Δ-aminolaevulinate Synthetase Research Articles

Red blood cell rhodanese: Its possible role in modulating δ-aminolaevulinate synthetase activity in mammals

1. 1. The optimum conditions for measuring rhodanese activity in human erythrocytes were established. The mean control values for males (112 nmol SCN/30 min/mg protein) and females (127 nmol SCN/30 min/mg protein) were determined. 2. 2. Rhodanese activity was measured in different porphyric patients. The activity was diminished in porphyria cutanea tarda (PCT), acute intermittent porphyria (AIP), variegate porphyria (VP) and lead intoxication (Pb), remaining in erythropoietic protoporphyria (EPP). 3. 3. δ-Aminolaevulinate synthetase (ALA-S) activity was increased in PCT, AIP, VP and Pb showing no changes in EPP. 4. 4. It is suggested that a similar scheme, to that proposed for the control of ALA-S in Rhodopseudomonas spheroides and soybean callus, is also operating in animals.

Porphyrin biosynthesis—immobilized enzymes and ligands. IX. Studies on δ-aminolaevulinate synthetase from cultured soybean cells

Soybean callus δ-aminolaevulinate synthetase (ALA-S) has been covalently attached to Sepharose 4B. The optimal conditions for binding have been determined. The water-insoluble ALA-S retained 40% of the activity of the original soluble preparation, the coupling yield was also high. Sepharose — ALA-S could be stored at 4°C for periods up to 40 days with only 25% loss of activity and it could be repeatedly used with little alteration of its enzymic activity. pH optima of the free and bound enzyme were the same.

Drug metabolism in cell culture. II. Studies on the metabolic route of DDC analogues.

To elucidate the major metabolic pathways of DDC analogues, the effect of inhibitors of oxidative and hydrolytic microsomal enzymes on the rate of metabolism of these compounds in chick embryo liver cell cultures has been examined. The metabolism of 3,5-diethoxycarbonyl-2,4,6-trimethylpyridine (Ox-DDC) was inhibited threefold by piperonyl butoxide and SKF-525A (β-diethylaminoethyl-diphenyl-n-propylacetate-HCl), but only onefold by bis(p-nitrophenyl)phosphate (BNPP). This indicates that this compound is metabolized predominately by an oxidative route and hydrolysis is a minor pathway. The half-life of 3,5-diethoxycarbonyl-2,6-dimethylpyridine (4-desmethyl-Ox-DDC) was increased 20-fold by BNPP, but no change was observed with piperonyl butoxide, indicating that this compound is metabolized exclusively by hydrolytic mechanisms. Since both piperonyl butoxide and BNPP produced similar changes in the half-life of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC), it appears that this compound is metabolized equally via oxidative and hydrolytic mechanisms. In contrast to the intact embryo where Ox-DDC is rapidly metabolized and the effect of the agent on δ-aminolevulinic acid (δ-ALA) synthetase (δ-aminolaevulinate synthetase; EC 2.3.1.37) activity is brief and hence porphyrin accumulation does not occur, in cell culture Ox-DDC is metabolized at a rate sufficiently slow so that induction of δ-ALA synthetase does occur.

Rat liver changes after subchronic exposition to polychlorinated biphenyls (PCB)of low chlorine content

Liver changes were studied after subchronic feeding a diet containing 2000 ppm PCB [chlorine content 42% corresponding to trichlorobiphenyls (triCB)]. The products differed in their content of highly chlorinated biphenyls (≥Cl5-biphenyl; 5%=triCB-5, 2%=triCB-2 and 0.4%=triCB-0.4). The most striking difference was observed in respect to their porphyrinogenic actions. TriCB-0.4 and triCB-2 caused an increase of porphyrins about 10-fold, whereas the liver porphyrin content of triCB-5 treated rats was 175-fold higher than normal. In all cases of porphyria the porphyrins consisted mainly of uro- and heptacarboxyporphyrin. In contrast to their different porphyrinogenic effects all triCB products caused an equal induction of δ-aminolaevulinate synthetase, but no increase of δ-aminolaevulinate dehydratase activity.

Effect of pyridoxal phosphate and cysteine on δ-aminolaevulinate synthetase and dehydratase in soya

The specific activity of ALA-S extracts prepared from lyophilized soya callus growing in light or dark showed a striking increase when tissue cultures were grown in the presence of pyridoxal phosphate (PyP) in order to complex aminothiols. By contrast ALA-D specific activity in the light grown callus was reduced on incorporation of PyP into the growth medium. Cysteine (Cy) added to the culture medium did not influence the specific activities of either enzyme.

Activities of haem synthetic enzymes in blood cells of pre-natal flexed-tailed (f/f) anaemic mice

Levels of activity of beta-aminolaevulinate synthetase, delta-aminolaevulinate dehydratase, porphyrinogen synthetase and haem synthetase in circulating reticulocytes of pre-natal FL4/Re +/+ and congenitally anaemic FL1/Re f/f mice have been determined. The activities of delta-aminolaevulinate synthetase and delta-aminolaevulinate dehydratase were found to be decreased in proportion to the hypochromia observed in mature liver-derived erythrocytes in neonatal f/f mice, but activities of enzymes later in the haem synthetic pathway were relatively undisturbed. No significant differences were found in levels of haem synthetic enzymes in foetal livers of +/+ and f/f mice. These results indicate that the severe anaemia of late prenatal and neonatal f/f mice is due both to reduction in haem synthesis expressed at the reticulocyte stage of erythroid differentiation and to restricted production of erythroid progenitor cells. Retarded foetal growth and skeletal abnormalities, both characteristic of the pre-natal expression of the f/f gene complement, can also be related to reduced levels of haem synthesis, but the abnormal distribution of pigment cells seen in f/f animals appears to be a secondary effect of reduced tissue oxygenation resulting from pre-natal anaemia.

Biochemical effects of 3,5-diethoxycarbonyl-1, 4-dihydrocollidine in mouse liver

3,5-Diethoxycarbonyl-1,4-dihydrocollidine (DDC) is a porphyrinogenic agent and is a powerful inducer of δ-aminolaevulinate synthetase, the first and rate-limiting enzyme of the haem-biosynthetic pathway, in mouse liver. However, DDC strikingly inhibits mitochondrial as well as microsomal haem synthesis by depressing the activity of ferrochelatase in vivo. The drug on repeated administration to female mice has been found to elicit hypertrophic effects in the liver microsomes initially, but the effects observed at later stages denote either hyperplasia or increase in polyploidal cells. The microsomal protein concentration shows a striking decrease with repeated doses of the drug. The rate of microsomal protein synthesis in vivo as well as in vitro shows an increase with two injections of DDC but decreases considerably with repeated administration of the drug. The activities of NADPH-cytochrome creductase and ribonuclease are not affected in the liver microsomes of drug-treated animals when expressed per mg of microsomal protein. DDC has also been found to cause degradation of microsomal haem, which is primarily responsible for the decrease in cytochrome P-450 content. The drug also leads to a decrease in mitochondrial cytochrome c levels due to inhibition of haem synthesis and also due to degradation of mitochondrial haem at later stages. The biochemical effects of the drug are compared and discussed with those reported for allylisopropylacetamide and phenobarbital.

Induction of delta-aminolaevulinate synthetase under environmental-stress conditions.

1. Exposure of rats to environmental-stress conditions of hypobaria, hypoxia and cold did not alter the activity of hepatic delta-aminolaevulinate synthetase. 2. Induction of the enzyme by diethoxycarbonyldihydrocollidine was inhibited when the rats were exposed to hypobaria before or during the treatment with the drug but not after the initial phase when the process of induction was initiated. Neither increased concentration of the drug nor the time of induction had any effect on the inhibition under hypobaria. 3. A period of 12-24h of pre-exposure to hypobaria gave the maximum inhibition, and on longer exposure the inhibitory effect was decreased. 4. The inhibition was not a permanent effect and could be substantially reversed in 12h of withdrawal to ambient pressure. 5. Inhibition of induction was found under hypobaria and hypoxia, but not on exposure to cold. This suggests a specific effect of lack of O(2) rather than a general effect of stress. 6. It appears possible that alteration of concentration of endogenous adenine nucleotides may control the process of diethoxycarbonyldihydrocollidine-mediated induction of delta-aminolaevulinate synthetase, since treatment with ATP, cyclic AMP or theophylline produced inhibition similar to that under hypobaria and hypoxia.

The use of exogenous δ-aminolaevulinic acid for the studies of the regulation of haem synthesis in rabbit reticulocytes

δ-Amino [4- 14C]laevulinate added to reticulocytes incubated in vitro is incorporated into haem. Exogenous δ-aminolaevulinate restores the incorporation of 59Fe into haem in reticulocytes which had been treated with isonicotinic acid hydrazide (INH) or penicillamine and were hence unable to synthesize δ-aminolaevulinate. On the other hand, the addition of δ-aminolaevulinate does not restore the incorporation of Fe into reticulocytes incubated with haemin. The inhibition of the incorporation of iron is neither restored by δ-aminolaevulinate in reticulocytes incubated with cycloheximide (which inhibits globin synthesis and thus elevates the free intracellular haem pool). These results suggest that in intact reticulocytes haemin does not inhibit δ-aminolaevulinate synthetase. This conclusion is further supported by the finding that the pattern of incorporation of [2- 14C]glycine and δ-amino[4- 14C]-laevulinate into haem differs in reticulocytes incubated with an inhibitor of δ-aminolaevulinate synthetase (INH) and in reticulocytes incubated with haemin and cycloheximide.

The effect of exogenous -aminolaevulinate on rat liver haem and cytochromes.

The activity of delta-aminolaevulinate synthetase is generally regarded as rate-limiting for hepatic haem biosynthesis. It has been suggested that cytochrome synthesis may also be regulated by changes in delta-aminolaevulinate synthetase activity. This hypothesis was studied by injecting product, delta-aminolaevulinate, into adult rats over a 4-240h period. The concentrations of hepatic mitochondrial cytochromes a, b, c and c(1) were unchanged by treatment with delta-aminolaevulinate, allylisopropylacetamide or phenobarbital. In control animals, total microsomal haem content equalled the sum of cytochromes b(5) plus P-450. After delta-aminolaevulinate administration the total amount of microsomal haem, measured as the pyridine haemochromogen, exceeded these components, indicating the formation of a ;free' haem pool. Haem synthesis does not appear rate-limiting for hepatic cytochrome synthesis in the adult rat.

Effect of hexachlorobenzene on haem synthesis.

Several drugs are known to induce the liver microsomal mixed-function oxidase system when administered in vivo or even in vitro in cell culture. A sequence of events has been suggested in which the drug is visualized to induce delta-aminolaevulinate synthetase, the first and rate-limiting enzyme of the haem-biosynthetic pathway, which is followed by enhanced haem synthesis and cytochrome P-450 content, facilitating the increase in the drug-metabolizing activity of the liver microsomal fraction. The present studies show that the fungicide hexachlorobenzene, when administered to female rats, can lead to enhanced amounts and rate of synthesis of cytochrome P-450 under conditions when the rate of total haem synthesis has not appreciably altered. The subsequent increase in the rate of total haem synthesis as well as the initial increase in amounts of cytochrome P-450 are brought about under conditions when delta-aminolaevulinate synthetase activity remains constant. However, manifestation of porphyria due to prolonged drug administration is accompanied by a twofold increase in delta-aminolaevulinate synthetase activity. The increase in enzyme activity appears to be due to a decreased degradation rate of the enzyme.

The relationship between delta-aminolaevulinate synthetase induction and the concentration of cytochrome P-450 and catalase in rat liver.

The porphyrinogenic drug 2-allyl-2-isopropylacetamide causes the degradation of microsomal cytochrome P-450 and inhibits the synthesis of catalase in rat liver. The inhibition of catalase synthesis follows the induction of delta-aminolaevulinate synthetase and the consequent overproduction of haem. The allylisopropylacetamide-mediated breakdown of cytochrome P-450 is a rapid event and has a reciprocal relationship to the pattern of delta-aminolaevulinate synthetase induction. Breakdown of cytochrome P-450 appears to be one of the conditions leading to the ;derepression' of delta-aminolaevulinate synthetase.

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.

The effect of carbohydrate on δ-aminolaevulinate synthetase: The role of ribonucleic acid

1. Studies are presented which indicate that the induction of δ-aminolaevulinate synthetase by allylisopropylacetamide or ethionine may be inhibited by the administration of metabolisable sugars. This effect is demonstrable both in vivo and in vitro. 2. The δ-aminolaevulinate synthetase-inducing effect of hepatic RNA from an animal fed allylisopropylacetamide or ethionine is considerably less if the animal is fed glucose at the same time. 3. It is suggested that the inhibitory effects of carbohydrates on the induction of the enzyme δ-aminolaevulinate synthetase is mediated through an RNA mechanism and may be due to an interference with the coding of messenger RNA for δ-aminolaevulinate synthetase.

Characterization and measurement of Δ-aminolaevulinate synthetase in bone marrow cell mitochondria

1. 1. Δ-Aminolaevulinate synthesis was demonstrated in normal rabbit bone marrow and the measurement of Δ-aminolaevulinate synthetase (succinyl-CoA: glycine succinyltransferase) was accomplished in mitochondria obtained from marrow cells ruptured by sonication. The micromethod used to quantify Δ-amino laevulinate was sensitive to the range of 10 −9 mole. The optimal concentrations of glycine, α-ketoglutarate, CoA, pyridoxal phosphate and NAD + for maximum enzyme activity at pH 7.5 were identified. Succinate and succinyl-CoA were less effective as substrates than α-ketoglutarate. Neither MgCl 2 nor EDTA stimulated enzyme activity. Inhibition of the enzyme by its product, Δ-aminolaevulinate, was observed. Neither heme nor protoporphyrin inhibited the production of Δ-aminolaevulinate. 2. 2. Electronmicroscopic study of the mitochondrial preparation disclosed that the mitochondria were partially damaged. The isolated mitochondria contained succinate dehydrogenase (EC 1.3.99.1) and ferrochelatase (EC 4.99.1.1) activities. The soluble enzymes of the heme synthetic pathway were absent. 3. 3. The rate of production of Δ-aminolaevulinate observed in the present experiments approximates the rate which would be required in vivo by the normal rabbit for hemoglobin synthesis.