Age-dependent decay of cytochrome b5 and cytochrome b5 reductase in human erythrocytes.

Age-dependent decrease in cytochrome b5 was observed in erythrocytes from both a normal person and a patient with hereditary methaemoglobinaemia without neurological symptoms. With aging, concentrations of cytochrome b5 in erythrocytes from the patient were almost the same as those in the control. Age-dependent decrease in cytochrome b5 reductase activity in the control erythrocytes was also shown; however, the reductase activity was very low in erythrocytes from the patient over the whole age range. Our studies show that methaemoglobin content of erythrocytes seems to be dependent on the content of cytochrome b5 in the cells, both in the control subject and in the patient.     

Interaction between cytochrome c and cytochrome b5.

The reduction of cytochrome c by cytochrome b5 was studied over a wide range of ionic strengths in four different buffer systems. The reaction rate decreased linearly as the I1/2 was increased, suggesting that electrostatic interactions are important in the interaction. The ionic strength dependence of the reaction rate was in quantitative agreement with the theory of Wherland & Gray [Wherland, S., & Gray, H.B. (1976) Proc. Natl. Acad. Sci U.S.A. 73, 2950] only if the effective radius of the interaction was 2 A. This indicates that the interaction between the two proteins is best described as the sum of n complementary charge interactions, each involving a specific lysine on cytochrome c and a specific carboxyl group on cytochrome b5. The number of complementary charge interactions, n, was calculated to be five to seven, in agreement with the results of our specific modification studies. Ultracentrifugation and gel permeation techniques were used to demonstrate that cytochrome b5 and cytochrome c formed a stable complex at low ionic strength.     

Modification of trypsin-solubilized cytochrome b5, apocytochrome b5, and liposome-bound cytochrome b5 by diethylpyrocarbonate

The interactions of diethylpyrocarbonate (DEP) with the various forms of cytochrome b5 were studied to gain a better understanding of the factors that influence the extent of modification of the axial histidines of cytochrome b5. Very low concentrations of DEP were able to decrease the heme binding capacity of apocytochrome b5. Moreover, it was shown that two additional histidines, presumed to be the axial ligands (His 39 and 63), were modified in the apo but not the holo form of a given preparation of cytochrome b5. Trypsin-solubilized bovine cytochrome b5 was resistant to the effects of DEP. A 200-fold molar excess of DEP displaced only 15% of the heme in the trypsin-solubilized protein in contrast to an 84% displacement of the heme in the detergent-solubilized protein. However, detergent-solubilized cytochrome b5 which had been incorporated into phospholipid vesicles exhibited the same reactivity with DEP as did the trypsin-solubilized protein. This is attributed to the fact that the two resistant preparations of cytochrome b5 are monomeric in their respective environments while detergent-solubilized cytochrome b5 is known to exist as an octamer in aqueous solutions. Our studies suggest that dissociation of the octamer to the monomer results in a conformational change that decreases the reactivity of the axial ligands of the hydrophilic heme-containing domain of cytochrome b5. Examination of the cytochrome b5 molecule by computer graphics indicates that a tunnel leads from the surface of the molecule to axial histidine 63 and that axial histidine 39 is buried.     

Fluorescence studies of cytochrome b5 topography. Incorporation of cytochrome b5 into brominated phosphatidylcholine vesicles by deoxycholate

Cytochrome b5 was incorporated into large vesicles of 1-palmitoyl-2-dibromostearoylphosphatidylcholine by mixing lipid, protein, and deoxycholate followed by removal of the detergent by gel filtration. The tryptophan fluorescence emanating from the hydrophobic membrane-binding domain was quenched more effectively when the bromine atoms were in the 6,7-positions than when they were in the 15,16-positions of the acyl chain. To more precisely define the position of the quenchable tryptophan, the experiment was repeated with lipids with the bromine atoms at the 4,5-, 6,7- or 9,10-positions. Again the 6,7 species was the most efficient quencher. The cytochrome b5 bound to these vesicles would not transfer to small unilamellar sonicated vesicles and so was in the "tight" configuration. If the cytochrome were added to the vesicles after the detergent was removed, the same order of quenching was seen but the cytochrome would transfer to other vesicles. These data indicate that the quenching of the tryptophan fluorescence is greatest when the bromines are at the 6,7-positions whether the vesicles are large or small and whether the cytochrome is in the tight or "loose" configuration and so place the tryptophan 0.7 nm below the vesicle surface in all of these membranes.     

Purification and properties of plant cytochrome b5.

Microsomal cytochrome b5 was 352-fold purified from potato tubers with a yield of 10.4%. To our knowledge, this is the first report relating the purification of higher-plant cytochrome b5. Its Mr (16 700) and absorption spectrum are similar to those of animal and yeast cytochrome b5.     

Interaction of cytochrome b5 with surfactant vesicles.

Lysates of protoplasts from the endosperm of developing grains of wheat (Triticum aestivum) were fractionated on density gradients of Nycodenz to give amyloplasts. Enzyme distribution on the gradients suggested that: (i) starch synthase and ADP-glucose pyrophosphorylase are confined to the amyloplasts; (ii) pyrophosphate: fructose-6-phosphate 1-phosphotransferase and UDP-glucose pyrophosphorylase are confined to the cytosol; (iii) a significant proportion (23-45%) of each glycolytic enzyme, from phosphoglucomutase to pyruvate kinase inclusive, is in the amyloplast. Starch synthase, ADP-glucose pyrophosphorylase and each of the glycolytic enzymes showed appreciable latency when assayed in unfractionated lysates of protoplasts. No activity of fructose-1,6-bisphosphatase was found in amyloplasts or in homogenates of endosperm. Antibody to plastidic fructose-1,6-bisphosphatase did not react positively, in an immunoblot analysis, with any protein in extracts of wheat endosperm. It is argued that wheat endosperm lacks significant plastidic fructose-1,6-bisphosphatase and that carbon for starch synthesis does not enter the amyloplast as a C-3 compound but probably as hexose phosphate.     

Simultaneous purification and characterization of cytochrome b5 reductase and cytochrome b5 from sheep liver

Cytochrome b5 was purified from detergent solubilized sheep liver microsomes by using three successive DEAE-cellulose, and Sephadex G-100 column chromatographies. It was purified 54-fold and the yield was 23.5% with respect to microsomes. The apparent Mr of cytochrome b5 was estimated to be 16,200 +/- 500 by SDS-PAGE. Absolute absorption spectrum of the purified cytochrome b5 showed maximal absorption at 412 nm and dithionite-reduced cytochrome b5 gave peaks at 557, 526.5 and 423 nm. The ability of the purified sheep liver cytochrome b5 to transfer electrons from NADH-cytochrome b5 reductase to cytochrome c was investigated. The K(m) and Vmax values were calculated to be 0.088 microM cytochrome b5 and 315.8 microM cytochrome c reduced/min/mg enzyme, respectively. Also the reduction of cytochrome b5 by reductase was studied and K(m) and Vmax values were determined to be 5 microM cytochrome b5 and 5200 nmol cytochrome b5 reduced/min/mg enzyme, respectively. The K(m) and Vmax values for the cofactor NADH in the presence of saturating concentration of cytochrome b5 were found to be 0.0017 mM NADH and 6944 nmol cytochrome b5 reduced/min/mg enzyme, respectively. NADH-cytochrome b5 reductase was also partially purified from the same source, detergent solubilized sheep liver microsomes, by using two successive DEAE-cellulose, and 5'-ADP-agarose affinity column chromatographies. It was purified 144-fold and the yield was 7% with respect to microsomes. The apparent monomer Mr of reductase was estimated to be 34,000 by SDS-PAGE. When ferricyanide was used as an electron acceptor, reductase showed maximum activity between 6.8 and 7.5. The K(m) and Vmax values of the enzyme for ferricyanide were calculated as 0.024 mM ferricyanide and 673 mumol ferricyanide reduced/min/mg enzyme, respectively. The K(m) and Vmax values for the cofactor NADH in the presence of saturating amounts of ferricyanide were found to be 0.020 mM NADH and 699 mumol ferricyanide reduced/min/mg enzyme, respectively.     

The opposite effect of bivalent cations on cytochrome b5 reduction by NADH:cytochrome b5 reductase and NADPH:cytochrome c reductase.

The effects of bivalent cations on cytochrome b5 reduction by NADH:cytochrome b5 reductase and NADPH:cytochrome c reductase were studied with the proteinase-solubilized enzymes. Cytochrome b5 reduction by NADH:cytochrome b5 reductase was strongly inhibited by CaCl2 or MgCl2. When 1.2 microM-cytochrome b5 was used, the concentrations of CaCl2 and MgCl2 required for 50% inhibition (I50) were 8 and 18 mM respectively. The inhibition was competitive with respect to cytochrome b5. The extent of inhibition by CaCl2 or MgCl2 was much higher than that by KCl or other alkali halides. In contrast, cytochrome b5 reduction by NADPH:cytochrome c reductase was extremely activated by CaCl2 or MgCl2. In the presence of 5 mM-CaCl2, the activity was 24-fold higher than control when 4.4 microM-cytochrome b5 was used. The magnitude of activation by CaCl2 was 2-3-fold higher than that by MgCl2. The activation by these salts was much higher than that by KCl, indicating that bivalent cations play an important role in this activation. The mechanisms of inhibition and activation by bivalent cations of cytochrome b5 reduction by these two microsomal reductases are discussed.     

Thermodynamic investigations of cytochrome b5 unfolding. II. Detergent-solubilized cytochrome b5 in solution and in a reconstituted system with dimyristoyl phosphatidylcholine

Thermal unfolding of the detergent-solubilized cytochrome b5 was investigated by scanning calorimetry. The protein shows different thermostability in the presence and absence of detergent, and it achieves the maximal transition temperature after incorporation into dimyristoyl phosphatidylcholine liposomes. However, transition temperature and Gibbs energy change at unfolding are still lower than that of the tryptic fragment of cytochrome b5 in aqueous solution. Cytochrome b5 undergoes in aqueous solution in the absence of detergent an irreversible, complicated transition, but it remains in the associated state after thermal denaturation. Half transition temperature, enthalpy and heat capacity changes of cytochrome b5 unfolding under various external conditions are reported and compared with the corresponding values of the tryptic fragment of the protein. The thermodynamic data and independent results are suitable for detailing a model proposed by Tanford (The Hydrophobic Effect (1980), pp. 205-211, John Wiley & Sons, New York) for the spatial arrangement of the protein within the membrane.     

Redox properties of microsomal reduced nicotinamide adenine dinucleotide-cytochrome b5 reductase and cytochrome b5.

Hepatic NADH-cytochrome b5 reductase was reduced by 1 mol of dithionite or NADH per mol of enzyme-bound FAD, without forming a stable semiquinone or intermediate during the titrations. However, the addition of NAD+ to the partially reduced enzyme or illumination in the presence of both NAD+ and EDTA yielded a new intermediate. The intermediate had an absorption band at 375 nm and the optical spectrum resembled anionic semiquinones seen on reduction of other flavin enzymes. Electron paramagnetic resonance measurements confirmed the free-radical nature of the species. To explain the results, a disproportionation reaction between the oxidized and reduced NAD+ complexes (E-FAD-NAD+ + E-FADH2-NAD+ in equilibrium 2E-FADH.-NAD+) is assumed. Potentiometric titration of NADH-cytochrome b5 reductase at pH 7.0 with dithionite gave a midpoint potential of -258 mV; titration with NADH gave -160 mV. This difference may be due to a difference in the relative affinity of NAD+ for the reduced and oxidized forms of the enzyme. The effects of pH on the midpoint potential of the NAD+-free enzyme were very similar to those which have been measured with free FAD. At pH 7.0, midpoint potentials of trypsin-solubilized and detergent-solubilized cytochrome b5 were 13 and 0 mV, respectively.     

The subcellular localization of newly synthesized cytochrome b5.

The fate of newly synthesized cytochrome b₅ was studied in rat hepatocytes. Using an antibody specific for microsomal cytochrome b₅, we found newly synthesized microsomal cytochrome b₅ in both mitochondria and a mitochondria associated membrane fraction as well as in microsomes. Newly synthesized cytochrome b₅ was quickly removed from the site of synthesis on free ribosomes and inserted into membranes at random. No migration of newly synthesized cytochrome b₅ between cellular compartments was observed and therefore the assembly of the apoprotein with the heme moiety is apparently not taking place in any particular cellular compartment.     

Interaction of cytochrome P-450scc with cytochrome b5

Spectrophotometric, affinity chromatography and cross-linking experiments provided evidence that cytochrome P-450scc from bovine adrenocortical mitochondria forms a tight complex with cytochrome b5 from rabbit liver microsomes. In the reconstituted system cholesterol side chain activity of cytochrome P-450scc was enhanced by the addition of cytochrome b5.     

Studies on methemoglobin reductase. Immunochemical similarity of soluble methemoglobin reductase and cytochrome b5 of human erythrocytes with NADH-cytochrome b5 reductase and cytochrome b5 of rat liver microsomes.

An antibody preparation elicited against purified, lysosomal-solubilized NADH-cytochrome b₅ reductase from rat liver microsomes was shown to interact with methemoglobin reductase of human erythrocytes by inhibiting the rate of erythrocyte cytochrome b₅ reduction by NADH. The ferricyanide reductase activity of the enzyme was not inhibited by the antibody, suggesting that the inhibition of methemoglobin reductase activity may be due to interference with the binding of cytochrorme b₅ to the flavoprotein. Under conditions of limiting concentrations of flavoprotein, the antibody inhibited the rate of methemoglobin reduction in a reconstituted system consisting of homogeneous methemoglobin reductase and cytochrome b₅ from human erythrocytes. This inhibition was due to the decreased level of reduced cytochrome b₅ during the steady state of methemoglobin reduction while the rate of methemoglobin reduction per reduced cytochrome b₅ stayed constant, suggesting that the enzyme was not concerned with an electron transport between the reduced cytochrome b₅ and methemoglobin.An antibody to purified, trypsin-solubilized cytochrome b₅ from rat liver microsomes was shown to inhibit erythrocyte cytochrome b₅ reduction by methemoglobin reductase and NADH to a lesser extent than microsomal cytochrome b₅ preparations from rat liver (trypsin solubilized or detergent solubilized) and pig liver (trypsin solubilized). The results presented establish that soluble methemoglobin reductase and cytochrome b₅ of human erythrocytes are immunochemically similar to NADH-cytochrome b₅ reductase and cytochrome b₅ of liver microsomes, respectively.     

Cytochrome b5/cytochrome b5 reductase interaction in microsomes: kinetics at subzero temperature.

The cytochrome $\text{b}{}_5\text{b}{}_5$ reductase system solubilized from microsomes exhibits monophasic reduction kinetics over the temperature range 15 ° to ?25 °C in aqueous/ethylene glycol co-solvent, whereas in intact microsomes, the process becomes increasingly heterogeneous below 0 °C, reflecting heterogeneities in membrane structure observable as distributions in reaction rates and activation energies.