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PubMed1996

Roles of cytochrome b5 in the oxidation of testosterone and nifedipine by recombinant cytochrome P450 3A4 and by human liver microsomes.

NADH-dependent testosterone 6 beta-hydroxylation and nifedipine oxidation activities could be reconstituted in systems containing cytochrome b5 (b5), NADH-b5 reductase, and bacterial recombinant cytochrome P450 (P450) 3A4 with a synthetic phospholipid mixture, cholate, MgCl2, and reduced glutathione. Replacement of NADH-b5 reductase with NADPH-P450 reductase produced an eightfold increase in testosterone 6 beta-hydroxylation activity. Further stimulation could be obtained when NADPH was used as an electron donor instead of NADH. Removal of b5 from the NADH- and NADPH-supported systems caused a 90% loss of testosterone 6 beta-hydroxylation activities in the presence of NADPH-P450 reductase but resulted in complete loss of the activities in the absence of NADPH-P450 reductase. These results suggested that about 10% of the activities was due to electron flow from NADPH-P450 reductase to P450 3A4 in the absence of b5. In the presence of testosterone and MgCl2, P450 3A4 was reduced by b5 and NADH-b5 reductase, although the rate of P450 3A4 reduction was much slower than that by NADPH-P450 reductase. Anti-human b5 immunoglobulin G (IgG) (purified using rabbit b5 affinity chromatography) inhibited testosterone 6 beta-hydroxylation activity catalyzed by human liver microsomes more strongly in NADH- than in NADPH-supported reactions. However, anti-rat NADPH-P450 reductase IgG inhibited microsomal activities in both NADH- and NADPH-supported systems to similar extents. Addition of NADH enhanced NADPH-supported testosterone and nifedipine oxidations in human liver microsomes. MgCl2 stimulated rates of reduction of b5 by NADPH-P450 reductase, but not by NADH-b5 reductase, in reconstituted systems. These results suggest that b5 is an essential component in P450 3A4-catalyzed testosterone hydroxylation and nifedipine oxidation in human liver microsomes. Our previous observation that rates of reduction of ferric P450 3A4 by NADPH-P450 reductase are accelerated by complexation with substrates and b5 is supported in this study.

Animals↗
PubMed1984

Deficiency of cytochromes b and aa3 in muscle from a floppy infant with cytochrome oxidase deficiency.

A girl was presented suffering from generalised weakness and cardiorespiratory insufficiency. She succumbed at the age of 5 months. Lactate levels were elevated in serum, cerebrospinal fluid and urine. Histopathological examination revealed a mitochondrial myopathy. In muscle tissue the cytochrome oxydase activity was strongly reduced. The content of cytochromes b and aa3 was very low. At autopsy a cardiomyopathy was found.

Acidosis↗
PubMed1999

Lack of polymorphism of the conversion of losartan to its active metabolite E-3174 in extensive and poor metabolizers of debrisoquine (cytochrome P450 2D6) and mephenytoin (cytochrome P450 2C19).

OBJECTIVE: Losartan was given to subjects with known phenotypes of the polymorphic enzymes CYP2D6 and CYP2C19 to study any possible influence on the metabolism of the drug. METHODS: Plasma concentrations of losartan and E-3174 were studied after oral intake of 50 mg losartan in 24 healthy, male, Swedish Caucasian subjects who were extensive or poor metabolizers (EM/PM) of debrisoquine [cytochrome P450 2D6 (CYP2D6)] or mephenytoin [cytochrome P450 2C19 (CYP2C19)]. RESULTS: The areas under the curve (AUCinfinity) of losartan and E-3174 did not differ between poor and extensive metabolizers of debrisoquine or mephenytoin, respectively. CONCLUSION: About 14% of the antihypertensive drug losartan is metabolized to the active carboxylic acid metabolite E-3174, which contributes to the effect of losartan. The present study suggests that CYP2D6 and CYP2C19 are not involved to any major extent in the in vivo conversion of losartan to E-3174.

Adult↗
PubMed2004

Determination of cytochrome P450 1A2 and cytochrome P450 3A4 induction in cryopreserved human hepatocytes.

Freshly prepared human hepatocytes are considered as the 'gold standard' for in vitro testing of drug candidates. However, several disadvantages are associated with the use of this model system. The availability of hepatocytes is often low and consequently the planning of the experiments rendered difficult. In addition, the quality of the available cells is in some cases poor. As an alternative, cryopreserved human hepatocytes were validated as a model to study cytochrome P450 1A2 (CYP1A2) and cytochrome P450 3A4 (CYP3A4) induction. In a single blinded experiment, hepatocytes from three separate lots were incubated with three concentrations of different compounds, and compared to non-treated cells and cells incubated with omeprazole or rifampicin. CYP1A2 and CYP3A4 induction was determined by measuring 7-ethoxyresorufin-O-deethylation activity and 6beta-hydroxytestosterone formation, respectively. CYP1A2 and CYP3A4 mRNA and protein expression were analyzed by TaqMan QRT-PCR and immunodetection. Cells responded well to the prototypical inducers with a mean 38.8- and 6.2-fold induction of CYP1A2 and CYP3A4 activity, respectively. Similar as with fresh human hepatocytes, high batch-to-batch variation of CYP1A2 and CYP3A4 induction was observed. Except for 1 and 10 microM rosiglitazone, the glitazones did not significantly affect CYP1A2. A similar result was observed for CYP3A4 activity although CYP3A4 mRNA and protein expression were dose-dependently upregulated. In conclusion, cryopreserved human hepatocytes may be a good alternative to fresh hepatocytes to study CYP1A and 3A induction.

Cells, Cultured↗
PubMed1993

Effects of surface amino acid replacements in cytochrome c peroxidase on intracomplex electron transfer from cytochrome c.

Transient absorption techniques were used to measure the intracomplex electron transfer rates between four recombinant yeast cytochrome c peroxidases and iso-1 cytochrome c (cytc). The binding affinities and catalytic activities with cytc were previously examined [Corin et al. (1991) Biochemistry 30, 11585]. The four include a wild-type peroxidase (ECcP) and three others, each of which has one surface aspartic acid converted to lysine at position 37, 79, or 217. These sites have been suggested to be within or proximal to the recognition site for cytc. These mutants conduct electron transfer with cytc but differ with respect to the ionic strength profiles of their limiting rate constants. At pH and mu = 114 mM, ECcP and D217K show similar limiting rate constants for electron transfer with cytc, k(lim), of ca. 2000 s-1. In the same peroxidase concentration range, the D37K mutant exhibits a k(obs) of ca. 100 s-1. Instability of the compound I form of D79K prevented a complete study of the intracomplex kinetics of this mutant by this technique. At pH 6 and low ionic strength (8 mM), D37K exhibits a dramatic increase in k(obs) to ca. 800 s-1 while the other two recombinants show a marked decrease to values < 150 s-1. D37K displays much lower affinity for cytc than do the other peroxidases at higher ionic strengths [Hake et al. (1992) J. Am. Chem. Soc. 114, 5442], thus preventing adequate complexation necessary for efficient electron transfer. Variations in binding affinity do not explain the more subtle ionic strength kinetic profile observed for D217K.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1983

Cytochrome P-450 isozyme 1 from phenobarbital-induced rat liver: purification, characterization, and interactions with metyrapone and cytochrome b5.

Cytochrome P-450 isozyme 1 (PB-1) (Mr congruent to 53 000) was purified to apparent homogeneity from phenobarbital (PB)-induced rat liver microsomes, and its spectral, structural, immunochemical, and catalytic properties were determined. PB-1, present in significant amounts in uninduced rat liver microsomes, is induced approximately 2-4-fold by phenobarbital, as compared to the greater than 30-fold induction typical of the major PB isozymes characterized previously. PB-1 was distinguished from the major PB-induced isozymes PB-4 and PB-5 [Waxman, D. J., & Walsh, C. (1982) J. Biol. Chem. 257, 10446-10457] by the absence of a Fe2+-metyrapone P446 complex, by its unique NH2-terminal sequence and distinct peptide maps, by the lack of immuno-cross-reactivity to PB-4, and by its characteristic substrate-specificity profile. Metyrapone effected a saturable enhancement of several PB-1-catalyzed reactions in the reconstituted system [Km(metyrapone) congruent to 200 microM], which varied in magnitude with the substrate, with a maximal stimulation of 5-8-fold in the case of acetanilide 4-hydroxylation. That metyrapone enhanced the corresponding microsomal activities only in cases where the metyrapone-sensitive PB-4 did not catalyze the same reaction at significant rates suggested that PB-1 is probably responsible for the substrate-dependent stimulatory effects of metyrapone on microsomal monooxygenations. In contrast to PB-4 and PB-5, PB-1 was characterized by a marked, but not absolute, dependence on cytochrome b5 (b5) for catalytic activity, with 4-7-fold stimulations typically effected by inclusion of stoichiometric b5 in the reconstituted system. That these b5-stimulations were lipid dependent and were abolished with specific proteolytic fragments lacking b5's COOH-terminal membranous segment evidenced the importance of this segment for efficient, b5-mediated electron transfer to P-450 PB-1 in the reconstituted monooxygenase system.

Amino Acid Sequence↗
PubMed1987

Rabbit liver cytochrome P-450 LM2: roles of substrates, inhibitors, and cytochrome b5 in modulating the partition between productive and abortive mechanisms.

Cytochrome b5 (b5) enhanced the rate of 7-ethoxycoumarin deethylation by rabbit liver cytochrome P-450 LM2. The effect was saturable and can be analyzed as the sum of two effects: a decrease in the KM for the substrate and an increase in the Vmax. When two substrates were present simultaneously, they competed in a complex way depending on the presence of b5. Various substrates at low concentrations inhibited 7-ethoxycoumarin deethylation in a competitive-like way. Only a part of the P-450 activity was found to be affected by this mode of inhibition. Higher inhibitor concentrations caused a new kind of inhibition characterized by much higher half-effect values. The pattern seemed dependent on the ability of the inhibitors to be metabolized and was dramatically changed by the addition of b5. The relative rates of P-450-dependent NADPH oxidation and hydrogen peroxide and water formation were determined as well as their dependence on substrate and b5. A steady-state kinetic model that includes two branch points for water, hydrogen peroxide, and product formation is proposed. The model allows a full prediction of the b5 effects and seems consistent with most of the steady-state and rapid kinetic data available in the literature.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1988

Infrared spectroscopic analysis of salt bridge formation between cytochrome b5 and cytochrome c.

The infrared spectrum of a solution of a protein contains bands due to both the peptide backbone and the amino acid side chains. Generally, the bands due to the peptide backbone, between 1700 and 1600 cm-1, are analyzed to determine the secondary structure of the protein; the bands due to the amino acid side chains, between 1600 and 1500 cm-1, are largely ignored. When cytochrome b5 is mixed with cytochrome c, under conditions that favor ionic complex formation, changes are seen in protein secondary structure and also in a band at 1562 cm-1. The band at 1562 cm-1 is due to the side-chain carboxyl of Glu residues, rather than those of Asp residues that show a band at 1585 cm-1, and the changes in the band at 1562 cm-1 indicate that when the two proteins interact, three ionized carboxyl groups of Glu become involved in salt bridge formation. This result is identical with that obtained by previous theoretical studies and suggests that infrared spectroscopy may be a rapid and quantitative method for the study of ionic interactions between proteins.

Cytochrome b Group↗
PubMed1982

Spectrophotometric analysis of the interaction between cytochrome b5 and cytochrome c.

The interaction between cytochrome c and the tryptic fragment of cytochrome b5 has been found to produce a difference spectrum in the Soret region with a maximum absorbance at 416 nm. The intensity of this difference has been used to determine the stoichiometry of complex formation and the stability of the complex formed. At pH 7.0 [25 degrees C (phosphate), mu = 0.01 M], the two proteins were found to form a 1:1 complex with an association constant, KA, of 8(3) x 10(4) M-1. The stability of the complex was found to be strongly dependent on ionic strength with KA increasing to 4(3) x 10(6) M-1 at mu = 0.001 M [25 degrees C, pH 7.0 (phosphate)]. Analysis of the dependence of KA on pH from pH 6.5 to 8 demonstrated that this complex is maximally stable between pH 7 and 8 or about midway between the isoelectric points of the two proteins. Analysis of the temperature dependence of KA revealed that formation of the complex between the two proteins is largely entropic in origin with delta Ho = 1 +/- 3 kcal/mol and delta So = 33 +/- 11 eu [pH 7.0 (phosphate), mu = 0.001 M]. This result may be explained either by the model of Clothia and Janin [Clothia, C., & Janin, J. (1975) Nature (London) 256, 705] in terms of extensive solvent reorganization upon complexation or by the model of Ross and Subramanian [Ross, P. D., & Subramanian, S. (1981) Biochemistry 20, 3096] in which the negative enthalpic and entropic contributions of short-range protein-protein interactions are offset by proton release.

Animals↗
PubMed2000

A NapC/NirT-type cytochrome c (NrfH) is the mediator between the quinone pool and the cytochrome c nitrite reductase of Wolinella succinogenes.

Wolinella succinogenes can grow by anaerobic respiration with nitrate or nitrite using formate as electron donor. Two forms of nitrite reductase were isolated from the membrane fraction of W. succinogenes. One form consisted of a 58 kDa polypeptide (NrfA) that was identical to the periplasmic nitrite reductase. The other form consisted of NrfA and a 22 kDa polypeptide (NrfH). Both forms catalysed nitrite reduction by reduced benzyl viologen, but only the dimeric form catalysed nitrite reduction by dimethylnaphthoquinol. Liposomes containing heterodimeric nitrite reductase, formate dehydrogenase and menaquinone catalysed the electron transport from formate to nitrite; this was coupled to the generation of an electrochemical proton potential (positive outside) across the liposomal membrane. It is concluded that the electron transfer from menaquinol to the catalytic subunit (NrfA) of W. succinogenes nitrite reductase is mediated by NrfH. The structural genes nrfA and nrfH were identified in an apparent operon (nrfHAIJ) with two additional genes. The gene nrfA encodes the precursor of NrfA carrying an N-terminal signal peptide (22 residues). NrfA (485 residues) is predicted to be a hydrophilic protein that is similar to the NrfA proteins of Sulfurospirillum deleyianum and of Escherichia coli. NrfH (177 residues) is predicted to be a membrane-bound tetrahaem cytochrome c belonging to the NapC/NirT family. The products of nrfI and nrfJ resemble proteins involved in cytochrome c biogenesis. The C-terminal third of NrfI (902 amino acid residues) is similar to CcsA proteins from Gram-positive bacteria, cyanobacteria and chloroplasts. The residual N-terminal part of NrfI resembles Ccs1 proteins. The deduced NrfJ protein resembles the thioredoxin-like proteins (ResA) of Helicobacter pylori and of Bacillus subtilis, but lacks the common motif CxxC of ResA. The properties of three deletion mutants of W. succinogenes (DeltanrfJ, DeltanrfIJ and DeltanrfAIJ) were studied. Mutants DeltanrfAIJ and DeltanrfIJ did not grow with nitrite as terminal electron acceptor or with nitrate in the absence of NH4+ and lacked nitrite reductase activity, whereas mutant DeltanrfJ showed wild-type properties. The NrfA protein formed by mutant DeltanrfIJ seemed to lack part of the haem C, suggesting that NrfI is involved in NrfA maturation.

Amino Acid Sequence↗
PubMed1991

Direct electrochemistry of proteins. Investigations of yeast cytochrome c mutants and their complexes with cytochrome b5.

Direct electrochemistry of site-specific mutants of yeast iso-1-cytochrome c (cyt c) and their complexes with bovine cytochrome b5 (cyt b5) has been investigated at edge-plane pyrolytic graphite (EPG) and bis(4-pyridyl)-disulphide-modified gold electrodes. Structure/function relationships have been investigated with the particular aim of clarifying the factors controlling the interactions of proteins at electrode/electrolyte interfaces and the determinants for direct electrochemistry in ternary protein/protein/electrode adducts, e.g. cyt c/cyt b5/EPG. Investigations of the cyt c mutants alone revealed a variety of electrochemical responses: all the mutants show similar voltammetric reversibility at modified gold electrodes, whereas at EPG electrodes the reversibility follows the order: Asn52Ile-Cys102Thr greater than Cys102Thr greater than Asn52Ala-Cys102Thr. Mid-point potentials follow the order: Arg13Ile (+60 +/- 5 mV vs. standard calomel electrode) greater than Cys102Thr (+40 +/- 5 mV) greater than Lys27Gln (+30 +/- 5 mV) approximately Lys72Asp (+30 +/- 5 mV) greater than Asn52Ala-Cys102Thr (+15 +/- 5 mV) greater than Asn52Ile-Cys102Thr (-10 +/- 5 mV). The structural basis for these differences is briefly discussed. When these mutants are bound to cyt b5, the differences in electrochemical response are greatly enhanced in the ternary cyt c/cyt b5/EPG adducts. A minimal analysis of these differences supports a model of multiple overlapping binding and recognition domains on cyt c which may be finely tuned to allow ternary complex formation so that a single-site variation could modify or abolish direct electrochemistry in the ternary adduct.

Animals↗
PubMed2004

Topological changes in the CYP3A4 active site probed with phenyldiazene: effect of interaction with NADPH-cytochrome P450 reductase and cytochrome b5 and of site-directed mutagenesis.

The active site topology of heterologously expressed CYP3A4 purified from an Escherichia coli expression system was examined using phenyldiazene. Incubation of CYP3A4 with phenyldiazene and subsequent oxidation yielded all four potential N-phenylprotoporphyrin IX regioisomers derived from attack on an available nitrogen atom in pyrrole rings B, A, C, or D (N(B):N(A):N(C):N(D) = 6:73:7: 13). Further study using 28 active site mutants showed that substitution of residues closer to the heme, Ala-305, Thr-309, or Ala-370, with a larger residue caused the most drastic changes in regioisomer formation, which reflected the location of each amino acid residue replaced in a CYP3A4 homology model. Previous studies have suggested a conformational change in CYP3A4 upon binding of NADPH-cytochrome P450 reductase (CPR) or cytochrome b(5) (b(5)). Therefore, regioisomer formation was also compared in the absence of redox partners and in the presence of CPR, b(5), or both. Formation of all four regioisomers in CYP3A4 wild type, particularly the minor ones, was reduced in the presence of b(5). CPR also greatly decreased the three minor isomers but increased the major isomer significantly. The presence of b(5) and CPR restored minor isomer formation and suppressed the enhancement of N(A) formation caused by CPR alone. Interestingly, the effects of the redox partners differed among representative active site mutants. In particular, the increase in N(C) upon substitution of Ala-370 with Phe was significantly reversed in the presence of redox partners, strongly suggesting that a conformational change occurs around pyrrole ring C due to protein-protein interactions between CYP3A4 and CPR or b(5).

Animals↗
PubMed1988

Probing the mechanisms of macromolecular recognition: the cytochrome b5-cytochrome c complex.

The specificity of complex formation between cytochrome b5 (cyt b5) and cytochrome c (cyt c) is believed to involve the formation of salt linkages between specific carboxylic acid residues of cyt b5 with lysine residues on cyt c. Site-directed mutagenesis was used to alter the specified acidic residues of cyt b5 to the corresponding amide analogues, which resulted in a lower affinity for complex formation with cyt c. The dissociation of the complex under high pressure resulted in specific volume changes, the magnitude of which reflected the degree of solvation of the acidic residues in the proposed protein-protein interface.

Animals↗
PubMed2006

A Brownian dynamics study of the interactions of the luminal domains of the cytochrome b6f complex with plastocyanin and cytochrome c6: the effects of the Rieske FeS protein on the interactions.

The availability of the structures of the cytochrome b6f complex (cyt b6f), plastocyanin (PC), and cytochrome c6 (cyt c6) from Chlamydomonas reinhardtii allowed us, for the first time, to model electron transfer interactions between the luminal domains of this complex (including cyt f and the Rieske FeS protein) and its redox partners in the same species. We also generated a model structure in which the FeS center of the Rieske protein was positioned closer to the heme of cyt f than observed in the crystal structure and studied its interactions with both PC and cyt c6. Our data showed that the Rieske protein in both the original crystal structure and in our modeled structure of the cyt b6f complex did not physically interfere with binding position or orientation of PC or cyt c6 on cyt f. PC docked on cyt f with the same orientation in the presence or the absence of the Rieske protein, which matched well with the previously reported NMR structures of complexes between cyt f and PC. When the FeS center of the Rieske protein was moved close to the heme of cyt f, it even enhanced the interaction rates. Studies using a cyt f modified in the 184-191 loop showed that the cyt f structure is a more important factor in determining the rate of complex formations than is the presence or the absence of the Rieske protein or its position with respect to cyt f.

Animals↗
PubMed1999

Enhancement of cytochrome P-450 3A4 catalytic activities by cytochrome b(5) in bacterial membranes.

Activities of testosterone, nifedipine, and midazolam oxidation by recombinant cytochrome P-450 (P-450) 3A4 coexpressed with human NADPH-P-450 reductase (NPR) in bacterial membranes (CYP3A4/NPR membranes) were determined in comparison with those of other recombinant systems and of human liver microsomes with high contents of CYP3A4. Growth conditions for Escherichia coli transformed with the bicistronic construct affected expression levels of CYP3A4 and NPR; an excess of NPR over P-450 in membrane preparations enhanced CYP3A4-dependent testosterone 6beta-hydroxylation activities of the CYP3A4/NPR membranes. Cytochrome b(5) (b(5)) and apolipoprotein b(5) further enhanced the testosterone 6beta-hydroxylation activities of CYP3A4/NPR membranes after addition to either bacterial membranes or purified enzymes. NPR was observed to enhance catalytic activity when added to the CYP3A4/NPR membranes, either in the form of bacterial membranes or as purified NPR (in combination with cholate and b(5)). Apparent maximal activities of testosterone 6beta-hydroxylation in CYP3A4/NPR membranes were obtained when the molar ratio of CYP3A4/NPR/b(5) was adjusted to 1:2:1 by mixing membranes containing each protein. Testosterone 6beta-hydroxylation, nifedipine oxidation, and midazolam 4- and 1'-hydroxylation activities in CYP3A4/NPR membranes plus b(5) systems were similar to those measured with microsomes of insect cells coexpressing CYP3A4 with NPR and/or of human liver microsomes, based on equivalent CYP3A4 contents. These results suggest that CYP3A4/NPR membrane systems containing b(5) are very useful models for prediction of the rates for liver microsomal CYP3A4-dependent drug oxidations.

Anesthetics, Intravenous↗
PubMed1995

The reducing ability of iron chelates by NADH-cytochrome B5 reductase or cytochrome B5 responsible for NADH-supported lipid peroxidation.

We studied iron reduction, NADH oxidation, and lipid peroxidation in the presence of iron chelates with a chelator, such as nitrilotriacetate, ADP, citrate, and pyrophosphate, in NADH-supported reconstituted system. The results showed the selectivity of NADH-cytochrome b5 reductase or cytochrome b5 towards iron chelates and the subsequent ability of this system to promote peroxidation. The lipid peroxidation was partially inhibited by superoxide dismutase. In the presence of any iron chelate, hydrogen peroxide was produced in the systems including the reductase, and the production was accompanied with an increase in NADH oxidation.

Adenosine Diphosphate↗
PubMed1990

Monomer-dimer structure of cytochrome-c oxidase and cytochrome bc1 complex from the thermophilic bacterium PS3.

Molecular weights of three membrane proteins have been measured in the presence of 0.1% octaethylene glycol n-dodecyl ether (C12E8) by the measuring system in which a membrane protein eluted from a gel chromatography column is monitored sequentially for ultraviolet absorption, light scattering and refractive index. The relative molecular mass (Mr) and amount of bound detergent per protein (delta) can be calculated from these data, if instrumental constants are measured using a set of appropriate water-soluble proteins which does not bind nonionic surfactants. The molecular masses of cytochrome c oxidase and cytochrome bc1 complex from the thermophilic bacterium PS3 were determined to be 127 kDa and 185 kDa, respectively, indicating that the oxidase is monomeric, while the bc1 complex dimeric in the presence of C12E8. The larger apparent molecular mass of about 310 kDa of the PS3 oxidase obtained from the retention time of the gel chromatography (Sone, N., Sekimachi, M. and Kutoh, E. (1987) J. Biol. Chem. 262, 15386-15391) turned out to be due to a high binding ability with the detergent (delta = 1.25 g/g) of this very hydrophobic protein. Analyses of bovine heart cytochrome oxidase, on which monomer/dimer properties have been reported, showed that the enzyme is mainly dimeric (Mr = 374,000), while a small portion is monomeric (Mr = 191,000). Mild alkaline treatment of this enzyme caused monomerization of the enzyme with accompanying aggregate formation. These results, thus, show that this method is suitable to analyze monomer/dimer conversion of membrane protein as well as to estimate structure of membrane proteins.

Animals↗
PubMed1988

A Triton X-100-hydroxyapatite procedure for a rapid purification of bovine heart cytochrome-c oxidase. Characterization of the cytochrome-c oxidase/Triton X-100/phospholipid mixed micelles by laser light scattering.

Cytochrome-c oxidase (ferrocytochrome-c:oxygen oxidoreductase, EC 1.9.3.1) has been isolated from bovine heart mitochondria by the combined use of the non-ionic detergent Triton X-100 as solubilizing agent and hydroxyapatite chromatography as the most important step in the purification of this membrane protein. This method is fast and very reproducible. The enzymic complex, purified in the form of protein/detergent/phospholipid mixed micelles, contains 9.7 mumol heme a per mg protein, and has a high molecular activity (500 mol cytochrome c per s per mol enzyme). These mixed micelles have been studied by laser light scattering, which has shown that the average molecular weight of the micelles is 540 +/- 80 kDa. This implies that cytochrome-c oxidase is purified in the form of a dimer. The average quadratic radius of gyration of the micelles is 40 +/- 10 nm, corresponding in our case to an approximately spherical shape.

Animals↗