Effect of endogenous heme generation on delta-aminolevulinic acid synthase activity in rat liver mitochondria.

This study examined the possibility that generation of heme within mitochondria may provide a local concentration sufficient to inhibit the activity of delta-aminolevulinic acid (ALA) synthase, the enzyme that catalyzes the rate-limiting step in hepatic heme biosynthesis. This was accomplished by simultaneously running ALA synthase and heme synthase activities in intact mitochondria isolated from rat liver. Radiochemical assays were used to measure the enzyme activities. ALA synthase activity did not decrease as the rate of heme formation was increased by varying the concentration of substrates for heme synthase. Even at a rate of heme generation estimated to be at least 75 times the rate occuring in vivo, ALA synthase activity was unchanged. We conclude that end product inhibition of ALA synthase activity by heme is not an important physiological mechanism for regulation of hepatic heme biosynthesis.     

Effect of uremic plasma on mouse liver delta-aminolevulinic acid synthetase activity

delta-Aminolevulinic acid (delta-ALA) synthetase in mouse liver homogenate was significantly (p less than 0.001) higher in the presence of uremic compared with normal plasma, the ratio of the two values being 1.36 +/- 0.24 in 30 paired experiments. This effect does not seem to be due to increased concentrations of urea or creatinine nor to any possible dialyzable substances. Its relationship to the retention of an inducing factor or decreased production of erythropoietin in uremic patients is discussed. A possible inhibitory effect of erythropoietin on liver delta-ALA synthetase is suggested.     

delta-Aminolevulinic acid synthetase from rat liver mitochondria. Purification and properties.

delta-Aminolevulinic acid synthetase has been purified from liver mitochondria of young, uninduced rats. After nonionic detergent solubilization of mitochondrial inner membrane-matrix fractions, the enzyme was purified to a specific activity of approximately 2,000 nmol of delta-aminolevulinic acid formed/h/mg of protein at 30 degrees C, by means of ammonium sulfate precipitation, diethylaminoethyl cellulose chromatography, Sephacryl chromatography, and preparative gel electrophoresis. The purified enzyme preparation thus obtained was apparently homogeneous as judged by its migration as a single band with a molecular weight of 58,000 +/- 6,000 upon electrophoresis in sodium dodecyl sulfate polyacrylamide gels. The native enzyme probably exists as a dimer with a molecular weight of approximately 120,000. A pH optimum of 7.5 and an isoelectric point of 4.5 were also determined. Both monovalent cations and hemin strongly inhibited the activity of the purified enzyme.     

Studies on adrenal delta-aminolevulinic acid synthetase.

The presence of δ-aminolevulinic acid synthetase (EC 2.3.1.37) in rat and bovine adrenals has been demonstrated. When untreated animals are employed, the activity of δ-aminolevulinic acid synthetase in rat and bovine adrenal homogenates is comparable to the activity found in hepatic homogenates. Adrenal δ-aminolevulinic acid synthetase is localized in the mitochondrial fraction and appears to be refractory to induction by agents that induce the hepatic enzyme. Starvation of rats increased adrenal δ-aminolevulinic acid synthetase activity without altering the activity of the hepatic enzyme. Treatment of rats with adrenocorticotropin also dramatically increased adrenal δ-aminolevulinic acid synthetase activity. These results suggest that the adrenal enzyme may be controlled by factors that differ from those which regulate the activity of the hepatic enzyme.     

Effect of abnormal atmospheric pressure conditions upon mouse liver delta-aminolevulinic acid synthetase activity

Liver delta-aminolevulinic acid synthetase activity was measured in mice living under abnormal atmospheric pressure conditions for 15 h. In the group living under low atmospheric pressure (51 kPa) the enzymic activity, either basal or induced by starvation and/or allylisopropylacetamide, was significantly (p less than 0.001) lower than that of the control group. In the group living under high atmospheric pressure (153 kPa) the enzymic activity was significantly (p less than 0.001) higher than the one of the controls. Our results might possibly be explained by changes in the cellular redox state, the heme oxygenase activity or the serum erythropoietin levels.     

Distribution of delta-aminolevulinic acid synthetase and delta-aminolevulinic acid dehydratase in liver and kidney of rainbow trout (Salmo gairdnerii)

1. Activities of delta-aminolevulinic acid synthetase (ALA-S) and delta-aminolevulinic acid dehydratase (ALA-D) in trout liver and kidney were compared with those in the mouse. 2. ALA-S activity (per unit tissue fresh weight) exceeded ALA-D activity in trout liver and kidney. 3. In trout kidney, ALA-S activity slightly exceeded, and ALA-D activity far exceeded, their activities in trout liver. 4. In trout, heme synthesis differs from that in mammals in that appreciable synthesis occurs in the kidney, and in that ALA-S activity is not rate limiting.     

Studies on the synthesis of rat liver fatty acid synthetase.

The synthesis of the multienzyme complex rat liver fatty acid synthetase was investigated utilizing modifications of methods developed in the laboratory of Schimke (Schimke, R. T. (1964) J. Biol. Chem. 239, 3808-3817 and Arias, I. M., Doyle, D., and Schimke, R. T. (1969) J. Biol. Chem. 244, 3303-3315). The relative amounts of radioactivity from a pulse of labeled lysine appearing in polypeptides derived from purified synthetase complex can be measured compensating for the varying amounts of lysine per polypeptide chain. The results show that labeled amino acid is incorporated into polypeptides derived from the complex at heterogeneous rates. However, 10 to 15 hours after the administration of a pulse, the amount of label per lysine residue in these polypeptides is identical. The results support the previously proposed model of this multienzyme complex (Tweto, J., Dehlinger, P., and Larrabee, A. R. (1972) Biochem. Biophys. Res. Commun. 48, 1371-1377). The previous work and that reported here suggests the existence of a pool of synthetase subunits which is an obligatory intermediate in both synthesis and turnover of the complex. The results obtained in this work are consistent with this model if the exchange of subunits into the intact complex is a relatively slow process requiring several hours to reach equilibrium.     

The uptake of silicic acid by rat liver mitochondria.

1. To gain insight into a putative role for mitochondria in silicon metabolism, mitochondrial uptake (by which it is meant the removal from the medium) of silicic acid [Si(OH)4] was studied under conditions minimizing SI(OH)4 polymerization. 2. Measurements of mitochondrial respiration and swelling indicated indirectly a significant uptake of Si(OH)4 as a weak acid, but this was not confirmed when 31Si(OH)4 was used as a tracer. 31Si(OH)4 occupied a mitochondrial volume similar to that of 3H2O and was relatively unaffected by mitochondrial energy status and by the pH gradient across the mitochondrial inner membrane. 3. Uptake was directly proportional to Si(OH)4 concentration in the range 0-3 mM. 4. The uptake consisted of two components: under all conditions examined, the greater quantity, amounting to 1-2nmol of Si(OH)4/mg of mitochondrial protein, was bound, a major portion of it external to the inner membrane, with the lesser quantity free within the matrix space. 5. Equilibration of 31Si(OH)4 between medium and matrix was a slow process, having a half-time of approx. 10 min at 22 degrees C. 6. Mersalyl and N-ethylmaleimide inhibited the uptake by preferentially lowering the amount of Si(OH)4 bound. Their action was somewhat variable, depending on the precise nature of the suspending medium, and suggesting that the bound material may represent polymerized forms of Si(OH)4. 7. It is concluded that Si(OH)4 may penetrate the mitochondrial inner membrane by a simple diffusion mechanism.     

Cyclic oscillations in rat hepatic heme oxygenase and delta-aminolevulinic acid synthetase following intravenous heme administration.

Heme administration in vivo results in the suppression of synthesis of rat hepatic δ-aminolevulinic acid (ALA) synthetase and induction of rat hepatic heme oxygenase. Intravenous heme administration in vivo results in the appearance of cyclic progressively damped oscillations of both hepatic ALA synthetase activity and hepatic heme oxygenase activity. Heme oxygenase induction precedes in time the induction of ALA synthetase. ALA synthetase oscillations are observed in hepatic cell cytosol and mitochondrial fractions as well as in the total homogenate. Cycloheximide pretreatment abolishes both the ALA synthetase and heme oxygenase oscillations, while actinomycin D pretreatment has only a minimal effect on the induction of heme oxygenase. These results suggest that hepatic heme metabolism is closely regulated by rapid changes in the capacity to synthesize and catabolize heme, and the cyclic oscillations following intravenous heme may be a manifestation of the feedback regulation processes involved. This regulatory capacity is dependent on protein synthesis, and the primary site of regulation may be at the translational level on the endoplasmic reticulum.