ALA synthetase inducers

Esters and amides, which are sterically hindered so that hydrolysis is diminished, probably retain their ALA-synthetase-inducing activity for a longer time—for example, DDC [10,82]. Enzyme reactions that saturate the double bonds of such steroids as progesterone, and hydroxyl groups in steroids or terpenes that are oxidized to keto groups, will tend... 

The induction is specific, not general. The increase in ALA-synthetase induced by AlA or DDC is not due to an overall rate of synthesis of mRNA or protein, for neither radioactive orotic acid nor uridine, nor radioactive leucine was incorporated at a greater rate in the presence than in the absence of the inducers [25, 128]. 

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. 

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). 

C. Properties and Classes of Chemicals that Induce ALA-Synthetase... 

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]. 

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

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]. 

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). 

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. 

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. 

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]. 

The chemical inducers that bring about enhanced synthesis of ALA synthetase appear to cause both increases and decreases in the activity of the microsomal drug metabolizing system depending on the chemical used, the time of assay after administration of the chemical and the age of the animal, whether prenatal, young or adult. Present evidence suggests several interpretations. Tentative interpretations of these facts are ... 

Powerful inducers of ALA synthetase, as AIA or DDC appear to generate, presumably in connection with the microsomal oxygenase system, sufficient oxygen radicals and peroxides not only for hydroxylation of these substrates but even for the destruction of the heme of P450. For example, Meyer and Marver [85a] have shown that a rapid decrease occurred in the labeled heme of P450 when rats were treated... 

The inducing chemicals may have several actions on different enzyme systems (p. 112, Section K). For example, phenobarbital is a relatively poor inducer of ALA synthetase but a good one for cytochrome P450 ... 

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. 

Physiologically, microsomal oxidation via the cytochromes appears to serve for the lipophilic oxidation of steroids, fatty acids, heme, and drugs. The microsomal cytochromes and smooth endoplasmic reticulum may be controlled in part by steroids. For example, the relatively high production of heme by normal liver suggests that there may be natural 5/i-H steroids that normally serve to maintain a certain rate of heme synthesis by inducing the synthesis of ALA-synthetase. An... 

The enzyme itself is not affected by inducing agents. The chemicals did not increase or decrease the activity or lifetime of ALA-synthetase, whether in isolated mitochondria or in cells in culture. In the simultaneous presence of acetoxycycloheximide (to block protein synthesis) and an inducing chemical, the decay rate of ALA-synthetase was identical with that of the control without inducing chemical—that is, ti/2 = 3 hour (Table 111). When the inducer was given for 14 hours to... 

Actions of Inhibitors and Inducers on ALA-Synthetase Formation and Decay ... 

Chick embryo liver cells, grown in monolayers, were treated with AIA for 14 hours to induce an increase in ALA-synthetase. Then the medium was changed, additions were made as noted, and the decay in activity of the enzyme was determined during the following 4 to 10 hours [21]. 

Direct evidence supporting the idea that chemical induction results in an increased synthesis of mRNA for ALA-synthetase has been reported by Hickman et al. [129]. After induction in the rat with AIA, the RNA was isolated from the liver and added to a culture medium containing chick embryo liver fragments. After incubation for 18 hours, the induced RNA, as compared to noninduced RNA, had caused a... 

The dominant Mendelian inheritance of acute intermittent porphyria is most simply explained as a defect in an operator gene that controls the operon for the synthesis of mRNA of ALA-synthetase. On the basis of this assumption, an operator-repressor mechanism at the transcriptional level is proposed (Fig. 8). Inducers like the 5p H steroid, etio-cholanolone, or chemicals like DDC would react with the repressor and derepress or unblock the operator. Then mRNA for ALA-synthetase could be made (Reaction 1). 

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