The effect of beta-adrenergic blocking agents on experimental porphyria induced by 3,5-diethoxycarbonyl-1,4-dihydrocollindine (DDC) in vivo and in vitro

The effect of DL-propranolol on 3′,5′-diethoxycarbonyl-1,4-dihydrocollidine-induced experimental porphyria was studied.dl-Propranolol, a beta-adrenergic blocking agent with non-specific membrane effects, partially inhibited 3′,5′-diethoxycarbonyl-1,4-dihydrocollidine-induced delta-aminolevulinate synthetase activity both in rats and in chick embryo liver cells in culture.In rats, DL-propranolol decreased urinary delta-aminolevulinate and porphobilinogen but no change occurred in the 24-h urinary excretion of total porphyrins and in the concentration of porphyrins in the liver. In cultured chick embryo liver cells treated with 3′,5′-diethoxycarbonyl-1,4-dihydrocollidine, DL-propranolol decreased accumulation of porphyrins in the medium.d-Propranolol, oxprenolol and quinidine acted like dl-propranolol in chick embryo liver cells in culture treated with 3′,5′-diethoxycarbonyl-1,4-dihydrocollidine. Pindolol, practolol and lidocaine had no effect.Phenobarbitone had a synergistic effect on the induction of delta-aminolevulinate synthetase by 3′,5′-diethoxycarbonyl-1,4-dihydrocollidine in cultures of chick embryo liver cells. This induction was partially inhibited by propranolol. However, the increased accumulation of porphyrins in the medium caused by 3′,5′-diethoxycarbonyl-1,4-dihydrocollidine was inhibited by the addition of phenobarbitone. This inhibited induction was further decreased by propranolol.Most of our results indicate that the drugs tested act mainly by their effects on membranes.     

Cobalt regulation of heme synthesis and degradation in avian embryo liver cell culture

Inorganic cobalt was found to induce heme oxygenase activity in primary cultures of embryonic chick liver cells and to inhibit the induction of delta-aminolevulinate synthetase by the porphyrinogenic compounds allylisopropylacetamide, dicarbethoxy-1,4-dihydrocollidine, etiocholanolone, phenobarbital, Aroclor (R)1254, and secobarbital. Much smaller concentrations of Co2+ (5 muM) were required to inhibit delta-aminolevulinate synthetase than to induce heme oxygenase activity (50 muM). These effects of Co2+ on heme synthesis and heme degradation were potentiated by depletion of cellular glutathione content as a result of treatment with diethyl maleate. Cobalt inhibition of the induction of delta-aminolevulinate synthetase was of the same magnitude and probably involved the same mechanism as that produced by cobalt heme dimethyl ester and iron heme. The induction of heme oxygenase by cobalt could be blocked by cycloheximide. Plasma protein synthesis was not inhibited in the presence of concentrations of Co2+ which produced inhibition of delta-aminolevulinate synthetase or induction of heme oxygenase. Other metals such as Cd2+ and Cu2+ also inhibited the induction of delta-aminolevulinate synthetase by allylisopropylacetamide. These findings indicate that Co2+ can regulate heme metabolism directly in liver cells without intermediate actions on extrahepatic tissues. It is suggested that regulation of production of delta-aminolevulinate synthetase and heme oxygenase is mediated through the action of the metal ion rather than the metal in the form of a tetrapyrrole chelate.     

Inhibition of experimental porphyria with colchicine

Colchicine at the concentrations of 5 X 10(-7) - 5 X 10(-6) M decreased significantly both delta-aminolevulinic acid synthase activity and accumulation of porphyrins in monolayers of chick embryo liver cells induced by allyl-isopropylacetamide, by 3,5-diethoxycarbonyl-1,4-dihydrocollidine or by phenobarbitone. No effect was noted in non-induced cells. In rats, colchicine 0.3 mg/kg, reduced significantly the allyl-isopropylacetamide induced increase in the activity of delta-aminolevulinic acid synthase in the liver and the concentration of urinary porphyrins while it did not affect these parameters in non-induced rats.     

Steroid induction of delta-aminolevulinic acid synthase and porphyrins in liver. Structure-activity studies and the permissive effects of hormones on the induction process.

Quantitative aspects and structure-activity relationships of the inducing effects of natural steroids on delta-aminolevulinic acid (ALA) synthase and porphyrins have been investigated in monolayer cultures of chick embryo liver cells maintained in a serum-free medium as well as in the chick embryo liver in ovo. Many 5 alpha and 5 beta metabolites of neutral C-19 and C-21 hormones and hormone precursors stimulated porphyrin formation and ALA-synthase induction in the cultured liver cells as we have previously described. In these inducing actions a number of 5 beta epimers (A:B cis) were found to be more potent than their corresponding 5 alpha epimers (A:B trans). The structure-activity relationship between 5 beta and 5 alpha steroid epimers with respect to ALA-synthase induction in culture was also found to prevail with respect to induction of this enzyme in chick embryo liver in ovo. Hemin in concentrations of 2 x 10(-7) M inhibited steroid induction of porphyrin formation, and CaMgEDTA enhanced the responsiveness of the cultured liver cells to steroids by approximately 10 times. The addition of insulin, or insulin plus hydrocortisone or insulin plus hydrocortisone plus triiodothyronine, was important for the maintenance of protein synthesis and essential for maximal expression of the ability of steroids to induce porphyrins and ALA-synthase in the "permissive" effect which insulin, hydrocortisone, and triiodothyronine exert on allylisopropylacetamide induction of porphyrins and ALA-synthase also extends to the induction process which is elicited by natural steroids. These findings also strongly suggest that the regulation of hepatic porphyrin-heme biosynthesis by endogenous as well as exogenous chemicals is significantly influenced by the internal hormonal milieu.     

The influence of carbamazepine on the heme biosynthetic pathway

Carbamazepine, a drug which is widely used in neurological diseases, has a porphyrogenic effect in chick embryo liver cells in culture. It increased the concentration of cellular porphyrins by 80-fold and delta-aminolevulinate synthase activity by 4-fold. The increase in the accumulation of porphyrins preceded that of ALAS activity. Measurements of the activities of aminolevulinate dehydrase, porphobilinogen deaminase, and uroporphyrinogen decarboxylase showed that C inhibits UROD up to nearly 50% and PBGD activity up to 20%, but does not affect the activity of ALAD. The pattern of accumulation of porphyrins, mainly uro- and heptacarboxylporphyrin, is compatible with an inhibition of UROD. We may, therefore, conclude that the porphyrogenic effect of C in monolayers of chick embryo liver cells is the result of its inhibitory effect on the activity of UROD.     

Decreased liver activity of porphyrin–metal chelatase in hepatic porphyria caused by 3,5-diethoxycarbonyl-1,4-dihydrocollidine. Studies in rats and mice

1. A difference has been found between rats and mice in their sensitivity to the porphyrogenic effect of drugs. Mice are more sensitive than rats to 3,5-diethoxycarbonyl-1,4-dihydrocollidine, but less sensitive than rats to 2-allyl-2-isopropylacetamide. 2. Use has been made of this difference in sensitivity to ascertain the importance of the decrease of liver porphyrin-metal chelatase activity in porphyria caused by 3,5-diethoxycarbonyl-1,4-dihydrocollidine. Mice, which are more sensitive than rats to the stimulation of 5-aminolaevulinate caused by this drug, are also more sensitive with respect to the decrease of chelatase activity. 3. In both species, after treatment with 3,5-diethoxycarbonyl-1,4-dihydrocollidine, the ratio between chelatase activity and 5-aminolaevulinate activity is linear with respect to the reciprocal of the liver porphyrin concentration. This suggests that under these conditions the degree of porphyrin accumulation depends on the balance between rate of porphyrin formation and rate of porphyrin utilization. 4. Compound SKF 525-A (2-diethylaminoethyl 3,3-diphenylpropylacetate) when given before 3,5-diethoxycarbonyl-1,4-dihydrocollidine prevents the appearance of porphyria in the rat and also largely prevents the decrease of chelatase activity. In the mouse it is much less effective in preventing porphyria and it is almost completely inactive in protecting the chelatase from a decrease in activity. 5. Cycloheximide, when given before 3,5-diethoxycarbonyl-1,4-dihydrocollidine also inhibits the induction of 5-aminolaevulinate synthetase and the appearance of porphyria in the rat, but does not prevent the decrease of chelatase activity. These results suggest that two successive stages can be distinguished in the induction process: a first stage leading to inhibition of haem synthesis and a second stage requiring synthesis of protein in the liver and leading to stimulation of 5-aminolaevulinate synthetase.     

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.     

Inhibition of steroid-mediated induction of δ-aminolevulinic acid synthase by 2-diethylaminoethyl-2,2-diphenylvalerate HCl (SKF 525-A)

The inhibition of the steroid-mediated induction of δ-aminolevulinate synthase, the rate-limiting enzyme in hepatic porphyrin-heme biosynthesis, by 2-diethylaminoethyl-2,2-diphenylvalerate HCl (SKF 525-A) as studied in cultured chick embryo liver cells. The formation of porphyrins in response to cyproterone, a synthetic steroid, was inhibited in a time-dependent manner by SKF 525-A, an inhibitor of several drug metabolizing enzyme systems. This action is a result of an inhibitory effect of SKF 525-A on the cyproterone-mediated induction of δ-aminolevulinate synthase; SKF 525-A laso inhibited the induction of the enzyme by the naturally occurring 5β-H steroids, etiocholanolone and pregnanolone. Employing [³H]etiocholanolone, we provide evidence that this inhibition is not associated with either decreased uptake or an altered metabolism of the steroid. Moreover, approx. 4–6-fold more radioactivity was associated with [³H]etiocholanolone-treated cells cultured in the presence of SKF 525-A. Alternative mechanisms for the induction of δ-aminolevulinate synthase by steroids are proposed which do not require the interaction of steroid-receptor complex with the genome.     

Inhibition of chemical induction of porphyrin synthesis in chick embryo liver cells by partially purified human chorionic gonadotropin

Commercial preparations of human chorionic gonadotropin (hCG) inhibited chemical induction of δ -aminolevulinic acid synthetase and porphyrin formation in chick embryo liver cell cultures. The inhibition was not attributable to hCG since highly purified preparations of the hormone were not inhibitory. After fractionation of crude hCG on Sephadex G-100, inhibitory activity was found in two fractions, one of slightly smaller and one of much smaller molecular weight than hCG. Thus partially purified hCG may have other biologic effects than those caused by the hormone itself. Moreover, the occurrence of substances in crude hCG which at low concentrations can interfere with drug and hormone effects on liver cells is of biologic and potential clinical interest.     

Stimulation of liver 5-aminolaevulinate synthetase by drugs and its relevance to drug-induced accumulation of cytochrome P-450. Studies with phenylbutazone and 3,5-diethoxycarbonyl-1,4-dihydrocollidine

The relevance of the stimulation of 5-aminolaevulinate synthetase to the accumulation of cytochrome P-450 after administration of drugs was examined in rats treated with phenylbutazone and with 3,5-diethoxycarbonyl-1,4-dihydrocollidine. 3,5-Diethoxycarbonyl-1,4-dihydrocollidine alone stimulated 5-aminolaevulinate synthetase without increasing the concentration of cytochrome P-450, whereas phenylbutazone alone increased the microsomal cytochrome P-450 without significantly affecting the activity of the enzyme. When the two drugs were given together both effects were found. It is concluded that if an increased amount of 5-aminolaevulinate and haem must be made to provide for the accumulation of cytochrome P-450, it need only be a small amount. It is also concluded from these findings that stimulation of the drug-metabolizing system on the one hand and marked enhancement of 5-aminolaevulinate synthetase activity and porphyria on the other are likely to result from different actions of the drugs. Evidence is presented suggesting that porphyrogenic drugs stimulate markedly the activity of 5-aminolaevulinate synthetase by lowering the concentration of haem in the liver, thereby decreasing the normal feedback control. With 3,5-diethoxycarbonyl-1,4-dihydrocollidine a rapid inhibition of mitochondrial ferrochelatase and of liver haem synthesis may be the primary mechanism involved.     

Inhibition of ferrochelatase by N-methylprotoporphyrin IX is not accompanied by delta-aminolevulinic acid synthetase induction in chick embryo liver cell culture

N-Methylprotoporphyrin has been shown to markedly inhibit ferrochelatase activity in chick embryo liver cell culture without inducing delta-aminolevulinic acid (ALA) synthetase activity. This result supports the idea that the effects of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) on ALA synthetase activity and ferrochelatase activity are dissociated and that inhibition of ferrochelatase alone is not sufficient to cause induction of ALA synthetase. We conclude that the porphyrinogenic activity of DDC can be explained only in part by the actions of N-methylprotoporphyrin.     

The effects of dihydropyridine calcium antagonists on heme biosynthesis in chick embryo liver cell culture

A variety of 1,4-dihydropyridine calcium antagonists were tested for porphyrinogenic activity in chick embryo liver cell culture. 3,5-Dimethoxycarbonyl-1,4-dihydro-2, 6-dimethyl-4-(ortho-nitrophenyl)pyridine (nifedipine) was shown to be a potent porphyrinogenic agent. This activity was shared by a number of related analogues, viz., the 4-phenyl, 4-(meta-nitrophenyl), 4-(para-nitrophenyl), 4-(ortho-methoxyphenyl), 4-(meta-trifluoromethylphenyl), and 4-(para-trifluoromethylphenyl) analogues and nitrendipine; nicardipine exhibited very weak activity. The porphyrinogenic potency of the 1,4-dihydropyridines did not parallel their calcium antagonist activity. Nifedipine did not exhibit ferrochelatase-lowering activity in chick embryo liver cell culture and uroporphyrin and heptacarboxylic acid porphyrin were the major porphyrins to accumulate. Nifedipine did not cause suicidal destruction of cytochrome P-450 in chick embryo hepatic microsomes. Because nifedipine possesses comparable porphyrinogenic activity to sodium secobarbital in chick embryo liver cell culture, caution is required if nifedipine or related drugs are administered to patients with hereditary hepatic porphyria.     

The 1986 Upjohn award lecture. Interaction of chemicals with hemoproteins: implications for the mechanism of action of porphyrinogenic drugs and nitroglycerin

The ferrochelatase inhibitory activity of a variety of analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) was studied in chick embryo liver cells. The ferrochelatase inhibitory activity of the 4-butyl, 4-pentyl, and 4-hexyl analogues was considered to be due to catalytic activation by cytochrome P-450 leading to heme alkylation and formation of the corresponding N-alkylporphyrins. The relative ferrochelatase inhibitory activity of the DDC analogues has implications for a postulated model of the binding of porphyrins in the ferrochelatase active site. 3-[2-(2,4,6-Trimethylphenyl)thioethyl]-4-methylsydnone (TTMS) was shown to be a potent porphyrinogenic agent and to inhibit ferrochelatase in chick embryo liver cells. A related sydnone, 3-benzyl-4-phenylsydnone did not inhibit ferrochelatase activity. These results supported the idea that the porphyrinogenicity of TTMS was due to catalytic activation by cytochrome P-450 leading to heme alkylation and formation of N-vinylprotoporphyrin which inhibits ferrochelatase. Polychlorinated biphenyls, phenobarbital, nifedipine, and a large number of structurally different chemicals which are porphyrinogenic in chick embryo liver cells inhibit uroporphyrinogen decarboxylase by an unknown mechanism. Thus drug-induced porphyrin biosynthesis in chick embryo liver cell culture appears to be caused by inhibition of either ferrochelatase or uroporphyrinogen decarboxylase. The biotransformation of nitroglycerin by human red blood cells is due to a combination of a sulfhydryl-dependent enzymatic process and an interaction with reduced hemoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects by heme, insulin, and serum albumin on heme and protein synthesis in chick embryo liver cells cultured in a chemically defined medium, and a spectrofluorometric assay for porphyrin composition.

Primary chick embryo liver cells, which had been previously cultured in Eagle's medium containing 10% fetal bovine serum, had the same characteristics (inducibility of delta-aminolevulinic acid synthetase and synthesis of plasma proteins) when cultured in a completely defined Ham F-12 medium containing insulin. Insulin was active in the physiological range; 2 to 3 nM were sufficient to increase the induced delta-aminolevulinic acid synthetase to 50% of the maximum effect obtained with a saturating amount of insulin (30 nM). Serum albumin added to the Ham-insulin medium caused protoporphyrin but not uroporphyrin, generated in the cultured liver cells, to be transferred to the medium. As little as 10 mug of human serum albumin per ml caused the transfer of one-half of the protoporphyrin. Bovine serum albumin was only about 1/30 as effective. A spectrofluorometric method and calculation procedure are described for quantitation, in the nanomolar range, of total porphyrin and the percentage of this that is protoporphyrin or uroporphyrin plus coproporphyrin. The method is satisfactory for the measurement of porphyrins generated by 1 mg wet weight of cells in culture in 20 hours. Heme (0.1 to 0.3 muM), when added to the medium as hemin, human hemoglobin, or chicken hemoglobin, specifically inhibited the induction of delta-aminolevulinic acid synthetase by one-half. This high sensitivity for heme was observed under conditions in which the defined medium was free of serum and where a chelator of iron was added to the medium to diminish the synthesis of endogenous heme. Heme endogenously generated from exogenous delta-aminolevulinic acid also inhibited the induction; chelators of iron prevented this inhibition. The migration of heme from the mitochondria to other portions of the cell is discussed in terms of the affinities of different proteins for heme. A hypothesis of a steady state of liver heme metabolism, controlled by the concentration of "free" heme, is presented. The different effects of heme on the synthesis of a number of proteins are summarized.     

Induction of aminolevulinate synthase and porphyrins in cultured liver cells maintained in chemically defined medium. Permissive effects of hormones on induction process.

Primary liver cells, isolated from 16- 17-day-old chick embryos, were incubated in a serum-free chemically defined medium (Ham's F12) supplemented with hormones for up to 6 days. The culture method also includes the complete removal of contaminating red cells before the initiation of culture. On the 2nd day in cluture, the level of amino-levulinate (ALA) synthase activity in response to allylisopropylacetamide (AIA) was increased 6-fold in cells grown in F12. Insulin, hydrocortisone, and triiodothyronine alone had no appreciable effects on ALA synthase levels. On the other hand, when added with AIA, insulin, insulin plus hydrocortisone, insulin plus hydrocortisone triiodothyronine increased ALA synthase levels 17-, 50-, 110-fold, respectively. The maximally induced levels of ALA synthase activity by AIA in the presence of insulin, hydrocortisone, and triiodothyronine were approximately 15 nmol of ALA/mg of protein/h, 37 degrees or 3 micronmol of ALA/g of tissue/h, 37 degrees, a value similar to that found in ovo or at least 5 times greater than that found in rat liver. The morphology of hepatocytes was maintained for at least 6 days in culture, although the induction of ALA synthase was reduced after the 4th day unless triiodothyronine was present. Dibutyryl adenosine 3':5'-monophosphate (10(8) M) or glucagon (5x10(8) M) had little effect on the induced as well as noninduced levels of ALA synthase or porphyrins. These data demonstrate a "permissive" effect of insulin, hydrocortisone, and triiodothyronine on the induction of ALA synthase and porphyrins by AIA in cultured chick embryo liver cells. In the absence of insulin hydrocortisone, or triiodothyronine, AIA produces only a slight increase in ALA synthase activity or porphyrins (or both); on the other hand, it produces a marked increase in the enzyme activity and porphyrins when these hormones are added to the culture medium. The term "permissive" is applied to these hormone-dependent effects. A sensitive spectrofluorometric method for heme quantitation allowed us to follow changes in the cellular heme content in hemoglobin-free cultured liver cells. Heme content in the cultured liver cells was approximately 250 pmol/mg of protein at the initiation of culture but gradually declined to 175 pmol/mg of protein at the initiation of culture but gradually declined to 175 pmol/mg of protein during 48 h of incubation. The apparent decrease in heme content may be accounted for by the concomitant increase in protein content in these cells.     

Synergistic induction of delta-aminolevulinate synthase by glutethimide and iron: relationship to the synergistic induction of heme oxygenase

Relationships between activities of delta-aminolevulinate synthase and heme oxygenase, respectively the rate-limiting enzymes of heme biosynthesis and degradation, have been studied in chick embryo liver cell cultures following exposure of the cultures to glutethimide and iron, a combination known to produce a synergistic induction of both enzymes. In time-course experiments, synergistic induction of heme oxygenase activity by glutethimide and iron preceded that of delta-aminolevulinate synthase by 4 h. Effects of selective inhibitors of both heme synthesis and degradation have also been studied with respect to effects on delta-aminolevulinate synthase and heme oxygenase activities. The synergistic induction of heme oxygenase by glutethimide and iron appears to be dependent upon cellular heme synthesis because addition of inhibitors of heme biosynthesis, 4,6-dioxoheptanoic acid or N-methyl-mesoporphyrin abolishes this synergistic induction. Exposure of cultures to tin-mesoporphyrin, a potent inhibitor of heme oxygenase, prevented the synergistic induction of delta-aminolevulinate synthase produced by glutethimide and iron, or, when added after induction was already established, promptly halted any further induction. These results suggest that the level of activity of heme oxygenase can reciprocally modulate intracellular heme levels and thus activity of delta-aminolevulinate synthase.     

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.     

delta-Aminolevulinate synthetases in the liver cytosol fraction and mitochondria of mice treated with allylisopropylacetamide and 3,5-dicarbethoxyl-1,4-dihydrocollidine.

Hepatic delta-aminolevulinate (ALA) synthetase was induced in mice by the administration of allylisopropylacetamide (AIA) and 3,5-dicarbethoxy-1,4-dihydrocollidine (DDC). In both cases, a significant amount of ALA synthetase accumulated in the liver cytosol fraction as well as in the mitochondria. The apparent molecular weight of the cytosol ALA synthetase was estimated to be 320,000 by gel filtration, but when the cytosol ALA synthetase was subjected to sucrose density gradient centrifugation, it showed a molecular weight of 110,000. In the mitochondria, there were two different sizes of ALA synthetase with molecular weights of 150,000 and 110,000, respectively; the larger enzyme was predominant in DDC-treated mice, whereas in AIA-treated mice and normal mice the enzyme existed mostly in the smaller form. When hemin was injected into mice pretreated with DDC, the molecular size of the mitochondrial ALA synthetase changed from 150,000 to 110,000. The half-life of ALA synthetase in the liver cytosol fraction was about 30 min in both the AIA-treated and DDC-treated mice. The half-life of the mitochondrial ALA synthetase in AIA-treated mice and normal mice was about 60 min, but in DDC-treated mice the half-life was as long as 150 min. The data suggest that the cytosol ALA synthetase of mouse liver is a protein complex with properties very similar to those of the cytosol ALA synthetase of rat liver, which has been shown to be composed of the enzyme active protein and two catalytically inactive binding proteins, and that ALA synthetase may be transferred from the liver cytosol fraction to the mitochondria with a size of about 150,000 daltons, followed by its conversion to enzyme with a molecular weight of 110,000 within the mitochondria. The process of intramitochondrial enzyme degradation seems to be affected in DDC-treated animals.     

The reversal of experimental porphyria and the prevention of induction of hepatic mixed-function oxidase by acetate

The administration of acetate or sulfanilamide depressed the porphyric response of rats to 3,5-dicarbethoxy-1,4-dihydrocollidine. The induction of δ-aminolevulinate synthetase (EC 2.3.1.37) in porphyric rats was decreased by acetate administration and δ-aminolevulinate synthetase activity in hepatic homogenates was inhibited by acetate. Succinate reversed the inhibition by acetate in vitro. Since an alteration of heme biosynthesis by acetate was observed, the effect of acetate on the induction of hepatic microsomal cytochrome P-450 and microsomal mixed-function oxidase by phenobarbital was examined. Acetate prevented the induction of hepatic mixed-function oxidase and cytochrome P-450 by phenobarbital. Unlike the action of other inhibitors of hepatic heme biosynthesis, acetate also prevented the induction by phenobarbital of NADPH-cytochrome c reductase (EC 1.6.99.3). These findings suggest that acetate may be inhibiting heme biosynthesis by effects on δ-aminolevulinate synthetase, the rate-limiting step in heme biosynthesis, by alteration of the induction of this enzyme and by a direct effect on the enzymic reaction itself. It is suggested that acetate may be involved in the glucose effect related to the inhibition of the induction of δ-aminolevulinate synthetase.