Δ-aminolevulinic Acid Synthetase Activity Research Articles

Antiporphyric activity of 3,5-dimethylpyrazole in allylisopropylacetamide-treated rats.

Abstract The AIA administration to rats increased δ-aminolevulinic acid synthetase activity and porphyrin levels in the liver and elevated plasma cholesterol and triglyceride values. 3,5-Dimethylpyrazole markedly decreased liver ALA synthetase and porphyrin values and also lowered plasma cholesterol and triglyceride levels in AIA-treated rats.

Antiporphyric activity of thiophene-2-carboxylic acid in allylisopropylacetamide-treated rats

Allylisopropylacetamide (AIA) administration to rats increases δ-aminolevulinic acid (ALA) synthetase activity and porphyrin levels in liver.

Purification and Properties of Solubilized Mitochondrial δ-Aminolevulinic Acid Synthetase and Comparison with the Cytosol Enzyme

Abstract δ-Aminolevulinic acid synthetase extracted from porphyric liver mitochondria by sonication or freeze-drying was found to be excluded from Sephadex G-200, and other studies revealed that activity was associated with a large aggregate. In contrast, aminoacetone synthetase behaved as a soluble enzyme with a molecular weight of 64,000. By treating the aggregate with both sodium chloride and dithioerythritol, δ-aminolevulinic acid synthetase activity was released to yield a soluble form of enzyme of molecular weight 77,000, as determined by Sephadex gel chromatography. This form of the enzyme was purified 40-fold from rat liver mitochondria by a method involving affinity chromatography. Antibodies prepared against the purified mitochondrial enzyme cross-react with the cytosol enzyme, although Sephadex chromatography indicates its molecular weight to be 178,000. No enzymically inactive, immunologically reactive precursor of δ-aminolevulinic acid synthetase could be detected in normal rat liver. End product inhibition by hemin was found to be 50% at 10 µm.

Decreased red cell uroporphyrinogen I synthetase activity in intermittent acute porphyria.

Intermittent acute porphyria has recently been distinguished biochemically from other genetic hepatic porphyrias by the observation of diminished hepatic uroporphyrinogen I synthetase activity and increased delta-aminolevulinic acid synthetase activity. Since deficient uroporphyrinogen I synthetase may be reflected in nonhepatic tissues, we have assayed this enzyme in red cell hemolysates from nonporphyric subjects and from patients with genetic hepatic porphyria. Only patients with intermittent acute porphyria had decreased erythrocyte uroporphyrinogen I synthetase activity which was approximately 50% of normal. The apparent K(m) of partially purified uroporphyrinogen I synthetase was 6 x 10(-6)m in both nonporphyrics and patients with intermittent acute porphyria. These data provide further evidence for a primary mutation affecting uroporphyrinogen I synthetase in intermittent acute porphyria. Further-more, results of assay of red cell uroporphyrinogen I synthetase activity in a large family with intermittent acute porphyria suggest that this test may be a reliable indicator of the heterozygous state.

Chemically induced porphyria: Protection by treatment with nicotinic acid

Administration of allylisopropylacetamide to rats caused an increase of liver δ-aminolevulinic acid synthetase activity and porphyrin levels. Administration of nicotinic acid to allylisopropylacetamide-treated rats significantly lowered liver δ-aminolevulinic acid synthetase activity and porphyrin levels. The antiporphyric activity of nicotinic acid was probably due to a change of the redox state, altered by administration of allylisopropylacetamide.

Soluble δ-Aminolevulinic Acid Synthetase of Rat Liver

Abstract The present study describes some characteristics of soluble hepatic δ-aminolevulinic acid synthetase, including end product inhibition by hemin. The enzyme is shown to contain a —SH group at its active center which is associated with the initial binding of the cofactor pyridoxal-5'-PO4 to the enzyme. In addition, the —SH group may influence the activation of δ-aminolevulinic acid synthetase by divalent cations. A mechanism is suggested for the catalytic formation of δ-aminolevulinic acid from glycine and succinyl-CoA. From data derived from isotopic studies with [1 -14C]glycine, and studies utilizing specific inhibitors, the following sequence of reactions resulting in the formation of δ-aminolevulinic acid is proposed. The initial formation of a thiohemiacetal between the enzyme and pyridoxal-5'-PO4; the subsequent formation of a Schiff base with glycine which is followed by its condensation with succinyl-CoA; the enzymatic reaction is terminated with the decarboxylation of the glycyl carboxyl group in association with the release of δ-aminolevulinic acid from the enzyme. End product inhibition of the mammalian enzyme was confirmed using hemin, which was demonstrated to have a Ki of 2 x 10-5 m. In addition, various metalloporphyrins, porphyrins, and bilirubin also inhibit δ-aminolevulinic acid synthetase activity. The relative potencies of the inhibitors appear to be dependent on the net charge present on the tetrapyrrole.

Studies of the perinatal differences in the activity of hepatic δ-aminolevulinic acid synthetase

Hepatic δ-aminolevulinic acid (ALA) synthetase activity in prenatal rats, rabbits and guinea pigs is four to eight times the adult level. During the period of elevated activity, ALA synthetase is relatively much less responsive to induction by 3,5-dicarbethoxy-1,4-dihydrocollidine (DDC) or to repression by hemin than is observed in adult rats and rabbits. No induction of ALA synthetase could be demonstrated in fetal rats. In newborn rabbits, hemin caused no significant reduction in ALA synthetase activity, whereas the adult enzyme activity was reduced to 32 per cent of control. This difference in response to hemin was also demonstrated in fetal and newborn rats. Refractoriness to induction decreases as enzyme activity decreases to adult levels. These studies indicate that the neonate may not regulate the activity of hepatic ALA synthetase at the same level as the adult. It is suggested that increasing repression of the enzyme to lower basal levels occurs as the animals mature, bringing the activity and response to DDC and hemin to the levels observed in adults. Perinatal alterations in the regulation of hepatic ALA synthetase activity may account for some of the problems associated with heme metabolism in the newborn and the toxic effects of bilirubin and various drugs.

Radiochemical microassay of delta-aminolevulinic acid synthetase in hepatic and erythroid tissues.

Abstract A specific radiochemical microassay for δ-aminolevulinic acid synthetase is described. The assay is rapid and 10 to 500 times more sensitive than currently available methods. The assay measures the incorporation of radioactive succinate into δ-aminolevulinic acid and requires a succinyl CoA generating system; alternate pathways of succinate utilization are blocked by specific metabolic inhibitors. The δ-aminolevulinic acid formed is isolated by ion-exchange chromatography. The increased sensitivity of this assay has permitted the direct measurement of δ-aminolevulinic acid synthetase activity in cell cultures of avian hepatocytes, needle biopsy specimens of human liver, peripheral erythrocytes, and human fibroblasts.

A case of primary acquired sideroblastic anemia with deficient -aminolevulinic acid synthetase activity in bone marrow erythroblasts.

A case of primary acquired sideroblastic anemia is described, in whom erythroblasts showed markedly reduced δ-aminolevulinic acid synthetase activity. The patient, 68-year-old Japanese female, was admitted with a history of anemia and congestive heart failure of 3 year duration. By hematological study, marked hypochromic anemia without apparent morphologic aberration was noted. Examination of bone marrow disclosed the intensive erythroid hyperplasia with minor megaloblastoid changes and maturation arrest. Iron stores in bone marrow were greatly increased with predominating sideroblasts. Serum iron was elevated and ferrokinetic study indicated normal rate of iron clearance and depressed red cell iron utilization. In the studies on porphyrin metabolism, incorporation of 14C-glycine into heme and activity of S-aminolevulinic acid (ALA) synthetase were found to be greatly reduced. Although there have been several reports on the defects in the heme synthetic pathway in sideroblastic anemia, this is a first case of sideroblastic anemia with deficient ALA synthetase activity demonstrated by a direct method.

-Aminolevulinic acid synthetase activity of human bone marrow erythroid cells in various hematological disorders.

A method to measure the δ-aminolevulinic acid (ALA) synthetase activity in human bonemarrow erythroid cells by incubating the bone-marrow cell homogenate with 14Ca-ketoglutarate has been presented. Studies representing an attempt to obtain the optimal conditions for the enzyme assay and to confirm the reliability of the results of the assay were performed. ALA-synthetase activity in erythroblasts was found decreased in all 4 cases of iron deficiency anemia. In the cases of sideroblastic anemia, the enzyme activity was markedly decreased to less than one-tenth of normal in one case, moderately decreased in 2 cases, and normal in 2 cases. In a case with β-thalassemia, a marked decrease in ALA-synthetase activity was in evidence. In these cases of the sideroblastic anemia or thalassemia with markedly decreased ALA-synthetase activity, a substantial diminution of 14C-glycine incorporation into heme in erythroblasts was also evident. Erythroblasts cf these cases, however, showed only a slight decrease in the heme-synthetase activity. In erythroblasts of patients with erythropoietic protoporphyria, ALA-synthetase activity was found to be within normal limits in 2 cases, and apparently increased in one case.

δ-Aminolevulinic Acid Synthetase Activity in Human Plasma: Relation to Erythropoiesis and Evidence of Induction in Erythropoietic Porphyria

Abstract Measurement of δ-aminolevulinic acid synthetase (ALA-S) activity in human plasma has been carried out with samples from normal individuals, cases of erythropoietic porphyria (EP) and erythropoietic protoporphyria (EPP), and of the three principal forms of hepatic porphyria—acute intermittent porphyria, variegate porphyria, and porphyria cutanea tarda. The method of measurement depends on formation of 14C-ALA when the plasma is incubated with 14C-succinic acid, succinyl-Co A synthetase, glycine, and other essential substances. The normal samples, as well as those from the hepatic porphyria cases, had small but significant activity of the same extent; those from the erythropoietic group showed consistently higher values, especially in the two cases of congenital type. A remarkably high value in one of these cases in which there was outspoken erythropoiesis was believed to be related to the presence of many fluorescing normoblasts in the peripheral blood. Following multiple transfusions these disappeared concomitantly with striking reduction of the porphyria. The plasma ALA-S activity declined to 1.4% of the pretransfusion value. These results are considered in respect to the question of induction of ALA-S in the developing red cells of the disease, special attention being given to the minor increase of ALA-S activity in the plasma of a nonporphyric individual whose peripheral blood contained large numbers of circulating normoblasts.

HUMAN RESPONSES TO LEAD IN SPECIAL REFERENCES TO PORPHYRIN METABOLISM IN BONE MARROW ERYTHROID CELLS, AND CLINICAL AND LABORATORY STUDY

Enzymatic studies on heme biosynthesis in erythroid cells of bone marrows ob-tained from ten lead workers were carried out after the establishment of methods.A remarkable reduction of the activities of δ-aminolevulinic acid (ALA) dehydrase and heme synthetase, a slight elevation of ALA synthetase activity, and a parallel reduction of 14C-glycine incorporation into heme and globin were observed. Abnor-malities found in hematological examinations and in urinary or erthrocyte porphyrin metabolism were discussed in connection with both the above mentioned results and the human response to lead.Clinical and laboratory examinations on these workers revealed a commonly exist-ing hypertension, ECG abnormalities, reduced renal functions, especially those of renal blood circulation and of renal tubules, EMG abnormalities, a reduced conduction velo-city of long nerves, and elevated concentrations of urea-N and uric acid in serum. The pathogenesis of these findings were also discussed.

Delta-aminolevulinic acid synthetase and adenosine triphosphate activity in acute saturnine intoxication in rabbits.

In experimentally induced lead poisoning in rabbits, the activity of δ-aminolevulinic acid (ALA) synthetase in the mitochondria isolated from liver and bone marrow remains within normal limits. The concentration of adenosine triphosphate in the liver is also normal, and the concentration of this nucleotide in circulating erythrocytes is moderately (significantly) increased. These findings support the view that the biosynthesis of porphyrins is not enhanced during intoxication by lead, but that utilization of porphyrins and their precursors is diminished by reduction in activity of heme synthetase.

Polychlorinated biphenyls as inducers of hepatic porphyria in Japanese quail, with special reference to δ-aminolevulinic acid synthetase activity, fluorescence, and residues in the liver

Male Japanese quail were orally dosed, daily for 7 days, with 1000, 500, 250, 100, 50, and 0 mg PCB (Aroclor 1260) per kg body weight. The birds receiving 1000 mg/kg died. A dose-related increase in liver and liver: body weight ratio was found. Macroscopic and microscopic examination under ultraviolet light were used for the detection of porphyrin fluorescence. Mitochondrial ALA synthetase activity demonstrated a large increase (20-fold at the 500 mg/kg level; 10-fold at the 50 mg/kg level). The doubling of mitochondrial protein, the increase of ALA synthetase activity, the liver enlargement, and the pyroninophilic staining of the hepatic nucleoli suggest an increased protein synthesis. In a second experiment, 5 groups of female quail received 100, 10, 1, 0.1, and 0 mg PCB per kg body weight. ALA synthetase activity was increased about 20-fold in the 100 mg/kg group. This increase, together with the high PCB content of the livers (mean 478 ppm) and the incidence of fluorescence, indicating porphyrins, were correlated with the loss of weight in this group (mean 8%). Mobilization of PCB from storage fat is considered to be responsible for the increased porphyria. The dose-response relationship and liver residue relationship of PCB and ALA synthetase activity are given.

Adaptive increase in δ-aminolevulinic acid synthetase by ethchlorovynol and certain related drugs in the guinea-pig and rabbit

1. 1. Prolonged treatment of guinea-pigs and rabbits with ethchlorovynol, methylparafynol, or ethinamate resulted in an enhancement of the activity of δ-aminolevulinic acid (ALA) synthetase as judged by the increase in the amounts of hepatic ALA, although to different extents. 2. 2. A similar but less pronounced effect was noticed after acute treatment with the drugs. 3. 3. Evidence is produced to support the thesis that the observed enzyme-induction was mainly due to de novo synthesis of enzyme protein and possibly to increased quantities of enzyme substrate. 4. 4. The drugs were not specific in this respect, since they were also capable of enhancing the activity of other enzymes such as glutamic dehydrogenase and glutamic-oxaloacetic transaminase.

δ-Aminolevulinic acid synthetase activity in erythroblasts of patients with sideroblastic anemia

A new method to assay the ALA-synthetase activity in erythroid cells by measuring the incorporation of 14C-α-ketoglutarate into ALA in the bone-marrow cell homogenate was developed. By using this method, ALA-synthetase activity in erythroblasts of 5 patients with sideroblastic anemia was measured. The enzyme activity was markedly decreased in one case, moderately decreased in 3 cases and normal in one case. In this case with decreased ALA-synthetase activity, incorporation of 14C-glycine into heme in erythroblasts was also markedly reduced. A decrease in heme-synthetase activity in erythroblasts of this case, however, was slight as compared to the decrease in ALA-synthetase activity.

Induction of Hepatic δ-Aminolevulinic Acid Synthetase Activity in Strains of Inbred Mice

Abstract δ-Aminolevulinic acid synthetase activity is increased in hepatic homogenates from mice that have been fasted or treated (or both) with 3,5-dicarbethoxy-1,4-dihydro-2,4,6-trimethylpyridine (DDC). Comparison of δ-aminolevulinic acid synthetase activity in livers from adult female mice of 15 inbred strains showed significant differences (p l 0.01) among the strains in the amount of enzyme activity normally present and in the amount present after induction. After DDC administration, δ-aminolevulinic acid synthetase activity increased at a linear rate for 12 hours and then reached a plateau at a level that was maintained until at least 20 hours after drug administration. Strain differences were maintained over a range of DDC doses from 28 mg per kg to 500 mg per kg (the highest dose tested). No significant differences in plasma DDC levels were noted between strains of and induced activity. Representative strains with high and low DDC-induced enzyme activity were compared in detail. Neither modification of the glycine, pyridoxal 5'-phosphate, or EDTA concentrations nor substitution of other buffers for the usual Tris buffer changed the relative enzyme activities of homogenates. Addition of succinyl coenzyme A or a succinyl CoA generating system increased total homogenate activity to the same extent in high and low strains. In animals from both classes, added succinate inhibited δ-aminolevulinate production. When hepatic homogenates of DDC-treated mice were fractionated by differential centrifugation, no substantial differences were noted among strains in the subcellular distribution of δ-aminolevulinic acid synthetase activity. Our data suggest that among inbred strains of mice there are either differences in the number of δ-aminolevulinic acid synthetase enzyme molecules present or differences in the catalytic activity of a structurally modified enzyme produced in response to fasting or DDC administration (or both) and that genetic factors are responsible for this heterogeneity of response.

Chemical induction of hepatic porphyria in inbred strains of mice

The activities of δ-aminolevulinic acid (ALA) synthetase, 1 1 The following ABBREVIATIONS are used in the text: ALA, δ-aminolevulinic acid; PBG, porphobilinogen; Uro, uroporphyrinogen; Copro, coproporphyrinogen; Proto, protoporphyrin; DDC, 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine; Tween 80, polyoxyethylene sorbitan monooleate; SEM, standard error of the mean. ALA dehydratase, uroporphyrinogen synthetase, and the quantities of the metabolites, porphobilinogen (PBG), coproporphyrin, and protoporphyrin were serially assayed in the livers of inbred mice which had received repeated injections of 3,5-diethoxycarbonyl-1,4-dihydro-2, 4,6-trimethylpyridine (DDC). During 48 hr of drug treatment, ALA synthetase activity increased 5- to 20-fold above a basal activity of 30–100 nmoles ALA formed/ hr/g liver. Hepatic uroporphyrinogen synthetase activity did not increase above a basal level of 35–60 nmoles PBG consumed/hr/g liver and appeared to represent the rate-limiting step during most of the course of induction of porphyria. ALA dehydratase activity was dependent upon genotype at the Lv locus and was strain specific, but in all strains this enzyme was present in excess (700–4800 nmoles/hr/g liver). The activity of the dehydratase increased 30% during 48 hr of drug treatment. PBG, proto- and, to a lesser extent, coproporphyrins accumulated, but much less porphyrin was found in livers of C57L/J mice than in those of A/HeJ, C57BL/6J, DBA/2J, SWR/J or C3H/HeJ. Hepatic protoporphyrin accumulation was directly correlated with ALA synthetase activity provided that the protoporphyrin level was less than 50 nmoles/g liver. When hepatic protoporphyrin exceeded 100 nmoles/g liver, there was an inverse correlation between ALA synthetase activity and protoporphyrin content suggesting that either feedback inhibition or feedback repression of the synthetase had occurred.

Further studies on the relationship of the stimulatory effects of phenobarbital and 3,4-benzpyrene on hepatic heme synthesis to their effects on hepatic microsomal drug oxidations

The administration of either phenobarbital or 3,4-benzpyrene to rats resulted in the rapid and marked induction of δ-aminolevulinic acid synthetase (EC 2.3.1.13), the proposed initial and rate-limiting enzyme in the hepatic heme biosynthetic pathway. Enhanced formation of δ-aminolevulinic acid was followed sequentially by an enhancement of the liver's capacity to synthesize microsomal heme in vivo, increases in the content of cytochrome P-450 and protoheme in hepatic microsomes and stimulation of certain hepatic microsomal drug oxidations. Changes in the hepatic microsomal levels of cytochrome P-450 paralleled, in part, changes in the activity of hepatic δ-aminolevulinic acid synthetase and in the capacity of the liver to synthesize microsomal heme in vivo, suggesting that the rate of hepatic heme synthesis may control the rate of synthesis of hepatic microsomal cytochome P-450. Increases in the hepatic microsomal content of cytochrome b 5, however, followed a different time course from that observed from either cytochrome P-450 or protoheme. The simultaneous administration of maximum stimulatory doses of phenobarbital and 3,4-benzpyrene did not result in an additive stimulation of δ-aminolevulinic acid synthetase activity, indicating that phenobarbital and 3,4-benzpyrene induce δ-aminolevulinic acid synthetase by the same or closely related mechanisms. However, the stimulatory effects of these agents on cytochrome P-450 and on the N-demethylation of 3-methyl-4-monomethylaminoazobenzene were additive, suggesting that differences may exist in the mechanism by which phenobarbital and 3,4-benzpyrene induce hepatic microsomal cytochrome P-450 and enhance certain hepatic microsomal drug oxidations.

Studies on the induction of hepatic δ-aminolevulinic acid synthetase in rat liver mitochondria

Administration of allylisopropylacetamide (AIA) to rats caused an initial induction of hepatic δ-aminolevulinic acid (ALA) synthetase activity in the postmitochondrial supernatant fraction of the cell. The activity in this fraction decreased 50% from 1–3 hr after injection, while during this time period, the ALA synthetase activity of the mitochondrial fraction continued to increase at a nearly linear rate. Cycloheximide blocked the induction of ALA synthetase in both subcellular fractions. Two injections of chloramphenicol inhibited 50–60% the induction of ALA synthetase activity in the mitochondrial fraction but not in the postmitochondrial supernatant fraction. The apparent half-life of the enzyme was 66 min after cycloheximide administration and 118 min after chloramphenicol administration. A slower rate of turnover of the enzyme was observed in animals 1 hr after AIA injection. These results support the suggestion that ALA synthetase is synthesized in the microsomes and is transferred to the mitochondrial matrix in a subsequent step. The content of cytochromes a–a 3, b, c–c 1 was increased 50% in mitochondria which contained high ALA synthetase activity.