Solution conformation of lasalocid and lasalocid-Na+ (X-537A)

The complete unraveling of the proton nuclear magnetic resonance spectra at high field strength of Lasalocid (1) and its sodium salt (2) in different solvents allowed the definition of their solution conformations. The free acid, a lozengeshaped molecule of 17 × 13 × 9 Å is prefolded in a way almost identical to that of its sodium derivative, where the ion lies in the centre, surrounded by all the oxygen atoms in the molecule except for the phenolic one. Although the upper side of the molecule is rather accessible to hydrophilic approach, some alkyl substituents are arranged out- and upwards, thus shielding the ion from possible contact with lipophilic surroundings. The spatial picture suggests that the ion is trapped upon arrival at one side (the flat upper side) and is released either at the same or at the opposite side.     

Mode of action of the antibiotic X-537A on mitochondrial glutamate oxidation

At 0.05 to 0.01 μM concentrations the monocarboxylic acid antibiotic X-537A inhibits ADP or 2,4-dinitrophenol-activated oxidation of glutamate but has no appreciable effect on state 4 respiration. ATP synthetase activity is also inhibited. There is no efflux of Mg²⁺ or Ca²⁺ from the mitochondria under these conditions. Dissociation of membrane bound Mg²⁺ induced by X-537A is reversed and prevented by Mg²⁺ + ATP but inhibitory effects of the antibiotic are not. Half maximal effects of X-537A occur when the ratio of X-537A to mitochondrial non-diffusible Mg²⁺ is $\text{1}\text{800}$ to $\text{1}\text{400}$. It is proposed that this small fraction of membrane associated Mg²⁺ may be at the catalytic site of energy transfer and irreversible inhibition by X-537A is due to hydrophobic complexation of Mg²⁺ in situ.     

The circular polarization of fluorescence of the ionophore lasalocid A (X-537A) and some of its metal complexes

The conformation of the ionophore lasalocid A (X-537A) and its complexes with metal ions was probed by the circular polarization of their luminescence (CPL). The CPL of each complex in methanol was found to be different than when in n-hexane. Furthermore, the different metal ion complexes investigated had a different CPL spectrum in each solvent. These findings indicate wide variability in the conformation of the complexes depending on the metal ion and the solvent. From the spectral behaviour of the CPL it was concluded that at least some of the complexes exist in more than one form in solution. A comparison between the CPL and CD spectra indicates a change in the conformation of the ionophore in the vicinity of the salicylate chromophore upon electronic excitation.     

Gas-liquid chromatographic determination of human fecal bile acids

A method for the determination of total bile acids in human feces that is suitable for routine application is described and discussed. Bile acids are extracted from freeze-dried feces with acetic acid and toluene, in the presence of the internal standard 23-nordeoxycholic acid. After saponification of the extract, bile acids and the internal standard are methylated and converted by mild chromic acid oxidation into their ketonic derivatives. The resultant mixture of a few stable compounds can be separated and measured quantitatively by gas-liquid chromatography on a methylsiloxane polymer. A reference bile acid mixture including the internal standard is also taken through the entire procedure with each series of samples. It has been demonstrated that, in spite of the omission of the usual purification steps, the method is specific for bile acids.     

Gas-liquid chromatographic determination of total phenylacetic acid in urine

A gas-liquid chromatographic procedure to measure total phenylacetic acid in urine is described. The method is simple, rapid, and reliable. Normal subjects (N = 48) excreted 141.1 ± 10.1 mg/24 h. Untreated depressed patients (N = 42) excreted 102.77 ± 15.9 mg/24 h. The difference in the means is significant and supports the role of phenylacetic acid as a biological marker in certain kinds of mental illnesses.     

Alterations in mitochondrial Ca2+ flux by the antibiotic X-537A (lasalocid-A).

A previous communication (Pereira da Silva, L., Bernardes, C.F. and Vercesi, A.E. (1984) Biochem. Biophys. Res. Commun. 124, 80-86) presented evidence that lasalocid-A, at concentrations far below those required to act as a Ca2+ ionophore, significantly inhibits Ca2+ efflux from liver mitochondria. In the present work we have studied the mechanism of this inhibition in liver and heart mitochondria. It was observed that lasalocid-A (25-250 nM), like nigericin, promotes the electroneutral exchange of K+ for H+ across the inner mitochondrial membrane and as a consequence can cause significant alterations in delta pH and delta psi. An indirect effect of these changes that might lead to inhibition of mitochondrial Ca2+ release was ruled out by experiments showing that the three observed patterns of lasalocid-A effect depend on the size of the mitochondrial Ca2+ load. At low Ca2+ loads (5-70 nmol Ca2+/mg protein), under experimental conditions in which Ca2+ release is supposed to be mediated by a Ca2+/2H+ antiporter, the kinetic data indicate that lasalocid-A inhibits the efflux of the cation by a competitive mechanism. The Ca2+/2Na+ antiporter, the dominant pathway for Ca2+ efflux from heart mitochondria, is not affected by lasalocid-A. At intermediate Ca2+ loads (70-110 nmol Ca2+/mg protein), lasalocid-A slightly stimulates Ca2+ release. This effect appears to be due to an increase in membrane permeability caused by the displacement of a pool of membrane bound Mg2+ possibly involved in the maintenance of membrane structure. Finally, at high Ca2+ loads (110-140 nmol Ca2+/mg protein) lasalocid-A enhances Ca2+ retention by liver mitochondria even in the presence of Ca2(+)-releasing agents such as phosphate and oxidants of the mitochondrial pyridine nucleotides. The maintenance of a high membrane potential under these conditions may indicate that lasalocid-A is a potent inhibitor of the Ca2(+)-induced membrane permeabilization. Nigericin, whose chemical structure resembles that of lasalocid-A, caused similar results.     

Inhibition of ruthenium red-induced Ca2+ efflux from liver mitochondria by the antibiotic X-537A

It has been reported (Becker, G.L., Fiskum, G. and Lehninger, A.L. (1980) J. Biol. Chem. 255, 9009-9012) that respiring rat liver mitochondria suspended in KC1 medium containing ATP, Mg2+ and phosphate, maintain a steady state extramitochondrial free Ca2+ concentration of about 0.5 microM. The results reported here show that the addition of the antibiotic X-537A, at concentrations far below those required for ionophorous activity, caused a perturbation in this steady state, lowering the extramitochondrial free Ca2+ concentration by about 0.20 microM. This shift in steady state was clarified by a study of X-537A inhibition of the Ca2+ efflux induced by ruthenium red; a half-maximum effect was observed at approximately 25 nM X-537A. No effect on Ca2+ transport through the influx uniporter was observed. The possibility of a generalized stabilizing action of the antibiotic on the mitochondrial membrane seems to be ruled out by its effectiveness at very low concentrations.     

Golgi organelle response to the antibiotic X537A

The effects of the ionophoric antibiotic X537A on cell structure were studied with phase-contrast, fluorescence, and electron microscopy. X537A induced selective vacuolation of the Golgi apparatus of vascular and intestinal smooth muscle, epithelium, plasma cells, and cultured chick heart and guinea pig vascular smooth muscle cells. The swelling of the Golgi apparatus induced by X537A was reversible in the systems examined for reversibility: vascular smooth muscle and cultured chick heart. Myelin figures were common in the Golgi apparatus vacuolated by X537A. Fluorescence microscopy of cultured cells incubated with X537A showed the characteristic blue X537A fluorescence associated with lipid globules in the cultured cells. Incubation of cultured chick heart cells with X537A reduced the beating rate and, after 24-72 h, abolished the sarcomere pattern. The swelling of the Golgi membranes produced by X537A in cultured vascular smooth muscle was associated with inhibition of D-[6-3H]glucosamine and [35S]sulfate incorporation into glycosaminoglycans.     

Gas-liquid chromatographic determination of lidocaine in cat plasma using mepivacaine as internal standard

A method for the gas—liquid chromatographic determination of lidocaine in cat plasma with mepivacaine as internal standard is described. The investigations demonstrated a high reliability in the method, although the precautions required are relatively few. Under the cited conditions the plasma concentrations determined with the method after lidocaine treatment of cats were proportional to the infusion rates and obeyed a logarithmic normal distribution.     

Gas-liquid chromatographic assay of serum bile acids

A sensitive method has been established for the analysis of serum bile acids by gas-liquid chromatography (glc). Bile acids are extracted from 0.5–2 ml of serum and analysed as methyl ester trifluoroacetates following enzymatic hydrolysis of the taurine and glycine conjugates. The method as described has been used to estimate serum bile acid levels in health and disease although bile acid sulphates are not detected. Inclusion of a solvolysis procedure before enzymatic hydrolysis would allow their measurement.     

Some effects of the ionophore X-537A on the isolated rat tail artery

The effects of micromolar concentrations of the ionophore X-537A (RO 2-2985) were studied using isolated preparations of the rat tail artery. The ionophore causes complete release of catecholamines from adrenergic nerves, which is the sole cause of the transient contractile response. The amines are released by a nonexocytotic process which seems to be related to the ability of X-537A to act as an efficient transmembrane carrier of Na+, k+, and H+. The ionophore also causes an almost complete and irreversible loss of the cocaine-sensitive component of metaraminol uptake by the tissue. X-537A dissipates the transmembrane concentration gradients of Na and K in the smooth muscle component of the preparation. This effect is unrelated to the release of endogenous catecholamines, and it can also be observed after the Na pump has been inhibited with ouabain. It is fully reversible, though not readily, and it can be induced repeatedly. In catecholamine-depleted strips, X-537A dissipates the transmembrane Na+ and K+ gradients without causing any change in tension. Stimulation of the rate of O2 consumption by X-537A in catecholamine-depleted tissue is reversible, and it is unaffected by ouabain and (or) removal of external Ca2+.     

The activation of sea urchin eggs by the divalent ionophores A23187 and X-537A

The divalent ionophores A23187 and X-537A induce parthenogenesis in sea urchin eggs. This results from their ability to mobilize intracellular Ca²⁺, which is implicated in both artificial parthenogenesis as well as the natural fertilization process. A23187 causes expulsion of cortical granules and elevation of the fertilization membrane within 0.5–9 min followed by an initiation of cell cleavage. The broader spectrum ionophore X-537A is less potent, but the production of cytoplasmic aberrations are more apparent. In contrast to the sperm-activated egg, the initial phase of ionophore induced activation is accompanied either by relatively insignificant changes in membrane resistance, or an increase.     

A differential scanning calorimetry study of the interaction of lasalocid antibiotic with phospholipid bilayers

Interaction of lasalocid sodium salt (Las-Na) with dipalmitoylphosphatidylcholine (DPPC) as a membrane model was investigated by highly-sensitive differential scanning calorimetry (DSC). The insertion properties of the antibiotic were studied both in multilamellar suspensions and unilamellar vesicles, for Las-Na/DPPC molar ratios (r) ranging from 0.005 to 0.1. The effect of the antibiotic on the lipid thermotropic behavior is concentration dependent and drastically changes at a critical r of 0.04 in both model membranes. Below this ratio, Las-Na molecules interact with DPPC bilayers without disrupting the global organization of the membrane. In the multilamellar systems only the transition cooperativity is affected whereas for the mixed vesicles, a decrease in the enthalpy change suggests a different mode of insertion. Above this ratio, implantation of the antibiotic give rise to lateral phase separation in multilamellar systems. These structural modifications have repercussions on the formation of mixed LAS-Na/DPPC vesicles which seems limited to an r value of 0.04.     

On the effect of the ionophore X-537A on neuromuscular transmission in the rat

In the rat phrenic nerve-diaphragm muscle preparation, X-537A at 6×10^?6 to 3×10^?5 M (1) depolarized muscle fibre membranes, (2) caused an occasional transient increase in and ultimate block of spontaneous transmitter release, (3) did not increase the amplitude of the end-plate potential (epp) but abruptly blocked stimulus-evoked transmitter release, and (4) produced an increase in the occurrence of “giant” miniature epp's (mepp's). The possibility is discussed that the sporadically raised mepp frequency was due to an ionophore-induced depolarization of nerve terminals. The increased occurrence of “giant” mepp's apparently reflected a X-537A-induced spontaneous multiquantal release of acetylcholine. This was not dependent on extracellular calcium but appeared to be of presynaptic origin.     

Gas-liquid chromatographic analysis of cyasin in cycad flour

A method for the quantitative determination of cycasin from cycad flour by gas-liquid chromatography is described. The flour is extracted with 70% ethanol and the residue from the dried extract is directly trimethylsilylated. Androsterone was found to be an excellent internal standard. The average content of cycasin from ten separate analyses of one lot of flour was $\text{0.429 gm}\text{100 gm}$. The method is rapid, sensitive, and not hindered by contaminating compounds.     

Utilization of X-537A to distinguish between intravesicular and membrane-bound calcium ions in sarcoplasmic reticulum

The Ca²⁺ ionophore X-537A is employed as a tool to distinguish between intravesicular Ca²⁺ and surface membrane-bound Ca²⁺ in sarcoplasmic reticulum isolated from rabbit skeletal muscle. When sarcoplasmic reticulum is incubated in 20 mM Ca²⁺ in the absence of ATP, 10–12 h are necessary for measurable amounts of Ca²⁺ to penetrate into the vesicular space, as determined by the fact that X-537A releases Ca²⁺ from ‘loaded’ vesicles only after this period of incubation. A fraction of Ca²⁺ of 50–60nmol/mg protein, rapidly taken up by sarcoplasmic reticulum, exchanges with Mg²⁺ and K⁺ in the medium and is readily released by ethyleneglycol-bis-(β-aminoethyl ether)-N,N′-tetraacetic acid, but it is not released by X-537A. The slow-penetrating fraction of Ca²⁺ (30–40 nmol/mg protein) is rapidly released by X-537A. The results indicate that most of the Ca²⁺ retained by sarcoplasmic reticulum under conditions of passive uptake is bound to the external side of the membrane. The fraction of Ca²⁺ that slowly penetrates the vesicles remains essentially free inside the vesicles and only a small part is bound to the internal side of the membrane.