Lutropin stimulation of RNA synthesis in corpus luteum chromatin.

Lutropin and human choriogonadotropin stimulated the endogenous chromatin-associated polymerase activity in purified chromatin prepared from nuclei of bovine corpus luteum. Chromatin was incubated in two different buffer systems: one that mainly supports the activity of polymerase I, another that supports the activity of polymerase II and is largely alpha-amanitin sensitive. The hormones lutropin and chorigonadotropin stimulated an increase in the rate of incorporation of [14C]ATP or [14C]UTP into RNA in both buffer systems. Follitropin, prolactin and beta-corticotropin had no stimulatory effect. Neither the alpha nor beta subunit of lutropin stimulated RNA synthesis. When premixed, the subunits rapidly formed the active molecule. A maximum response to RNA synthesis was achieved by a 10(-9) M concentration of human choriogonadotropin. Considerable activity was obtained at 10(-11) M human choriogonadotropin. There was no lutropin stimulation to RNA synthesis using calf thymus DNA and Escherichia coli RNA polymerase.     

A relation between (NAD+)/(NADH) potentials and glucose utilization in rat brain slices.

Changes in several parameters involved in the control of metabolism were correlated with changes in glucose utilization in rat brain slices incubated under conditions which reduced glucose oxidation by 40 to 70%. The parameters included: the concentrations of ATP, ADP, AMP, and the adenylate energy charge; the cytoplasmic oxidation-reduction state ([NAD+]/[NADH]), determined from the [pyruvate]/[lactate] equilibrium; the mitochondrial oxidation-reduction state, determined from the [NH4+] ]2-oxoglutarate]/[glutamate] Equilibrium; the cytoplasmic and mitochondrial oxidation-reduction potentials (in volts), calculated from the respective [NAD+]/ [NADH] ratios using the Nernst equation; and the difference between the cytoplasmic and mitochondrial [NAD+]/[NADH] potentials. The conversion of [3, 4-14C] glucose to 14CO2 and of [U-14C] glucose to acetylcholine and to lipids, proteins, and nucleic acids by the brain slices were also determined. The values obtained by subtracting the mitochondrial from the cytoplasmic [NAD+1/[NADH] potentials correlated more closely with glucose utilization than did other parameters, under the conditions studied. For the synthesis of acetylcholine, the correlation coefficient was 0.96, and for the production of 14CO2 from [3, 4-14C] glucose it was 0.82.     

Inhibition of the conversion of cholesterol to pregnenolone in bovine adrenocortical preparations.

The inhibition of the conversion of [4-14C] cholesterol to [4-14C] pregnenolone by a number of steroids has been studied in bovine adrenocortical mitochondrial acetonedried preparations. At equimolar substrate and inhibitor concentrations (3.3 muM) the most potent inhibitors were cholesterol derivatives containing a nitrogen function at c-22, followed by derivatives containing oxygen functions at c-22 or c-20 or both. The presence of a hydroxyl group at c-17 or the replacement of the 3beta-hydroxyl group by fluorine reduced the inhibitory efficacy. In the presence of inhibitors that were also relatively good substrates of the cholesterol side-chain cleavage system, such as some cholesterol derivatives hydroxylated in the side-chain,the rate of [4-14C] pregnenolone formation increased with time as the inhibitor was consumed. (20S)-20,21-Dihydroxycholesterol exerted such an effect on the kinetics of [4-14C]pregnenolone formation, and yielded 21-hydroxypregnenolone which was identified by gas chromatography-mass spectrometry. The synthesis of (20R)-22-ketocholesterol, of (20R,22R)-22hydroxycholesterol, (20R,22S)-hydroxycholesterol, and of (20S)-desmosterol is described.     

Metabolism of pyruvate by isolated rat mesenteric lymphocytes, lymphocyte mitochondria and isolated mouse macrophages.

1. The activities of pyruvate dehydrogenase in rat lymphocytes and mouse macrophages are much lower than those of the key enzymes of glycolysis and glutaminolysis. However, the rates of utilization of pyruvate (at 2 mM), from the incubation medium, are not markedly lower than the rate of utilization of glucose by incubated lymphocytes or that of glutamine by incubated macrophages. This suggests that the low rate of oxidation of pyruvate produced from either glucose or glutamine in these cells is due to the high capacity of lactate dehydrogenase, which competes with pyruvate dehydrogenase for pyruvate. 2. Incubation of either macrophages or lymphocytes with dichloroacetate had no effect on the activity of subsequently isolated pyruvate dehydrogenase; incubation of mitochondria isolated from lymphocytes with dichloroacetate had no effect on the rate of conversion of [1-14C]pyruvate into 14CO2, and the double-reciprocal plot of [1-14C]pyruvate concentration against rate of 14CO2 production was linear. In contrast, ADP or an uncoupling agent increased the rate of 14CO2 production from [1-14C]pyruvate by isolated lymphocyte mitochondria. These data suggest either that pyruvate dehydrogenase is primarily in the a form or that pyruvate dehydrogenase in these cells is not controlled by an interconversion cycle, but by end-product inhibition by NADH and/or acetyl-CoA. 3. The rate of conversion of [3-14C]pyruvate into CO2 was about 15% of that from [1-14C]pyruvate in isolated lymphocytes, but was only 1% in isolated lymphocyte mitochondria. The inhibitor of mitochondrial pyruvate transport, alpha-cyano-4-hydroxycinnamate, inhibited both [1-14C]- and [3-14C]-pyruvate conversion into 14CO2 to the same extent, and by more than 80%. 4. Incubations of rat lymphocytes with concanavalin A had no effect on the rate of conversion of [1-14C]pyruvate into 14CO2, but increased the rate of conversion of [3-14C]pyruvate into 14CO2 by about 50%. This suggests that this mitogen causes a stimulation of the activity of pyruvate carboxylase.     

Crabtree effect in tumoral pancreatic islet cells

The relationship between glycolysis and respiration was examined in a model of pancreatic B-cell dysfunction, namely in tumoral insulin-producing cells of the RINm5F line. A rise in D-glucose concentration from 2.8 to 16.7 mM increased the utilization of D-[5-3H]glucose and production of [14C]lactate from D-[U-14C]glucose, whereas decreasing the oxidation of either D-[U-14C]glucose or D-[6-14C]glucose. Whereas 2.8 mM D-glucose augmented O2 uptake above basal value, a further rise in D-glucose concentration to 16.7 mM decreased respiration, which remained higher, however, than basal value. Whether at low or high concentration, D-glucose exerted a pronounced sparing action upon the oxidation of endogenous nutrients in cells prelabeled with either L-[U-14C]glutamine or [14C]palmitate and, nevertheless, augmented above basal value the rate of lipogenesis, ATP/ADP content, adenylate charge, and cytosolic NADH/NAD+ and NADPH/NADP+ ratios. The generation of ATP resulting from the catabolism of either exogenous D-glucose or endogenous nutrients was not affected by the rise in hexose concentration from 2.8 to 16.7 mM. Thus, in sharp contrast with the situation found in normal islet cells, a rise in D-glucose concentration, instead of stimulating mitochondrial oxidative events, caused, through a Crabtree effect, inhibition of hexose oxidation and O2 consumption in tumoral islet cells.     

The effect of calcium on cholesterol side-chain-cleavage enzyme in superovulated rat ovaries

Cholesterol side-chain-cleavage enzyme activity in mitochondria isolated from heavily luteinized rat ovaries was determined using isocitrate as an electron donor and [4-14C]cholesterol to start the reaction. The activity of the enzyme was reduced when more than 10 microM calcium was added to the mitochondrial preparations. When 100 microM EGTA or 2 mM ATP was added to the reaction an increase in enzyme activity was observed and ATP was able to partially overcome the calcium-induced inhibition.     

Hexose metabolism in pancreatic islets: preferential utilization of mitochondrial ATP for glucose phosphorylation

The respective contribution of exogenous and intramitochondrially formed ATP to D-glucose phosphorylation by mitochondria-bound hexokinase was examined in both rat liver and pancreatic islet mitochondria by comparing the generation of D-glucose 6-[32P]phosphate from exogenous [gamma-32P]ATP to the total rate of D-[U-14C]glucose phosphorylation. In liver mitochondria, the fractional contribution of exogenous ATP to D-glucose phosphorylation ranged from 4 to 74%, depending on the availability of endogenous ATP formed by either oxidative phosphorylation or in the reaction catalyzed by adenylate kinase. Likewise, in islet mitochondria exposed to exogenous ATP but deprived of exogenous nutrient, about 60% of D-glucose phosphorylation was supported by mitochondrial ATP. Such a fractional contribution was further increased in the presence of ADP and succinate, and suppressed by mitochondrial poisons. It is concluded that, in islet like in liver mitochondria, mitochondrial ATP is used preferentially to exogenous ATP as a substrate for D-glucose phosphorylation by mitochondria-bound hexokinase. This may favour the maintenance of a high cytosolic ATP concentration in glucose-stimulated islet cells.     

Functional relationship between the ADP/ATP-carrier and the F1-ATPase in mitochondria.

1. The distribution of labeled and unlabeled adenine-nucleotides inside and outside mitochondria was followed after addition of [14C]ADP to rat liver mitochondria. Two types of mitochondria were used: 1, respiring mitochondria which were carrying out oxidative phosphorylation and which had been replenished in ATP by incubation in a medium supplemented with succinate and phosphate; 2, non-respiring mitochondria which had been partially depleted of ATP by incubation in a medium supplemented with rotenone and phosphate. During the first minute following addition of [14C]ADP to the respiring mitochondria, the pre-existing intramitochondrial (internal) [12C]ATP was released into the medium and replaced by newly synthesized [14C]ATP. No [14C]ADP accumulated in the mitochondria. It is suggested that extramitochondrial (external) ADP entering respiring mitochondria in exchange for internal ATP is phosphorylated to ATP before its complete release in the matrix space. In non-respiring mitochondria, the entry of [14C]ADP into the mitochondria was accompanied by the appearance in the external space of [12C]ADP and [12C]ATP, with a marked predominance of [12C]ADP. Thus in non-respiring mitochondria, the residual internal ATP is dephosphorylated to ADP in the inner membrane before being released outside the mitochondria. 2. When mitochondria were incubated with glutamate, ADP and [32P]phosphate, the [32P]ATP which accumulated in the matrix space became rapidly labeled in both the P gamma and P beta groups of the ATP, due to the presence of a transphosphorylation system in the mitochondrial matrix. The [32P]ATP which accumulated outside the mitochondria was also labeled in the P beta group, although less rapidly than the internal ATP. Our data show that a large fraction (75-80%) of the ATP produced by phosphorylation of added ADP within the inner mitochondrial membrane is released into the matrix space before being transported out from the mitochondria; only a small part (20-25%) is released directly outside the mitochondria without penetrating the matrix space. 3. In respiring and phosphorylating mitochondria, the value of the Km of the ADP-carrier for external ADP was 2-4 times lower than its value in non-respiring and non-phosphorylating mitochondria. 4. The above experimental data are discussed with reference to the topological and functional relationships between the ADP-carrier and the oxidative phosphorylation complex in the inner mitochondrial membrane. They strongly suggest that the ADP-carrier comes to the close neighbourhood of the ATP synthetase on the matrix side of the inner membrane.     

Acetoacetate metabolism in infant and adult rat brain in vitro

1. Acetoacetate or dl-beta-hydroxybutyrate increases the rate of oxygen consumption to a smaller extent than that brought about by glucose or pyruvate in adult rat brain-cortex slices but to the same extent as that in infant rat brain-cortex slices. 2. The rate of (14)CO(2) evolution from [1-(14)C]glucose considerably exceeds that from [6-(14)C]glucose in respiring infant rat brain-cortex slices, in contrast with adult brain-cortex slices, suggesting that the hexose monophosphate shunt operates at a greater rate in the infant rat brain than in the adult rat brain. 3. The rate of (14)CO(2) evolution from [3-(14)C]acetoacetate or dl-beta-hydroxy[3-(14)C]butyrate, in the absence of glucose, is the same in infant rat brain slices as in adult rat brain slices. It exceeds that from [2-(14)C]glucose in infant rat brain but is less than that from [2-(14)C]glucose in adult rat brain. 4. Acetoacetate is oxidized in the brain through the operation of the citric acid cycle, as shown by the accelerating effect of glucose on acetoacetate oxidation in adult brain slices, by the inhibitory effects of malonate in both infant and adult brain slices and by its conversion into glutamate and related amino acids in both tissues. 5. Acetoacetate does not affect glucose utilization in adult or infant brain slices. It inhibits the rate of (14)CO(2) formation from [2-(14)C]glucose or [U-(14)C]-glucose the effect not being wholly due to isotopic dilution. 6. Acetoacetate inhibits non-competitively the oxidation of [1-(14)C]pyruvate, the effect being attributed to competition between acetyl-CoA and CoA for the pyruvate-oxidation system. 7. Acetoacetate increases the rate of aerobic formation of lactate from glucose with both adult and infant rat brain slices. 8. The presence of 0.1mm-2,4-dinitrophenol diminishes but does not abolish the rate of (14)CO(2) formation from [3-(14)C]acetoacetate in rat brain slices. This points to the participation of ATP in the process of oxidation of acetoacetate in infant or adult rat brain. 9. The presence of 5mm-d-glutamate inhibits the rate of (14)CO(2) formation from [3-(14)C]acetoacetate, in the presence or absence of glucose. 10. Labelled amino acids are formed from [3-(14)C]acetoacetate in both adult and infant rat brain-cortex slices, but the amounts are smaller than those found with [2-(14)C]glucose in adult rat brain and greater than those found with [2-(14)C]glucose in infant rat brain. 11. Acetoacetate is not as effective as glucose as a precursor of acetylcholine in adult rat brain but is as effective as glucose in infant rat brain slices. 12. Acetoacetate or beta-hydroxybutyrate is a more potent source of acetyl-CoA than is glucose in infant rat brain slices but is less so in adult rat brain slices.     

Transport and metabolism of acetate in rat brain cortex in vitro

1. [1-(14)C]Acetate undergoes metabolism when incubated aerobically at 37 degrees in the presence of rat brain-cortex slices, forming (14)CO(2) and (14)C-labelled amino acids (glutamate, glutamine, aspartate and relatively small quantities of gamma-aminobutyrate). In the absence of glucose the yield of (14)C-labelled aspartate exceeds that of (14)C-labelled glutamate and glutamine. The addition of glucose brings about a doubling of the rate of formation of (14)CO(2) and a greatly increased yield of (14)C-labelled glutamate or glutamine, whereas that of (14)C-labelled aspartate is diminished. 2. The addition of potassium chloride (100mm) to the incubation medium causes an increased rate of (14)CO(2) formation in the presence or absence of glucose and an increased rate of utilization of acetate. 3. The addition of 2,4-dinitrophenol (0.1mm) suppresses the rate of utilization of [1-(14)C]acetate. 4. The presence of ouabain (10mum) suppresses the rate of formation of (14)CO(2) from [1-(14)C]acetate and the rate of acetate utilization. Acetate conversion into carbon dioxide in the rat brain cortex is both Na(+)- and K(+)-dependent and controlled by operation of the active sodium-transport process. Only the Na(+)-stimulated rate is suppressed by ouabain. 5. Sodium fluoroacetate (1mm) decreases the rate of (14)CO(2) evolution from [1-(14)C]acetate in the presence of rat brain cortex without affecting the respiratory rate. The results are consistent with the conclusion that fluoroacetate competes with, or blocks, a transport carrier for acetate, so that in its presence only the passive diffusion rate of acetate takes place. 6. The presence of sodium propionate or sodium butyrate suppresses the utilization of [1-(14)C]acetate in rat brain cortex and leads to a concentration ratio (tissue/medium) of [1-(14)C]-acetate greater than unity. 7. The presence of NH(4) (+) diminishes acetate utilization, this being attributed to a diminished ATP concentration. Glycine is also inhibitory. It is concluded that acetate transport into the brain is carrier-mediated and dependent on the operation of the sodium pump.     

The metabolic route by which oleate is converted into cholesterol in rat hepatocytes.

The effect of (-)-hydroxycitrate on the conversion of [1-14C]oleate into cholesterol was dependent on the time of day at which the cells were prepared and on the extracellular oleate concentration. In hepatocytes prepared during the light phase of the diurnal cycle (L2-hepatocytes), (-)-hydroxycitrate inhibited the conversion of L-[U-14C]lactate (2 mM) and of 0.13 mM-[1-14C]oleate into cholesterol. However, when [1-14C]oleate was present at 1.3 mM, most of the sterol carbon was derived from this source, and under these conditions (-)-hydroxycitrate had no inhibitory effect on [14C]cholesterol formation. In these cells, non-radioactive acetoacetate blocked the conversion of 1.3 mM-[1-14C]oleate, but not of 0.13 mM-[1-14C]oleate, into cholesterol. In cells prepared during the dark phase of the diurnal cycle (D6-hepatocytes), irrespective of the concentration of [1-14C]oleate, (-)-hydroxycitrate decreased its conversion into cholesterol. In both types of cell preparation, the inhibitory effect of (-)-hydroxycitrate on the conversion of L-[U-14C]lactate into cholesterol was greater than that on the overall rate of cholesterol production from all endogenous sources. These results provide evidence for the following. (1) The major metabolic route by which oleate is converted into cholesterol is dependent on its extracellular concentration. (2) When oleate is the major source of hepatic sterol carbon, the flux of substrate through citrate into cholesterol is dependent on the nutritional state of the animal. (3) When endogenous substrates are the sole source of sterol carbon, a substantial proportion of the carbon enters the cholesterol pathway through routes not involving citrate cleavage.     

Role of the intramitochondrial adenine nucleotides as intermediates in the uncoupler-induced hydrolysis of extramitochondrial ATP.

1. A formula is given that describes the appearance of [14C]ATPADP outside the mitochondria after the addition of [14C] 1atp during the steady-state uncoupler-induced hydrolysis of extramitochondrial ATP. If the transported adenine nucleotides equilibrate with the intramitochondrial pool, [14C]ADP0 would be expected to appear with a lag phase that corresponds with the time needed for the radioactive labelling of the intramitochondrial adenine nucleotide pool. 2. The rates of formation of [14C]ADP outside the mitochondria after addition of [14C]ATP during the steady-state uncoupler-induced ATP hydrolysis catalysed by rat-liver mitochondria at 0 degree C were measured. 3. In the presence of carbonyl cyanide m-chlorophenylhydrazone the time course of the [14]ADPo formation was the same as that predicted on the basis of the above assumption. 4. In the presence of the less effective uncoupler, 2,4-dinitrophenol, the time course of [14C]ADPo formation was not consistent with the theoretical predictions: no lag phase was present and the measured rate was higher than the maximal calculated rate. These results can be explained by assuming a functional interaction between the adenine nucleotide translocator and the mitochondrial ATPase (F1). 5. It is concluded that under phosphorylating as well as dephosphorylating conditions, the adenine nucleotide translocator and the mitochondrial ATPase can be functionally linked to catalyse phosphorylation or dephosphorylation of extramitochondrial ADP or ATP, without participation of the intramitochondrial adenine nucleotides.     

Partial characterization of steryl ester biosynthesis in spinach leaves

Acetone powders of a 20,000g pellet fraction from spinach leaves (Spinacia oleracea L.) synthesized [4-(14)C]cholesteryl esters when incubated with [4-(14)C]cholesterol. The reaction was inhibited by digitonin. There was a reciprocal relationship between the decline of label in cholesterol and its incorporation into cholesteryl ester, indicating that free cholesterol was the direct precursor for cholesteryl ester biosynthesis. The hydrolysis of cholesteryl [1-(14)C]palmitate into free cholesterol and [1-(14)C]palmitate was not detected in these acetone powder preparations. Exogenous cholesteryl palmitate had no effect on the esterification of [4-(14)C]cholesterol. The data indicate that an esterase-type mechanism was not involved in the biosynthesis of these steryl esters. Label from [1-(14)C]palmitoyl-CoA was incorporated into steryl esters when incubated with spinach leaf acetone powder preparations. The optimal buffer for steryl ester biosynthesis was 2-(N-morpholino)ethanesulfonate and the optimal pH was 6. Iodoacetamide, N-ethylmaleimide, and dithiothreitol had no effect on the esterification reaction. Ethylenediaminetetraacetate, MgCl(2), CaCl(2), MnCl(2), and ZnSO(4) inhibited at concentrations of 10 to 30 mm.     

Hormonal steroidogenesis in liver and small intestine of the green frog, Rana esculenta L.

We have investigated the potential of autonomous hormonal steroidogenesis in liver and small intestine of male and female frogs, Rana esculenta, during the recovery phase. After incubation of mitochondrial fractions with [4-14C]cholesterol, female liver and intestine formed pregnenolone at a rate of 0.63 and 2.3 pmol/mg protein/h, respectively, whereas conversion by male organs was only c. 0.03 pmol/mg protein/h. Minced tissues preparations transformed [4-14C]pregnenolone into progesterone and 17alpha-hydroxypregnenolone, the former prevailing in the liver, the latter in the intestine. Moreover, both tissues produced 20alpha-dihydropregnenolone, 20alpha-dihydroprogesterone and dehydroepiandrosterone. From incubates with [4-14C]dehydroepiandrosterone, androstenedione and androst-5-ene-3beta, 17beta-diol were identified, the former being more abundant in the liver, the latter in the intestine. These results indicate that both liver and intestine in frog can be independent sources of hormonally active steroids in both sexes.     

Characterization of the subunit structure of the maize tonoplast ATPase. Immunological and inhibitor binding studies

Gradient purified preparations of the maize 400-kDa tonoplast ATPase are enriched in two major polypeptides, 72 and 62 kDa. Polyclonal antibodies were prepared against these two putative subunits after elution from sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel slices and against the solubilized native enzyme. Antibodies to both the 72- and 62-kDa polypeptides cross-reacted with similar bands on immunoblots of a tonoplast-enriched fraction from barley, while only the 72-kDa antibodies cross-reacted with tonoplast and tonoplast ATPase preparations from Neurospora. Antibodies to the 72-kDa polypeptide and the native enzyme both strongly inhibited enzyme activity, but the 62-kDa antibody was without effect. The identity and function of the subunits was further probed using radiolabeled covalent inhibitors of the tonoplast ATPase, 7-chloro-4-nitro[14C]benzo-2-oxa-1,3-diazole ([14C]NBD-Cl) and N,N'-[14C]dicyclohexylcarbodiimide ([14C]DCCD). [14C]NBD-Cl preferentially labeled the 72-kDa polypeptide, and labeling was prevented by ATP. [14C]DCCD, an inhibitor of the proton channel portion of the mitochondrial ATPase, bound to a 16-kDa polypeptide. Venturicidin blocked binding to the mitochondrial 8-kDa polypeptide but did not affect binding to the tonoplast 16-kDa polypeptide. Taken together, the results implicate the 72-kDa polypeptide as the catalytic subunit of the tonoplast ATPase. The DCCD-binding 16-kDa polypeptide may comprise the proton channel. The presence of nucleotide-binding sites on the 62-kDa polypeptide suggests that it may function as a regulatory subunit.     

Cell-free transfer of sterols from dictyosome-like structures to plasma membrane vesicles of guinea pig testes

Summary Transfer of radiolabeled lipids from dictyosome-like structures (DLS) from testis tubules of the guinea pig as donor to unlabeled plasma membrane from testis tubules immobilized on nitrocellulose as acceptor was studied in a completely cell-free system. As a general label for lipids of the donor DLS, isolated testis tubules were incubated with [¹⁴C]acetate. Time- and temperature-dependent transfer of [¹⁴C]acetate labeled constituents was observed in the cellfree system. However, despite the fact that phospholipids and other constituents were highly labeled in the donor fraction, primarily radioactive sterols were transferred to the plasma membrane acceptor vesicles. Transfer at 37°C represented 0.4 to 0.7% of the total radiolabeled cholesterol at 37°C but little or no transfer occurred at 4°C. The sterols transferred exhibited Chromatographic mobilities corresponding to those of cholesterol and lanosterol. Similar results were obtained with [¹⁴C]mevalonic acid. In subsequent experiments, cholesterol transfer from DLS to plasma membrane was demonstrated by incubation of DLS with [³H]squalene which was converted into sterol or with [¹⁴C]cholesterol. Transfer of sterols required ATP, but not cytosol, and was both time- and temperature-dependent. DLS were more effective than either endoplasmic reticulum or plasma membrane as the donor fraction. The results from the cell-free analysis suggest a possible functional role of the DLS in sterol biogenesis and transfer to the plasma membrane during spermatid development.Abbreviations DLS dictyosome-like structure(s) - PBS phosphatebuffered saline - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - BSA bovine serum albumin     

Organic mercurial diuresis: inhibition of glutamine utilization in the acidotic rat.

Organic mercurials inhibit mitochondrial glutamine metabolism in vitro while metabolic acidosis, a condition in which the predominant renal fuel is glutamine, potentiates mercurial diuresis. The following studies were undertaken to determine whether potentiation of diuresis reflects mercurial inhibition of glutamine utilization. (1) All three mercurials employed (mersalyl, chlormerodrin, and p-chloromercuribenzoate) are diuretics in the rat and this effect was potentiated by NH4Cl. (2) Despite reabsorbing less sodium, mercurial-treated rats had lower kidney ATP content (4.35 +/- 0.26 and 3.84 +/- 0.43 mumol/g dry weight (mercurial plus NH4Cl) than did controls (4.95 +/- 0.31 and 4.87 +/- 0.39 mumol/g dry weight (NH4Cl). (3) Isolated kidneys from NH4Cl and NH4Cl plus mercurial treated rats were perfused with 1 mM L-[U-14C]glutamine to determine rates of extraction and oxidation. Mercurial-treated acidotic rat kidneys had a reduced rate of glutamine uptake (40.8 +/- 7.4 vs. 64.8 +/- 5.8 mumol/h per kidney), a diminished rate of glutamine conversion to CO2 (14.8 +/- 3.6 vs. 26.4 +/- 5.2 mumol/h per kidney), and a reduction in glucose production (16 +/- 5 vs. 27 +/- 4 mumol/h per kidney). These results are consistent with an effect of organic mercurials upon glutamine utilization, limiting ATP availability, and thereby reducing tubular active sodium reabsorption.     

Functioning of mitochondria-bound hexokinase in rat brain in accordance with generation of ATP inside the organelle.

The function of mitochondria-bound hexokinase, the enzymatic form peculiar to the brain, in utilization of ATP generated inside the organelles, was examined by incubating rat brain mitochondrial fraction with [14C]glucose under various conditions. Addition of succinate and ADP to the incubation medium increased glucose 6-phosphate formation by the mitochondrial hexokinase and caused a smaller increase in ATP concentration in the mitochondria. The glucose phosphorylation was markedly inhibited by the addition of dinitrophenol, potassium cyanide, and oligomycin, and the ATP concentration was decreased. On the other hand, addition of atractyloside suppressed the glucose phosphorylation without affecting the mitochondrial hexokinase activity, whereas addition of antiserum against the mitochondrial hexokinase inhibited both glucose 6-phosphate formation and hexokinase activity. A part of both the glucose phosphorylation and hexokinase activities, however, remained even in the presence of the maximum dose of the anti-hexokinase serum and atractyloside. These results indicate the active utilization of intrinsically generated ATP by the mitochondria-bound hexokinase, a part of which may be located away from the surface of the mitochondrial membrane.     

Leucine degradation in cell-free extracts of skeletal muscle.

Since skeletal muscle is the major site in the body for oxidation of leucine, isoleucine and valine, the pathway and control of leucine oxidation were investigated in cell-free preparations of rat muscle. Leucine was found to be transaminated to 4-methyl-2-oxopentanoate, which was then oxidatively decarboxylated. On differential centrifugation 70--80% of the transaminase activity was recovered in the soluble fraction of the cell, and the remaining amount in the mitochondrial fraction. The transaminase, from both fractions had similar pH optima and both were markedly inhibited by Ca2+. Thus changes in cellular Ca2+ concentration may regulate transaminase activity. Both transaminases had a much higher affinity for 2-oxoglutarate than for pyruvate. Therefore the utilization of amino groups from leucine for the biosynthesis of alanine in muscle [Odessey, Khairallah & Goldberg (1974) J. Biol. Chem. 249, 7623--7629] in vivo involves transamination with 2-oxoglutarate to produce glutamate, which is then transaminated with pyruvate to produce alanine. The dehydrogenase activity assayed by the decarboxylation of methyl-2-oxo[1-14C]pentanoate was localized exclusively in the fraction containing mitochondria and required NAD+, CoA and thiamin pyrophosphate for optimal activity. Measurements of competitive inhibition suggested that the oxo acids of leucine, isoleucine and valine are all decarboxylated by the same enzyme. The enzyme activity was decreased by 90% upon freezing or sonication and was stimulated severalfold by Mg2+, K+ and phosphate ions. In addition, it was markedly inhibited by ATP, but not by non-metabolizable analogues. This observation suggests that splitting of ATP is required for inhibition. The oxidative decarboxylation of 4-methyl-2-oxopentanoate by the dehydrogenase appears to be the rate-limiting step for leucine oxidation in muscle homogenates and also in intact tissues. In fact, rat muscles incubated with [1-14C]leucine release 1-14C-labelled oxo acid into the medium at rates comparable with the rate of decarboxylation. Intact muscles also released the oxo acids of [1-14C]valine or [1-14C]isoleucine, but not of other amino acids. These findings suggest that muscle is the primary source of the branched-chain oxo acids found in the blood.