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H A Dailey

Publications and source records attributed to H A Dailey.

At least 73 records · Page 4Linked to original sources
PubMed1987

Reconstitution of the two terminal enzymes of the heme biosynthetic pathway into phospholipid vesicles.

Purified mouse protoporphyrinogen oxidase (EC 1.3.3.4) and ferrochelatase (EC 4.99.1.1), the two terminal enzymes of the heme biosynthetic pathway, have been reconstituted into phospholipid vesicles, and the kinetics of the enzymes in the reconstituted systems were compared with the values obtained with the free enzymes. The apparent Km for free protoporphyrinogen oxidase in detergent solution is 5.61 +/- 0.62 microM for free protoporphyrinogen. The Km was lower when the enzyme was inserted into phospholipid vesicles (0.78 +/- 0.28 microM) and when both enzyme and substrate were incorporated into phospholipid vesicles (0.61 +/- 0.14 microM). In the presence of cardiolipin, a phospholipid present mainly in the inner mitochondrial membrane, the value of the Km for the substrate decreased 3-fold (0.20 +/- 0.02 microM). For reconstituted ferrochelatase similar kinetic analyses were carried out and it was found that the apparent Km values were only weakly affected by the lipid environment. Studies on the orientation of ferrochelatase demonstrated that approximately 50% of the enzyme in the reconstituted system had the active site located in the inner face of the phospholipid vesicle. This is in contrast to intact mitochondria where the active site is located on the matrix side of the inner mitochondrial membrane. The activation energies for both enzymes were determined for free and reconstituted enzymes. It was found that for both enzymes the activation energies were lower for the reconstituted systems than for the free enzymes.

Animals↗
PubMed1987

Metal inhibition of ferrochelatase.

Ferrochelatase activity was examined both in growing MEL cells and in in vitro assays of the purified enzyme to determine what effect a variety of divalent cations would have. Data obtained with the purified enzyme demonstrated that Mn2+ strongly inhibits the activity in a competitive fashion with respect to Fe2+ with a calculated Ki of 15 microM. Cadmium ion is also inhibitory (Ki: 50 microM), as is Hg2+ and arsenite, but Pb2+ is a poor inhibitor. All other metals tested had no effect. When these same metal ions were tested on differentiating MEL cells it was found that their ability to inhibit both heme formation and ferrochelatase activity mimicked their in vitro effect on purified ferrochelatase.

Animals↗
PubMed1986

The role of arginyl residues in porphyrin binding to ferrochelatase.

The role of cationic amino acid residues in the binding of porphyrin substrates by purified bovine ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1) have been examined via chemical modification with camphorquinone-10-sulfonic acid, phenylglyoxal, butanedione, and trinitrobenzene sulfonate. The data obtained show that modification of arginyl, but not lysyl, residues results in the rapid inactivation of ferrochelatase. The 2,4-disulfonate deuteroporphyrin, which is a competitive inhibitor of mammalian ferrochelatase, protects the enzyme against inactivation. Ferrous iron has no protective effect. Reaction with radiolabeled phenylglyoxal shows that modification of 1 arginyl residue causes maximum inhibition of enzyme activity. The inactivation does not follow simple pseudo-first order reaction kinetics, but is distinctly biphasic in nature. Comparison of the enzyme kinetics for modified versus unmodified enzyme show that modification with camphorquinone-10-sulfonic acid has no effect on the Km for iron but does alter the Km for porphyrin.

Amino Acids↗
PubMed1986

Ferrochelatase from Rhodopseudomonas sphaeroides: substrate specificity and role of sulfhydryl and arginyl residues.

Purified ferrochelatase (protoheme ferrolyase; EC 4.99.1.1) from the bacterium Rhodopseudomonas sphaeroides was examined to determine the roles of cationic and sulfhydryl residues in substrate binding. Reaction of the enzyme sulfhydryl residues with N-ethylmaleimide or monobromobimane resulted in a rapid loss of enzyme activity. Ferrous iron, but not porphyrin substrate, had a protective effect against inactivation by these two reagents. Quantitation with 3H-labeled N-ethylmaleimide revealed that inactivation required one to two sulfhydryl groups to be modified. Modification of arginyl residues with either 2,3-butanedione or camphorquinone 10-sulfonate resulted in a loss of ferrochelatase activity. A kinetic analysis of the modified enzyme showed that the Km for ferrous iron was not altered but that the Km for the porphyrin substrate was increased. These data suggested that arginyl residues may be involved in porphyrin binding, possibly via charge pair interactions between the arginyl residue and the anionic porphyrin propionate side chain. Modification of lysyl residues had no effect on enzyme activity. We also examined the ability of bacterial ferrochelatase to use various 2,4-disubstituted porphyrins as substrates. We found that 2,4-bis-acetal- and 2,4-disulfonate deuteroporphyrins were effective substrates for the purified bacterial enzyme and that N-methylprotoporphyrin was an effective inhibitor of the enzyme. Our data for the ferrochelatase of R. sphaeroides are compared with previously published data for the eucaryotic enzyme.

Arginine↗
PubMed1985

Spectroscopic examination of the active site of bovine ferrochelatase.

Spectrofluorometric techniques have been employed to examine the active site of the terminal enzyme of the heme biosynthetic pathway, ferrochelatase (protoheme ferrolyase, EC 4.99.1.1). The fluorescence of both endogenous tryptophan and exogenous 2-(4-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) has been examined. The fluorescence emission of the enzyme's active site bound MIANS is at 428 nm while the enzyme tryptophan(s) yielded a single fluorescence emission maximum at 347 nm. These values are characteristic of a polar environment for tryptophan and a relatively nonpolar environment for the MIANS. The dynamic fluorescence quenching constants for acrylamide of MIANS and tryptophan are 3.00 M-1 and 1.85 M-1, respectively. Quenching constants for KI of both fluorescent centers were approximately 1 M-1. These data suggest that both fluorophores are poorly accessible to the external anionic contact quencher but that an unchanged quencher, while larger, is still better able to penetrate the enzyme's active site. The extrapolated anisotropies (r0) for ferrochelatase-bound MIANS and tryptophan are 0.198 and 0.307. The dissociation constant (KD) determined by fluorescence anisotropy of protoporphyrin was 1.5 microM with the calculated number of porphyrin binding sites as 1.0 per 40000 daltons. A model is presented for the active site of ferrochelatase based upon the data presented here and previously. This model proposes that the active site is a hydrophobic pocket similar in nature to the heme binding crevices found in many hemoproteins.

Anilino Naphthalenesulfonates↗
PubMed1985

Orientation of ferrochelatase in bovine liver mitochondria.

The orientation of ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1), the terminal enzyme of the heme biosynthetic pathway, was examined in bovine liver mitochondria. The ability of a membrane-impermeable sulfhydryl reagent, 4,4'-dimaleimidylstilbene-2,2'-disulfonic acid, to inactivate ferrochelatase in intact or disrupted mitochondria and mitoplasts was examined. Using succinate dehydrogenase as an internal marker, it was found that ferrochelatase was inactivated only in disrupted mitochondria and mitoplasts, suggesting an internal location for the active site of the enzyme. In addition, antibodies raised against purified ferrochelatase were found to inhibit activity only in disrupted but not in intact mitoplasts. These data demonstrate that in bovine liver mitochondria ferrochelatase is located on the matrix side of the inner mitochondrial membrane. Data obtained with the membrane-impermeable amino reagent isethionyl acetimidate indicate that ferrochelatase physically spans the inner mitochondrial membrane with portions of the protein exposed on both sides of the membrane.

Animals↗
PubMed1985

Iron transport and its relation to heme biosynthesis in Rhodopseudomonas sphaeroides.

The uptake of iron supplied as ferric citrate or ferric parabactin was examined in aerobically grown whole cells and vesicles of Rhodopseudomonas sphaeroides. Inner and outer membrane fractions from R. sphaeroides contained no membrane proteins which were inducible by growth in low-iron medium. Vesicles composed of the inner membrane and devoid of outer membrane and periplasmic proteins were able to transport iron supplied as ferric citrate and ferric parabactin. This uptake required the presence of NADH. When the electrical component of the proton motive force was depleted in whole cells, the uptake of iron supplied as ferric parabactin was completely inhibited. The uptake of iron supplied as ferric citrate was inhibited by gallium citrate; however, Ga3+ was not transported. The relationship between iron uptake and heme synthesis was examined by treating whole cells with N-methylprotoporphyrin which inhibits ferrochelatase, the enzyme which inserts ferrous iron into protoporphyrin to form heme. This treatment reduced ferrochelatase activity by 82% but had no effect on iron uptake, indicating that iron uptake and heme synthesis are not directly coupled. The fate of transported iron was investigated by measuring intracellular concentrations of heme and nonheme iron. It was determined that newly transported iron exists primarily as nonheme iron.

Aerobiosis↗
PubMed1985

Ferric iron reductase of Rhodopseudomonas sphaeroides.

Ferric iron reductase activity was examined in the facultative photosynthetic bacterium Rhodopseudomonas sphaeroides. The specific activities of extracts from cells grown under phototrophic and aerobic conditions were similar and not affected by the concentration of iron in the growth media. The activity was resolved by ion-exchange column chromatography into two fractions, designated iron reductase A and iron reductase B, with molecular weights of 41,000 and 32,000, respectively. Both of these soluble cytoplasmic enzymes required the presence of flavin mononucleotide for activity and utilized NADH to reduce iron supplied as ferric citrate. Iron reductase B was responsible for the majority of activity in crude extracts and was purified 556-fold by conventional protein purification techniques. The apparent Km values of iron reductase B for NADH, Fe3+, and flavin mononucleotide were determined to be 18.2, 8.3, and 3.2 microM, respectively.

FMN Reductase↗
PubMed1984

Differential interaction of porphyrins used in photoradiation therapy with ferrochelatase.

The mechanism of porphyrin accumulation by tumours is not yet established. If metabolism aids porphyrin elimination, tumours, unlike normal tissues, may not metabolize porphyrins used clinically, such as proto-, haemato-, OO'-diacetyl-haemato- and monohydroxyethyl-monovinyl-deutero-porphyrin. Proto-, haemato- and monohydroxyethyl-monovinyl-deutero-porphyrin are substrates for the mitochondrial enzyme ferrochelatase (EC 4.99.1.1), which can form haem analogues from exogenous porphyrins. The Km values for proto-, haemato- and monohydroxyethyl-monovinyl-deutero-porphyrin are 11, 22 and 23 microM respectively. However, OO'-diacetyl-haematoporphyrin is an effective competitive inhibitor with Ki of 11 microM. Hepatic ferrochelatase specific activity is 5.9 and 5.5 nmol of haem/h per mg of protein respectively in normal Buffalo rat and in those bearing the extrahepatic Morris 7288C hepatoma, and is only 0.13 nmol/h per mg in the hepatomas. Therefore low ferrochelatase activity in cancerous cells may provide one means whereby some porphyrins accumulate in tumours, and the ability of certain porphyrins to act as ferrochelatase inhibitors may provide another.

Animals↗
PubMed1984

Purification and characterization of chicken erythrocyte ferrochelatase.

Ferrochelatase (EC 4.99.1.1) was purified 2000-fold to apparent homogeneity from isolated chicken erythrocyte mitochondria. The purified enzyme yields a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with an apparent Mr of 42 000. The enzyme utilizes proto-, meso- and deutero-porphyrin with Km values of 37, 51 and 80 microM respectively. The disubstituted porphyrins 2,4-bisglycol deutero-porphyrin and 2,4-disulphonic deuteroporphyrin were not substrates. Mn2+, Hg2+, Pb2+ and Co2+ were strong inhibitors of the purified enzyme. Palmitic acid and oleic acid stimulated activity, whereas linoleic acid inhibited and phospholipids had variable effects. Chicken ferrochelatase was inhibited by N-ethylmaleimide and iodoacetamide. Inhibition by iodoacetamide was pseudo-first-order, but inhibition by N-ethylmaleimide appeared to be biphasic in nature with an initial high rate followed by a much lower rate of inactivation. The characteristics of the chicken erythrocyte enzyme are compared with those previously reported for mammalian liver ferrochelatase.

Animals↗
PubMed1984

Effect of sulfhydryl group modification on the activity of bovine ferrochelatase.

The role of sulfhydryl groups in the activity of the terminal enzyme of the heme biosynthetic pathway, ferrochelatase (protoheme ferrolyase, EC 4.99.1.1), has been examined by using a variety of sulfhydryl group-specific reagents. The enzyme is rapidly inactivated in a pseudo-first order reaction by N-ethylmaleimide and monobromobimane and more slowly by iodoacetamide and bromotrimethylammoniobimane. Reaction with [3H]N-ethylmaleimide indicates that modification of a single sulfhydryl group is sufficient to inactivate bovine ferrochelatase. The enzyme is protected from inactivation by one substrate, ferrous iron, but not by the porphyrin substrate. Mercury and arsenite are reversible inhibitors. The fluorescence of the bound bimane is blue shifted 8 nm from that obtained in aqueous solutions and is sensitive to quenching by iodide.

Animals↗
PubMed1984

Siderophore utilization and iron uptake by Rhodopseudomonas sphaeroides.

The growth of Rhodopseudomonas sphaeroides in iron-deficient medium did not result in the production of detectable levels of siderophores of either the catechol or hydroxamate type. Iron-limited cultures of R. sphaeroides were not able to remove iron from ferric transferrin unless supplemented with 2,3-dihydroxybenzoic acid. R. sphaeroides was shown to take up 59Fe+3 when it was supplied as ferric chloride, ferric citrate, or ferric parabactin, but not when supplied as ferric rhodotorulate or ferric Desferal. When iron was supplied as ferric citrate, citrate was not taken up by the cells. The growth rate of R. sphaeroides under iron-limiting conditions was decreased by the addition of either Desferal or rhodotorulic acid, while the addition of citrate or parabactin did not affect growth.

Biological Transport↗
PubMed1983

Bovine ferrochelatase. Kinetic analysis of inhibition by N-methylprotoporphyrin, manganese, and heme.

The terminal enzyme of the heme biosynthetic pathway, ferrochelatase (protoheme ferrolyase EC 4.99.1.1), has been purified to apparent homogeneity from bovine liver mitochondria using a scheme similar to that reported by Taketani and Tokunaga (Taketani, S. and Tokunaga, R. (1981) J. Biol. Chem. 256, 12748-12753) for purification of the enzyme from rat liver. The final yield was 49% with a 2000-fold purification. Ferrochelatase has an apparent molecular weight of approximately 40,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and column chromatography on Sepharose CL-6B in the presence of 0.5% sodium cholate. The purified enzyme was only slightly stimulated by added lipid and was inhibited by Mn2+, Pb2+, and Hg2+. Bovine ferrochelatase utilized proto-, meso-, and deuteroporphyrin, but not disubstituted porphyrins (2,4-disulfonic and 2,4-bisglycol deuteroporphyrin). N-Methylprotoporphyrin, a toxic by-product of the metabolism of some drugs, was found to inhibit ferrochelatase in a competitive fashion with respect to porphyrin with a Ki of 7 nM and uncompetitive with respect to iron. Manganese inhibits ferrochelatase competitively with respect to iron (Ki = 15 microM) and noncompetitively with respect to the porphyrin substrate. Heme, one of the products, is a noncompetitive inhibitor with respect to iron. These findings lead to a sequential Bi Bi kinetic model for ferrochelatase with iron binding occurring prior to porphyrin binding and heme being released prior to the release of two protons.

Animals↗
PubMed1983

Aerobic ferrisiderophore reductase assay and activity stain for native polyacrylamide gels.

The reduction of ferric iron from microbial iron-binding compounds (siderophores) releases the iron from the siderophore so that it may be utilized by the microorganism. A method to detect aerobic ferrisiderophore reductase activity using ferrozine as a ferrous iron trap is shown to be applicable to cytoplasmic fractions from Rhodopseudomonas sphaeroides and four other different species of bacteria. The ferrisiderophore reductase uses reduced nicotinamide cofactors as reducing agents, and activity is stimulated by flavins. This assay has been adapted as a staining method to locate ferrisiderophore reductase activity in native polyacrylamide gels.

Aerobiosis↗
PubMed1982

Purification and characterization of membrane-bound ferrochelatase from Rhodopseudomonas sphaeroides.

Ferrochelatase (protohaem ferro-lyase EC 4.99.1.1) has been purified to apparent homogeneity from the facultative photosynthetic bacterium Rhodopseudomonas sphaeroides. The enzyme has been purified 1,640-fold with 43% recovery from isolated membrane fragments. The enzyme has a molecular weight of approximately 115,000 as estimated by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography through Sephadex G-150 in the presence of 0.5% sodium deoxycholate. The purification procedure involves solubilization of ferrochelatase with sodium deoxycholate off of salt-washed membranes, followed by ammonium sulfate fraction, ion exchange chromatography on DEAE-Sephacel, followed by chromatography on Amicon dye matrix blue B, and finally Sephadex G-150. The enzyme has an extinction coefficient of 90,000 at 278 nm, and the absorption spectrum reveals no chromophoric cofactors. Purified ferrochelatase is inhibited by iodoacetamide, N-ethylmaleimide, Hg, Pb, Cu, and hemin. The apparent Km values are for mesoporphyrin IX, 20 microM; deuteroporphyrin IX, 95 microM; and iron, 20 microM.

Cations↗
PubMed1981

Orientation of the carboxyl and NH2 termini of the membrane-binding segment of cytochrome b5 on the same side of phospholipid bilayers.

The present data show that the carboxyl terminal end of the membrane binding segment (nonpolar peptide) of cytochrome b5 is present on the same side of phospholipid bilayers as the hydrophilic, heme-containing, NH2-terminal segment. This orientation was determined by observing rapid ionization of both tyrosyl residues at positions 5 and 8 from the carboxyl terminus upon addition of sodium hydroxide to the outer aqueous phase of vesicle preparations, and the reaction of one of these residues with a polar, impermeant reagent, diazotized sulfanilic acid. The rate of ionization of both aromatic residues occurred at least 1 order of magnitude faster than ionization of indigo trisulfonate trapped in the inner aqueous compartment of the vesicles. These data and consideration of our earlier characterization of cytochrome b5 structure and binding to membranes support a model for the membrane binding segment that is highly structured, penetrates to the middle of the bilayer, and loops back to the outer surface to place both the NH2 and the carboxyl termini on the same surface of the bilayer.

Amino Acid Sequence↗