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M L Tomaro

Publications and source records attributed to M L Tomaro.

At least 37 records · Page 2Linked to original sources
PubMed1986

The regulation of porphobilinogen oxygenase and porphobilinogen deaminase activities in rat bone marrow under conditions of erythropoietic stress.

Porphorbilinogen oxygenase (EC 4.2.1.24) was associated with the microsomal fraction of bone marrow in normal rats and in rats submitted to erythropoietic stress, while porphobilinogen deaminase (EC 4.3.1.8) of the same origin was present in the cytosol. An NADPH-dependent electron-donor system for the oxygenase was also present in the microsomes of the bone marrow. Under conditions of erythropoietic stress caused by hypoxia, the activities of both enzymes were found to be inversely correlated. While the oxygenase showed minimum activity between the 4th and 8th day of hypoxia, porphobilinogen deaminase reached its maximum activity during this period. After the 8th day of hypoxia, oxygenase activity increased while deaminase activity decreased. The NADPH-dependent electron-transport system necessary for the microsomal oxygenase activity was largely inactivated after the 10th day of hypoxia, while oxygenase activity was not affected. The particulate porphobilinogen oxygenase could be solubilized from the bone marrow microsomes with 1% deoxycholate or 0.5 M KCl. In addition, the oxygenase was also released by freezing and thawing the microsomes isolated from bone marrow of rats which had been submitted to an erythropoietic stress (hypoxia or phenylhydrazine). The enzyme solubilized with deoxycholate or KCl showed a high molecular weight form and a low molecular weight form (Mr 25 000). The former could be transformed into the latter either by treatment with 2 M KCl or by succinylation. When the oxygenase was solubilized by freezing and thawing a third molecular weight form (Mr 50 000) also appeared. The solubilized enzyme could be succinylated without loss of its catalytic activity, while the membrane-bound enzyme could not be succinylated.

Ammonia-Lyases↗
PubMed1986

The chemical and enzymatic oxidation of hematohemin IX. Identification of hematobiliverdin IX alpha as the sole product of the enzymatic oxidation.

Oxidative cleavage of hematohemin IX in pyridine solution in the presence of ascorbic acid (coupled oxidation), followed by esterification of the products with boron trifluoride/methanol produced the four possible hematobiliverdin dimethyl esters in 11.1% overall yield. Transetherifications took place simultaneously with the esterification reaction and resulted in the formation of the dimethyl ester of hematobiliverdin IX gamma 8a,13a-dimethyl ether (1.8%), the dimethyl ester of hematobiliverdin IX beta 13a,18a-dimethyl ether (1.9%), the dimethyl ester of hematobiliverdin IX delta 8a-monomethyl ether (1.4%), and the dimethyl ester of hematobiliverdin IX alpha 18a-monomethyl ether (0.4%). The latter was the sole product obtained after the enzymatic oxidation of hematohemin with heme oxygenase, after esterification of the reaction product with boron trifluoride/methanol. When the esterification step was omitted hematobiliverdin IX alpha was obtained from the enzymatic oxidation. The structures of the hematobiliverdin derivatives were secured by their NMR and mass spectra data. Saponification of the dimethyl esters afforded the hematobiliverdin methyl ethers, which were excellent substrates of biliverdin reductase and were readily reduced to the corresponding bilirubins. Hematobiliverdin IX alpha was also a good substrate of biliverdin reductase. It is concluded that the enzymatic oxidation of hematohemin IX by heme oxygenase is alpha-selective, while biliverdin reductase shows no selectivity in the reduction of the four hematobiliverdin isomers.

Animals↗
PubMed1984

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 (Fischer's 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.

Animals↗
PubMed1984

The specificity of biliverdin reductase. A study with different biliverdin types.

The specificity of rat liver biliverdin reductase was examined with the help of a series of synthetic biliverdins. The mixture of the four biliverdin isomers obtained by the chemical oxidation of protohemin I, protohemin XI, protohemin XIV and harderohemin were used as substrates of biliverdin reductase and were compared with the mixture of biliverdins IX alpha-delta. Biliverdin reductase (molecular form 1) from rat liver efficiently reduced the isomer mixtures of biliverdins I, XI, XIV and harderobiliverdins to the bilirubins in the presence of NADPH. The enzymatic reduction of the different biliverdin types was studied in the presence of different NADPH analogues. NADPH could be replaced by NADH, 3-acetyl NADPH and deamino-NADPH with retention of a good substrate activity only in the case of biliverdins of types I and IX and harderobiliverdins. Biliverdins XI and XIV were efficiently reduced only in the presence of NADPH and an excess of NADH. Bactobilin III-alpha was also very efficiently reduced by biliverdin reductase in the presence of both NADPH and NADH but not in the presence of the other analogues. These results indicate that biliverdin reductase reduced bilitriene acids substituted with non-polar and polar residues.

Animals↗
PubMed1984

The specificity of biliverdin reductase. The reduction of biliverdin XIII isomers.

The substrate specificity of the different molecular forms of biliverdin reductase (bilirubin:NAD(P)+ oxidoreductase, EC 1.3.1.24) using biliverdin XIII alpha, XIII beta and XIII gamma was examined. It was found that molecular form 1 (the major form in normal rat liver) reduced biliverdin XIII alpha at a much higher rate than the other two isomers. Molecular form 2 (the minor form) reduced isomers XIII alpha and XIII beta at similar rates, while molecular form 3 (the major form induced by CoCl2 treatment) reduced the XIII beta isomer at a slightly higher rate than the XIII alpha isomer. Molecular forms 2 and 3, both reduced isomer XIII gamma more slowly than they reduced the XIII alpha and XIII beta isomers. These results are similar to those obtained previously using biliverdins IX alpha, IX beta and IX gamma, suggesting that biliverdin reductase specificity is related to the type of the isomer rather than to the series (IX or XIII) of the isomer.

Animals↗
PubMed1984

Isolation from rat liver of a peroxisomal enzyme which converts molecular form 1 of biliverdin reductase into molecular form 3.

Cobaltous chloride induced in rat liver an enzyme which converted biliverdin reductase molecular form 1 into the molecular form 3. This conversion involves the oxidation of two sulfhydryl groups of form 1 giving rise to a disulfide bond in form 3. The converting enzyme was isolated from the liver peroxisomal fraction (which was devoid of biliverdin reductase activity), and was absent in liver peroxisomes of control rats. The enzyme was solubilized by treatment of the peroxisomes with 0.1% sodium deoxycholate, and partially purified by DEAE-cellulose and Sephadex G-100 filtration. It is a NAD+ dependent enzyme which was inactivated by trypsin and heat treatments. It did not oxidize either reduced glutathione or cysteine. The converting enzyme had a molecular weight of about 54,000 daltons. The oxidation of biliverdin reductase molecular form 1 mediated by the converting enzyme did not affect the latter's molecular weight or activity.

Animals↗
PubMed1983

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.

Animals↗
PubMed1982

Specific alpha-bridge cleavage by heme oxygenase of [alpha-14C]deuterohemin IX, [alpha-14C]hematohemin IX and 2,4-diacetyl[alpha-14C]deuterohemin IX.

The enzymatic oxidations of [alpha-14C]deuterohemin IX, [alpha-14C]hematohemin IX and 2,4-diacetyl[alpha-14C]deuterohemin IX were carried out by using a microsomal heme oxygenase system from rat liver in combination with the biliverdin reductase from the same origin. In every case the bilirubins formed were devoid of radioactivity, indicating the alpha-selective oxidation of the three hemins. Hematohemin IX was oxidized at the highest rate, followed by deuterohemin IX and 2,4-diacetyldeuterohemin. When the three hemins were preincubated with microsomal heme oxygenase in the absence of NADPH, and the latter was added after the preincubation period, it was found that the enzymatic oxidation of the hemins was inhibited. Therefore, for the maximal rate of oxidation both hemin and NADPH must be present simultaneously. In the presence of hemin IX (the natural substrate), the enzymatic oxidation of the synthetic hemins was inhibited. The oxidation of 2,4-diacetyldeuterohemin IX was the most inhibited, while the oxidation of hematohemin IX was affected to a much lesser degree. These results are in agreement with the higher affinity (Km = 150 microM) of hematohemin IX for the enzyme, as compared to 2,4-diacetyldeuterohemin IX (Km = 660 microM) and deuterohemin IX (Km = 330 microM).

Animals↗
PubMed1981

Specificity of heme oxygenase: a study with synthetic hemins.

A large number of synthetic iron porphyrins were enzymatically oxidized by a microsomal heme oxygenase preparation from rat liver. They all had in common two vicinal propionic acid residues at C6 and C7. Iron porphyrins of type I were not substrates of the enzyme. Iron porphyrins that carried electron-withdrawing substituents (acyl residues) at C2 and C4 were substrates of heme oxygenase, although the product yields were reduced. Several iron porphyrins, such as hemin XIII (4) and hem III (5), were better substrates of heme oxygenase than the natural substrate hemin IX (1). The enzymatic oxidation was selective for the alpha-methine bridge, and the alpha-biliverdins obtained were reduced by Biliverdin reductase to the corresponding alpha-bilirubins. Preincubation of the enzymatic system with hemin IX (1) and hemin XIII (4) in the absence of NADPH resulted in an inhibition of their oxidation. The iron-free porphyrins which carried two vicinal propionic acid residues at C6 and C7 were also found to be inhibitors of the enzymatic system when preincubated with the latter. The presence of hematochemin IX (18) suppressed the enzymatic oxidation of hemin IX (1).

Animals↗
PubMed1981

Induction of porphobilinogen oxygenase and porphobilinogen deaminase in rat blood under conditions of erythropoietic stress.

Porphobilinogen is the substrate of two enzymes: porphobilinogen deaminase and porphobilinogen-oxygenase. The first one transforms it into the metabolic precursors of heme and the second diverts it from this metabolic pathway by oxidizing porphobilinogen to 5-oxopyrrolinones. Rat blood is devoid of porphobilinogen-oxygenase under normal conditions while it carries porphobilinogen-deaminase activity. When the rats were submitted to hypoxia (pO2 = 0.42 atm) for 18 days, the activity of porphobilinogen-oxygenase appeared at the tenth day of hypoxia and reached the maximum at the 14-16th day. It decreased to a half after 2 days (half-life of the enzyme) and disappeared after 4 days of return to normal oxygen pressure. Porphobilinogen-deaminase activity increased after the first day of hypoxia, reached a maximum at the 14-16th day and did not decrease to normal values until the 15th day after return to normal oxygen pressure. The activities of both porphobilinogen-oxygenase and porphobilinogen-deaminase were induced by administration of erythropoietin. When rats were made anaemic with phenylhydrazine, porphobilinogen-oxygenase activity also appeared in the blood cells. Although the reticulocyte concentration was higher when compared to that obtained under hypoxia, the activities of the oxygenase obtained under both conditions were comparable. Porphobilinogen-deaminase activity was always closely related to the reticulocyte content. The appearance of porphobilinogenase-oxygenase under the described erythropoietic conditions was due to a de novo induction of the enzyme, as shown by its inhibition with actinomycin D and cycloheximide. Porphobilinogen-oxygenase as well as porphobilinogen-deaminase were present in the rat bone marrow under normal conditions. Their activities increased in phenylhydrazine treated rats. The properties and kinetics of porphobilinogen-oxygenase from the rat blood and bone marrow were determined and found it differ in several aspects.

Ammonia-Lyases↗
PubMed1979

Porphobilinogen oxygenase from human erythrocytes.

Porphobilinogen oxygenase was isolated from red cells of healthy persons and of patients with disturbances in porphyrin metabolism. The hemolysates were purified by DEAE-cellulose chromatography and the oxygenase was eluted with either 3 mmol/l phosphate buffer (pH 7.4) or with 10 mmol/l Tris-HCl buffer (pH 7.6). The oxygenase can also be isolated by filtration of the hemolysate through Sephadex G-100. In healthy persons a mean value of 84.1 +/- 29.7 nmol of porphobilinogen consumed in 30 min per ml of blood was obtained. In patients with hepatic porphyrias (acute intermittent porphyria and porphyria cutanea tarda) the activity of porphobilinogen oxygenase was very low. In the case with acute intermittent porphyria the activity was increased when measured after storage at 4 degrees C but never reached normal values. In the cases of porphyria cutanea tarda, oxygenase activity increased after recovery and reached normal values. In patients with erythropoietic porphyria and in anemias the activity of porphobilinogen oxygenase gave high values. The erythrocyte enzyme was found to be heterogeneous when compared with the enzyme of other sources. It was only partially succinylated and was inactivated after storage for a few days at 4 degrees C. Some preparations showed the usual allosteric kinetics (n = 1.6--2.0), although Michaelian kinetics were also often observed. The enzyme was inhibited by alpha, alpha'-dipyridyl and EDTA as well as by several metals.

Animals↗