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.     

Haem synthase and cobalt porphyrin synthase in various micro-organisms

1. The preparation of a crude extract of Clostridium tetanomorphum containing cobalt porphyrin synthase but little haem-synthase activity is described. 2. The properties of cobalt porphyrin synthase in the clostridial extracts is compared with the properties of a haem synthase present in crude extracts of the yeast Torulopsis utilis. 3. Cobalt porphyrin synthase in extracts of C. tetanomorphum inserts Co²⁺ ions into the following dicarboxylic porphyrins in descending order of rate of insertion: meso-, deutero- and proto-porphyrins. Esterification renders meso- and deutero-porphyrins inactive as substrates. Neither the tetracarboxylic (coproporphyrin III) nor the octacarboxylic (uroporphyrin III) compounds are converted into cobalt porphyrins by the extract, but the non-enzymic incorporation of Co²⁺ ions into these two porphyrins is rapid. These extracts are unable to insert Mn²⁺, Zn²⁺, Mg²⁺ or Cu²⁺ ions into mesoporphyrin. 4. Crude extracts of T. utilis readily insert both Co²⁺ and Fe²⁺ ions into deutero-, meso, and proto-porphyrins. Unlike the extracts of C. tetanomorphum, these preparations catalyse the insertion of Co²⁺ ions into deuteroporphyrin more rapidly than into mesoporphyrin. This parallels the formation of haems by the T. utilis extract. 5. Cobalt porphyrin synthase is present in the particulate fraction of the extracts of C. tetanomorphum but requires a heat-stable factor present in the soluble fraction. This soluble factor can be replaced by GSH. 6. Cobalt porphyrin synthase in the clostridial extract is inhibited by iodoacetamide and to a smaller extent by p-chloromercuribenzoate and N-ethylmaleimide. The haem synthases of T. utilis and Micrococcus denitrificans are also inhibited by various thiol reagents.     

The effects of bromocriptine and prolactin on porphyrin biosynthesis in the Harderian gland of the male hamster,Mesocritecus auratus

Porphyrin biosynthesis was examined in the Harderian gland of the male golden hamster by fluorometric assays of gland porphyrin content and by measuring the activity of a rate-limiting enzyme for haem biosynthesis, 5-aminolaevulinic acid synthase. Both porphyrin content and enzyme activity are low in normal male glands but were greatly raised in males castrated for 6 weeks. However, porphyrin synthesis remained at basal levels in castrates given the dopamine agonist bromocriptine; this suppression could be reversed by simultaneous prolactin administration, and castrated males receiving prolactin alone exhibited very high enzyme activity and porphyrin content. Bromocriptine also prevents the morphological feminisation of the Harderian gland which would normally occur after castration; again, the simultaneous administration of prolactin permits feminisation to occur. The results support the hypothesis that, while androgens have an inhibitory effect on porphyrin synthesis within this model, other factors, including prolactin, are permissive.Abbreviations ALA 5-aminolaevulinic acid - ALA-s 5-aminolaevulinate synthase - GH growth hormone     

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.     

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.     

Brain 5-aminolaevulinate synthase. Developmental aspects and evidence for regulatory role.

1. Brain 5-aminolaevulinate synthase showed a peak of increased activity in the first few weeks of life, which preceded and accompanied the development of brain cytochromes. 2. In the brain of the adult rat the activity of the enzyme was only 20% of that in the liver (on a per g wet wt. basis), but it was still probably sufficient to maintain the turnover of brain cytochromes. 3. The brain synthase activity could be decreased by treatment of rats with cycloheximide or with large doses of 5-aminolaevulinate, especially when this precursor was given as the methyl ester. 4. Injected haematin and CoCl2 markedly inhibited the synthase activity in the liver but failed to affect the brain enzyme; neither was taken up by the brain in vivo. 5. It is concluded that the brain can itself produce the haem required for the synthesis and turnover of its own haemoproteins and that 5-aminolaevulinate synthase may regulate the pathway in brain as in other tissues. 6. The relevance of the present findings to the pathogenesis of the neurological symptoms of acute porphyria and to the beneficial effect of exogenous haematin in porphyric patients is briefly considered.     

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.     

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.     

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).     

The effects of acetate, metal cations, phenobarbitone, porphyrogens and substrates of glycine acyltransferase on the utilization of haem by rat liver apo-(tryptophan pyrrolase).

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.     

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.     

The effects of bromocriptine and prolactin on porphyrin biosynthesis and morphology in the female hamster Harderian gland

Porphyrin biosynthesis was examined in the Harderian gland of the female golden hamster by fluorometric assays of gland porphyrin content and by measuring the activity of a rate-limiting enzyme for haem biosynthesis, -aminolaevulinic acid synthase. Both porphyrin content and enzyme activity are high in normal female glands. Enzyme activity was lowered in females ovariectomised for 6 weeks, and both enzyme activity and porphyrin content were greatly lowered in ovariectomised females given the dopamine agonist bromocriptine; this suppression could be prevented by simultaneous prolactin administration. Bromocriptine (but not ovariectomy alone) also masculinised the morphology of the Harderian gland, resulting in the appearance of type II cells and polytubular complexes; again, the simultaneous administration of prolactin prevented masculinisation. The results support the hypothesis that while androgens have an inhibitory effect on porphyrin synthesis within this model, prolactin may have a major facilitatory role.Abbreviations ALA -aminolaevulinic acid - ALA-s -aminolaevulinate synthase     

Isolation and characterization of PZ-peptidase in developing rat brain

Changes in the activity of a collagen peptidase, PZ-peptidase, acting on a synthetic substrate [4-Phenylazobenzyloxycarbonyl(PZ)-l-Pro-l-Leu-Gly-l-Pro-l-Arg] for bacterial collagenase were examined in developing rat brain regions. The hypothalamus, pons-medulla, colliculi, cerebellum, ceerbrum, midbrain and pituitary gland were studied in rats ranging in age from 1 week to adult; PZ-peptidase activity continuously decreased with maturation in all of the brain regions examined except the hypothalamus. The pituitary gland showed the highest activity in all of the brain regions. PZ-peptidase activity in crude mitochondrial and supernatant fractions from rat whole brain had an optimum pH between 7.5–8.0. It was strongly inhibited by p-chloromercuribenzoic acid, N-ethylmaleimide or EDTA. whereas iodoacetic acid did not affect the enzyme activity. Among various metal ions, the enzyme activity was inhibited by Zn⁺² or Cu⁺² but not by Mn⁺², Ca⁺², Mg⁺² or Na⁺. There is no inhibition of the activity by serine protease inhibitors, including diisopropylfluorophosphate and phenylmethylsulphonyl fluoride. An approximate molecular weight of this enzyme was estimated to be 68,000 by gel filtration. Since these properties of rat brain PZ-peptidase were similar to those of other peripheral PZ-peptidases, we suppose that PZ-peptidase in the brain may be the same molecule as the enzyme which hydrolyses collagen peptides in peripheral tissues, but it may have some different physiological roles.     

Tetrapyrrole biosynthesis in greening etiolated barley seedlings

Allyl isopropylacetamide (AIA) does not stimulate porphyrin biosynthesis in greening barley; AIA inhibits the synthesis of 5-aminolaevulinate (ALA) in plants and does not overcome the repression of ALA-synthetase. This indicates that the ALA synthesis system of green plants is regulated differently from ALA synthetase of mammalian systems. Laevulinic acid (LA) inhibited the biosynthesis of tetrapyrrole pigments in greening barley and diminished the insertion of ⁵⁵Fe into extractable protohaem, confirming that haem was synthesized at a time of little net increase in protohaem. ALA feeding increased iron incorporation into protohaem without increasing either extractable protohaem or cytochromes b and f. Since ALA feeding greatly increased the protochlorophyllide content of darkgrown plants and subsequent chlorophyll levels in the light, the regulation of haem pigment synthesis in plants occurs after protoporphyrin and protohaem synthesis and is likely to involve the turnover of protohaem produced in excess of haem protein requirements.     

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.     

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

To assess whether the synthesis of haem can be studied in small amounts of human liver, we measured kinetics of the conversion of 5-aminolaevulinate into haem and haem precursors in homogenates of human livers. We used methods previously developed in our laboratory for studies of rat and chick-embryo livers [Healey, Bonkowsky, Sinclair & Sinclair (1981) Biochem. J. 198, 595-604]. The maximal rate at which homogenates of human livers converted 5-aminolaevulinate into protoporphyrin was only 26% of that for rat, and 58% of that for chick embryo. In the absence of added Fe2+, homogenates of fresh human liver resembled those of chick embryos in that protoporphyrin and haem accumulated in similar amounts, whereas fresh rat liver homogenate accumulated about twice as much haem as protoporphyrin. However, when Fe2+ (0.25 mM) was added to human liver homogenates, mainly haem accumulated, indicating that the supply of reduced iron limited the activity of haem synthase, the final enzyme in the haem-biosynthesis pathway. Addition of the potent iron chelator desferrioxamine after 30 min of incubation with 5-amino[14C]laevulinate stopped further haem synthesis without affecting synthesis of protoporphyrin. Thus the prelabelled haem was stable after addition of desferrioxamine. Since the conversion of 5-amino[14C]laevulinate into haem and protoporphyrin was carried out at pH 7.4, whereas the pH optimum for rat or bovine hepatic 5-aminolaevulinate dehydratase is about 6.3, we determined kinetic parameters of the human hepatic dehydrase at both pH values. The Vmax was the same at both pH values, whereas the Km was slightly higher at the lower pH. Our results indicate that the synthesis of porphyrins and haem from 5-aminolaevulinate can be studied with the small amounts of human liver obtainable by percutaneous needle biopsy. We discuss the implications of our results in relation to use of rat or chick-embryo livers as experimental models for the biochemical features of human acute porphyria.     

Specificity and mutational analysis of the metal-dependent 3-deoxy-d-manno-octulosonate 8-phosphate synthase from Acidithiobacillus ferrooxidans

3-Deoxy-d-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyzes the reaction between phosphoenol pyruvate and d-arabinose 5-phosphate to generate KDO8P. This reaction is part of the biosynthetic pathway to 3-deoxy-d-manno-octulosonate, a component of the lipopolysaccharide of the Gram-negative bacterial cell wall. Two distinct groups of KDO8PSs exist, differing by the absolute requirement of a divalent metal ion. In this study Acidithiobacillus ferrooxidans KDO8PS has been expressed and purified and shown to require a divalent metal ion, with Mn²⁺, Co²⁺ and Cd²⁺ (in decreasing order) being able to restore activity to metal-free enzyme. Cd²⁺ significantly enhanced the stability of the enzyme, raising the T_m by 14 °C. d-Glucose 6-phosphate and d-erythrose 4-phosphate were not substrates for A. ferrooxidans KDO8PS, whereas 2-deoxy-d-ribose 5-phosphate was a poor substrate and there was negligible activity with d-ribose 5-phosphate. The ²⁴³AspGlyPro²⁴⁵ motif is absolutely conserved in the metal-independent group of synthases, but the Gly and Pro sites are variable in the metal-dependent enzymes. Substitution of the putative metal-binding Asp243 to Ala in A. ferrooxidans KDO8PS gave inactive enzyme, whereas substitutions Asp243Glu or Pro245Ala produced active enzymes with altered metal-dependency profiles. Prior studies indicated that exchange of a metal-binding Cys for Asn converts metal-dependent KDO8P synthase into a metal-independent form. Unexpectedly, this mutation in A. ferrooxidans KDO8P synthase (Cys21Asn) gave inactive enzyme. This finding, together with modest activity towards 2-deoxy-d-ribose 5-phosphate suggests similarities between the A. ferrooxidans KDO8PS and the related metal-dependent 3-deoxy-d-arabino-heptulosonate phosphate synthase, and highlights the importance of the AspGlyPro loop in positioning the substrate for effective catalysis in all KDO8P synthases.     

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.     

Purification and properties of the glucose-6-phosphate-dependent form of human placental glycogen synthase.

Glycogen synthase in the glucose-6-phosphate (glucose-6-P)-dependent form was purified over 10,000-fold from an extract of term human placenta. The purified enzyme shows a single protein band on polyacry1amide-gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme activity in the presence of glucose-6-P is increased by the single addition of Mg²⁺, Ca²⁺, or Mn²⁺ and is reduced by the addition of either sulfate or phosphate. Addition of either Mg²⁺, Ca²⁺, or Mn²⁺ relieves the inhibition by sulfate or phosphate. The enzyme activity in the absence of glucose 6-P is greatly increased by the addition of MnSO₄, CoSO₄, and NiSO₄ and is increased to a lesser extent by MgSO₄, CaSO₄, and FeSO₄. The activation of the glucose-6-P-dependent form of the enzyme by these metal sulfates in the absence of glucose-6-P has never been reported. MnSO₄, which shows homotropic cooperativity, is the best activator among the various metal sulfates tested. The human placental glucose-6-P-dependent form of glycogen synthase (D form) can be converted to the glucose-6-P-independent form (I form) of the enzyme by incubating the partially purified glycogen synthase, which is copurified with synthase phosphatase, with Mn²⁺. This conversion can be reversed by the addition of cyclic AMP-dependent protein kinase. The synthase D to synthase I converting system from human placenta is unique in its stringent requirement for Mn²⁺.