Dataset Information

Regulation of production of delta-aminolaevulinate synthase in tissues of chick embryos. Effects of porphyrogenic agents and of haem precursors.

ABSTRACT: 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.

SUBMITTER: Drew PD

PROVIDER: S-EPMC1133346 | biostudies-other | 1989 Sep

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

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.

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.

Dataset Information

Regulation of production of delta-aminolaevulinate synthase in tissues of chick embryos. Effects of porphyrogenic agents and of haem precursors.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure