Donald E. Hultquist

Soluble cytochrome b5 reductase from human erythrocytes

SUMMARY I. An enzyme that catalyzes the reduction of erythrocyte cytochrome b 5 has been isolated from the supernatant fraction of erythrocyte hemolysates by chromatography on DEAE-cellulose, Amberlite CG-5 o, and Bio-Gel P-6o. 2. Erythrocyte cytochrome b 5 reductase has been shown to contain FAD. Incubation of the reductase in the absence of EDTA results in both the appearance of flavin fluorescence and the loss of enzymatic activity with time. 3. The reductase catalyzes the reduction of erythrocyte cytochrome b 5 5o times faster with NADH than with NADPH. The apparent Km of NADH was calculated to be 1.6. IO -~ M and the turnover number is 128o moles of cytochrome b 5 per min per mole of flavin. The reduction of electron acceptors proceeded in the following decreasing order of rate: K~Fe(CN)6, 2,6-dichlorophenolindophenol (DCIP), cytochrome b 5, methylene blue, cytochrome c, 03, oxidized glutathione, and methemoglobin. 4- The FAD prosthetic group, the substrate specificity, and the effect of ionic strength, pH, and EDTA on activity suggest that the reductase is the same enzyme as NADH dehydrogenase I, the enzyme lacking in many cases of congenital methemoglobinemia. The properties of the reductase, including its molecular weight, are very similar to those of the cytochrome b 5 reductases solubilized from microsomes and mitochondria of other tissues.

The preparation and characterization of phosphorylated derivatives of histidine

Abstract 1. Phosphorylated derivatives of histidine were synthesized and studied in order to provide a better understanding of the chemistry of phosphohistidine-containing proteins. 1-Phosphohistidine, 1,3-diphosphohistidine, α, 1,3-triphosphohistidine, and phosphorylated derivatives of α-N-acetylhistidine were prepared by reactions with POCl3 or phosphoramidate. The compounds were purified by ion-exchange chromatography and characterized by the phosphate: histidine ratio, electrophoretic behavior, and ultraviolet spectra. 2. The rates of hydrolysis of these compounds, as well as 3-phosphohistidine, phosphoimidazole, and 1,3-diphosphoimidazole, were determined over a wide range of H+ concentrations. In strongly acidic solution all of the compounds appeared to be hydrolyzed by the same mechanism. In all cases the N-phosphoryl bond was hydrolyzed more readily if a proton or phosphoryl group were substituted on the other ring nitrogen, and histidine derivatives were less stable than the corresponding imidazole derivatives. 3. For both mono- and diphosphoryl derivatives of histidine, the N-1 phosphoryl group was hydrolyzed and transferred to other imidazole compounds more readily than the N-3 phosphoryl group. The greater reactivity at the N-1 position is partially due to the presence of the protonated α-amino group. 4. In aqueous solution, 1-phosphohistidine is rapidly converted to 3-phosphohistidine, histidine and inorganic phosphate.

Soluble cytochrome b5 from human erythrocytes

Abstract 1. A hemeprotein with properties similar to microsomal cytochrome b5 has been detected in the supernatant fraction of hemolysates of human, beef, and rabbit erythrocytes. A method has been developed for determining the amount of this soluble cytochrome in small volumes of blood. The amount of the protein decreases during cell storage at 4 °C. Blood cells rich in reticulocytes contain more of the protein than do mature cells. 2. The cytochrome has been purified from human erythrocytes by a procedure which employs chromatography on Amberlite CG-50 and DETE-cellulose, ultrafiltration, and gel filtration. The purified protein sedimented in the ultracentrifuge as a single peak with an s20, w of 1.40. However, minor impurities were detected by polyacrylamide disc electrophoresis. 3. The molecular weight of the purified protein has been calculated to be 14600 from sedimentation and diffusion measurements and 18400 as determined by gel filtration. The prosthetic group has been identified as protoheme IX. The spectral properties of the hemeprotein are those of a low spin heme complex. The EPR spectrum of the oxidized form shows g values of 3.03, 2.21, and 1.39 and the visible spectrum has a Soret absorbance maximum at 413 nm. The protein is reducible by dithionite or NADH plus cytochrome b5 reductase and the reduced form shows absorbance maxima at 423, 527, and 556 nm with a shoulder at 560 nm. 4. The cytochrome b5 differs from the other B-type cytochrome of erythrocyte, S-protein (hemeprotein 559), and is not derived from this protein. The erythrocyte cytochrome b5 is similar to the cytochrome b5 solubilized from liver microsomes in terms of spectral properties, molecular weight, prosthetic group, and reactivity.

Protection by vitamin B2 against oxidant-mediated acute lung injury.

The effect of vitamin B2 (riboflavin) on oxidant-mediated acute lung injury has been examined in three different rat models. Pulmonary injury was induced by intravenous injection of cobra venom factor (CVF), by the intrapulmonary deposition of IgG immune complexes, or by hind limb ischemia-reperfusion. In each of the three models, injury was characterized by increases in vascular permeability (leakage of 125I-labeled bovine serum albumin), alveolar hemorrhage (extravasation of 51Cr-labeled rat erythrocytes), and neutrophil accumulation (myeloperoxidase activity). Intraperitoneal administration of riboflavin at a dose of 6 μmoles/kg body weight reduced vascular leakage by 56% in the CVF model, by 31% in the immune complex model, and by 53% in the lung injury model following ischemia-reperfusion of the hind limbs. Similar treatment reduced hemorrhage by 76%, 51%, and 70% in the three models of lung injury. In the CVF model, riboflavin was also shown to decrease products of lipid peroxidation (conjugated dienes) in lungs (by 45%) and in plasma (by 74%). Neutrophil accumulation in the lungs was not influenced by riboflavin administration in any of the three models. The studies demonstrate that riboflavin can mount a significant protection against oxidant-mediated inflammatory organ injury.

Catalysis of Methemoglobin Reduction

Publisher Summary This chapter focuses on the catalysis of methemoglobin reduction. It discusses the results of studies in which photometry and magnetic circular dichroism (MCD) spectroscopy were used to follow the kinetics of methemoglobin reduction. The chapter discusses an experimental study in which methemoglobin reduction in hemolysates was assayed. The hemolysate differs from conditions within the normal erythrocytes in that nitrite was added to convert hemoglobin to methemoglobin. The components of the cytoplasm were diluted by a factor of 4.8, the membranous fraction was removed, and NADH or NADPH was added to a concentration of 4 m M . In this system, the dependence of rate on the concentrations of the various components was studied by addition to the hemolysate of purified human methemoglobin, human erythrocyte cytochrome b 5 reductase, or bovine erythrocyte cytochrome b 5 . Direct reduction of methemoglobin by the addition of isolated ferrocytochrome b 5 was measured photometrically. The reaction was carried out at 37°C in a cuvette, which was already positioned in an Aminco recording spectrophotometer. Photochemical reduction mediated by lumiflavin 3-acetate was employed to reductively titrate mixtures of cytochrome b 5 and methemoglobin. MCD spectra were obtained with a Jasco J-40C spectropolarimeter equipped with an electromagnet. Spectra were acquired, stored, corrected, and analyzed by using a Nova III computer, a Tracor 1500N signal analyzer, an Ohio Scientific Challenger II microcomputer, and an ADM 3a terminal retrofitted with Retro-Graphics plotting hardware.

Riboflavin Reduces Edema in Focal Cerebral Ischemia

Oxidized irwN has been proposed as a mediator of the free radical-induced damage that occurs during cerebral ischemia. Dihydroriboflavin, a compound produced from riboflavin (B2) by NADPH-dependent flavin reductase, rapidly reduces oxidized iron. Since treatment with riboflavin offers protection from ischemic injury in other tissues, we tested the effect of pretreatment with B2 on brain edema formation during focal ischemia. Two different models of middle cerebral artery occlusion (MCAO) in rats were tested: transcranial electrocautery and intracarotid occlusion with a nylon thread. Groups of 6-8 animals were treated with 7.5 mg of B2/kg or saline vehicle 1 h before MCAO and brain water content was determined after 4 h of ischemia. Pretreatment with B2 reduced total hemisphere edema formation from 0.37 +/- 0.05 to 0.19 +/- 0.05 mg/g dry wt. (48% protection, p < 0.01) following transcranial MCAO. Edema was greater following MCAO with the intra-carotid thread (0.54 +/- 0.05 ml/g) but protection by B2 was less (21%). We conclude that pretreatment with B2 reduces ischemic brain injury, perhaps by reacting with oxidized iron. However, the larger stroke produced by the thread MCAO method makes it more difficult to observe protection following brief ischemia in this model.

Effects of hemolysate concentration, ionic strength and cytochrome b5 concentration on the rate of methemoglobin reduction in hemolysates of human erythrocytes

An assay for determining the rate of methemoglobin reduction in hemolysates of human erythrocytes has been developed. The rates obtained by this assay, when corrected for dilution, are comparable to those obtained with intact cells. Increased ionic strength inhibits the reaction, whereas EDTA increases the rate of reduction. The rate with NADPH as electron donor is 65-70% of the rate with NADH. Added cytochrome b5 stimulates the reaction. The assay has been used to examine erythrocytes from two methemoglobinemic sisters and their asymptomatic mother. Hemolysates of the two patients have both decreased dichlorophenolindophenol reductase activity and decreased ability to reduce methemoglobin. Hemolysates from the heterozygous mother have intermediate dichlorophenolindophenol reductase activity and intermediate methemoglobin reduction ability. The data presented in this paper indicate that the concentrations of cytochrome b5 and cytochrome b5 reductase determine the rate of methemoglobin reduction in hemolysates.

Homogeneous cytochrome b5 from human erythrocytes.

Abstract Homogeneous cytochrome b 5 has been isolated from large volumes of human erythrocytes by sequential chromatography on DEAE-cellulose, Amberlite CG-50, Bio-Gel P-60, and DEAE-Sephadex A-50. A molecular weight of 15,300 was determined by SDS disc gel electrophoresis. Trypsin converted the protein to a smaller hemepeptide which was indistinguishable from trypsin-cytochrome b 5 of human liver microsomes by disc gel electrophoresis. The data suggest that erythrocyte cytochrome b 5 has the same structure as a segment of liver microsomal cytochrome b 5 and is intermediate in size between the trypsin- and detergent-solubilized forms of the liver protein.

Isolation of an acid protease from rabbit reticulocytes and evidence for its role in processing redox proteins during erythroid maturation

A protease which generates a soluble hemepeptide from bovine liver microsomal cytochrome b5 has been isolated from the membrane fraction of rabbit reticulocytes. Inhibition by pepstatin and an acidic pH optimum indicate that the protease belongs to the acid protease class. Little cytochrome b5-processing activity is observed in rabbit erythrocytes. We suggest that the protease may be involved in the processing which generates the proteins of the methemoglobin reduction system from their membrane-bound precursors during the maturation of the erythroid cell.

Demonstration that bovine erythrocyte cytochrome b5 is the hydrophilic segment of liver microsomal cytochrome b5

Abstract A structural comparison has been made between bovine erythrocyte cytochrome b5 and solubilized forms of bovine hepatic microsomal cytochrome b5. Two soluble forms of microsomal cytochrome b5 (designated Forms A and B) were generated by digestion of microsomes with a crude hepatic lysosomal cathepsin preparation and purified by successive chromatography on DEAE-cellulose,Bio-Gel P-60 and DEAE-Sephadex A-50.Amino acid analyses and terminal residue analyses identified Form A as the segment corresponding to residues 1∗95 of the native microsomal protein and Form B as the segment corresponding to residues 1∗107. Erythrocyte cytochrome b5 I was shown to be a protein which corresponds to a segment of the hepatic microsomal molecule containing residues 1∗97, whereas erythrocyte cytochrome b5 II is a protein corresponding to residues 1∗95. Like the native microsomal cytochrome and the cathepsin-solubilized forms of the cytochrome, no amino terminal residue could be detected in the erythrocyte cytochrome.Carboxypeptidases A and B released from erythrocyte Form I a residue eluting at the position of serine, but released no residue from Form II. The results are consistent with serine being the residue at position 97 of the native microsomal protein, and proline and serine being the residues in positions 94 and 95, respectively. The maps of the tryptic peptides derived from the apoprotein forms of erythrocyte cytochrome b5 I and II and cathepsin-solubilized microsomal Forms A and B were very similar, with eight of the expected twelve peptides displaying the same mobility on every map. Amino acid analyses of the isolated tryptic peptides from erythrocyte Form I and hepatic Form B confirmed the structural assignments of these proteins. These data demonstrate that the soluble forms of erythrocyte cytochrome b5 correspond to hydrophilic segments of the native membrane-bound microsomal cytochrome b5 and suggest that the hepatic lysosomal proteases serve as a good model for the putative erythroid proteases which solubilize microsomal cytochrome b5 during erythroid maturation.