Purified cytochrome b561 catalyzes transmembrane electron transfer for dopamine beta-hydroxylase and peptidyl glycine alpha-amidating monooxygenase activities in reconstituted systems

Cytochrome b561 from bovine adrenal medulla chromaffin granules has been purified by fast protein liquid chromatography chromatofocusing. The purified cytochrome was reconstituted into ascorbate-loaded phosphatidylcholine vesicles. With this reconstituted system transmembrane electron transfer for extravesicular soluble dopamine beta-hydroxylase activity was demonstrated. In accordance with the model proposed by Njus et al. (Njus, D., Knoth, J., Cook, C., and Kelley, P. M. (1983) J. Biol. Chem. 258, 27-30), catalytic amounts of a redox mediator were necessary to achieve electron transfer between cytochrome and soluble dopamine beta-hydroxylase. Our observations also showed that when membranous dopamine beta-hydroxylase was reconstituted on cytochrome containing vesicles, electron transfer occurred only in the presence of a redox mediator. Since cytochrome b561 has been found in secretory vesicles associated with peptidyl glycine alpha-amidating monooxygenase, electron transfer to this enzyme was also examined. Analogous to the results obtained for dopamine beta-hydroxylase, transmembrane electron transfer to peptidyl glycine alpha-amidating monooxygenase appears to require a redox mediator between cytochrome and this monooxygenase. These observations indicate that purified cytochrome b561 is capable of providing a transmembrane supply of electrons for both monooxygenases. Since no direct protein to protein electron transfer occurs, the results support the hypothesis that the ascorbate/semidehydroascorbate redox pair serves as a mediator for these enzymes in vivo.     

Cytochrome b561 catalyzes transmembrane electron transfer

Purified cytochrome b561 from bovine adrenal medulla chromaffin vesicles has been reconstituted into phosphatidylcholine vesicles by a detergent-dialysis method. When the reconstituted cytochrome-containing vesicles were preloaded with ascorbic acid and cytochrome c was added to the external medium, the internal ascorbic acid was able to reduce the external cytochrome c. This reduction of cytochrome c was dependent on the presence of cytochrome b561 in the membrane and was not due to leakage of ascorbate from the vesicles. These results demonstrate that cytochrome b561 catalyzes a transmembrane electron transfer.     

Rate of electron transfer between cytochrome b561 and extravesicular ascorbic acid

Cytochrome b561 transfers electrons across secretory vesicle membranes in order to regenerate intravesicular ascorbic acid. To show that cytosolic ascorbic acid is kinetically competent to function as the external electron donor for this process, electron transfer rates between cytochrome b561 in adrenal medullary chromaffin vesicle membranes and external ascorbate/semidehydroascorbate were measured. The reduction of cytochrome b561 by external ascorbate may be measured by a stopped-flow method. The rate constant is 450 (+/- 190) M-1 s-1 at pH 7.0 and increases slightly with pH. The rate of oxidation of cytochrome b561 by external semidehydroascorbate may be deduced from rates of steady-state electron flow. The rate constant is 1.2 (+/- 0.5) x 10(6) M-1 s-1 at pH 7.0 and decreases strongly with pH. The ratio of the rate constants is consistent with the relative midpoint reduction potentials of cytochrome b561 and ascorbate/semidehydroascorbate. These results suggest that cytosolic ascorbate will reduce cytochrome b561 rapidly enough to keep the cytochrome in a mostly reduced state and maintain the necessary electron flux into vesicles. This supports the concept that cytochrome b561 shuttles electrons from cytosolic ascorbate to intravesicular semidehydroascorbate, thereby ensuring a constant source of reducing equivalents for intravesicular monooxygenases.     

Properties of two distinct heme centers of cytochrome b561 from bovine chromaffin vesicles studied by EPR, resonance Raman, and ascorbate reduction assay

Cytochrome b(561) from bovine adrenal chromaffin vesicles contains two hemes b with different midpoint potentials (+150 and +60 mV) and participates in transmembrane electron transport from extravesicular ascorbate to an intravesicular monooxygenase, dopamine beta-hydroxylase. Treatment of oxidized cytochrome b(561) with diethylpyrocarbonate caused a downshift of midpoint potential for the lower component, and this shift was prevented by the presence of ascorbate during the treatment. Present EPR analyses showed that, upon the treatment, the g(z) = 3.69 heme species was converted to a non-ascorbate-reducible form, although its g(z)-value showed no appreciable change. The treatment had no effect on the other heme (the g(z) = 3.13 species). Raman data indicated that the two heme b centers adopt a six-coordinated low-spin state, in both the reduced and oxidized forms. There was no significant effect of diethylpyrocarbonate-treatment on the Raman spectra of either form, but the reducibility by ascorbate differed significantly between the two hemes upon the treatment. The addition of ferrocyanide enhanced both the reduction rate and final reduction level of the diethylpyrocarbonate-treated cytochrome b(561) when ascorbate was used as a reductant. This observation suggests that ferrocyanide scavenges monodehydroascorbate radicals produced by the univalent oxidation of ascorbate and, thereby, increases both the reduction rate and the final reduction level of the heme center on the intravesicular side of the diethylpyrocarbonate-treated cytochrome. These results further clarify the physiological role of this heme center as the electron donor to the monodehydroascorbate radical.     

Stopped-flow analyses on the reaction of ascorbate with cytochrome b561 purified from bovine chromaffin vesicle membranes

Cytochrome b(561) in adrenal chromaffin vesicle membranes conveys electron equivalents from extravesicular ascorbate to the intravesicular monodehydroascorbate radical. We conducted a stopped-flow study on the reaction of ascorbate with purified cytochrome b(561) in the detergent-solubilized state for the first time. The time course of the reduction of oxidized cytochrome b(561) with ascorbate could not be fitted with a single exponential but with a linear combination of at least four exponential functions. This result is consistent with the notion that cytochrome b(561) contains two hemes b, each having a distinct redox potential and a function upon reactions with ascorbate and monodehydroascorbate radical. The fastest phase, which was assigned to the first one-electron donation from ascorbate to heme b on the extravesicular side, was further analyzed by transient phase kinetics employing a two-step bi-uni sequential ordered mechanism. The result showed K(s) = 2.2 mM for ascorbate at pH6.0. At a region below pH5.5, there was a significant lag before the reduction of hemes b occurred. This time lag was interpreted as due to a pH-dependent transient state before the first electron transfer to take place. The fastest phase was completely lost by N-carbethoxylation of heme-coordinating histidyl residues (His88 and His161) and Lys85 upon treatment with diethylpyrocarbonate. The presence of ascorbate during the treatment inhibited the N-carbethoxylation of the histidyl residues and, thereby, restored the final reduction level of hemes b. But the reduction rate was still only one-twentieth of the native form. This result suggested an important role of the conserved Lys85 for the interaction with ascorbate.     

Diethyl pyrocarbonate modification abolishes fast electron accepting ability of cytochrome b561 from ascorbate but does not influence electron donation to monodehydroascorbate radical: identification of the modification sites by mass spectrometric analysis

Cytochrome b(561) from bovine adrenal chromaffin vesicles contains two heme B prosthetic groups and transports electron equivalents across the vesicle membranes to convert intravesicular monodehydroascorbate radical to ascorbate. To elucidate the mechanism of the transmembrane electron transfer, effects of the treatment of purified cytochrome b(561) with diethyl pyrocarbonate, a reagent specific for histidyl residues, were examined. We found that when ascorbate was added to the oxidized form of diethyl pyrocarbonate-treated cytochrome b(561), less than half of the heme iron was reduced but with a very slow rate. In contrast, radiolytically generated monodehydroascorbate radical was oxidized rapidly by the reduced form of diethyl pyrocarbonate-modified cytochrome b(561), as observed for untreated cytochrome b(561). These results indicate that the heme center specific for the electron acceptance from ascorbate was perturbed by the modification of amino acid residues nearby. We identified the major modification sites by mass spectrometry as Lys85, His88, and His161, all of which are fully conserved and located on the extravesicular side of cytochrome b(561) in the membranes. We suggest that specific N-carbethoxylation of the histidyl ligands of the heme b at extravesicular side abolishes the electron-accepting ability from ascorbate.     

A kinetic analysis of electron transport across chromaffin vesicle membranes

Some types of secretory vesicles, such as the chromaffin vesicles of the adrenal medulla, have cytochrome b561 which is believed to mediate the transfer of electrons across the vesicle membrane. To characterize the kinetics of this process, we have examined the rate of electron transfer from ascorbate trapped within chromaffin vesicle ghosts to external ferricyanide. The rate of ferricyanide reduction saturates at high ferricyanide concentrations. The reciprocal of the rate is linearly related to the reciprocal of the ferricyanide concentration. The internal ascorbate concentration affects the y intercept of this double-reciprocal plot but not the slope. These observations and theoretical considerations indicate that the slope is associated with a rate constant k1 for the oxidation of cytochrome b561 by ferricyanide. The intercept is associated with a rate constant k0 for the reduction of cytochrome b561 by internal ascorbate. From k0 and standard reduction potentials, the rate constant k-0 for the reduction of internal semidehydroascorbate by cytochrome b561 can be calculated. Under conditions prevailing in vivo, this rate of semidehydroascorbate reduction appears to be much faster than the expected rate of semidehydroascorbate disproportionation. This supports the hypothesis that cytochrome b561 functions in vivo to reduce intravesicular semidehydroascorbate thereby maintaining intravesicular ascorbic acid.     

Rate of transmembrane electron transfer in chromaffin-vesicle ghosts

The chromaffin vesicle of the adrenal medulla contains a transmembrane electron carrier that may provide reducing equivalents for dopamine beta-hydroxylase in vivo. This electron-transfer system can be assayed by trapping ascorbic acid inside resealed membrane vesicles (ghosts), adding an external electron acceptor such as ferricytochrome c or ferricyanide, and following the reduction of these acceptors spectrophotometrically. Cytochrome c reduction is more rapid at high pH and is proportional to the amount of chromaffin-vesicle ghosts, at least at low ghost concentrations. At pH 7.0, ghosts loaded with 100 mM ascorbic acid reduce 60 microM cytochrome c at a rate of 0.035 +/- 0.010 mu equiv min-1 (mg of protein)-1 and 200 microM ferricyanide at a rate of 2.3 +/- 0.3 mu equiv min-1 (mg of protein)-1. The rate of cytochrome c reduction is accelerated to 0.105 +/- 0.021 mu equiv min-1 (mg of protein)-1 when cytochrome c is pretreated with equimolar ferrocyanide. Pretreatment of cytochrome c with ferricyanide also causes a rapid rate of reduction, but only after an initial delay. The ferrocyanide-stimulated rate of cytochrome c reduction is further accelerated by the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), probably because FCCP dissipates the membrane potential generated by electron transfer. These rates of electron transfer are sufficient to account for electron transfer to dopamine beta-hydroxylase in vivo and are consistent with the mediation of electron transfer by cytochrome b-561.     

Reaction of ascorbic acid with cytochrome b561. Concerted electron and proton transfer.

Rate constants for reduction of cytochrome b561 by internal ascorbate (k0A) and oxidation by external ferricyanide (k1F) were determined as a function of pH from rates of steady-state electron transfer across chromaffin-vesicle membranes. The pH dependence of electron transfer from cytochrome b561 to ferricyanide (k1F) may be attributed to the pH dependence of the membrane surface potential. The rate constant for reduction by internal ascorbate (k0A), like the previously measured rate constant for reduction by external ascorbate (k-1A), is not very pH-dependent and is not consistent with reduction of cytochrome b561 by the ascorbate dianion. The rate at which ascorbate reduces cytochrome b561 is orders of magnitude faster than the rate at which it reduces cytochrome c, despite the fact that midpoint reduction potentials favor reduction of cytochrome c. Moreover, the rate constant for oxidation of cytochrome b561 by ferricyanide (k1F) is smaller than the previously measured rate constant for oxidation by semidehydroascorbate, despite the fact that ferricyanide has a higher midpoint reduction potential. These results may be reconciled by a mechanism in which electron transfer between cytochrome b561 and ascorbate/semidehydroascorbate is accelerated by concerted transfer of a proton. This may be a general property of biologically significant electron transfer reactions of ascorbic acid.     

Functional coupling between enzymes of the chromaffin granule membrane

The reactions of cytochrome b561 with other redox-active components of the adrenal chromaffin granule were examined using optical difference spectroscopy. It was shown that there is no direct electron transfer between the cytochrome and dopamine beta-hydroxylase, but that in the presence of ascorbate, turnover of dopamine beta-hydroxylase causes an oxidation of the cytochrome, which is partially reversed by the action of the mitochondrial NADH:A-. oxidoreductase. Thus, these three proteins may be functionally coupled via ascorbate. A quantitative study of the relationship between the redox state of the cytochrome and the ascorbate radical concentration measured by EPR showed that ascorbate reduces the cytochrome in a one-electron transfer reaction. Generation of a proton electrochemical gradient across the granule membrane causes only a small (20 mV) increase in the cytochrome midpoint potential suggesting the cytochrome is not a proton pump. The data are consistent with a model in which cytochrome b561, by reacting with ascorbate or ascorbate free radical on either side of the granule membrane, could couple the ascorbate-consuming reaction of the dopamine beta-hydroxylase inside the chromaffin granule to the ascorbate-regenerating reaction of the NADH:A-. oxidoreductase on the outer mitochondrial membrane. The H+-ATPase of the granule membrane could both drive the flow of electrons in the direction from cytosol to granule and replenish protons consumed by the turnover of dopamine beta-hydroxylase inside the granule.     

Selective perturbation of the intravesicular heme center of cytochrome b561 by cysteinyl modification with 4,4'-dithiodipyridine

Cytochrome b(561) from bovine adrenal chromaffin vesicles contains two hemes b with EPR signals at g(z) = 3.69 and 3.14 and participates in transmembrane electron transport from extravesicular ascorbate to an intravesicular monooxygenase, dopamine beta-hydroxylase. Treatment of purified cytochrome b(561) in an oxidized state with a sulfhydryl reagent, 4,4'-dithiodipyridine, caused the introduction of only one 4-thiopyridine group per b(561) molecule at either Cys57 or Cys125. About half of the heme centers of the modified cytochrome were reduced rapidly with ascorbate as found for the untreated sample, but the final reduction level decreased to approximately 65%. EPR spectra of the modified cytochrome showed that a part of the g(z) = 3.14 low-spin EPR species was converted to a new low-spin species with g(z) = 2.94, although a considerable part of the heme center was concomitantly converted to a high-spin g = 6 species. Addition of ascorbate to the modified cytochrome caused the disappearance or significant reduction of the EPR signals at g(z) = 3.69 and 3.14 of low-spin species and at g = 6.0 of the high-spin species, but not for the g(z) approximately 2.94 species. These results suggested that the bound 4-thiopyridone at either Cys57 or Cys125 affected the intravesicular heme center and converted it partially to a non-ascorbate-reducible form. The present observations suggested the importance of the two well-conserved Cys residues near the intravesicular heme center and implied their physiological roles during the electron donation to the monodehydroascorbate radical.     

Electron transfer in chromaffin-vesicle ghosts containing peroxidase.

In chromaffin vesicles, the enzyme dopamine beta-monooxygenase converts dopamine to norepinephrine. It is believed that reducing equivalents for this reaction are supplied by intravesicular ascorbic acid and that the ascorbate is regenerated by importing electrons from the cytosol with cytochrome b-561 functioning as the transmembrane electron carrier. If this is true, then the ascorbate-regenerating system should be capable of providing reducing equivalents to any ascorbate-requiring enzyme, not just dopamine beta-monooxygenase. This may be tested using chromaffin-vesicle ghosts in which an exogenous enzyme, horseradish peroxidase, has been trapped. If ascorbate and peroxidase are trapped together within chromaffin-vesicle ghosts, cytochrome b-561 in the vesicle membrane is found in the reduced form. Subsequent addition of H2O2 causes the cytochrome to become partially oxidized. H2O2 does not cause this oxidation if either peroxidase or ascorbate are absent. This argues that the cytochrome is oxidized by semidehydroascorbate, the oxidation product of ascorbate, rather than by H2O2 or peroxidase directly. The semidehydroascorbate must be internal because the ascorbate from which it is formed is sequestered and inaccessible to external ascorbate oxidase. This shows that cytochrome b-561 can transfer electrons to semidehydroascorbate within the vesicles and that the semidehydroascorbate may be generated by any enzyme, not just dopamine beta-monooxygenase.     

Role of ascorbic acid in dopamine beta-hydroxylation. The endogenous enzyme cofactor and putative electron donor for cofactor regeneration

The role(s) of ascorbic acid in dopamine beta-hydroxylation was studied in primary cultures of bovine adrenomedullary chromaffin cells and in isolated bovine adrenomedullary chromaffin vesicles. Dopamine beta-hydroxylase activity was assessed by measuring the rate of conversion of tyramine to octopamine. The ascorbic acid content of chromaffin cells declined with time in culture and the dopamine beta-hydroxylase activity of ascorbate-depleted cells was low. Ascorbate additions to ascorbate-depleted cells increased both the intracellular ascorbate concentrations and the rates of dopamine beta-hydroxylation. Ascorbate uptake into the cells was rapid; however, the onset of enhanced octopamine synthesis by added ascorbate was delayed by several hours and closely followed the time course for accumulation of the newly taken up ascorbate into the chromaffin vesicle. The amount of octopamine synthesized by the chromaffin cells exceeded the intracellular ascorbate content and ascorbate levels were maintained during dopamine beta-hydroxylation in the absence of external ascorbate. This suggests an efficient recycling of ascorbate. In contrast to intact cells, ascorbic acid was depleted during octopamine synthesis in isolated chromaffin vesicles. The molar ratio of octopamine formed to ascorbate depleted was close to unity. Thus, the recycling of intravesicular ascorbate depends on an extravesicular factor(s). The depletion of intravesicular ascorbate during dopamine beta-hydroxylation was prevented by the addition of nonpermeant extravesicular electron donors such as ascorbate or glucoascorbate. This suggests that intravesicular ascorbate is maintained in the reduced state by electron transport across the vesicle membrane. These results are compatible with the hypothesis that both intra- and extravesicular ascorbate participate in the regulation of dopamine beta-hydroxylase. Intravesicular ascorbate is the cofactor for the enzyme. Cytosolic ascorbate is most likely the electron donor for the vesicle-membrane electron transport system which maintains the intravesicular cofactor concentration.     

Cytochrome b561 spectral changes associated with electron transfer in chromaffin-vesicle ghosts

The involvement of cytochrome b561, an integral membrane protein, in electron transfer across chromaffin-vesicle membranes is confirmed by changes in its redox state observed as changes in the absorption spectrum occurring during electron transfer. In ascorbate-loaded chromaffin-vesicle ghosts, cytochrome b561 is nearly completely reduced and exhibits an absorption maximum at 561 nm. When ferricyanide is added to a suspension of these ghosts, the cytochrome becomes oxidized as indicated by the disappearance of the 561 nm absorption. If a small amount of ferricyanide is added, it becomes completely reduced by electron transfer from intravesicular ascorbate. When this happens, cytochrome b561 returns to its reduced state. If an excess of ferricyanide is added, the intravesicular ascorbate becomes exhausted and the cytochrome b561 remains oxidized. The spectrum of these absorbance changes correlates with the difference spectrum (reduced-oxidized) of cytochrome b561. Cytochrome b561 becomes transiently oxidized when ascorbate oxidase is added to a suspension of ascorbate-loaded ghosts. Since dehydroascorbate does not oxidize cytochrome b561, it is likely that oxidation is caused by semidehydroascorbate generated by ascorbate oxidase acting on free ascorbate. This suggests that cytochrome b561 can reduce semidehydroascorbate and supports the hypothesis that the function of cytochrome b561 in vivo is to transfer electrons into chromaffin vesicles to reduce internal semidehydroascorbate to ascorbate.     

Evidence for an essential histidine residue in the ascorbate-binding site of cytochrome b561

Cytochrome b(561) mediates equilibration of the ascorbate/semidehydroascorbate redox couple across the membranes of secretory vesicles. The cytochrome is reduced by ascorbic acid and oxidized by semidehydroascorbate on either side of the membrane. Treatment with diethyl pyrocarbonate (DEPC) inhibits reduction of the cytochrome by ascorbate, but this activity can be restored by subsequent treatment with hydroxylamine, suggesting the involvement of an essential histidine residue. Moreover, DEPC inactivates cytochrome b(561) more rapidly at alkaline pH, consistent with modification of a histidine residue. DEPC does not affect the absorption spectrum of cytochrome b(561) nor does it change the midpoint reduction potential, confirming that histidine modification does not affect the heme. Ascorbate protects the cytochrome from inactivation by DEPC, indicating that the essential histidine is in the ascorbate-binding site. Further evidence for this is that DEPC treatment inhibits oxidation of the cytochrome by semidehydroascorbate but not by ferricyanide. This supports a reaction mechanism in which ascorbate loses a hydrogen atom by donating a proton to histidine and transferring an electron to the heme.     

Ascorbate inhibits the carbethoxylation of two histidyl and one tyrosyl residues indispensable for the transmembrane electron transfer reaction of cytochrome b561

Cytochrome b(561) from bovine adrenal chromaffin vesicles contains two heme B prosthetic groups and transports electron equivalents across the vesicle membranes to convert intravesicular monodehydroascorbate radical to ascorbate. We found previously that treatment of oxidized cytochrome b(561) with diethyl pyrocarbonate caused specific N-carbethoxylation of three fully conserved residues (His88, His161, and Lys85) located at the extravesicular side. The modification lead to a selective loss of the electron-accepting ability from ascorbate without affecting the electron donation to monodehydroascorbate radical [Tsubaki, M., Kobayashi, K., Ichise, T., Takeuchi, F., and Tagawa, S. (2000) Biochemistry 39, 3276-3284]. In the present study, we found that these modifications lead to a drastic decrease of the midpoint potential of heme b at the extravesicular side from +60 to -30 mV. We found further that the O-carbethoxylation of one tyrosyl residue (Tyr218) located at the extravesicular side was significantly enhanced under alkaline conditions, leading to a very slow reduction process of the oxidized heme b with ascorbate. On the other hand, the presence of ascorbate during the treatment with diethyl pyrocarbonate was found to suppress the carbethoxylation of His88, His161, and Tyr218, whereas the modification level of Lys85 was not affected. Concomitantly, the final reduction level of heme b with ascorbate was protected, although the fast reduction phase was not fully restored. These results suggest that the two heme-coordinating histidyl residues (His88 and His161) are also a part of the ascorbate binding site. Tyr218 and Lys85 may have a role in the recognition/binding process for ascorbate and are indispensable for the fast electron transfer reaction.     

The in situ kinetics of dopamine beta-hydroxylase in bovine adrenomedullary chromaffin cells. Intravesicular compartmentation reduces apparent affinity for the cofactor ascorbate

The Km of dopamine beta-hydroxylase for its cofactor, ascorbic acid, was determined in situ in primary cultures of bovine adrenomedullary chromaffin cells and in isolated chromaffin vesicles. A range of intravesicular ascorbate concentrations in chromaffin cell cultures (1.1-31.2 mM) was achieved by varying the number and concentration of ascorbate additions to the culture media. The rate of octopamine synthesis from tyramine displayed a Michaelis-Menten relationship with respect to ascorbate concentration and an apparent Km of dopamine beta-hydroxylase for ascorbate of 15.0 +/- 2.0 mM was determined. In isolated chromaffin vesicles, with an initial intravesicular ascorbate concentration of approximately 10 mM, ascorbate consumption during beta-hydroxylation occurred as a first order process. This indicated that dopamine beta-hydroxylase was not saturated at this initial ascorbate concentration. When isolated chromaffin vesicles were prepared with different intravesicular ascorbate concentrations, the rate of octopamine synthesis displayed a Michaelis-Menten relationship with respect to ascorbate with an apparent Km of 17.0 +/- 5.0 mM. Ascorbate consumption also occurred as a first order process in ascorbate-loaded chromaffin-vesicle ghosts which had initial ascorbate concentrations of approximately 30 mM but which were depleted of other small molecules such as catecholamines. These results indicate that the in situ Km of dopamine beta-hydroxylase for ascorbate (approximately 15 mM) is 25-fold higher than it is for the purified or partially purified enzyme assayed under optimal conditions in vitro (0.6 mM). The factor(s) which decreases the enzyme affinity for ascorbate, relative to in vitro, resides in the chromaffin vesicle interior and is also retained in chromaffin-vesicle ghosts. The mechanism of this effect remains to be determined. The Km value determined in these experiments is close to the estimated intravesicular ascorbate concentration of bovine chromaffin granules in vivo (4), suggesting that the availability of ascorbate could become a factor in regulating the rate of dopamine beta-hydroxylation.     

Electron transfer across posterior pituitary neurosecretory vesicle membranes

Secretory vesicles from the neurohypophysis have a transmembrane electron carrier very similar to that found in adrenal medullary chromaffin granules. Two different tests show that ascorbic acid contained in the vesicles will reduce an external electron acceptor. First, reduction of cytochrome c or ferricyanide in the medium by a neurosecretory vesicle suspension can be followed spectrophotometrically. Second, the membrane potential (inside positive) generated by electron transfer can be monitored using the membrane potential-sensitive optical probe Oxonol VI. As in chromaffin granules, this electron transfer is probably mediated by cytochrome b561. It may function to regenerate internal ascorbic acid and to provide reducing equivalents needed by the intravesicular amidating enzyme.     

Conversion of soluble dopamine beta-hydroxylase to a membrane binding form

We have shown that purified bovine soluble dopamine beta-hydroxylase can reconstitute onto preformed phosphatidylserine containing vesicles. The binding is dependent on pH and vesicle phosphatidylserine composition but does not require calcium. Reconstitution appears to be irreversible, with the lipid-bound enzyme possessing hydroxylase activity. Additionally, [14C] phosphatidylserine binds to soluble dopamine beta-hydroxylase and remains bound after several detergent washes. Thus the reconstituted soluble form of the enzyme appears to be functionally analogous to the membranous form. Both the reconstitution data and the lipid binding data suggest that multiple phosphatidylserine molecules bind to the soluble hydroxylase. We propose that noncovalently bound phosphatidylserine moieties, which copurify with the membrane bound form of the enzyme, alone are responsible for anchoring membranous dopamine beta-hydroxylase to chromaffin granule and model membranes.