Δ-aminolevulinic Acid Synthetase Activity Research Articles

Prevention of barbiturate-mediated increases of rat hepatic ornithine decarboxylase and δ-aminolevulinic acid synthetase activities by nicotinic acid and nicotinamide

Administration of secobarbital to rats produced dramatic increases in the activities of hepatic ornithine decarboxylase and δ-aminolevulinic acid synthetase at three hours which were reversed by administration of either nicotinic acid or nicotinamide. No appreciable alterations of the activity of either enzyme were observed following administration of nicotinic acid. Administration of nicotinamide produced a significant increase of ornithine decarboxylase activity, but failed to alter the activity of δ-aminolevulinic acid synthetase.

Hemin Control of Heme Biosynthesis and Catabolism in a Human Leukemia Cell Line

Hemin treatment of the Philadelphia chromosome positive leukemia cell line, K562, accentuates a number of erythroid phenotypic characteristics. The nature of this hemin effect was investigated by examining heme production and heme biosynthetic and catabolic enzyme activity in untreated and 0.05 mM hemin-treated cells. Activities of δ-aminolevulinic acid synthetase (ALAS), the rate limiting heme synthetic enzyme, and δ-aminolevulinic dehydratase (ALAD) were detectable in both uninduced and induced K562 cells. However, cells treated with hemin showed a significant increase in ALAS and ALAD activity by 2.5 days of treatment compared to untreated cells, and both enzyme activities further increased over the remainder of the incubation period. Incorporation of 3H-ALA into heme was also monitored. 3H-ALA was incorporated into heme in both untreated and treated cultures, but this incorporation was significantly greater in hemin-treated cells. Incorporation of 3H-ALA into heme indicates that cellular heme biosynthesis accounts for a portion of the heme utilized for hemoglobin production and that hemin stimulates heme synthesis by increasing enzyme activities distal to ALAS in the heme biosynthetic pathway. Heme oxygenase, the rate-limiting enzyme in mammalian heme degradation, was also studied and found to be present in large amounts in treated and untreated K562 cells. However, heme oxygenase activity was significantly decreased following hemin treatment. The demonstration of heme oxygenase activity in K562 cells documents the presence of this heme catabolic enzyme in human erythropoietic stem cells. The fall in heme oxygenase activity that accompanies hemin treatment might reflect an effort by erythroid cells to conserve heme during erythroid differentiation. These studies indicate the central role that heme synthesis and catabolism play during human erythroid differentiation.

Depressed guinea pig testicular microsomal cytochrome P-450 content by 2,3,7,8-tetrachlorodibenzo-p-dioxin

Human exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can result in hirsutism and chloracne, symptoms that suggest an alteration in endocrine regulation. Consequently, the effect of TCDD on guinea pig testicular cytochrome P-450 has been investigated. Twelve hours after a single, oral dose of TCDD (1 μg/kg), testicular microsomal cytochrome P-450 content was depressed by approximately 36%. Microsomal cytochrome P-450 content reached a maximal depression at approximately 1 day (52% of control) and remained at this level for 9 days. No appreciable alterations of testicular microsomal heme levels or activity of testicular δ-aminolevulinic acid (ALA) synthetase were observed.

Measurement and regulation of rat testicular δ-aminolevulinic acid synthetase activity

δ-Aminolevulinic acid synthetase (EC 2.3.1.37) activity has been detected in the rat testis. Optimal substrate and coenzyme concentrations and assay conditions have been determined. The majority of enzyme activity is localized in the mitochondrial fraction, with a small amount being detected in the cytosol. Fasting, which is known to increase activity in the adrenal and decrease activity in the heart, had no effect on testicular enzyme activity. Administration of the known hepatic inducers 3,5-dicarbethoxy-1,4-dihydrocollidine or allylisopropylacetamide to rats did not alter the activity of testicular δ-aminolevulinic acid synthetase. Administration of human chorionic gonadotropin to rats increased enzyme activity at 24 and 48 h, which indicates that testicular δ-aminolevulinic acid synthetase and the synthesis of heme may be under the control of pituitary gonadotropins.

Sulfonamide inhibition of rat hepatic uroporphyrinogen I synthetase activity and the biosynthesis of heme

Sulfonamides are known to provoke attacks of acute intermittent porphyria, which is characterized by a deficiency of uroporphyrinogen I synthetase (EC 4.3.1.8) activity. Various sulfonamides were examined for their potential as inhibitors of purified rat hepatic uroporphyrinogen I synthetase activity in vitro, and 10 were found to be inhibitory. Inhibition of uroporphyrinogen I synthetase activity by sulfonamides is reversible by dialysis and exhibits noncompetitive kinetics. Inhibition constants ( K i ) ranged from 100 μ m for sulfaguanidine to 270 μ m for sulfadiazine. These observations were extended to in vivo studies using sulfamerazine ( K i = 250 μm). Twenty-four hours after administration of sulfamerazine (orally; 1 g/kg) to male rats, hepatic microsomal heme and cytochrome P-450 contents were decreased approximately 33 and 42%, respectively. This was reflected by the inhibition of both aniline- p-hydroxylase and amino pyrine- N-demethylase activities, which are P-450 mediated. Heme oxygenase activity was not altered by sulfamerazine treatment. However, the relative rate of heme biosynthesis, as measured by the incorporation of δ-[4- 14C]aminolevulinic acid into heme, was 28% lower in sulfamerazine-treated rats. Thus, these results indicate that sulfamerazine acts to inhibit heme biosynthesis. Hepatic δ-aminolevulinic acid synthetase (EC 2.3.1.37) activity was increased 2.2-fold in the sulfamerazine-treated group, which is probably a compensatory increase caused by depression of the heme content. Consequently, it is suggested that sulfamerazine inhibits the synthesis of heme at the Uroporphyrinogen I synthetase step to impair hemoprotein formation and cytochrome P-450-mediated hepatic drug metabolism.

Cardiac δ-aminolevulinic acid synthetase activity: Effects of fasting, cobaltous chloride and hemin

Cardiac δ-aminolevulinic acid (ALA) synthetase activity was studied in animals that had been fasted. The kinetics of the decline in cardiac ALA synthetase activity were measured for 48 hr after food was withdrawn from rats. A marked rate of decrease in activity was observed between 12 and 24 hr. Cardiac ornithine decarboxylase activity also decreased in fasted rats, but cardiac tyrosine aminotransferase activity remained unchanged. Cardiac ALA synthetase activity also decreased in fasted mice but did not decrease significantly in fasted guinea pigs or fasted rabbits whose activites were low even in the fed state. Cobaltous chloride administration caused a marked decrease in cardiac ALA synthetase activity which remained low for a longer duration of time than has been observed in rat liver. The administration of hemin, which causes a marked decrease in hepatic ALA synthetase activity. had no effect on cardiac ALA synthetase activity. The difference in the responses of hepatic and cardiac ALA synthetase activities to hemin administration and food deprivation suggests differential regulation of ALA synthetase activity in heart and liver.

Regulation of heme metabolism during senescence: Activity of several heme-containing enzymes and heme oxygenase in the liver and kidney of aging rats

The activities of several heme-containing enzymes plus succinate dehydrogenase, the content of mitochondrial cytochromes, the amount of microsomal cytochrome P-450, and the activity of heme oxygenase, the major enzyme of heme degradation, have been compared in young, mature and senescent rats. Measurements were made in liver, a tissue previously reported to have an age-related decrease in δ-aminolevulinic acid synthetase, and in kidney, a tissue previously reported to have no age-related change in this enzyme, the rate-limiting enzyme of heme biosynthesis (Paterniti, Lin and Beattie, Arch. Biochem. Biophys., 191 (1978) 792–797). The activity of cytochrome oxidase in liver mitochondria did not decrease with age while this activity in kidney mitochondria was highest in animals one year old and decreased in the two-year-olds. By contrast, succinate dehydrogenase of both kidney and liver mitochondria decreased markedly in the aging rats. No age-related change in the content of cytochromes b, c or aa 3 was observed in liver mitochondria; however, a marked age-related decrease in cytochrome aa 3 was observed in kidney mitochondria. Similarly no change in cytochrome P-450 levels was observed in either tissue obtained from aging animals. In the liver, catalase activity increased while in the kidney it decreased in senescent as compared to mature animals. Heme degradation does not decrease with age as the activity of heme oxygenase increased in both liver and kidney of two-year-old rats as compared to one-year-olds. These results suggest that the lower activity of δ-aminolevulinic acid synthetase observed in the aging rat may not be correlated with a decrease in the activity of heme-containing proteins and that the regulation of the heme pool used for the synthesis of various intracellular hemo-proteins may be complex.

Studies on the Influence of Polychlorinated Biphenyls (PCB) on Aquatic Organisms. VIII. Influence of PCB on Drug-Metabolizing Enzyme System in Carp

The effect of Aroclor 1248 (PCB) on the hepatopancreatic microsomal enzymes of carp was examined. The carp were fed a diet containing 250 ppm PCB for 7 days, and activities of NADPH-cytochrome c reductase, NADH-cytochrome b5 reductase, aminopyrine N-demethylase, and aryl hydrocarbon hydroxylase, along with the amount of cytochrome P-450, cytochrome b5, and microsomal protein increased significantly by ingestion of PCB. These results showed that Aroclor 1248 induced hepatopancreatic drug-metabolizing enzymes of carp which are similar to hepatic enzymes in mammals. The induction, however, seemed to be a mixed type, also induced by phenobarbital and 3-methylcholanthrene. By measuring the substrate-binding difference spectra of hepatopancreatic microsomes from the carp pretreated with Aroclor 1248, the PCB was found to be bound to cytochrome P-450 in a "modified type II" pattern as classified by Schenkman and others. The spectral changes induced by hexobarbital and aniline, and an increase in the δ-aminolevulinic acid synthetase activity also suggested that Aroclor 1248 increased the synthesis of cytochrome P-450 in the carp hepatopancreas. SDS-polyacrylamide gel electrophoresis, further demonstrated that the hepatopancreatic microsomes contained four kinds of heme protein and that the synthesis of the heme was induced by treatment of the carp with Aroclor 1248.

Studies on the mechanism of experimental porphyria and ferrochelatase inhibition produced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine

1. 1. The porphyrogenic agent, 3,5-diethoxycarbonyl-l,4-dihydrocollidine (DDC) produces a marked inhibition of hepatic ferrochelatase activity when injected in vivo. It has no effect in vitro. 2. 2. Following the administration of DDC to mice or rats, an inhibitor of ferrochelatase activity has been isolated from livers and resolved and purified by LH-20 column chromatography. Mice which are more sensitive to DDC produce more inhibitor substance than rats and control livers possess small but measurable quantities of the inhibitor substance. 3. 3. The inhibitor has porphyrin-like properties with characteristic visible and fluorescence spectra and can be separated from protoporphyrin IX. When [ 14C]aminolevulinic acid was injected 2 hr prior to DDC, the purified inhibitor substance contained high radioactivity. 4. 4. Cycloheximide pretreatment of animals did not affect the amount of inhibitor formed, although this treatment is known to markedly reduce δ-aminolevulinic acid synthetase activity and the porphyria produced by DDC. 5. 5. These results indicate that the formation of an inhibitor after the administration of DDC is due to the catabolism of hepatic heme to a substance with porphyrin-like properties and with profound inhibitory characteristics toward ferrochelatase activity.

Sex-related effect of cadmium on hepatic cytochrome p-450, drug-metabolizing enzymes and 5-aminolevulinic acid synthetase and heme oxygenase in the rat

The effect of cadmium on hepatic cytochrome P-450 containing drugmetabolizing enzymes, δ-aminolevulinic acid synthetase (ALA synthetase) and heme oxygenase activity in both male and female rats was examined to define the nature of sex differences in response. In male rats injected i.p. with a single dose of cadmium chloride (1.25 mg Cd/kg body weight) there was a reciprocal relationship between the decrease of cytochrome P-450 levels and drug-metabolizing enzymes and the increase of heme oxygenase activity for up to 120 h. Hepatic ALA synthetase activity was initially decreased and returned to control levels at 24 h. In female rats, cadmium initially produced similar effects to those seen in male rats, but these were of shorter duration except for the increase of heme oxygenase activity. In addition, ALA synthetase activity was slightly increased at 24 h and 48 h. The results suggest that the sex differences in response reside mainly in the duration of the metal effect on these hepatic enzymes.

Assay of δ-aminolevulinic acid synthetase in homogenates of mouse, rat, and human liver: Species differences in requirement for an exogenous succinyl-CoA-generating system

Conditions required for optimal assay of low levels of activity of hepatic δ-aminolevulinic acid synthetase have been studied, comparing dilute homogenates of mouse, rat, and human livers. The assay method used was a modification of that described by Ebert et al. (Biochim. Biophys. Acta (1970) 208, 236–250), and livers were studied from both untreated animal and human subjects and subjects pretreated with porphyrinogenic compounds. In homogenates of mouse and human but not rat liver, maximal rates of δ-aminolevulinic acid formation required addition to the incubation mixture of an exogenous system for succinyl-CoA generation. The requirement for this generating system was increased if livers from pretreated subjects were frozen and stored prior to assay, suggesting that the endogenous capacity for succinyl-CoA generation was more labile than δ-aminolevulinic acid synthetase under these conditions. Of the metabolic inhibitors tested (F −, malonate, and arsenite), only F − (100 m m final concentration) enhanced activity. Increasing the permeability of mitochondria by quick freezethawing of fresh homogenates just before assay did not increase the rate of δ-aminolevulinic acid formation.

Effect of host-mediating anti-tumor drugs on hepatic δ-aminolevulinic acid synthetase and heme oxygenase activity in mice

Effect of host-mediating anti-tumor drugs, OK-432 and PSK, on hepatic δ-aminolevulinic acid synthetase (ALA synthetase) and heme oxy genase activity was examined. Both OK-432 and PSK were shown to inhibit ALA synthetase activity when administered in vivo to mice. Both OK-432 and PSK also inhibited the 3,5-dicarboethoxy-1,4-dihydro collidine (DDC)-mediated induction of ALA synthetase when administered 12 and 16 h prior to the inducer. Additionally, the administration of OK-432 and PSK resulted in a significant increase in hepatic heme oxy genase activity. These data suggest that the decrease of hepatic drug-metabolizing enzymes as well as cytochrome P-450 levels seen on the administration of OK-432 and PSK is the result of the inhibition of ALA synthetase and of the increase of heme oxygenase activity leading to a decreased heme moiety required for the synthesis of cytochrome P-450 in the liver.

Detection and regulation of δ-aminolevulinic acid synthetase activity in the rat brain

The presence of δ-aminolevulinic acid synthetase (EC 2.3.1.37) in homogenates and mitochondria from rat brain has been observed. Optimal conditions for the measurement of this activity are described. The enzyme activity appears to be localized in the mitochondria, although a small amount of activity is also present in the cytosol. The activity of brain δ-aminolevulinic acid synthetase was measured under conditions which are known to affect the enzyme activity in other tissues. The activity of δ-aminolevulinic acid synthetase in the brain was unaffected by a 24-h fast or by the presence of inducers such as allylisopropylacetamide, 3,5-dicarbethoxy-1,4-dihydrocollidine, or ethanol. A decrease in enzyme activity of more than 50% was observed when δ-aminolevulinic acid synthetase was measured in brain homogenates derived from rats of increasing age from 1 month to 2 years.

Heat inhibition of reticulocyte protein synthesis Evidence for a mechanism independent of the hemin-controlled repressor

Incubation of rabbit reticulocytes at 45°C results in a prompt but reversible decrease in protein synthesis and a concomitant conversion of polyribosomes to smaller aggregates. These effects occur even in the presence of 100 μM hemin in the incubation medium. There is also inhibition of heme synthesis, but this occurs at a later time than the effect on protein synthesis. The inhibition of heme synthesis results from a decrease in activity of δ-aminolevulinic acid synthetase. This decrease of heme synthesis appears to be secondary to the inhibition of protein synthesis with resultant accumulation of intramitochondrial heme (which will decrease δ-aminolevulinic acid synthetase activity). An inhibitor of reticulocyte cell-free protein synthesis formed in the post-ribosomal supernatants of cells incubated at both 45 and 37°C, but not at 0°C. No temporal or quantitative differences in the amount of this inhibitor from cells treated at either 37 or 45°C was apparent. The inhibitor was not found in the fraction where the hemin-controlled repressor is isolated. It is concluded that heat inactivation of intact reticulocyte protein synthesis does not depend upon a decrease in heme synthesis, heme concentration or generation of the hemin-controlled repressor. Furthermore, it appears that the inhibitor formed in the post-ribosomal supernatant cannot be the sole cause of the heat inhibition of protein synthesis.

Influence of sex hormones on free erythrocytes protoporthyrin response to lead in rats

Following oral administration of lead a difference in free erythrocyte protoporphyrin (FEP) increase was found between adult male, adult female and suckling rats: young animals are more susceptible than adult female rats which in turn exhibit a greater FEP increase than adult, male animals. This observation parallels that made previously in humans. Possible difference in iron metabolism does not appear to explain the sex-linked difference in FEP response to lead. Sex hormones mainly progesterone, seem to play a role but their interaction with lead on the FEP response is restricted to female rats and apparently is not mediated through changes in δ-aminolevulinic acid synthetase activity.

Hemin feedback inhibition at reticulocyte δ-aminolevulinic acid synthetase and δ-aminolevulinic acid dehydratase

Addition of hemin (5–200 μM) to a rabbit reticulocyte iron-free incubation medium, resulted in a progressive inhibition of heme synthesis as measured by incorporation of ( 14C)-glycine. In contrast when ( 14C) δ-aminolevulinic acid incorporation into heme was studied, significant inhibition below that of the ( 14C)-glycine control only occurred with hemin concentrations greater than 100 μM. Hemin progressively inhibited cellular and mitochondrial δ-aminolevulinic acid synthetase activity, as well as cellular δ-aminolevulinic acid dehydratase activity. The results indicated that elevated levels of hemin initially control heme synthesis by feedback inhibition at the rate-limiting enzyme of heme synthesis, δ-aminolevulinic acid synthetase. Hemin inhibition of δ-aminolevulinic acid dehydratase is only significant for the entrire heme synthetic pathway when greater than one-third of this enzyme's activity is inhibited.

Drug-induced porphyrin biosynthesis—XVI. Effects of hydrocortisone, adenosine 3′:5′-cyclic monophosphoric acid, its dibutyryl derivative and a phosphodiesterase inhibitor on allylisopropylacetamide-induced porphyrin biosynthesis in chick embryo liver cells maintained in serum-free waymouth medium

Chick embryo liver cells were maintained in serum-free Waymouth medium containing insulin and thyroxine. Hydrocortisone (20.6 μM) enhanced allylisopropylacetamide (AIA)-induced δ-aminolevulinic acid (ALA) synthetase activity, as it was previously demonstrated to do in rat liver. It is likely that previous failures to observe a hydrocortisone effect in hepatic cell culture were due to the presence of hydrocortisone in serum added to the medium. Adenosine 3′: 5′-cyclic monophosphoric acid (cAMP; 0.3 mM) and its dibutyryl derivative (61 μM), although not essential for AIA induction of porphyrin biosynthesis, were observed to enhance the accumulation of porphyrins. In contrast, cAMP has been reported to be essential for AIA induction of ALA-synthetase activity in rat liver cell suspensions. The cyclic nucleotide phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine (135 μM), was shown to exert a similar effect on AIA-induced porphyrin biosynthesis as cAMP. It was concluded that cAMP enhances but does not function as a “second messenger” in AIA-induced porphyrin biosynthesis.

An assay for δ-aminolevulinic acid synthetase based on a specific, semiautomatic determination of picomole quantitites of δ-[ 14C]aminolevulinate

δ-Aminolevulinic acid (ALA) synthetase activities in the range of 0.1 to 100 U/ml of enzyme are routinely assayed using a modified Beckman Model 121 amino acid analyzer. THis method reproducibly and specifically quantitates [ 14C]aminolevulinate from a mixture of [ 14C]succinate metabolites known to interfere in other methods. Mitochondrial ALA synthetase activity in livers of normal adult guinea pigs is determined to be approximately 3 U/g of liver. This activity is only 1 100 th the activity in guinea pigs treated with the porphyrinogenic drug 3,5-dicarbethoxy-1,4-dihydrocollidine.

Effect of piperonyl butoxide on hepatic δ-aminolevulinic acid synthetase activity in mice

Effect of piperonyl butoxide on hepatic δ-aminolevulinic acid synthetase activity in mice

Cytochrome P-450 heme and the regulation of δ-aminolevulinic acid synthetase in the liver

Hepatic δ-aminolevulinic acid synthetase was induced in rats injected with allylisopropylacetamide. The induction process was studied in relation to experimental perturbation of cytochrome P-450 in the liver. Animals were treated with either administered endotoxin or exogenous heme, both of which accelerate degradation of cytochrome P-450 heme. These manipulations were effective in blocking induction of δ-aminolevulinic acid synthetase, and the effect of each compound was proportional to its ability to stimulate degradation of cytochrome P-450 heme. The findings suggest that the heme moiety of cytochrome P-450 dissociates reversibly from its apoprotein and, prior to its degradation, mixes with endogenously synthesized heme to form a pool that regulates δ-aminolevulinic acid synthetase activity. A similar or identical heme fraction appears to mediate stimulation of heme oxygenase, which suggests that the regulation of δ-aminolevulinic acid synthetase and of heme oxygenase in the liver are closely interrelated.