1. delta-Aminolaevulate dehydratase, the enzyme catalysing the condensation of delta-aminolaevulic acid to porphobilinogen, has been prepared from cow liver and its properties have been studied. The enzyme has been purified 310-fold. 2. The purified preparation behaves as a single protein under gel filtration on Sephadex and Bio-Gel columns; it migrates as a single band in disk and starch-gel electro-phoresis at different pH values and it sediments as a single symmetrical peak in the ultracentrifuge. 3. The pH optimum for the pure enzyme was 6.8, the K(m) at pH 6.8 and 38 degrees was 1.5x10(-4)m, the isoelectric point was about pH 4.9 and the molecular weight was 140000+/-14000 by the gel-filtration method. Maximal enzyme activity was observed at 65 degrees . 4. The presence of thiol groups in the enzyme system, essential for its activity, was indicated and the total number of thiol groups was determined. 5. After the first steps of purification the enzyme required cysteine or reduced glutathione for activity.

Animals↗

PubMed1967

Estimation of molecular weights of proteins by Bio-Gel P gel filtration.

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Animals↗

PubMedDate not supplied

Regulation of heme pathway in regenerating mouse liver.

1. delta-Aminolevulinic acid synthetase (ALA-S), rhodanese and microsomal heme oxygenase (MHO), were quantitated in Cl4C induced regenerating mouse liver. 2. Maximal hepatomegalia was observed at 48 hr after i.p. injection of a single dose of the toxin. 3. ALA-S activity decreased on day 2, and then significantly increased (50%) between days 3 and 7, returning afterwards to control values. 4. Cytoplasmic rhodanese, as well as MHO activities, exhibited a clear correlation as compared with the ALA-S activity profile. 5. Porphyrin biosynthesis from precursor delta-aminolevulinic acid (ALA) was significantly increased even after 15 days of intoxication. 6. Present results would indicate that Cl4C is acting in a dual fashion.

5-Aminolevulinate Synthetase↗

PubMedDate not supplied

S-adenosyl-L-methionine: its effect on aminolevulinate dehydratase and glutathione in acute ethanol intoxication.

The effect of disulfiram and S-adenosyl-L-methionine (SAM) administration to acute ethanol intoxicated mice on the hepatic glutathione (GSH) concentration and aminolevulinic and dehydratase (ALA-D) activity was investigated. It was found that both GSH levels and ALA-D activity were decreased, and evidence suggested that the toxic action of ethanol was due to its conversion into acetaldehyde. Administration of SAM reverses the effects of acute alcohol abuse by increasing liver GSH availability. In vitro, hepatic ALA-D activity was not modified by ethanol; instead it was non-competitively inhibited by acetaldehyde. This inhibition was efficiently reversed by GSH and cysteine (CySH). Therefore, a mechanism for the action of ethanol on ALA-D, based on the inhibitory effect of acetaldehyde, is proposed.

Acetaldehyde↗

PubMedDate not supplied

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.

Amino Acids↗

PubMedDate not supplied

Yeast porphobilinogen deaminase also forms enzyme-pyrrole intermediates.

The enzyme porphobilinogen deaminase (PBG deaminase, EC 4.3.1.8) catalyzes the condensation of four molecules of PBG to give the linear tetrapyrrol, hydroxymethylbilane. It has been shown that this enzyme forms stable mono-, di-, tri- and tetrapyrrole-enzyme covalent complexes. When the enzyme, partially purified in the absence or presence of phenylmethylsulfonyl fluoride (PMSF) and preincubated with PBG, was applied on DEAE-cellulose columns, three peaks with PBG deaminase activity were detected. Using Ehrlich's reagent, it was found that the active peaks corresponded to mono-, di- and tri-pyrrylmethane-enzyme complexes. Therefore, the mechanism of action of PBG deaminase from Saccharomyces cerevisiae also involves the sequential addition of four PBG units, leading to the formation of the enzyme-substrate intermediate complexes, as has already been described for the same enzyme from other sources.

Chromatography, DEAE-Cellulose↗

PubMedDate not supplied

Studies on porphobilinogen-deaminase from Saccharomyces cerevisiae.

Porphobilinogen-deaminase from Saccharomyces cerevisiae has been isolated and partially purified 80- and 230-fold in the absence or presence of phenylmethylsulphonyl fluoride, respectively. Some properties of the isolated enzyme were studied. Porphyrin formation was linear with time and protein concentration. Optimum pH was about 7.5-7.8. Molecular mass of the protein was 30,000 +/- 3000 Dalton when the enzyme was purified in the presence of phenylmethylsulphonyl fluoride. A less active and unstable 20,000 Da molecular mass species was obtained when purification was performed in the absence of the protease inhibitor. Porphobilinogen-deaminase exhibited classical Michaelis-Menten kinetics. The apparent Km for uroporphyrinogen formation was 19 microM; Vmax was 3.6 nmol uroporphyrin/h and the Hill coefficient was n = 1. Also the action of several reagents on the activity was studied. Protective thiol agents had no effect. Heavy metals inhibited both porphyrin formation and porphobilinogen consumption, but known sulphydryl inactivating chemicals inhibit the former without modifying the latter. Ammonium ions had no effect on the activity while hydroxylamine completely inhibited both porphyrin formation and porphobilinogen consumption.

Chromatography, Gel↗

PubMedDate not supplied

Studies on uroporphyrinogen biosynthesis in pig liver.

Porphobilinogen-deaminase (PBG-D) and PBG-D-isomerase complex (PBG-D-I) from pig liver were isolated and partially purified. Uroporphyrinogen I and III formation was found to be linear with time and protein concentration. Optimal pH was about 7.4 and 7.6-7.8 for PBG-D and PBG-D-I complex, respectively. Some properties of the isolated enzymes were studied. Molecular mass determination gave a value of 40,000 Da for PBG-D and 50,000 Da for the complex. Both enzymes exhibited classical Michaelis-Menten kinetics. Km and Vmax parameters were estimated. The effect of several divalent cations, ammonia and thiol reagents was also investigated. The differential action of some of these chemicals on PBG-D and PBG-D-I system would suggest that PBG-D and isomerase may not be only physically adjacent but actually associated.

Ammonia↗

PubMedDate not supplied

Non-essential activation of rat liver porphobilinogen-deaminase by folic acid.

This report demonstrates the ability of folic acid to activate rat liver porphobilinogen-deaminase (PBG-D). Lineweaver-Burk analysis revealed an increase in Vmax (38%) without affecting the Km. In the concentration range assayed, secondary replots of 1/delta slope and 1/delta intersect versus 1/[folic acid] yielded straight lines, indicating the binding of a single molecule of activator to the enzyme PBG-D, with a KA = 1.66 mM. Results presented here show that folic acid acts as a non-essential activator (alpha = 1; beta = 1.6). The activating effect of folic acid has been observed employing the 35-70% ammonium sulphate precipitated fraction, desalted by dialysis or gel filtration, whereas no action was detected when other partially purified PBG-D preparations were utilized as the enzyme source, suggesting either the presence of sites saturated for the activator, or the existence of a different structural protein conformation, or both.

Animals↗

PubMedDate not supplied

[Saccharomyces cerevisiae: porphobilinogenase activity in a wild-type strain and its heme-deficient mutant].

Properties of Porphobilinogenase (PBGase), the enzyme complex converting porphobilinogen (PBG) into uroporphyrinogens, were comparatively studied in a wild strain D273-10B and its mutant B231 of Saccharomyces cerevisiae, Figure 1 shows the growth curves for both strains. The basic pattern of growth was observed but, although S. cerevisiae is a facultative aerobe and was grown on dextrose, a diauxic growth curve was not observed. The beginning of the exponential phase was slightly delayed for the mutant, so, its generation time (G = 3.20 h) was greater than that for the wild strain (G = 1.26 h). Optimum conditions for extracting the enzyme from both strains were found to be sonication at 10 mu for 3 min (Table 1). Table 2 shows the effect of centrifugation at 24,000 xg for 30 min on activity. For both strains the amount of porphyrins formed was the same either in the absence or presence of air. It was found (Figure 2) that urogen formation was linear with protein over a wide range of concentrations and with incubation time up to 2h in agreement with previous results for the enzyme of different sources. Figure 3 shows the effect of pH on PBGase activity. An optimum pH of 7.4 was found for both strains employing sodium phosphate buffer pH 8.0. The shape of the pH curve as well as optimum pH were the same in both Tris-HCl and phosphate buffer, however PBGase was 15% less active in the former. When plots of velocity against PBG concentration were analyzed for PBGase, it was found that measuring the rate of the reaction on the basis of total urogen formation, saturation curves for wild and mutant strains harvested at the exponential phase, followed classical Michaelis-Menten kinetics. Saturation was reached at PBG concentration of about 70-90 microM. Therefore, double reciprocal plots (Figure 4) were linear and from these plots apparent Km's values of 20 and 14 microM were obtained for the wild and mutant strain respectively. It is known that in some organisms, the activity of the enzyme of heme synthesis is significantly influenced by the days of growing; therefore the effect of time growing on PBGase activity was studied (Figure 5). A well defined maximum of enzyme activity was observed for the mutant strain after 20h of growing; while activity of wild strain did not significantly vary during growth.

Aerobiosis↗