Δ-aminolevulinic Acid Synthetase Research Articles

Relationship between chemical structure and activity. I. Effects of the number of chlorine atoms in chlorinated benzenes on the components of drug-metabolizing system and the hepatic constituents.

To elucidate the relationship between chemical structure and their biological activities, the contents of cytochromes and hepatic constituents in addition to the activities of drug-metabolizing enzymes and δ-aminolevulinic acid (δ-ALA) synthetase were examined in rats treated with various chlorinated benzenes, i.e., monochlorobenzene (MCB), p-dichlorobenzene (p-DCB), 1, 3, 5-trichlorobenzene (1, 3, 5-TRCB), 1, 2, 4, 5-tetrachlorobenzene (1, 2, 4, 5-TECB), pentachlorobenzene (PECB) and hexachlorobenzene (HCB). 1) The content of cytochrome P-450 and activities of aminopyrine demethylase and aniline hydroxylase were increased by oral administration of all chlorobenzenes except MCB at daily dose of 250 mg/kg, once daily, for 3 days. The contents of microsomal protein and phospholipids also showed a similar tendency to those as described above. The activity of δ-ALA synthetase was increased by treatment with all compounds used. 2) The content of cytochrome P-450 and activity of aminopyrine demethylase were decreased in 24 hr after a single administration of MCB in doses of 125, 250, 500 and 1000 mg/kg, whereas the activity of δ-ALA synthetase was increased markedly by all doses used. The activity of aniline hydroxylase was increased by a dosing of 1000 mg/kg MCB. 3) In the time-course after a single administration of MCB in a dose of 250 mg/kg, the activity of δ-ALA synthetase was decreased in 6 hr after administration, and subsequently restored to normal levels in 12 hr, and then increased markedly in 24 hr. The opposite changes were noted in the content of cytochrome P-450.

Relationship between chemical structure and activity. II. Influences of isomers in dichlorobenzene, trichlorobenzene, and tetrachlorobenzene on the activities of drug-metabolizing enzymes.

Hepatic constituents and the cytochrome contents in addition to activities of drugmetabolizing enzymes and δ-aminolevulinic acid (δ-ALA) synthetase were examined in rats treated with each isomer of dichlorobenzene (DCB), trichlorobenzene (TRCB) and tetrachlorobenzene (TECB) in an oral dose of 250 mg/kg once daily for 3 days. 1) In the studies on DCB-isomers, activities of aminopyrine demethylase and aniline hydroxylase were enhanced markedly by treatment with m-DCB, whereas cytochrome content was not altered significantly by treatment with any DCB-isomers. δ-ALA synthetase activity was enhanced 63, 32 and 42% by treatment with o-, m- and p-DCB, respectively, but these enhancement were not paralleled with the changes in cytochrome P-450 content. 2) In the administration of TRCB-isomers, enzyme activities were enhanced markedly by treatment with 1, 2, 4-TRCB. Cytochrome P-450 content was also increased by treatment with 1, 2, 4-TRCB, and this increase could be partly related with the enhancement of δ-ALA synthetase activity. 3) By treatment with all TECB-isomers, activity of aminopyrine demethylase was enhanced, whereas aniline hydroxylase was not altered. Cytochrome P-450 content was increased by treatment with all TECB-isomers. 4) Microsomal protein content was increased with all isomers of DCB, TRCB and TECB treatment. Microsomal Pi was increased markedly 36, 70 and 91% by treatment with m-DCB, 1, 2, 4-TRCB and 1, 2, 3, 5-TECB, respectively. Hepatic glycogen content was decreased only by 1, 2, 3, 5-TECB, but triglyceride content was not altered. 5) The spectral change induced by substrate-cytochrome P-450 binding was characterized by type I in each treatment, but conversion of the type occurred in treatment with 1, 2, 4, 5-TECB in high concentration of 1 mM.

Metabolic controls in precancerous liver—VI. Studies in vivo and in vitro on feedback regulation of Δ-aminolevulinic acid synthetase in ethionine-fed rats

We have studied induction of δ-aminolevulinic acid (ALA) synthetase by the drug allylisopropylacetamide and the ability of exogenous heme to repress such induction in normal rat liver, in the livers of rats fed the hepatocarcinogen, ethionine and in host livers and hepatomas of rats bearing Morris hepatomas 5123C, 7794A and 9618A. We also measured changes in tyrosine aminotransferase. In experiments in vivo, ALA synthetase was shown to be inducible in normal liver, in ethionine-treated liver, in host livers of hepatoma-bearing rats and in hepatoma 5123C. Under our conditions it was non-inducible in hepatomas 9618A and 7794A. In normal and host livers injected heme repressed drug-stimulated induction, but in ethionine-treated livers and in hepatoma 5123C this feedback-type repression by heme was absent. This apparent defect in metabolic control in both cancerous and precancerous liver parallels the similar loss of feedback control of cholesterol synthesis previously reported. In contrast to findings in vivo for ethionine-fed rats, heme repression was found to be fully operative when studied in vitro, i.e. in liver cells isolated from ethionine-treated rats. The possible significance of this difference between findings in vivo and in vitro, in relation to the nature of ethionine-induced changes in precancerous liver, is discussed.

Induction of δ-aminolevulinic acid synthetase in the kidney of chicks treated with porphyrinogenic drugs

1. 1. The administration of allylisopropylacetamide induced an increase in δ-aminolevulinic acid synthetase activity in both kidney and liver of one-day-old chicks. 2. 2. The induction of δ-aminolevulinic acid synthetase in chick kidney was measurable by colorimetric assay and was comparable in activity to the increase observed in the liver. 3. 3. One of the most potent inducers of δ-aminolevulinic acid synthetase in chick liver, 3,5-dicarbethoxy-1,4-dihydrocollidine, did not increase the activity of chick kidney although the activity in liver was higher than that obtained with allylisopropylacetamide. Ferrochelatase activity was decreased in both liver and kidney by dicarbethoxy-1,4-dihyrocollidine.

Determination of the sites of synthesis of chlorophyll synthesizing enzymes in cell cultures of nicotiana tabacum

The activity of a sequence of enzymes involved in chlorophyll biosynthesis (δ-aminolevulinic acid synthetase (ALAS), δ-aminolevulinic acid dehydratase, porphobilinogenase and chlorophyllase) was followed during greening of tobacco cell cultures under the influence of chloramphenicol (CAP). The photosynthetic enzymes ribulose diphosphate carboxylase (RuDPCO) and NADP linked glyceraldehyde dehydrogenase (NADP-GDH) were used as markers for penetration and action of the inhibitor. RuCPCO was inhibited at concentrations of CAP which still allowed good chlorophyll accumulation. The enzymes of chlorophyll biosynthesis, the activity of which increased during illumination and CAP treatment, behaved like NADP-GDH which is known to be synthesized in the cytoplasm. The results suggest that synthesis of enzymes of chlorophyll biosynthesis takes place in the cytoplasm. Decreasing light induced increment of ALAS activity caused by CAP may possibly be taken as an indication that things are more complicated with this enzyme.

Drug-induced porphyrin biosynthesis--XII. Levels of cytochrome P-450 in chick embryo liver following administration of allylisopropylacetamide and propylisopropylacetamide.

Allylisopropylacetamide caused a decrease in the level of chick embryo liver cytochrome P-450 1 h after administration, followed by an elevation above control levels at a later time period. Propylisopropylacetamide on the other hand did not produce an early decrease in cytochrome P-450 but produced an elevation of cytochrome P-450 at a later time period. Since propylisopropylacetamide is an inducer of δ-aminolevulinic acid synthetase activity and porphyrin accumulation in chick embryo liver, it was concluded that a loss of cytochrome P-450 is not a prerequisite for ALA-synthetase induction as is thought to be the case in rats.

Induction of delta-aminolevulinic acid synthetase during erythroid differentiation of cultured leukemia cells.

SummaryA cloned line of Friend erythroleukemia cells (T-3-Cl-2) can be induced to produce hemoglobin when grown in the presence of 1%-2% DMSO. Such DMSO-treated cells become 30%-60% benzidine-positive in 5-7 days. The control enzyme for hemoglobin synthesis, Δ-aminolevulinic acid (ALA) synthetase becomes elevated after 28 hr in DMSO-treated cells. Furthermore, this synthetase activity increased 2-6 times the level of control cells during the following 3 days. Maximal ALA synthetase activity was found when the culture media contained 2% DMSO. Allylisopropylacetamide alone was found to be as effective an inducer of ALA synthetase as 1% DMSO, and could augment enzyme activity when present with DMSO.

Measurement of delta-aminolevulinic acid synthetase activity in human erythroblasts.

A new, specific, and simple method for the determination of delta-aminolevulinic acid (ALA) synthetase activity in human bone marrow cells has been developed. ALA synthetase of erythroblasts was partially purified so as to permit the use of [(14)C]succinyl-CoA as a substrate for this enzyme. In this enzyme preparation there were negligible activities of succinyl-CoA hydrolase, alpha-ketoglutarate dehydrogenase, and succinyl-CoA synthetase and there was no activity of ALA dehydrase. The ALA formed from [(14)C]succinyl-CoA has been isolated by column chromatography. Radioactivity in the eluate from the column has been proved by paper chromatography to be exclusively that of [(14)C]ALA. The entire assay can be completed within 4 h, and [(14)C]succinyl-CoA was incorporated into [(14)C]ALA on the order of several percent. Moderate to marked decreases of ALA synthetase activity have been demonstrated in the erythroblasts of all cases of sideroblastic anemia. In the cases of iron deficiency anemia, on the other hand, normal or slightly elevated activity has been obtained.

The effect of progesterone on activities of δ-aminolevulinic acid synthetase and δ-aminolevulinic acid dehydratase in estrogen-primed avian oviduct

The effect of a single dose of progesterone, administered to estradiol-primed chicks, on the activities in oviduct and liver of the first two enzymes of the porphyrin-heme biosynthetic pathway, δ-aminolevulinic acid synthetase (ALAS) and δ-aminolevulinic acid dehydratase (ALAD), has been investigated. The specific activity of ALAS in pooled glandular tissue of uterus and isthmus became significantly elevated at 18–24 hr after hormone injection. ALAD activity in uterine glandular tissue was not significantly increased after such treatment. Progesterone had no effect on hepatic activities of these two enzymes.

Antiporphyric effect exerted by caffeine administration in allylisopropylacetamide-treated rats

Allylisopropylacetamide (AIA) administration to rats increases δ-aminolevulinic acid (ALA) synthetase activity and at the same time markedly decreases cytochrome P 450 levels. Caffeine injection to animals treated with AIA markedly lowers ALA synthetase activity and causes an increase of cytochrome P 450 amount. Caffeine inhibits ALA synthetase activity, protecting heme, the natural repressor of this enzyme, from being destructed by AIA, as it appears from cytochrome P 450 levels reported in AIA treated animals.

Experimental hepatic porphyria induced by polychlorinated biphenyls

Aroclor 1254, which consists of a mixture of polychlorinated biphenyls (PCBs) containing 54% chlorine, produced an experimental hepatic porphyria in rats resembling hexachlorobenzene poisoning and human porphyria cutanea tarda. The PCB-induced porphyria is characterized by delayed development, increased excretion of urinary uroporphyrins, accumulation of 8- and 7-carboxyporphyrins in the liver and increased drug-metabolizing capacity of the liver. Cytochrome P-450 and microsomal heme were increased maximally at 1 week, in the absence of an increase in the rate-limiting enzyme in heme synthesis, δ-aminolevulinic acid (ALA) synthetase. Induction of ALA synthetase and porphyria occurred later, after 2–7 months' exposure to PCBs. No induction of ALA synthetase could be demonstrated prior to the onset of porphyria. Marked induction of ALA synthetase occurred 5 hr after large single doses of Aroclor 1254; however, the doses required were larger than those used to produce porphyria when administered chronically, and induction appeared to be related to the marked increase in cytochrome P-450 seen 24 hr after administration of the drug.

Induction of δ-Aminolevulinic Acid Synthetase in Isolated Rat Liver Cell Suspensions: ADENOSINE 3′ : 5′-MONOPHOSPHATE DEPENDENCE OF INDUCTION BY DRUGS

Abstract The control of total δ-aminolevulinic acid (ALA) synthetase activity in mammalian liver has been studied in isolated rat liver cell suspensions. Induction of ALA synthetase by the drug, allylisopropyl-acetamide (AIA) at a rate comparable to that of drug-stimulated induction in fasted rats could be achieved in the isolated cells; induction was largely dependent on the presence of adenosine 3' : 5'-monophosphate (cyclic AMP) or its dibutyryl derivative. At optimal concentrations either AIA (1.2 mm) or dibutyryl cyclic AMP (50 µm) alone caused little induction, but in combination caused a 4- to 5-fold increase of ALA synthetase activity in isolated cells over a 6-hour period. It is suggested that the cyclic AMP dependence of induction by drugs, also demonstrated for the drug 3,5-dicarbethoxydihydrocollidine, explains glucose repression of ALA synthetase observed in vivo. Glucose had no effect on induction by AIA ± dibutyryl cyclic AMP in isolated cells. Despite reports that glucocorticoids are required for induction in vivo, addition of hydrocortisone succinate to concentrations maximally effective in inducing hepatic tyrosine aminotransferase had little effect on the increase in total ALA synthetase activity stimulated by AIA ± dibutyryl cyclic AMP in isolated cells. Induction was prevented by addition of cycloheximide or actinomycin D to cell suspensions.

Studies on δ-Aminolevulinic Acid Synthetase Activity in Bone Marrow in Experimental Lead Poisoned Rats

Urinary increase of δ-aminolevulinic acid and coproporphyrin as well as basophilic stippled cells in the peripheral blood are the characteristic signs of lead poisoning. The biochemical lesion of porphyrin biosynthesis in lead poisoning is the partial inhibition of δ-aminolevulinic acid dehydratase, but whether or not δ-aminolevulinic acid synthetase is induced (stimulated) or not by lead is still unknown. This paper reports that δ-aminolevulinic synthetase activity in bone marrow in lead poisoned rats was 6.8 times higher than that of normal rats. Activity was found to increase within three or four days after lead administration and in parallel with the urinary increase of δ-aminolevulinic acid.The levels of urinary δ-aminolevulinic acid and coproporphyrin in lead poisoned rats decreased markedly following the administration of mitomycin c. This phenomenon were also observed in the case of hepatic porphyria caused by diethyl-1, 4-dihydro-2, 4, 6-trimethylpyridine-3, 5-dicarboxylate poisoned rats when treated with mitomycin c.These results strongly suggest that δ-aminolevulinic acid synthetase activity is induced either directly or indirectly by lead. Excessive δ-aminolevulinic acid thus formed seems to overcome the partial blocking of δ-aminolevulinic acid dehydratase resulting in the increased formation of coproporphyrin in lead poisoning.

Drug metabolism in cell culture—I: Importance of steric factors for activity in porphyrin-inducing drugs

3,5-Diethoxycarbonyl-2,4,6-trimethylpyridine- 14C, a compound in which the ester groups are sterically hindered from hydrolysis, was synthesized and found to be slowly metabolized in chick embryo liver cell cultures. On the other hand, however, 3,5-diethoxycarbonyl-2,6-dimethylpyridine- 14C, a compound in which the ester groups are not sterically hindered from hydrolysis was rapidly metabolized in this system. Differences in δ-aminolevulinic acid synthetase induction and porphyrin accumulation can be explained on the basis of the rates of metabolism of these compounds.

The role of substrates for glycine acyltransferase in the reversal of chemically induced porphyria in the rat

Experimental porphyria in the rat induced by the porphyrogenic agent, 3,5-dicarbethoxy-1,4-dihydrocollidine, was reversed by sodium benzoate or p-aminobenzoate treatment. In porphyric rats, benzoate and p-aminobenzoate markedly decreased the urinary excretion of the heme precursors, δ-aminolevulinic acid, porphobilinogen, and porphyrins, as well as the levels of tissue and blood porphyrins. The administration of glycine prevented the reversal of the porphyria. Neither benzoate, p-aminobenzoate, nor their respective metabolites, hippurate and p-aminohippurate, had an effect on δ-aminolevulinic acid synthetase in vivo or in vitro, indicating that the reversal of porphyria could not be explained by an effect on the rate limiting enzyme for heme biosynthesis. Hippurate, administered intraperitoneally, had no effect on the porphyric state. These results indicate that benzoate and p-aminobenzoate, substrates for glycine acyltransferase (EC 2.3.1.13), promote the diversion of glycine from the heme biosynthetic pathway to hippurate biosynthesis, thereby altering the biochemical pattern associated with the porphyric state.

The δ-aminolevulinic acid synthetase activity and the effect of exogenous δ-aminolevulinate on the synthesis of cytochrome c in the thoracic muscles of the tobacco horn worm during adult development

Activity of δ-aminolevulinic acid synthetase was measured in homogenates of flight muscles of the tobacco horn worm moth ( Manduca sexta) during adult development. The activity in pupae prior to 15-days old is below the limit of detection. The activity in the 16–17-day-old pupae is minimal but it increases rapidly thereafter, reaches the highest level at the emergence of adult moths, and then drastically falls to a very low level within 18 h. The rise in activity prior to the adult emergence was inhibited by puromycin and actinomycin D. However, it was found that injected δ-aminolevulinic acid did not stimulate de novo synthesis of cytochrome c.

Cellular compartmentation of two species of δ-aminolevulinic acid synthetase in a facultative photohetero-trophic bacterium ( [formula omitted

Both species of δ-aminolevulinic acid (ALA) synthetase appear compartmentalized in Rps. spheroides Y. The ALA synthetase, “F I”, activity is correlated with dark respiratory metabolism and is a cytoplasmic “soluble” enzyme. The other enzyme, “F II”, induced only in light, is in chromatophores and its activity is correlated with photometabolism.

Drug-induced porphyrin biosynthesis. 8. Investigation of the importance of an allyl group for activity in substituted acetamides.

Allylisopropylacetamide (AIA) was found to be considerably more potent than propylisopropylacetamide (PIA) in inducing hepatic δ-aminolevulinic acid (ALA) synthetase activity and hepatic porphyrin accumulation in the 17-day-old chick embryo, chickens, and mice of the CBA/J strain. AIA and PIA were shown to be approximately equipotent in inducing ALA synthetase activity and porphyrin accumulation in chick embryo liver cell culture. It is likely that the unusual responsiveness of chick embryo liver cell culture to PIA is not due to species or developmental differences but to the large amount of PIA available to the cultured liver cells.

Drug-induced porphyrin biosynthesis. X. Potentiation of propanidid-induced elevation of delta-aminolevulinic acid synthetase and porphyrins in chick embryo liver by the carboxylesterase inhibitor bis-(p-nitrophenyl) phosphate.

Propanidid, an ultra short-acting non-barbiturate anesthetic containing an ester group, induces δ-aminolevulinic acid (ALA)-synthetase and porphyrin accumulation in 17-day-old chick embryo liver. The potency and duration of action of propanidid in inducing ALA-synthetase activity and porphyrin accumulation was markedly increased when administered to chick embryos which had been pretreated with bis-[p-nitrophenyl] phosphate, an inhibitor of liver carboxylesterase.

Theophylline suppression of Δ-aminolevulinic acid synthetase induction in chick embryo and rat livers

Theophylline was found to be a potent inhibitor of induction of hepatic δ-aminolevulinic acid synthetase (ALAS). The inhibitory effect of theophylline was observed in chick embryo and rat livers with allylisopropylacetamide (AIA), a strong inducer of ALAS. The effect of other inducers of ALAS in chick embryo (phenobarbital and steroids) was also suppressed by theophylline, which exerted its effect when administered either simultaneously with the inducer or 3 to 4 hours following the induction. Theophylline had no direct inhibitory effect on ALAS activity in vitro. The effect of xanthine derivatives other than theophylline on ALAS induction was also tested; however, theophylline and aminophylline were the only significantly potent suppressors of ALAS in both chick embryo and rat livers.