Pyruvate uptake is inhibited by valproic acid and metabolites in mitochondrial membranes

The pyruvate uptake rate in inverted submitochondrial vesicles prepared from rat liver was optimized and further characterized; the potential inhibitory effects of the anticonvulsive drug valproic acid or 2-n-propyl-pentanoic acid (VPA), Delta4-valproic acid or 2-n-propyl-4-pentenoic acid and the respective coenzyme A (CoA) conjugates were studied in the presence of a proton gradient. All tested VPA metabolites inhibited the pyruvate uptake, but the CoA esters were stronger inhibitors (40% and 60% inhibition, respectively, for valproyl-CoA and Delta4-valproyl-CoA, at 1mM). At the same concentration, the specific inhibitor 2-cyano-4-hydroxycinnamate decreased the pyruvate uptake rate by 70%. The reported inhibition of the mitochondrial pyruvate uptake may explain the significant impairment of the pyruvate-driven oxidative phosphorylation induced by VPA.     

Similarities between rat liver mitochondrial and cytosolic glutathione reductases and their apoenzyme accumulation in riboflavin deficiency

Glutathione reductase has been purified 5,500-fold from rat liver mitochondrial matrix in a yield of 30%. The mitochondrial enzyme was immunochemically indistinguishable from that of the cytosol and the subunit molecular weight was apparently similar to that of the cytosolic enzyme, that is, 50,000 daltons. The optimum pH and kinetic properties investigated were not significantly different from those of the cytosolic enzyme. When rats were fed a riboflavin-deficient diet, the enzyme activity in the mitochondria decreased to a greater extent than that in the cytosol, and greater accumulation of apo-enzyme in the former than that in the latter was confirmed by the amount of immunoprecipitable protein, activation by FAD addition in vitro, and the enzyme activity recovery after injection of riboflavin, into riboflavin-deficient rats.     

The riddle of mitochondrial caspase-3 from liver

Caspase-3 is one of the main executors of apoptosis. Its zymogen procaspase-3 was localized to cytosol, mitochondria and nuclei. The subcellular location of procaspase-3 in liver was reported by several studies to be either cytosolic or cytosolic and mitochondrial. Our aim was to investigate these separate procaspase-3 pools to differentiate the pathways of their activation. By cell fractionation, immunocytochemistry, and confocal microscopy we report that there is a single procaspase-3 pool located to the cytosol in primary hepatocytes and in fractions of rat liver. In contrast, it depends on the isolation purity whether procaspase-3 is located in mitochondria of non-parenchymal liver cells, or not. All preparations with mitochondrial procaspase-3 fractions contain traces of haemoglobin, indicating the presence of some erythrocytes, which are the source of mitochondrial procaspase-3. Since erythrocytes migrate with mitochondria in subcellular fractionations, it is important to check for haemoglobin, before localizing the protein to mitochondria.     

Effect of clofibrate on lipid peroxidation in rats treated with aspirin and 4-pentenoic acid

The in vivo hepatic lipid peroxide content of rats was increased by aspirin or 4-pentenoic acid (4-PA) administration but was decreased by clofibrate (CPIB) administration. The increase by aspirin or 4-PA treatment was depressed by simultaneous administration of CPIB. However, the in vitro formation of lipid peroxide in liver mitochondria and microsomes of rats treated with CPIB as well as aspirin and 4-PA was also elevated compared to that of control rats. The formation of lipid peroxide in mitochondria and microsomes of control rats in vitro was depressed by the addition of cytosols obtained from untreated (control), aspirin-treated, 4-PA-treated, and CPIB-treated rats, but was not depressed by the addition of albumin or heated cytosols. The most effective depression was obtained by the addition of cytosol obtained from CPIB-treated rats. In addition, glutathione peroxidase activity and nonprotein sulfhydryl content in cytosol obtained from CPIB-treated rats were elevated compared to those from control, aspirin, and 4-PA-treated rats. The results suggest that the action of CPIB may be mainly related to the increase of cytosolic glutathione peroxidase activity and nonprotein sulfhydryl content. Hepatic triglyceride and phospholipid contents of rats treated with aspirin or 4-PA were increased compared to those of control rats. These increases were also reversed by simultaneous administration of CPIB.     

Influence of valproic acid on hepatic carbohydrate and lipid metabolism

Valproic acid (dipropylacetic acid), an antiepileptic agent known to be hepatotoxic in some patients, caused inhibition of lactate gluconeogenesis, fatty acid oxidation, and fatty acid synthesis by isolated hepatocytes. The latter process was the most sensitive to valproic acid, 50% inhibition occurring at ca. 125 microM with cells from meal-fed female rats. The medium-chain acyl-CoA ester fraction was increased whereas coenzyme A (CoA), acetyl-CoA, and the long chain acyl-CoA fractions were decreased by valproic acid. The increase in the medium chain acyl-CoA fraction was found by high-pressure liquid chromatography to be due to the accumulation of valproyl-CoA plus an apparent CoAester metabolite of valproyl-CoA. Salicylate inhibited valproyl-CoA formation and partially protected against valproic acid inhibition of hepatic metabolic processes. Octanoate had a similar protective effect, suggesting that activation of valproic acid in the mitosol is required for its inhibitory effects. It is proposed that either valproyl-CoA itself or the sequestration of CoA causes inhibition of metabolic processes. Valproyl-CoA formation also appears to explain valproic acid inhibition of gluconeogenesis by isolated kidney tubules. No evidence was found for the accumulation of valproyl-CoA in brain tissue, suggesting that the effects of valproic acid in the central nervous system are independent of the formation of this metabolite.     

High-performance liquid chromatographic analysis of the sulfation of 4-hydroxypropranolol enantiomers by monkey liver cytosol

We developed a new high-performance liquid chromatographic method using an ODS column and a chiral column for the assay of racemic 4-OH-PL sulfate and enantiomeric 4-OH-PL sulfates, respectively. The method was successfully applied to measure phenolsulfotransferase (PST) activities for 4-OH-PL in cytosolic fractions from livers of Japanese monkeys (Macaca fuscata) and for comparison with its activity of cytosolic fractions from rat, rabbit, dog, and human livers and Hep G2 cells. The activity was ranked as Hep G2 cells > monkeys = humans = dogs = rats > rabbits. To evaluate the Japanese monkey as a nonhuman animal model in drug metabolism studies, we further characterized sulfation of 4-OH-PL as a further metabolic pathway in monkey livers to compare that with human livers. Inhibition studies in which cytosolic fractions were preincubated at 43 degrees C or 2,6-dichloro-4-nitrophenol (DCNP) used as a PST inhibitor indicated that two kinds of PSTs, thermolabile, low-Km and DCNP-resistant PST and thermostable, high-Km and DCNP-sensitive PST were involved in 4-OH-PL sulfation by monkey liver cytosol, which is very similar to the reported profile of 4-OH-PL sulfation by human liver cytosol. Sulfation kinetics in a low concentration range of 4-OH-PL enantiomers demonstrated that apparent Km values were similar between human and monkey liver cytosolic fractions, but the Vmax values were different, so that intrinsic clearance values (Vmax/Km, Clint) were higher in monkeys than in humans. Furthermore, enantiomer selectivity of [R(+)-4-OH-PL > S(-)-4-OH-PL] was observed in the Vmax and CLint values of monkey liver cytosol. These results indicate that the profile of sulfation of 4-OH-PL by liver cytosolic fractions is similar in humans and Japanese monkeys.     

Effects of valproic acid on cardiac metabolism

We investigated whether the antiepileptic valproic acid (VPA) might interfere with oxidative metabolism in heart, as it does in liver. We administered VPA to working rat hearts perfused with radiolabeled carbohydrate and fatty acid fuels. Measurements included oxidation rates of (i) glucose, pyruvate, or lactate in the presence of palmitate and (ii) palmitate, octanoate, or butyrate in the presence of glucose. Oxidation rates were quantified as the rate of appearance of 14CO2 or 3H2O from 14C- or 3H-labeled substrates. In hearts perfused with palmitate, VPA (1 mmol/L) strongly inhibited the oxidation of pyruvate and lactate but slightly stimulated the oxidation of glucose. VPA also inhibited lactate or pyruvate uptake into erythrocytes in vitro. In hearts perfused with glucose, VPA strongly inhibited the oxidation of palmitate and octanoate but had no effect on butyrate oxidation. The absence of valproate CoA ligase activity in cell-free homogenates indicated that the inhibition of fatty acid oxidation by VPA did not require prior activation to valproyl-CoA. The results are consistent with the hypothesis that VPA selectively interferes with myocardial fuel oxidation by mechanisms that are independent of conversion to the CoA thioester.     

Cytosolic activation of 2-aminoanthracene: implications in its use as diagnostic mutagen in the Ames test.

The metabolic activation of 2-aminoanthracene to mutagens in the Ames test was investigated using hepatic S9, microsomal and cytosolic fractions from control and Aroclor 1254-treated rats as activation systems. Microsomal and S9 preparations from control animals could activate 2-aminoanthracene, but the efficiency of activation was suppressed by pretreatment of animals with Aroclor 1254. Cytosolic fractions from Aroclor 1254-treated rats could readily activate the promutagen more readily than microsomes. The cytosolic activation of 2-aminoanthracene required NADPH and could not be accounted for by possible microsomal contamination. The molybdenum oxygenases appear not to contribute to the cytosolic activation of this promutagen. It is concluded that (a) the microsomal activation of 2-aminoanthracene is catalysed more effectively by enzyme systems other than the P450 I family and (b) an enzyme system capable of activating this carcinogen in vitro is present in the hepatic cytosol. The implications of these findings in the use of 2-aminoanthracene as a positive control in the Ames test are discussed.     

The role of sterol carrier protein 2 in stimulation of steroidogenesis in rat adrenal mitochondria by adrenal cytosol

Cholesterol side-chain cleavage (CSCC) in isolated rat adrenal mitochondria is enhanced by prior corticotropin (ACTH) stimulation in vivo (8-fold). Part of this stimulation is retained in vitro by addition of cytosol from ACTH-stimulated adrenals to mitochondria from unstimulated rats (2.5- to 6-fold). In vivo cycloheximide (CX) treatment fully inhibits the in vivo response and resolves the in vitro cytosolic stimulation into components: (i) ACTH-sensitive, CX-sensitive; (ii) ACTH-sensitive, CX-insensitive; and (iii) ACTH-insensitive, CX-insensitive. These components contribute approximately equally to stimulation by ACTH cytosol. Components (i) and (iii) most probably correspond to previously identified cytosolic constituents steroidogenesis activator peptide and sterol carrier protein 2 (SCP2). SCP2, as assayed by radioimmunoassay or ability to stimulate 7-dehydrocholesterol reductase, was not elevated in adrenal cytosol or other subcellular fractions by ACTH treatment. Complete removal of SCP2 from cytosol by treatment with anti-SCP2 IgG decreased cytosolic stimulatory activity by an increment that was independent of ACTH or CX treatment. Addition of an amount of SCP2, equivalent to that present in cytosol, restored activity to SCP2-depleted cytosol but had no effect alone or when added with intact cytosol, suggesting the presence of a factor in cytosol that potentiates SCP2 action. Pure hepatic SCP2 stimulated CX mitochondrial CSCC 1.5- to 2-fold (EC50 0.7 microM) but was five times less potent than SCP2 in adrenal cytosol. Two pools of reactive cholesterol were distinguished in these preparations characterized, respectively, by succinate-supported activity and by additional isocitrate-supported activity. ACTH cytosol and SCP2 each stimulated cholesterol availability to a fraction of mitochondrial P450scc that was reduced by succinate but failed to stimulate availability to additional P450scc reduced only by isocitrate.     

Organ and intracellular localization of short-chain acyl-CoA synthetases in rat and guinea-pig.

1. Homogenates of rat epididymal fat pad, heart, kidney, lactating mammary gland, liver, skeletal muscle and small intestinal mucosa have been partitioned into a particulate and supernatant fraction. With reliable marker enzymes for the mitochondrial matrix and the cytosol: propionyl-CoA carboxylase and pyruvate kinase, the distributions of the acyl-CoA synthetase activities measured at 1 and 10 mM C2, C3 and C4 over mitochondria and cytosol have been calculated. From these values an estimate was made of the K0.5 of the fatty acids. 2. A distinct fatty acid-activating enzyme was assumed to be present in one of the compartments when that fatty acid was activated with a K0.5 less than or equal to 1.5 mM in an amount of greater than 13% of the total cellular activity. Adipose tissue, gut, liver and mammary gland, all organs of a high lipogenetic capacity, contained a cytosolic acetyl-CoA synthetase. At 1 mM acetate 60, 31, 77 and 83% of the total cellular activities in these organs were cytosolic in nature, with activities of 0.021, 0.32, 0.37 and 1.16 mumol C2 activated per min per g wet weight, respectively. 3. Mitochondrial acetyl-CoA and butyryl-CoA synthetases were found in adipose tissue, gut, heart, kidney, mammary gland and muscle. They were absent in liver. Adipose tissue and liver contained a mitochondrial propionyl-CoA synthetase with activities at 1 mM C3 of 0.014 and 1.50 mumol C3 activated per min per g wet weight, respectively. 4. At 1 mM, C2 was activated with decreasing rates by kidney, heart, mammary gland and gut (7.6-1.0 mumol C2 activated per min per g wet weight). C3 (1 mM) activation was about equal (1.6-1.9 mumol C3 activated per min per g wet weight) in liver, kidney and heart. C4 (1 mM) was activated with decreasing rates by heart, liver, kidney and gut (4.0-0.5 mumol C4 activated per min per g wet weight) in the order given. 5. The influence of the isolation method and the diet on fatty acid activation in small intestinal mucosal scrapings have been studied. To demonstrate the existence of cytosolic acetyl-CoA synthetase in fed animals a pre-treatment of everted intestine by low amplitude vibration has been found essential. Also C16 activation was highly (95%) decreased in a non-pre-vibrated preparation. 24 h starvation lowered cytosolic C2 and total C16 activation by 90 and 80%, respectively. Refeeding of starved rats with a balanced fat-free diet, and not with sucrose only, gave the same cytosolic C2 and total C16 activation as normally fed rats. 6. In guienea-pig heart, kidney, liver and muscle about the same partitions have been found as in the respective rat organs. The acetate activation in liver was factor 6 lower. Acetate and butyrate activation in guinea-pig muscle was much higher (6 and 37 times, respectively).     

The binding of gold(I) to metallothionein

The binding of gold(I) to metallothionein, MT, has been unambiguously established by the reaction of Na₂AuTM with purified horse kidney MT. Zinc was displaced more readily than cadmium although the latter could be displaced using large Au/Cd ratios. The metal exchange reactions were complete within 2 hr of mixing. Further evidence that such reactions might be physiologically significant were obtained by studying in vitro metal displacements in the liver cytosol of in vivo metal treated rats: When Na₂AuTM was added to the cytosol of rats administered CdCl₂ in vivo, zinc, copper and cadmium were displaced in 2/1/1 ratios from the metallothionein fraction. The zinc and cadmium displacement provide direct evidence that the gold was binding to MT. Addition of Cd⁺² to liver cytosol of gold-treated rats resulted in displacement of copper and zinc, but not gold, from the MT fractions. When liver MT is prepared from rats exposed to Au or Cd, the Cd/protein ratio increased during the preparation, but the Au/protein ratio decreased. The Mt-bound metals account for 95% of the cytosolic Cd but only 15%–30% of the cytosolic gold in these studies. Thus, the nonspecific binding of gold to MT in vivo should be considered as one aspect in its equilibration among protein binding sites, which include, inter alia, metallothionein. Gold was found to coelute with zinc and cadmium in the MT fraction of rat kidney cytosol, when both Cd and Na₂AuTM were administered to the rats. The possible significance of gold binding to MT in the treatment of rheumatoid arthritis-chrysotherapy-is briefly discussed.     

Extraction of corticosterone from cell homogenates and subcellular fractions of the rat adrenal cortex. III. ACTH-induced temporal subcellular redistributions of steroid precursors to corticosterone

Cholesterol, pregnenolone, progesterone, 11-deoxycorticosterone (11-DOC) and corticosterone were quantitated in subcellular fractions isolated from in vivo adrenocorticotropin (ACTH)-stimulated rat adrenal zona fasciculata/reticularis. Six adrenal subcellular fractions separated by discontinuous sucrose gradient centrifugation (lipid, 0.125 M sucrose, cytosolic, microsomal, mitochondrial and nuclear) were extracted with alkaline ether/ethanol and assayed by high pressure liquid chromatography (HPLC). Lipid fractions contained the major cholesterol stores, while most pregnenolone and progesterone was found in lipid, microsomal and mitochondrial fractions. The 0.125 M sucrose and cytosol fractions together contained approximately 75% of the total 11-DOC and corticosterone. The five steroids were only present in small amounts in organelle fractions containing steroidogenic enzymes. Homogenate and lipid fraction cholesterol decreased between 10 and 15 min and again 30 min after ACTH injection. In the homogenate, lipid, microsomal and mitochondrial fractions, pregnenolone and progesterone were increased after ACTH injection; peak pregnenolone and progesterone concentrations were often measured in adrenal gland sucrose, cytosolic, microsomal and mitochondrial fractions 15 to 20 min after rats were injected with ACTH. Although ACTH increased 11-DOC and corticosterone in all but the mitochondrial and nuclear fractions, the sucrose, cytosolic and microsomal 11-DOC, and cytosolic corticosterone increased most dramatically. In many fractions, peak 11-DOC and corticosterone concentrations were most often observed between the 10 and 15 min periods and again at 30 min.     

Tissue distribution of avian pancreatic polypeptide-degrading activity

Degradation of avian pancreatic polypeptide (APP) by subcellular fractions from homogenates of chicken kidney, liver, and brain was characterized in this study. Chicken kidney cytosol exhibited the highest degrading activity of all kidney subcellular fractions studied including nuclear, mitochondrial, and microsomal. The cytosolic kidney APP-degrading activity was inhibited in a dose-dependent manner by bacitracin, serine protease inhibitors, and dithiothreitol, and eluted in the void volume of a Sephadex G-100 column, indicating that it is a soluble, serine protease-like activity with a Mr greater than 100,000 kDa and with some dependence on disulfide bonds. Soluble cytosol fractions from chicken liver, kidney, and brain all exhibited greater APP-degrading activity than that of corresponding membrane fractions and, furthermore, were similar in activity between one another. It is concluded that APP degradation by tissue homogenates occurs via a soluble, cytosolic protease which is inhibited by selected serine protease inhibitors; the activity does not differ among liver, kidney, and brain, three tissues which show different receptivity for APP.     

Subcellular distribution of tissue radiocopper following intravenous administration of 67Cu-labeled Cu-PTSM

The subcellular distribution of radiocopper in the brain and liver of rats has been determined following i.v. administration of Cu-PTSM, pyruvaldehyde bis(N⁴-methylthiosemicarbazonato)copper(II), labeled with copper-67. Homogenized tissue samples were separated by differential centrifugation into four subcellular fractions: (I) cell membrane + nuclei; (II) mitochondria; (III) microsomes; and (IV) cell cytosol. Upon sacrifice at 10 min post-Cu-PTSM injection, brain fractions, I, II, III and IV contain 35 ± 12, 11 ± 3, 2.8 ± 1.3 and 51 ± 7% of brain activity, respectively (n = 4). In animals sacrificed 24 h post-injection the subcellular fractions of brain tissue show little change from the radiocopper distribution seen at 10 min post-injection, although the mitochondrial fraction may contain slightly more tracer and the cytosolic fraction slightly less (I, 40 ± 10%; II, 18 ± 5%; III, 3.4 ± 1.5%; and IV, 38 ± 5%; n = 5). Subcellular fractions I, II, III and IV of liver contain 25 ± 5, 12 ± 3, 17 ± 4 and 46 ± 6% of ⁶⁷Cu tracer in animals sacrificed 10 min post-Cu-PTSM injection. An identical subcellular distribution of ⁶⁷Cu, was found in the liver following i.v. administration of ionic radiocopper (as Cu-citrate). The liver and brain cytosolic fractions at 10 min post-injection were further separated by Sephadex column chromatography. In liver cytosol, three different radiocopper components with molecular weights of about 140,000, 41,000–46,000 and 10,000–16,000 Da were found. In the brain supernatant fraction, most of the radiocopper was bound to a single low molecular weight cytosolic component (14,000–16,000 Da). These results suggest that the intracellular decomposition of tracer Cu-PTSM may result in the radiocopper entering the normal cellular pools for copper ions.     

Subcellular distribution and properties of aldehyde dehydrogenase from 2-acetylaminofluorine-induced rat hepatomas

The subcellular distribution and properties of four aldehyde dehydrogenase isoenzymes (I-IV) identified in 2-acetylaminofluorene-induced rat hepatomas and three aldehyde dehydrogenases (I-III) identified in normal rat liver are compared. In normal liver, mitochondria (50%) and microsomal fraction (27%) possess the majority of the aldehyde dehydrogenase, with cytosol possessing little, if any, activity. Isoenzymes I-III can be identified in both fractions and differ from each other on the basis of substrate and coenzyme specificity, substrate K(m), inhibition by disulfiram and anti-(hepatoma aldehyde dehydrogenase) sera, and/or isoelectric point. Hepatomas possess considerable cytosolic aldehyde dehydrogenase (20%), in addition to mitochondrial (23%) and microsomal (35%) activity. Although isoenzymes I-III are present in tumour mitochondrial and microsomal fractions, little isoenzyme I or II is found in cytosol. Of hepatoma cytosolic aldehyde dehydrogenase activity, 50% is a hepatoma-specific isoenzyme (IV), differing in several properties from isoenzymes I-III; the remainder of the tumour cytosolic activity is due to isoenzyme III (48%). The data indicate that the tumour-specific aldehyde dehydrogenase phenotype is explainable by qualitative and quantitative changes involving primarily cytosolic and microsomal aldehyde dehydrogenase. The qualitative change requires the derepression of a gene for an aldehyde dehydrogenase expressed in normal liver only after exposure to potentially harmful xenobiotics. The quantitative change involves both an increase in activity and a change in subcellular location of a basal normal-liver aldehyde dehydrogenase isoenzyme.     

Subcellular localization of acetaldehyde dehydrogenase in human liver

The subcellular distribution of aldehyde dehydrogenase activity was determined in human liver biopsies by analytical sucrose density-gradient centrifugation. There was bimodal distribution of activity corresponding to mitochondrial and cytosolic localizations. At pH 9.6 cytosolic aldehyde dehydrogenase had a lower apparent Kappm for NAD (0.03 mmol l-1), than the mitochondrial enzyme (Kappm NAD = 1.1 mmol l-1). Also, the pH optimum for cytosolic aldehyde dehydrogenase activity (pH 7.5) was lower than that for the mitochondrial enzyme activity (pH 9.0), and the cytosolic enzyme activity was more sensitive to inhibition by disulfiram in vitro. Disulfiram (40 mumol l-1) caused a 70% reduction in cytosolic aldehyde dehydrogenase activity, but only a 30% reduction in mitochondrial enzyme activity after 10 min incubation. The liver cytosol may therefore be the major site of acetaldehyde oxidation in vivo in man.     

Metabolism of tritiated water in the cell nucleus and intracellular substances of the rat

Tritiated water was given to rats in single oral doses, and the cell fractions for each organ were prepared by ultracentrifugation for measurement of the concentration of tissue-bound tritium. The concentration of tissue-bound tritium reached a peak relatively soon after intubation, 1-4 days after administration. The initial concentration of tissue-bound tritium in liver and kidney was high in the mitochondrial and microsomal fractions but low in the nuclear and cytosol fractions. The initial tissue-bound tritium concentration in the brain was high in the mitochondrial and microsomal fractions but low in the nuclear and cytosol fractions. The initial concentration of tissue-bound tritium in the testes was high in the mitochondrial, microsomal, and cytosol fractions but low in the nuclear fraction. The half-life for the long component was larger in the nuclear, mitochondrial, and microsomal fractions of the brain than in the other organs according to an interorgan comparison of each fraction. As for the testes, the values for the mitochondrial and microsomal fractions were larger than those for the other organs.     

Properties of cytosolic components activating rat hepatic 5'' [corrected]-deiodination in the presence of NADPH.

The effects of cytosol, NADPH and reduced glutathione (GSH) on the activity of 5'-deiodinase were studied by using washed hepatic microsomes from normal fed rats. Cytosol alone had little stimulatory effect on the activation of microsomal 5'-deiodinase. NADPH had no stimulatory effect on the microsomal 5'-deiodinase unless cytosol was added. 5'-deiodinase activity was greatly enhanced by the simultaneous addition of NADPH and cytosol (P less than 0.001); this was significantly higher than that with either NADPH or cytosol alone (P less than 0.001). GSH was active in stimulating the enzyme activity in the absence of cytosol, but the activity of 5'-deiodinase with 62 microM-NADPH in the presence of cytosol was significantly higher than that with 250 microM-GSH in the presence of the same concentration of cytosol (P less than 0.001). The properties of the cytosolic components essential for the NADPH-dependent activation of microsomal 5'-deiodinase independent of a glutathione/glutathione reductase system were further assessed using Sephadex G-50 column chromatography to yield three cytosolic fractions (A, B and C), wherein A represents pooled fractions near the void volume, B pooled fractions of intermediate Mr (approx. 13 000), and C of low Mr (approx. 300) containing glutathione. In the presence of NADPH (1 mM), the 5'-deiodination rate by hepatic washed microsomes is greatly increased if both A and B are added and is a function of the concentrations of A, B, washed microsomes and NADPH. A is heat-labile, whereas B is heat-stable and non-dialysable. These observations provide the first evidence of an NADPH-dependent cytosolic reductase system not involving glutathione which stimulates microsomal 5'-deiodinase of normal rat liver. The present data are consistent with a deiodination mechanism involving mediation by a reductase (other than glutathione reductase) in fraction A of an NADPH-dependent reduction of a hydrogen acceptor in fraction B, followed by reduction of oxidized microsomal deiodinase by the reduced acceptor (component in fraction B).     

Enzymatically inactive forms of acetyl-CoA carboxylase in rat liver mitochondria.

Biotinyl proteins were labelled by incubation of SDS-denatured preparations of subcellular fractions of rat liver with [14C]methylavidin before polyacrylamide-gel electrophoresis. Fluorographic analysis showed that mitochondria contained two forms of acetyl-CoA carboxylase [acetyl-CoA:carbon dioxide ligase (ADP-forming) EC 6.4.1.2], both of which were precipitated by antibody to the enzyme. When both forms were considered, almost three-quarters of the total liver acetyl-CoA carboxylase was found in the mitochondrial fraction of liver from fed rats while only 3.5% was associated with the microsomal fraction. The remainder was present in cytosol, either as the intact active enzyme or as a degradation product. The actual specific activity of the cytosolic enzyme was approx. 2 units/mg of acetyl-CoA carboxylase protein while that of the mitochondrial enzyme was about 20-fold lower, indicating that mitochondrial acetyl-CoA carboxylase was relatively inactive. Fractionation of mitochondria with digitonin showed that acetyl-CoA carboxylase was associated with the outer mitochondrial membrane. The available evidence suggests that mitochondrial acetyl-CoA carboxylase represents a reservoir of enzyme which can be released and activated under lipogenic conditions.     

Chronic Sodium Valproate Selectively Decreases Protein Kinase C α and ε In Vitro

Abstract: Valproic acid (VPA) is a fatty acid antiepileptic with demonstrated antimanic properties, but the molecular mechanism or mechanisms underlying its therapeutic efficacy remain to be elucidated. In view of the increasing evidence demonstrating effects of the first-line antimanic drug, lithium, on protein kinase C (PKC), we investigated the effects of VPA on various aspects of this enzyme. Chronic exposure (6–7 days) of rat C6 glioma cells to "therapeutic" concentrations (0.6 m M ) of VPA resulted in decreased PKC activity in both membrane and cytosolic fractions and increased the cytosol/membrane ratio of PKC activity. Western blot analysis revealed isozyme-selective decreases in the levels of PKC α and ε (but not δ or ζ) in both the membrane and cytosolic fractions after chronic VPA exposure; VPA added to reaction mixtures did not alter PKC activity or ³H-phorbol ester binding. Together, these data suggest that chronic VPA indirectly lowers the levels of specific isozymes of PKC in C6 cells. Given the pivotal role of PKC in regulating neuronal signal transduction and modulating intracellular cross-talk between neurotransmitter systems, the specific decreases in PKC α and ε may play a role in the antimanic effects of VPA.