5-aminolaevulinate Synthase Activity Research Articles

From chemistry to genomics: A concise history of the porphyrias.

We describe developments in understanding of the porphyrias associated with each step in the haem biosynthesis pathway and the role of individuals whose contributions led to major advances over the past 150 years. The first case of erythropoietic porphyria was reported in 1870, and the first with acute porphyria in 1889. Photosensitisation by porphyrin was confirmed by Meyer-Betz, who self-injected haematoporphyrin. Günther classified porphyrias into haematoporphyria acuta, acuta toxica, congenita and chronica. This was revised by Waldenström into porphyria congenita, acuta and cutanea tarda, with the latter describing those with late-onset skin lesions. Waldenström was the first to recognise porphobilinogen's association with acute porphyria, although its structure was not solved until 1953. Hans Fischer was awarded the Nobel prize in 1930 for solving the structure of porphyrins and the synthesis of haemin. After 1945, research by several groups elucidated the pathway of haem biosynthesis and its negative feedback regulation by haem. By 1961, following the work of Watson, Schmid, Rimington, Goldberg, Dean, Magnus and others, aided by the availability of modern techniques of porphyrin separation, six of the porphyrias were identified and classified as erythropoietic or hepatic. The seventh, 5-aminolaevulinate dehydratase deficiency porphyria, was described by Doss in 1979. The discovery of increased hepatic 5-aminolaevulinate synthase activity in acute porphyria led to development of haematin as a treatment for acute attacks. By 2000, all the haem biosynthesis genes were cloned, sequenced and assigned to chromosomes and disease-specific mutations identified in all inherited porphyrias. These advances have allowed definitive family studies and development of new treatments.

Photosynthesis gene expression in Rhodobacter sphaeroides is regulated by redox changes which are linked to electron transport

The effect of the respiratory electron acceptor nitrous oxide on the synthesis of the photosynthetic apparatus of Rhodobacter sphaeroides was examined. In phototrophically grown cells, the addition of nitrous oxide caused a reduction in the level of light-harvesting complex I and light-harvesting complex II under conditions of high light intensity (200 W m−2) and low light intensity (16 W m−2). 5-Aminolaevulinate synthase activity was decreased during growth in the presence of nitrous oxide and this limited production of spectral complexes since addition of exogenous 5-aminolaevulinic acid partially suppressed the effect of nitrous oxide. The effect of nitrous oxide on the expression of the operons encoding the pigment-binding proteins of light-harvesting complex I (puf), light-harvesting complex II (puc) and the two isoenzymes of 5-aminolaevulinate synthase (hemA and hemT) were measured using transcriptional fusions to lacZ. Nitrous oxide caused a decrease in puf and hemA transcription. However, there was an apparent increase in the expression of puc and hemT transcriptional fusions. The level of light-harvesting complexes in cells grown in the dark with different electron acceptors was also examined. Cells grown anaerobically with DMSO had a higher level of light-harvesting complexes than those grown anaerobically with nitrous oxide as electron acceptor. Cells grown aerobically had the lowest level of light-harvesting complexes. It is proposed that FnrL-dependent and FnrL-independent expression of photosynthesis genes is modulated by redox changes elicited by nitrous oxide respiration.

Erythroid 5‐aminolaevulinate synthase activity during normal and iron deficient erythropoiesis

Reduced erythroblast 5-aminolaevulinate (ALA) synthase activity was observed during iron/haem deficient erythropoiesis. Enzyme activity was reduced approximately threefold to levels similar to those previously detected during sideroblastic erythropoiesis. This response would appear to be erythroblast specific as haem deficiency is known to stimulate hepatic ALA synthase activity. It is, however, unclear as to whether this reduced enzyme activity relates to iron deficiency or to the consequent haem deficiency.

Measurement of 5-aminolaevulinate synthase activity by gas-liquid chromatography with electron-capture detection

A specific assay for 5-aminolaevulinate synthase activity is described, with a sensitivity comparable with that of radiochemical assays. It is based on measurement by g.l.c. with electron-capture detection of the pentafluorobenzyl ester of the ethyl acetoacetate pyrrole derivative of 5-aminolaevulinic acid, and of the corresponding compound from 6-amino-5-oxohexanoic acid used as internal standard. Enzyme activity has been measured in homogenates of rat liver, spleen, kidney and brain, and in human lymphocytes.

High-performance liquid chromatographic analyses of porphyrins in hamster Harderian glands

Porphyrin content adn 5-aminolaevulinate synthase activity of the Harderian gland were measured in intact and gonadectomised male and female hamsters; porphyrin profiles were analysed by high-pressure liquid chromatography. The total porphyrin content of the two female groups was similar, but enzyme activity in females ovariectomised for 20 weeks significantly decreased. Intact males have low porphyrin content and enzyme activity, while in castrates (6 weeks) both increased to female levels. Protoporphyrin IX formed 93% of total porphyrins in intact females, compared with 70% of total porphyriins in intact males. The remainder in both sexes was chiefly penta- and hexacarboxylic porphyrins and coproporphyrin and (in females) Harderoporphyrin. Gonadectomy in either sex resulted in protoporphyrin levels intermediate between male and female values.

Effects of acute ethanol administration on rat liver 5-aminolaevulinate synthase activity.

1. Liver 5-aminolaevulinate (ALA) synthase activity of 24 h-starved rats is maximally increased at 4 h after intraperitoneal administration of a 1.6 g/kg body wt. dose of ethanol. Larger doses cause a dose-dependent decrease in the extent of this stimulation, exhibiting a reciprocal relationship with an elevation of hepatic haem concentration, as suggested by the simultaneous increase in the haem saturation of tryptophan pyrrolase. 2. ALA synthase induction by ethanol is abolished if the above increase in pyrrolase saturation with haem is enhanced by theophylline, but is potentiated when the increase in the haem saturation is inhibited by anti-lipolytic agents. 3. ALA synthase induction by ethanol is also inhibited by inhibitors of alcohol dehydrogenase and aldehyde dehydrogenase. Acetaldehyde and acetate are, however, not responsible; they both decrease ALA synthase activity and increase the haem saturation of tryptophan pyrrolase. These latter effects of acetaldehyde are not mediated by acetate. 4. ALA synthase activity is also stimulated by succinate, which, however, also increases the haem saturation of tryptophan pyrrolase. 5. Ethanol does not influence the rate of ALA synthase degradation. 6. It is suggested that ethanol increases rat liver ALA synthase activity as a result of its own metabolism by the alcohol dehydrogenase-dependent pathway by a mechanism not involving decreased degradation of the former enzyme or the participation of the metabolites acetaldehyde and acetate.

Haem synthesis from exogenous 5-aminolaevulinate in cultured chick-embryo hepatocytes. Effects of inducers of cytochromes P-450.

The effects of inducers of cytochrome P-450 on haem biosynthesis from 5-aminolaevulinate were examined by using cultured chick-embryo hepatocytes. Cultures treated with either 2-propyl-2-isopropylacetamide or 3-methylcholanthrene contained increased amounts of cytochrome P-450 and haem. After treatment for 3 h with 5-amino[4-14C]laevulinate, the relative amounts of radioactivity accumulating as haem corresponded to the relative amounts of total cellular haem, but not to increases in the amounts of cytochrome P-450. Treatment with 5-aminolaevulinate did not alter cellular haem or cytochrome P-450 concentrations in either control or drug-treated cultures. The mechanism of the enhanced accumulation of radioactivity in haem was investigated. Although 2-propyl-2-isopropylacetamide enhanced the uptake of 5-aminolaevulinate and increased the cellular concentration of porphobilinogen 1.5-fold, these changes did not account for the increases in haem radioactivity. The inducing drugs had no effect on the rates of degradation of radioactive haem, but appeared to enhance conversion of protoporphyrin into haem. This latter effect was shown by: (1) a decreased accumulation of protoporphyrin from 5-aminolaevulinate in cells treated with inducers, and (2) complete prevention of this decrease if the iron chelator desferrioxamine was present. We conclude that inducers of cytochrome P-450 may increase haem synthesis not only by increasing activity of 5-aminolaevulinate synthase, but also by increasing conversion of protoporphyrin into haem.

Uroporphyrin accumulation produced by halogenated biphenyls in chick-embryo hepatocytes. Reversal of the accumulation by piperonyl butoxide.

Cultures of chick-embryo hepatocytes were used to study the mechanism by which 3,4,3',4'-tetrachlorobiphenyl and 2,4,5,3',4'-pentabromobiphenyl cause accumulation of uroporphyrin. In a previous paper, an isoenzyme of cytochrome P-450 induced by 3-methylcholanthrene had been implicated in this process [Sinclair, Bement, Bonkovsky & Sinclair (1984) Biochem. J. 222, 737-748]. Cells treated with 3,4,3',4'-tetrachlorobiphenyl and 5-aminolaevulinate accumulated uroporphyrin and heptacarboxyporphyrin, whereas similarly treated cells accumulated protoporphyrin immediately after piperonyl butoxide was added. Piperonyl butoxide also restored haem synthesis as detected by incorporation of radioactive 5-aminolaevulinate into haem, and decrease in drug-induced 5-aminolaevulinate synthase activity. The restoration of synthesis of protoporphyrin and haem by piperonyl butoxide was not affected by addition of cycloheximide, indicating recovery was probably not due to protein synthesis de novo. Piperonyl butoxide also reversed uroporphyrin accumulation caused by 3,4,5,3',4',5'-hexachlorobiphenyl, mixtures of other halogenated biphenyls, lindane, parathion, nifedipine and verapamil. The effect of piperonyl butoxide was probably not due to inhibition of metabolism of these compounds, since the hexachlorobiphenyl was scarcely metabolized. Other methylenedioxyphenyl compounds, as well as ellipticine and acetylaminofluorene, also reversed the uroporphyrin accumulation caused by 3,4,3',4'-tetrachlorobiphenyl. SKF-525A (2-dimethylaminoethyl-2,2-diphenyl valerate) did not reverse the uroporphyrin accumulation caused by the halogenated biphenyls, but did reverse that caused by phenobarbital and propylisopropylacetamide. We conclude that the mechanism of the uroporphyrin accumulation cannot be due to covalent binding of activated metabolites of halogenated compounds to uroporphyrinogen decarboxylase.

PORPHYRINOGENICITY OF ETOMIDATE AND KETAMINE AS CONTINUOUS INFUSIONS: Screening in the DDC-primed Rat Model

The porphyrinogenicity of etomidate and ketamine administered as continuous i.v. infusions was screened in the DDC-primed rat model of latent variegate porphyria. Ketamine produced no change from control in 5-aminolaevulinate synthase (ALAs) activity and haem intermediate production in either untreated or DDC-primed rats, and would appear to be safe for use in the patient with genetic porphyria. Etomidate, while producing no significant changes in these parameters in untreated rats, caused a statistically significant 47% increase in hepatic ALAs activity with a corroborative 85% increase in coproporphyrin and a 40% increase in protoporphyrin content, in DDC-primed rats. On these grounds, etomidate must be regarded as potentially porphyrinogenic when administered as a continuous infusion for total i.v. anaesthesia.

Modified uroporphyrinogen decarboxylase activity in a yeast mutant which mimics porphyria cutanea tarda

The isolation of a new mutant Sm1 strain of yeast, Saccharomyces cerevisiae, is described: this strain was partially defective in haem formation and accumulated large amounts of Zn-porphyrins. Genetic analysis showed that the porphyrin accumulation was under the control of a single nuclear recessive mutation. Biochemical analysis showed that the main porphyrins accumulated in the cells were uroporphyrin and heptacarboxyporphyrin, mostly of the isomer-III type. The excreted porphyrins comprised mainly dehydroisocoproporphyrin. Analysis of uroporphyrinogen decarboxylase activity in the cell-free extract revealed a 70-80% decrease of activity in the mutant and showed that the relative rates of the different decarboxylation steps were modified with the mutant enzyme. A 2-3-fold increase in 5-aminolaevulinate synthase activity was measured in the mutant. The biochemical characteristics of the Sm1 mutant are very similar to those described for porphyria cutanea tarda.

Iron loading of cultured hepatocytes. Effect of iron on 5-aminolaevulinate synthase is independent of lipid peroxidation.

Cultured chick embryo hepatocytes were iron-loaded with ferric nitrilotriacetate. Iron-loading was confirmed by both quantitative cellular iron determinations and ultrastructural studies. With iron-loading, lipid peroxidation, as detected by malonaldehyde released into the medium, occurred at a linear rate for 12h, after which time the rate of malonaldehyde production decreased. No cell toxicity, as detected by lactate dehydrogenase release, was noted. The amount of malonaldehyde recovered in the medium after 18h of exposure to iron represented 24-33% of the total malonaldehyde that could be produced by incubating lysed cells with iron and ascorbate. Cellular glutathione was not affected by iron-stimulated lipid peroxidation, but was increased by allylisopropylacetamide. Although iron-loading by itself had no effect on activity of 5-aminolaevulinate synthase, the first and rate-limiting step in haem synthesis, iron-loading in the presence of the porphyrogenic drug allylisopropylacetamide increased levels of 5-aminolaevulinate synthase 6-fold over levels induced by the drug alone. The antioxidant, butylated hydroxytoluene, totally inhibited iron-stimulated lipid peroxidation, but did not interfere with the effect of iron-loading to potentiate an increase in 5-aminolaevulinate synthase. After 18h of exposure to iron, followed by a change to fresh medium, the iron remaining within the cells did not stimulate further lipid peroxidation over the following 18h, but did potentiate an increase in 5-aminolaevulinate synthase on exposure to allylisopropylacetamide. It therefore appears that lipid peroxidation is not the mechanism by which iron potentiates induction of hepatic 5-aminolaevulinate synthase.

Does naloxone always act as an opiate antagonist?

Evidence for the ability of the opiate antagonist naloxone to block a variety of metabolic effects exerted by morphine and non-opiate drugs is reviewed. Naloxone prevents or reverses the following effects in the rat: (a) the chronic morphine-induced increase in liver [NADPH]; (b) the consequent chronic morphine-induced inhibition of liver tryptophan pyrrolase activity; (c) the resultant chronic morphine-induced enhancement of brain 5-hydroxytryptamine synthesis; (d) the similar effects on liver and brain tryptophan metabolism exerted chronically by other drugs of dependence (ethanol, nicotine and phenobarbitone); (e) the acute ethanol-induced increase in the hepatic [NADH]/[NAD] ratio. Naloxone also (f) inhibits basal and stimulated lipolysis in fed and 24hr-starved rats. This leads to prevention of (g) the consequent increase in the availability of circulating free tryptophan, and (h) the resultant tryptophan-mediated decrease in liver 5-aminolaevulinate synthase activity. The question of how many of these effects involve changes in endogenous opiates or at opiate receptors is not clearly understood at present and thus merits investigation. However, because most of the above effects are explained on biochemical grounds, and in view of evidence from behavioural and pharmacological studies [see (1)], the possibility must be considered that many of the actions of naloxone may be unrelated to its opiate-receptor-antagonistic properties.

Pyridoxine‐Responsive Primary Acquired Sideroblastic Anaemia

The activity of 5-aminolaevulinate (ALA) synthase, the first and rate-limiting of haem synthesis, was markedly reduced (13% of controls) in erythroblasts of a patient with acquired, primary sideroblastic anaemia (PASA). The reduced activity of ALA synthase could not be restored in vitro with 1 mmol/l pyridoxal-5-phosphate (PLP). Treatment of the patient with pyridoxine for several months increased the ALA synthase activity from 13% to 50% of controls in the absence and to 100% in the presence of PLP in the incubation medium. These studies suggest that both increased degradation of apo-ALA synthase and decreased affinity of ALA synthase for PLP may be involved in pyridoxine-responsive PASA.

Tryptophan pyrrolase in haem regulation. The mechanisms of enhancement of rat liver 5-aminolaevulinate synthase activity by starvation and of the glucose effect on induction of the enzyme by 2-allyl-2-isopropylacetamide.

1. Rat liver tryptophan pyrrolase activity is enhanced by a hormonal-type mechanism during the first 2 days of starvation and by a substrate-type mechanism during the subsequent 2 days. 5-Aminolaevulinate synthase activity is also enhanced during the first 2 days of starvation, but returns thereafter to values resembling those observed in the fed rat. Treatments that prevent or reversé the enhancement of tryptophan pyrrolase activity in 24-48h-starved rats also abolish that of 5-aminolaevulinate synthase activity. Starvation of guinea pigs, which does not enhance the pyrrolase activity, also fails to alter that of the synthase. It is suggested that the decrease in 5-aminolaevulinate synthase activity in 72-96h-starved rats represents negative-feedback repression of synthesis, possibly involving tryptophan participation, whereas the enhancement observed in 24-48h-starved animals is caused by positive-feedback induction secondarily to increased utilization of the regulatory-haem pool by the newly synthesized apo-(tryptophan pyrrolase). 2. Glucose, fructose and sucrose abolish the 24h-starvation-induced increases in rat liver tryptophan pyrrolase and 5-aminolaevulinate synthase activities. Cortisol reverses the glucose effect on 5-aminolaevulinate synthase activity, presumably by enabling pyrrolase to re-utilize the regulatory-haem pool after induction of synthesis of this latter enzyme. 3. The impaired ability of 2-allyl-2-isopropylacetamide to enhance markedly 5-aminolaevulinate synthase activity in 24h-starved rats treated with glucose is associated with a failure of the porphyrogen to cause loss of tryptophan pyrrolase haem. Cortisol restores the ability of the porphyrogen to destroy tryptophan pyrrolase haem and to enhance markedly 5-aminolaevulinate synthase activity, presumably by enhancing tryptophan pyrrolase synthesis and, thereby, its re-utilization of the regulatory-haem pool. It is tentatively suggested that 2-allyl-2-isopropylacetamide destroys the above pool only after it has become bound to (or utilized by) apo-(tryptophan pyrrolase).

Tryptophan and tryptophan pyrrolase in haem regulation. The role of lipolysis and direct displacement of serum-protein-bound tryptophan in the opposite effects of administration of endotoxin, morphine, palmitate, salicylate and theophylline on rat liver 5-aminolaevulinate synthase activity and the haem saturation of tryptophan pyrrolase.

1. The increase in the haem saturation of rat liver tryptophan pyrrolase caused by tryptophan administration was previously shown to be associated with a decrease in 5-aminolaevulinate synthase activity. 2. It is now shown that similar reciprocal effects are caused by palmitate and salicylate, both of which increase tryptophan availability to the liver by direct displacement of the serum-protein-bound amino acid. 3. The reciprocal effects on the former two parameters caused by endotoxin and morphine are associated with an increase in liver tryptophan concentration produced by a lipolysis-dependent, non-esterified fatty acid-mediated, displacement of the serum-protein-bound amino acid. 4. All these changes and those caused by another lipolytic agent, theophylline, are prevented by the beta-adrenoceptor-blocking agent propranolol and by the opiate-receptor antagonist naloxone, whose anti-lipolytic nature is demonstrated. 5. High correlation coefficients have been obtained for one or more pairs of the following parameters: serum non-esterified fatty acid concentration, free serum tryptophan concentration, liver tryptophan concentration, liver 5-aminolaevulinate synthase activity, liver holo-(tryptophan pyrrolase) activity and the haem saturation of liver tryptophan pyrrolase. 6. It is suggested that liver tryptophan concentration may play an important role in the regulation of 5-aminolaevulinate synthase synthesis, and that the latter may be subject to control by changes in lipid metabolism and may be influenced by pharmacological agents that affect tryptophan disposition. 7. Preliminary evidence suggests that tryptophan may be bound in the liver and that such a possible binding may control its availability for its hepatic functions.

Formation of cobalt protoporphyrin by chicken hepatocytes in culture: Relationship to decrease of 5-aminolaevulinate synthase caused by cobalt

Cobalt protoporphyrin generated from 5-amino[4- 14C]laevulinate by homogenates or primary cultures of chick embryo liver exposed to CoCl 2 was found to be radioactivity unextractable by acid/ acetone, when extra protein was added. The activity of ferrochelatase was required for formation of cobalt protoporphyrin since inhibition of ferrochelatase with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (in the presence of cycloheximide) inhibited formation of cobalt protoporphyrin and resulted in accumulation of protoporphyrin. Cobalt protoporphyrin was detected spectrophotometrically in hepatocyte cultures exposed to the combination of 2-allyl-2-isopropylacetamide and CoCl 2: (1) as the pyridine haemochrome of the protein pellet remaining after acid-acetone extraction of the cells, or (2) as the material extracted from the protein pellet with acetic acid-pyridine-chloroform. The amount of cobalt protoporphyrin increased with increasing CoCl 2 concentration as cellular haem declined. The decrease in haem was about equal to the amount of cobalt protoporphyrin that accumulated. 2-Allyl-2-isopropylacetamide and polychlorinated biphenyls were both powerful inducers of 5-aminolaevulinate synthase. The former led to protoporphyrin accumulation, whereas with the latter, uroporphyrin accumulated, probably due to a concomitant decrease in activity of uroporphyrinogen decarboxylase. The decrease in activity of 5-aminolaevulinate synthase produced by administration of CoCl 2 was greater after treatment with 2-allyl-2-isopropylacetamide than after treatment with allylisopropylacetamide and 3,4,3',4'-tetrachlorobiphenyl. We conclude: (a) that cobalt protoporphyrin is readily formed in cultured hepatocytes, and (b) that its formation accounts for the action of cobalt on 5-aminolaevulinate synthase.

Role of haem in the induction of cytochrome P-450 by phenobarbitone. Studies in chick embryos in ovo and in cultured chick embryo hepatocytes

The role of haem synthesis during induction of hepatic cytochrome P-450 haemoproteins was studied in chick embryo in ovo and in chick embryos hepatocytes cultured under chemically defined conditions. 1. Phenobarbitone caused a prompt increase in the activity of 5-aminolaevulinate synthase, the rate-limiting enzyme of haem biosynthesis, and in the concentration of cytochrome P-450. This induction response occurred without measurable initial destruction of the haem moiety of cytochrome P-450. 2. When intracellular haem availability was enhanced by exogenous haem or 5-aminolaevulinate, phenobarbitone-medicated induction of cytochrome P-450 was not affected in spite of the well known repression of 5-aminolaevulinate synthase by haem. These data are consistent with the concept that haem does not regulate the synthesis of cytochrome P-450 haemoproteins. 3. Acetate inhibited haem biosynthesis at the level of 5-aminolaevulinate formation. When intracellular haem availability was diminished by treatment with acetate, phenobarbitone-medicated induction was decreased. 4. This inhibitory effect of acetate on cytochrome P-450 induction was reversed by exogenous haem or its precursor 5-aminolaevulinate. These data suggest that inhibition of haem biosynthesis does not decrease synthesis of apo-cytochrome P-450. Moreover, they indicate that exogenous haem can be incorporated into newly formed aop-cytochrome P-450.

Tryptophan pyrrolase in haem regulation. The mechanism of the opposite effects of tryptophan on rat liver 5-aminolaevulinate synthase activity and the haem saturation of tryptophan pyrrolase.

1. Administration of tryptophan to starved rats causes a rapid decrease in liver 5-aminolaevulinate synthase activity associated with an increase in the haem saturation of tryptophan pyrrolase. Both effects are maximally produced at 30 min by a 100 mg/kg body wt. dose of tryptophan. 2. Pb2+ prevents both effects. 3. Prevention by allopurinol or benserazide of the tryptophan-induced increase in the haem saturation of tryptophan pyrrolase renders this haem available for further repression of synthase synthesis. 4. The opposite effects on synthase activity and pyrrolase saturation with haem caused by administration of 5-aminolaevulinate, but not those by that of haematin, are potentiated by tryptophan. 5. It is suggested that tryptophan decreases 5-aminolaevulinate synthase activity and causes the initial increase in the haem saturation of tryptophan pyrrolase by enhancing the conversion of 5-aminolaevulinate into haem by a process requiring protein synthesis.

Effect of allylisopropylacetamide on glutathione metabolism in the rat liver. The possible role of glutathione in the induction of 5-aminolaevulinate synthase.

Administration of allylisopropylacetamide to rats caused a marked decline in the concentrations of reduced and oxidized glutathione in the liver. However, this decrease occurred in the presence of uninhibited activities of gamma-glutamylcysteine synthase and glutathione reductase, and unaltered activities of glutathione transferases A, B and C. The administration of cysteine, the rate-limiting precursor of glutathione formation, to rats treated with allylisopropylacetamide potentiated the inductive effects of the agent on 5-aminolaevulinate synthase, and markedly decreased the extent of decrease in glutathione concentrations by the agent. Conversely, the administration of diethyl maleate, which depletes the hepatic glutathione concentrations, to allylisopropylacetamide-pretreated rats (1h) diminished the extent of 5-aminolaevulinate synthase induction and the production of porphyrins by nearly 50%, when measured at 16h. This treatment did not alter the extent of non-enzymic degradation of liver haem by allylisopropylacetamide. When diethyl maleate was administered to the animals possessing high 5-aminolaevulinate synthase activity (at 3, 7 and 15h after allylisopropylacetamide), in 1h the enzyme activity was markedly decreased. Diethyl maleate had no effect on induction of 5-aminolaevulinate synthase by 3,5-diethoxycarbonyl-1,4-dihydrocollidine, also a potent porphyrinogenic agent. Diethyl maleate alone neither inhibited 5-aminolaevulinate synthase activity nor decreased the cellular content of porphyrins and haem. The data suggest that the decreases observed in the glutathione concentrations after allylisopropylacetamide administration are not the result of decreased production of the tripeptide. Rather, they most likely reflect the increased utilization of glutathione. The findings further suggest that the inhibition by diethyl maleate of allylisopropylacetamide-stimulated 5-aminolaevulinate synthase involves the inhibition of induction processes.

Tryptophan pyrrolase in haem regulation. Experiments with administered haematin and the relationship between the haem saturation of tryptophan pyrrolase and the activity of 5-aminolaevulinate synthase in rat liver.

1. Administration of haematin to rats decreases 5-aminolaevulinate synthase activity in whole liver homogenates. 2. An inverse relationship between this decrease and the increase in saturation of apo-(tryptophan pyrrolase) with haem is observed during the initial phase of treatment with haematin. 3. Significant changes in both functions are caused by a 1 mg/kg dose of haematin, whereas the maximum effects are achieved by the 5 mg/kg dose. 4. Prevention by allopurinol of the conjugation of exogenously administered haematin with apo-(tryptophan pyrrolase) renders this haem available for further repression of 5 aminolaevulinate synthase. 5. The various aspects of the relationship between synthase activity and the haem saturation of tryptophan pyrrolase are discussed.