Affinity of folic acid for the folate-binding protein of choroid plexus.

The affinity of folic acid for the folate-binding protein of rabbit choroid plexus was determined by equilibrium dialysis at 4 °C. All solutions contained 0.02% Triton X-100 to maintain the binder in solution. At pH 7.0, the apparent dissociation constant (K_a) at a binder concentration of 0.36 nm was 9.4 pm with slight positive cooperativity (Hill coefficient = 1.19). The K_a increased at pH 6.0 and when a higher concentration of binder (3.25 nm) was used to 30.1 and 46.0 pm, respectively. However, the maximal binding capacity per milligram of protein did not change. At pH 5.0, the K_a was greater than 20 nm. These results show that the affinity of the choroid plexus folate-binding protein (when solubilized in Triton X-100) for folic acid depends on both the concentration of binder and the pH.     

An investigation of the ligand-binding properties of Pseudomonas aeruginosa nitrite reductase.

The low-temperature e.p.r. and m.c.d. (magnetic-circular-dichroism) spectra of Pseudomonas aeruginosa nitrite reductase, together with those of its partially and fully cyanide-bound derivatives, were investigated. The m.c.d. spectra in the range 600-2000 nm indicate that the native axial ligands to haem c are histidine and methionine, and furthermore that it is the methionine ligand that must be displaced before cyanide binding at this haem. The m.c.d. spectra in the range 1000-2000 nm contain no charge-transfer bands arising from low-spin ferric haem d1, a chlorin. New optical transitions in the region 700-850 nm were found for the cyanide adduct of haem d1. The g-values of haem d1 in the native enzyme are 2.51, 2.43 and 1.71, suggesting co-ordination by two histidine ligands in the oxidized state. There is clear evidence in the e.p.r. data of an interaction between the c and d1 haem groups. This is not apparent in the optical spectra. The results are interpreted in terms of haem groups that are remote from each other, their interaction being mediated through protein conformational changes. The possible implications of this in relation to reduction processes catalysed by the enzyme are considered.     

Ligand binding to cytochrome c and other related haem proteins and peptides. Part III. Temperature dependence studies

The temperature dependency of ligand binding processes lend support to the proposed mechanisms and the factors affecting ligand binding reported earlier in this series. The free energy contribution from each factor affecting ligand binding was estimated for a number of haem proteins. The structures of the haem proteins used, as conveyed from ligand binding data, are in agreement with the structures of these haem proteins as determined by other methods (e.g. X-ray crystallography, NMR, etc.). Therefore, ligand binding could be used as a facile probe to investigate some of the structural and functional properties of haem proteins. In this respect, it was concluded that the structure of native cytochrome c at pH 10 is similar to the structure of carboxymethyl-Met 80 cytochrome c between pH 7 and 10.     

Potential intercellular futile cycling of carbohydrates in diabetes.

A simple and rapid method for the isolation of a large quantity of cytochrome c oxidase from bovine heart mitochondria was developed, based on selective solubilization of mitochondrial protein with first Triton and then lauryl maltoside. Gel filtration shows that the lauryl maltoside-solubilized oxidase preparation is in a hydrodynamically homogeneous state with a Stokes radius of 7.5 +/- 0.2 nm. It contains 8.0 mumol of haem (with an a/a3 ratio of 1)/g of protein. The catalytic constant (maximum turnover number) with respect to cytochrome c approaches 600 S-1. After further purification of the solubilized enzyme on a sucrose-gradient centrifugation, the purified enzyme has a haem content of 10.3 mumol/g of protein and eight major polypeptide bands shown on SDS/polyacrylamide-gel electrophoresis.     

Near-infrared magnetic and natural circular dichroism of cytochrome c oxidase.

A detailed study is presented of the room-temperature absorption, natural and magnetic circulation-dichroism (c.d. and m.c.d.) spectra of cytochrome c oxidase and a number of its derivatives in the wavelength range 700-1900 nm. The spectra of the reduced enzyme show a strong negative c.d. band peaking at 1100nm arising from low-spin ferrous haem a and a positive m.c.d. peak at 780nm assigned to high-spin ferrous haem a3. Addition of cyanide ion doubles the intensity of the low-spin ferrous haem c.d. band and abolishes reduced carbonmonoxy derivative the haem a32+-CO group shows no c.d. or m.c.d. bands at wavelengths longer than 700nm. A comparison of the m.c.d. spectra of the oxidized and cyanide-bound oxidized forms enables bands characteristic of the high-spin ferric form of haem a33+ to be identified between 700 and 1300nm. At wavelengths longer than 1300nm a broad positive m.c.d. spectrum, peaking at 1600nm, is observed. By comparison with the m.c.d. spectrum of an extracted haem a-bis-imidazole complex this m.c.d. peak is assigned to one low-spin ferric haem, namely haem a3+. On binding of cyanide to the oxidized form of the enzyme a new, weak, m.c.d. signal appears, which is assigned to the low-spin ferric haem a33+-CN species. A reductive titration, with sodium dithionite, of the cyanide-bound form of the enzyme leads to a partially reduced state in which low-spin haem a2+ is detected by means of an intense negative c.d. peak at 1100 nm and low-spin ferric haem a33+-CN gives a sharp positive m.c.d. peak at 1550nm. The c.d. and m.c.d. characteristics of the 830nm absorption band in oxidized cytochrome c oxidase are not typical of type 1 blue cupric centres.     

Comparative studies on intrinsic factor and cobalophilin in different parts of the gastrointestinal tract of the pig

The vitamin B₁₂ binders in the pig pyloric mucosa gastric and intestinal juice from the upper gastrointestinal tract were fractionated into only two molecular forms, classified as intrinsic factor and cobalophilin. The unsaturated vitamin B₁₂-binding power due to cobalophilin was lower in the intestinal than in the gastric juice. Electrofocusing revealed that intrinsic factor and cobalophilin in the intestinal juice contained more of the `neutral'-type isoproteins, and the suggestion is made that this is due to enzyme activity. The gastric-juice intrinsic factor contained more acidic isoproteins, which supports the hypothesis that carbohydrate is added on to the polypeptide chain of this protein before it is secreted into gastric juice. The gastric- and intestinal-juice cobalophilins, studied also by electrofocusing, differed from that of pyloric mucosa and they appeared to be of salivary origin. With regard to molecular dimensions there was no significant difference between the intrinsic factors and cobalophilins from all sources studied. All cobalophilins had molecular weights by the formula of Svedberg of approx. 92500, Stokes radii of 4.62nm and sedimentation coefficients of 5.15S. The corresponding values for the intrinsic factors were 63600, 3.57nm and 4.38S. In addition, the intrinsic factors exhibited similar avidities for binding to the solubilized ileal intrinsic-factor receptor. Also the intrinsic factors and cobalophilins, irrespective of their source, bound to the analogous specific xenoantibodies with the same avidity. The present results demonstrate that intrinsic factor remains practically unaltered during its passage through the proximal intestine and render unlikely the speculations made about the presence of an endogenous binder for intrinsic factor as well as the existence of a `pancreatic intrinsic factor'. In addition, they are compatible with the theory that the interference by undegraded cobalophilin may be the reason for the abnormal vitamin B₁₂ absorption observed in patients with pancreatic insufficiency.     

Physical characterization of the transferrin receptor in human placentae

The physical properties and binding characteristics of the solubilized transferrin receptor isolated from the placental brush-border membrane of a human trophoblast cell were investigated. The receptor protein was isolated from solubilized 125I-labeled membranes by immunoprecipitation with anti-human transferrin in the presence of saturating amounts of human transferrin. Gel filtration on acrylamide agarose (AcA-22) at 23 degrees C in the absence of transferrin indicates the transferrin receptor has a Stokes radius of 4.6 nm. In the presence of transferrin, the Stokes radius of the receptor shifts to 6.3 nm. Sucrose density centrifugation studies indicate that it has a sedimentation coefficient of 9.8 S in the absence of transferrin and 11.2 S in the presence of transferrin. The molecular weight for the transferrin free receptor is calculated to be 213,000. Upon incubation with transferrin, it increases to 364,000. This is consistent with the idea that the active form of the solubilized receptor is a dimer and the dimer is in turn capable of binding two transferrin molecules.     

Comparison of microsomal and solubilized monooxygenases from rat and rabbit by proton magnetic relaxation.

The paper presents results of a comparative study of the haem environment, by proton magnetic relaxation, in P-450 and P-448 monooxygenases from rat and rabbit, induced by phenobarbital and 3-methylcholanthrene, in both species. It was established that the method yields information on the accessibility of the haem iron for solvent molecules (protons), both in microsomes and in solubilized samples of various degrees of purification, i.e. association. The state of micelles in the solutions does not alter the haem iron accessibility. A slight difference was found for the microsomes suspended in a phosphate vs. pyrophosphate buffer, but this is without any consequence with regard to the species and form differences. The correlation time for the highly purified LM2 fraction of rabbit P-450 could not be determined more precisely than before for a sample of lower purity, because the relaxation rates are frequency independent. The correlation time for the rat P-448 monooxygenase was determined by dispersion measurements to be (4.1 +/- 0.4) x 10(-11) s. It was found that the PMRx behaviours of rabbit and rat monooxygenases are more alike in microsomes than in the partially purified solubilized form. The solubilization produces a pronounced alteration of the PMRx temperature dependence only for the rat 3-MC induced monooxygenase P-448. For the P-450 form the haem iron becomes less accessible on solubilization, both for the rabbit and the rat liver monooxygenases, whereas in case of rat liver P-448 the accessibility is considerably enhanced on solubilization. There is a substantial structural specificity of the haem environments from the two animal species, the one from rat being tighter. The reduced, NO-bound rabbit liver monooxygenase was studied also, but the results are not yet conclusive, except the fact that the unpaired spin from NO is thoroughly shielded from the solvent compared with the haem iron from the original sample. The following series of increased haem-iron accessibility emerges from the PMRx studies known so far: rat (P-448) less than rabbit (P-448) less than rat (P-450) less than rabbit (P-450) in microsomes, and rabbit (P-448, with 3-MC bound?) less than Pseudomonas putida (P-450) rat less than (P-448), less than rat (P-450) less than rabbit (P-450) from solubilized samples. For the latter, it appears that increased enzymic specificity goes along with a closing of the haem cleft.     

Phenylmethylsulfonyl fluoride (PMSF) inhibits nerve growth factor binding to the high affinity (type I) nerve growth factor receptor

Dorsal root ganglia were extirpated from 9-day old embryonic chickens and solubilized in phosphate buffered saline containing 0.5% Noniodet P 40 detergent. When nerve growth factor binding studies are performed on these samples, the expected curvilinear Rosenthal (Scatchard) plot is obtained. However, when the solubilized cell sample is made 1-2 mM in phenylmethylsulfonyl fluoride and nerve growth factor binding is determined, a linear Rosenthal (Scatchard) plot is obtained. The equilibrium dissociation constant obtained from the slope of the line is 1.9 X 10(-9) M, identical to the equilibrium dissociation constant of the low affinity receptor. A similar phenomenon is observed when rat pheochromocytoma cells are solubilized in the non-ionic detergent and nerve growth factor binding is determined. No high affinity binding can be detected for either cell type when detergent solubilized cells are incubated with phenylmethylsulfonyl fluoride.     

Net hepatic and splanchnic metabolism of lactate, pyruvate and propionate in dairy cows in vivo in relation to lactation and nutrient supply.

The significance of the exposed haem edge in cytochrome c was directly probed by chemically modifying the partially exposed haem propionate in the crevice region around residues threonine-78 and threonine-49. Reaction of tuna heart cytochrome c with a water-soluble carbodi-imide at pH 3.7 in the absence of any added nucleophilic base leads to the covalent addition of substituted N-acylureas to the protein at two sites. One site has been shown to be a haem propionate by isotope-tracer and i.r.-spectral analysis of haem purified from the apoprotein. The other site is aspartial acid-62 on the back of the molecule. The modified cytochrome c demonstrates abnormal properties, including auto-oxidizability, a reduction potential of + 105mV, a reversible transition to a high-spin species below pH 5.3, no 695 nm charge-transfer band in the ferric state and abnormal binding to mitochondrial membranes. The derivative does react with cytochrome oxidase in deoxycholate-treated submitochondrial particles or in purified preparations with a specific activity of 43-65% compared with that obtained with native cytochrome c. The results are consistent with the view that an intact haem crevice is essential for normal values for physiochemical characteristics, but the significant residual enzymic activity suggests that the electron-transfer interface and/or the cytochrome oxidase-binding site cannot be localized solely in the region of the exposed haem propionate.     

Solubilization and characterization of the beta-bungarotoxin-binding protein of chick brain membranes

The previously characterized ( Rehm , H., and Betz, H. (1982) J. Biol. Chem. 257, 10015-10022) neuronal binding protein for the presynaptic neurotoxin beta-bungarotoxin (beta-BuTx) was solubilized from synaptic membrane fractions of chick brain using the nonionic detergent Triton X-100. 125I-beta-BuTx bound to the solubilized protein with a dissociation constant (KD) of 1.9 +/- 0.1 nM. This binding of 125I-beta-BuTx was Ca2+-dependent and pharmacologically specific. From different basic proteins tested, only unlabeled beta-BuTx and its antagonist dendrotoxin inhibited 125I-beta-BuTx binding. Potassium ions were required during solubilization and binding in order to detect 125I-beta-BuTx-binding activity. Sedimentation in sucrose/H2O and sucrose/D2O gradients and gel exclusion chromatography on Sepharose 6B indicated a s20,w of 12.8 +/- 0.6 S and a Stokes radius of 8.6 +/- 0.2 nm for the solubilized beta-BuTx-binding component. From these data, the protein molecular weight of the beta-BuTx binding site was calculated to be 431,000 +/- 45,000.     

Characteristics of solubilized human-somatotropin-binding protein from the liver of pregnant rabbits.

A specific binding site for somatotropin was solubilized by 1% (v/v) Triton X-100 from a crude particulate membrane fraction of pregnant rabbit liver, partially purified and characterized. The solubilized binding site retained many of the characteristics observed in the original particulate fraction, indicating that extraction of the binding site with Triton X-100 does not cause any major changes in its properties. The binding of human 125I-labelled-somatotropin to the solubilized binding site is a saturable and reversible process, depending on temperature, incubation time, pH and ionic environment. Analysis of the kinetic data revealed a finite number of binding sites with an affinity constant of 0.32 x 10(10)M-1. The binding activity for human 125I-labelled-somatotropin was adsorbed to a concanavalin-A-Sepharose column and was dissociated from the column with alpha-methyl-D-glucoside, suggesting that the binding protein may be a glycoprotein. Using affinity chromatography on concanavalin-A-Sepharose, ion-exchange chromatography on DEAE-cellulose and gel filtration on Sepharose 6B, the binding protein was purified 1000-4000-fold from the original liver homogenate. When the partially purified preparation was chromatographed on Sepharose 6B, the binding protein eluted as a molecule with an apparent molecular weight of 200000, with a Stokes' radius of 4.9 nm. Sucrose-density-gradient centrifugation of the preparation showed that the sedimentation coefficient of the binding protein was 7.2S. Isoelectric focusing experiments revealed that a major part of the protein has an acidic pI (4.2-4.5). Exposure of the protein to trypsin decreased the binding activity for human 125I-labelled-somatotropin or bovine 125I-labelled-somatotropin, whereas ribonuclease, deoxyribonuclease, phospholipase C or neuraminidase had little or no effect.     

Haem accessibility in cytochrome P-450 from rabbit liver. A proton magnetic relaxation study by stereochemical probes.

Cytochrome P-450 was solubilized from phenobarbital induced rabbit liver and purified by affinity chromatography. The longitudinal proton magnetic relaxation rates of this ferric, low-spin sample (as confirmed by ESR) in 20% glycerol aqueous solution are very large compared with low-spin methaemoglobin and myoglobin derivatives. Similarly high rates were measured in a deuterated solution using the aliphatic protons of glycerol as stereochemical markers, which strongly suggests that the haem iron in cytochrome P-450 is much more accessible to the solvent than in harmoglobin or myoglobin. Type I substate (Spasman) produced small but significant increases in NMR rates both in the H2O and in the 2H2O solution, while binding of aniline (Type II substrate) doubled the rates.     

Characterization of Cupriavidus metallidurans CYP116B1--a thiocarbamate herbicide oxygenating P450-phthalate dioxygenase reductase fusion protein

The novel cytochrome P450/redox partner fusion enzyme CYP116B1 from Cupriavidus?metallidurans was expressed in and purified from Escherichia coli. Isolated CYP116B1 exhibited a characteristic Fe(II)CO complex with Soret maximum at 449 nm. EPR and resonance Raman analyses indicated low-spin, cysteinate-coordinated ferric haem iron at both 10 K and ambient temperature, respectively, for oxidized CYP116B1. The EPR of reduced CYP116B1 demonstrated stoichiometric binding of a 2Fe-2S cluster in the reductase domain. FMN binding in the reductase domain was confirmed by flavin fluorescence studies. Steady-state reduction of cytochrome c and ferricyanide were supported by both NADPH/NADH, with NADPH used more efficiently (K(m[NADPH]) = 0.9 ± 0.5 μM and K(m[NADH]) = 399.1 ± 52.1 μM). Stopped-flow studies of NAD(P)H-dependent electron transfer to the reductase confirmed the preference for NADPH. The reduction potential of the P450 haem iron was -301 ± 7 mV, with retention of haem thiolate ligation in the ferrous enzyme. Redox potentials for the 2Fe-2S and FMN cofactors were more positive than that of the haem iron. Multi-angle laser light scattering demonstrated CYP116B1 to be monomeric. Type I (substrate-like) binding of selected unsaturated fatty acids (myristoleic, palmitoleic and arachidonic acids) was shown, but these substrates were not oxidized by CYP116B1. However, CYP116B1 catalysed hydroxylation (on propyl chains) of the herbicides S-ethyl dipropylthiocarbamate (EPTC) and S-propyl dipropylthiocarbamate (vernolate), and the subsequent N-dealkylation of vernolate. CYP116B1 thus has similar thiocarbamate-oxidizing catalytic properties to Rhodoccocus erythropolis CYP116A1, a P450 involved in the oxidative degradation of EPTC.     

Multiple roles of haem in cystathionine β-synthase activity: implications for hemin and other therapies of acute hepatic porphyria

The role of haem in the activity of cystathionine β-synthase (CBS) is reviewed and a hypothesis postulating multiple effects of haem on enzyme activity under conditions of haem excess or deficiency is proposed, with implications for some therapies of acute hepatic porphyrias. CBS utilises both haem and pyridoxal 5′-phosphate (PLP) as cofactors. Although haem does not participate directly in the catalytic process, it is vital for PLP binding to the enzyme and potentially also for CBS stability. Haem deficiency can therefore undermine CBS activity by impairing PLP binding and facilitating CBS degradation. Excess haem can also impair CBS activity by inhibiting it via CO resulting from haem induction of haem oxygenase 1 (HO 1), and by induction of a functional vitamin B₆ deficiency following activation of hepatic tryptophan 2,3-dioxygenase (TDO) and subsequent utilisation of PLP by enhanced kynurenine aminotransferase (KAT) and kynureninase (Kynase) activities. CBS inhibition results in accumulation of the cardiovascular risk factor homocysteine (Hcy) and evidence is emerging for plasma Hcy elevation in patients with acute hepatic porphyrias. Decreased CBS activity may also induce a proinflammatory state, inhibit expression of haem oxygenase and activate the extrahepatic kynurenine pathway (KP) thereby further contributing to the Hcy elevation. The hypothesis predicts likely changes in CBS activity and plasma Hcy levels in untreated hepatic porphyria patients and in those receiving hemin or certain gene-based therapies. In the present review, these aspects are discussed, means of testing the hypothesis in preclinical experimental settings and porphyric patients are suggested and potential nutritional and other therapies are proposed.     

Inhibitor effects on redox-linked protonations of the b haems of the mitochondrial bc1 complex

The effects of pH and inhibitors on the spectra and redox properties of the haems b of the bc1 complex of beef heart submitochondrial particles were investigated. The major findings were: (1) both haems have a weakly redox-linked protonatable group with pKox and pKred of around 6 and 8; (2) at pH values above 7, haem bH becomes heterogeneous in its redox behaviour. This heterogeneity is removed by the Qi site inhibitors antimycin A, funiculosin and HQNO, but not by the Qo site inhibitors myxothiazol or stigmatellin; (3) of all inhibitors tested only funiculosin had a large effect on the Em/pH profile of either haem b. In all cases where definite effects were found, the haem most affected was that thought to be closest to the site of inhibitor binding; (4) spectral shifts of haem groups caused by inhibitor binding were usually, but not always, of the haem group closest to the binding site; (5) titrations with succinate/fumarate were in reasonable agreement with redox-mediated data provided that strict anaerobiosis was maintained. Apparent large shifts of haem midpoint potentials with antimycin A and myxothiazol could be produced in aerobic succinate/fumarate titrations in the presence of cyanide, as already reported in the literature, but these were artefactual; (6) the heterogeneous haem bH titration behaviour can be simulated with a model similar to that proposed by Salerno et al. (J. Biol. Chem. (1989) 264, 15398-15403) in which there is redox interaction between haem bH and ubiquinone species bound at the Qi site. Simulations closely fit both the haem bH data and known semiquinone data only if it is assumed that semiquinone bound to oxidised haem bH is EPR-silent.     

Cytochrome c maturation: a complex pathway for a simple task?

Post-translational maturation of c-type cytochromes involves covalent attachment of haem to the apocytochrome polypeptide by two thioether bonds. In bacteria, haem attachment occurs in the periplasm, after the separate translocation of haem and the polypeptide across the cytoplasmic membrane. In Escherichia coli, delivery and attachment of the cofactor requires eight or nine specific proteins, which are believed to be organized in a membrane protein complex. After transport across the membrane, haem is attached covalently to the haem chaperone CcmE in an unusual way at a single histidine residue. However, haem binding to CcmE is transient and is succeeded by a further transfer to apocytochrome c. Both haem binding to and release from CcmE involve integral membrane proteins, CcmC and CcmF respectively, which carry a conserved tryptophan-rich motif in a periplasmic domain. Apocytochrome c polypeptides are synthesized as precursors and reach the periplasm by sec-dependent translocation. There they are prepared for haem binding by reduction of the cysteine residues in the motif Cys-Xaa-Xaa-Cys-His, which is characteristic of such proteins. This reduction is achieved in a thio-reduction pathway, whereby electrons are passed from cytoplasmic thioredoxin to the transmembrane protein DsbD, across the membrane, and on to the specific reductases CcmG/CcmH. The merging of the haem delivery and the thio-reduction pathways leads to the stereospecific insertion of haem into various type c cytochromes.     

Binding assay and physicochemical characteristics of solubilized intrinsic factor receptor in ileal mucosal homogenates using phenyl-Sepharose to separate the saturated receptor from free intrinsic factor.

A radioisotopic assay was set to determine the physicochemical properties of the solubilized intrinsic factor receptor in pig mucosal extracts. In this assay, phenyl-Sepharose was used to separate the receptor-intrinsic factor-labelled cobalamin complex from the free saturated intrinsic factor. The association constant (at pH 7.4) of the receptor-intrinsic factor complex was estimated at 3.4 +/- 0.3 nM-1. Adsorption of the apo-receptor to phenyl-Sepharose allowed its binding site to be made accessible to intrinsic factor with an association constant in order of 6 nM-1. The receptor binding activity obtained with five mucosal extracts was closely correlated with that obtained by gel filtration of the intrinsic factor-receptor complex (r = 0.99). The radioisotope assay was used to detect the unsaturated receptor (apo-receptor) in sucrose density ultracentrifugation and in superose 6 gel filtration. The sedimentation coefficient was 9.5 s. The apo-receptor was eluted in three peaks in gel filtration, corresponding to the formation of oligomers. The peak of the monomer was increased in presence of EDTA. Its molecular mass was estimated at 270 kDa and its Stokes radius at 5.9 nm. It was concluded that calcium is involved in the oligomerisation of the apo-receptor.