Effect of lead ions on chick-embryo liver mitochondrial delta-aminolaevulinate synthase.
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ABSTRACT:
SUBMITTER: Pirola BA
PROVIDER: S-EPMC1144223 | biostudies-other | 1984 Sep
REPOSITORIES: biostudies-other
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Project description:2-Allyl-2-isopropylacetamide-mediated induction of hepatic porphyria was studied in isolated chick-embryo liver cells. Increased delta-aminolaevulinate synthase activity occurred within 1h of induction and continued to increase for 8h. Protoporphyrins synthesized during this time accumulated to a concentration 10-fold greater than that in the control. Removal of 2-allyl-2-isopropylacetamide from the cells by washing at 3h immediately inhibited further increases in delta-aminolaevulinate synthase synthesis. However substitution of 2-allyl-2-isopropylacetamide at 3h by deferoxamine methane-sulphonate, an inhibitor of haem synthesis, allowed continued delta-aminolaevulinate synthase induction at an unaltered rate, even though this agent did not, by itself, induce enzyme synthesis. Exogenously added haemin was shown completely to inhibit 2-allyl-2-isopropylacetamide-mediated delta-aminolaevulinate synthase induction at concentrations as low as 20nm, a value that is less than the reported physiological one. The duration of inhibition was dependent on the concentration of added haemin and was followed by a period of delta-aminolaevulinate synthase synthesis at a rate similar to that of the control. These data are consistent with the hypothesis that delta-aminolaevulinate synthase synthesis is regulated by the concentration of intracellular haem and that induction is initiated by 2-allyl-2-isopropylacetamide-mediated destruction of haem. Induction of delta-aminolaevulinate synthase was shown to be dependent on both RNA and protein synthesis, and a study of the comparative effects of cordycepin, cycloheximide and haem has shown that, at haemin concentrations up to 50nm, the inhibition of delta-aminolaevulinate synthase synthesis followed kinetics similar to the effect of cordycepin, with no synergism between cordycepin and 50nm-haemin. However, at a haemin concentration of 2mum, the inhibition of delta-aminolaevulinate synthase synthesis followed similar kinetics to the effect of cycloheximide. These data demonstrate the control of delta-aminolaevulinate synthase synthesis by low concentrations of haemin and suggests that the primary effect of haemin is at the level of transcription.
| S-EPMC1161962 | biostudies-other
Project description:The effect of haemin on the biogenesis of delta-aminolaevulinate synthase (ALA synthase) was investigated in primary cultures of embryonic-chick liver. The activity of the enzyme and the amount of the enzyme detected by 'immune-blotting' were determined in hepatocytes incubated with the porphyrogenic agent allylisopropylacetamide. The results of these studies indicated that the loss in ALA synthase activity in cells incubated in the presence of haemin (10 microM) was roughly proportional to a loss in the immune-reactive mass of the enzyme. Haemin was as effective as cycloheximide in causing depletion of ALA synthase in hepatocytes. We had previously established that haemin blocked the maturation of the precursor of ALA synthase [Ades (1983) Biochem. Biophys. Res. Commun. 110, 42-47]. From results reported in the present paper on analyses of immune-precipitated ALA synthase after pulse-labelling with [35S]methionine in the presence and in the absence of haemin, we determined that the inhibition of processing of pre-ALA synthase in cells by haemin was concentration-dependent. A concentration of 2 microM in the culture medium blocked the processing of pre-ALA synthase by 50% in hepatocytes. We also determined that, after inhibition of its maturation by haemin, pre-ALA synthase turned-over with a half-time of 30 min; on the other hand, mature ALA synthase turned-over with a half-time of 120 min.
| S-EPMC1152339 | biostudies-other
Project description:We presented evidence indicating that the established procedure for purifying delta-aminolaevulinate (ALA) synthase from embryonic-chick liver yielded an enzyme with a partially degraded subunit of molecular weight 51000 [Ades & Harpe (1981) J. Biol. Chem. 256, 9329-9333]. We now report the purification from livers of porphyric embryos of a preparation of ALA synthase which consisted primarily of a 63000-Da polypeptide and a component migrating as a smear of polypeptides with a minimum molecular weight of 52 000. Neither component could be recovered from liver mitochondria of normal embryos, where the amounts of ALA synthase were relatively low. The 52 000-Da component had been established to be the partially degraded subunit of the enzyme. Peptide-mapping analyses indicated that the 63 000- and the 52 000-Da components possessed significant structural homologies, and it was concluded that the 63 000-Da polypeptide represented the mature subunit of ALA synthase.
| S-EPMC1158476 | biostudies-other
Project description:1. Postmitochondrial supernatants were prepared from the livers of chick embryos and were incubated under conditions that supported protein synthesis. delta-Aminolaevulinate synthase (EC 2.3.1.37) was synthesized by supernatants from livers treated with the porphyrinogenic drugs 2-allyl-2-isopropylacetamide and/or 3,5-diethoxycarbonyl-1,4-dihydrocollidine, but synthesis by supernatants from normal livers could not be detected. Synthesis of enzyme released from polyribosomes was measured by immunoprecipitation with specific antibody to the mitochondrial enzyme, and the specificity of the reaction was established by electrophoresis of dissociated immunoprecipitates on sodium dodecyl sulphate/polyacrylamide gels. 2. The relative synthesis of delta-aminolaevulinate synthase in vitro was comparable with that previously measured in vivo, and was correlated with the enzyme activity of the liver. 3. Enzyme synthesis in vitro occurred predominantly on free rather than membrane-bound polyribosomes. 4. The mol.wt. of the product synthesized in vitro was 7000 +/- 7000 by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. However, pulse-labelling of the enzyme in vivo confirmed its mol.wt. to be 49000 +/- 5000 when isolated from the mitochondrion. A small amount of immunoprecipitable enzyme of mol.wt. 70000 was detected in the cytosol in vivo. In chick embryo liver, delta-aminolaevulinate synthase therefore appears to be synthesized on cytoplasmic polyribosomes as a polypeptide of mol.wt. 70000, which in vivo is rapidly incorporated into the mitochondrion, and is then extracted as a lower-molecular-weight form. 5. Haemin added to the postmitochondrial supernatant-containing incubation mixture at concentrations up to 10 muM had no effect on general protein synthesis or the synthesis of delta-aminolaevulinate synthase. On the other hand, haemin treatment of induced chick embryo livers in vivo for 3h markedly decreased the relative synthesis of delta-aminolaevulinate synthase in vitro. These results suggest that haemin represses the synthesis of delta-aminolaevulinate synthase by decreasing the amount of mRNA for the enzyme available for translation.
| S-EPMC1163982 | biostudies-other
Project description: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.
| S-EPMC1163307 | biostudies-other
Project description: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.
| S-EPMC1144919 | biostudies-other
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.
| S-EPMC1165006 | biostudies-other
Project description:Studies conducted by several groups have established that porphyrogenic agents which caused elevations in chick-embryo liver delta-aminolaevulinate (ALA) synthase activity also increased the concentrations of the enzyme's RNA, and that haemin inhibited these elevations. We have determined in this study, using immune-blot analyses, that administration in ovo of allylisopropylacetamide (AIA) in combination with diethyl 1,4-dihydro-2,4,6-trimethyl,3,5-pyridinedicarboxylate (DDC) increased the mass of ALA synthase in intestine and kidney of chick embryos. Furthermore, the molecular mass of the subunit of the enzyme in those tissues appeared identical with that of liver ALA synthase. Using a synthetic oligonucleotide complementary to ALA synthase mRNA, we determined by solution hybridization and Northern-blot analyses that AIA and DDC also increased the concentrations of ALA synthase mRNA in intestine and kidney and that testosterone elevated the concentration of the RNA in kidney. In analyses of RNA obtained from chick-embryo liver, intestine, kidney, heart, brain and lung, the probe bound primarily in each case to a single 2.3 kb RNA. Finally, the haem precursors ALA and FeCl3, when injected together into the fluid surrounding embryos, inhibited both the elevations in ALA synthase mass and RNA concentration brought about by porphyrogenic agents in liver, kidney and intestine. Thus the results indicated that: (1) certain porphyrogenic agents increased ALA synthase mass and RNA in chick-embryo intestine and kidney, in addition to liver; (2) ALA and FeCl3 inhibited the elevations; and (3) the sizes of ALA synthase's subunit as well as the enzyme's mRNA appeared identical, in each case, in all tissues examined.
| S-EPMC1133346 | biostudies-other
Project description:The synthesis of haem has been postulated to be a key regulatory step in muscle mitochondrial biogenesis. We examined the expression of delta-aminolaevulinate synthase (ALAs), the regulatory enzyme of haem metabolism, in 10 Hz electrically stimulated and non-stimulated control rat tibialis anterior (TA) muscle. ALAs activity and mRNA levels were measured at 0, 18 and 48 h of recovery after 3 h of acute stimulation, or after 7 days of stimulation (3 h/day). ALAs activity in control muscles averaged 7.8 +/- 0.8 nmol/h per g (n = 30). After 3 h of stimulation and during recovery, no change in ALAs activity occurred. ALAs mRNA during the same time was unchanged except at 48 h of recovery, when it increased 1.3-fold above control (P < 0.05). After 7 days of stimulation, ALAs activity was unchanged at 0 h, but increased at 18 and 48 h of recovery to 2.0- and 1.8-fold above control (P < 0.05). ALAs mRNA was also increased, but to a level averaging 1.6-fold above control (P < 0.05) at all times, indicating an increased mRNA stability or synthesis. No change in the haem-containing enzyme cytochrome c oxidase (CYTOX) activity occurred after 3 h of stimulation in the red section of the TA. After 7 days of stimulation, the increase in CYTOX activity averaged 1.7-fold above control (P < 0.05) at all times. Thus the induction of ALAs during recovery after 7 days was regulated by factors which not only change ALAs mRNA content, but which also affect ALAs mRNA at translational or post-translational steps. This induction occurred despite a 1.7-fold increase in CYTOX, implying that a precursor-product relationship does not always exist.
| S-EPMC1132505 | biostudies-other
Project description: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.
| S-EPMC1146579 | biostudies-other