Porphyrin biosynthesis. VIII. Avian erythrocyte porphobilinogen deaminase-uroporphyrinogen III cosynthetase, its purification, properties and the separation of its components

1. 1. A method for the isolation and purification of porphobilinogenase, porphobilinogen deaminase and uroporphyrinogen isomerase from avian erythrocytes is described. 2. 2. Some properties of the isolated enzymes were studied. The optimal pH for porphobilinogenase and deaminase was 7.4. Purified porphobilinogenase was resolved into three bands on starch gel electrophoresis. The molecular weight of the purified enzymes was determined by gel filtration. The presence of porphobilinogen or NH4 + at certain concentrations afforded protection against heat inactivation of isomerase, the heat labile enzyme. Initial porphyrin formation by porphobilinogenase was linear with time. 3. 3. The action of various compounds added to the system was studied. Thiol reagents inhibited both porphobilinogenase and deaminase, indicating the presence of thiol groups essential for activity. NH4 +, hydroxylamine, adenine, ADP, ATP, some dicarboxylic acids and 2-methoxy-5-nitrotropone inhibited deaminase. © 1971.Fil:Llambías, E.B.C. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Del C. Batlle, A.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Porphyrin biosynthesis in soybean callus. V. The porphobilinogen deaminase-uroporphyrinogen cosynthetase system. Kinetic studies

Kinetic studies were carried out using purified porphobilinogenase and deaminase preparations in the presence and absence of ammonium ions. It has been found in plots of v versus [S] that a deviation from the Michaelis-Menten hyperbola occurs with both enzymes; double-reciprocal plots were concave downward; Rs values were greater than 81; and in some cases the Hill coefficient was less than 1, indicating negative homotropic kinetics. Evidence also suggested that porphobilinogenase contains at least two substrate-binding sites per molecule of enzyme. It has also been found that ammonium ions act competitively on the first reaction of the porphobilinogenase. © 1970.Fil:Llambías, E.B.C. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Del C. Batlle, A.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Thiol groups of soybean callus succinyl CoA synthetase

1. 1. The reaction between 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) and the SH groups of soybean callus succinyl-CoA synthetase has been investigated. At pH 8-6 and 25° C, four SH groups are titrable with DTNB in the native enzyme. No additional thiol groups have been revealed after unfolding of the protein with 8 M urea. 2. 2. The loss of enzymatic activity paralleled the decrease in the number of free SH groups. As with p-mercuribenzoate and other mercurials, reaction with DTNB also resulted in dissociation of the enzyme into subunits. © 1974.Fil:Del C. Batlle, A.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Porphyrin biosynthesis in Rhodopseudomonas palustris-XII. δ-aminolevulinate synthetase switch-off/on regulation

The high levels of δ-aminolevulinate synthetase (ALA-S) in Rhodopseudomonas palustris cells grown anaerobically in the light (Ph) decrease to those found in cells grown aerobically in the dark (A), when the former cultures were vigorously oxygenated; simultaneously bacteriochlorophyll (Bchl) synthesis abruptly halted leading to diminished steady-state specific Bchl content. When flushing oxygen was interrupted, enzymic activity increased, whether chloramphenicol was present or not in the medium; if the protein synthesis inhibitor was added when oxygenation started, ALA-S declined in the same fashion as in its absence, but thereafter reactivation of the enzyme was lower than before. Succinyl-CoA-synthetase and ALA-dehydratase activities were also measured under the conditions described, and no changes at all have been observed. The existence of different forms of ALA-S in R. palustris depending on growth conditions is postulated along with the formation of low molecular weight factors which can modulate ALA-S activity by binding to the enzyme; a widespread mechanism in the adaptation of micro-organisms to changes in environment. It is also proposed that this particular regulatory phenomenon, could be referred to as a switch off/on mechanism controlling ALA-S activity in R. palustris. © 1987.Fil:Viale, A.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Del C. Batlle, A.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

δ-aminolaevulinate synthetase in extracts of cultured soybean cells

1. 1. δ-Aminolaevulinate synthetase has been detected in extracts of soybean callus tissues and the enzyme activity reached its maximum when callus were 11 days old. 2. 2. The presence of a compound which seems to control δ-aminolaevulinate synthetase activity was demonstrated. The enzyme was present in the soluble fraction and was very labile. 3. 3. When crude extracts or 500 × g supernatant were stored at 4-6°, the apparent activity of δ-aminolaevulinate synthetase increased by as much as 3-6 times, while the activities of δ-aminolaevulinate dehydratase and succinyl-CoA synthetase did not significantly change during the storage. Activation was dependent on concentrations of cells suspensions during disruption and aging. 4. 4. Gel filtration with Sephadex G-25 of 2000 × g supernatants produced an enzyme fraction 30% more active. An increase in enzyme activity was observed when dark-grown callus were exposed to light. 5. 5. The addition of ATP, gibberellic acid and δ-aminolaevulinate to the culture media diminished activity; iron deficiency also produced an δ-aminolaevulinate synthetase less active. © 1971.Fil:Del C. Batlle, A.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Tigier, H.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Tissue distribution and kinetics of endogenous porphyrins synthesized after topical application of ALA in different vehicles

The use of 5-aminolaevulinic acid (ALA) is gaining increasing attention for photosensitization in photodynamic therapy of superficially localized tumours. The aim of this work was to determine the kinetics of porphyrin generation in tissues after topical application of ALA delivered in different vehicles on the skin overlying the tumour and normal skin of mice. Maximal accumulation was found in tumour 3 h after ALA application in both cream and lotion preparations. Normal and overlying tumour skin tissues showed different kinetic patterns, reflecting histological changes when the latter is invaded by tumour cells. Liver, kidney, spleen and blood porphyrins also raised from basal levels, showing that ALA and/or ALA-induced porphyrins reach all tissues after topical application. During the first 24 h of ALA topical application, precursors and porphyrins are excreted by both urine and faeces. ALA lotion applied on the skin overlying the tumour induced higher accumulation of tumoural porphyrins than cream, and lotion applied on normal skin appeared to be the most efficient upon inducing total body porphyrins. This work has demonstrated the great influence of the formulation of ALA vehicle on penetration through the skin. Knowledge of the kinetics of porphyrin generation after different conditions of ALA application is needed for the optimization of diagnosis and phototherapy in human tumours. © 1999 Cancer Research Campaig

Porphyrin biosynthesis in Rhodopseudomonas palustris-IX. PBG-deaminase. Kinetic studies

1. 1. PBG-Deaminase obtained from Rp. palustris exhibited classical Michaelis-Menten kinetics in the absence or presence of different ions. 2. 2. Detailed kinetic studies were carried out in the presence of ammonium, phosphate and magnesium ions. 3. 3. It has been found that the different effects observed are dependent on both the substrate and the ion concentration. © 1987.Fil:Kotler, M.L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Fumagalli, S.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Juknat, A.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Batlle, C. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Porphyrin biosynthesis in rhodopseudomonas palustris-V. Purification of porphyrinogen decarboxylase and some unusual properties

1. 1. Uroporphyrinogen decarboxylase (EC 4.1.1.37) has been purified 16-fold from Rp. palustris to a specific activity of 210 nmol of total decarboxylated porphyrinogens III formed/hr per mg of protein and about 50% yield. The Rp. palustris enzyme exhibits some unusual properties as compared with URO-D from other sources. 2. 2. The purified enzyme is a monomer with a molecular weight of ∼46,000, an isoelectric point of 4.6 and an optimum pH of 6.9 and 6.8 with urogen III and I substrate. Neither GSH nor EDTA seem to be necessary for activity, and the decarboxylation rate and the distribution of the reaction products was not affected either by the presence or absence of oxygen. 3. 3. The Rp. palustris enzyme is a thermo-stable protein, heating at 60°C for 15 min enhanced several times activity. This is the first time that the heat treatment is included as one of the steps to purify URO-D. 4. 4. Thermal activation followed an identical profile using either substrate. The ratios of specific activity for the type III and I isomer of urogen remained constant throughout the purification. These findings are indicating that a single enzyme catalyzes the four decarboxylations occurring from urogen to coprogen. 5. 5. Kinetic data employing urogen III and I as substrate showed that the pattern of accumulated intermediates was rather different depending on whether type III or I isomer was used. 6. 6. While decarboxylation of urogen III responds to the usual scheme: {A figure is presented} where v1≫v2 and decarboxylation of heptagen III is the rate-controlling step. 7. 7. Decarboxylation of urogen I revealed a completely different and characteristic picture fitting the scheme: {A figure is presented} where again v′1≫v′2 and the removal of the final carboxyl group from pentagen I becomes the rate-limiting step. © 1986.Fil:Juknat de Geralnik, A.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:del C. Batlle, A.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Heme regulation in mouse mammary carcinoma and liver of tumor bearing mice-I. Effect of allyl-isopropylacetamide and veronal on δ -aminolevulinate synthetase, cytochrome P-450 and cytochrome oxidase

1. 1. Basal levels and allyl-isopropylacetamide (AIA) or veronal induced levels of δ-aminolevulinate synthetase (ALA-S), cytochrome P-450 (cyt P-450) and cytochrome oxidase were determined in tumor (T) and liver of both normal mice (NM) and T bearing mice (TBM). 2. 2. Basal levels of ALA-S were nearly the same in either source. The amount of cyt P-450 was lower in TBM liver than in NM liver, and no detectable in T. While the basal activity of cytochrome oxidase in TBM liver and T were higher than those of NM liver. 3. 3. In AIA intoxicated animals there was a lower induction of ALA-S in liver of TBM than in NM liver. There was no induction in T ALA-S. The loss of cyt P-450 was less in TBM liver when compared with NM liver. 4. 4. The induction level of cyt P-450 after veronal administration was nearly the same in liver of both TBM and NM. 5. 5. We conclude that lower induction of liver ALA-S activity in TBM liver is due to correspondingly lower drug metabolism ability of TBM liver. Otherwise our results suggest that the control mechanism operating in T and probably in its original tissue are different from those described for normal liver. © 1990.Fil:Del C. Batlle, A.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Porphyrin biosynthesis in the soybean callus tissue system-XV. The effect of growth conditions

1. 1. Effects of various factors on chlorophyll, porphyrin and protein content, growth and on the activities of the enzymes involved in the earlier stages of tetrapyrrole synthesis, in cultured soybean cells, were studied. 2. 2. When dark-grown callus was exposed to light, it was found that the amount of porphyrins formed was not altered, but chlorophyll content as well as Succinyl CoA Synthetase (Suc.CoA-S), Aminolevulic Acid Synthetase (ALA-S), Aminolevulic Acid Dehydratase (ALA-D) and Porphobilinogenase (PBGase) activities increased. 3. 3. Addition of Aminolevulic Acid (ALA) to the medium culture, was found to stimulate porphyrin accumulation and to prevent growth; however chlorophyll content was not significantly modified. ALA-S was inhibited while both ALA-D and PBGase activities were enhanced. The action of puromycin and mitomycin added along with ALA to the media, was also studied, but neither of these inhibitors modified much the effects produced by ALA. 4. 4. Addition of Porphobilinogen (PBG), showed accumulation of uroporphyrin in the tissue; except inhibition of ALA-S, enzymes activities, protein and chlorophyll content were not modified. Evidence obtained would indicate that callus tissue was not permeable to PBG. 5. 5. Omission of iron from the culture medium, produced porphyrin accumulation and prevented growth. It has been consistently found that, the higher the content of porphyrins, the less the callus growth. Coproporphyrin was the major component of the porphyrins formed in ALA supplemented or iron deficient media. ALA-S and ALA-D were reduced under iron deficiency. 6. 6. The addition of ATP to the media, did not affect porphyrin, protein, and chlorophyll synthesis, growth or ALA-D, but Suc.CoA-S, ALA-S and PBGase activities diminished. 7. 7. Gibberelic acid produced a measurable increase of PBGase, while diminished Suc.CoA-S and ALA-S. 8. 8. Succinate increased growth and inhibited ALA-S and ALA-D. 9. 9. Carbonyl cyanide m-chlorophenyl hydrazine (CCCP), added to the medium produced accumulation of porphyrins, consequently, ALA-S was greatly inhibited and growth prevented. PBGase was also diminished. 10. 10. Coproporphyrinogenase and Decarboxylases activities were hardly detected in most experiments, and are limiting. 11. 11. The complex pattern of results obtained is discussed. © 1975.Fil:Del C. Batlle, A.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Llambias, E.B.C. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Tigier, H.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina