemical oxidation of the substances. The chromatographic analysis showed that the levorin degradation products were identical to the polyenic products of the antibiotic oxidative destruction. As for mycoheptin and amphotericin B, destruction of their molecules to non-polyenic products was observed. It was found that toxicity of the levorin aromatic heptaen did not practically change after gamma-irradiation in high doses. The toxicity of mycoheptin and amphotericin B, non-aromatic heptaens increased after exposure to high doses of gamma-rays.     

Electrolyte effects on the activity of mutant enzymes in vivo and in vitro.

All temperature-sensitive histidine auxotrophs of Salmonella typhimurium tested are corrected by addition of neutral salts to their growth medium. The correctability seems to result from direct electrolyte effects on mutant protein stability since several of the mutant proteins are also salt correctable in vitro.     

Solubilization of the [8-lysine]vasopressin receptor and adenylate cyclase from pig kidney plasma membranes.

Adenylate cyclase and the [8-lysine]vasopressin receptor were solubilized from pig kidney medulla membranes using the nonionic detergent Triton X-100. Optimal conditions for solubilization were under continuous stirring in a medium containing 0.5% (/v) Triton X-100, 100 mM Tris-HCl, pH 8, and 10 mM MgCl2. Both adenylate cyclase activity and [3H][8-lysine]vasopressin binding activity were recovered in a -26,000 X g supernatant of detergent-treated membranes. The yield of solubilized adenylate cyclase was nearly 100%. The soluble enzyme was no longer sensitive to antidiuretic hormone but was slightly activated by sodium fluoride. The affinity of the soluble receptor for [8-lysine]vasopresin was les than that of the membrane-bound receptor (mean apparent Km values, respectively 10(-7) M and 2 X 10(-8) M), however binding cooperativity was preserved. Hill coefficients were 1.42 for the soluble receptor and 1.50 for the membrane receptor. The soluble receptor discriminated as efficiently as did the membrane receptor between [8-lysine-a1vasopressin and oxytocin. The yield of spolubilized receptor was only 30% despite the fact that all binding activity had disappeared from the residual pellet of detergent-treated membranes. When the membranous receptors were occupied before solubilization and the latter was performed under conditions in which dissociation of the hormone-receptor comples is slow, i.e. at low temperature, 65% to 100% of the hormone-receptor complex was recovered in the soluble fraction. The soluble hormone-receptor complex partially dissociated on rewarming whereas the free hormone concentration was kept unchanged in the medium. The residual binding capacity, which was 30% of the initial value, was identical with that determined when the receptor was solubilized in free form before incubation with labeled hormone. It was concluded that (a) solubilization of the receptor molecules was complete, (b) during solubilization two forms of the receptor appear, of which only one is accessible to the hormone, (c) occupancy of the receptor by the hormone prevented the formation of the nonaccessible form, and (d) some component or components of the soluble fraction might be responsible for the loss in apparent affinity.