Evidence that inhibitors of anion exchange induce a transmembrane conformational change in band 3

The transport inhibitor, eosin 5-maleimide, reacts specifically at an external site on the membrane-bound domain of the anion exchange protein, Band 3, in the human erythrocyte membrane. The fluorescence of eosin-labeled resealed ghosts or intact cells was found to be resistant to quenching by CsCl, whereas the fluorescence of labeled inside-out vesicles was quenched by about 27% at saturating CsCl concentrations. Since both Cs+ and eosin maleimide were found to be impermeable to the red cell membrane and the vesicles were sealed, these results indicate that after binding of the eosin maleimide at the external transport site of Band 3, the inhibitor becomes exposed to ions on the cytoplasmic surface. The lifetime of the bound eosin maleimide was determined to be 3 ns both in the absence and presence of CsCl, suggesting that quenching is by a static rather than a dynamic (collisional) mechanism. Intrinsic tryptophan fluorescence of erythrocyte membranes was also investigated using anion transport inhibitors which do not appreciably absorb light at 335 nm. Eosin maleimide caused a 25% quenching and 4,4'-dibenzamidodihydrostilbene-2,2'-disulfonate) caused a 7% quenching of tryptophan fluorescence. Covalent labeling of red cells by either eosin maleimide or BIDS (4-benzamido-4'-isothiocyanostilbene-2,2'-disulfonate) caused an increase in the susceptibility of membrane tryptophan fluorescence to quenching by CsCl. The quenching constant was similar to that for the quenching of eosin fluorescence and was unperturbed by the presence of 0.5 M KCl. Neither NaCl nor Na citrate produced a large change in the relative magnitude of the tryptophan emission. The tryptophan residues that can be quenched by CsCl appear to be different from those quenched by eosin or BIDS and are possibly located on the cytoplasmic domain of Band 3. The results suggest that a conformational change in the Band 3 protein accompanies the binding of certain anion transport inhibitors to the external transport site of Band 3 and that the inhibitors become exposed on the cytoplasmic side of the red cell membrane.     

Fluorescence quenching of human orosomucoid. Accessibility to drugs and small quenching agents.

The fluorescence behaviour of human orosomucoid was investigated. The intrinsic fluorescence was more accessible to acrylamide than to the slightly larger succinimide, indicating limited accessibility to part of the tryptophan population. Although I- showed almost no quenching, that of Cs+ was enhanced, and suggested a region of negative charge proximal to an emitting tryptophan residue. Removal of more than 90% of sialic acid from the glycan chains led to no change in the Cs+, I-, succinimide or acrylamide quenching, indicating that the negatively charged region originates with the protein core. Quenching as a function of pH and temperature supported this view. The binding of chlorpromazine monitored by fluorescence quenching, in the presence and in the absence of the small quenching probes (above), led to a model of its binding domain on orosomucoid that includes two tryptophan residues relatively shielded from the bulk solvent, with the third tryptophan residue being on the periphery of the domain, or affected allotopically and near the negatively charged field.     

Transverse location of anthracyclines in lipid bilayers. Paramagnetic quenching studies

Quenching of anthracycline fluorescence by a series of spin-labeled fatty acids was used to probe the transverse location of the drug in phosphatidylcholine bilayers in the form of small unilamellar vesicles. Stern-Volmer plots of the quenching data indicate that the fluorophore moiety of the anthracycline is intercalated into the hydrocarbon region of the bilayer, with deeper penetration observed in fluid-phase than in solid-phase vesicles. 31P-NMR parameters (T1 and nuclear Overhauser enhancement (NOE] are unaffected by the presence of drug, consistent with a binding site removed from the interfacial region. Comparison of intensity (F0/F) plots with lifetime (tau 0/tau) data shows that the predominant mechanism of anthracycline quenching by membrane-bound nitroxides is static. Since the membrane-bound drug is also accessible to quenching by I-, the binding site in the membrane must create a channel which is accessible to solvent. Two other fluorescent probes, 12-(9-anthroyloxy)stearate (12-AS) and diphenylhexatriene (DPH), were employed to confirm the results obtained with the anthracyclines, giving quenching data representative of their location in the bilayer.     

Deoxygenation affects fluorescence photobleaching recovery measurements of red cell membrane protein lateral mobility.

We have used the fluorescence photobleaching recovery technique to study the dependence on oxygen tension of the lateral mobility of fluorescently labeled band 3, the phospholipid analogue fluorescein phosphatidylethanolamine, and glycophorins in normal red blood cell membranes. Band 3 protein and sialic acid moieties on glycophorins were labeled specifically with eosin maleimide and fluorescein thiosemicarbazide, respectively. The band 3 diffusion rate increased from 1.7 x 10(-11) cm2 s-1 to 6.0 x 10(-11) cm2 s-1 as oxygen tension was decreased from 156 to 2 torr, and a further increase to 17 x 10(-11) cm2 s-1 occurred as oxygen tension was decreased from 2 to 0 torr. The fractional mobility of band 3 decreased from 58 to 32% as oxygen tension was decreased from 156 to 0 torr. The phospholipid diffusion coefficient remained constant as oxygen tension was decreased from 156 to 20 torr, but increased from 2.3 x 10(-9) cm2 s-1 to 7.1 x 10(-9) cm2 s-1 as oxygen tension was decreased from 20 to 0 torr. Neither the diffusion coefficient nor the fractional mobility of glycophorins changed significantly at low oxygen tension. Under non-bleaching excitation conditions, intensities of fluorescence emission were identical for oxygenated and deoxygenated eosin-labeled RBCs. Deoxygenated eosin-labeled RBCs required 160-fold greater laser intensities than did oxygenated RBCs to achieve comparable extents of photobleaching, however. Oxygen seems to act as a facilitator of fluorophore photobleaching and may thereby protect the fluorescently labeled red cell membrane from photodamage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in the membrane surface charge density and/or membrane potential of the porcine intestinal brush-border membrane vesicles induced by treatment with neuraminidase

The effects of neuraminidase treatment on the membrane surface charge density and/or membrane potential of the porcine intestinal brush-border membrane vesicles were studied by using three fluorescent dyes, 1,6-diphenyl-1,3,5-hexatriene (DPH), 1-anilino-8-naphthalene sulfonate (ANS), and 3,3'-dipropyl-2,2'-thiadicarbocyanine iodide (DiS-C3(5]. The results of quenching studies of DPH-labeled membranes using cationic (T1+) and anionic (I-) quenchers suggested an increase of negative charge on the membrane surface by desialylation upon neuraminidase treatment. This interpretation was further supported by a decrease of ANS-binding affinity of the membranes after treatment with the enzyme. In addition, the degree of valinomycin-induced fluorescence change of DiS-C3(5)-probed membranes in the presence of various concentrations of KCl was reduced by treatment of the membranes with neuraminidase. This suggests that penetration of the dye molecules into the vesicle interior is facilitated by the treatment. The membrane potentials estimated from the null point of valinomycin-induced changes in the DiS-C3(5) fluorescence of the control and neuraminidase-treated membranes were -25 to -29.7 and -40 to -48.8 mV, respectively. From these results, it is suggested that the membrane surface charge density and/or membrane potential of the intestinal brush-border membranes are susceptible to modification of carbohydrate moieties on the membrane surface by neuraminidase treatment.     

Domain structural flexibility in rhodanese examined by quenching of a phosphorescent probe

Rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) is an enzyme composed of two domains with the catalytic site located in the bottom of the crevice formed by the two domains. In this work, rhodanese was labeled at its catalytic site with the phosphorescence probe eosin isothiocyanide. The accessibility of molecules to the probe was determined by phosphorescence lifetime-quenching studies. The phosphorescent probe was much more accessible to small molecules (I- and thiosulfate, radius about 3-5 A) than to a larger molecule (spin-label probe TEMPO, radius about 8-10 A). It was observed that a temperature-induced change in the rate of quenching occurred at around 28 degrees C. The results are interpreted in terms of structural fluctuations and displacement in the domains.     

Oxonol-v as a probe of chromaffin granule membrane potentials

The dye, oxonol-V (bis(3-phenyl-5-oxoisoxazol-4-yl)pentamethine oxonol), can be used to estimate the transmembrane potential of chromaffin granules. The potentials result either from a resting-state Donnan equilibrium (inside negative at pH 6.6) or from an ATP-driven proton pump. The fluorescence and absorption changes generated by ATP addition depended on the pH of the medium and the dye-to-vesicle ratio. Energization resulted in an increase in the number of oxonol-V binding sites, the new binding sites having the same dissociation constant. The rate of dye association was higher with resting than with energized chromaffin granules. The absorption change was associated with a red shift whereas the fluorescence change involved a quenching due to the increase in dye concentration on the membrane. The absorption and fluorescence changes varied linearly with the transmembrane potential difference when the interior potential was positive relative to the medium.     

Interaction of dipyridamole with micelles of lysophosphatidylcholine and with bovine serum albumin: fluorescence studies.

The interaction of the coronary vasodilator dipyridamole with biological systems, protein and membranes has been studied through optical absorption and fluorescence spectroscopies. Using the analysis of the spectra and fluorescence intensity of dipyridamole (DIP) in solution, the interaction of this compound with the transport protein albumin (BSA) and with a model of cell membranes, namely micelles of lysophosphatidylcholine (L-PC), was investigated. Measurements were performed at pH 5.0 and pH 7.0 where the molecule of DIP is fully protonated and partially protonated, respectively. The quenching of fluorescence with nitroxide-stable radicals 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) as well as with acrylamide and iodide allowed the localization of the drug in the polar interface of micelles. Quenching by acrylamide and iodide in L-PC micelles demonstrated the effect of micelle protonation which increased the accessibility of iodide to the chromophore. An effective association constant was obtained both at pH 7.0 (7.5 x 10(3) M-1) and pH 5.0 (2.5 x 10(3) M-1) and a very good agreement with the proposed binding model was observed. The quantum yields of fluorescence data agree very well with the fluorescence lifetimes. The measurement of lifetimes was important to understand the kinetic data obtained from Stern-Volmer plots both of radical, acrylamide and iodide quenching of fluorescence. It was observed that, in the presence of micelles, the kq value increased for TEMPO while decreased for TEMPOL. This result, together with the vanishing solubility of DIP in saturated hydrocarbons and the preferential partition of TEMPO in micelles, suggested the localization of DIP in the polar micellar interface. This is also supported by the enhanced iodide quenching at pH 5.0, constancy of acrylamide quenching in the range of pH 7.0-5.0 and the partition of TEMPO and TEMPOL in SDS micelles. The association constant of DIP to BSA was also estimated both at pH 7.0 (2 x 10(4) M-1) and pH 5.0 (4 x 10(3) M-1). Quenching studies with nitroxide radicals, acrylamide and iodide also suggested the binding of the drug to a hydrophobic region of the protein. At pH 5.0, the protein undergo a conformational change which leads to a loosening of the overall structure so that the accessibility of the nitroxide radicals for DIP is increased at this pH. The differences in kq values at pH 7.0 and pH 5.0 suggested that at pH 7.0 the chromophore is protected in the protein site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Triplet-state detection of labeled proteins using fluorescence recovery spectroscopy

The experimental procedures for detecting the triplet states of chromophores in solutions (cuvettes) by fluorescence recovery spectroscopy (FRS) are described in detail, together with applications in studies of protein structure and protein-cell interactions in the microsecond to millisecond time domain. The experimental configuration has been characterized by measuring the emission intensities and anisotropies of eosin and erythrosin immobilized in poly(methyl methacrylate). The fluorescence data are compared with those from phosphorescence emission measurements and with theoretical predictions. Triplet-state lifetimes were obtained in 5 mM phosphate buffer, pH 7.0, of concanavalin A labeled with eosin, tetramethylrhodamine, and fluorescein and of alpha 2-macroglobulin labeled with the first two probes. In the case of labeled concanavalin A, iodide quenching measurements gave bimolecular rate constants of approximately 10(9) M-1 s-1. The usefulness of FRS for studying protein-cell interactions is exemplified with eosin-labeled concanavalin A bound to living A-431 human epidermoid carcinoma cells. Finally, the advantages and disadvantages of the technique are compared to those of the alternative phosphorescence emission method.     

Solvent accessibility of the adenosine 5'-triphosphate catalytic site of sarcoplasmic reticulum CaATPase

The CaATPase of rabbit skeletal sarcoplasmic reticulum was labeled at or near the ATP catalytic site with fluoresceinyl isothiocyanate (FITC), and the accessibility of the attached probe to the bulk solvent was determined by I- quenching of its fluorescence. The quenching of free FITC was also measured. In both cases, the quenching was of the Stern-Volmer type and collisional quenching rate constants were obtained over the pH range 5-8 in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and with added Ca2+, vanadate, or phosphate. The fluorescence intensity and susceptibility to quenching by I- of free FITC were insensitive to the added ligands. In all cases, the intensity decreased with pH, as predicted from the known properties of FITC mono- and dianions. The collisional quenching rate constants increased at lower pH, as expected for I- quenching of a molecule with decreasing negative charge due to protonation. When FITC was attached to the CaATPase, the FITC fluorescence intensity and I- collisional quenching rate constants were sensitive to ligand binding as well as pH. The changes in fluorescence intensity with acidity, when compared to the results for free FITC, indicated the pKa of the FITC was reduced 0.6 unit when it was attached to the CaATPase. Excited-state lifetime measurements indicated that ligand effects at constant pH were not due to protonation-induced changes in FITC quantum yield but to conformational changes of the CaATPase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quenching of tryptophanyl fluorescence of human growth hormone by iodide.

Quenching of tryptophanyl fluorescence of human growth hormone by I- followed saturation kinetics and was abolished by KSCN. In the presence of 6 M guanidine hydrochloride quenching was linear between 0 to 0.2 M KI. These results suggest that I- quenched the fluorescence of the native hormone by binding at or near the single tryptophanyl residue. Quenching by 0.1 M KI decreased exponentially with increasing concentrations of human and bovine growth hormones. Acidification did not have a significant effect on quenching of the human hormone, but it markedly decreased quenching of the bovine hormone. Conformational differences at the vicinity of the lone tryptophanyl residue that could be inferred by these and other experiments may be contributing to the biological specificity of native human and bovine growth hormones.     

Internal motions of band 3 of human erythrocytes

Band 3 was labeled with N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonate either exofacially in the intact washed erythrocytes or endofacially by treating inside-out vesicles. Exo labeling resulted in the labeling of several other proteins, besides band 3, which could not be removed from the membrane. Therefore, the exo-labeled band 3 was extracted and purified by chromatography on DEAE-cellulose in Triton X-100. The endo labeling also resulted in the labeling of several other proteins. In this case, washing with NaOH removed all labeled material except band 3 from the vesicles. The lifetime of bound N-[(acetylamino)ethyl]-5-naphthylamine-1-sulfonate was heterogeneous, suggesting the positioning of the label in different environments either because different sites were labeled or because of positional freedom of the label at the same point of attachment. The main fraction of emission intensity had a lifetime near 20 ns, as expected for a hydrophobic environment. The rest showed a lifetime of about 3 ns in the exo-labeled band 3 and 9 ns in the endo-labeled band 3. Both lifetimes appeared to be independent of temperature between 5 and 25 degrees C, suggesting shielding of the probe from the solvent. Quenching phenomena must be responsible for both the 3- and 9-ns lifetimes, not due to residual heme, as proven by the persistence of such quenching in the Triton X-100 extracted protein. The correlation times indicated the presence of a short component, between 2 and 4 ns in the different systems, probably due to the presence of a flexible portion in the structure of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measurement and analysis of triplet-state lifetimes by multifrequency cross-correlation phase and modulation phosphorimetry.

In this paper we describe a novel approach to study the triplet-state lifetimes by a conventional multifrequency cross-correlation phase and modulation apparatus. The analysis of phase and modulation data of eosin-labeled band 3 erythrocyte ghosts revealed the existence of two phosphorescence lifetime values of 2700 and 750 microseconds, with a fractional contribution of 78 and 22%, respectively, which are in good agreement with those reported in the literature. Differential polarization phase analysis, which facilitates the study of the rotational properties of band 3, provided data in good agreement with those reported in the literature. The method proposed in this paper to study the radiative emission from the triplet state may represent a convenient alternative to the pulse laser flash technique.     

Pore formation by the Bordetella adenylate cyclase toxin in lipid bilayer membranes: role of voltage and pH

The bifunctional adenylate cyclase toxin (ACT or CyaA) of Bordetella pertussis invades target cells via transport through the cytoplasmic membrane. The membrane potential represents thereby an important factor for the uptake in vivo. Previous studies demonstrated that adenylate cyclase (AC) delivery into cells requires a negative membrane potential inside the cells. The results of lipid bilayer experiments with ACT presented here indicated that two different types of pore-like structures are formed by ACT dependent on the orientation of the electrical potential across the membranes. Pore formation at a positive potential at the cis side of the membranes, the side of the addition of the toxin, was fast and its conductance had a defined size, whereas at negative potential the pores were not defined, had a reduced pore-forming activity and a very short lifetime. Fluctuations inserted at positive potentials showed asymmetric current-voltage relationships for positive and negative voltages. Positive potentials at the cis side resulted in an increasing current, whereas at negative potentials the current decreased or remained at a constant level. Calcium ions enhanced the voltage dependence of the ACT pores when they were added to the cis side. The single-pore conductance was strongly affected by the variation of the pH value and increased in 1M KCl with increasing pH from about 4 pS at pH 5 to about 60 pS at pH 9. The ion selectivity remained unaffected by pH. Experiments with ACT mutants revealed, that the adenylate cyclase (AC) and repeat (RT) domains were not involved in voltage and pH sensing.     

Pore formation by the Bordetella adenylate cyclase toxin in lipid bilayer membranes: Role of voltage and pH

The bifunctional adenylate cyclase toxin (ACT or CyaA) of Bordetella pertussis invades target cells via transport through the cytoplasmic membrane. The membrane potential represents thereby an important factor for the uptake in vivo. Previous studies demonstrated that adenylate cyclase (AC) delivery into cells requires a negative membrane potential inside the cells. The results of lipid bilayer experiments with ACT presented here indicated that two different types of pore-like structures are formed by ACT dependent on the orientation of the electrical potential across the membranes. Pore formation at a positive potential at the cis side of the membranes, the side of the addition of the toxin, was fast and its conductance had a defined size, whereas at negative potential the pores were not defined, had a reduced pore-forming activity and a very short lifetime. Fluctuations inserted at positive potentials showed asymmetric current-voltage relationships for positive and negative voltages. Positive potentials at the cis side resulted in an increasing current, whereas at negative potentials the current decreased or remained at a constant level. Calcium ions enhanced the voltage dependence of the ACT pores when they were added to the cis side. The single-pore conductance was strongly affected by the variation of the pH value and increased in 1M KCl with increasing pH from about 4 pS at pH 5 to about 60 pS at pH 9. The ion selectivity remained unaffected by pH. Experiments with ACT mutants revealed, that the adenylate cyclase (AC) and repeat (RT) domains were not involved in voltage and pH sensing.     

A fluorescence study of A23187 interaction with phospholipid vesicles

The fluorescence of the ionophore A23187 has been monitored in suspensions of egg yolk phosphatidylcholine (EYPC) and dipalmitoyl phosphatidylcholine (DPPC) vesicles. Both the protonated form of A23187 and the Ca²⁺ complex exhibit fluorescence enhancement when extracted into a hydrophobic environment. Measurements of fluorescence intensity versus lipid concentration were thus used to establish lower limits to the lipid/ water partition coefficients. Values obtained in this way were ? 50 ml water/mg phosphatidylcholine. Quenching of A23187 fluorescence by the spin labels 5NMS (methyl ester of 5-nitroxyl stearate), 12NMS, 16NMS, and TEMPO stearamide in EYPC and DPPC vesicles was also investigated. In EYPC all the labels yielded fairly linear Stern-Volmer plots, with TEMPO stearamide quenching about half as strong as the other probes. Quenching in DPPC was generally much stronger than in EYPC, but 12 NMS and 16NMS gave hyperbolic Stern-Volmer plots, apparently due to clustering of the labels. In all the cases the protonated form of A23187 was quenched approximately twice as efficiently as the Ca²⁺ complex, possibly due to a longer fluorescence lifetime for the former. Calculations based on measured spectral properties were performed which indicate that the Förster transfer mechanism extends the nitroxides' quenching range to ～- 10 Å.     

Fluorescence studies on a membrane-embedded peptide from the carboxy terminus of lipophilin

Fluorescence of an intramembranous polypeptide (T-3) derived from the carboxy-terminal sequence of lipophilin was studied in aqueous solution, detergent micelles, and lipid vesicles. In all cases, the fluorescence of the only Trp (211) was indicative of a hydrophobic, buried residue. Addition of lysophosphatidylcholine (LPC) or phosphatidylcholine (PC) gave Trp-211 a more hydrophobic, less quenching environment as compared to that in aqueous solution. Energy transfer between Trp and Tyr observed in aqueous solution was decreased by the addition of lipid or detergent. There was limited quenching by acrylamide both in the aqueous and in the lipid or detergent environments. However, PC or LPC further decreased this quenching. Cs+ and I- were even less accessible than acrylamide to Trp, further proving that the Trp was located inside the lipid bilayer. The quenching indicated that I- binds to positive charges of the protein located on the surface of the membrane. This, combined with knowledge of the sequence of lipophilin, suggested that Trp-211 was located within the membrane but was close to amino acid residues that are external to the bilayer.     

ATPase activity and anion transport across the peribacteroid membrane of isolated soybean symbiosomes

Addition of ATP to intact symbiosomes isolated from soybean nodules, resulted in generation of a membrane potential (positive inside) across the peribacteroid membrane (PBM). This energisation was monitored as oxonol fluorescence quenching. The rate of fluorescence quenching was inhibited by the inclusion of permeant anions in the reaction medium. Using this inhibition as a measure of anion uptake across the PBM, the presence of a phthalonate-sensitive dicarboxylate carrier on the PBM was confirmed. Following dissipation of the membrane potential by a permeant anion, a pH gradient, measured using [¹⁴C]methylamine uptake, was slowly established across the PBM. This pH was abolished by addition of an uncoupler but was insensitive to inhibitors of bacteroid respiration. The difference in pH between the external medium and the symbiosome interior was estimated to be in the range of 1–1.6 pH units. The magnitude in planta will depend on the concentrations of ATP and permeant anions in the cytosol of the host cell.Abbreviations PBM peribacteroid membrane - electrical membrane potential - MA methylamine The term symbiosome refers to the peribacteroid unit consisting of bacteroids enclosed in the host-derived peribacteroid membrane     

Quenching of the triplet state of Safranine-O by aliphatic amines in AOT reverse micelles studied by transient absorption spectroscopy

The photophysics of Safranine-O (3,6-diamino-2,7-dimethyl-5 phenyl phenazinium chloride) (SfH(+)Cl(-)) was investigated in reverse micelles (RMs) of AOT (sodium bis(2-ethylhexyl)sulfosuccinate) with special emphasis on the triplet state processes. The triplet is formed in its monoprotonated form, independently of the pH of the water used to prepare the RMs. While the intersystem crossing quantum yields in RMs are similar to those in organic solvents, the triplet lifetime is much longer. Since the pH in the water pool of AOT RMs is close to 5 and the triplet state of the dye is subjected to proton quenching, the long lifetime indicates that the dye resides in a region where it cannot be reached by protons during its lifetime. All the measurements indicate that the dye is localized in the interface, sensing a medium of micropolarity similar to EtOH : water (3:1) mixtures. The quenching by aliphatic amines was also investigated. While the quenching by the hydrophobic tributylamine is similar to that in methanol, the hydro-soluble triethanolamine is one order of magnitude more effective in RMs than in homogeneous solution. In the latter case the quenching process is interpreted by a very fast intramicellar quenching, the overall kinetics being controlled by the exchange of amine molecules between RMs. Semireduced dye is formed in the quenching process in RMs in the di-protonated state with a comparable quantum yield to the monoprotonated state formed in homogeneous solvents. The results point to the advantage of the reverse micellar system for the generation of active radicals for the initiation of vinyl polymerization, since a much lower concentration of amine can be employed with similar quantum yields.     

Delta pH, H+ diffusion potentials, and Mg2+ ATPase in neurosecretory vesicles isolated from bovine neurohypophyses

The proton gradient (delta pH) and electrical potential (delta psi) across the neurosecretory vesicles were measured using the optical probes 9-aminoacridine and Oxanol VI, respectively. The addition of neurosecretory vesicles to 9-aminoacridine resulted in a rapid quenching of the dye fluorescence which was reversed when the delta pH was collapsed with ammonium chloride or K+ in the presence of nigericin. From fluorescence quenching data and the intravesicular volume, delta pH across the membrane was calculated. Mg2+ ATP caused a marked carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive change in the membrane potential measured using Oxanol VI (plus 100 mV inside positive), presumably due to H+ translocation across the neurosecretory vesicle membrane. Imposition of this membrane potential was responsible for the lysis of vesicles in the presence of permeant anions. The effectiveness of these anions to support lysis reflected the relative permeability of the anion which followed the order acetate greater than I- greater than Cl greater than F- greater than SO4- = isethionate = methyl sulfate. These data showed that the neurosecretory vesicles possess a membrane H+-translocating system and prompted the study of Mg2+-dependent ATPase activities in the vesicle fractions. In intact vesicles a Mg2+ ATPase appeared to be coupled to electrogenic proton translocation, since the enzyme activity was enhanced by uncoupling the electrical potential, using proton ionophores. Inhibition of this enzyme with dicyclohexylcarbodiimide also inhibited the carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive delta psi across the vesicle membrane caused by H+ translocation. A second Mg2+ ATPase was also found on the vesicle membranes which is sensitive to vanadate. Complete inhibition of this enzyme with vanadate had little effect on the proton ionophore-uncoupled ATPase activity or on the Mg2+ ATP-induced membrane potential change.