Conversion of 5-aminolaevulinate into haem by liver homogenates. Comparison of rat and chick embryo.

1. We have studied the kinetics of the conversion of 5-aminolaevulinate into haem and haem precursors in homogenates of livers of rats and chick embryos. Homogenates of fresh liver from both species efficiently convert 5-aminolaevulinate into haem. After frozen storage for 1 year, homogenates of rat, but not chick, liver have decreased rates of formation of haem with accumulation of more protoporphyrin. The rate of haem formation after storage is restored by addition of Fe2+ and menadione. 2. At all initial concentrations of 5-aminolaevulinate tested (2 microM-1 mM), homogenates of rat liver accumulate less protoporphyrin than haem. In contrast, homogenates of chick embryo liver accumulate more protoporphyrin than haem at concentration of 5-aminolaevulinate greater than 10 microM. Conversion of protoporphyrin into haem by homogenates of fresh or frozen chick embryo liver is not increased by addition of Fe2+. 3. Homogenates of liver from both species accumulate porphobilinogen; the kinetic parameters for this process reflect those of 5-aminolaevulinate dehydratase. 4. The results show that the rate-limiting enzyme for the hepatic conversion of 5-aminolaevulinate into protoporphyrin is porphobilinogen deaminase. In addition, chick liver, compared with rat liver, has only about one-fifth the activity of ferrochelatase, the final enzyme of the haem biosynthetic pathway, which inserts Fe2+ into protoporphyrin to form haem. 5. Comparison of these results with previous studies indicates that the homogenate system described here provides physiologically and clinically relevant information for study of hepatic haem synthesis and its control.     

Haem synthesis from exogenous 5-aminolaevulinate in cultured chick-embryo hepatocytes. Effects of inducers of cytochromes P-450.

The effects of inducers of cytochrome P-450 on haem biosynthesis from 5-aminolaevulinate were examined by using cultured chick-embryo hepatocytes. Cultures treated with either 2-propyl-2-isopropylacetamide or 3-methylcholanthrene contained increased amounts of cytochrome P-450 and haem. After treatment for 3 h with 5-amino[4-14C]laevulinate, the relative amounts of radioactivity accumulating as haem corresponded to the relative amounts of total cellular haem, but not to increases in the amounts of cytochrome P-450. Treatment with 5-aminolaevulinate did not alter cellular haem or cytochrome P-450 concentrations in either control or drug-treated cultures. The mechanism of the enhanced accumulation of radioactivity in haem was investigated. Although 2-propyl-2-isopropylacetamide enhanced the uptake of 5-aminolaevulinate and increased the cellular concentration of porphobilinogen 1.5-fold, these changes did not account for the increases in haem radioactivity. The inducing drugs had no effect on the rates of degradation of radioactive haem, but appeared to enhance conversion of protoporphyrin into haem. This latter effect was shown by: (1) a decreased accumulation of protoporphyrin from 5-aminolaevulinate in cells treated with inducers, and (2) complete prevention of this decrease if the iron chelator desferrioxamine was present. We conclude that inducers of cytochrome P-450 may increase haem synthesis not only by increasing activity of 5-aminolaevulinate synthase, but also by increasing conversion of protoporphyrin into haem.     

Formation of cobalt protoporphyrin in the liver of rats. A mechanism for the inhibition of liver haem biosynthesis by inorganic cobalt.

1. Treatment of rats with small doses of CoCl2 decreases liver 5-aminolaevulinate synthase (EC 2.3.1.37) activity and impairs incorporation of 5-amino[14C]laevulinate into liver haem. Salts of other metals (cadmium, nickel, manganese and zinc) are all relatively inactive. 2. The dose-response curves obtained for both these effects closely mirror the accumulation in the liver of a compound that is labelled by 5-amino[14C]laevulinate and is unextractable by acetone/HCl. 3. Incorporation of 5-amino[14C]laevulinate into unextractable compound is also obtained in vitro by incubating liver homogenates with label in the presence of cobalt:isotope-dilution experiments show that the radioactivity passes through pools of porphobilinogen and protoporphyrin, but not of haem. 4. The unextractable compound is not covalently bound to protein and possesses the same extraction and spectral properties as authentic cobalt protoporphyrin. 5. It is concluded (a) that cobalt protoporphyrin is readily formed not only in vitro, but also in vivo, and (b) that its formation accounts for the impaired incorporation of 5-aminolaevulinate into haem and may also be responsible for the action of cobalt on 5-aminolaevulinate synthase.     

Uroporphyrin accumulation produced by halogenated biphenyls in chick-embryo hepatocytes. Reversal of the accumulation by piperonyl butoxide.

Cultures of chick-embryo hepatocytes were used to study the mechanism by which 3,4,3',4'-tetrachlorobiphenyl and 2,4,5,3',4'-pentabromobiphenyl cause accumulation of uroporphyrin. In a previous paper, an isoenzyme of cytochrome P-450 induced by 3-methylcholanthrene had been implicated in this process [Sinclair, Bement, Bonkovsky & Sinclair (1984) Biochem. J. 222, 737-748]. Cells treated with 3,4,3',4'-tetrachlorobiphenyl and 5-aminolaevulinate accumulated uroporphyrin and heptacarboxyporphyrin, whereas similarly treated cells accumulated protoporphyrin immediately after piperonyl butoxide was added. Piperonyl butoxide also restored haem synthesis as detected by incorporation of radioactive 5-aminolaevulinate into haem, and decrease in drug-induced 5-aminolaevulinate synthase activity. The restoration of synthesis of protoporphyrin and haem by piperonyl butoxide was not affected by addition of cycloheximide, indicating recovery was probably not due to protein synthesis de novo. Piperonyl butoxide also reversed uroporphyrin accumulation caused by 3,4,5,3',4',5'-hexachlorobiphenyl, mixtures of other halogenated biphenyls, lindane, parathion, nifedipine and verapamil. The effect of piperonyl butoxide was probably not due to inhibition of metabolism of these compounds, since the hexachlorobiphenyl was scarcely metabolized. Other methylenedioxyphenyl compounds, as well as ellipticine and acetylaminofluorene, also reversed the uroporphyrin accumulation caused by 3,4,3',4'-tetrachlorobiphenyl. SKF-525A (2-dimethylaminoethyl-2,2-diphenyl valerate) did not reverse the uroporphyrin accumulation caused by the halogenated biphenyls, but did reverse that caused by phenobarbital and propylisopropylacetamide. We conclude that the mechanism of the uroporphyrin accumulation cannot be due to covalent binding of activated metabolites of halogenated compounds to uroporphyrinogen decarboxylase.     

Decrease in hepatic cytochrome P-450 by cobalt. Evidence for a role of cobalt protoporphyrin.

Exposure of cultured chick-embryo hepatocytes to increasing concentrations of CoCl2 in the presence of allylisopropylacetamide results in formation of cobalt protoporphyrin, with a reciprocal decrease in haem and cytochrome P-450. Treatment of rats with CoCl2 (84 mumol/kg) and 5-aminolaevulinate (0.2 mmol/kg) also results in formation of cobalt protoporphyrin and a decrease in cytochrome P-450 in the liver. Hepatic microsomal fractions from rats treated with phenobarbital, CoCl2 and 5-aminolaevulinate were analysed by polyacrylamide gel electrophoresis. Cobalt protoporphyrin was associated mainly with proteins of 50000-53000 mol.wt. The results suggest that the formation of cobalt protoporphyrin occurred at the expense of the synthesis of haem, leading to a decrease in cytochrome P-450. Furthermore, the cobalt protoporphyrin that was formed may itself have been incorporated into apocytochrome P-450.     

Inhibition of haem synthesis caused by cobalt in rat liver. Evidence for two different sites of action.

Cobalt inhibits liver haem synthesis in vivo by acting at least two different sites in the biosynthetic pathway: (1) synthesis of 5-aminolaevulinate and (2) conversion of 5-amino-laevulinate into haem. The first effect is largely, if not entirely, due to inhibition of the activity of 5-aminolaevulinate synthase, rather than to inhibition of the formation of the enzyme. The second effect results from diversion of 5-aminolaevulinate into an unidentified liver pool with solubility properties similar to those of cobalt protoporphyrin.     

Studies on Cerebellar Haem Metabolism in the Rat In Vivo

Abstract: Rats were injected intraventricularly with 5-amino[4-¹⁴C]laevulinate and the radioactivity recovered in the total cerebellum homogenate and in its haem and porphyrin fractions was determined in time. Two phases could be distinguished in the decline of haem radioactivity, suggesting labelling of at least two pools of widely different turnover rates. Succinyl acetone, when injected intraventricularly, caused a marked and long-lasting inhibition of cerebellar 5-aminolaevulinate dehydratase activity and a corresponding inhibition of the incorporation of [¹⁴C]5-aminolaevulinate into cerebellar haem in vivo. Inhibition of cerebellar haem biosynthesis by succinylacetone was followed by stimulation of the first enzyme of the pathway, 5-aminolaevulinate synthase, whereas intraventricular injection of haematin led to a significant depression of the activity of the enzyme. This suggested that the cerebellar 5-aminolaevulinate synthetase is regulated by haem through a negative feedback mechanism. Rats given repeated doses of succinylacetone, so as to maintain 80% inhibition of their cerebellar 5-aminolaevulinate dehydratase activity for 5 days, failed to exhibit any obvious symptoms of toxicity but became more sensitive to the neurotoxic effects of large intraventricular doses of 5-aminolaevulinate.     

Mechanism of action of 5-aminolaevulinate dehydratase from human erythrocytes.

Purified 5-aminolaevulinate dehydratase (porphobilinogen synthase, EC 4.2.1.24) from human erythrocytes was incubated initially with limiting amounts of 5-amino [5-14C]laevulinate in a rapid-mixing apparatus. The single-turnover reaction with respect to the bound labelled 5-aminolaevulinate was completed by the addition of unlabelled 5-aminolaevulinate and the resulting radioactive porphobilinogen was isolated and degraded. The 14C label was found to be located predominantly at C-2 of the product, demonstrating that, of the two substrate molecules participating in the reaction, the 5-aminolaevulinate molecule initially bound to the enzyme provides the propionic acid 'side' of the porphobilinogen. The same enzyme-[14C]substrate species that yields regiospecific porphobilinogen may be trapped by reaction with NaBH4, showing that the substrate molecule initially bound to the enzyme does so in the form of a Schiff base. A conventional incubation with 5-amino[5-14C]laevulinate yielded porphobilinogen with an equal distribution of the label between C-2 and C-11. The reaction mechanism of the human erythrocyte 5-aminolaevulinate dehydratase thus follows the same course as that of other dehydratases studied in our laboratory by using single-turnover techniques.     

Induction of 5-aminolaevulinate synthase by two- to five-carbon alcohols in cultured chick-embryo hepatocytes. Relationship to induction of cytochrome P-450.

The induction of 5-aminolaevulinate synthase and of cytochrome P-450 by short-chain aliphatic alcohols was compared in primary cultures of chicken-embryo hepatocytes. Isopropyl alcohol, isobutanol, pentan-1-ol and isopentanol alone caused up to a 4-fold increase in 5-aminolaevulinate synthase, whereas ethanol and propan-1-ol did not. Induction of the synthase by isopentanol was maximal at 8 h, and reached a plateau thereafter, whereas the activity induced by 2-propyl-2-isopropylacetamide continued to increase for 20 h. In the presence of 3,4,3',4'-tetrachlorobiphenyl, an inhibitor of haem synthesis at the uroporphyrinogen decarboxylase step, synergistic induction of 5-aminolaevulinate synthase was observed with all the alcohols except ethanol. Ethanol, but not isopentanol, decreased the extent of induction of 5-aminolaevulinate synthase by 2-propyl-2-isopropylacetamide and 3,4,3',4'-tetrachlorobiphenyl (50% decrease at 112 mM-ethanol). Total protein synthesis was not inhibited by ethanol in these cells. The composition of porphyrins was determined after treatment of cells with ethanol, isopentanol or 2-propyl-2-isopropylacetamide. Untreated cells, when incubated with 5-aminolaevulinate for 6 h, accumulated mainly protoporphyrin. However, when cells were pretreated with ethanol, isopentanol or 2-propyl-2-isopropylacetamide for 20 h, and 5-aminolaevulinate was added, 8- and 7-carboxyporphyrins increased, whereas protoporphyrin decreased. The dose responses for induction of either 5-aminolaevulinate synthase or cytochrome P-450 after a 20 h exposure to 3- to 5-carbon alcohols were identical. The results indicate that: simple alcohols can induce both enzymes; hydrophobicity increases their effectiveness; and induction of both enzymes are probably mediated by a common mechanism.     

Regulation of rat liver tryptophan pyrrolase by its cofactor haem: Experiments with haematin and 5-aminolaevulinate and comparison with the substrate and hormonal mechanisms.

1. The administration of haematin or 5-aminolaevulinate to rat enhances the activity of liver tryptophan pyrrolase; both endogenous and newly formed apoenzymes become strongly haem-saturated. Haem activation does not stabilize tryptophan pyrrolase. 2. Actinomycin D, puromycin or cycloheximide prevent the activation of the enzyme by 5-aminolaevulinate but not that by haematin. The latter is inhibited by haem-destroying porphyrogens. 3. The combined injection of either haematin or 5-aminolaevulinate with cortisol does not produce an additive effect, whereas potentation is observed when tryptophan is jointly given with either the cofactor or the haem precursor. 4. Further experiments on the substrate (tryptophan) mechanism of pyrrolase regulation are reported, and a comparison between this and the cofactor and hormonal mechanisms is made. 5. It is suggested that the substrate mechanism may also involve increased haem synthesis. 6. The role of tryptophan pyrrolase in the utilization of liver haem, and as a possible model for the exacerbation by drugs of human hepatic porphyrias, is discussed.     

Role of haem in the induction of cytochrome P-450 by phenobarbitone. Studies in chick embryos in ovo and in cultured chick embryo hepatocytes.

The role of haem synthesis during induction of hepatic cytochrome P-450 haemoproteins was studied in chick embryo in ovo and in chick embryos hepatocytes cultured under chemically defined conditions. 1. Phenobarbitone caused a prompt increase in the activity of 5-aminolaevulinate synthase, the rate-limiting enzyme of haem biosynthesis, and in the concentration of cytochrome P-450. This induction response occurred without measurable initial destruction of the haem moiety of cytochrome P-450. 2. When intracellular haem availability was enhanced by exogenous haem or 5-aminolaevulinate, phenobarbitone-medicated induction of cytochrome P-450 was not affected in spite of the well known repression of 5-aminolaevulinate synthase by haem. These data are consistent with the concept that haem does not regulate the synthesis of cytochrome P-450 haemoproteins. 3. Acetate inhibited haem biosynthesis at the level of 5-aminolaevulinate formation. When intracellular haem availability was diminished by treatment with acetate, phenobarbitone-medicated induction was decreased. 4. This inhibitory effect of acetate on cytochrome P-450 induction was reversed by exogenous haem or its precursor 5-aminolaevulinate. These data suggest that inhibition of haem biosynthesis does not decrease synthesis of apo-cytochrome P-450. Moreover, they indicate that exogenous haem can be incorporated into newly formed aop-cytochrome P-450.     

Bile pigment synthesis in plants. Incorporation of haem into phycocyanobilin and phycobiliproteins in Cyanidium caldarium.

A procedure was developed whereby haem was taken up by dark-grown cells of the unicellular rhodophyte Cyanidium caldarium. These cells were subsequently incubated either in the dark with 5-aminolaevulinate, which results in excretion of phycocyanobilin into the suspending medium or incubated in the light, which results in synthesis and accumulation of phycocyanin and chlorophyll a within the cells. Phycocyanobilin was isolated from phycocyanin by cleavage from apoprotein in methanol. Phycocyanobilin prepared from phycocyanin or excreted from cells given 5-aminolaevulinate was methylated and purified by t.l.c. By using 14C labelling either in the haem or in 5-aminolaevulinate administered, haem incorporation into phycocyanobilin was demonstrated in both dark and light systems. Since chlorophyll a synthesized in the light in the presence of labelled haem contained no radioactivity, it was clear that haem was directly incorporated into phycocyanobilin and not first converted into protoporphyrin IX. These results clearly demonstrate phycocyanobilin synthesis via haem and not via magnesium protoporphyrin IX as has also been postulated.     

The use of exogenous δ-aminolaevulinic acid for the studies of the regulation of haem synthesis in rabbit reticulocytes

δ-Amino [4-¹⁴C]laevulinate added to reticulocytes incubated in vitro is incorporated into haem. Exogenous δ-aminolaevulinate restores the incorporation of ⁵⁹Fe into haem in reticulocytes which had been treated with isonicotinic acid hydrazide (INH) or penicillamine and were hence unable to synthesize δ-aminolaevulinate. On the other hand, the addition of δ-aminolaevulinate does not restore the incorporation of Fe into reticulocytes incubated with haemin. The inhibition of the incorporation of iron is neither restored by δ-aminolaevulinate in reticulocytes incubated with cycloheximide (which inhibits globin synthesis and thus elevates the free intracellular haem pool). These results suggest that in intact reticulocytes haemin does not inhibit δ-aminolaevulinate synthetase. This conclusion is further supported by the finding that the pattern of incorporation of [2-¹⁴C]glycine and δ-amino[4-¹⁴C]-laevulinate into haem differs in reticulocytes incubated with an inhibitor of δ-aminolaevulinate synthetase (INH) and in reticulocytes incubated with haemin and cycloheximide.     

The effects of chemical porphyrogens and drugs on the activity of rat liver tryptophan pyrrolase

1. Drugs such as phenobarbitone and phenylbutazone, which increase the concentration of microsomal haem and cytochrome P-450, also increase the saturation of rat liver apo-(tryptophan pyrrolase) with its haem activator, as does the haem precursor 5-aminolaevulinate. 2. At 4h after the administration of the porphyrogens 2-allyl-2-isopropylacetamide, 3,5-diethoxycarbonyl-1,4-dihydrocollidine and griseofulvin, the total pyrrolase activity is increased whereas the haem saturation of the apoenzyme is decreased. This decreased saturation is prevented by pretreatment of the animals with the inhibitor of drug-metabolizing enzymes, SKF 525-A. 3. Pretreatment of rats with the above porphyrogens inhibits the rise in holo-(tryptophan pyrrolase) activity produced by subsequent administration of cortisol, tryptophan and 5-aminolaevulinate with two single exceptions, the possible reasons for which are discussed. 4. At 24h after the administration, in starved rats, of a single daily injection of the above porphyrogens for 1 or 2 days, the holoenzyme activity is significantly increased. 5. It is suggested that the saturation of rat liver apo-(tryptophan pyrrolase) with its haem activator can be modified by treatment known to cause destruction, inhibition of synthesis, increased utilization and enhanced synthesis of liver haem. The possible involvement of the latter phenomenon in the aetiology of mental disorders in some patients with porphyria is discussed.     

Formation of cytochrome P-450 containing haem or cobalt-protoporphyrin in liver homogenates of rats treated with phenobarbital and allylisopropylacetamide.

The potent porphyrogen allylisopropylacetamide and related compounds decrease hepatic concentrations of cytochrome P-450. This decrease occurs particularly in phenobarbital-induced cytochrome P-450 and is caused by suicidal breakdown of the haem of cytochrome P-450. Quantitative rocket immunoelectrophoresis showed that the protein moiety of the major phenobarbital-inducible form of hepatic cytochrome P-450 was not diminished up to 1 h, but was markedly decreased (to 43% of that of the phenobarbital-treated control) at 20 h after allylisopropylacetamide treatment. In contrast, the concentration of total cytochrome P-450, measured spectrophotometrically, decreased to 30-40% of the control at both 1 and 20 h after allylisopropylacetamide. Cytochrome P-450-dependent demethylations of ethylmorphine and benzphetamine decreased to a similar extent. When liver homogenates from rats treated with allylisopropylacetamide 1 h before being killed were incubated with haem, functional holocytochrome P-450 could be reconstituted from the apoprotein. Incubation with haem increased spectrophotometrically measurable cytochrome P-450 to 69%, ethylmorphine demethylase to 64% and benzphetamine demethylase to 93% of the activities in rats treated with phenobarbital alone. At 20 h after allylisopropylacetamide treatment, however, little or no reconstitution of cytochrome P-450 occurred after incubation with haem. When liver homogenates were incubated with cobalt and protoporphyrin, and microsomal proteins were then subjected to polyacrylamide-gel electrophoresis, cobalt-protoporphyrin was found specifically associated with proteins of Mr 50 000-53 000. When homogenates from rats given allylisopropylacetamide for 1 h or 20 h were compared, it was found that the extent of this association was higher in livers from the rats containing more apocytochrome P-450, suggesting that cobalt-protoporphyrin had associated with the apocytochrome. The data provide insight into the association of haem with the protein moiety of cytochrome P-450 and factors affecting breakdown of this protein.     

Tryptophan pyrrolase in haem regulation. Experiments with administered haematin and the relationship between the haem saturation of tryptophan pyrrolase and the activity of 5-aminolaevulinate synthase in rat liver.

1. Administration of haematin to rats decreases 5-aminolaevulinate synthase activity in whole liver homogenates. 2. An inverse relationship between this decrease and the increase in saturation of apo-(tryptophan pyrrolase) with haem is observed during the initial phase of treatment with haematin. 3. Significant changes in both functions are caused by a 1 mg/kg dose of haematin, whereas the maximum effects are achieved by the 5 mg/kg dose. 4. Prevention by allopurinol of the conjugation of exogenously administered haematin with apo-(tryptophan pyrrolase) renders this haem available for further repression of 5 aminolaevulinate synthase. 5. The various aspects of the relationship between synthase activity and the haem saturation of tryptophan pyrrolase are discussed.     

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.     

Tryptophan pyrrolase in haem regulation. The mechanisms of enhancement of rat liver 5-aminolaevulinate synthase activity by starvation and of the glucose effect on induction of the enzyme by 2-allyl-2-isopropylacetamide

1. Rat liver tryptophan pyrrolase activity is enhanced by a hormonal-type mechanism during the first 2 days of starvation and by a substrate-type mechanism during the subsequent 2 days. 5-Aminolaevulinate synthase activity is also enhanced during the first 2 days of starvation, but returns thereafter to values resembling those observed in the fed rat. Treatments that prevent or reversé the enhancement of tryptophan pyrrolase activity in 24–48h-starved rats also abolish that of 5-aminolaevulinate synthase activity. Starvation of guinea pigs, which does not enhance the pyrrolase activity, also fails to alter that of the synthase. It is suggested that the decrease in 5-aminolaevulinate synthase activity in 72–96h-starved rats represents negative-feedback repression of synthesis, possibly involving tryptophan participation, whereas the enhancement observed in 24–48h-starved animals is caused by positive-feedback induction secondarily to increased utilization of the regulatory-haem pool by the newly synthesized apo-(tryptophan pyrrolase). 2. Glucose, fructose and sucrose abolish the 24h-starvation-induced increases in rat liver tryptophan pyrrolase and 5-aminolaevulinate synthase activities. Cortisol reverses the glucose effect on 5-aminolaevulinate synthase activity, presumably by enabling pyrrolase to re-utilize the regulatory-haem pool after induction of synthesis of this latter enzyme. 3. The impaired ability of 2-allyl-2-isopropylacetamide to enhance markedly 5-aminolaevulinate synthase activity in 24h-starved rats treated with glucose is associated with a failure of the porphyrogen to cause loss of tryptophan pyrrolase haem. Cortisol restores the ability of the porphyrogen to destroy tryptophan pyrrolase haem and to enhance markedly 5-aminolaevulinate synthase activity, presumably by enhancing tryptophan pyrrolase synthesis and, thereby, its re-utilization of the regulatory-haem pool. It is tentatively suggested that 2-allyl-2-isopropylacetamide destroys the above pool only after it has become bound to (or utilized by) apo-(tryptophan pyrrolase).     

Studies of cytochrome synthesis in rat liver

The incorporation of radioactive amino acids and of δ-amino[2,3-³H₂]laevulinate into rat liver cytochromes b₅ and c and cytochrome oxidase has been examined with and without protein-synthesis inhibitors. Cycloheximide promptly inhibits labelling of both haem and protein for cytochrome c in parallel fashion. Although incorporation of ¹⁴C-labelled amino acid into microsomal cytochrome b₅ is also rapidly inhibited, cycloheximide incompletely inhibits haem labelling of cytochrome b₅ and cytochrome a+a₃, and inhibition occurs only after repeated antibiotic injections. The possibility of apo-protein pools, or of haem exchange, with a rapidly renewed `free' haem pool, is considered. Consistent with this model is the observation of non-enzymic haem exchange in vitro between cytochrome b₅ and methaemoglobin. Chloramphenicol, injected intravenously over 5h, results in a 20–40% decrease in incorporation of δ-amino[2,3-³H₂]laevulinate into haem a+a₃ and haem of cytochromes b₅ and c. With the dosage schedule of chloramphenicol studied, amino acid labelling of total liver protein and of cytochrome c was not inhibited. Similarly, ferrochelatase activity was not decreased.