Tryptophan pyrrolase in haem regulation. The mechanism of the opposite effects of tryptophan on rat liver 5-aminolaevulinate synthase activity and the haem saturation of tryptophan pyrrolase.
ABSTRACT: 1. Administration of tryptophan to starved rats causes a rapid decrease in liver 5-aminolaevulinate synthase activity associated with an increase in the haem saturation of tryptophan pyrrolase. Both effects are maximally produced at 30 min by a 100 mg/kg body wt. dose of tryptophan. 2. Pb2+ prevents both effects. 3. Prevention by allopurinol or benserazide of the tryptophan-induced increase in the haem saturation of tryptophan pyrrolase renders this haem available for further repression of synthase synthesis. 4. The opposite effects on synthase activity and pyrrolase saturation with haem caused by administration of 5-aminolaevulinate, but not those by that of haematin, are potentiated by tryptophan. 5. It is suggested that tryptophan decreases 5-aminolaevulinate synthase activity and causes the initial increase in the haem saturation of tryptophan pyrrolase by enhancing the conversion of 5-aminolaevulinate into haem by a process requiring protein synthesis.
Project description: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.
Project description:1. The increase in the haem saturation of rat liver tryptophan pyrrolase caused by tryptophan administration was previously shown to be associated with a decrease in 5-aminolaevulinate synthase activity. 2. It is now shown that similar reciprocal effects are caused by palmitate and salicylate, both of which increase tryptophan availability to the liver by direct displacement of the serum-protein-bound amino acid. 3. The reciprocal effects on the former two parameters caused by endotoxin and morphine are associated with an increase in liver tryptophan concentration produced by a lipolysis-dependent, non-esterified fatty acid-mediated, displacement of the serum-protein-bound amino acid. 4. All these changes and those caused by another lipolytic agent, theophylline, are prevented by the beta-adrenoceptor-blocking agent propranolol and by the opiate-receptor antagonist naloxone, whose anti-lipolytic nature is demonstrated. 5. High correlation coefficients have been obtained for one or more pairs of the following parameters: serum non-esterified fatty acid concentration, free serum tryptophan concentration, liver tryptophan concentration, liver 5-aminolaevulinate synthase activity, liver holo-(tryptophan pyrrolase) activity and the haem saturation of liver tryptophan pyrrolase. 6. It is suggested that liver tryptophan concentration may play an important role in the regulation of 5-aminolaevulinate synthase synthesis, and that the latter may be subject to control by changes in lipid metabolism and may be influenced by pharmacological agents that affect tryptophan disposition. 7. Preliminary evidence suggests that tryptophan may be bound in the liver and that such a possible binding may control its availability for its hepatic functions.
Project description:1. The utilization of haem by rat liver apo-(tryptophan pyrrolase) under basal conditions and after enhancement of the enzyme activity by various mechanisms was studied under the influence of treatments affecting various aspects of liver haem metabolism. 2. These treatments were: benzoate and p-aminobenzoate as substrates of glycine acyltransferase, acetate as an inhibitor of 5-aminolaevulinate synthase activity, enhancement of 5-aminolaevulinate dehydratase by aluminium, destruction of haem and inhibition of ferrochelatase by porphyrogens, increased haem utilization by phenobarbitone and enhancement of haem oxygenase activity by metal cations. 3. The results show that the haem saturation of the apoenzyme is sensitive to all these treatments. 4. The possible usefulness of tryptophan pyrrolase in studying the regulation of liver haem is suggested.
Project description: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).
Project description:Endotoxin was administered to rats at a dose shown previously to stimulate hepatic haem oxygenase activity and to block induction of delta-aminolaevulinate synthase, apparently by causing redistribution of haem from cytochrome P-450 to a regulatory haem pool in the liver. Within 5h of the administration of endotoxin (at a time when the effect of the compound on cytochrome P-450 is maximal) the relative saturation of tryptophan pyrrolase with intrinsic haem rose, from a basal value of 50% to 90%, indicating that 'free' haem had become available. Concurrently, the activity of delta-aminolaevulinate synthase was decreased to 25% of its basal value. Haem oxygenase reached peak activity 13h after endotoxin administration. These findings provide new evidence for the existence of an 'unassigned' hepatic haem fraction, which exchanges with cytochrome P-450 haem and regulates these three enzyme functions.
Project description: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.
Project description: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.
Project description:Rat liver tryptophan pyrrolase haem is maximally depleted at 30 min after administration of a 400 mg/kg dose of 2-allyl-2-isopropylacetamide. This depletion lasts for 24 h, by which time 5-aminoleevulinate synthase activity becomes maximally enhanced. 2. though the above maximum depletion of pyrrolase haem (at 0.5h) is also produced by a 100 mg/kg dose of the porphyrogen, this does not enhance synthase activity at 24 h. It and smaller doses, however, cause a smaller but earlier enhancement of synthase activity (maximum at 2 h) and produce a similarly short-lived deplation of pyrrolase haem. 3. The depletion of pyrrolase haem and the enhancement of synthase activity by the porphyrogen are inhibited by compound SKF 525-A and phenazine methosulphate, and are potentiated by nicotinamide but not by phenobarbitone. Phenazine methosulphate and nicotinamide also exert opposite effects on hexobarbital sleeping-time. 4. 2-Allyl-2-isopropylacetamde also the depletes pyrrolase haem in vitro. It does so in liver homogenates of control rats in the presence, and in those of phenobarbitone-treated rats in the absence of added NADPH. 5. A discussion of the present results in relation to previous work with other haemoproteins suggests that, whereas cytochrome P-450 (haem) is primarily involved in the production of the active (porphyrogenic) metabolite(s) of 2-allyl-2-isopropylacetamide, the haem pool used by tryptophan pyrrolase may play an important role in the effects of this compound on haem biosynthesis.
Project description:1. The importance of the early depletion of liver haem in the production of porphyria is discussed and further supporting evidence is presented from experiments with tryptophan pyrrolase, under conditions of exacerbation of experimental porphyria by therapeutic and other agents. 2. In addition to the early depletion of pyrrolase haem by porphyrogens, a further depletion is produced when rats are given a porphyrogen plus an analogue or one of 19 drugs known to exacerbate the human disease. 3. Non-exacerbators of human porphyrias do not cause a further early depletion of pyrrolase haem and it is suggested that this system may be used as a screening test for possible exacerbation of the disease by new and existing drugs. 4. A similar further early depletion of haem is produced by combined administration of lead acetate plus phenobarbitone, thus suggesting that the depletion is a more general phenomenon in experimental porphyria. 5. The relationship between tryptophan pyrrolase and the regulatory free haem is discussed. It is suggested that pyrrolase may play an important role in the regulation of haem biosynthesis.
Project description:1. Liver 5-aminolaevulinate (ALA) synthase activity of 24 h-starved rats is maximally increased at 4 h after intraperitoneal administration of a 1.6 g/kg body wt. dose of ethanol. Larger doses cause a dose-dependent decrease in the extent of this stimulation, exhibiting a reciprocal relationship with an elevation of hepatic haem concentration, as suggested by the simultaneous increase in the haem saturation of tryptophan pyrrolase. 2. ALA synthase induction by ethanol is abolished if the above increase in pyrrolase saturation with haem is enhanced by theophylline, but is potentiated when the increase in the haem saturation is inhibited by anti-lipolytic agents. 3. ALA synthase induction by ethanol is also inhibited by inhibitors of alcohol dehydrogenase and aldehyde dehydrogenase. Acetaldehyde and acetate are, however, not responsible; they both decrease ALA synthase activity and increase the haem saturation of tryptophan pyrrolase. These latter effects of acetaldehyde are not mediated by acetate. 4. ALA synthase activity is also stimulated by succinate, which, however, also increases the haem saturation of tryptophan pyrrolase. 5. Ethanol does not influence the rate of ALA synthase degradation. 6. It is suggested that ethanol increases rat liver ALA synthase activity as a result of its own metabolism by the alcohol dehydrogenase-dependent pathway by a mechanism not involving decreased degradation of the former enzyme or the participation of the metabolites acetaldehyde and acetate.