Rosalia B. Frydman

Heme oxygenase induction by CoCl2, Co-protoporphyrin IX, phenylhydrazine, and diamide: Evidence for oxidative stress involvement

The induction of heme oxygenase in rat liver by cobaltous chloride (CoCl2) and Co-protoporphyrin IX is entirely prevented by the administration of alpha-tocopherol and allopurinol. CoCl2 was converted in the liver into Co-protoporphyrin IX before it induced heme oxygenase activity. Actinomycin and cycloheximide affected to a similar degree the induction of heme oxygenase by both CoCl2 and Co-protoporphyrin IX. Administration of either CoCl2 or Co-protoporphyrin strongly decreased the intrahepatic GSH pool, a decrease which was completely prevented by the administration of either alpha-tocopherol or allopurinol. The latter compounds prevented heme oxygenase induction as well as the decrease in hepatic GSH when administered 2 h before, together with, or 2 h after CoCl2. However, when given 5 h after administration of CoCl2, alpha-tocopherol and allopurinol showed no preventive effect. Similar results were obtained when Co-protoporphyrin IX was used, with the difference that when alpha-tocopherol and allopurinol were given 2 h after administration of the inducer, they showed no protective effect. Phenylhydrazine and diamide also induced heme oxygenase activity in rat liver. This inductive effect was preceded by a decrease in the intrahepatic GSH pool, which took place several hours before induction of the oxygenase. Administration of alpha-tocopherol and allopurinol prevented induction of the oxygenase but had no effect on the decrease in GSH levels. These results suggest that the induction of heme oxygenase by phenylhydrazine and the diamide is preceded by an oxidative stress which very likely originates in the depletion of GSH. The induction of heme oxygenase by hemin was not prevented by administration of alpha-tocopherol or allopurinol. Coprotoporphyrin IX did not affect the pattern of the molecular forms of hepatic biliverdin reductase, at variance with CoCl2, which is known to convert molecular form 1 of the enzyme into molecular form 3.

Studies on porphobilinogen deaminase and uroporphyrinogen III cosynthase from human erythrocytes

Abstract 1. 1. Porphobilinogen deaminase and uroporphyrinogen III cosynthase from human erythrocytes were isolated and partially purified. 2. 2. The deaminase had a mol.wt of 25 000 ± 5000 and behaved as a single protein. It was found to be inactivated by photooxidation and chemical oxidations, by sulphydryl reagents and by divalent metals such as Ca2+, Cd2+ and Mg2+. 3. 3. The cosynthase was inhibited by sulphydryl reagents, and was not inhibited by the oxidation procedures which inhibited the deaminase. Dithiothreitol was an effective protecting agent of cosynthase. 4. 4. Deaminase and deaminase-cosynthase showed Michaelian kinetics. Uroporphyrinogen III formation increased with time and porphobilinogen concentration. 5. 5. A number of porphobilinogen analogues were examined as substrates of the system. None of them behaved as such, while several were effective inhibitors. 6. 6. Deaminase was bound to a Sepharose support and the immobilized enzyme associated with cosynthase in the absence of porphobilinogen. The whole complex formed uroporphyrinogen III.

A phosphorylase involved in starch biosynthesis

Abstract A novel phosphorylase, which has no requirement for primer addition, has been isolated from Solanum tuberosum . The properties of this enzyme were found to differ from those of the classical potato phosphorylase. The results are discussed in terms of the possible role of this enzyme in starch biosynthesis.

Concerning the specificity of heme oxygenase: The enzymatic oxidation of synthetic hemins

Abstract Hemin XIII 4 ∼ , hemin III 5 ∼ , and iron 1,4-di(β-hydroxyethyl)porphyrin 6 ∼ were enzymatically oxidized by a microsomal heme oxygenase preparation from rat liver. These are all better substrates of the oxygenase than the natural substrate, hemin IX 1 ∼ . The enzymatic oxidation was selective for the α-methine bridge and in every case only the α-biliverdins were obtained. The latter were readily reduced by biliverdin reductase to the corresponding α-bilirubins. The absence of isomers in addition to the α-bilirubins was established by preparing the derived azopigments and by using [α-14C] 6 ∼ and [α-14C] 4 ∼ as substrates. The chemical oxidation of 4 ∼ , 5 ∼ , and 6 ∼ gave the expected mixture of biliverdins. It is concluded that heme oxygenase is not specific for hemin IX. On the other hand, the enzyme is highly selective for the α-methine bridge, defined as the methine opposed to that flanked by the 6,7-propionic acid residues.

Purification and properties of porphobilinogen deaminase from wheat germ

Abstract Porphobilinogen deaminase from wheat germ was purified about 1000-fold. A series of DEAE-cellulose fractionations gave varying yields of uroporphyrin formation. The properties of the purified wheat germ enzyme were studied and compared with PBG deaminases from other origins such as Swiss chard leaves, human erythrocytes, and Rhodospirillum rubrum . A series of inhibitory effects on the enzyme were examined and the results are discussed in terms of enzyme structure and reactivity. The substrate specificity of the enzyme toward several 2-methylamino pyrroles was examined. A dithionite-activated protein factor was also isolated from wheat germ and Swiss chard and was found to consume PBG in a special way and to interact with the PBG-deaminase by inhibiting its activity. An hypothesis of the mode of action of deaminase is discussed in light of the data presented.

Pyrrolooxygenases: Isolation, properties, and products formed

Abstract 1. 1.|A new group of enzymes was isolated from wheat germ and rat liver which oxidized the pyrrole ring of indoles affording o -formamidophenacyl derivatives. They behaved as mixed-function oxidases and were named pyrrolooxygenases. Tryptophan, ethyl N -acetyltryptophan, skatole, 3-indoleacetic acid, 3-indolepropionic acid and indole were substrates of the pyrrolooxygenases. The enzymatic oxidations were catalyzed by at least two enzymes within the group; one acting on tryptophan and its derivatives, and the other one acting on skatole and the other indoles. 2. 2.|The pyrrolooxygenases had an absolute requirement for oxygen and an exogenous reducing agent. The reducing agents were illuminated chloroplasts for the plant enzymes, and NADPH and a microsomal transport system for the mammalian enzymes. Both could be replaced by sodium dithionite. 3. 3.|Chelating agents, such as α,α′-dipyridyl and EDTA inhibited the enzymatic activity, while sodium cyanide had no effect. The enzymes were also inactivated by dithiothreitol and mercaptoethanol. 4. 4.|A natural heat-labile macromolecular inhibitor of the pyrrolooxygenases was present in the crude extracts and was separated during the successive purification steps. 5. 5.|Formamidase (EC 3.5.1.9) activity was present in the extracts together with the pyrrolooxygenases and transformed the o -formamidophenacyl derivatives into the corresponding o -amino derivatives. During the purification steps the formylase activity was partly removed.

Interconversion of the molecular forms of biliverdin reductase from rat liver.

Rat liver biliverdin reductase exists in two molecular forms. The major molecular form 1 has a high reduction rate for biliverdin IX alpha, while the minor molecular from 2 has high reduction rates for both biliverdins IX alpha and IX beta. The major molecular form 1 was gradually transformed into a second major form (form 3) by treatment of the rats with CoCl2. Form 1 reduces biliverdin IX alpha at two and a half times the rate of biliverdin IX beta while form 3 reduces both isomers at about the same rate. This transformation involves a de novo mRNA and protein synthesis since it could be prevented by cycloheximide and actinomycin D. Molecular form 3 can be transformed back into molecular form 1 by an in vitro treatment with reduced thioredoxin. Phenylhydrazine treatment also induced the transformation of molecular form 1 into molecular form 3 in rat liver. Biliverdin reductase from rat spleen and kidney exists only in molecular form 1 and is not transformed into molecular form 3 by either CoCl2 or phenylhydrazine treatments.

The oxidation of hemins by microsomal heme oxygenase: Structural requirements for the retention of substrate activity

The substrate specificity of microsomal heme oxygenase from rat liver was studied by introducing systematic structural changes in the array of substituents of the protohemin IX rings. Replacement of the vinyls by methyl groups resulted in hemins which were excellent substrates of the heme oxygenase. Replacement of the 4-vinyl group by a propionic acid chain (harderohemin), decreased substrate activity to 40%. The replacement of the vinyls by formyl residues strongly decreased substrate activity but the hemins were still substrates of heme oxygenase. The oxidation rates of Spirographis hemin and of 2,4-diformyldeuterohemin IX showed a time lag which was absent when isoSpirographis hemin was used as a substrate. This lag could be attributed to the formation of a transient hemiacetal between the 2-formyl group and the alpha-mesohydroxy residue. The isomeric protohemins I, XI, and XIV (Fischers notation) were examined as possible substrates of microsomal heme oxygenase. In these protohemins the array of substituents of rings A and B was the same as in protohemin IX, but the methyl and propionic acid residues of rings C and D were at different positions from those of protohemin IX. None of them had substrate activity, indicating that the presence of two vicinal propionic acid side-chains at C6 and C7 was necessary for substrate activity. A hemin with only one propionic acid residue at C5 was not a substrate of the enzyme, either. When the propionic acid residues of protohemin IX were replaced by butyric acid residues, substrate activity decreased to 50% (as compared to protohemin IX), while when they were replaced by acetic acid residues, the substrate activity was entirely suppressed. The addition of dimethyl sulfoxide (25 mM) to the incubation mixture enhanced the oxidation of hemins with non-polar substituents in rings A and B by about 35%, while it was without effect on hemins with polar substituents in the same rings.

The enzymatic incorporation of a dipyrrylmethane into uroporphyrinogen III.

Uroporphyrinogen III 2 is the biosynthetic precursor of heme, chlorophylls and all the natural porphyrins. During the enzymatic conversion of porphobilinogen 1 into uroporphyrinogen III 2 an intramolecular rearrangement takes place (see [l] and references therein for a literature survey). The polymerization of four units of porphobilinogen by the combined action of the enzymes porphobilinogen deaminase and uroporphyrinogen III cosynthetase does not afford uroporphyrinogen I 3 as could be expected from a repetitive head-to-tail condensation of porphobllinogen but the isomeric uroporphyrinogen III 2 where an inversion in the order of the p substituents took place. Since no chemically defined pyrrylmethanes were isolated during the enzymatic reaction that could help explain that inversion, the synthetic 2-aminomethyldipyrrylmethane 4 resulting from the formal head-to-tail condensation of two units of porphobilinogen was examined as a first intermediate of the enzymatic system involved in porphobilinogen polymerization [l] . It was found that in the presence of porphoblllnogen it was incorporated exclusively into uroporphyrinogen I, and not into uroporphyrinogen III. We then proposed [l] that both isomers originate by different pathways from the start of the polymerization and that the dipyrrylmethane 5 resulting from the formal head-to-head condensation of two units of

The enzymatic oxidation of porphobilinogen

All the natural porphyrins and chlorins originate metabolically in a single monopyrrole porphobilinogen I. Porphobilinogen is thus a crucial intermediate in porphyrin metabolism, and is overproduced and excreted in large amounts during the pathological derangements of this metabolism which result in the metabolic and acquired diseases known as por. phyrias [1]. Its only known metabolic pathway was its enzymatic transformation in either uroporphyrinogen III under normal metabolic conditions, or into uroporphyrinogen III and uroporphyrinogen I under pathological conditions, where the overproduction of porphobilinogen leads to the in vivo formation of large amounts of porphyrins [1]. In this report we describe the existence of a new enzyme present in plants and animals which efficiently oxidizes porphobilinogen transforming it into 5-oxo-porphobilinogen 2, a product which is not transformed into porphyrins any longer. The enzyme, for which we propose the tentative name of porphobilinogen oxygenase, belongs to the group of the pyrrolooxygenases, a new type of enzyme recently described [2, 3].