Granule Ghosts Research Articles

Role of Phosphatidylserine and Diacylglycerol in the Fusion of Chromaffin Granules with Target Membranes

The role of phosphatidylserine and diacylglycerol in the fusion of chromaffin granules with target membranes was investigated in vitro, monitoring the mixing of membrane lipids with the octadecyl rhodamine B fluorescence dequenching assay. Polylysine was able to induce fusion of chromaffin granule ghosts with plasma membrane vesicles in the absence of Ca 2+. This fusion was maximal at 1.6 μM polylysine and the kinetics were dependent on the amount of plasma membrane added. Polylysine lowered the Ca 2+ threshold concentration for inducing fusion, increased the extent of fusion at a given Ca 2+ concentration and acted synergistically with Ca 2+ when added prior to the cation. Removal of membrane proteins by trypsinization of chromaffin granule ghost and/or plasma membranes increased the extent of polylysine induced fusion. Incorporation of diacylglycerol into the plasma membrane promoted Ca 2+-induced fusion. Chromaffin granule ghosts were induced to fuse with model membranes of different complexities from one resembling the inner monolayer of the erythrocyte membrane to one composed solely of pure phosphatidylserine. The characteristics of the fusion with model membranes were qualitatively similar to that observed with target plasma membrane, although differences in kinetics and stoichiometries were found. Interestingly, considerably low (μM) Ca 2+ concentrations were able to induce fusion of chromaffin granule ghosts with diacylglycerol containing phosphatidylserine vesicles in the presence of polylysine. Our results indicate that acidic phospholipid-like phosphatidylserine may have an important role in the process of fusion and suggest that diacylglycerol, as a destabilizing lipid, may have a role by itself in promoting exocytosis in chromaffin cells.

Indirect coupling of calcium transport in chromaffin granule ghosts to the proton pump

In chromaffin granule ghosts, the Na+/Ca2+ exchanger in the granule membrane can provide a high affinity (Km 1-3 microM) and high capacity (Vmax 50-100 nmol mg min-2) mechanism for Ca2+ accumulation. The activity of the Na+/H+ antiporter can be used to couple Ca2+ uptake via Na+/Ca2+ exchange to ATP-dependent proton translocation via the granule membrane H(+)-ATPase. Therefore, Ca2+ uptake can be indirectly linked to the proton pump. However, under conditions designed to mimic the environment of a granule in the cytosol of a chromaffin cell, measured rates of Ca2+ uptake are low, a free Ca2+ concentration of about 5 microM in the ghost matrix being attained. Under such circumstances, the granules seem unlikely to play a major role in calcium homeostasis in the intact cell.

Chapter 21 Synexin (annexin VII) and membrane fusion during the process of exocytotic secretion

Publisher Summary Synexin is a cytosolic protein, which in the presence of increased calcium can undergo a conformational change that renders it highly hydrophobic. This chapter discusses some of the assumptions to model the fusion process in vitro, the ways in which synexin promotes different aspects of the model, and some of the physical and chemical evidence underlying the present understanding of synexin action during membrane fusion. The ability of synexin to fuse target membranes lies in the fact that once activated by calcium, the synexin monomers form oligomers, which are capable of interacting with two membranes simultaneously. While the hydrophobic bridge hypothesis has been developed to explain the process of fusion driven by synexin, it need not be limited in principle to just this fusion event. It may be anticipated that any fusion event in which a hydrophobic environment is introduced to the potential fusion site, may proceed by the hydrophobic bridge mechanism and is exemplified by studies on calcium-dependent, synexin driven membrane contact and fusion of chromaffin granules, chromaffin granule ghosts, and phospholipid bilayer liposomes.

Activity of membranous dopamine beta-monooxygenase within chromaffin granule ghosts. Interaction with ascorbate

The role of intra- and extravesicular ascorbate has been investigated in dopamine beta-monooxygenase (D beta M) turnover using adrenal medulla chromaffin granule ghosts. Resealing of vesicle ghosts with high levels of intravesicular ascorbate leads to viable vesicles, as evidenced from the high rates of the ATP-dependent accumulation of tyramine, Vmax = 14 +/- 1 nmol/min.mg and Km = 20 +/- 6 microM. However, the D beta M-catalyzed conversion of tyramine to octopamine occurs slowly, Vmax = 0.50 +/- 0.13 nmol/min.mg and Km = 29 +/- 18 mM. When ascorbate is present instead in the external buffer, the D beta M rate increases 3.6-fold for a final Vmax = 1.8 +/- 0.2 and Km = 1.2 +/- 0.3 mM. This relatively high rate of enzyme turnover is retained in ghosts resealed with a large excess of ascorbate oxidase, ruling out contamination by intravesicular ascorbate as the source of enzyme activity. The synergistic effect of intravesicular ascorbate was examined under conditions of 2 mM external ascorbate, showing that the enzymatic rate increases 2.7-fold, from 1.2 (0 internal ascorbate) to 3.2 +/- 0.4 nmol/min.mg (saturating internal ascorbate). This result confirms that high levels of internal ascorbate are not damaging to intravesicular D beta M. These studies demonstrate very clearly that external ascorbate is the preferred reductant for the membranous form of D beta M in chromaffin granule ghosts.

The effect of calcium channel blockers on the H +-ATPase and bioenergetics of catecholamine storage vesicles

A number of commonly used calcium channel blockers have been compared with respect to their effects on the bioenergetics of catecholamine storage vesicles. Chromaffin granule ghosts with a well-preserved ability to actively transport and store catecholamines, were used as a model for adrenergic synaptic vesicles due to their functional similarity. Nicardipine, verapamil, terodiline and diltiazem were found to have effects comparable to that of prenylamine (Grønberg, M., O. Terland, E.S. Husebye and T. Flatmark, 1990. Biochem. Pharmacol. 40, 351) by inhibiting the generation of a transmembrane proton electrochemical gradient driven by the vesicular H(+)-ATPase, mainly by loose-coupling/uncoupling of this ATPase. Amlodipine inhibited the internal acidification of the vesicles in a tyramine-like manner and increased the steady-state membrane potential (positive inside) generated by the MgATP-dependent proton translocation. Nifedipine and felodipine also inhibited the efficiency of the proton pump, but their mechanisms of action require further investigation. The concentrations giving 50% inhibition of the H(+)-ATPase-dependent generation of a pH-gradient were found to be: 12 microM felodipine, 16 microM nicardipine, 25 microM terodiline, 50 microM nifedipine, 60 microM verapamil, 65 microM amlodipine and 150 microM diltiazem. The effects of the calcium channel blockers on the bioenergetics of chromaffin granules explain the release of catecholamines from sympathetic nerves and ganglia in vitro by the calcium channel blockers.

Biomembrane-modulated, lysosomal phospholipase A 2 contamination of chromaffin granule ghosts

It was reported that subcellular fractionation of bovine adrenal medulla results in the separation of distinct, non-calcium-dependent phospholipases A 2 — one associated with chromaffin granule ghosts, another with lysosomes. The basis of this distinction is pH optimum: in routine assays utilizing neat liposomal substrates, the chromaffin granule ghost-associated enzyme is alkaline-active whereas the lysosomal enzyme is acid-active (Husebye, E.S. and Flatmark, T. (1987) Biochim. Biophys. Acta 920, 120–130). We now report that biomembranes alter liposomal substrates and/or lysosomal phospholipase A 2 such that the enzyme now hydrolyzes them (at low cation concentration) with an alkaline pH optimum. In a lysosomal membrane fraction, phospholipase A 2 activity at pH 7.5 relative to activity at pH 5.0 increases as increasing amounts of lysosomal membranes are assayed. The pH optimum of chromaffin granule ghost-associated phospholipase A 2 toward liposomal substrates is likewise biomembrane-dependent and, when assayed carefully, is indistinguishable on the basis of optimal pH from the lysosomal enzyme. Although chromaffin granule ghost-associated phospholipase A 2 is most likely a lysosomal contaminant, its broad, biomembrane-modulated pH range may still allow it to participate in catecholamine secretion. More importantly, however, sensitivity of adrenal medullary lysosomal phospholipase A 2 to biomembranes broadens its potential physiologic pH range and may also play a role in the regulation of this potentially deleterious activity.

The effect of prenylamine and organic nitrates on the bioenergetics of bovine catecholamine storage vesicles

We have compared the cardioprotective agents prenylamine and glyceryl trinitrate (GTN) with respect to their effects on the bioenergetics of catecholamine storage vesicles. Chromaffin granule ghosts, which have a well preserved ability to actively transport and store catecholamines, were used as a model for adrenergic synaptic vesicles due to their functional similarity. Prenylamine, which partially and reversibly deplete the endogenous stores of noradrenaline in adrenergic nerves and ganglia, was found to inhibit the generation of the transmembrane proton electrochemical gradient driven by a H +-lATPase, mainly by acting as an uncoupler of this ATPase. The inhibition of the energy dependent dopamine uptake (and noradrenaline biosynthesis) by prenylamine could be accounted for by its effect on the bioenergetics of the storage vesicles. The organic nitrates glyceryl trinitrate and isosorbide dinitrate also partly inhibited the catecholamine uptake in parallel with their effects on the proton electrochemical gradient. It is concluded that GTN is a weak catecholamine depletor. Experiments with 3-morpholinosydnonimin-hydrochloride, a source of nitric oxide (NO), opens up the possibility that the mechanism of inhibition of the bioenergetics of chromaffin granule ghosts by GTN is mediated by NO.

Purification and kinetic properties of a membrane-bound phosphatidylinositol kinase of the bovine adrenal medulla

Phosphatidylinositol (PI) kinase (EC 2.7.1.67), an integral membrane protein of chromaffin granule ghosts of the bovine adrenal medulla, was found to phosphorylate PI in the 4-position of the inositol ring. The PI kinase was purified about 200-fold from a membrane fraction containing chromaffin granules and microsomes by extraction with Triton X-114, followed by phase partition (clouding) and heparin Sepharose chromatography. The PI kinase preparation (specific activity of 5.1 nmol PIP/mg protein per min) was free from other enzymatic activities that metabolize polyphosphoinositides. K m values of 55 μM and 40 μM for ATP and PI, respectively, were estimated for the purified enzyme. Concentrations of Triton X-100 above the critical micellar concentration (0.01%, w/v) were necessary to support significant enzyme activity, which was optimal at about 0.1% (w/v). Its dependence of pH was similar to that of the membrane-bound enzyme, with a broad optimum around pH 7. Mes in the millimolar concentration range was found to strongly inhibit the activity of the purified PI kinase ( I 50 at about 4 mM). The enzyme was almost totally inhibited by low micromolar concentrations of free calcium, and stimulated by hydrophilic cations, e.g., Mg 2+ and poly( l-lysine), with the same potencies as for the membrane-bound enzyme. The amphiphilic cation trifluoperazine, however, stimulated the activity of purified PI kinase less effectively than the membrane-bound enzyme (Husebye, E.S. and Flatmark, T. (1988) Biochem. Pharmacol. 37, 449–456), whereas the inhibitory effect of near millimolar concentrations of trifluoperazine was the same for the two forms of the enzyme. It is concluded that the membrane-bound PI kinase of this tissue is of type II according to the classification of Cantley and co-workers (Whitman et al. (1987) Biochem. J. 247, 165–174).

Nature of Rate-limiting Steps in a Compartmentalized Enzyme System: Quantitation of dopamine transport and hydroxylation rates in resealed chromaffin granule ghosts

Abstract Using isolated chromaffin granule ghosts from bovine adrenal medullae, we have studied the kinetics of dopamine beta-monooxygenase (D beta M) activity as it is linked to dopamine transport. Measurements of the initial rates of transport and of transport-linked norepinephrine formation suggested that enzyme activity may be partially rate-limiting in the coupled carrier/enzyme system. This was confirmed by (i) measurements of initial rates of norepinephrine formation using deuterated substrate, which gave isotope effects greater than 2.0, and (ii) kinetic measurements using ghosts pulsed with varying concentrations of labeled dopamine, which indicated substantial substrate accumulation in the vesicle interior as a function of time. Initial rates of product formation, when combined with approximations of internal substrate concentrations, allowed estimates of Kcat and Km for intravesicular D beta M. Activation by external reductant was apparent in both initial rate parameters and the measurements of transients. Under conditions of optimal D beta M activity, the enzyme rate parameters (kcat = 0.31 nmol/s.mg and Km = 2 mM) indicated partial rate limitation compared to dopamine transport (kcat = 0.38 nmol/s.mg and Km = 32 microM). Compartmental analysis of the time curves, performed using numerical nonlinear least squares methods, gave least squares estimates of rate constants for a simple carrier mechanism and kcat values for D beta M which were consistent with estimates from initial rates.

Purification and kinetic properties of a soluble phosphatidylinositol-4-phosphate kinase of the bovine adrenal medulla with emphasis on its inhibition by calcium ions

A phosphatidylinositol-4-phosphate kinase (PIP kinase, EC 2.7.1.68) was purified about 1000-fold from the cytosolic fraction of bovine adrenal medulla by ammonium sulfate precipitation, anion exchange, phosphocellulose and gel permeation chromatography. The PIP kinase preparation was free from other polyphosphoinositide metabolizing activities, and some of its catalytic properties were studied using both membrane-bound and liposomal/micellar phosphatidylinositol 4-phosphate (PIP) as the substrate. Using a native substrate (chromaffin granule ghosts) enriched in PIP, the Km value for MgATP was about 130 microM at 5 mM Mg2+, and optimum activity was obtained at pH 7.2. ADP inhibited the PIP kinase activity in a mixed non-competitive manner. The enzyme was stimulated by Mg2+ and spermidine, whereas free calcium effectively inhibited the PIP kinase activity (I50 at about 0.1 microM Ca2+). This inhibition was independent of calmodulin and CaATP (80 microM) was not a substrate for the PIP kinase. The presence of near intracellular concentrations of potassium decreased the sensitivity to calcium ions (I50 of about 50 microM), but the inhibitory effect was still evident in the physiologically interesting concentration range. The concentration for half-maximal inhibition was three orders of magnitude higher (I50 of about 200 microM) using a liposomal/micellar suspension of purified PIP as the substrate, which demonstrates the importance of using a native substrate in studying the regulatory properties of this enzyme. It is concluded that the high sensitivity of the PIP kinase activity to calcium ions is likely to be physiologically significant, as recently proposed for the phosphatidylinositol (PI) kinase (Husebye, E.S. and Flatmark, T. (1988) Biochim. Biophys. Acta 968, 261-265), and may represent a negative feedback control of the cytosolic calcium concentration. The cationic amphiphile trifluoperazine was a potent inhibitor of PIP kinase activity (I50 at about 15 microM), and may represent a useful tool to study PI kinase selectively in membranes or cells containing both kinases.

Effects of dopamine β-monooxygenase substrate analogs on ascorbate levels and norepinephrine synthesis in adrenal chromaffin granule ghosts

Chromaffin granule ghosts from bovine adrenal medullae have been used to investigate the effects of prototypic dopamine beta-monooxygenase substrate analogs of two distinct classes on intravesicular reduced ascorbic acid (AscH2) levels and on norepinephrine synthesis. Phenyl-2-aminoethyl sulfide (PAES), a sulfur-containing substrate, was shown to concentrate within ghosts, a process that was time and ATP dependent, but reserpine insensitive. Dopamine beta-monooxygenase oxygenation of PAES resulted in accumulation of the oxygenation product, PAESO, without affecting intravesicular levels of AscH2. Similarly, incubations of ghosts with phenyl-2-aminoethyl selenide (PAESe) also resulted in rapid, time- and ATP-dependent, but reserpine-insensitive uptake. However, oxygenation of PAESe by dopamine beta-monooxygenase within ghosts was found to cause a marked decrease in intravesicular AscH2, without buildup of the oxygenated product, phenyl 2-aminoethyl selenoxide. These results illustrate two basic differences between the consequences of PAES and PAESe turnover: while PAES accumulation proceeds concomitant with PAESO production and without AscH2 depletion, PAESe accumulation proceeds with a marked lowering of internal AscH2 but without observable product formation. Both PAES and PAESe were capable of competing with dopamine, the physiological substrate, for enzymatic oxygenation and/or vesicular uptake, and were capable of significantly reducing norepinephrine synthesis. In experiments where ghosts were preincubated with either PAES or PAESe with delayed addition of dopamine, it was clear that neither compound nor their oxygenated products interfered with electron transport via cytochrome b561. These results are consistent with the hypothesis that the physiological activity observed with both PAES and PAESe may be related to their ability to gain entrance to adrenergic neurons and decrease norepinephrine synthesis within neurotransmitter storage vesicles.

Phosphatidylinositol kinase of bovine adrenal chromaffin granules modulation by hydrophilic and amphiphilic cations

The effects of hydrophilic and amphiphilic cations on the activity of phosphatidylinositol (PI) kinase (EC 2.7.1.67) of chromaffin granule ghosts were investigated. The cations studied can be divided into two groups, i.e. (i) compounds with a biphasic response (stimulation and inhibition), and (ii) those with a selective stimulatory effect on the enzyme activity. The cationic amphiphile trifluoperazine belongs to the first group, and stimulated the enzyme activity, maximal at 80 μM (2-fold), with a progressive inhibition at higher concentrations. This biphasic response was shared by a number of structurally related cationic ampniphiles, i.e. the tricyclic antidepressants, imipramine and desipramine, the phenothiazine, chlorpromazine, the miconazole derivative, calmidazolium, the β-adrenergic agonist, propranolol, compound 48/80, as well as by the hydrophilic cations neomycin and poly-L-lysine. On the other hand, a pure stimulatory effect was observed with the amphiphilic polypeptide mastoparan and the polycationic compound spermidine, whereas ACTH 1–39 and ACTH 1–24 (peptides structurally related to mastoparan) revealed a slight inhibitory effect. We conclude that all the cations tested including Mg 2+, stimulate PI kinase activity rather unspecifically by binding of the positively charged groups to a membrane component, probably the PI kinase itself. This site is different from that mediating the specific inhibition by calcium (Husebye ES and Fiatmark T, Biochim Biophys Acta 968: 261–265,1988). The inhibitory effect of cationic amphiphiles is correlated to their lipid solubility, and represents a perturbation of the membrane structure, but not a solubilization of enzyme or phosphoinositide from the membrane. The inhibitory effect of hydrophilic cations is due to complexation of ATP.

A molecular basis for synexin-driven, calcium-dependent membrane fusion.

Membranes of secretory vesicles fuse with each other and with plasma membranes during exocytosis in many different cell types. The probable role of calcium in the process is now widely accepted, and it is possible that at least one cytosolic mediator of calcium action is synexin. Synexin is a 47,000 Mr calcium-binding protein, initially discovered in the bovine adrenal medulla, which binds to granule membranes and to inner aspects of chromaffin cell plasma membranes. Synexin causes chromaffin granules to aggregate, and such aggregates can be caused to fuse in the additional presence of arachidonic acid. Synexin also mediates the direct fusion of liposomes and chromaffin granule ghosts. To understand better the mechanisms of membrane fusion promoted by synexin we have attempted to define the primary sequence of the protein. Our initial efforts were directed towards purification of bovine synexin in sufficient amounts to allow us to sequence tryptic peptides. However, as the project progressed we also directed our attention to human synexin, preparing peptides from this protein as well. From analysis of bovine peptides we learned that the synexin molecule might be closely related to a class of proteins including lipocortin I, calpactin (p36), endonexin II, protein II and calelectrin 67K. Complete analysis of a human synexin cDNA clone revealed strong homology with bovine synexin. The analysis also showed that synexin contained a unique, long, highly hydrophobic N-terminal leader sequence followed by a characteristic four-fold repeat homologous with those found in other members of the synexin gene family. The highly hydrophobic character of synexin seems consistent with information previously obtained that synexin is able to insert directly into the interior of bilayers prepared not only from purified phosphatidylserine but also from biological membranes. The evidence for such insertions is a dramatic increase in the capacitance of the membrane, formed at the tip of a patch pipette, when calcium-activated synexin is applied to the bilayer. Additional evidence is the fact that synexin also forms calcium-selective channels when the protein is applied to the cytosolic aspect of the plasmalemma when that side is also exposed to calcium at sub-millimolar concentrations. Thus, the synexin molecule not only enters the membrane, but also spans it. From these and other data we have developed the concept that the fusion process may involve synexin forming a 'hydrophobic bridge' between two fusing membranes. Lipid movement across this bridge may then be the material basis for final fusion.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochemical and immunological evidence for a calcium pump in chromaffin granules

ATP stimulated the accumulation of 45Ca2+ by chromaffin granule ghosts which contained 5 mM oxalate to trap transported calcium within the lumen. Inasmuch as the ATP-dependent 45Ca2+ transport was resistant to 25 mM ammonium acetate as well as the proton ionophore, carbonylcyanide-m-chlorophenylhydrazone, the chromaffin granule proton translocating ATPase does not provide the energy for this process. Instead, we suggest that chromaffin granules contain a calcium translocating ATPase which catalyzes the 45Ca2+ uptake directly. The observation that chromaffin granules bind to a monoclonal antibody raised against a calcium pump from bovine brain supports this hypothesis.

Effects of the sulfhydryl reagent N-ethylmaleimide on reserpine binding to the catecholamine transporter in chromaffin granule membranes.

1. Catecholamines are transported into chromaffin granules via a carrier-mediated, active-transport process which is inhibited by micromolar concentrations of the sulfhydryl reagent, N-ethylmaleimide (NEM). Reserpine is a very potent, competitive inhibitor of the catecholamine transporter and can be used to investigate the characteristics of the catecholamine transporter. 2. The purpose of this study was to determine whether [3H]reserpine binding to the catecholamine transporter present in chromaffin granule membranes isolated from bovine adrenal glands was also inhibited by NEM and, if so, whether this was a direct or an indirect effect of NEM on the catecholamine transporter. 3. Both [3H]norepinephrine transport into and [3H]reserpine binding to the chromaffin granule ghosts isolated from bovine adrenal glands are inhibited by NEM, with IC50 values of 0.63 +/- 0.02 and 2.8 +/- 0.66 microM, respectively. 4. Mg and ATP protected both the [3H]norepinephrine transport into the ghosts and the [3H]reserpine binding to the transporter from inhibition by NEM, shifting the IC50 values to 260 +/- 43 and 120 +/- 29 microM, respectively. 5. NEM inhibition of the catecholamine transport and reserpine binding appears to be due to an action on the proton translocator associated with the Mg ATPase enzyme rather than a direct action on the catecholamine transporter since (a) the concentration of NEM required to inhibit formation of a membrane potential is similar to that required to inhibit [3H]norepinephrine transport into and [3H]reserpine binding to the ghosts and (b) Mg and ATP protected the proton translocation and [3H]norepinephrine transport into the ghosts, and [3H]reserpine binding to the ghosts, from inhibition by NEM.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of the H +-ATPase in bovine adrenal chromaffin granule ghosts by diethylstilbestrol Evidence for a tight coupling between ATP hydrolysis and proton translocation

Diethylstilbestrol (DES) was found to inhibit reversibly the hydrolysis of MgATP (80% at 100μM) and proton pump activity ( I 50⋍15 μM, complete at 100 μM) in chromaffin granule ghosts. The parallel inhibition suggests a tight kinetic coupling between the two activities. The Mg 2+-ATPase activity, but not proton pumping, was partially restored by N, N′-dicyclohexylcarbodiimide,indicating that the two inhibitors in combination cause a partial uncoupling. The non-competitive type of inhibition shows that the action of DES is distal to the site of ATP binding and hydrolysis. Although unspecific, the interaction of DES with the chromaffin granule membrane seems primarily to affect the H +-ATPase.

Phosphatidylinositol kinase of bovine adrenal chromaffin granules: Kinetic properties and inhibition by low concentrations of Ca 2+

The kinetics and regulatory properties of phosphatidylinositol (PI) kinase were studied in chromaffin granule ghosts isolated from the bovine adrenal medulla. Phosphatidylinositol 4-phosphate (PIP) was the major 32P-labelled phospholipid formed when the isolated membranes were phosphorylated by [γ- 32P]ATP. The PI kinase activity was rather independent of pH, but highly dependent on Mg 2+ with a maximal stimulation at 60 mM Mg 2+. By contrast, KCl and NaCl had a slight inhibitory effect. The K m value for MgATP was 44 and 62 μM in the presence of 1 and 20 mM MgCl 2, respectively. The PI kinase was almost fully and reversibly inhibited by free Ca 2+ (calmodulin-independent) in the nanomolar and low micromolar range, depending on the concentration of Mg 2+. The inhibition was not dependent on Ca 2+-stimulated protein phosphorylation, and it could not be explained by a dephosphorylation of PIP.

Demonstration of the ascorbate dependence of membrane-bound dopamine beta-monooxygenase in adrenal chromaffin granule ghosts.

Chromaffin granule ghosts from bovine adrenal medullae have been used to examine the ability of membrane-bound dopamine beta-monooxygenase to interact directly with intravesicular ascorbate and to investigate vectorial electron transfer from external ascorbate across the ghost membrane. Ghosts prepared by a modification of published procedures were shown to be fully active in both dopamine uptake and norepinephrine production. Dopamine uptake is dependent on the presence of a magnesium and ATP ionic complex, is abolished by reserpine, and reaches a steady-state level in the presence of dopamine beta-monooxygenase, ascorbate, catalase, and fumarate. Omission of ascorbate either inside or outside the ghosts greatly enhances dopamine accumulation, which reaches levels of approximately 30 nmol/mg under these conditions. Correspondingly, in the presence of all components, norepinephrine production reached approximately 100 nmol/mg in 30 min of incubation. Norepinephrine production was strictly magnesium-ATP-dependent, inhibited by either reserpine or dopamine beta-monooxygenase inactivation, and was markedly reduced when ascorbate was omitted from either inside or outside the ghosts. In the presence of limiting amounts of internal ascorbate, rapid norepinephrine production occurred which corresponded to the amount of initial ascorbate present, followed by a much slower endogenous norepinephrine production observable after complete depletion of internal ascorbate. The endogenous rate of norepinephrine production likely represents epinephrine-supported dopamine beta-monooxygenase turnover. Taken together, the data demonstrate that facile norepinephrine production by membrane-bound dopamine beta-monooxygenase occurs only when internal ascorbate is present, terminates upon depletion of internal ascorbate, and can only be sustained at a significant rate when reducing equivalents from external ascorbate are available.

Synexin-mediated fusion of bovine chromaffin granule ghosts. Effect of pH

Synexin induces chromaffin granule ghosts to fuse one to another, a process which is followed continuously and quantitatively by monitoring the mixing of the intragranular aqueous compartments. A freeze-thaw technique was used for preparing chromaffin granule ghosts loaded with a self-quenching concentration of the fluorescent, high molecular weight probe FITC-Dextran. When the loaded ghosts were mixed with empty ghosts in the presence of synexin, the two compartments fused, resulting in the dilution of the probe with the concomitant increase in fluorescence. So as to suppress possible leakage signals, anti-fluorescein antibodies which quench probe fluorescence were present in the reaction media. Synexin-mediated fusion of freeze-thaw (F/Th) ghosts and binding of 125I-synexin to these membranes were found to be dependent on Ca 2+ concentration, but only in a partial manner. However, these two synexin-mediated properties were demonstrably sensitive to [H +] in the medium. A detailed pH profile of fusion revealed an apparent midpoint of activation at approx. pH 5.2, with asymptotic value at pH 4 (maximum) and pH 7.2 (minimum). In our attempt to determine whether the pH effect was on the synexin or on the membranes, we found that fusion was blocked only by treatment of the membranes with the membrane-impermeant carboxyl group modifier 1-ethyl-3(4-azonia-4,4-dimethylpentyl)carbodiimide. These data suggest that membrane fusion evoked by synexin seems to be promoted by rendering the F/Th membranes relatively less negatively charged while the synexin becomes more positively charged. The fusion process was entirely dependent upon synexin concentration; the k 1/2 under optimal conditions of p Ca and pH was 85 nM. Similar to what has been previously found with intact granules, an anti-synexin polyclonal antibody partially (48%) blocked fusion, as did pretreatment of the chromaffin granules ghosts with trypsin (30%). We conclude that the coincident p Ca and pH sensitivity of synexin-mediated binding to chromaffin granule membranes and their subsequent fusion might be associated with physiological changes in the concentration of both cations in the cytoplasm of secreting chromaffin cells.

Effect of synexin on aggregation and fusion of chromaffin granule ghosts at pH 6

Fusion of chromaffin granule ghosts was induced by synexin at pH 6, 37° C, in the presence of 10 −7 M Ca 2+. To study the kinetics and extent of this fusion process we employed two assays that monitored continuously mixing of aqueous contents or membrane mixing by fluorescence intensity increases. In both assays chromaffin granule ghosts were either labeled on the membrane or in the included aqueous phase. The ratios of blank to labeled chromaffin granule ghosts were varied from 1 to 10. The results were analyzed in terms of a mass action kinetic model, which views the overall fusion reaction as a sequence of a second-order process of aggregation followed by a first-order fusion reaction. The model calculations gave fare simulations and predictions of the experimental results. The rate constants describing membrane mixing are more than 2-fold larger than those for volume mixing. The analysis also indicated that the initial aggregation and fusion processes, up to dimer formation, were extremely fast. The rate constant of aggregation was close to the limit in diffusion-controlled processes, whereas the fusion rate constant was about the same as found in fastest virus-liposome fusion events at pH 5. A small increase in volume was found to accompany the fusion between chromaffin granule ghosts. Using ratios of synexin to chromaffin granule ghost protein of 0.13, 0.41 and 1.15 indicated that the overall fusion rate was larger for the intermediate (0.41) case. The analysis showed that the main activity of synexin was an enhancement of the rate of aggregation. At intermediate or excessive synexin concentrations it, respectively, promoted moderately, or inhibited the actual fusion step.