ALA-Synthetase in Liver

In animals the control of heme biosynthesis appears to be primarily a control on the rate of biosynthesis of the enzyme ALA-synthetase. Recent experiments [Sassa and Granick, 21] suggest that control of this enzyme occurs at both the transcription and the translation levels. Because these controls have been studied mainly in the liver, we shall discuss the evidence for the control mechanisms in this tissue, particularly the more recent work using chick embyro liver cells grown in primary culture. In later sections we shall discuss the ALA-synthetase control mechanism in the red cells for heme and hemoglobin synthesis, and the control mechanisms for chlorophyll synthesis in plants. 

A number of diverse findings have elucidated the control mechanisms for heme in the liver cell. These findings had their origin in studies of hepatic porphyria—acute intermittent porphyria (AIP), a relatively rare disease of humans. They led to studies of drug-induced or chemical porphyria and to the more recent findings that certain steroids are active in the control of heme. 

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IV. SOME PROPERTIES OF THE INDUCING SYSTEM OF ALA-SYNTHETASE IN LIVER... 

The chemicals induce only in liver, not in other tissues. The steroids, as will be discussed later, induce ALA-synthetase in liver cells as well as in erythroid cells of the chick blastoderm. Although the kidneys of the chick embryo may rapidly convert ALA to porphyrins, neither steroids nor other inducing chemicals have an inducing effect on this tissue. Thus, response to inducing compounds depends on the tissue. In the liver, at least of the chick embryo, both steroids and other chemicals induce in erythropoietic blastoderm only steroids induce and in kidneys and other tissues none of these compounds induce. Steroid induction in liver and erythropoietic tissue appears to involve a transcriptional mechanism induction by chenucals appears to involve mainly the translational mechanism (see Section V). 

Normally, the activity of ALA-synthetase in mitochondria is very low, and in the cytosol it is not detectable. When ALA-synthetase is induced in an animal the activity rises ten to forty times, and the enzyme is then found also outside the mitochondria [Granick and Urata, 35]. Studies by Hayashi et al. [33] have shown that as much as 35% of the enzyme may be obtained in the supernatant from an induced rat liver homogenate after centrifugation at 77,000 g for 2 hours. The soluble enzyme may represent newly synthesized enzyme on the way to being incorporated into the mitochondrion, as suggested by studies with marker enzymes of the mitochondria. When the activities were determined in vivo using inhibitors of protein synthesis, the soluble enzyme was estimated to have a half-life of 20 minutes, compared to 1 hour for the mitochondrial enzyme. ALA-synthetase is relatively more stable in the isolated mitochondrion [35] than in vivo. Whether its first-order decay in vivo occurs inside or outside the mitochondrion is unknown, nor is it known whether the enzyme, once transported into the mitochondrion, can be transported out again. More recently Beattie and Stuchell [35a] have shown that after induction with AIA, ALA synthetase activity in the rat liver first accumulates in the cytosol and then is transferred into the mitochondria. 

Applications of these assays to the determination of ALA-synthetase in biopsied specimens of human liver (20 to 40 mg fresh weight) have required further modifications [56-58], The use of semimicrocuvettes of 50-mm length by Levere et aL [58] has made possible the colorimetric determination of ALA in the range of 10 mole. 

After it had been shown in chick embryo liver culture treated with inhibitors that RNA and protein synthesis were required for induction of ALA-synthetase [24,25], these results were confirmed in whole-animal studies [77-79]. Narisawa and Kikuchi [79] were able to detect an increase of ALA-synthetase in the liver 1 to 2 hours after a single subcutaneous dose of 400 mg of ALA per kilogram. It was also shown that not only porphyrins but heme synthesis was increased in chemical porphyria. DeMatteis and Rimington [80], using 2- " C-glycine or Fe, found an increased heme labeling in the presence of the inducers sedormid, AIA, and griseofulvin. It was not determined whether total heme was increased or whether heme turnover had increased. 

A biphasic curve of ALA-synthetase activity of liver mitochondria of rats, induced by AlA and possible involving repression by heme, was first observed by Narisawa and Kikuchi [107]. The enzyme activity increased slightly during the first 12 hours, then remained unchanged for several hours, and then continued to increase during the next 24 hours to tenfold the control value. No biphasic curve was observed with DDC by Wada et al. [86]. Waxman et al. [108] reported that, after the establishment of induced ALA-synthetase in the rat for 3 days, the administration of 2 mg of hemin initiated a series of cyclic oscillations of ALA-synthetase activity with a period of 8 to 12 hours the oscillations continued for 3 days. To explain these oscillations they postulated a repression by heme of the synthesis of ALA-synthetase and thence an overshoot of abnormally increased synthesis. 

The inducing effect of chemicals that bring about a three- to four-fold increase in liver ALA-synthetase in 6 hours was found by Marver [109] to be nullified if simultaneously with the inducing chemical the large dose of 12 mg of hemin chloride (per 100 gm wt) was injected into the rat. 

Factors Affecting the Synthesis of ALA-Synthetase IN Chick Embryo Liver Cells in Culture"... 

Increased activity of ALA-synthetase in the liver and a limiting coproporphyrinogenase and iron chelatase. Excess iron may enhance the porphyria at the translational level [128]... 

It was found by Levere et ah [137] that the same 5)6-H steroids that actively stimulated the synthesis of ALA-synthetase in chick embryo liver cells in culture also stimulated hemoglobin synthesis in chick embryo blastoderm cells (Fig. 9). It is inferred that the stimulation by the steroids is brought about by the same mechanism as for the liver cells that is, the steroids induce the synthesis of ALA-synthetase at the transcriptional level. ALA-synthetase is the rate-limiting enzyme in early red cell precursors, and its increase causes an earlier increase in the formation of ALA the ALA, once made, is rapidly converted to heme, and once heme is made globin is synthesized. Thus the controlling step in hemoglobin synthesis appears to be one that is turned on by a 5y -H steroid. Stimulation of hemoglobin synthesis by Sfi-H steroids has recently also been demonstrated in mice and in humans [140,142],... 

A role for heme in the repression of ALA synthetase has been proposed by Burnham and Lascelles (1963) and by Graniek and Kappas (1967). Heme has been shown to repress the synthesis of ALA synthetase in Khodopseudomonas spheroides (Bmnham and Lascelles, 1963). In liver cells and erythroid cells of the chick embryo, Gio and C21 j8-H steroids induce heme synthesis. This induction can be blocked by actinomycin D, by puromycin, and by added hemin (Graniek and Kappas, 1967). Graniek and his associates have proposed that heme... 

Various minor hematological effects have been noted in animals. Rats exposed to 50-800 ppm of trichloroethylene continuously for 48 or 240 hours showed time- and dose-related depression of delta-aminolevulinate dehydratase activity in liver, bone marrow, and erythrocytes (Fujita et al. 1984 Koizumi et al. 1984). Related effects included increased delta-aminolevulinic acid (ALA) synthetase activity, reduced heme saturation of tryptophan pyrrolase and reduced cytochrome P-450 levels in the liver and increased urinary excretion of... 

Commercial PCB Mixtures. Urinary coproporphyrin levels were increased in rats that ingested 0.3 or 1.5 mg/kg/day Aroclor 1242 in the diet for 2-6 months (Bruckner et al. 1974). Rats treated with 5 mg/kg/day Aroclor 1254 in the diet had maximum increases in liver microsomal P-450 concentration and liver weight after 1 week, but onset of porphyria and induction of 5-aminolevulinic acid (ALA) synthetase was delayed until 2-7 months of treatment (Goldstein et al. 1974). A marked accumulation of uroporphyrins occurred in the liver, and urinary excretion of coproporphyrin and other porphyrins was increased the largest increase was in uroporphyrins. The uroporphyrins in the liver and urine of the treated rats consisted primarily of 8- and 7-carboxyporphyrins. 

Synthesis of succinyl-CoA in mammalian cells such as the red cell and liver cell can be accomplished either from a-KG or from succinate. The formation of succinyl-CoA occurs in the mitochondria as part of the citric acid cycle reactions. The requirement for a citric acid cycle to form ALA or protoporphyrin or heme has been shown by tracer studies with acetate and succinate [39], and by inhibition studies with malonate, Ira j -aconitate, fluoracetate, and arsenite [49]. The requirement for an electron transfer system from the citric acid cycle to O2 has been shown by inhibition studies with anaerobiosis and CO. The requirement for oxidative phosphorylation has been shown by dinitrophenol inhibition of ALA synthesis dinitrophenol may also inhibit ALA-synthetase [3,49]. 

One should also mention the cytological changes that appeared in the liver of phenobarbital-treated rats. Phenobarbital is normally a weak inducer of ALA-synthetase. It induced a fourfold increase in microsomal NADPH cjTochrome reductase by 6 to 8 hours, although electron microscope pictures did not reveal an increase in the endoplasmic reticulum [Kuriyama et ah, 64] until several days later, (see page 104, Section C). 

One of the most active inducing compounds is Lindane. It is about 100 times as active as AIA in porphyrin induction in chick embyro liver culture as well as in the increase in ALA-synthetase activity [21]. The relative activities of isomers of hexahydrohexachlorobenzene for porphyrin production, are Lindane, the y-isomer = 1, a =0.5, P = 0.3, E =0.25, 5 =0.15. These results may in part reflect the relative stability of the isomers whose structures are given by Hornstein [67b]. The hexahydrobromo derivative had no activity [67a]. 

The use of chick embryo liver culture in vitro as contrasted to induction in the whole animal permitted the inference that the inducing chemicals acted directly on the hepatic cells and not via other stimuli generated from other organs. The porphyrins formed could be seen by fluorescence microscopy to accumulate in the c)doplasm of the liver parenchyma cells and were identified mainly as coproporphyiin with some protoporphyrin [25]. However, Doss [70] found mainly protoporphyrin. This difference may depend on conditions of culture the older cultures had more coproporphyrin and uroporphyrin. The increase in porphyrins in tissue culture has been assumed to be a result of an increase in ALA-synthetase. With the aid of 13-cm-diameter petri dishes, it has been possible to grow sufficient numbers of cells and demonstrate with inducing chemicals an increase in vitro of ALA-synthetase activity of seven to ten times that of controls, in a 20-hour period [21]. 

ALA-synthetase has one of the shortest half-lives yet reported for any mammalian liver enzyme, even the inducible ones. In contrast, the half-lives of two other mitochondrial enzymes, alanine and ornithine-amino transferases, inducible by the corticosteroid prednisolone acetate in a concentration of 0.5 mg/rat/day was 17 to 24 hours [75]. The short half-life of ALA-synthetase apparently depends on other cell constituents. When mitochondria from induced guinea pigs were isolated, the activity of their ALA-synthetase was maintained constant over a period of at least 5 hours [35] this activity may have finally not been limited by the stability of the enzyme itself but by the succinyl-CoA-synthesizing mechanism of the mitochondria. These facts suggest that there may be an active process of ALA-synthetase destruction which occurs in the cells but not necessarily in the isolated mitochondria, and which is not affected by the inducing chemicals such as DDC or AIA. 

In this section, the activities of inducing chemicals are discussed in relation to their destruction by the liver microsomal oxygenase system the rate of heme synthesis and breakdown is considered in relation to the inhibitory properties of heme on the synthesis of ALA-synthetase and the contrasting effects of glucose and starvation are summarized in relation to their effects on induction. 

Many of the lipophilic chemicals that induce the synthesis of ALA-synthetase also induce a set of concomitant reactions which help to dispose of these chemicals. Those chemicals that cannot be readily converted to a water-soluble form are as a last resort oxidized in the liver by a microsomal oxygenase system that primarily uses NADPH and O2 to generate OH for hydroxylation [Guroffc al., 83]. 

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