Δ-aminolevulinic Acid Synthetase Research Articles

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.

δ-Aminolevulinic Acid Synthetase: III. SYNERGISTIC EFFECT OF CHELATED IRON ON INDUCTION

Abstract Orally administered ferric citrate exerts a pronounced synergistic effect on the induction of hepatic δ-aminolevulinic acid synthetase produced by two compounds. The effect appears to be specific for iron citrate, as opposed to other metals in complex with citrate, and specific also in affecting the level of δ-aminolevulinic acid synthetase as compared with another induced enzyme, tyrosine transaminase. Inorganic iron salts did not produce augmentation of the induction of hepatic δ-aminolevulinic acid synthetase. The data suggest that iron augments the rate of synthesis of the enzyme during induction rather than activating or stabilizing the enzyme.

Induction of Hepatic δ-Aminolevulinic Acid Synthetase by Oral Contraceptive Steroids

ABSTRACT The ability of oral contraceptive steroids to induce the hepatic enzyme, δ-aminolevulinic acid synthetase (ALAS), has been examined in vivo in a model test system utilizing 15- to 16-day-old chick embryos. ALAS controls the rate-limiting step in the biosynthesis of porphyrins and heme. All the progestational components of the contraceptive mixtures studied had potent ALAS inducing activity except for chlormadinone acetate. The inducing capacity of the different progestational agents varied, but all showed positive dose-response relationships. Neither of the estrogenic components of the oral contraceptives, mestranol or ethinyl estradiol, induced the enzyme significantly by itself, nor did these estrogens alter the degree of induction by the progestational compounds. The possible relevance of these findings to the increased urinary excretion of δ-aminolevulinic acid reported in some normal women receiving oral contraceptives, and to the experimental hormonal therapy of selected patients with acute...

Erythropoietic activity of steroid metabolites in mice.

The per cent Fe(59) incorporation into circulating erythrocytes as an indicator for new hemoglobin production was used in the polycythemic exhypoxic mouse system to study the effects of certain steroid hormone metabolites on erythropiesis. Enhanced Fe(59) incorporation was observed after the administration of several metabolites with a 5beta-H configuration, while those with a 5alpha-H configuration had no stimulatory effect. The stimulatory effect in avian systems has been shown by others to be due to an increased activity of delta-aminolevulinic acid synthetase, the limiting enzyme in heme biosynthesis. These in vivo studies thus indicate that in this mouse system, as in previously reported studies with avian and human bone marrow cells, some steroid metabolites stimulate hemoglobin synthesis. The observation that the same structure-junction relationship exists in the currently described system as in the avian suggests that these steroids probably induce delta-aminolevulinic acid synthetase in the mouse. The erythropoietic action of these nonandrogenic steroid metabolites may prove to be clinically useful.

A model for calculating enzyme synthetic rates during induction: Application to the synergistic effect of ferric citrate on the induction of hepatic δ-aminolevulinic acid synthetase

A model for calculating enzyme synthetic rates during induction: Application to the synergistic effect of ferric citrate on the induction of hepatic δ-aminolevulinic acid synthetase

Methylmalonyl CoA as an abnormal substrate for δ-aminolevulinic acid synthetase

The effect of methylmalonyl CoA on δ-aminolevulinic acid synthetase has been investigated. Methylmalonyl CoA has been shown to act as an abnormal substrate for this enzyme but does not inhibit the formation of δ-aminolevulinic acid from succinyl CoA. The product of the condensation of methylmalonyl CoA and glycine appears to be an aminoketoacid and it is suggested that it is a chain isomer of δ-aminolevulinic acid.

The role of heme synthesis during the induction of hepatic microsomal cytochrome P-450 and drug metabolism produced by benzpyrene

δ-Aminolevulinic acid synthetase, the initial and rate limiting step in hepatic heme synthesis, is induced by both benzpyrene and phenobarbital. Induction of this enzyme by benzpyrene results in the stimulation of glycine-2- 14C incorporation into hepatic microsomal heme in vivo and in the induction of cytochrome P-450 and N-demethylase activity. 3-Amino-1,2,4-triazole, an inhibitor of the second step in hepatic heme synthesis, prevents the stimulation of hepatic heme synthesis and the induction of P-450 and N-demethylase activity. It is suggested that induction of δ-aminolevulinic acid synthetase leading to increases in hepatic heme synthesis may be the mechanism by which benzpyrene induces cytochrome P-450 and certain hepatic microsomal oxidations.

Regulation of Heme Biosynthesis in Neurospora crassa

Abstract The mold Neurospora crassa does not accumulate porphyrins in iron deficiency but instead accumulates the sideramine desferricoprogen. In iron deficiency there is an accumulation of δ-aminolevulinic acid and the δ-aminolevulinic acid dehydratase level is very low. A similar situation exists in cobalt toxicity and zinc deficiency. The δ-aminolevulinic acid synthetase and ferroprotoporphyrin chelatase comparatively show only marginal changes under these conditions. Addition of iron and zinc to the respective metal-deficient cultures results in an induction of δ-aminolevulinic acid dehydratase. The induction of δ-aminolevulinic acid dehydratase is repressed by protoporphyrin and less effectively by hemin and hemoglobin. Iron deficiency, zinc deficiency, and cobalt toxicity have been found to interfere with the conversion of protoporphyrin into heme, thus rendering protoporphyrin available to repress the enzyme. This repression can be counteracted by iron and much more effectively by coprogen. A model has been proposed in which protoporphyrin has been visualized as the corepressor for the enzyme δ-aminolevulinic acid dehydratase. It is held that iron in the form of coprogen converts protoporphyrin to heme, the latter having a lesser affinity for the aporepressor. Coprogen can inhibit heme binding to the aporepressor and thus render the repressor nonfunctional. This will lead to a derepression of the enzyme δ-aminolevulinic acid dehydratase.

Soluble hepatic delta-aminolevulinic acid synthetase: end-product inhibition of the partially purified enzyme.

The present study confirms the existence of hepatic delta-aminolevulinic acid synthetase in the cytosol of the liver, suggests that this enzyme may be in transit to the mitochondria, and defines some of the characteristics of the partially purified enzyme. The substrate and cofactor requirements are similar to those of mitochondrial delta-aminolevulinic acid synthetase. Heme strongly inhibits the partially purified enzyme. A number of proteins that bind heme block this inhibition, which explains previous failures to demonstrate heme inhibition in crude systems. End-product inhibition of delta-aminolevulinic acid synthetase in the mitochondria may play an important role in the regulation of heme biosynthesis in eukaryotic cells.

Methyl 5-diazolevulinate intervention in chemically induced porphyria of rats

We have found that methyl 5-diazolevulinate (diazoester) abolishes the excretion of porphobilinogen from rats made porphyric with allylisopropylacetamide. At the same time, the high levels of δ-aminolevulinic acid found in the urine of porphyric rats are not significantly altered by diazoester. Induction of convulsions in porphyric rats by 4-methoxymethylpyridoxine is unaffected by diazoester, indicating that porphobilinogen is not a factor in the neurological consequences of porphyria, and that a precursor of porphobilinogen must be responsible. Only one precursor, δ-aminolevulinic acid, in a reasonable choice as the agent involved in the neurological changes. It was postulated that pyridoxal derivatives of δ-aminolevulinic acid strongly inhibit enzymes controlling the metabolism of γ-aminobutyric acid, a neurotransmitter, and that the neurological effects observed arise from an imbalance in γ-aminobutyric acid concentrations. It was suggested that a strong inhibitor for δ-aminolevulinic acid synthetase might be useful in the treatment of acute intermittent porphyria. The diazoester represents an example of a potentially useful class of compounds, the diazoketones, and its use in rats at relatively high dosages demonstrates that such compounds, given the appropriate specificities, are not very toxic on a short-term basis.

Effect of friend leukemia virus infection upon polycythemia and δ-aminolevulinic acid synthetase activity in murine spleen and liver

Effect of friend leukemia virus infection upon polycythemia and δ-aminolevulinic acid synthetase activity in murine spleen and liver

Mechanism and Control of Pyrrole Synthesis

This chapter discusses the enzymatic control of pyrrole synthesis (porphobilinogen). It focuses on the mechanism of the reaction and the nature and conformation of the enzyme involved in a particular biological system. Porphobilinogen is enzymatically synthesized (δ-aminolevulinic acid dehydratase) from two molecules of δ-aminolevulinic acid that arise from the condensation of succinyl-CoA and glycine (δ-aminolevulinic acid synthetase). The synthesis of the aminoketone, δ-aminolevulinic acid, can be considered to be the first committed step in the series of reactions that lead to the synthesis of porphyrins and related compounds, whereas the synthesis of the pyrrole is the step in which aromatization occurs. The latter reaction may also be considered a distinct committed step, because an aliphatic compound is changed into an aromatic ring compound. If these two enzymatic reactions are committed steps, they may both be subjected to metabolic control by feedback inhibition and/or by repression.

A Sensitive Radiochemical Assay Method for δ-Aminolevulinic Acid Synthetase

Abstract 1. A new sensitive method for measuring δ-aminolevulinic acid (ALA) synthetase activity has been developed based on the incorporation of 14C-succinate into ALA. The method is extremely sensitive and will permit assay of the enzyme in normal tissues and in small amounts of tissue for which the existing colorimetric methods are inadequate. Difficulties due to aminoacetone synthesis are eliminated. 2. The procedure has been applied to liver homogenates and mitochondrial suspensions, to reticulocytes, and to bacterial extracts. 3. It has been found that the normal level of ALA synthetase activity in guinea pig liver mitochondria is even less than hitherto believed from colorimetric assays and cannot with confidence be said to be significantly above zero. 4. An inhibitor of ALA synthetase activity has been shown to be present in the insoluble fraction of normal liver mitochondria.

The effect of diet in variegate (South African genetic) porphyria

Studies of the effect of diet on porphyrin and porphyrin precursor excretion in two patients with variegate (mixed or South African genetic) porphyria demonstrate that these are inversely related to the intake of carbohydrate and/or protein in the diet. During the course of severe dietary restriction, symptoms developed, suggesting an acute attack of the disease in both patients. In one patient, this was accompanied by the syndrome of “inappropriate release of antidiuretic hormone” into the circulation. Symptoms and biochemical findings rapidly reversed with intravenous glucose administration. These data imply that hepatic δ-aminolevulinic acid synthetase is induced in variegate porphyria and is subject to what has previously been called the “glucose effect.”

Enzyme and nucleic acid formation during synchronous growth of Rhodopseudomonas spheroides.

The synthesis of various cell components was examined during the anaerobic photosynthetic growth of synchronous populations of Rhodopseudomonas spheroides. Net deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein increased continuously as did the rate of incorporation of radioactive precursors into protein. The rates of incorporation of radioactive precursors into RNA and DNA were marked by abrupt discontinuities. It is not clear whether these discontinuities represent changes in rates of synthesis or fluctuations in precursor pools. Although the synthesis of bacteriochlorophyll occurred in a continuous manner, those enzymes examined which are involved in the synthesis of tetrapyrroles, i.e., succinyl CoA thiokinase, delta-aminolevulinic acid synthetase, and delta-aminolevulinic acid dehydrase, increased discontinuously. Two other enzymes not involved in tetrapyrrole biosynthesis were examined. Alkaline phosphatase increased in a stepwise manner during the division cycle, whereas the synthesis of ornithine transcarbamylase increased rapidly before leveling off for a period of time until synthesis began again. In each instance of discontinuous enzyme synthesis, increases occurred at regular and characteristic times during the division cycle. Ammonium sulfate precipitation was employed to remove low molecular weight end product inhibitors from enzyme preparations. These studies suggested that the stepwise increases in enzyme activity observed in the present investigation were not affected by periodic end product inhibition. A temporal map of enzyme synthesis during the division cycle was constructed. Both delta-aminolevulinic acid synthetase and delta-aminolevulinic acid dehydrase appeared early in the division cycle, whereas alkaline phosphatase and succinyl CoA thiokinase appeared later on.

Steroid Induction of Porphyrin Synthesis in Liver Cell Culture: II. THE EFFECTS OF HEME, URIDINE DIPHOSPHATE GLUCURONIC ACID, AND INHIBITORS OF NUCLEIC ACID AND PROTEIN SYNTHESIS ON THE INDUCTION PROCESS

1. Steroid induction of porphyrin synthesis in liver cell culture is, in its time of onset and course of development, in all respects similar to the induction process evoked by certain foreign chemicals and drugs. Steroids and foreign chemicals have additive-inducing effects when added to cultures in suboptimal amounts; however, when added in individually optimal amounts, the intensity of induction is not enhanced nor is its latent period shortened. These results suggest that natural steroids and porphyria-inducing drugs act at the same intracellular site or sites. 2. Actinomycin D, mitomycin C, puromycin, acetoxycyclo-heximide, and other agents which inhibit nucleic acid and protein synthesis block the porphyrin-inducing effect of natural steroids. These results are consistent with the view that steroids, like porphyria-inducing drugs, stimulate porphyrinogenesis by inducing the formation de novo of δ-aminolevulinic acid synthetase, the rate-limiting enzyme in heme biosynthesis. 3. Certain metalloporphyrins, including iron-protoporphyrin orч eme, inhibit the steroid-induction process. The ability of heme to block steroid induction supports the idea that heme and natural steroids may compete for a binding site on a hypothetical repressor protein which ultimately regulates the activity of the structural gene which codes for δ-aminolevulinic acid synthetase. 4. Uridine diphosphate glucuronic acid also blocks porphyrin induction by steroids. The blocking effect of UDP-glucuronic acid is considered to reflect the enhanced conversion of free steroid inducers to the glucuronidated or inactive derivatives. It is postulated that the ability of large amounts of glucose to inhibit the porphyric process in vivo reflects, in part or in whole, conversion of this sugar to UDP-glucuronic acid, resulting in enhanced glucuronidation and inactivation of inducing substances. 5. The clinical implications of this steroid action on the liver are discussed, and a working hypothesis on the mechanism of steroid control of porphyrin and heme synthesis is proposed. It is considered likely that endogenously derived inducing steroids are the physiological agents which account for the periodic, spontaneous, i.e. not drug related, exacerbations of hepatic porphyria in certain patients carrying the genetic lesion of this disease.

The origin of the increased protoporphyrin in erythrocytes of mice with experimentally induced porphyria.

Abstract Although the administration of 3,5-dicarbethoxy-1, 4-dihydrocollidine (DDC) and griseofulvin increased the circulating erythrocyte and plasma protoporphyrin concentration in mice, activities of δ-aminolevulinic acid (ALA) synthetase, heme synthetase in the spleen, and formation of porphyrins from ALA in erythrocytes were unchanged. After an intraperitoneal injection of protoporphyrin, protoporphyrin concentration in the circulating erythrocytes increased. The overproduction of protoporphyrin in the liver induced by the porphyria-inducing chemicals caused a rise in the concentration of plasma protoporphyrin, which was apparently taken up by the red blood cells of mice, resulting in an increased erythrocyte protoporphyrin concentration.

Steroid Induction of Porphyrin Synthesis in Liver Cell Culture: I. STRUCTURAL BASIS AND POSSIBLE PHYSIOLOGICAL ROLE IN THE CONTROL OF HEME FORMATION

Abstract 1. A number of steroid metabolites strongly stimulate porphyrin biosynthesis in chick embryo liver cells growing in primary culture. These metabolites are of the 5β-H type; they are derived from the biotransformation of C 19 and C 21 hormones, such as testosterone and progesterone, and they are active in inducing porphyrinogenesis in concentrations at least as low as 10-6 to 10-8 m. Typical inducing steroids are etiocholanolone, pregnandiol, 11-ketopregnanolone, and pregnantriol. Their activities in liver cell culture equal or exceed those of the most potent porphyria-inducing drugs and chemicals previously described. 2. Cholesterol, the major bile acids, and C 21-hydroxylated steroids such as cortisol and aldosterone are inactive in induction; other primary hormones and intermediates have weak activity, as do metabolites of the 5α-H type. 3. The glucuronide derivatives of even potent inducers are devoid of activity. Steroid induction of porphyrin synthesis in vivo, therefore, may be expected to depend, among other factors, on the rate at which the free inducing steroid is glucuronidated and the rate at which the steroid glucuronide is hydrolyzed. 4. These porphyrinogenic steroids are the first recognized general class of physiological agents which have potent porphyrin-inducing action in liver cells. Because they also stimulate heme synthesis in chick blastoderm erythroid cells, it is suggested as a working hypothesis that they may normally play a role in the control of heme biosynthesis in all cells through regulation of the formation of δ-aminolevulinic acid synthetase, the initial and rate-limiting enzyme in the heme biosynthetic pathway. 5. These porphyrinogenic steroids may also be involved in the pathogenesis of hepatic porphyria in man, particularly at times when uridine diphosphoglucuronic acid formation or uridine diphosphoglucuronic acid-transferase activity for steroids becomes inhibited or limiting.

Porphyrin synthesis in hepatic cirrhosis: Increase in δ-aminolevulinic acid synthetase

The activity of the heme biosynthetic chain is limited by δ-aminolevulinic acid synthetase, the first enzyme in this metabolic pathway. Elevated levels of this enzyme have been described in various congenital and acquired porphyrias. Since porphyrinuria and occasionally clinical porphyria occur in hepatic cirrhosis, the levels of δ-aminolevulinic acid synthetase were measured in patients with cirrhosis of the liver. Five of seven patients with hepatic cirrhosis demonstrated five to fifteenfold elevations of hepatic δ-aminolevulinic acid synthetase. Minimal elevations of this enzyme were found in one of the two remaining patients. It is unlikely that these cirrhotic patients had congenital hepatic porphyria as the basis for the elevated enzyme levels. Therefore, while hepatic injury may lead to an exacerbation of congenital porphyria, it seems that hepatic dysfunction alone may cause the development of abnormalities in porphyrin metabolism, which are indistinguishable from the inherited disease. Elevations of δ-aminolevulinic acid synthetase may be secondary to the inability of the diseased liver to metabolize certain endogenous substances such as sex steroids which are capable of inducing this enzyme.

Concurrent decrease of enzymic activities concerned with the synthesis of coenzyme B 12 and of propionic acid in Propionibacteria

Cells of Propionibacterium shermanii grown aerobically produce far less of the corrin ring of vitamin B 12 than those cells grown anaerobically. The decreased amount of the corrin ring is in part explained by the diminished activities of enzymes concerned with the synthesis of the porphyrin-like ring of B 12. It has been found that the activity of δ-aminolevulinic acid synthetase in extracts of aerobically grown cells is about 60% of that found in extracts of anaerobically grown cells. Furthermore, the δ-aminolevulinic acid dehydrase activity in aerobic extracts is either not demonstrable, or only about 10% of that in extracts of anaerobically grown cells. The diminished concentration of coenzyme B 12 in aerobically grown cells is reflected in the fatty acid composition of the fermentation products. Propionic acid, the predominant fatty acid among the products of the anaerobic fermentation, occurs in much smaller quantities as a product of aerobic fermentation. This is consistent with the well-known dependence of the methylmalonyl CoA-succinyl CoA isomerase reaction on coenzyme B 12. The addition of coenzyme B 12 to extracts of aerobically grown cells restores the propionate-succinate conversion to only about 30–50% of that found in extracts of anaerobically grown cells. It appears, therefore, that along with the decreased activities of enzymes concerned with B 12 synthesis, there is a concurrent decreased activity of an enzyme system which depends in part on coenzyme B 12. Since the cellular biotin concentration appears to be dependent on the amount of the biotin-dependent enzymes, the finding that extracts of aerobically grown cells contain less biotin than those of cells grown anerobically may be the result of the decreased amount of the functional biotin dependent enzyme.