Evidence of increased synthesis of δ-aminolevulinic acid dehydratase in experimental lead-poisoned rats

δ-Aminolevulinic acid dehydratase (porphobilinogen synthase; 5-aminolevulinate hydro-lyase, EC 4.2.1.24) was purified from rat and rabbit erythrocytes to a homogeneous state. Specific activities were 26.0 and 26.6 units/mg protein for the rat and rabbit enzymes, respectively, and their estimated molecular weight was 280 000, each consisting of 8 subunits of M_r 35 000. In order to quantitate rat δ-aminolevulinic acid dehydratase at several stages of lead-poisoning, a radioimmunoassay technique using goat antiserum against the rat enzyme was developed for the first time. This technique was specific, reproducible and high sensitive allowing determination of 1 ng enzyme. When drinking water containing 25 mM lead acetate was given daily to rats ad lib. the δ-aminolevulinic acid dehydratase activity in the blood, assayed without any pretreatment, decreased to 8% of the control level on the next day. On the contrary, the restored enzyme activity, assayed in the presence of Zn²⁺ and dithiothreitol, was greater than normal by the fourth day of lead administration in bone-marrow cells and by the ninth day in the peripheral blood. The increased activity level stayed the same from the ninth day onward. The enzyme content as determined directly by the radioimmunoassay technique at this stage was about 2-fold above that the control. There was no significant difference in the number of reticulocytes and the distribution profile of different types of reticulocytes between the lead-exposed and non-exposed rats. Therefore, the increase in the amount of δ-aminolevulinic acid dehydratase in erythrocytes of lead-poisoned rats was suggested to be due to an increased rate of synthesis in the bone-marrow cells.     

Delta-aminolevulinate dehydratase, a zinc dependent enzyme

Erythrocyte and liver tissue δ-aminolevulinate dehydratase activity was determined in rats fed a semipurified diet under controlled nutritional intake of zinc and copper. A significant decrease in enzymatic activity was observed in animals fed low zinc diet, while dietary copper had no effect. $\text{In vitro}$ addition of zinc to the erythrocyte preparations obtained from rats on low zinc diet produced a slight increase in enzymatic activity. It appears that, even though zinc may be the metal ion activator of δ-aminolevulinate dehydratase, the requirement of this metal is at the site of synthesis of this enzyme.     

Control of the initial steps in heme biosynthesis in free-livingRhizobium sp. by culture conditions

Cell-free extracts obtained from free-livingRhizobium sp. in early stationary phase had three times as much 5-aminolevulinate synthase activity as did similar extracts from log phase cells. The level of 5-aminolevulinate dehydratase was also elevated at this point. The presence of 0.1 mM hemin in the culture medium prevented the transitory increase in enzyme activities during this early stationary phase. The effect of hemin was counteracted by 1 mg bovine serum albumin per milliliter medium. This control of the development of 5-aminolevulinate synthase and 5-aminolevulinate dehydratase activities by free hemin suggests a mechanism by which heme and globin formation might be coordinated for the synthesis of leghemoglobin in legume root nodules.     

Affinity labelling of 5-aminolevulinic acid dehydratase with 2-bromo-3-(5-imidazolyl)propionic acid

2-Bromo-3-(5-imidazolyl)propionic acid, a zinc-directed thiol reagent, inactivates the enzyme 5-aminolevulinic acid dehydratase from bovine liver (5-aminolevulinate hydro-lyase (adding 5-aminolevulinate and cyclizing, EC 4.2.1.24). The substrate, 5-aminolevulinic acid, completely protects against inactivation. The reagent inhibits the zinc-containing enzyme to a greater extent than the zinc-deprived enzyme; and it competes with the zinc chelator 1,10-phenanthroline. The reagent alkylates essential sulfhydryl groups of the enzyme, since the extent of the inactivation depends on the reduction of the enzyme protein by thiol compounds. It is concluded that the zinc site, the substrate site and the essential sulfhydryl groups are in close proximity in the active site.     

Molecular cloning of DNA complementary to mRNA of rat liver serine dehydratase

A cDNA clone containing sequences complementary to the mRNA cording for rat hepatic serine dehydratase was isolated to study the multihormonal regulation of this enzyme. Serine dehydratase mRNA was partially purified (50-fold enrichment, 8.2% of the total mRNA activity) from the liver of rats fed high protein diet by polysome immunoadsorption followed by oligo(dT)-cellulose column chromatography. This preparation was used as template for synthesis of cDNA. Double-stranded cDNA sequences were inserted into the plasmid pBR322 and cloned in Escherichia coli DH1. Of 860 transformants screened, 6 clones containing DNA complementary to serine dehydratase mRNA were identified by differential colony hybridization and hybrid-selected translation. The length of serine dehydratase mRNA was estimated to be 1,500 bases by Northern blot analysis. One cloned cDNA comprised about 1,000 base pairs, or 65% of the length of the mRNA. The amount of the mRNA was greatly increased in the liver of rats given high protein diet.     

An immunological study of delta-aminolevulinic acid dehydratase specificity consistent with the phylogeny of species

Rabbit antibody directed to homogeneously purified mouse liver delta-aminolevulinic acid dehydratase cross-reacted with the enzyme in erythrocytes, spleen, kidney and brain in the mouse. The antibody also cross-reacted with the enzyme in the rat, hamster and gerbil, but not in the rabbit, guinea pig, cattle, chick embryo, and human. In contrast, rabbit antibody against the human enzyme partially recognized the monkey enzyme, but not the enzyme in the other species. The species specificity of delta-aminolevulinic acid dehydratase in this study was consistent with the phylogenetic evolution of the species examined.     

An immunological study of δ-aminolevulinic acid dehydratase specificity consistent with the phylogeny of species

Rabbit antibody directed to homogeneously purified mouse liver δ-aminolevulinic acid dehydratase cross-reacted with the enzyme in erythrocytes, spleen, kidney and brain in the mouse. The antibody also cross-reacted with the enzyme in the rat, hamster and gerbil, but not in the rabbit, guinea pig, cattle, chick embryo, and human. In contrast, rabbit antibody against the human enzyme partially recognized the monkey enzyme, but not the enzyme in the other species. The species specificity of δ-aminolevulinic acid dehydratase in this study was consistent with the phylogenetic evolution of the species examined.     

Effects of hemin on rat liver cyclic AMP-dependent protein kinases in cell extracts and intact hepatocytes

Cyclic AMP-dependent protein kinases I and II, partially purified from rat liver cytosol, were inhibited 50% by 40 microM hemin and 100 microM hemin, respectively. With the purified catalytic subunit of cyclic AMP-dependent protein kinase, hemin caused non-competitive inhibition with respect to the peptide substrate and mixed inhibition with respect to ATP. Hemin also inhibited purified phosphorylase b kinase, indicating that hemin concentrations above 10 microM markedly inhibit multiple protein kinases. In isolated intact hepatocytes, hemin inhibited the glucagon-dependent activation of cyclic AMP-dependent protein kinases and the activation of glycogen phosphorylase. For both effects, high heme concentrations (40-60 microM) were required for 50% inhibition. Similar high levels of exogenous hemin inhibited total hepatocyte protein synthesis. By contrast, 5 microM hemin or less was sufficient to raise intracellular heme levels, as indicated by the relative heme-saturation of tryptophan oxygenase in hepatocytes. Hemin, 5 microM, completely repressed induction of 5-aminolevulinate synthase by dexamethasone in hepatocyte primary cultures. Such repression is unlikely to be mediated by inhibition of protein kinases.     

A new biosynthetic route of porphyrin precursors in common between animals and plants

An additional enzyme, 4-oxo-5-hydroxyvalerate (OHV) dehydrogenase was identified and characterized. This enzyme catalyzes the conversion of OHV to 4,5-dioxovalerate, a direct precursor of 5-aminolevulinate. The enzyme was partially purified from rat liver supernatant as two isoenzyme (ca. 40,000 and 70,000 dalton). 5-Aminolevulinate was formed from OHV via 4,5-dioxovalerate by this dehydrogenase and alanine-4,5-dioxovalerate aminotransferase (EC 2.6.1.43). This dehydrogenase required NADP of NAD as a hydrogen acceptor. The enzyme was heat sensitive and catalyzed the reaction reversibly. The dehydrogenase was present in the high speed supernatants of liver and kidney of rat, rabbit and human, and that of spinach leaf.     

Cloning of the Escherichia coli K-12 hemB gene.

An Escherichia coli heme-requiring, heme-permeable mutant had no detectable 5-aminolevulinate dehydratase or porphobilinogen deaminase activities. The gene which complemented this mutation was cloned to a high-copy-number plasmid, and porphobilinogen deaminase activity was restored to normal levels, but the synthesis of 5-aminolevulinate dehydratase increased 20- to 30-fold. A maxicell procedure confirmed that the gene cloned was hemB.     

A specific inhibitor to δ-aminolevulinate dehydratase in rat bone marrow cells

A factor that specifically inhibited δ-aminolevulinate dehydratase was found in rat bone marrow cells. The inhibitor, which was located in the supernatant fraction of the bone marrow hemolysate, was purified about 12-fold by ammonium sulfate fractionation and column chromatography on Sephadex G-75. The partially purified inhibitor was heat labile and sensitive to trypsin and was denatured by urea. It had a pH optimum of 7.5–8.0, and a molecular weight of 28,000. It inhibited the activity of δ-aminolevulinate dehydratase noncompetitively.     

Stimulation par la lumière rouge lointain de la synthèse de la δ-aminolévulinate déshydratase des cotylédons de radis

Stimulation of de novo synthesis of δ-aminolevulinate dehydralasc of radishes grown under far-red light .
Density labelling studies of δ-aminolevulinate dehydratase (ALAD) in cotyledons of radish ( Raphanus sativus L. cv. Longue Rave Saumonée) seedlings demonstrate that far-red light stimulates de novo synthesis of ALAD and that the turn-over of this enzyme is very poor. Cycloheximide reduces considerably both the increase of ALAD activity and the incorporation of deuterium in ALAD, which indicates that ALAD synthesis depends upon cytoplasmic ribosomes.     

Studies on the mechanism of 5-fluoroorotic acid inhibition of serine dehydratase induction

Administration of glucagon to rats fed a protein-free diet caused a significant induction of the liver enzyme, serine dehydratase. This effect of glucagon is inhibited by the concomitant administration of fluoroorotic acid. This inhibition was enhanced by pretreatment with glucosamine or galactosamine, probably through depletion of the intracellular uridine pools. Although less than a doubling of enzyme activity was observed after glucagon plus fluoroorotic acid administration, the amount of protein precipitable by antisera specifically reactive against serine dehydratase increased 4.5 times. Ouchterlony double-diffusion analysis showed a completely cross-reacting single precipitin band from liver extracts of untreated animals and rats treated with the analog. Analysis of the antigen-antibody complex by Na dodecyl sulfate-gel electrophoresis indicated that a single protein was being immunochemically precipitated from both the glucagon- and glucagon plus fluoroorotic acid-treated rats. In the latter, the precipitated protein had a molecular weight similar to purified serine dehydratase. These results are consistent with the concept that the incorporation of fluoroorotic acid into mRNA results in the synthesis of a protein with characteristics similar to authentic serine dehydratase but without normal enzymatic activity. Other possible mechanisms to explain the production of this abnormal protein are discussed.     

Inhibitory effect of gabaculine on 5-aminolevulinate dehydratase activity in radish seedlings

We have compared the activity of 5-aminolevulinate dehydratase (5-ALAD) with the amount of protein detected by specific antibodies in rocket immunoelectrophoresis. Parallel kinetic evolutions of enzymic activity and amount of antigen were observed in radish (Raphanus sativus L.) cotyledons, both in complete darkness or under standard far red light involving phytochrome. However, the treatment of seedlings with gabaculine leads to an important decrease in enzymic activity, while the specific protein content is maintained. This inhibition is not overcome by the addition of glutamic acid, but by 5-aminolevulinic acid which points to a specific control of 5-ALAD activity by its substrate. As there is no discrepancy between the enzymic activity and the amount of antigen during the time course development of seedlings, this could confirm a coordinate cellular control between 5-aminolevulinic acid formation and 5-ALAD protein synthesis, both being amplified by the action of phytochrome.     

Protective Effect of Sclerin on a Lead-acetate Injury Affecting the Growth and Porphyrin Metabolism in Rat

While a continuous ingestion of lead acetate added in drinking water suppressed the rat growth, depressing in some degree the level of hepatic δ-aminolevulinate (ALA) dehydratase, a very small amount of sclerin (SCL) added simultaneously in the water restored the growth and dehydratase level. Moreover, subcutaneous injection of SCL to the rat not only maintained the ALA dehydratase level, but prevented a marked depression of the level of mitochondrial ALA synthetase in liver caused by intraperitoneal injection of lead acetate. Injection of SCL alone increased tolerably (about 1.8 times) the mitochondrial ALA synthetase, but little the extramitochondrial synthetase. The treatment by SCL was attended by a initial decrease, then a gradual increase in the activity of microsomal drug metabolizing enzyme.     

Immunochemical studies of serine dehydratase and ornithine aminotransferase regulation in rat liver in vivo.

Previous studies of serine dehydratase (EC 4.2.1.13) and ornithine aminotransferase (EC 2.6.1.13) adaptation in rat liver showed that in rats on a high protein diet, glucocorticoid administration increased serine dehydratase activity while simultaneously reducing the activity of ornithine aminotransferase. The present study examines the role of enzyme synthesis in the expression of these and other dissimilar adaptive characteristics of the two enzymes. Both enzymes were purified to crystallinity and used to prepare specific antibodies. Changes in the rate of synthesis of each enzyme during adaptation were then measured immunochemically. In rats fed ad libitum, the synthetic rates for both enzymes exhibited circadian rhythm, although enzyme levels remained relatively constant. The circadian cycle for ornithine aminotransferase synthesis was in phase with the cycles for body weight and relative liver weight (maxima at 9 a.m., minima at 9 p.m.) but was approximately 12 hours out of phase with the cycle for serine dehydratase synthesis. 9alpha-Fluoro-11beta, 21-dihydroxy-16alpha, 17alpha-isopted at 9 a.m., increased serine dehydratase synthesis and simultaneously decreased the synthesis of ornithine aminotransferase. When triamcinolone was injected at 9 p.m., however, serine dehydratase synthesis was not stimulated, although the reduction of ornithine aminotransferase synthesis was still produced. These results suggest that: (a) circadian cycling of synthesis may be a general phenomenon in enzyme regulation even though for enzymes with relatively long half-lives, such cycling may not be reflected as fluctuations in enzyme levels; (b) such circadian rhythmicity may also involve cyclic changes in the responsiveness of the enzyme-forming system to regulatory stimuli; (c) whereas the adaptive behavior of serine dehydratase typifies that of amino acid-catabolizing enzymes in general, the responses of ornithine aminotransferase denote a functional association of this enzyme with anabolic processes. On this basis, the possibility that ornithine aminotransferase plays a pivotal role in the regulation of urea cycle activity and nitrogen balance is discussed.     

In situ assay for 5-aminolevulinate dehydratase and application to the study of a catabolite repression-resistant Saccharomyces cerevisiae mutant.

To facilitate the study of the effects of carbon catabolite repression and mutations on 5-aminolevulinate dehydratase (EC 4.2.1.24) from Saccharomyces cerevisiae, a sensitive in situ assay was developed, using cells permeabilized by five cycles of freezing and thawing. Enzymatic activity was measured by colorimetric determination of porphobilinogen with a modified Ehrlich reagent. For normal strains, porphobilinogen production was linear for 15 min, and the reaction rate was directly proportional to the permeabilized cell concentration up to 20 mg (dry weight) per ml. The reaction exhibited Michaelis-Menten-type kinetics, and an apparent Km of 2.6 mM was obtained for 5-aminolevulinic acid. This value is only slightly higher than the value of 1.8 mM obtained for the enzyme assayed in cell extracts. The in situ assay was used to assess catabolite repression-dependent changes in 5-aminolevulinate dehydratase during batch culture on glucose medium. In normal S. cerevisiae cells, the enzyme is strongly repressed as long as glucose is present in the medium. In contrast, a strain bearing the hex2-3 mutation exhibits derepressed levels of enzyme activity during growth on glucose. Synthesis of cytochromes by this strain is also resistant to catabolite repression. Similar studies employing a strain containing the glc1 mutation, which enhances porphyrin accumulation, did not reveal any significant phenotypic change in catabolite regulation of 5-aminolevulinate dehydratase.     

Effects of hemin on rat liver cyclic AMP-dependent protein kinases in cell extracts and intact hepatocytes

Cyclic AMP-dependent protein kinases I and II, partially purified from rat liver cytosol, were inhibited 50% by 40 μM hemin and 100 μM hemin, respectively. With the purified catalytic subunit of cyclic AMP-dependent protein kinase, hemin caused non-competitive inhibition with respect to the peptide substrate and mixed inhibition with respect to ATP. Hemin also inhibited purified phosphorylase b kinase, indicating that hemin concentrations above 10 μM markedly inhibit multiple protein kinases. In isolated intact hepatocytes, hemin inhibited the glucagon-dependent activation of cyclic AMP-dependent protein kinases and the activation of glycogen phosphorylase. For both effects, high heme concentrations (40–60 μM) were required for 50% inhibition. Similar high levels of exogenous hemin inhibited total hepatocyte protein synthesis. By contrast, 5 μM hemin or less was sufficient to raise intracellular heme levels, as indicated by the relative heme-saturation of tryptophan oxygenase in hepatocytes. Hemin, 5 μM, completely repressed induction of 5-aminolevulinate synthase by dexamethasone in hepatocyte primary cultures. Such repression is unlikely to be mediated by inhibition of protein kinases.     

The effects of succinylacetone (4,6-dioxoheptanoic acid) on δ-aminolevulinate synthase activity and the content of heme in monolayers of chick embryo liver cells

Succinylacetone was shown to inhibit aminolevulinate dehydratase (5-aminolevulinate hydro-lyase (adding 5-aminolevulinate and cyclizing), EC 4.2.1.24) to reduce cellular heme and porphyrins and to induce δ-aminolevulinate synthase (succinyl-CoA:glycine C-succinyltransferase (decarboxylating), EC 2.3.1.37) in monolayers of chick embryo liver cells. Marked synergistic effects on δ-aminolevulinate synthase activity were obtained by combining succinylacetone with levulinate and porphyrogenic drugs. The time course of δ-aminolevulinate synthase activity showed a delayed synergistic response.