Reversely-oriented cytochrome b561 in reconstituted vesicles catalyzes transmembrane electron transfer and supports extravesicular dopamine beta-hydroxylase activity

Cytochrome b561 from bovine adrenal chromaffin vesicles contains two heme B prosthetic groups. We verified that purified cytochrome b561 can donate electron equivalents directly to cytochrome c. The purified cytochrome b561 was successfully reconstituted into cholesterol-phosphatidylcholine-phosphatidylglycerol vesicles by a detergent-dialysis and extrusion method. When ascorbate-loaded vesicles with cytochrome b561 were mixed with ferricytochrome c, the intravesicular ascorbate was able to reduce external thiazole blue or cytochrome c. The reduction of thiazole blue or cytochrome c was dependent on the presence of cytochrome b561 in the vesicle membranes. Pre-treatment of cytochrome b561 with diethylpyrocarbonate suppressed the reduction of extravesicular cytochrome c significantly, confirming that the reduction was not due to leakage of ascorbate from the vesicles. The topology of the reconstituted cytochrome b561 in the vesicle membranes was examined by treatment with trypsin followed by SDS-PAGE and MALDI-TOF-MS analyses. Only one major cleavage site at Lys191 was identified, indicating that cytochrome b561 was reconstituted into the membranes in an inside-out orientation irrespective of the modification with diethylpyrocarbonate. The addition of a soluble form of dopamine beta-hydroxylase to the external medium resulted in the successful reconstitution of the hydroxylation activity towards tyramine, an analogue of dopamine, suggesting that a direct electron transfer via complex formation occurred. This activity was enhanced significantly upon the addition of ferricyanide as a mediator between cytochrome b561 and dopamine beta-hydroxylase.     

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.     

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.     

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.     

Planarian cytochrome b(561): conservation of a six transmembrane structure and localization along the central and peripheral nervous system

Cytochrome b(561) is a major transmembrane protein of catecholamine and neuropeptide secretory vesicles in the central and peripheral nervous systems of higher animals. We succeeded in cloning a full-length cDNA encoding planarian cytochrome b(561). The deduced amino acid sequence shows a very similar six transmembrane topology to those of cytochromes b(561) of higher vertebrates and contains both putative ascorbate- and monodehydro ascorbate-binding sites. Among the six totally-conserved His residues of cytochrome b(561) in higher vertebrates, one is substituted with an Asn residue, indicating that His88 and His161 of bovine cytochrome b(561) play roles as heme b ligands at the extravesicular side. Northern- and Western-blot analyses confirmed the expression of the mRNA and protein with the expected sizes in planarians. The distributions of the mRNA and apoprotein were analyzed by in situ hybridization and immunohistochemical staining, respectively, showing two morphologically distinct structures, a pair of ventral nerve cords and the cephalic ganglion cluster in the head region. The present results suggest that the usage of ascorbate to supply electron equivalents to neuroendocrine-specific copper-containing monooxygenases is likely to have originated in organisms with a very simple nervous system.     

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.     

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.     

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.     

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.     

The asymmetric orientation of cytochrome b561 in bovine chromaffin granule membranes

The topological arrangement of cytochrome b561 in the bovine adrenal medullary chromaffin granule membrane was investigated by radiolabeling and immunoprecipitation techniques using antibody raised against the purified cytochrome. The first labeling procedure involved a membrane-permeable amino group labeling reagent, ethyl acetimidate, and two membrane-nonpermeable amino group labeling reagents, isethionyl acetimidate and trinitrobenzenesulfonic acid. The second radiolabeling procedure involved lactoperoxidase-catalyzed iodination of the exposed tyrosines on the membrane-bound proteins. The labeled cytochrome b561 was isolated by immunoprecipitating detergent extracts of treated membranes, followed by electrophoresis of the precipitated cytochrome in polyacrylamide-dodecyl sulfate. From the analysis of both labeling techniques, cytochrome b561 appeared to be a transmembrane protein and a major portion of this protein was cytoplasmically exposed.     

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.     

Purification and characterization of bovine adrenal cytochrome b561 expressed in insect and yeast cell systems

Bovine adrenal chromaffin granule cytochrome (cyt) b561 is a transmembrane hemoprotein that plays a key role in transporting reducing equivalents from ascorbate to dopamine-beta-hydroxylase for catecholamine synthesis. We have developed procedures for expression and purification of functional bovine adrenal cyt b561 in insect and yeast cell systems. The bovine cyt b561 coding sequence, with or without a hexahistidine-tag sequence at the C-terminus, was cloned into the pVL1392 transfer vector under the control of the polyhedrin promoter to generate recombinant baculovirus for protein expression in Sf9 insect cells (approximately 0.5 mg detergent-solubilized cyt b561/L culture). For the yeast system, the cyt b561 cDNA was modified with a hexahistidine-tag sequence at the C-terminus, and inserted into the pPICZB vector under the control of the alcohol oxidase promoter. The recombinant plasmid was transformed into Pichia pastoris GS115 competent cells to give methanol-inducible cyt b561 expression (approximately 0.7 mg detergent-solubilized cyt b561/L culture). Recombinant His-tagged cyt b561 expressed in Sf9 or Pichia cells was readily solubilized from membrane fractions with dodecyl maltoside and purified to electrophoretic homogeneity by one-step chromatography on Ni-NTA affinity resin. The purified recombinant cytochrome from both systems had a heme to protein ratio close to two and was fully functional, as judged by comparison with the spectroscopic and kinetic parameters of the endogenous cytochrome from chromaffin granules. A novel procedure for isolation of chromaffin granule membranes was developed to utilize frozen adrenal glands instead of fresh tissue.     

Retrieving vesicles in secretion-induced rat chromaffin cells contain fodrin

We examined the distribution of fodrin and cytochrome b561 in secretion-induced rat chromaffin cells (epinephrine cells) by immunofluorescence and immunoelectron microscopy. Fasted rats injected with a large dose of insulin were perfusion-fixed and frozen sections of the adrenal medulla were immunolabeled. Fodrin, a peripheral membrane protein, was distributed only in the cell periphery in control cells, but was observed in the cell interior after the insulin treatment; many of the markers were found around small vesicles, 50-200 nm in diameter, and large vacuoles, more than 500 nm in diameter. On the other hand, cytochrome b561, an integral membrane protein, was seen only in the chromaffin granules in control cells, and appeared in small vesicles in the stimulated cells but not in large vacuoles. By double immunolabeling it was shown that cytochrome b561 coexisted with fodrin in the small vesicles. The coexistence of the two proteins was confirmed by the labeling of subcellular particles immunoadsorbed from the insulin-treated adrenal medulla homogenate; vesicles immunoisolated with anti-fodrin antibody on polyacrylamide beads were positively immunolabeled with anti-cytochrome b561 antibody. The present results show that during massive secretion fodrin is taken into the cell interior by vesicles, which may be a mechanism that retrieves the secretory granule membrane from the cell surface.     

Cytochrome b561 is fatty acylated and oriented in the chromaffin granule membrane with its carboxyl terminus cytoplasmically exposed

Two polyclonal antibodies were raised to synthetic peptides corresponding to amino acids Ser21-Tyr35 and Lys247-Phe261 of cytochrome b561. These antibodies were used to test the native orientation of the amino and carboxyl termini of this transmembrane electron transport protein. Carboxyl-terminal epitopes were lost when intact chromaffin granules were treated with Pronase. This result indicates that the carboxyl terminus is cytoplasmically exposed and confirms a theoretical prediction obtained from hydropathy plots. Epitopes that were recognized by an amino-terminal antipeptide antibody were not removed under the same conditions. This finding implied that the amino terminus was not proteolytically accessible on the exterior of the granule. The abundance of threonine and serine residues in the amino-terminal region suggested that the amino terminus could be held in the membrane by covalent fatty acylation. Treatment of purified delipidated cytochrome b561 with hydroxylamine resulted in the release of a fatty acid hydroxamate. Sulfhydryl analysis of purified cytochrome b561 showed that all 3 cysteine residues were in the free sulfhydryl form. These observations indicate that cytochrome b561 is covalently fatty acylated and that the lipid is bound through ester linkages of serine or threonine residues.     

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.     

Existence of two gamma subunits of the G proteins in brain

Although amino acid sequences have been determined for the alpha and beta subunits of Gs, Gi, and Go, sequences have not been reported for the gamma subunits of these G proteins. In the present paper, we determined the sequences of peptides prepared by partial proteolysis of two different forms of the gamma subunit of Gs, Gi, and Go from bovine brain. Using oligonucleotide probes based on the sequences of two of these peptides, a cDNA clone was isolated from a bovine adrenal cDNA library. This clone contained a 0.9-kilobase cDNA insert that included an open reading frame of 213 bases encoding a 71-amino acid polypeptide with an estimated Mr of 7850. The amino acid sequence predicted for the adrenal cDNA clone was identical to that determined for one form of the gamma subunit from brain. In addition, an antibody to a peptide based on the predicted amino acid sequence of this cDNA clone reacted specifically with one of the brain gamma subunits, indicating the adrenal cDNA clone encodes a gamma subunit present in both adrenal gland and brain. Also, evidence is presented, demonstrating the existence of a second, structurally distinct, form of the gamma subunit of Gs, Gi, and Go in brain.     

Heterologous production and characterisation of two distinct dihaem-containing membrane integral cytochrome b(561) enzymes from Arabidopsis thaliana in Pichia pastoris and Escherichia coli cells

Cytochrome (cyt) b(561) proteins are dihaem-containing membrane proteins, belonging to the CYBASC (cytochrome-b(561)-ascorbate-reducible) family, and are proposed to be involved in ascorbate recycling and/or the facilitation of iron absorption. Here, we present the heterologous production of two cyt b(561) paralogs from Arabidopsis thaliana (Acytb(561)-A, Acytb(561)-B) in Escherichia coli and Pichia pastoris, their purification, and initial characterisation. Spectra indicated that Acytb(561)-A resembles the best characterised member of the CYBASC family, the cytochrome b(561) from adrenomedullary chromaffin vesicles, and that Acytb(561)-B is atypical compared to other CYBASC proteins. Haem oxidation-reduction midpoint potential (E(M)) values were found to be fully consistent with ascorbate oxidation activities and Fe(3+)-chelates reductase activities. The ascorbate dependent reduction and protein stability of both paralogs were found to be sensitive to alkaline pH values as reported for the cytochrome b(561) from chromaffin vesicles. For both paralogs, ascorbate-dependent reduction was inhibited and the low-potential haem E(M) values were affected significantly by incubation with diethyl pyrocarbonate (DEPC) in the absence of ascorbate. Modification with DEPC in the presence of ascorbate left the haem E(M) values unaltered compared to the unmodified proteins. However, ascorbate reduction was inhibited. We concluded that the ascorbate-binding site is located near the low-potential haem with the Fe(3+)-chelates reduction-site close to the high-potential haem. Furthermore, inhibition of ascorbate oxidation by DEPC treatment occurs not only by lowering the haem E(M) values but also by an additional modification affecting ascorbate binding and/or electron transfer. Analytical gel filtration experiments suggest that both cyt b(561) paralogs exist as homodimers.     

Sequence analysis of the catalytic subunit of H(+)-ATPase from porcine renal brush-border membranes.

The catalytic subunit of the H(+)-ATPase from brush-border membranes of porcine renal proximal tubules was labeled with the hydrophobic SH-group reagent 10-N-(bromoacetyl)amino-1-decyl-beta-glucopyranoside (BADG) which irreversibly inhibits proton pump activity in the absence but not in the presence of ATP. The labeled protein was purified and digested with proteinases. After isolation and sequencing of proteolytic peptides two BADG-labeled cysteines were identified. The amino acid sequences of the obtained proteolytic peptides were homologous to the catalytic subunit of V-ATPases. From mRNA of porcine kidney cortex a catalytic H(+)-ATPase subunit was cloned. 181 of the 183 amino acids which overlap in the sequence derived from the cDNA and the proteolytic peptides were identical, and the two deviations are due to single base exchanges. A comparison of the amino acid sequence derived from the cloned cDNA with sequences of catalytic H(+)-ATPase subunits communicated by other laboratories revealed 98%, 96% and 94% identity with sequences from bovine adrenal medulla, from bovine kidney medulla and from clathrin-coated vesicles of bovine brain. Between 64% and 69% identity was obtained with sequences from fungi and plants. The data show that the catalytic subunit of V-ATPases is highly conserved during evolution. They indicate organ and species specificity in mammalians.     

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.