A.M. del C. Batlle

High δ-aminolevulinic acid uptake in rat cerebral cortex: effect on porphyrin biosynthesis

Abstract The response of nerve cells to high exogenous aminolevulinic acid (ALA) concentrations was studied by examining the changes in its uptake and in porphyrin biosynthesis. ALA was shown to be taken up by cerebral cortex particles by a non-saturable process. As opposed to other previously described experimental systems, it was also observed that 84–87% of porphyrins formed was found within the cells. Exposure of cerebral cortex particles to high exogenous ALA (0.8–4.0 mM) showed that ALA can be accumulated in relatively high concentrations in brain cells (21.04 ± 1.05 nmol/mg protein). Under these experimental conditions, porphyrin biosynthesis was found to be markedly inhibited (52%). 2.4 mM ALA caused an initial stimulation of glucose uptake after 1 hr incubation and a later fall to below control values, being consistent with the fact that acute porphyric crisis could be precipitated by the action of ALA on energy metabolism. ALA toxicity could be due both to its accumulation in the cells and to deficient heme concentrations, with an additional effect on glucose metabolism. These findings provide the basis for a useful brain tissue model to investigate the nature of the metabolic mechanisms occurring in acute intermittent porphyria (AIP) patients.

Uroporphyrinogen III cosynthetase. Evidence for the existence of a polypyrrolic substrate in soybean callus tissue.

The details of the reactions by which porphobilinogen (PBG) is converted into uroporphyrinogen III are still the subject of speculation. Two enzymes are required together to bring about this conversion, PBG dearninase and uroporphyrinogen III cosynthetase. Deaminase alone converts PBG into uroporphyrinogen I while cosynthetase alone has no action on either PBG or uroporphyrinogen I. Both enzymes have been separated and partially purified from a number of sources (see [l] for a comprehensive bibliography). No intermediate in the overall reaction has yet been identified, although the existence of a dior tri-pyrrol has been postulated [2] and dipyrrolic compounds with uro-type side chains have been reported in the urine of patients with porphyria [3] . Kinetic data [ 1,2] suggest that cosynthetase requires two substrates, PBG and some product of the action of deaminase on PBG. j Here we report for the first time the formation and partial purification of a pyrrolic intermediate formed by the action of porphobilinogenase** on PBG. This intermediate acts as the second substrate, with PBG, in the formation of uroporphyrinogen III by purified cosynthetase preparations from various sources. When it is incubated with PBG-and ptrrrfied deaminase a

The porphobilinogen deamise - uroporphyrinogen III cosynthetase system (porphobilinogenase) from bovine liver. Kinetic studies☆

The conversion of prophobilinogen (PBG) into uroporphyrinogens is enzymically catalysed by the porphobilinogenase enzyme system which is widely distributed [l-lo]. In plant tissue extracts [ 1 l] the formation of uroporphyrinogen I from PBG is brought about by PBG deaminase (Uroporphyrinogen I synthetase); in the presence of both PBG deaminase and uroporphyrinogen III cosynthetase (isomerase) uroporphyrinogen III, the physiological intermediate in haem biosynthesis, is formed. Here we report kinetic studies using purified PBG deaminase and porphobihnogenase. The effects of NH: ion concentrations on the type of uroporphyrinogen formed are also reported. The results indicate that bovine liver porphobilinogenase shows cooperative effects.

Altered 5-aminolevulinic acid metabolimn leading to pseudoporphyria in hemodialysed patients

Hemodialysed patients with no history of porphyria may present neurological symptoms similar to those seen in acute porphyrias. Porphyria has been associated with an increase in plasma levels of 5-aminolevulinic acid and porphobilinogen. Our aim was to evaluate these parameters and the activities of the enzymes involved in the first steps of heme metabolism in non-porphyric hemodialysed patients. The activities of 5-aminolevulinate dehydratase and deaminase were determined in red blood cells (RBC) from 78 hemodialysed patients, before and after dialysis. Plasma levels of 5-aminolevulinic acid, porphobilinogen and zinc were also measured. These parameters were also measured in 40 volunteers to obtain controls levels. The levels of 5-aminolevulinic acid (0.98 +/- 0.09 microgram/ml) and porphobilinogen (1.32 +/- 0.13 micrograms/ml) were raised in non-porphyric patients prior to hemodialysis (P < 0.001) compared with controls (5-aminolevulinic acid 0.13 +/- 0.02 microgram/ml; porphobilinogen 0.90 +/- 0.09 microgram/ml). After dialysis there was a decrease in both 5-aminolevulinic acid (to 0.61 +/- 0.05 microgram/ml) and porphobilinogen (to 1.10 +/- 0.16 micrograms/ml) although both parameters remained higher than controls (P < 0.001). The activities of both 5-aminolevulinate dehydratase (0.550 +/- 0.095 U/ml RBC), and deaminase (54.13 +/- 9.13 U/ml RBC) were diminished in blood samples of patients before dialysis (P < 0.001) compared to controls (dehydratase 0.975 +/- 0.115 U/ml RBC; deaminase 77.32 +/- 10.00 U/ml RBC). After dialysis 5-aminolevulinate dehydratase activity was partially recovered (to 0.666 +/- 0.100 U/ml RBC) while deaminase returned to normal values (73.45 +/- 9.46 U/ml RBC). The plasma zinc concentration in hemodialysed patients (44 +/- 12 micrograms/100 ml) was significantly lower than controls (105 +/- 30 micrograms/100 ml, P < 0.001). Addition of 22.5 mM zinc to the dehydratase reaction mixture raised the activity of 5-aminolevulinate dehydratase in blood samples of hemodialysed patients taken before and after dialysis. The study reports a partial loss of activity of 5-aminolevulinate dehydratase and deaminase activities in red blood cells from non-porphyric patients undergoing hemodialysis. Since plasma zinc levels were below normal in hemodialysed patients, and the activity of 5-aminolevulinate dehydratase could be restored by the addition of zinc, it is suggested that these abnormalities in heme metabolism may be explained by altered zinc and associated antioxidant status following dialysis.

Porphyrin-induced protein structural alterations of heme enzymes

Some alterations in the protein structure of delta-aminolevulinic acid dehydratase (ALA-D) and porphobilinogen deaminase (PBG-D) induced by uroporphyrin (URO) and prototoporphyrin (PROTO) have been observed previously. To obtain further evidence of these phenomena, the absorption and fluorescence spectra of ALA-D and PBG-D and the total protein content of sulfhydryl and free amino groups were analyzed after exposure of the enzymes to URO I and PROTO IX, ALA-D and PBG-D were partially purified from bovine liver and exposed to URO I or PROTO IX, both in the dark and under UV light. All experiments were performed in the enzyme solutions after removing the porphyrins. Absorbance spectra changes in the region of 220-300 nm were registered, indicating the interaction of the porphyrins with the molecular structure of the enzymes. The main changes in the fluorescence spectra were observed in the spectral region of 555 nm, and only slight modifications in the spectral region of 340-360 nm; moreover, alterations were stronger upon UV irradiation and in the presence of URO I when compared with darkness and PROTO IX. Variations in total SH groups would suggest the formation of disulfur bridges induced by URO I and the rupture of some S-S groups induced by PROTO IX. The effect of porphyrins on free amino groups would reflect a combination of cross-linking and fragmentation of proteins. Structural changes were observed when the enzymes were exposed to the porphyrin both in the dark or under UV light; however, they were stronger in the latter condition. These results suggest that porphyrins per se could act directly on the protein structure and that this action would be enhanced upon UV irradiation.

Porphyrin biosynthesis in Rhodopseudomonas palustris—VIII. Purification and properties of deaminase

Abstract 1. 1. Uroporphyrinogen I synthetase from Rhodopseudomonas palustris has been isolated and purified. 2. 2. Assay conditions were determined. 3. 3. Sephadex G-100 column chromatography was found to increase 4-fold the degree of purification yielding a deaminase that was purified 72-fold. 4. 4. Some properties of the isolated enzyme were studied. The optimal pH was about 7.6–7.8. Porphyrin formation was linear with time. The presence of several thiol reagents was found to be no essential for deaminase activity. 5. 5. Deaminase exhibited classical Michaelis-Menten kinetics K m and V max were estimated. 6. 6. Molecular weight determinations, by means of gel filtration on a calibrated Sephadex G-100 column, gave values of 74,000 ± 7400.

Porphyrin biosynthesis in normal and haem mutants of Saccharomyces cerevisiae. Studies on the inheritance of the HEM R+ phenotype☆

Heme biosynthesis was studied in the segregants of Saccharomyces cerevisiae (DW10 tetrade 2) from D27 and D27/C6 mating, as a function of the carbon source in the growth medium and the physiological state of the cells. The effects of the HEM R+ gene on the 5-aminolevulinate synthase (ALA-S) and 5-aminolevulinate dehydratase (ALA-D) activities of heme biosynthesis in cells grown on nonfermentable and fermentable carbon sources were compared. Profiles obtained for both strains grown on a fermentable carbon source (glucose) were identical. However, in the presence of a nonfermentable carbon source (ethanol), they behave quite different, as if the mutation could only be expressed under these growth conditions. Moreover, their behavior is similar to that found for the parental strains, indicating that for the mutant its particular behavior might be inheritedly linked to the HEM R+ gene, which in turn affects some regulatory aspects of ALA synthesis explaining its characteristic phenotype.