Moisés Grinstein

Porphyrin biosynthesis. VII. Porphyrinogen carboxy-lyase from avian erythrocytes. Purification and properties.

Abstract 1. 1. Uroporphyrinogen carboxy-lyase (EC 4.1.1.d), the enzyme catalysing the decaroxylation of uroporphyrinogen to coproporphyrinogen, has been isolated from normal chicken erythrocytes. The enzyme was purified 220-fold with a yield of 24% from haemolysate supernatant by DEAE-cellulose batch treatment, (NH4)2SO4 fractionation and chromatography on DEAE-cellulose. 2. 2. The purified material appears to be homogeneous in polyacrylamide gel disc electrophoresis. 3. 3. The enzyme was heat labile and inhibited by sodium salt; the activity was enhanced by EDTA, GSH and boiled rat-liver extract. 4. 4. The influence of these chemical and physical agents on the removal of the first and second carboxyl groups from uroporphyrinogen was compared; the second group was more susceptible to these agents. 5. 5. The possibility that one or several enzymes were involved in the stepwise decarboxylation of uroporphyrinogen is discussed. 6. 6. The general name of porphyrinogen carboxy-lyase for the enzyme system is proposed because of the different porphyrinogens it can decarboxylate.

Porphyrin biosynthesis: VI. Separation and purification of porphobilinogen deaminase and uroporphyrinogen isomerase from cow liver. Porphobilinogenase an allosteric enzyme

Abstract 1. 1.|Porphobilinogenase has been isolated and purified from cow liver and its components, porphobilinogen deaminase and uroporphyrinogen isomerase, have been separated from each other and purified. 2. 2.|The effect of NH 4 + was studied. The deaminase exhibited classical Michaelis-Menten kinetics in the absence or presence of NH 4 + , which at high concentrations behaved as a noncompetitive inhibitor of the deaminase. As expected from Hill plots, n = 1 both in the absence or presence of NH 4 + . Instead, when activity of porpho bilinogenase is plotted versus porphobilinogen concentration, sigmoid curves are obtained; but the presence of NH 4 + at different concentrations altered the kinetic parameters of this enzymic system, again showing normal kinetics. In addition, n values were found to be 2 for porphobilinogen per porphobilinogenase molecule and 1 in the presence of NH 4 + which behaves as a competitive inhibitor of the isomerase. Results are discussed in relation to the allosteric theories of monod et al. 1,2 and liver porphobilinogenase is proposed to be an allosteric protein. 3. 3.|The presence of an ultrafiltrable factor which stimulates uroporphyrinogen formation from porphobilinogen has been revealed.

Porphyrin biosynthesis III. Porphyrin metabolism in experimental lead poisoning

Abstract Porphyrinogen and heme synthesis in whole blood, hemolysates, and supernatant of hemolysates from lead-poisoned rabbits and control rabbits were studied in vitro. [2-14C]Glycine, δ-aminolaevulic acid, δ-amino[4-14C]laevulic acid, porphobilinogen, [14C]uroporphyrinogen III, [14C]coproporphyrinogen III and Fe2+ ions were the basic substrates employed. With [2-14C]glycine as the subtrate, free porphyrinogen synthesis by teh lead-poisoned system was depressed as was depressed as was incorporation of the substrate into heme. When starting with δ-aminolaevulic acid, there was a general decrease in all porphyrinogens synthesized, but most particularly for uro- and phyriaporphyrinogens. Incorporation of δ-amino[4-14C]laevulic acid into heme was also diminished. In contrast, when porphobilinogen and [24C]uroporphyrinogen III were the substrates, there were no significant differences in porphyrin synthesis between the lead-poisoned and control systems and comparable amounts of uroporphyrinogen III and phyriaporphyrinogen III were metabolized. Nevertheless, an accumulation of phyriaporphyrinogen III, relative to the amounts of coproprophyrinogen III, occured. These results demonstrate that one of the principal blocks in the biosynthetic pathway of protorporphyrin in lead poisoning is at the δ-aminolaevulic acid dehydrase (EC 4.2.1.24) level, and that a second block, quantitatively less important, occurs between phyriaporphyrinogen III and coproporhyrinogen III. [14C]Coproporphyrinogen III inorporation into protopohyrin 9 and heme was decreased in the lead-poisoned systems. The well-documented accumulation of both these intermediates could be satisfactorily explained on the basis of lead impairing the transport of iron or other metabolites through the mitochondrial membrane.

Porphyrin biosynthesis I. Studies on erythrocyte preparations

Abstract Further studies have been reported on the identification of the biosynthetic porphyrins which are formed on incubation of supernatant preparations obtained from chicken red-cell haemolysates and δ-aminolaevulic acid or porphobilinogen under both anaerobic and aerobic conditions. Pseudo-uroporphyrin was identified as the so-called “208” porphyrin isolated from porphyria material. It is suggested that this porphyrin should be called phyria-porphyrin III, as was proposed in 1955. Chromatography on columns of CaCO 3 has proved to be a very sensitive tool for the fractionation of mixtures of biosynthetic porphyrins.

Porphyrin biosynthesis: IV. 5- and 6-COOH porphyrinogens (type III) as normal intermediates in haem biosynthesis

1. 1. The role of 5- and 6-COOH porphyrinogens III in haem biosynthesis was studied in vitro. 2. 2. When haemolysates of normal chicken erythrocytes were incubated in the presence of 5- or 6-COOH porphyrinogens III, a marked isotopic dilution was observed served on the [2-12C]glycine incorporation into haem. This effect was similar and even greater than that produced by uroporphyrinogen III and coproporphyrinogen III. To confirm these results 14C-labelled porphyrinogens of 5- and 6-COOH were incubated with chicken erythrocyte haemolysates. Labelling of the haem took place in a way similar to that when [14C]uroporphyrinogen III was used. 3. 3. These results are consistent with the idea that 5-COOH porphyrinogen III and 6-COOH porphyrinogen III are normal intermediates in haem biosynthesis with a stepwise decarboxylation process of uroporphyrinogen III to coproporphyrinogen III according to the following scheme: Uroporphyrinogen III → 7-COOH porphyrinogen III → 6-COOH porphyrinogen III → 5-COOH porphyrinogen III → coproporphyrinogen III

Porphyrin biosynthesis. X. Porphyrinogen carboxy-lyase from avian erythrocytes futher properties

Abstract Several properties of porphyrinogen carboxy-lyase from normal chicken erythrocytes were studied. 1. 1. The utilization of the substrate uroporphyrinogen (8-COOH), and the formation of intermediate products (porphyrinogens of 7-, 6- and 5-COOH) and the final product coproporphyrinogen (4-COOH) were investigated as function of time and substrate concentration. The results confirm a two-stage hypothesis involving firstly, the elimination of the first carboxyl group from uroporphyrinogen and secondly the elimination of the further three carboxyl groups to form coproporphyrinogen. The elimination of the first carboxyl group is not the rate-limiting step in this multiple decarboxylation because large amounts of 7 COOH porphyrinogen were accumulated. The effect of temperature on the stepwise decarboxylation process also suggests and easier elimination of the first carboxyl group. 2. 2. Cysteine and glutathione inhibited the decarboxylation process at low concentrations, but at higher concentrations cysteine continues to inhibit while glutathione allows the recovery of enzyme activity. 3. 3. Studies of the effect of uroporphyrinogen concentration on the first and second stages; and of 7-COOH porphyrinogen concentration on the second stage revealed both substrates as inhibitors of their own decarboxylations. Furthermore, when 7-COOH porphyrinogen was incubated in the presence of uroporphyrinogen, it further inhibited the first decarboxylation of uroporphyrinogen, 7-COOH porphyrinogen was a stronger inhibitor than 8 COOH porphyrinogen.

Human porphyria cutanea tarda. Isolation and properties of the urinary porphyrins.

Abstract Porphyrins with 8-, 7-, 6-, 5-, and 4-COOH were isolated from urine of patients with porphyria cutanea tarda and were studied by column and paper chromatographic techniques both qualitatively and quantitatively, and their absorption spectra and melting points were determined. It was found that the relative excretion pattern was uroporphyrin > coproporphyrin > phyriaporphyrin > 5-COOH porphyrin > 6-COOH porphyrin. Studies of the isomeric composition of these different porphyrins showed that: the uroporphyrin was a mixture of isomers I and III in a 4:1 proportion; phyriaporphyrin and 6-COOH porphyrin were only type III; 5-COOH porphyrin and coproporphyrin were a mixture of isomers I and III.

[19] Preparation of porphobilinogen

Publisher Summary This chapter illustrates the preparation of porphobilinogen (PBG). PBG is a normal intermediate in the biosynthesis of heme. An enzymatic method has been considered for the preparation of porphobilinogen with good yield using both δ-aminolevulinic acid and the enzyme aminolevulinic acid dehydratase. PBG can be isolated and purified by either of the two procedures of isolation and can be essentially identified by different methods. By paper chromatography, a biosynthetic PBG runs as a single spot having the same R f as that for the markers. A solution of biosynthetic PBG when mixed with an equal volume of Ehrlichs reagent gives maxima at 555 m μ and 525 m μ . PBG can be enzymatically converted into uroporphyrinogens by means of the deaminase–isomerase enzymatic complex.

Effect of glucose on induction of δ-aminolevulinic acid synthase, ferrochelatase and cytochrome P-450 hemoproteins in isolated rat hepatocytes by phenobarbital and lead

In the present work we have been able to demonstrate that phenobarbital and lead exert an inducing effect on the biosynthesis of delta-aminolevulinic acid synthase, ferrochelatase and cytochrome P-450 hemoproteins in isolated rat hepatocytes of normal adult rats. Dibutyryl cyclic AMP enhances the induction effect produced by phenobarbital in this in vitro system. Glucose inhibits the induction of delta-aminolevulinic acid synthase and ferrochelatase. This repression effect can be reversed with increasing concentrations of dibutyryl cyclic AMP. No glucose effect was observed on the phenobarbital- and lead-mediated inductions of cytochrome P-450. he present results add more experimental evidence to support the concept that the last enzyme of the heme pathway is inducible, and as such may have a significant role in regulatory mechanisms of porphyrin and heme biosynthesis.

Porphyrin biosynthesis. Phyriaporphyrinogen III, a normal intermediate in the biosynthesis of protoporphyrin 9.

Abstract In the present paper we report experimental data, indicating that phyriaporphyrinogen III, the porphyrinogen of the porphyrin which was originally called “208” porphyrin, is a normal intermediate in protoporphyrin-9 biosynthesis. Similar results were obtained with the pseudouroporphyrinogen. Additional results have confirmed the scheme and suggest a stepwise decarboxylation in the metabolic pathway leading to the biosynthesis of protoporphyrin 9.