L.C. San Martín de Viale

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.

Studies on the active centre of rat liver porphyrinogen carboxylyase in vivo effect of hexachlorobenzene

Abstract 1. 1. Porphyrinogen carboxylyase from the liver of normal and hexachlorobenzene porphyric rats was subjeced to chemical modification using photo-oxidation with methylene blue, diethylpyrocarbonate, butane-2,3-dione, and phenylglyoxal. 2. 2. All of these chemicals inactivated the enzyme from both sources. 3. 3. Reversion of the diethylpyrocarbonate reaction with hydroxylamine as well as protection of the enzymes with uroporphyrinogen III indicated that histicline is involved at least in the first decarboxylation active site of the porphyrinogen carboxylyase, and perhaps in one or more sites where the removal of the other carboxyl groups take place. 4. 4. Arginine seems not to be at the active site of the enzyme but at its environment since two diketones alter the enzyme activity, however the substrate did not protect the enzyme from the butane-2,3-dione modification. 5. 5. Comparative studies between the enzyme from normal and porphyric animals suggest that the low enzyme activity from intoxicated animals could be due to alterations of its active centre environment produced by hexachlorobenzene treatment. This treatment seems to partially protect the active site of the porphyrinogen carboxylyase from the modification reactions.

Comparison between crab hepatopancreas and rat liver uroporphyrinogen decarboxylase

We characterized Uroporphyrinogen decarboxylase (UroD) (E.C. 4.1.1.37) in hepatopancreas of the crab Chasmagnathus granulatus as a first step to establish this enzyme as a possible biomarker for environmental contamination. We performed a comparative study of crab UroD with the enzyme UroD present in Wistar rat liver, which is known as a useful indicator of intoxication by polyhalogenated aromatic hydrocarbons (PAHs). The final products were the same in crab and rat UroD: the remaining substrate (8-carboxyl-porphyrinogen), the final product Coproporphyrinogen (4-COOH) and intermediate compounds with 7-, 6- and 5-COOH. The elimination of the second carboxyl group seems to be the rate-limiting step in this multiple decarboxylation, because large amounts of 7-COOH porphyrinogen are accumulated. The V(max)/K(m) ratio was 100-fold higher for rat liver UroD than for crab hepatopancreas UroD, suggesting a higher efficiency of the rat enzyme. Optimum pH for enzyme activity was 7.2 and 6.8 for crab and rat, respectively. Although both systems showed the same optimum temperature (47 degrees C), the activation energy was clearly different, 51.5 kJ/mol for C. granulatus and 5.4 kJ/mol for Rattus norvegicus (Wistar strain). Superdex 75 gel chromatography yielded a single symmetrical peak with an apparent molecular mass of 48+/-3 kDa for crab hepatopancreas UroD, suggesting the existence of only one enzymatic species in C. granulatus.

Porphyria‐induced hepatic porphyrinogen carboxy‐lyase inhibitor and its interaction with the active site(s) of the enzyme

Porphyrinogen carboxy-lyase is an enzyme that sequentially decarboxylates uroporphyrinogen III (8-COOH) to yield coproporphyrinogen III (4-COOH). In mammals this enzyme activity is impaired by hexachlorobenzene treatment, through generation of an enzyme inhibitor. The interaction of porphyrinogen carboxy-lyase inhibitor, extracted from the liver of hexachlorobenzene-treated rats, with substrate decarboxylation sites on the enzyme, was studied using four different carboxylated substrates belonging to the isomeric III series of naturally-formed porphyrinogens containing 8-,7-,6- and 5-COOH. Similar inhibitor effects were elicited against all the substrates assayed, with the exception of pentacarboxyporphyrinogen III in which decarboxylation was not inhibited to same extent. Enzyme protection assays in the presence of the different substrates, indicated that each porphyrinogen protects its own decarboxylation from inhibitor action. Preincubation of the inhibitor with normal enzyme increased its inhibitory effect. On the other hand, preincubation of both enzyme and inhibitor with superoxide dismutase or mannitol, did not alter inhibitory activity. Preincubation of the inhibitor with a number of amino acids showed that only arginine and its derivative N alpha-Benzoyl-L-Arginine ethyl ester interact with the inhibitor, noticeably reducing its ability to inhibit porphyrinogen carboxy-lyase. Albumin, histidine, serine, cysteine and imidazol, were unable to quench inhibitor activity. The present results indicate that the inhibitor acts at the binding site of each porphyrinogen. Taking into account that arginine is related to enzyme activity, and that histidine is found at the binding site of the substrates, the results suggest that the inhibitor could bind to arginine residues, blocking the access of substrates to histidine and altering the adequate orientation for decarboxylation by masking the positively charged active site necessary for porphyrinogen binding to the enzyme. In addition an indirect effect of the inhibitor mediated through free radicals could be discarded.

Studies on uroporphyrinogen decarboxylase from chicken erythrocytes.

Uroporphyrinogen (URO’gen*) is decarboxylated stepwise to coproporphyrinogen (COPRO’gen) by the uroporphyrinogen decarboxylase (URO’gen carboxy-lyase E.C.4.l.l.d), the intermediate products being porphyrinogens with 7-, 6-, and 5carboxylic groups [l-3] . This enzyme has been partially purified from rabbit reticulocytes [4,5] and from Rhodopseudomonas spheroides [6] . To study the yet unsolved problems related to the enzymatic transformations of URO’gen to COPRO’gen and to determine whether one or more enzymes are involved in this process, a pu;i

Heme metabolism after discontinued hexachlorobenzene administration in rats: possible irreversible changes and biomarker for hexachlorobenzene persistence.

ed URO’gen decarboxylase from chicken erythrocytes was obtained. The differential action of several chemical and physical agents on the different steps involved in the enzymic decarboxylation of URO’gen has been studied. The experimental this communication. results are reported in

Liver ferrochelatase from normal and hexachlorobenzene porphyric rats studies on their properties

The aim of the present study was to determine whether short-term administration of hexachlorobenzene (HCB) (1 g/kg body wt., suspended in water, 5 days/week), could cause and maintain marked porphyria in the absence of the exogenous drug, and whether porphyria parameters can be useful as biomarkers of HCB persistence in rats. Hepatic uroporphyrinogen decarboxylase activity, its inhibitor formation, porphyrin content and composition were studied in Wistar rats treated with the fungicide for 1, 2, 3, or 4 weeks and then withdrawn for a 20-week period. The time course of urinary porphyrin excretion was studied for 7 weeks either by continuous treatment for the entire period, or a 1-week HCB administration. The degree of porphyria achieved by rats after 20 weeks of suspended HCB administration was severe, independent of the length of the treatment, and even higher than that observed in animals analysed immediately at the end of each treatment. Rats treated with HCB for 1 week showed a modest decrease in uroporphyrinogen decarboxylase and low inhibitor formation, and exhibited a greater enzyme inhibition, inhibitor formation, hepatic porphyrin accumulation, and an altered pattern of porphyrin composition in the absence of the exogenous drug. Independent of the treatment, urinary porphyrins rose after a delay of 5 weeks. Substantial amounts of HCB were still found in fat of rats treated with HCB for 1 week, after a withdrawal period of 20 weeks. These results suggest that the high persistence of HCB in tissues acts as a continuous source of the xenobiotic, and stimulus for heme biosynthesis derangement. The alterations induced by HCB within 1 week of treatment could be regarded as an initial trigger for irreversible damage on heme metabolism. Thus, abnormalities in heme biosynthesis can be considered effective markers of HCB persistence in rats or of irreversible HCB-induced damage. Taking into account the delayed and enhanced metabolic effects of HCB, it is advisable that porphyria parameters should be evaluated not only immediately after exposure, but also some time afterwards, especially in susceptible and occupationally-exposed populations.

Erythrocyte porphyrinogen carboxy-lyase activity in porphyria cutanea tarda and certain other human porphyrias

1. The aim of the present work is to shed light on the way of action of hexachlorobenzene (HCB) on hepatic ferrochelatase the mitochondrial enzyme which catalyzes the last step of haem biosynthetic pathway. 2. Some properties of this enzyme from normal and HCB porphyric rat liver were studied. 3. The present findings indicate that HCB treatment would modify the configuration of the enzyme perhaps allowing the active center of the porphyric ferrochelatase to be more exposed. 4. As a consequence it would show: (a) its higher affinity for the iron; (b) the shorter time necessary to form the intermediate enzyme-substrate, reflected both by the existence of a shorter lag and consequently a shorter pre incubation time. 5. However this modification elicited by the fungicide does not alter the submitochondrial distribution of the enzyme nor the optimal conditions for its measurement.

Studies on the active centre(s) of rat liver porphyrinogen carboxy-lyase. in vivo effect of hexachlorobenzene on decarboxylation site(s) of porphyrinogens

Red cell porphyrinogen carboxy-lyase activity was measured using uroporphyrinogen III as substrate in 18 normal persons, 7 male patients with porphyria cutanea tarda, 3 female patients with erythropoietic protoporphyria and 2 female patients with variegate porphyria. The mean values obtained in normal subjects were 0.151 nmol of uroporphyrinogen disappeared in 30 min per mg of protein, and 0.038 nmol of coproporphyrinogen formed in 30 min per mg of protein. We have not been able to detect significant differences between males and females. In porphyria cutanea tarda the enzyme activity was the same as in normal subjects considering either substrate disappearance or end product formation. The differences were not significant at the p less than 0.05 level. Patients with variegata porphyria also exhibited normal erythrocyte porphyrinogen carboxy-lyase activity. The enzyme activity of erythrocytes from patients with erythropoietic protoporphyria was higher than in normals; mean values for specific activities being 0.204 nmol of uroporphyrinogen disappeared, and 0.071 nmol of coproporphyrinogen formed. The significance of the results with respect to the chemical picture of different porphyrias is discussed.