The stimulatory effects of Hofmeister ions on the activities of neuronal nitric-oxide synthase. Apparent substrate inhibition by l-arginine is overcome in the presence of protein-destabilizing agents.
A variety of monovalent anions and cations were effective in stimulating both calcium ion/calmodulin (Ca2+/CaM)-independent NADPH-cytochrome c reductase activity of, and Ca2+/CaM-dependent nitric oxide (NO.) synthesis by, neuronal nitric oxide synthase (nNOS). The efficacy of the ions in stimulating both activities could be correlated, in general, with their efficacy in precipitating or stabilizing certain proteins, an order referred to as the Hofmeister ion series. In the hemoglobin capture assay, used for measurement of NO. production, apparent substrate inhibition by L-arginine was almost completely reversed by the addition of sodium perchlorate (NaClO4), one of the more effective protein-destabilizing agents tested. Examination of this phenomenon by the assay of L-arginine conversion to L-citrulline revealed that the stimulatory effect of NaClO4 on the reaction was observed only in the presence of oxyhemoglobin or superoxide anion (generated by xanthine and xanthine oxidase), both scavengers of NO. Spectrophotometric examination of nNOS revealed that the addition of NaClO4 and a superoxide-generating system, but neither alone, prevented the increase of heme absorption at 436 nm, which has been attributed to the nitrosyl complex. The data are consistent with the release of autoinhibitory NO. coordinated to the prosthetic group of nNOS, which, in conjunction with an NO. scavenger, causes stimulation of the reaction. (+info)
Reactivity of cyanate with valine-1 (alpha) of hemoglobin. A probe of conformational change and anion binding.
The 3-fold increase in the carbamylation rate of Val-1 (alpha) of hemoglobin upon deoxygenation described earlier is now shown to be a sensitive probe of conformational change. Thus, whereas this residue in methemoglobin A is carbamylated at the same rate as in liganded hemoglobin, upon addition of inositol hexaphosphate its carbamylation rate is enhanced 30% as much as the total change in the rate between the CO and deoxy states. For CO-hemoglobin Kansas in the presence of the organic phosphate, the relative increase in the carbamylation rate of this residue is about 50%. These results indicate that methemoglobin A and hemoglobin Kansas in the presence of inositol hexaphosphate do not assume a conformation identical with deoxyhemoglobin but rather form either a mixture of R and T states or an intermediate conformation in the region around Val-1 (alpha). Studies on the mechanism for the rate enhancement in deoxyhemoglobin suggest that the cyanate anion binds to groups in the vicinity of Val-1 (alpha) prior to proton transfer and carbamylation of this NH2-terminal residue. Thus, specific removal with carboxypeptidase B of Arg-141 (alpha), which is close to Val-1 (alpha) in deoxyhemoglobin, abolishes the enhancement in carbamylation. Chloride, which has the same valency as cyanate, is a better competitive inhibitor of the carbamylation of deoxyhemoglobin (Ki = 50 mM) compared with liganded hemoglobin. Nitrate and iodide are also effective inhibitors of the carbamylation of Val-1 (alpha) of deoxyhemoglobin (Ki = 35 mM); inorganic phosphate, sulfate, and fluoride are poor competitive inhibitors. The change in pKa of Val-1 (alpha) upon deoxygenation may be due to its differential interaction with chloride. (+info)
Distinct sensitivities of OmpF and PhoE porins to charged modulators.
The inhibition of the anion-selective PhoE porin by ATP and of the cation-selective OmpF porin by polyamines has been previously documented. In the present study, we have extended the comparison of the inhibitor-porin pairs by investigating the effect of anions (ATP and aspartate) and positively charged polyamines (spermine and cadaverine) on both OmpF and PhoE with the patch-clamp technique, and by comparing directly the gating kinetics of the channels modulated by their respective substrates. The novel findings reported here are (1) that the activity of PhoE is completely unaffected by polyamines, and (2) that the kinetic changes induced by ATP on PhoE or polyamines on OmpF suggest different mechanisms of inhibition. ATP induces a high degree of flickering in the PhoE-mediated current and appears to behave as a blocker of ion flow during its presumed transport through PhoE. Polyamines modulate the kinetics of openings and closings of OmpF, in addition to promoting a blocker-like flickering activity. The strong correlation between sensitivity to inhibitors and ion selectivity suggests that some common molecular determinants are involved in these two properties and is in agreement with the hypothesis that polyamines bind inside the pore of cationic porins. (+info)
Regulation of a volume-sensitive anion channel in rat pancreatic beta-cells by intracellular adenine nucleotides.
1. The patch-clamp technique in the whole-cell configuration was used to measure the effects of intracellular adenine nucleotides on activity of the volume-sensitive anion channel in single, isolated rat pancreatic beta-cells. 2. In the absence of intracellular nucleotides, swelling of cells with a hypertonic pipette solution failed to activate the conductance. Addition of ATP over the range 2-10 mM maintaining the same degree of hypertonicity caused a progressive activation of the conductance. An increase in ATP produced a similar activation of the conductance in non-swollen cells, albeit with reduced current amplitudes. 3. Activation of the conductance was also observed in the presence of ATPgammaS, adenylyl imidophosphate (AMP-PNP), ADP, diadenosine tetraphosphate and GTPgammaS. Neither ADP nor GDPbetaS inhibited activation of the conductance by ATP. 4. It is concluded that activity of the beta-cell volume-sensitive anion channel can be modulated by changes in intracellular concentrations of ATP within the physiological concentration range by a mechanism that does not require nucleotide hydrolysis. Activity of the channel does not appear to be modulated by a G protein-coupled mechanism. (+info)
Phospholipid-subclass-specific partitioning of lipophilic ions in membrane-water systems.
Herein, we systematically investigate phospholipid-subclass-specific alterations in the partitioning of both cationic and anionic amphiphiles to identify the importance of ester, ether and vinyl ether linkages at the sn-1 position of phospholipids in the partitioning of charged amphiphiles. The results demonstrated that the membrane-water partition coefficient of a prototypic cationic amphiphile (i.e. 3,3'-dipropylthiadicarbocyanine iodide) was approximately 2.5 times higher in membranes comprised of plasmenylcholine in comparison with membranes comprised of either phosphatidylcholine or plasmanylcholine. In striking contrast, the membrane-water partition coefficient of a prototypic anionic amphiphile [i.e. bis-(1,3-dibutylbarbituric acid)trimethine oxonol] in membranes comprised of plasmenylcholine was approximately 2.5 times lower than that manifest in membranes comprised of phosphatidylcholine or plasmanylcholine. Utilizing theseexperimentally determined partition coefficients,the relative membrane dipole potential of membranes comprised of plasmenylcholine was calculated and found to be approximately 25 mV lower than in membranes comprised of phosphatidylcholine or plasmanylcholine. This lower membrane dipole potential in membranes comprised of plasmenylcholine is equivalent to the membrane potential induced by incorporation of approximately 25 mol% of anionic phospholipids in membranes comprised of phosphatidylcholine. Collectively, these results demonstrate that phospholipid-subclass-specific differences in the membrane dipole potential contribute to alterations in the partitioning of lipophilic ions in membrane bilayers comprised of distinct phospholipid subclasses. Moreover, they suggest that these physicochemical differences can be exploited to facilitate the targeting of charged lipophilic drugs to specific cells and subcellular membrane compartments. (+info)
Evidence for an anion exchange mechanism for uptake of conjugated bile acid from the rat jejunum.
Absorption of conjugated bile acids from the small intestine is very efficient. The mechanisms of jejunal absorption are not very well understood. The aim of this study was to clarify the mechanism of absorption of conjugated bile acid at the apical membrane of jejunal epithelial cells. Brush-border membrane vesicles from intestinal epithelial cells of the rat were prepared. Absorption of two taurine-conjugated bile acids that are representative of endogenous bile acids in many variate vertebrate species were studied. In ileal, but not jejunal brush-border membrane vesicles, transport of conjugated bile acids was cis-stimulated by sodium. Transport of conjugated bile acids was trans-stimulated by bicarbonate in the jejunum. Absorption of conjugated dihydroxy-bile acids was almost twice as fast as of trihydroxy-bile acids. Coincubation with other conjugated bile acids, bromosulfophthalein, and DIDS, as well as by incubation in the cold inhibited the transport rate effectively. Absorption of conjugated bile acids in the jejunum from the rat is driven by anion exchange and is most likely an antiport transport. (+info)
Anion efflux from cytotrophoblast cells derived from normal term human placenta is stimulated by hyposmotic challenge and extracellular A23187 but not by membrane-soluble cAMP.
The regulation of placental anion transport influences fetal accretion and placental homeostasis. We investigated whether efflux of 125I- or 36Cl- from multinucleated cytotrophoblast cells derived from human term placenta is regulated by one of three stimuli: (a) the calcium ionophore A23187, (b) a 'cocktail' of agents designed to raise intracellular levels of cAMP, (c) a hyposmotic solution. After loading with the appropriate isotope for 2 h and thorough washing, cells were exposed to sequential aliquots of buffer applied and removed each minute. Following an equilibration period of 5 min one of the stimuli was applied at room temperature At the end of the experiment the cells were lysed to give a lysate count which was used to express the count obtained from each aliquot as percentage efflux of that possible for that minute. The cAMP 'cocktail' and A23187 were applied for 5 min; the hyposmotic solution was applied for 10 min. The results for 125I- at 7 min showed that the mean efflux in the presence of hyposmotic shock was greater than control (5.7 +/- 1.0% min-1 versus 2.2 +/- 0.1% min-1, respectively; mean +/- S.E.M., n = 4 placentas). Similarly mean efflux at 6 min in the presence of A23187 was also significantly greater than control (6.5 +/- 1.9% min-1 versus 2.6 +/- 1.0% min-1, respectively, n = 3 placentas). The mean efflux in the presence of the cAMP cocktail was not different from control at any time point. The results were qualitatively the same if 36Cl- was used in the place of 125I- and when the experiment was performed with 36Cl- in a HCO3- buffer gassed with CO2. Mean 125I- efflux at 6 min in response to hyposmotic challenge was 33% less (P < 0.01) in the presence of 1 mM 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 37% less (P < 0.005) in the presence of 10 microM tamoxifen but no different if the hyposmotic solution was nominally calcium free. We conclude that there are differential effects of second messengers on anion efflux from the differentiated cytotrophoblast cells. (+info)
Thermodynamic studies on anion binding to apotransferrin and to recombinant transferrin N-lobe half molecules.
Equilibrium constants for the binding of anions to apotransferrin, to the recombinant N-lobe half transferrin molecule (Tf/2N), and to a series of mutants of Tf/2N have been determined by difference UV titrations of samples in 0.1 M Hepes buffer at pH 7.4 and 25 degrees C. The anions included in this study are phosphate, sulfate, bicarbonate, pyrophosphate, methylenediphosphonic acid, and ethylenediphosphonic acid. There are no significant differences between anion binding to Tf/2N and anion binding to the N-lobe of apotransferrin. The binding of simple anions like phosphate appears to be essentially equivalent for the two apotransferrin binding sites. The binding of pyrophosphate and the diphosphonates is inequivalent, and the studies on the recombinant Tf/2N show that the stronger binding is associated with the N-terminal site. Anion binding constants for phosphate, pyrophosphate, and the diphosphonates with the N-lobe mutants K206A, K296A, and R124A have been determined. Anion binding tends to be weakest for the K296A mutant, but the variation in log K values among the three mutants is surprisingly small. It appears that the side chains of K206, K296, and R124 all make comparable contributions to anion binding. There are significant variations in the intensities of the peaks in the difference UV spectra that are generated by the titrations of the mutant apoproteins with these anions. These differences appear to be related more to variations in the molar extinction coefficients of the anion-protein complexes rather than to differences in binding constants. (+info)