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.     

Purification and analysis of phospholipids in the inner mitochondrial membrane fraction of bovine corpus luteum, and properties of cytochrome P-450scc incorporated into vesicles prepared from these phospholipids

Cytochrome P-450scc, which catalyses the conversion of cholesterol to pregnenolone in steroidogenic tissues, can be incorporated into artificial phospholipid vesicles and cholesterol binding to the cytochrome is affected by the composition of the vesicles. We have purified the phospholipids from the inner mitochondrial membrane fraction of the bovine corpus luteum where the cytochrome is located. The composition in mol % was 49% phosphatidylcholine, 34% phosphatidylethanolamine, 8.7% cardiolipin, 6.4% lysophosphatidylethanolamine and 1.5% phosphatidylinositol. The ratio of cholesterol to phospholipid (mol/mol) in the inner membrane fraction was 0.14 to 1. The Km for cholesterol of purified luteal cytochrome P-450scc incorporated into vesicles prepared from the total inner mitochondrial membrane phospholipids was 0.063 mol of cholesterol per mol of phospholipid. Removal of the cardiolipin component of the inner mitochondrial membrane phospholipids prior to preparation of vesicles caused a four fold increase in the Kd of cytochrome P-450 for cholesterol and a two fold increase in Km. The data suggests that in the inner mitochondrial membrane of the bovine corpus luteum the cholesterol concentration is less than saturating for cytochrome P-450scc.     

Immunocytochemical demonstration of dopamine-beta-hydroxylase and cytochrome B561 on the axonal reticulum in bovine sympathetic neurons.

In sympathetic neurons the axonal reticulum can be considered an extension of the secretory pole of the Golgi apparatus. If this tubular system indeed represents the neurosecretory apparatus, it would likely contain on its membranes the enzymes involved in catecholamine synthesis. To test this hypothesis, we investigated the distribution of dopamine-beta-hydroxylase and cytochrome b561 in bovine splenic nerve and nerve terminals in the vas deferens with an immunogold procedure after glycolmethacrylate embedding. Counterstaining with phosphotungstic acid at low pH selectively revealed the axonal reticulum elements. With antibodies against both enzymes, gold labeling was observed over the large dense-cored vesicles, the Golgi-associated axonal reticulum, the reticulum within axons, and the tubular complex at the nerve terminal. From our results it can be concluded that in sympathetic neurons the axonal reticulum represents a tubular neurosecretory system, extending from the Golgi apparatus in the cell soma to the nerve terminal. This concept emphasizes the local production of neurosecretory vesicles and may be of importance in the interpretation of neuronal transmission in normal and diseased states.     

Fluorescent probe study of temperature-induced conformational changes in cytochrome oxidase in lecithin vesicle and solubilized systems

A protein-bound label, N-(1-anilinonaphthyl-4)-maleimide (ANM), was used to investigate conformational changes in bovine heart cytochrome oxidase. The fluidity of cytochrome oxidase vesicles was monitored by a lipophilic probe, 1,6-diphenyl-1,3,5-hexatriene. The fluroescence intensity and emission anisotropy of these probes were examined between 4 and 60 degrees C in enzyme--dipalmitoyllecithin vesicles, in enzyme--dimyristoyllecithin vesicles, in enzyme--dioleoyllecithin vesicles, and in the soluble enzyme. The temperature-dependent changes in these quantities indicated that there were two types of conformational changes in oxidized cytochrome oxidase: one was attributed to an intrinsic enzyme conformation change which occurred around 20 degrees C, and the other was attributed to a conformational change induced by the lipid phase transition. Although ANM-reactive subunits of cytochrome oxidase in these four lecithin vesicle and solubilized systems were different from each other, subunit I always reacted with ANM in preference to other subunits.     

Functional equivalence of monomeric (shark) and dimeric (bovine) cytochrome c oxidase

Cytochrome c oxidase isolated from hammerhead shark red muscle is monomeric in relation to the dimeric form of isolated bovine cytochrome c oxidase but in other ways bears a close resemblance to the enzyme isolated from mammalian tissue [1, 2]. Comparative studies of shark and bovine cytochrome c oxidase were extended to address the degree of functional similarity between the monomeric (shark) and dimeric (bovine) enzymes in the kinetics of peroxide binding and in the extent to which the catalytic action of the enzymes in vesicles can establish a proton gradient. Although the kinetics of peroxide binding and the proton pumping processes are complex, the dimeric and monomeric forms are quite similar with respect to these functional attributes. The kinetic heterogeneity of the process of peroxide binding is expressed in the shark enzyme as well as in the bovine enzyme, and both types of enzymes in vesicles can generate transmembrane proton gradients. On this basis we conclude that the dimeric state of isolated cytochrome c oxidase from mammalian sources is not essential for its function in vitro.     

Light-activated proton-motive force generation in lipid vesicles containing cytochrome b-c1 complex and bacterial reaction centres

(1) Purified bovine heart mitochondrial cytochrome b-c1 complex (ubiquinone-cytochrome c oxidoreductase) and photosynthetic reaction centres isolated from Rhodopseudomonas sphaeroides strain R-26 have been incorporated into lipid vesicles. In the presence of cytochrome c and ubiquinone-2, light activation caused a cyclic electron transfer involving both components. (2) Since cytochrome c is added outside the vesicles, it is both reduced by the cytochrome b-c1 complex and oxidised by the reaction centre on the outside of the vesicles. Ubiquinone-2, however, is reduced by the reaction centres at a site in contact with the inside of the vesicles, but the reduced form, ubiquinol-2, is oxidised by the cytochrome b-c1 complex at a site in contact with the outer aqueous phase. (3) In the presence of valinomycin plus K+, initiation of cyclic electron flow causes protons to move from inside the vesicles to the outer medium and the H +/2e- ratio was calculated to be close to 4.     

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.     

Synaptin/synaptophysin, p65 and SV2: their presence in adrenal chromaffin granules and sympathetic large dense core vesicles.

The subcellular distribution of three proteins of synaptic vesicles (synaptin/synaptophysin, p65 and SV2) was determined in bovine adrenal medulla and sympathetic nerve axons. In adrenals most p65 and SV2 is confined to chromaffin granules. Part of synaptin/synaptophysin is apparently also present in these organelles, but a considerable portion is found in a light vesicle which does not contain significant concentrations of typical markers of chromaffin granules (cytochrome b-561, dopamine beta-hydroxylase or the amine carrier). An analogous finding was obtained for sympathetic axons. The large dense core vesicles contain most p65 and also SV2 but only a smaller portion of synaptin/synaptophysin. A lighter vesicle containing this latter antigen and some SV2 has also been found. These results establish that in adrenal medulla and sympathetic axons three typical antigens of synaptic vesicles are not restricted to light vesicles. Apparently, a varying part of these antigens is found in chromaffin granules and large dense core vesicles. On the other hand, the light vesicles do not contain significant concentrations of functional antigens of chromaffin granules. Thus, the biogenesis of small presynaptic vesicles which contain all three antigens as well as functional components like the amine carrier is likely to involve considerable membrane sorting.     

The structure of cytochrome b561, a secretory vesicle-specific electron transport protein

Cytochrome b561 is a transmembrane electron transport protein that is specific to a subset of secretory vesicles containing catecholamines and amidated peptides. This protein is thought to supply reducing equivalents to the intravesicular enzymes dopamine-beta-hydroxylase and alpha-peptide amidase. We have purified cytochrome b561 from bovine adrenal chromaffin granules by reverse phase chromatography and have determined internal amino acid sequences from peptides. Complementary oligonucleotides were used to isolate two cDNA clones from a bovine brain library. The structure predicted by the sequences of these cDNAs suggests a highly hydrophobic protein of 273 amino acids which spans the membrane six times with little extramembranous sequence. Cytochrome b561 is not homologous to any other cytochrome and thus represents a new class of electron carriers. RNA blotting experiments indicate that cytochrome b561 is expressed in the adrenal medulla and all brain regions of the cow, but not in visceral organs. This result agrees well with the putative function of this unique cytochrome and with the notion that this protein is localized to large dense-core synaptic vesicles.     

Differences in the Distribution of Cytochrome b561 and Synaptophysin in Dog Splenic Nerve: A Biochemical and Immunocytochemical Study

Abstract: Compared with neurons of the CNS, the organization of the peripheral adrenergic axon and nerve terminal is more complex because two types of neurotrarismitter-containing vesicles, i.e., large (LDVs) and small densecore vesicles, coexist with the axonal reticulum (AR) and the well-characterized small synaptic vesicles. The AR, which is still poorly examined, is assumed to play some role in neurosecretion. We have studied the subcellular localization of noradrenaline, cytochrome b₅₆₁, and synaptophysin in control and ligated dog splenic nerve using both biochemical and ultrastructural approaches. Noradrenaline and cytochrome b₅₆₁ coaccumulated proximal to a ligation, whereas distally only the latter was found. Despite a codistribution with noradrenaline at high densities in sucrose gradients, Synaptophysin did not accumulate on either side of the ligation. At the ultrastructural level, cytochrome b₅₆₁ immunoreactivity was found on LDVs and AR elements, both accumulating proximal to the ligation. Distally, the multivesicular bodies (MVBs), immunolabeled for cytochrome b₅₆₁, account for the retrograde transport of LDVs and AR membranes retrieved at the nerve terminal. No Synaptophysin immunoreactivity could be detected on LDVs, AR, or MVBs. The results obtained from the ligation experiments together with the ultrastructural data Clearly illustrate that Synaptophysin is absent from LDVs and AR elements in adrenergic axons.     

Lipid-protein interactions in membrane models. Fluorescence polarization study of cytochrome b5-phospholipids complexes.

According to previous authors, cytochrome b5, when extracted from bovine liver by a detergent method, is called cytochrome d-b5. On the other hand, the protein obtained after trypsin action, which eliminates an hydrophobic peptide of about 54 residues, is called cytochrome t-b5. Fluorescence polarization of the dansyl phosphatidylethanolamine probe inserted into phospholipid vesicles is very sensitive to the binding of proteins, and so is a useful method to study lipid-protein interactions. The chromophore mobility, R, decreases markedly when dipalmitoyl phosphatidylcholine vesicles are incubated with cytochrome d-b5, whereas R does not change for cytochrome c and cytochrome t-b5. This can be interpreted as a strengthening of bilayer, only due to the interaction of the hydrophobic peptide tail. Interaction of dipalmitoly phosphatidylcholine vesicles with cytochrome d-b5 occurs either below or above the melting temperature of the aliphatic chains (41 degrees C). Even for a high protein to lipid molar ratio (1 molecule of protein for 40 phospholipid molecules), the melting temperature is apparently unaffected. Phosphatidylserine and phosphatidylinositol do not interact at pH 7.7 with cytochrome d-b5, because electrostatic forces prevent formation of complexes. At low pH, the interaction with the protein occurs, but the binding is mainly of electrostatic nature.     

Structure and vectorial properties of proteoliposomes containing cytochrome oxidase in the submitochondrial orientation

Cytochrome oxidase proteoliposomes were prepared from bovine heart oxidase. Size distributions determined by quasi-elastic light scattering (QELS) showed that there was a small population of large vesicles (120-200-nm diameter) and a large population of small vesicles (50-100-nm diameter). Trapping cytochrome c inside the proteoliposomes did not significantly alter this size distribution. Separation of the vesicles by gel filtration, however, revealed that the cytochrome c/cytochrome a ratio is higher in the larger vesicles. Internally trapped cytochrome c can be reduced by the membrane-permeable reductants 2,3,5,6-tetramethyl-p-phenylenediamine (DAD) or N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). Respiration on internal cytochrome c generated a membrane potential of 53 mV (positive inside) and a pH gradient of 0.2 (acid inside) as monitored by the optical probes oxonol V and pyranine, respectively. But the true magnitude of these gradients in individual proteoliposomes is complicated by vesicle heterogeneity. The membrane potential increased biphasically with increasing concentration of reductant. Ionophore sensitivity was higher for the "low Km" phase, and respiration became increasingly uncoupled as the reductant concentration was increased. These findings are consistent with a kinetic heterogeneity such that vesicles respiring at lower reductant concentrations generate a higher proton motive force than those with a larger Km. The steady-state internal acidification induced by turnover of the internally facing enzyme is probably maintained by both cytochrome oxidase proton translocation and a TMPD+/H+ antiport present in these vesicles [Cooper, C. E., & Nicholls, P. (1987) FEBS. Lett. 223, 155-160].     

Calmodulin stimulation of adenylate cyclase of intestinal epithelium

The effect of dicyclohexylcarbodiimide (DCCD) on the proton pumping two-subunit cytochrome c oxidase from Paracoccus denitrificans was investigated. Purified Paracoccus oxidase was reconstituted into phospholipid vesicles by cholate dialysis. Following incubation with increasing amounts of DCCD, proton ejection was recorded in response to reductant pulses with reduced cytochrome c. Concentrations of DCCD which greatly reduced proton pumping by bovine cytochrome c oxidase used as a control were found to exert only a minor effect on proton translocation by Paracoccus oxidase. Similarly, incubation of the bacterial enzyme with [14C]DCCD failed to reveal the specific covalent interaction previously demonstrated to occur with bovine cytochrome c oxidase, and here also shown for the oxidase of yeast. Thus, Paracoccus oxidase differs in its interaction with DCCD from the functionally analogous eukaryotic enzymes.     

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.     

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.     

Orientation of cytochrome c oxidase molecules in the two populations of reconstituted vesicles resolved by column chromatography on DEAE-Sephacryl

Vesicles reconstituted with bovine heart cytochrome c oxidase and dioleoylphosphatidylcholine can be resolved into two populations by column chromatography in DEAE-Sephacryl (Madden, T.D. and Cullis, P.R. (1984) J. Biol. Chem. 259, 7655-7658). These two fractions (I and II) were treated with two proteases. These are trypsin, which has been found to cleave subunit IV in the M domain of the cytochrome c oxidase molecule, and chymotrypsin, which has been found to cleave subunit III in the C domain. These studies show that fraction I vesicles contain cytochrome c oxidase orientation with the M domain outside, i.e., in the same topology as in submitochondrial particles, while fraction II vesicles contain enzyme molecules with their C domain outside, and thus in the same orientation as in mitochondria.     

Electron transfer across the chromaffin granule membrane

Membrane vesicles (ghosts) containing ascorbic acid were prepared from bovine chromaffin granules. When ferricyanide or ferricytochrome c were added to the external medium, a membrane potential (interior positive) developed across the ghost membrane. This membrane potential could not be elicited from ascorbate-free ghosts or by ferrocyanide added instead of ferricyanide. These results indicate that the chromaffin-granule membrane has a transmembrane electron carrier with a midpoint potential between that of ascorbate (+85 mV) and that of cytochrome c (+255 mV). The most likely candidate is cytochrome b-561 (+140 mV).     

ON THE DEVELOPMENT OF SYMPATHETIC NERVE TRUNK VESICLES DURING AXONAL TRANSPORT: DENSITY GRADIENT ANALYSIS OF DOPAMINE β-HYDROXYLASE IN BOVINE SPLENIC NERVE

—Dopamine β-hydroxylase was used as a marker enzyme for sympathetic nerve vesicles which were studied by density gradient technique in bovine splenic nerves. The enzyme analyses were complicated by the occurrence of inhibitors which had to be carefully neutralized with copper. The inhibitor was mainly found in the soluble fraction and no evidence for the occurrence of endogenous inhibitors in the nerve vesicles was obtained. A great variation in density of the dopamine β-hydroxylase containing particles was observed. This was probably mainly due to the variation in vesicle maturation since dopamine β-hydroxylase was distributed more towards the lighter gradient fractions in the proximal nerve segment preparations compared with intrasplenic nerve segment preparations. Noradrenaline/protein and noradrenaline/dopaminc β-hydroxylase ratios were found to be increased about 1·7-fold in the vesicle fraction isolated from the proximal nerve segments to those from the intrasplenic segments. A further increase of the noradrenaline/dopamine β-hydroxylase ratio was observed in a fraction with the same density isolated from the spleen. On the basis of these findings the noradrenaline/protein ratio was calculated to be about 500-600 nmol/mg in the nerve terminal vesicles.     

Microvesicles of the neurohypophysis are biochemically related to small synaptic vesicles of presynaptic nerve terminals

Nerve endings of the posterior pituitary are densely populated by dense- core neurosecretory granules which are the storage sites for peptide neurohormones. In addition, they contain numerous clear microvesicles which are the same size as small synaptic vesicles of typical presynaptic nerve terminals. Several of the major proteins of small synaptic vesicles of presynaptic nerve terminals are present at high concentration in the posterior pituitary. We have now investigated the subcellular localization of such proteins. By immunogold electron microscopy carried out on bovine neurohypophysis we have found that three of these proteins, synapsin I, Protein III, and synaptophysin (protein p38) were concentrated on microvesicles but were not detectable in the membranes of neurosecretory granules. In addition, we have studied the distribution of the same proteins and of the synaptic vesicle protein p65 in subcellular fractions of bovine posterior pituitaries obtained by sucrose density centrifugation. We have found that the intrinsic membrane proteins synaptophysin and p65 had an identical distribution and were restricted to low density fractions of the gradient which contained numerous clear microvesicles with a size range the same as that of small synaptic vesicles. The peripheral membrane proteins synapsin I and Protein III exhibited a broader distribution extending into the denser part of the gradient. However, the amount of these proteins clearly declined in the fractions preceding the peak of neurosecretory granules. Our results suggest that microvesicles of the neurohypophysis are biochemically related to small synaptic vesicles of all other nerve terminals and argue against the hypothesis that such vesicles represent an endocytic byproduct of exocytosis of neurosecretory granules.     

Lipid regulation of bovine cytochrome P450(11) beta activity

Purified bovine adrenocortical cytochrome P450(11) beta has been reconstituted into phospholipid vesicles using a detergent dialysis procedure. Using this reconstituted system, we have examined the effect of changes in the fatty acyl substituents of the lipids on the catalytic activity of the enzyme. The studies reported here show that cytochrome P450(11) beta exhibits a completely different response to changes in the fatty acyl groups from that shown by cytochrome P450scc. Cytochrome P450(11) beta displays maximal activity in lipid vesicles composed of saturated lipids, such as dipalmitoyl and dimyristoyl phosphatidylcholines, with turnover numbers ranging from 35 to 60 min-1. Incremental increases of phospholipids such as diphytanoyl and dioleoyl phosphatidylcholines result in a progressive inhibition of 11 beta hydroxylase activity; most of this kinetic effect is attributable to a significant decrease in Vmax accompanied by modest changes in Km for the steroid substrate deoxycorticosterone. Diphosphatidyl glycerol (cardiolipin), which has been previously shown to activate cytochrome P450scc, is a potent inhibitor of the 11 beta hydroxylase activity of cytochrome P450(11) beta, with half maximum inhibition observed in vesicles containing 4-5 mol% diphosphatidyl glycerol. Kinetic analysis demonstrates that this inhibition by diphosphatidyl glycerol is reflected in both a decrease in Vmax and relatively large increases (up to sevenfold) in Km for the steroid substrate. These effects on the 11 beta hydroxylase activity may have important implications for the in vivo regulation of not only the 11 beta hydroxylase activity, but also the other catalytic activities of this enzyme, particularly 18- and 19-hydroxylase and oxidase activities.