Iain A. Borthwick

Control of 5-aminolevulinate synthase in animals

The proposed mechanism by which hepatic ALV-synthase mitochondrial levels are regulated is outlined in Fig. 2. ALV-synthase catalyzes the first and rate-limiting step in the heme pathway and is normally present in low amounts. A cytosolic, regulatory free heme pool tightly controls the amount of ALV-synthase in two ways. In the primary mechanism of regulation, heme is proposed to inhibit the synthesis of ALV-synthase mRNA. Most likely this would be mediated through the action of specific heme-binding protein(s) which recognize regulatory control regions of the ALV-synthase gene. Gene activity therefore is significantly repressed most of the time. When there is an increased demand for heme by newly synthesized cellular hemoproteins, the free heme pool is reduced, leading to a derepression of ALV-synthase mRNA synthesis. Once the need for increased heme synthesis is satisfied, inhibitory heme levels build up again. When drugs such as phenobarbital are administered to animals, there is a rapid induction in the liver of both cytochrome P-450 and ALV-synthase. It is proposed that the heme pool governing ALV-synthase levels is lowered by the increased heme demand due to cytochrome P-450 apoprotein formation. The primary event in the drug induction of ALV-synthase is therefore the increased synthesis of cytochrome P-450 apoprotein. However, the mechanism by which this occurs is unknown, although drugs do increase the synthesis of mRNA for cytochrome P-450 (Fig. 2). (There is evidence that for the aromatic hydrocarbons a specific cytosolic receptor exists.) In the acute hepatic porphyria diseases, uncontrolled synthesis of hepatic ALV-synthase occurs. The various forms are characterized by reduced levels of one of the heme pathway enzymes other than ALV-synthase. Attacks of the disease are commonly precipitated by drugs which induce cytochrome P-450, and the uncontrolled accumulation of ALV-synthase which accompanies these attacks results from the combined action of the block in the heme pathway and the increased cytochrome P-450 levels. A major challenge which now exists is to understand at the molecular level how the genes for ALV-synthase and cytochrome P-450 are regulated in the liver and other tissues.(ABSTRACT TRUNCATED AT 400 WORDS)

Hemin inhibits transfer of pre-δ-aminolevulinate synthase into chick embryo liver mitochondria

Abstract Pulse labelling studies in chick embryo livers show that hemin prevents the transfer of drug induced pre-δ-aminolevulinate synthase from the cytosol into the mitochondria, leading to an accumulation of precursor in the cytosol. No effect of hemin was observed on the transfer of pre-pyruvate carboxylase into mitochondria. These results eliminated a general toxic effect of hemin on mitochondrial import of proteins and are consistent with the view that hemin specifically inhibits the transfer of ALA synthase.

Evidence for a cytosolic precursor of chick embryo liver mitochondrial δ-aminolevulinate synthase

Following the recent demonstration [Borthwick, I.A., Srivastava, G., Brooker, J.D., May, B.K. and Elliott, W.H. (1982) Eur. J. Biochem. in press] that chick embryo liver mitochondrial delta-aminolevulinate synthase has a minimum molecular weight of 68,000 (rather than the hitherto accepted value of 49,000), we have shown that the primary translation product of delta-aminolevulinate synthase mRNA is a protein of molecular weight 74,000. This protein has for the first time been shown to occur in the cytosol fraction of drug-treated chick embryo livers. This form does not occur in mitochondria nor does the smaller mitochondrial form occur in the cytosol. It is concluded that the 74,000 molecular weight protein is a precursor which is processed during transport into the mitochondria. In vivo labelling experiments are consistent with this conclusion.

Purification of rat liver mitochondrial δ-aminolaevulinate synthase

Abstract Mitochondrial δ-aminolaevulinate synthase was purified from drug-induced adult rat liver and identified for the first time as a protein of minimum molecular weight 70,000. The enzyme was also identified in mitochondria by pulse-labelling and immunoprecipitation and shown to have a molecular weight of 70,000. The purified enzyme was degraded by papain to smaller forms of molecular weight about 56,000 with no loss of enzyme activity. In vitro translation experiments suggest that the enzyme is synthesized initially as a larger precursor of molecular weight 76,000.

Effect of heme on the activity of chick embryo liver mitochondrial δ-aminolevulinate synthase

We have examined the effect of heme on the activity of native δ‐aminolevulinate synthase isolated from drug‐induced chick embryo liver mitochondria. The enzyme was not inhibited by concentrations of heme up to 1nM and this finding makes it improbable that heme acts physiologically to control mitochondrial δ‐aminolevulinate synthase activity.

Purification of hepatic mitochondrial 5-aminolevulinate synthase.

Publisher Summary The mitochondrial enzyme 5-aminolevulinate (ALA) synthase (EC 2.3.1.37) is the first and rate-limiting enzyme in the hepatic heme biosynthetic pathway. This chapter describes a purification protocol for chick embryo liver mitochondrial ALA synthase that can also be used for isolation of highly purified rat liver mitochondrial ALA synthase. Hepatic ALA synthase from drug-induced chick embryos and rats is detected by a colorimetric assay. In this assay succinyl-CoA is generated by an endogenous system. The product ALA is converted to a pyrrole which condenses with modified Ehrlichs reagent and the resulting pink-colored ALA pyrrole complex is measured spectrophotometrically. A potential source of error in the assay is the presence of mitochondrial aminoacetone synthase which generates aminoacetone and hence aminoacetone pyrrole. For determination of rat liver ALA synthase activity, separation of aminoacetone pyrrole from ALA pyrrole is necessary. Chick embryo liver ALA synthase is purified 200-fold under conditions which minimize proteolytic degradation and aggregation of the enzyme. The steps of the purification procedure are performed at 4°, and include isolation of mitochondria, preparation of mitoplasts, sephacryl S-200 chromatography, chromatofocusing, and CoA-agarose affinity chromatography. The purification of rat ALA synthase is also outlined in the chapter.