Response of mitochondrial ATPase activity to uncouplers in isolated organelles and whole cells of Zajdela hepatoma

ATPase activity of coupled Zajdela hepatoma mitochondria was rendered uncoupler-sensitive by decreasing free fatty acids content in mitochondria or by preincubation of mitochondria with ATP prior to the addition of an uncoupler. The latter treatment resulted in an accelerated transport of ATP into the organelles. The effect of carbonylcyanide-m-chlorophenylhydrazone and oligomycin on the decrease of the ATP content in whole Zajdela hepatoma cells indicated that the hepatoma mitochondrial ATPase is stimulated by uncouplers $\text{in}\text{vivo}$. The conclusion is that the uncoupler-insensitive ATPase activity of coupled Zajdela hepatoma mitochondria is exhibited only by isolated organelles and results from a reduced $\text{ATP}\text{ADP}$ translocase activity.     

Characterization of shuttle mechanisms for the transport of reducing equivalents into mitochondria

The malate-aspartate, fatty acid, and α-glycerophosphate shuttles for the transport of reducing equivalents into mitochondria were reconstituted, using isolated hepatic mitochondria and the extramitochondrial components of the shuttles. Clofibrate and thyroxin increased, while propylthiouracil treatment decreased, the activity of mitochondrial α-glycerophosphate dehydrogenase. Despite these changes, the activity of the reconstituted α-glycerophosphate shuttle was similar in mitochondria from control rats and those from rats treated with clofibrate and propylthiouracil. There was an increase in the activity of the shuttle using mitochondria from thyroxin-treated rats. Rotenone caused 60–90% inhibition of this shuttle, suggesting that rotenone-sensitive NADH dehydrogenase participates in the pathway of oxidation of extramitochondrial hydrogen. Palmitate, oleate, and octanoate were equally effective in reconstituting a cyclic fatty acid shuttle. The shuttle was inhibited by various compounds affecting mitochondrial metabolism, including oligomycin, dinitrophenol, cyanide, rotenone, atractyloside, and α-bromopalmitate. Carnitine and several dicarboxylic and tricarboxylic acids which stimulate fatty acid elongation, augmented fatty acid shuttle activity. The malate-aspartate shuttle was inhibited by cycloserine, amino-oxyacetic acid, and hydrazine, and stimulated by pyridoxal phosphate, at the same concentrations which affected the activities of cytoplasmic and mitochondrial glutamic oxalacetic transaminase. This shuttle was inhibited by uncouplers, antimycin, azide, cyanide, rotenone, amobarbital, oligomycin, and several inhibitors of anion transport including iodobenzylmalonate and avenaciolide. The reconstituted shuttle is sufficiently active to provide about 70–80% of the oxalacetate required for maximal rates of gluconeogenesis. Extrapolations based on the rates of mitochondrial oxidation of acetaldehyde and the activity of the microsomal ethanol oxidizing system suggest that any one of the shuttles could account for the rate of ethanol metabolism in vitro by the alcohol dehydrogenase pathway.     

Acetylaspartate as an extramitochondrial physiological carrier of acetyl coA for fatty acid synthesis

Aminooxyacetate, a transaminase inhibitor, suppresses the enrichment in radioactivity found in the fatty acids of animals receiving 2, 4-¹⁴C citrate in comparison with 1, 5-¹⁴C citrate. On the other hand 3H from N-acetyl-³H aspartate is significantly incorporated into fatty acids $\text{in vivo}$ or in presence of liver supernatant fractions. Our results indicate that citrate seems to be an effective carrier of acetyl CoA for fatty acid synthesis mainly in the rat liver and that acetylaspartate may be an other physiological carrier of acetyl CoA outside the mitochondria.     

The influence of diet on the lipogenic response to thyroxine in rat liver.

ATP citrate lyase, acetyl-CoA synthetase, malic enzyme and hexose monophosphate dehydrogenase activities and rates of $\text{denovo}$ synthesis of long chain fatty acids from labeled acetate and citrate were measured in cell-free fractions of liver from rats fed various diets, with and without D- or L- thyroxine. Diets containign sucrose (vs. isocaloric glucose) or lard (vs. isocaloric corn oil) stimulated hepatic lipogenesis both in control and in thyroxine-treated rats. The lipogenic response to thyroxine was greatly modified by diet, except for an invariable rise in malic enzyme activity. With diets providing less than 6% of calories as linoleic acid, thyroxine increased fatty acid synthesis, depleted liver glycogen and retarded growth; when linoleic acid was increased to 16% of calories, thyroxine had no effect on fatty acid synthesis or growth and liver glycogen depletion was significantly attenuated. This response to dietary linoleic acid suggests that these phenomena may be largely secondary to the increased requirement for essential fatty acid in thyrotoxicosis. Further study should reveal the extent to which observed effects of excess thyroid hormone are amenable to control by dietary polyunsaturated fat.     

Oxaloacetate transport into plant mitochondria

The properties of oxaloacetate (OA) transport into mitochondria from potato (Solanum tuberosum) tuber and pea (Pisum sativum) leaves were studied by measuring the uptake of ¹⁴C-labeled OA into liposomes with incorporated mitochondrial membrane proteins preloaded with various dicarboxylates or citrate. OA was found to be transported in an obligatory counterexchange with malate, 2-oxoglutarate, succinate, citrate, or aspartate. Phtalonate inhibited all of these countertransports. OA-malate countertransport was inhibited by 4,4′-dithiocyanostilbene-2,2′-disulfonate and pyridoxal phosphate, and also by p-chloromercuribenzene sulfonate and mersalyl, indicating that a lysine and a cysteine residue of the translocator protein are involved in the transport. From these and other inhibition studies, we concluded that plant mitochondria contain an OA translocator that differs from all other known mitochondrial translocators. Major functions of this translocator are the export of reducing equivalents from the mitochondria via the malate-OA shuttle and the export of citrate via the citrate-OA shuttle. In the cytosol, citrate can then be converted either into 2-oxoglutarate for use as a carbon skeleton for nitrate assimilation or into acetyl-coenzyme A for use as a precursor for fatty acid elongation or isoprenoid biosynthesis.     

Iron enhances the bactericidal action of streptonigrin

The lethal action of streptonigrin on strains of $\text{Escherichia}\text{coli}$ is greatly enhanced by citrate (10^?2 M). Desferrioxamine (2×10^?4 M), when added with streptonigrin and citrate, eliminates the citrate enhancement. These observations point to a role for iron in the bactericidal mechanism of streptonigrin. Extracellular citrate is known to promote the acquisition of iron by $\text{E.}\text{coli}$ by delivering it as a ferric citrate complex to a specific transport apparatus on the cell envelope. Therefore, it may promote action of streptonigrin by increasing the intracellular concentration of available iron. Desferrioxamine, which forms a much stronger complex with ferric ion than does citrate, would be expected to suppress the ferric citrate effect, and this was observed.     

Adenine nucleotide control of the rate of oxygen uptake by rat heart mitochondria over a 15- to 20-fold range

Rat heart mitochondria oxidizing pyruvate (in the presence of 20% as much malate) took up nearly the amount of oxygen required for complete oxidation to CO₂. Thus pyruvate, a physiological substrate of the citrate cycle, is oxidized through the entire cycle in these mitochondria, and they seem suitable for study of regulation of integrated mitochondrial energy transduction. By addition of graded amounts of hexokinase or pyruvate kinase to the suspending medium (in the presence of excess glucose or phosphoenolpyruvate), a wide range of steady-state values of the $\text{ATP}\text{ADP}$ concentration ratio was obtained. At a constant concentration of phosphate, the steady-state rate of oxygen uptake by rat heart mitochondria oxidizing pyruvate was a function of the adenylate energy charge or of the $\text{ATP}\text{ADP}$ ratio, and relatively independent of the absolute concentrations of these nucleotides. The oxygen uptake rates typically spanned a range of about 20-fold. At very high values of the $\text{ATP}\text{ADP}$ ratio, the rate of oxygen uptake is much lower than the “state 4” rate seen after added ADP has been phosphorylated. This result suggests that “state 4” respiration, at least in these freshly prepared mitochondria, measures the rate at which ADP is made available by ATPase activity, rather than indicating uncoupling of electron transport from phosphorylation. The concentration of orthophosphate affected the rate of oxygen uptake and the pattern of response to the $\text{ATP}\text{ADP}$ ratio or the energy charge, but the effects did not seem interpretable in terms of the mass-action expression for hydrolysis of ATP, $\text{(ATP}\text{ADP)}$ (P_i.     

Evidence for participation of the inner membrane in the import of sulfite oxidase into the intermembrane space of liver mitochondria

Sulfite oxidase, a soluble enzyme in mitochondrial intermembrane space, was synthesized as a precursor protein larger than the authentic enzyme when rat liver RNA was translated $\text{in}\text{vitro}$ using reticulocyte lysate. When the $\text{in}\text{vitro}$ translation products were incubated with isolated rat liver mitochondria, the precursor of sulfite oxidase was converted to the size of the mature enzyme. The $\text{in}\text{vitro}$ processed mature enzyme was no longer susceptible to externally added proteases and was extractable by a hypotonic treatment of the mitochondria, suggesting its location in the intermembrane space. When mitochondria were subfractionated, most of the processing activity was recovered in the mitoplast fraction. The import-processing activity of mitochondria was inhibited by CCCP, oligomycin, or atractyloside in the presence of KCN. These results suggest that the import of sulfite oxidase into mitochondrial intermembrane space requires the participation of inner membrane.     

Ethanol metabolism in guinea pig: Ethanol oxidation and its effect on NADNADH ratios,oxygen consumption,and ketogenesis in isolated hepatocytes of fed and fasted animals

Ethanol metabolism was studied in isolated hepatocytes of fed and fasted guinea pigs. Alcohol dehydrogenase (EC 1.1.1.1) activities of fed or fasted liver cells were 2.04 and 1.88 μmol/g cells/min, respectively. Under a variety of in vitro conditions, alcohol dehydrogenase operates in fed hepatocytes at 34–74% and in fasted liver cells at 23–61% of its maximum velocity, respectively. Hepatocytes of fed animals, incubated in Krebs-Ringer bicarbonate buffer, oxidized ethanol at an average rate of 0.69 μmol/g wet weight cells/min, whereas cells of 48-h fasted animals consumed only 0.44 μmol/g/min under identical conditions. Various substrates and metabolites of intermediary metabolism significantly enhanced ethanol oxidation in fed liver cells. Maximum stimulatory effects were achieved with alanine (+138%) and pyruvate (+102%), followed in decreasing order by propionate, lactate, fructose, dihydroxyacetone, and galactose. In contrast to substrate couples such as lactate/pyruvate and glycerol/dihydroxyacetone, sorbitol with or without fructose significantly inhibited ethanol oxidation. The addition of hydrogen shuttle components such as malate, aspartate, or glutamate to fasted hepatocytes resulted in significantly higher stimulation of ethanol uptake than in fed hepatocytes. Also, the degree of inhibition of shuttle activity by n-butylmalonate was more pronounced in fasted liver cells (77% inhibition) than in fed cells (59% inhibition). These data as well as oxygen kinetic studies in intact guinea pig hepatocytes utilizing uncouplers (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, dinitrophenol), electron-transport inhibitors (rotenone, antimycin), and malate-aspartate shuttle inhibitors (aminooxyacetate, n-butylmalonate) strongly suggested that the malate-aspartate shuttle is the predominant hydrogen transport system during ethanol oxidation in guinea pig liver.Comparison of the alcohol dehydrogenase-inhibitors 4-methylpyrazole and pyrazole on ethanol oxidation demonstrated that the alcohol dehydrogenase system is quantitatively the most important alcohol-metabolizing pathway in guinea pig liver. Supporting this conclusion, it was found that the H₂O₂-forming substrate glycolate slightly increased ethanol oxidation in liver cells of control animals (+26%), but prior inhibition of catalase by 3-amino-1,2,4-triazole resulted in a significant increase (+25%) instead of a decrease in alcohol oxidation. This finding does not support a quantitatively important role of peroxidatic oxidation of ethanol by catalase in liver.Cytosolic $\text{NAD}\text{NADH}$ ratios were greatly shifted toward reduction during ethanol oxidation. These reductive shifts were even more pronounced when cells were incubated in the presence of fatty acids (octanoate, oleate) plus ethanol. Inhibitor studies with 4-methylpyrazole demonstrated that the decrease of the cytosolic $\text{NAD}\text{NADH}$ ratio during fatty acid oxidation was due to an inhibition of hydrogen transport from cytosol to mitochondria and not the result of transfer of hydrogen, generated by fatty acid oxidation, from mitochondria to cytosol. Lactate plus pyruvate formation was slightly inhibited by ethanol in fed hepatocytes but greatly accelerated in fasted cells; this latter effect was mostly the result of increased lactate formation. Such regulation may represent a hepatic mechanism of alcoholic lactic acidosis as observed in human alcoholics. The ethanol-induced decrease of the mitochondrial $\text{NAD}\text{NADH}$ ratio was prevented by addition of 4-methylpyrazole. Endogenous ketogenesis was greatly increased (+80%) by ethanol in fed liver cells. This effect of ethanol was blunted in the presence of glucose. Propionate, by competing with fatty acid oxidation, was strongly antiketogenic. This effect was alleviated by ethanol. In 48-h fasted hepatocytes, endogenous ketogenesis was enhanced by 84%. Although ethanol did not further stimulate endogenous ketogenesis under these conditions, alcohol significantly increased ketogenesis in the presence of octanoate or oleate. This stimulatory effect of ethanol was almost completely prevented by 4-methylpyrazole. These findings demonstrate that the syndrome of alcoholic ketoacidosis may be due, at least partially, to the additional stimulation of ketogenesis by or from ethanol during fatty acid oxidation in the fasting state.     

Effect of long chain unsaturated fatty acids on the calcium transport of sarcoplasmic reticulum

The effect of archidonic, oleic and linoleic acid on calcium uptake and release by sarcoplasmic reticulum isolated from longissimus dorsi muscle was investigated using a Ca²⁺ electrode. All three long chain fatty acids stimulated the release of Ca²⁺ from sacroplasmic reticulum when added after exogenous Ca²⁺ was accumulated by the vesicles, and also inhibited Ca²⁺ uptake when added before Ca²⁺. This inhibitory effect on the calcium transport by arachidonic, oleic and linoleic acid was prevented by bovine serum albumin through its ability to bind with the fatty acid. The order of effectiveness of the fatty acids in inhibiting calcium transport by isolated sarcoplasmic reticulum was $\text{arachidonic acid> oleic acid >}$ linoleic acid. Similar inhibition of calcium uptake and induction of calcium release by arachidonic acid was observed in muscle homogenate sarcoplasmic reticulum preparations. Both arachidonic and oleic acid stimulated the (Ca²⁺ + Mg²⁺)-ATPase activity of sarcoplasmic reticulum at low concentrations, but inhibited the (Ca²⁺ + Mg²⁺)-ATPase activity at high concentrations. The maximal (Ca²⁺ + Mg²⁺-ATPase activity observed with arachidonic acid was twice that obtained with oleic acid, but the concentration of arachidonic acid required was 3–4-times greater than that of oleic acid. The concentration of arachidonic acid required to give maximum stimulation of the (Ca²⁺ + Mg²⁺)-ATPase activity was 3.6-times greater than that needed for complete inhibition of calcium accumulation by the sacroplasmic reticulum. With oleic acid, however, the concentration required to give maximum stimulation of the (Ca²⁺ + Mg²⁺)-ATPase activity inhibited the sarcoplasmic reticulum Ca²⁺ accumulation by 72%. The present data support our hypothesis that, in porcine malignant hyperthermia, unsaturated fatty acids from mitochondrial membranes released by endogenous phospholipase A₂ would induce the sarcoplasmic reticulum to release calcium (Cheah K.S. and Cheah, A.M. (1981) Biochim. Biophys. Acta 634, 70–84).     

A shuttle system for the transfer of reducing equivalents in mouse sperm mitochondria.

Studies have been carried out on an $\text{in vitro}$ reconstituted system composed of mouse lactate dehydrogenase isozyme X or C4, branched chain amino acid aminotransferase, NAD, alpha-hydroxy isocaproate, glutamate and mouse sperm mitochondria. This system demonstrated capacity for the oxidation of extramitochondrial NADH. It is proposed that a branched chain alpha-hydroxyacid / amino acid shuttle for the transfer of reducing equivalents from cytosol to mitochondria may be functional in mouse spermatozoa.     

Pathway of carbon flow during fatty acid synthesis from lactate and pyruvate in rat adipose tissue

The metabolism of pyruvate and lactate by rat adipose tissue was studied. Pyruvate and lactate conversion to fatty acids is strongly concentration-dependent. Lactate can be used to an appreciable extent only by adipose tissue from fasted-refed rats. A number of compounds, including glucose, pyruvate, aspartate, propionate, and butyrate, stimulated lactate conversion to fatty acids. Based on studies of incorporation of lactate-2-(3)H and lactate-2-(14)C into fatty acids it was suggested that the transhydrogenation sequence of the "citrate-malate cycle"(1) was not providing all of the NADPH required for fatty acid synthesis from lactate. An alternative pathway for NADPH formation involving the conversion of isocitrate to alpha-ketoglutarate via cytosolic isocitrate dehydrogenase was proposed. Indirect support for this proposal was provided by the rapid labeling of glutamate from lactate-2-(14)C by adipose tissue incubated in vitro, as well as the demonstration that glutamate can be readily metabolized by adipose tissue via reactions localized largely in the cytosol. Furthermore, isolated adipose tissue mitochondria convert alpha-ketoglutarate to malate, or in the presence of added pyruvate, to citrate. Glutamate itself can not be metabolized by these mitochondria, a finding in keeping with the demonstration of negligible levels of NAD-glutamate dehydrogenase activity in adipose tissue mitochondria. Pyruvate stimulated alpha-ketoglutarate and malate conversion to citrate and reduced their oxidation to CO(2). It is proposed that under conditions of excess generation of NADH malate may act as a shuttle carrying reducing equivalents across the mitochondrial membrane. Malate at low concentrations increased pyruvate conversion $$Word$$ citrate and markedly decreased the formation of CO(2) by isolated adipose tissue mitochondria. Malate also stimulated citrate and isocitrate metabolism by these mitochondria, an effect that could be blocked by 2-n-butylmalonate. This potentially important role of malate in the regulation of carbon flow during lipogenesis is underlined by the observation that 2-n-butylmalonate inhibited fatty acid synthesis from pyruvate, but not from glucose and acetate, and decreased the stimulatory effect of pyruvate on acetate conversion to fatty acids.     

Effects of free fatty acids as membrane components on permeability of drugs across bilayer lipid membranes. A mechanism for intestinal absorption of acidic drugs

Studies of the influence of fatty acids, which were the component of intestinal mucosal lipids, on the permeability of several drugs across bilayer lipid membranes generated from egg phosphatidylcholine and intestinal lipid have been pursued. The permeability coefficients of $\text{p-}\text{aminobenzoic}$ acid, salicylic acid and $\text{p-}\text{aminosalicylic}$ acid (anionic-charged drug) increased when fatty acids such as lauric, stearic, oleic, linoleic and linolenic acid were incorporated into the bilayer lipid membranes generated from phosphatidylcholine. In the presence of methyl linoleate and oleyl alcohol, no enhancing effect on $\text{p-}\text{aminobenzoic}$ acid transfer was obtained. The effect of fatty acids was more marked at pH 6.5 than at pH 4.5. In contrast, upon the addition of fatty acids to intestinal lipid membranes which originally contained fatty acids, the permeability coefficient of $\text{p-}\text{aminobenzoic}$ acid tended to decrease, though the permeability through intestinal lipid membranes was larger than that of phosphatidylcholine membranes. The permeability of $\text{p-}\text{aminobenzoic}$ acid across bilayer lipid membranes from intestinal phospholipids was significantly decreased to about equal that of phosphatidylcholine membranes, and reverted to the value of intestinal lipid membranes when fatty acids were added to intestinal phospholipids. It seemed reasonable to assume that free fatty acids in the intestinal neutral lipid fraction could contribute to the increase in the permeability of $\text{p-}\text{aminobenzoic}$ acid. On the basis of above results, possible mechanisms for good absorbability of weakly acidic drugs from the intestine are discussed.     

Effects of glucagon, insulin and cyclic-AMP on mitochondrial calcium uptake in the liver.

The effects of glucagon and insulin administration $\text{in vivo}$ on hepatic mitochondrial Ca²⁺ uptake were compared with the effects of these hormones when they were added directly to the perfused liver. Glucagon administration increased mitochondrial calcium uptake both $\text{in vivo}$ and in the perfused liver. In contrast, while injection of insulin into rats stimulated, addition of insulin to the perfusate, inhibited Ca²⁺ uptake. Cyclic AMP, when added to the perfusate, also increased the uptake of Ca²⁺ by mitochondria, subsequently isolated. The possible implications of the results are discussed.     

Flexibility of coupling and stoichiometry of ATP formation in intact chloroplasts

Since coupling between phosphorylation and electron transport cannot be measured directly in intact chloroplasts capable of high rates of photosynthesis, attempts were made to determine $\text{ATP}\text{2}\text{e}$ ratios from the quantum requirements of glycerate and phosphoglycerate reduction and from the extent of oxidation of added NADH via the malate shuttle during reduction of phosphoglycerate in light. These different approaches gave similar results. The quantum requirement of glycerate reduction, which needs 2 molecules of ATP per molecule of NADPH oxidized was found to be pH-dependent. 9–11 quanta were required at pH 7.6, and only about 6 at pH 7.0. The quantum requirement of phosphoglycerate reduction, which consumes ATP and NADPH in a $\text{1}\text{1}$ ratio, was about 4 both at pH 7.6 and at 7.0. $\text{ATP}\text{2}\text{e}$ ratios calculated from the quantum requirements and the extent of phosphoglycerate accumulation during glycerate reduction were usually between 1.2 and 1.4, occasionally higher, but they never approached 2.Although the chloroplast envelope is impermeable to pyridine nucleotides, illuminated chloroplasts reduced added NAD via the malate shuttle in the absence of electron acceptors and also during the reduction of glycerate or CO₂. When phosphoglycerate was added as the substrate, reduction of pyridine-nucleotides was replaced by oxidation and hydrogen was shuttled into the chloroplasts to be used for phosphoglycerate reduction even under light which was rate-limiting for reduction. This indicated formation of more ATP than NADPH by the electron transport chain. From the rates of oxidation of external NADH and of phosphoglycerate reduction at very low light intensities $\text{ATP}\text{2}\text{e}$ ratios were calculated to be between 1.1 and 1.4.Fully coupled chloroplasts reduced oxaloacetate in the light at rates reaching 80 and in some instances 130 μmoles · mg^?1 chlorophyll · h^?1 even though ATP is not consumed in this reaction. The energy transfer inhibitor phlorizin did not significantly suppress this reduction at concentrations which completely inhibited photosynthesis. Uncouplers stimulated oxaloacetate reduction by factors ranging from 1.5 to more than 10. Chloroplasts showing little uncoupler-induced stimulation of oxaloacetate reduction were highly active in photoreducing CO₂. Measurements of light intensity dependence of quantum requirements for oxaloacetate reduction gave no indication for the existence of uncoupled or basal electron flow in intact chloroplasts. Rather reduction is brought about by loosely coupled electron transport. It is concluded that coupling of phosphorylation to electron transport in intact chloroplasts is flexible, not tight. Calculated $\text{ATP}\text{2}\text{e}$ ratios were obtained under conditions, where coupling should be expected to be optimal, i.e. at low phosphorylation potentials $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{]}\text{[}\text{P}{}_{\mathrm{<mtext>i</mtext>}}\text{]}$. Flexible coupling implies, that $\text{ATP}\text{2}\text{e}$ ratios should decrease with increasing phosphorylation potentials inside the chloroplasts.     

Mechanism of phospholipid biosynthesis in Escherichia coli: Acyl-CoA synthetase is not required for the incorporation of intracellular free fatty acids into phospholipid

Previous results have shown that acyl-CoA synthetase is required both for the incorporation of exogenous fatty acids into the phospholipids of $\text{E. coli}$ and for the transport of fatty acids into the cell. We have demonstrated that acyl-CoA synthetase is not required for the incorporation of intracellular free fatty acids into phospholipid. This finding indicates that the role of this enzyme in the incorporation of exogenously supplied fatty acids is primarily at the level of fatty acid transport.     

Control of membrane polar lipid composition in Acholeplasma laidlawii a by the extent of saturated fatty acid synthesis

The low level of endogenous fatty acid synthesis in Acholeplasma laidlawii A strain EF22 was found to be caused by a deficiency of pantetheine in the lipid-depleted growth medium. By supplementing the oleic acid-containing medium with increasing concentrations of pantetheine, saturated fatty acid synthesis was stimulated (having an apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 5 μM for pantetheine) and the incorporation of endogenously synthesized fatty acids in membrane lipids increased markedly. Furthermore, carotenoid biosynthesis was stimulated. Exogenous palmitic acid was found to inhibit partially the endogenous fatty acid synthesis. A gradual stimulation of fatty acid synthesis was accompanied by a linear increase in the molar proportion between the two dominating membrane glucolipids, monoglucosyldiacylglycerol and diglucosyldiacylglycerol. The total amount of charged membrane lipids decreased upon increasing the degree of fatty acid saturation. These regulations are discussed in terms of membrane stability, and influence of membrane molecular ordering and surface charge density on lipid polar head group synthesis.     

Methionine transport in S37 cells. Substrate-dependent function of amino acid transport system A in exchange processes

Methionine had been observed to interact with two principal transport systems for amino acids in mammalian cells, the A and L systems. The present study of methionine transport and of exchange processes through system A arose in the course of a study to define the specificity of a transinhibition effect caused by cysteine.Methionine uptake through two transport systems in the S37 cell was confirmed by the occurrence of a biphasic double-reciprocal plot for labeled methionine uptake. Preloading cells with methionine stimulated labeled histidine uptake through both systems A and L. Efflux of labeled methionine from cells was stimulated by histidine in a biphasic manner, so that both systems A and L can be used for exchange when methionine is the intracellular amino acid. Aminocycloheptanecarboxylic acid elicited exchange efflux of labeled methionine only through system L. α-Aminoisobutyric acid and $\text{N-}\text{methyl}\text{-α-}\text{aminoisobutyric acid}$ both stimulated efflux of labeled $\text{N-}\text{methyl}\text{-α-}\text{aminoisobutyric acid}$ from S37 cells. These findings are interpreted a showing that transport system A is capable of functioning as an exchange system depending upon the identity of intracellular and extracellular substrates available.     

The hormonal stimulation of ureogenesis in isolated hepatocytes through increases in mitochondrial ATP production

Isolated hepatocytes incubated with 2 mm ornithine-10 mm glutamine as substrates and challenged with either glucagon, epinephrine, or phenylephrine exhibited stimulated rates of urea production, and mitochondria isolated from these cells displayed an increased rate of energy-dependent citrulline formation. There was no change in the total carbamyl phosphate synthetase I activity, nor mitochondrial content of the positive effector N-acetyl glutamate after acute hormonal treatment. The time of onset of ureogenesis and its sensitivity to glucagon were compared with stimulation of glucose production from lactate-pyruvate. No apparent differences in time of onset or sensitivity of the responses were observed indicating both pathways may be stimulated by a common mechanism. Mitochondria prepared from cells treated with catecholamines exhibited increased rates of State 3 respiration and increased uncoupler-dependent ATPase activity, in addition to the increased rates of citrulline formation. There was also an elevated intramitochondrial content of ATP and an increased $\text{ATP}\text{ADP}$ ratio. The catecholamine-induced stimulation of ureogenesis was mediated by an α-adrenergic cyclic AMP independent mechanism. The addition of the α-adrenergic antagonist, dihydroergotamine, blocked both the epinephrine-induced stimulation of ureogenesis and also the stimulated functions in the isolated mitochondria. dl-Propranolol, a β-antagonist, inhibited the rise in cyclic AMP due to epinephrine, but had no effect on any of the other reactions measured. The effects of catecholamines on citrulline formation and urea production are correlated with the increased capacity of the mitochondria to generate ATP. It is suggested that both glucagon and catecholamines, acting via independent mechanisms, stimulate electron transport and the activity of the ATP-forming enzyme complex. The consequent elevated intramitochondrial ATP levels and $\text{ATP}\text{ADP}$ ratio enhance the rate of citrulline formation and hence ureogenesis.     

Motion of spin-labeled fatty acids in murine macrophages relation to cellular phagocytic activity

Macrophage membrane fluidity was investigated with respect to cellular phagocytic activity through the use of fatty acid spin labels.Spin-labeled fatty acid derivatives were incorporated into intact mouse peritoneal macrophages by exchange from bovine serum albumin. The electron spin resonance (ESR) spectra of the spin-labeled fatty acids in the macrophages showed a pronounced temperature dependence and a decrease in the hyperfine splittings ($\text{2T}{}_{\mathrm{|}}$) of the spectra as the nitroxide radical was moved away from the polar head group of the fatty acid derivatives.Spin-labeled macrophages underwent a time- and temperature-dependent decay, which was inhibited by preincubating the cells with mercuric chloride, heating at 56 °C, or by fixing them with 0.25 % glutaraldehyde.No correlation between the phagocytic activity of macrophages and membrane freedom of motion could be demonstrated. Treatment of macrophages with anti-macrophage serum or extended in vitro cultivation inhibited cellular phagocytic activity but exerted no effect on the motional freedom of the macrophage membrane. Enrichment of the fatty acid composition of the macrophage membrane with $\text{cis-}$ or $\text{trans-}\text{unsaturated}$ fatty acids had striking effects on cellular phagocytic activity, while no significant changes could be detected in the freedom of motion of incorporated fatty acid spin labels at the degree of specific enrichment achieved here. Thus no correlation between cellular phagocytic activity and lipid motion could be detected.