Incorporation of cytosol δ-aminolevulinate synthase of rat liver into the mitochondria in vitro

When ¹²⁵I-labeled cytosol δ-aminolevulinate synthase was incubated in suspensions of rat liver mitochondria, the enzyme was incorporated into the mitochondira at the rate that was linear with time and with the [¹²⁵I]enzyme added. Subfractionation of the mitochondria using a digitonin technique revealed that the [¹²⁵I]enzyme was incorporated into the innermembrane-matrix fraction where endogeneous δ-aminolevulinate synthase is located.     

Effect of an exogenous succinyl-CoA-generating system on the measurement of δ-aminolevulinic acid synthase activity in rat liver tissue by a radiochemical assay

Rat liver tissue was used to examine the effect of an exogenous succinyl-CoA-generating system on the radiochemical assay for δ-aminolevulinic acid synthase (succinyl-CoA:glycine C-succinyltransferase (decarboxylating), EC 2.3.1.37) activity developed by Ebert et al. (Ebert, P.S., Tschudy, D.P., Choudry, J.N. and Chirigos, M.A. (1970) Biochim. Biophys. Acta 208, 236–250). In the absence of exogenous succinate thiokinase, 34–62% (average 55%) of the radioactivity in the final column eluate could be attributed to δ-amino-[4-¹⁴C]levulinic acid, as assessed by conversion of δ-aminolevulinic acid in the eluate to a pyrrole. The addition of succinate thiokinase markedly enhanced the formation of the contaminant(s), as succinyl-CoA was metabolized to a compound or compounds that eluted chromatographically with δ-amino-levulinic acid. This effect was abolished by 10 mM EDTA, probably because the generation of succinyl-CoA was suppressed due to the chelation of Mg²⁺. These observations indicate that this radiochemical assay should be carefully examined for each set of assay conditions employed.     

Cytochrome P-450 heme and the regulation of δ-aminolevulinic acid synthetase in the liver

Hepatic δ-aminolevulinic acid synthetase was induced in rats injected with allylisopropylacetamide. The induction process was studied in relation to experimental perturbation of cytochrome P-450 in the liver. Animals were treated with either administered endotoxin or exogenous heme, both of which accelerate degradation of cytochrome P-450 heme. These manipulations were effective in blocking induction of δ-aminolevulinic acid synthetase, and the effect of each compound was proportional to its ability to stimulate degradation of cytochrome P-450 heme. The findings suggest that the heme moiety of cytochrome P-450 dissociates reversibly from its apoprotein and, prior to its degradation, mixes with endogenously synthesized heme to form a pool that regulates δ-aminolevulinic acid synthetase activity. A similar or identical heme fraction appears to mediate stimulation of heme oxygenase, which suggests that the regulation of δ-aminolevulinic acid synthetase and of heme oxygenase in the liver are closely interrelated.     

Studies on the binding of d-α-tocopherol to rat liver nuclei

The present study describes the nature and characteristics of the intranuclear binding sites of [³H]d-α-tocopherol in rat liver. When radioactively labeled d-α-tocopherol was intravenously administered to rats, approximately 55% of the nuclear radioactivity was associated with an intranuclear nucleoprotein complex. This complex, which was extractable by high concentrations of NaCl, was characterized by equilibrium density ultracentrifugation on a 30 to 60% linear sucrose gradient. About 50% of the high-salt-extracted radioactivity was coprecipitable with macromolecules by 10% ice-cold trichloroacetic acid (TCA). This TCA-precipitable radioactivity was completely ethanol soluble. Alkaline conditions favored the solubilization of the vitamin-receptor complex. Among various enzymes tested, only Pronase and trypsin were capable of dissociating the vitamin-receptor complex. Both ionic (sodium dodecyl sulfate) and nonionic (Triton X-100) detergents solubilized α-tocopherol from the nuclei and concomitantly released some of the associated macromolecules. In addition, treatment of nuclei with low concentrations of Triton X-100 showed that about 30% of the nuclear bound α-tocopherol is associated with inner core sites in the nucleoprotein complex with very high affinity for the vitamin. Dissociation of the nucleoprotein complex (chromatin) by high-salt solubilization and subsequent partial reassociation of the components by salting out procedures revealed the high affinity association of α-tocopherol with the reconstituted DNA-protein complex. Subfractionation of this complex further revealed that α-tocopherol is predominantly associated with the fraction containing phenol-soluble nonhistone proteins having a high affinity for DNA. In vitro binding studies also showed that there are specific saturable binding sites for d-α-tocopherol in rat liver nuclei.     

Biosynthesis of δ-aminolevulinic acid in Rhodopseudomonas sphaeroides

The biosynthesis of δ-aminolevulinic acid was investigated in three strains of Rhodopseudomonas sphaeroides. A wild-type strain (NCIB 8253) possessed both δ-aminolevulinic acid synthetase and γ,δ-dioxovaleric acid transaminase in the cytoplasmic and membrane cell fractions. δ-Aminolevulinic acid synthetase activities were not detected in extracts of mutant strains H5 and H5D. However, γ,δ-dioxovaleric acid transaminase was found in the cytoplasmic and membrane fractions of these latter two strains. Strain H5 required exogenously added δ-aminolevulinic acid for growth and bacteriochlorophyll synthesis. Strain H5D did not require this compound for growth and bacteriochlorophyll synthesis. γ,δ-Dioxovaleric acid added in the growth medium did not support the growth of H5, although it was actively transported into the cells. Addition of γ,δ-dioxovaleric acid to the growth medium did not enhance the growth of either the wild-type or H5D strains. These results indicate that ALA synthetase is not required for growth and bacteriochlorophyll synthesis in H5D and that γ,δ-dioxovaleric acid is probably not an intermediate in the formation of δ-aminolevulinic acid in the strains of Rhodopseudomonas sphaeroides studied. In strain H5D another pathway may function in the formation of δ-aminolevulinic acid other than that catalyzed by δ-aminolevulinic acid synthetase or γ,δ-dioxovaleric acid transaminase.     

Detection and regulation of δ-aminolevulinic acid synthetase activity in rat skeletal muscle

The presence of δ-aminolevulinic acid synthetase (ALAS) in mitochondria obtained from rat skeletal muscles has been observed. Optimal conditions for the meausurement of this activity are described. The activity of skeletal muscle ALAS was investigated under conditions known to affect the activity of this enzyme in other tissues. ALAS activity in skeletal muscle mitochondria was decreased 55% by a 48-h fast. Treatment with dexamethasone did not reverse the effect of starvation on ALAS activity and did not change the activity in the fed controls. ALAS activity was decreased 56% in skeletal muscle mitochondria obtained from rats in which diabetes mellitus had been induced by streptozotocin. Administration of insulin to the diabetic animals partially reversed the effect of diabetes on skeletal muscle ALAS; however, administration of insulin to control animals caused a 21% decrease in skeletal muscle ALAS activity. By contrast, treatment with inducers of hepatic ALAS such as allylisopropylacetamide or 3,5-dicarbethoxy-1,4-dihydrocollidine had no effect on skeletal muscle ALAS. These results confirm our previous suggestion that ALAS activity is regulated in a tissue-specific manner.     

Studies on the mechanism of estrogen biosynthesis in the rat ovary–I

Methods were evaluated for obtaining a reliable, active estrogen synthetase (aromatase) system from the rat ovary for mechanistic studies. Short terrn treatment with luteinizing hormone and follicle stimulating hormone in various combinations did not produce appreciable stimulation, whereas long term treatment (8–16 days) with pregnant mare's serum gonadotropin increased activity in homogenates up to nine fold per mg wet wt of tissue. A similar increase per mg protein was noted in the 105,000_g microsomal fraction where the bulk of the activity was found. Various conditions for preparing and incubating the aromatase were evaluated to obtain optimal enzyme activity. The potencies of six steroids as aromatase inhibitors were compared in the rat ovarian and human placental microsomal systems. In all cases except one the results were comparable.     

Influence of gabaculine on growth,chlorophyll synthesis,and δ-aminolevulinic acid synthase activity in Euglena gracilis

Euglena gracilis is capable of forming the heme and chlorophyll precursor δ-aminolevulinic acid (ALA) by two routes: from glutamate via the five-carbon path in the chloroplasts, and by ALA synthase-mediated condensation of succinyl-CoA and glycine, probably in the mitochondrion. 5-Amino-1,3-cyclohexadienyl carboxylic acid (gabaculine), a powerful inhibitor of ALA formation via the five-carbon path, was administered to E. gracilis Klebs strain Z Pringsheim cells growing in the light or dark, and its effects on growth, chlorophyll accumulation and extractable ALA synthase activity were measured. Gabaculine had no effect in vitro on ALA synthase or ALA dehydratase, even at 100 μM. Administration of 100 μM gabaculine to wild-type cells growing in the light slowed growth, inhibited chlorophyll accumulation, and induced an increase in extractable ALA synthase activity. Chlorophyll accumulation in the light was abolished by prior administration of the compound to growing cells for 6 h in the dark, whereas chlorophyll accumulation in cells without gabaculine began immediately after transfer to light. Extractable ALA synthase activity from gabaculine-pretreated dark-grown cells was initially lower than the activity from untreated cells, but it did not undergo a further decline after transfer of the cells to the light, whereas the activity from untreated cells dropped to less than one eighth the dark level after 2 h in the light, and by 4 h had fallen to a level five times lower than that extractable from gabaculine-treated cells. These results suggest that suppression of ALA synthase activity by light in untreated cells is related to light-induced activation of the five-carbon ALA biosynthetic pathway in the plastids, and may result from a contribution by a product of the five-carbon pathway to non-plastid tetrapyrrole pools in the light.     

The biosynthesis of δ-aminolevulinic acid in greening maize leaves

In greening maize leaves δ-aminolevulinic acid (ALA) was not formed from succinyl-CoA and glycine as shown by the incorporation of [¹⁴C]-labeled     

Lipogenesis in the brain of suckling rats. Studies on the mechanism of mitochondrial–cytosolic carbon transfer

1. Studies on the incorporation of [3-¹⁴C]pyruvate and d-3-hydroxy[3-¹⁴C]butyrate into the brain lipid fraction by brain homogenates of the suckling (7-day-old) rat have been carried out. 2. Whereas approximately twice as much CO₂ was evolved from pyruvate compared with 3-hydroxybutyrate metabolism, similar amounts of the radioactivity of these two precursors were incorporated into the lipid fraction. Furthermore, in both cases the incorporation into lipid was almost tripled when glucose (10mm) or NADPH (2.5mm) was added to the incubation media. 3. If 5mm-(—)-hydroxycitrate, an ATP–citrate lyase inhibitor, was added to the incubation the incorporation of carbon from pyruvate was inhibited to 39% of the control and from 3-hydroxybutyrate to 73% of the control, whereas CO₂ production from both precursors was not affected. 4. The incorporation from pyruvate or 3-hydroxybutyrate into lipids was not affected by the presence of 10mm-glutamate in the medium (to encourage N-acetylaspartate production). However, incorporation from pyruvate was inhibited by 21% in the presence of 5mm-amino-oxyacetate (a transaminase inhibitor) and by 83% in the presence of both hydroxycitrate (5mm) and amino-oxyacetate. 5. Incorporation from 3-hydroxybutyrate into brain lipids was inhibited by 20% by amino-oxyacetate alone, but by 55% in the presence of both hydroxycitrate and amino-oxyacetate. 6. It is concluded that the mechanism of carbon transfer from pyruvate into lipids across the mitochondrial membrane in the suckling rat brain is mainly via citrate and N-acetylaspartate. 3-Hydroxybutyrate, in addition to using these routes, may also be incorporated via acetoacetate formation and transport to the cytosol.     

Studies on the oxidation of ω-hydroxyprostaglandins by an NAD-dependent dehydrogenase from mammalian liver cytosol

The oxidation of 12-hydroxylauric acid methyl ester (12-OH-L-Me) and of ω-hydroxy-prostaglandins (ω-OH-PGs) such as 20-OH-PGB₁ and 20-OH-PGE₁, was demonstrated with liver cytosol from rat, rabbit, and guinea pig in the presence of NAD; however, NADP did not support this oxidation. (ω-1)-Hydroxy-compounds (11-OH-laurate and 19-OH-PGB₁) and PGE₁, PGF_1α, and PGB₁, all lacking the terminal (ω)-hydroxyl, did not reduce NAD. However, at pH 10, PGE₁ slightly enhanced NAD reduction, suggesting that at this pH PGE₁, could be a substrate for 15-hydroxy-PG dehydrogenase (PGDH). The oxidation products from incubations of 12-OH-L-Me, 20-OH-PGB₁-Me, and 20-OH-PGE₁ with guinea pig liver cytosol were isolated and identified by gas chromatography/mass fragmentation spectrometry as being the corresponding dicarboxylic acids. In contrast to the liver cytosol, guinea pig kidney cytosol had only a minimal effect on NAD reduction by 12-OH-L-Me but nevertheless did support the stimulation of NAD reduction by PGE₁, and PGF_1α, but not by PGB₁, indicating the participation of kidney cytosolic PGDH in PGE₁ and PGF_1α oxidation and demonstrating that the oxidation of ω-OH to the carboxylic acid is not mediated by PGDH. Though the in vivo rate of oxidation of ω-OH-PGs has not been established, these results suggest that the urinary dicarboxylic-PG metabolites involve a multiple sequentialstep oxidation of PGs involving ω-hydroxylation by an NADPH-cytochrome P-450 system in the endoplasmic reticulum and the subsequent oxidation of the ω-OH by an NAD-dependent dehydrogenase in the cytosol.     

Inhibition of δ-aminolevulinic acid synthetase and δ-aminolevulinic acid dehydrase by L-2-amino-4-methoxy-trans-3-butenoic acid in the rat

The rate limiting enzyme of heme biosynthesis, δ-aminolevulinic acid synthetase (ALA synthetase), and the second enzyme in the heme biosynthetic pathway, δ-aminolevulinic acid dehydrase (ALA dehydrase), were inhibited by the olefinic amino acid L-2-amino-4-methoxy - $\text{trans}$-3-butenoic acid (AMTB). Administration of AMTB (20 mg/kg; i.p.) to rats inhibited ALA synthetase and ALA dehydrase in control animals and in animals with markedly elevated activity of ALA synthetase which resulted from the administration of 3,5-dicarbethoxy-1,4-dimethyl-collidine (DDC, 200 mg/kg, i.p.) or allylisopropylacetamide (200 mg/kg, s.c.). AMTB also blocked the synthesis of rat hepatic porphyrins and inhibited the increase in the urinary excretion of δ-aminolevulinic acid and porphobilinogen following DDC (150 mg/kg, p.o.) administration. Preincubation of AMTB with liver mitochondria or a soluble fraction of liver decreased the activity of mitochondrial ALA synthetase and soluble ALA dehydrase, respectively.     

Assay of δ-aminolevulinic acid synthetase in homogenates of mouse,rat, and human liver: Species differences in requirement for an exogenous succinyl-CoA-generating system

Conditions required for optimal assay of low levels of activity of hepatic δ-aminolevulinic acid synthetase have been studied, comparing dilute homogenates of mouse, rat, and human livers. The assay method used was a modification of that described by Ebert et al. (Biochim. Biophys. Acta (1970)208, 236–250), and livers were studied from both untreated animal and human subjects and subjects pretreated with porphyrinogenic compounds. In homogenates of mouse and human but not rat liver, maximal rates of δ-aminolevulinic acid formation required addition to the incubation mixture of an exogenous system for succinyl-CoA generation. The requirement for this generating system was increased if livers from pretreated subjects were frozen and stored prior to assay, suggesting that the endogenous capacity for succinyl-CoA generation was more labile than δ-aminolevulinic acid synthetase under these conditions. Of the metabolic inhibitors tested (F^?, malonate, and arsenite), only F^? (100 mm final concentration) enhanced activity. Increasing the permeability of mitochondria by quick freezethawing of fresh homogenates just before assay did not increase the rate of δ-aminolevulinic acid formation.     

Studies on liver mitochondrial 5′-endonuclease activity in rats fed carcinogenic amines and on their binding to mitochondrial proteins

Mitochondrial 5′-endonuclease activity has been determined at regular time intervals in the livers of rats fed a diet containing 0.09% 2-aminofluorene (AF), 0.09% 2-acetylaminofluorene (AAF) or 0.06% N,N-dimethyl-4-aminoazobenzene (DAB). The results obtained indicate that the 5'-endonuclease activity was not affected significantly.The quantity of AF, AAF or DAB bound to liver homogenate and mitochondrial fraction proteins has also been measured at regular time intervals. The amount of AF and AAF bound to homogenate proteins after 4 weeks of carcinogen feeding is about 60-fold higher than that of DAB. The binding of the AF compounds to the mitochondrial fraction proteins is comparatively more important, reaching a level 300-fold higher than that of DAB. The amount of AF residues bound per mg of mitochondrial fraction proteins is higher than that of the homogenate while that of rats fed DAB is smaller. The present results suggest that no relation can be established between the total amount of these carcinogens bound to liver cellular proteins in vivo and their potential carcinogenic effect.