Delta-aminolevulinate Synthase Research Articles

The effect of delta-aminolevulinate on catalase T-messenger RNA levels in delta-aminolevulinate synthase-defective mutants of Saccharomyces cerevisiae.

Total RNA was isolated from mutants of Saccharomyces cerevisiae that lack active delta-aminolevulinate synthase and are therefore defective in heme biosynthesis. The RNAs were translated in the cell-free protein synthesis system from wheat germ, and the catalase T synthesized was isolated by immunoadsorption and polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. Little or no catalase T product was detected with RNA from mutant cells grown in the absence of a heme precursor. As judged from in vitro translational capacity, RNA fractions from mutant cells grown in the presence of delta-aminolevulinate contained at least 10 times more catalase T mRNA than RNA from unsupplemented cells.

Synthesis of the apoprotein of cytochrome b in heme-deficient yeast cells.

The presence of the apoprotein of cytochrome b has been demonstrated in a mutant of Sacchromyces cerevisiae lacking delta-aminolevulinic acid synthase and, hence, devoid of heme. The apoprotein of cytochrome b present in the mutant was identical with cytochrome b of control cells (mutant cells grown in the presence of delta-aminolevulinic acid) by the following criteria: similar apparent molecular weights in dodecyl sulfate-polyacrylamide gel electrophoresis; anomalous migration behavior during electrophoresis in polyacrylamide gels of different porosities; identical gel pattern obtained after immunoprecipitation with specific antiserum against cytochrome b; and identical fingerprints obtained after limited proteolysis with Staphylococcus aureus V8 protease. The kinetics of incorporation in vivo of [35S]methionine into apoprotein of cytochrome b in the mutant suggested that heme deficiency may affect assembly into the membrane of subunits of the cytochrome b.c1 complex rather than synthesis of cytochrome b.

Translocation of delta-aminolevulinate synthase from the cytosol to the mitochondria and its regulation by hemin in the rat liver.

Translocation of delta-aminolevulinate synthase from the cytosol to the mitochondria and its regulation by hemin in the rat liver.

Purification and some properties of delta-aminolevulinate synthase from the rat liver cytosol fraction and immunochemical identity of the cytosolic enzyme and the mitochondrial enzyme.

Purification and some properties of delta-aminolevulinate synthase from the rat liver cytosol fraction and immunochemical identity of the cytosolic enzyme and the mitochondrial enzyme.

Heme biosynthesis in Friend erythroleukemia cells: control by ferrochelatase.

The activities of the enzymes of heme biosynthesis (except protoporphyrin oxidase) have been followed during the induction of Friend cells in culture. All the enzyme activities increased after induction with dimethyl sulfoxide. The activities of the intermediate enzymes were much higher than those of delta-aminolevulinate synthase [succinyl-CoA:glycine C-succinyltransferase (decarboxylating), EC 2.3.1.37], the initial enzyme, or ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1), the final enzyme of the pathway. Ferrochelatase activity was not detectable in the uninduced cell. delta-Aminolevulinate synthase activity increased during the first 24 hr of induction; porphobilinogen deaminase activity began to increase after 48 hr and ferrochelatase activity, after 72 hr. However, the induction of heme synthesis followed the same time course as that of ferrochelatase activity, not that of delta-aminolevulinate synthase activity. The cellular growth medium was found to contain traces of protoporphyrins. Thus, ferrochelatase is shown to be rate limiting for heme synthesis during early stages of Friend cell induction. A Friend cell variant (Fw), which is not inducible except in the presence of exogenous hemin, was also studied. All the enzymes of heme synthesis except ferrochelatase were inducible by butyric acid. Ferrochelatase was not inducible by butyric acid or hemin plus butyric acid. These cells also excrete protoporphyrin, The failure to induce ferrochelatase activity is believed to be the cause of, not a consequence of, the noninducibility of this cell line.

Effects of administration of cobalt chloride and cobalt protoporphyrin on delta-aminolevulinate synthase in rat liver.

Cobalt protoporphyrin inhibited the drug-induced increase of delta-aminolevulinate synthase as well as its transfer from the cytosol fraction to the mitochondria in rat liver in a similar way to protoheme. Cobalt chloride given to animals in a large dose exhibited similar effects. Cobalt protoporphyrin was isolated from the liver of rats treated with cobalt chloride. The observed regulatory effects of cobalt chloride with respect to the induction and the intracellular translocation of delta-aminolevulinate synthase may be mediated by cobalt protoporphyrin synthesized in vivo.

Postulated deficiency of hepatic heme and repair by hematin infusions in the "inducible" hepatic porphyrias.

There is compelling, indirect evidence of hepatic heme deficiency due primarily to the respective genetic errors of the three inducible hepatic porphyrias, acute intermittent porphyria, porphyria variegata, and hereditary coproporphyria. The induction is enhanced by exogenous inducers such as barbiturate, estrogens and other "porphyrogenic" chemicals and factors, including glucose deprivation. The newer knowledge of the induction of delta-aminolevulinic acid synthetase [delta-aminolevulinate synthase; succinyl--CoA:glycine C-succinyltransferase (decarboxylating), EC 2.3.1.37] in relation to inadequate heme, and repression by heme, stimulated early trials of hematin infusions to overcome the acute relapse in the foregoing inducible porphyrias. Recently this experience has been considerably expanded, 143 infusions of hematin having been given in 22 cases. Studies of the effect on the serum concentrations of delta-aminolevulinic acid and porphobilinogen have shown a highly significant decline, often to 0, especially of delta-aminolevulinic acid. A distinct relationship to the clinical severity of the attack has been evident in the frequency and magnitude of decline of serum delta-aminolevulinic acid and porphobilinogen. This was regularly associated with objective clinical improvement.

Regulation of heme pathway enzymes and cellular glutathione content by metals that do not chelate with tetrapyrroles: blockade of metal effects by thiols.

The trace metals nickel and platinum, which are not substrates for ferrochelatase and thus do not form heme in biological systems, were found to act similaryl to cobalt, and heme itself, in regulating heme metabolism in liver and kidney. These metals induced heme oxygenase activity in both organs with the peak of induced enzyme activity reached approximately 16 hr after single injections in rats. Both metals caused transient depression of cellular glutathione content followed by increases above normal after 12 hr in liver. Nickel and platinum were more potent inducers of heme oxygenase in kidney than in liver (10-13 times normal versus 5-6 times normal). At high concentrations, they inhibited heme oxygenase [heme, hydrogen-donor:oxygen oxidoreductase (alpha-methene-oxidizing, hydroxylating), EC 1.14.99.3] in vitro. Both were active in regulating heme metabolism only when administered in the ionic form. Complexing of the metals with sulfhydryl agents completely blocked their actions on heme metabolism. Administration of cysteine orally prior to or shortly after administration of the metals had a similar blocking effect. Nickel and platinum produced depression of delta-aminolevulinate synthase [succinyl-CoA:glycine c-succinyltransferase (decarboxylating), EC 2.3.1.37] activity in liver, but neigther inhibited this rate-limiting ennzyme for heme synthesis in vitro. Furthermore, despite the substantial decreases in cellular heme and hemoprotein contents mediated by the metal, production of delta-amimolevulinate synthase did not undergo the compensatory increase that would be expected if there were a direct reciprocal feedback relationship between cellular heme level and synthesis of this enzyme. These findings indicate that it is not necessary for metal ions to be chelated in the porphyrin ring in order to regulate the enzymes of heme synthesis and heme oxidation. Accordingly, it is suggested that the iron atom of heme is the proximately active regulator of delta-aminolevulinate synthase and heme oxygenase--actions generally ascribed to the iron-tetrapyrrole complex itself--and that the tetrapyrrole moiety of the complex functions primarily as a means of transport of the metal to regulatory sites in cells.

Yeast mutants deficient in heme biosynthesis and a heme mutant additionally blocked in cyclization of 2,3-oxidosqualene.

Mutants of Saccharomyces cerevisiae were isolated which were blocked in heme biosynthesis and required heme for growth on a nonfermentable carbon source. They were rho+, and grew fermentatively on ergosterol or cholesterol and Tween 80, as a source of oleic acid. Cells grown on ergosterol and Tween 80 lacked cytochromes and catalase which were restored by growth on heme. The mutants comprised five nonoverlapping complementation groups. Tetrad analysis showed that the pleiotropic properties of each of the mutants resulted from a single mutation in one of five unlinked loci (hem1 to hem5) affecting heme biosynthesis. Biochemical studies confirmed that each mutation resulted in loss of a single enzyme activity. hem1 mutants grew on delta-aminolevulinate and lacked delta-aminolevulinate synthase activity, hem2 mutants lacked delta-aminolevulinate dehydratase, and hem3 mutants uroporphyrin I synthase. Mutants in hem1, hem2, and hem3 had an additional requirement for methionine on synthetic medium supplemented with either heme or ergosterol and Tween 80, owing to a lack of sulfite reductase which contains siroheme, a modified uroporphyrin III. Since hem4 and hem5 mutants have sulfite reductase activity under all growth conditions, they are blocked after uroporphyrin III. Cell extracts of a hem4 mutant incubated with delta-aminolevulinate accumulated coproporphyrin III suggesting a block in coproporphyrinogenase, the enzyme which converts coproporphyrinogen III to protoporphyrinogen. Cells and extracts of a hem5 mutant accumulated protoporphyrin IX. Since it was the only mutant that grew on heme but not on protoporphyrin IX, a block in ferrochelatase was suggested for this strain. Mutant strains grown on heme had the sterol composition of wild type cells, whereas without heme only squalene, small amounts of lanosterol, and added sterol was observed. A heme product therefore participates in the transformation of lanosterol to ergosterol. A hem3 mutant was isolated which was also blocked between 2,3-oxidosqualene and lanosterol (erg12). When grown on lanosterol or ergosterol (with Tween 80) it accumulated a compound which was identified as 2,3-oxidosqualene by comparison with the synthetic compound in thin layer and gas-liquid chromatography, and by proton magnetic resonance and mass spectroscopy. Supplementation with heme did not remove the requirement for sterol, but it enabled the mutant to convert lanosterol to ergosterol.

Quaternary structure of delta-aminolevulinic acid synthase from Rhodopseudomonas spheroides.

Delta-Aminolevulinic acid synthase (succinyl-CoA: glycine C-succinyltransferase (decarboxylating) EC 2.3.1.37) was purified from Rhodopseudomonas spheroides. The purity of the enzyme preparation was established by its behavior in disc electrophoresis in the presence and absence of sodium dodecyl sulfate and by analytical ultracentrifugation. The molecular weight of the enzyme as determined by sedimentation equilibrium was found to be about 80,300, a value similar to those obtained by gel filtration, polyacrylamide gel electrophoresis, and sucrose gradient centrifugation. The molecular weight of the enzyme, denatured with either sodium dodecyl sulfate or guanidine hydrochloride, was found to be about 45,000 and 41,000, respectively. The dimeric structure was supported by sedimentation in sucrose gradients. Further evidence for the dimetic nature of the enzyme was obtained by gel electrophoresis of the enzyme treated with dimethylsuberimidate and sodium dodecyl sulfate.

Selenium regulation of hepatic heme metabolism: induction of delta-aminolevulinate synthase and heme oxygenase.

Selenium was found to be a novel regulator of cellular heme methabolism in that the element induced both the mitochondrial enzyme delta-aminolevulinate synthase [succinyl-CoA:glycine C-succinyltransferase (decarboxylating); EC 2-3-1-37] and the microsomal enzyme heme oxygenase [heme, hydrogen-donor:oxygen oxidoreductase(alpha-methene-oxidizing, hydroxylating); EC 1-14-99-3] in liver. The effect of selenium on these enzyme activities was prompt, reaching a maximum within 2 hr after a single injection. Other changes in parameters of hepatic heme metabolism occurred after administration of the element. Thirty minutes after injection the cellular content of heme was significantly increased; however, this value slightly decreased below control values within 2 hr, coinciding with the period of rapid induction of heme oxygenase. At later peroids heme content returned to normal values. Selenium treatment caused only a slight decrease in microsomal cytochrome P-450 content. However, drug-metabolizing activity was severely inhibited by higher doses of the element. Unlike other inducers of delta-aminolevulinate synthase, which as a rule are also porphyrinogenic agents, selenium induction of this enzyme was not accompanied by an increase in the cellular content of prophyrins. When rats were pretreated with selenium 90 min before administration of heme, a potent inhibitor of delta-aminolevulinate synthase production, the inhibitory effect of heme of formation of this mitochondrial enzyme was completely blocked. Selenium, at high concentrations in vitro, was inhibitory to delta-aminolevulinate synthase activity. It is postulated that selenium may not be a direct inducer of heme oxygenase as is the case with trace metals such as cobalt, but may mediate an increase in heme oxygenase through increased production and cellular availability of "free" heme, which results from the increased heme synthetic activity of hematocytes. Subsequently, the increased heme oxygenase activity is in turn responsible for the lack of increase in the microsomal heme content, thus maintaining heme levels at normal values despite the highly increased activities of both heme oxygenase and delta-aminolevulinate synthase. It is further suggested that the increase in delta-aminolevulinate synthase activity is not due to a decreased rate of enzyme degradation or an activation of preformed enzyme, but to increased rate of synthesis of enzyme protein. Although selenium in trace amounts has been postulated to be involved in microsomal electron transfer process, the data from this study indicate that excess selenium can substantially inhibit microsomal drug metabolism.

Cobalt inhibition of synthesis and induction of delta-aminolevulinate synthase in liver.

Cobalt has complex actions on the metabolism of heme in the liver. In this organ the metal potently induces heme oxygenase (EC 1.14.99.3), and decreases cellular heme and hemoprotein content. The metal also displays biphasic effects on hepatic heme synthesis. These effects are reflected in the ability of cobalt to initially inhibit synthesis of delta-aminolevulinate synthase [succinyl-CoA:glycine C-succinyltransferase (decarboxylating) EC 2.3.1.37], the rate limiting enzyme of the heme pathway, following which a later enhanced rate of formation of this enzyme occurs. In this study, cobalt was shown to block almost entirely the ability of the barbiturate analogue allylisopropylacetamide to induce delta-aminolevulinate synthase in liver. The blocking effect of cobalt on the otherwise potent enzyme inducing action of this drug was time-dependent; if the metal was injected 30 min prior to allylisopropylacetamide, inhibition of enzyme induction was complete. When the metal was administered 1.5 or more hours after allylisopropylacetamide, inhibition of enzyme induction was incomplete. Cobalt did not block the ability of the drug to directly degrade heme to "green pigment" thus the enzyme inducing action of allylisopropylacetamide and its degradative action on heme are separately mediated.

Delta-Aminolevulinic acid synthase from chick embryo liver mitochondria. II. Immunochemical correlation between synthesis and activity in induction and repression.

A specific rabbit antibody was prepared against chick embryo liver mitochondrial delta-aminolevulinic acid synthase (EC 2.3.1.37) and used to quantitate the amount of enzyme present in liver mitochondria from normal and drug-treated chick embryos. When increases in enzyme activity were produced by the drugs 2-allylisopropylacetamide, or 1,4-dihydro-3,5-dicarbethoxycollidine, quantitative immunotitrations and the Laurell electroimmunoassay showed proportional increases occurred in the amount of enzyme antigen. Conversely, decreases of induced enzyme activity produced by hemin were accompanied by corresponding decreases in enzyme antigen. The relative rate of synthesis of delta-aminolevulinic acid synthase was measured by pulse-labeling of liver proteins with L-[4,5-3H]leucine. delta-Aminolevulinic acid synthase was isolated by quantitative immunoprecipitation followed by electrophoresis of the dissolved immunoprecipitate on a sodium dodecyl sulfate-polyacrylamide gel. In normal and drug-treated chick embryo liver mitochondria, enzyme activity was closely correlated with the relative rate of enzyme synthesis. When a 360-fold increase in enzyme activity was produced in vivo by a combination of 2-allylisopropylacetamide and 1,4-dihydro-3,5-dicarbethoxycollidine in 12 hours, there was a 500-fold increase in relative synthesis, such that delta-aminolevulinic acid synthase constituted over 1% of the total intracellular protein synthesis and over 3% of the total labeled protein in liver mitochondria. Hemin administered after inducing chemicals was able to completely block the induced synthesis of delta-aminolevulinc acid synthase with no significant change in general protein synthesis. Mitochondria from untreated chick embryo livers contain delta-aminolevulinic acid synthase that migrates on sodium dodecyl sulfate gels identically with the induced enzyme.

Bacteriochlorophyll and heme synthesis in Rhodopseudomonas spheroides: possible role of heme in regulation of the branched biosynthetic pathway.

Synthesis of heme, measured by incorporation of iron-59, and of bacteriochlorophyll was studied with wild-type and mutant strains of Rhodopseudomonas spheroides. The wild type formed heme from glycine and succinate at one-fortieth the rate of bacteriochlorophyll under anaerobic-light conditions. Added delta-aminolevulinate stimulated heme synthesis 10-fold without increasing bacteriochlorophyll production. Heme synthesis from glycine and succinate was increased when the magnesium branch of the biosynthetic path was curtailed by mutation or by p-fluorophenylalanine or 8-azaguanine. Synthesis of bacteriochlorophyll by the wild type from glycine and succinate stopped immediately after addition of puromycin, but heme production continued for a period. Porphyrins and other precursors did not appear upon addition of puromycin alone, but simultaneous addition of o-phenanthroline resulted in the accumulation of coproporphyrin. Production of this porphyrin by a mutant strain with impaired ability to form heme was unaffected by puromycin. Heme synthesis from glycine and succinate or from delta-aminolevulinate was decreased by limitation of methionine; it is suggested that coproporphyrin accumulation from glycine and succinate under conditions of methionine deficiency results from relief of feedback inhibition of delta-aminolevulinate synthase by heme. The development of delta-aminolevulinate synthase activity in response to low aeration is prevented by addition of delta-aminolevulinate. This repressive action of the latter is abolished when its conversion to heme is impeded by mutation or by methionine deficiency. It is suggested that heme, the quantitatively minor end product of the branched biosynthetic pathway, may regulate the flow of common intermediates when utilization of protoporphyrin by the magnesium branch is diminished. This regulation may be exerted by feedback inhibition of delta-aminolevulinate synthase and also by repression of enzyme formation.