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Summary Mitochondria from L1210 mouse leukemia cells show superstoichiometric → H + /2e − ejection and → Ca 2+ /2e − uptake ratios in the absence of phosphate. Kinetic analysis of Ca 2+ -induced respiratory jumps shows a very rapid, early burst of H + ejection and Ca 2+ uptake dependent on energy-coupled electron transport but with extraordinarily high stoichiometry (→H + /2e − > 20; →Ca 2+ /2e − > 10), followed by a slower phase with normal stoichiometry (→H + /2e − =4–5; →Ca 2+ /2e − =4.0). The early burst of H + ejection, which has a higher K M for Ca 2+ than respiratory stimulation, accounts for the superstoichiometry phenomenon; it requires energization by state 4 respiration.

Summary The rate of formation of the type I spectral change upon drug addition to a suspension of isolated rat liver cells was used to study factors that influence drug uptake by the hepatocytes. Although considerably slower than in liver homogenates and microsomes, drug combination with cellular cytochrome P-450 was still rapid and occurred within a few seconds. The effects of varying temperature and concentration and lipid solubility of the drugs studied as well as the lack of effect of preincubation of the cells with rotenone on the rate of formation of the type I spectral change, lead us to suggest that drug uptake into the hepatocytes occurs by a non-energy requiring diffusion process.

The oxidation of malate, citrate, and α-ketoglutarate by cauliflower (Brassica oleacea L.) bud mitochondria was inhibited by rotenone. This inhibition was relieved upon addition of NAD+ to the medium, and ADP/O values were lowered to less than 2 when both rotenone and NAD+ were present. Dinitrophenol did not affect the relief of rotenone inhibition by exogenous NAD+.

SUMMARY The yeast/mould (Y/M) dimorphism of three strains of the genus Mycotypha (Zygomycetes, Mucorales) was investigated. The Y-form is most readily induced in M. africana (CBSI22.64), whereas M. microspora (CBS230.32) has a strong tendency to grow as M-form. The Y-form is promoted by anaerobiosis, increased Pco 2, increased temperatures, pH values between 5-8 and 6-5, and 10% (w/v) glucose in the culture medium, as well as by the addition of certain inhibitors of the respiratory chain or mitochondrial protein synthesis. In contrast, M-form is stimulated by aerobic conditions, by temperatures below approximately 20 °C and at pH values below 4-5 and above 7-4. Ultraviolet-induced mutants with a defective respiratory system invariably grow as pure Y-form. Suppression of aerobic metabolism leads to a simultaneous suppression of mycelial growth. This correlation was confirmed by studies of fine structure with the electron microscope.

Rotenone, a potent inhibitor of mitochondrial respiration is also an effective antimitotic agent. The addition of either rotenone or Colcemid to exponentially growing Chinese hamster ovary cells resulted in a dramatic increase in mitotic index after 90 min. When the cultures were washed free of the drugs, mitosis was completed and the cells progressed into G 1 at approximately the same rate. Further similarity of rotenone-arrested cells to Colcemid-induced mitotic inhibition was apparent at the ultrastructural level. Mitotic cells treated by either drug contained monopolar spindles with chromosomes grouped around centriole pairs near the cell center. Occasional microtubules were seen near the kinetochore and centrioles. These observations, along with the fact that rotenone inhibited the binding of 3H-colchicine to isolated bovine brain tubulin, suggested that rotenone inhibited mitosis by binding directly to tubulin to prevent microtubule assembly.

The effect of three groups of metabolic inhibitors on the incorporation of Fe and release of bicarbonate from transferrin by rabbit reticulocytes was measured. Inhibitors which affect reticulocyte Fe and transferrin uptake to the same extent (sodium arsenite, N-ethylmaleimide and iodoacetamide); those which inhibit reticulocyte Fe uptake to a greater extent than transferrin uptake (NaN3, NaF, NaCN, rotenone, oligomycin, 2,4-dinitrophenol and cycloheximide); and compounds which after reticulocyte heme synthesis (CoCl2, isonicotinic acid hydrazide and hemin) were used. In each case the effect on Fe incorporation and bicarbonate release was the same Thus, additional evidence has been obtained for the idea that the reticulocyte-mediated release of Fe and bicarbonate from transferrin are tightly coupled. The results are consistent with the hypothesis that an enzymatic attack on transferrin-bound bicarbonate is involved in the removal of Fe from transferrin by erythroid cells.

Succinate-energized guinea pig heart mitochondria prepared by the Polytron technique take up Ca2+ under limited loading conditions in a manner similar to mitochondria prepared with bacterial proteinase, despite the associated loss of high affinity nonenergized Ca2+ binding by proteinase-prepared mitochondria. Phenethylbiguanide inhibits initial energized Ca2+ uptake; 50 % inhibition occurs at 2 mm biguanide in both Polytron and proteinase preparations and this inhibition is at least partly dissociated from inhibition of substrate oxidation. Energized heart mitochondria lose accumulated Ca2+ by a process dependent on temperature, Ca2+ to protein ratio, and monovalent cation. Phenethylbiguanide also inhibits this Ca2+ release. Nonenergized Ca2+ binding is 50 % inhibited by 1.0 mm biguanide, and inhibition is intermediate between competitive and non-competitive. Phenethylbiguanide also inhibits Ca2+ uptake into both energized and nonenergized guinea pig liver mitochondria; 50% inhibition occurs at 1.1 and 8.5 mm biguanide, respectively. In contrast to heart mitochondria, biguanide inhibition of energized Ca2+ uptake into liver mitochondria exactly parallels inhibition of Ca2+ and of ADP + Pi-stimulated respiration. Biguanide inhibition of nonenergized Ca2+ binding to liver mitochondria is competitive. [14C]Phenethylbiguanide is taken up by energized and nonenergized heart and liver mitochondria; binding capacity is about 60 nmoles per mg of protein. At 1 mm biguanide, nonenergized heart mitochondria bind about 6 nmoles per mg of protein; energized heart mitochondria accumulate labeled biguanide in a rapid phase, which corresponds to inhibition of initial 45Ca2+ uptake, and a slower, progressive uptake phase. The data suggest that both energized Ca2+ movement and biguanide binding occur at the locus which accounts for low affinity Ca2+ binding in the absence of energy. Site-specific inhibition of coupled respiration may result from biguanide binding primarily in or near this low affinity Ca2+ locus. The high affinity locus may play a modulator role in energized Ca2+ transport and in respiratory inhibition by biguanides. Interference with mitochondrial Ca2+ transport may in part explain the cellular effects of guanidine derivatives.

The capability of ascites tumour mitochondria to oxidize externally formed NADH has been investigated in intact cells. Lactate has been used as the source of reducing equivalents and the oxidation of this substrate to pyruvate has been estimated. Ascites cells, under conditions of endogenous metabolism, are able to produce pyruvate upon addition of lactate. This effect is prevented by aminooxyacetate, an inhibitor of glutamate—oxalacetate transaminase (EC 2.6.1.1). Half-maximal inhibition by aminooxyacetate is attained at a concentration of approx. 30 μM. Oxidation of lactate is also sensitive to inhibitors of mitochondrial electron and energy transfer and it is enhanced by α-oxoglutarate plus aspartate. These data demonstrate that reducing equivalents can be transported across the mitochondrial membrane of intact Ehrlich ascites tumour cells by the malate—aspartate shuttle.

The distributions of phosphate and of a dicarboxylate were compared in rat liver mitochondria. The phosphate can be divided into an ionised part, assuming a Donnan relation holds between it and the other anion, and a non-ionised part which is found to be stoichiometrically related in the ratio 1:1.8 to the mitochondrial Ca content. When arsenate and phosphate distributions are compared, the distribution calculated for ionised phosphate corresponds to that measured for the arsenate. The ionised phosphate was deduced from the total by deduction of an amount calculated to be equivalent to the Ca content. Both sets of results support the concept of mitochondria normally carrying non-ionised phosphate as the Ca salt. A competition between arsenate and ionised phosphate for accumulation is described.

The uptake of [N-14C methyl]morphine into rat, mouse, or guinea pig brain slices does not meet the criteria for an active transport process. (1) Methods are described for the extraction and TLC of morphine at 96% recovery levels, and for the determination of14C at 70–90% counting efficiency with brain tissue samples. (2) After morphine had been in the tissue for 30 min, 96% of the radioactivity was extractable, and 99% of this was unmetabolized morphine. (3) The ratio of the concentration in the tissue to that in the medium was2.22 ± 0.16 e.s.e., from3 × 10−8 Mto2 × 10−3 M. Therefore, the uptake is not saturable. (4 Strong metabolic inhibitors,e.g. cyanide, at concentrations that completely block amino acid transport, did not markedly reduce the uptake of morphine. (5) The uptake into and efflux from brain tissue was not affected by concentrated morphine, analogs, or narcotic antagonists. Therefore, the uptake of morphine showed no signs of the competitive inhibition that is typical of active transport processes. In addition, morphine uptake was not affected by acute or chronic morphine injections in the mouse or guinea pig. (6) The uptake of morphine into brain tissue was temperature dependent, but it occurred at 0.5°C and also after the tissue was kept at 95°C for 10 min.

SYNOPSIS Drug sensitivities of a lower trypanosomatid, Leptomonas sp. from the hemipteran Dysdercus, to antitrypanosomatid agents were estimated by growth inhibition and inhibition of polarographically measured O2 uptake of cells and particulate fractions. Several standard trypanocides, antimalarials, and electron-transport inhibitors were used. Acriflavine, quinacrine, primaquine, pentamidine, and ethidium, each at ∼1 mM, inhibited cellular O2 uptake by 50% after 3-hr incubation. Suramin and melarsen were inactive. Crude particulate fractions oxidized L-α-glycerophosphate at 80% the rate of succinate oxidation and L-proline at 55% the succinate rate, leading to choice of succinate as standard respiratory substrate. Concentrations for 50% inhibition of growth were (μg/ml): ethidium bromide 0.009, acriflavine HCl 0.12, Antrycide 0.83, pentamidine diisethionate 1.5, and stilbamidine isethionate 1.1. Possible use of this trypanosomatid as model organism for screening antitrypanosomatid agents, and use of ethidium and acriflavine as initial model compounds are discussed.

The effects of 2-phenylisatogen and menadione on some mitochondrial reactions have been studied. The respiration with NAD+-linked substrates was stimulated, but respiration with succinate was not affected. The stimulation of respiration was inhibited by p-hydroxymercuribenzoate, but not by other respiratory chain inhibitors. Potassium cyanide and sodium sulphide stimulated NADH oxidation in the presence of 2-phenylisatogen or menadione. 2-Phenylisatogen was found to be converted to 2-phenylindolone. It is concluded that both 2-phenylisatogen and manadione are acting as electron acceptors in the NADH dehydrogenase region of the respiratory chain. Possible mechanisms for the reaction and for the stimulation observed in the presence of cyanide are discussed.

The interaction of La3+ with rat liver mitochondria was examined with a wide variety of techniques permitting measurement of respiratory and structural responses. It is concluded that La3+ is accumulated by mitochondria in a process that is at least partially energy-dependent, and is bound with quite high affinity to membrane-associated sites both external and internal to the inner membrane. The relative insensitivity of the accumulation process to respiratory inhibitors and to the permeant anion acetate has interesting implications for the mechanism of active cation transport.

A multiculture system employing confluent monolayers of embryonic human skin fibroblasts has been developed to provide a model to evaluate the Cytograf, registering the cytotoxic effects of actinomyci

Diclidophora merlangiwas found to consume oxygen under a variety of experimental conditions. TheQo2of homogenates was 0.415 μl oxygen consumed/mg wet wt/h. Oxygen uptake was reduced in the presence of inhibitors: cyanide 0.1 mM and 5.0 mM, 100%; rotenone 10−6M, 56%; antimycin A10−6M, 63 %; malonate 10 mM, 36 %; arsenite 5 mM, 46 %; fluoroacetate 5 mM, 47 %. Worms died after 1 h incubation in 1 mM cyanide. It is suggested that these data provide evidence for the existence of a functional TCA cycle and respiratory chain of the classical pattern in this fluke.

Bone cells were isolated by collagenase-hyaluronidase digestion of 19–20 day-old fetal rat calvaria. It is shown that contamination from calcified matrix can give erroneously high values for total cell calcium and can mask intracellular calcium exchange. Filtration of the cell suspension through 35 μ mesh nylon net and a five minute treatment with a cold pH 6.0 isotonic salt solution removed the contamination. Total calcium for cells prepared in this manner was 16.8 mmoles/kg wet weight. 45Ca uptake studies revealed an active component of calcium exchange.

Rotenone, carbonyl cyanide chloromethoxyphenylhydrazone and 2,4-dinitrophenol cause parallel falls in ox pituitary ATP content and growth-hormone release in the presence of 65mm-K+. Carbonyl cyanide chloromethoxyphenylhydrazone (0.4μg/ml) prevented the rise in growth-hormone release, but not the rise in 3′:5′-cyclic AMP content after addition of prostaglandin E2 (1μg/ml). The rate of hormone release from unstimulated slices and from slices in the presence of Ba2+ at 2.3 or 6.9mm was proportional to the ATP content of the slices. Carbonyl cyanide chloromethoxyphenylhydrazone at concentrations that inhibited release in the presence of the three secretagogues did not alter the pituitary contents of Na+, K+ or Ca2+. The implications of these observations are discussed in terms of the mechanism of stimulus–secretion coupling.

Virulent Treponema pallidum has been shown to consume O 2 at a rate similar to that of the known aerobic spirochaete, Leptospira. Such O 2 uptake is cyanide sensitive, indicating a functioning cytochrome oxidase. Inhibition of O 2 uptake by azide, chlorpromazine, and amytal further suggests a functioning electron transport system for the oxidation of nicotinamide adenine dinucleotide (reduced) to O 2 . Evidence is consistent with the probability that this terminal electron-transport system is coupled to oxidative phosphorylation. The potential significance of these findings is discussed.

1. The application of metabolic inhibitors to nerve-muscle synapses on ‘white’ and ‘red’ fibres in the retractor unguis muscles of P. americana and B. giganteus resulted in a dramatic increase in the spontaneous miniature potential discharge and was accompanied by a summation of the miniature potentials to form ‘composite’ potentials. 2. Axon terminals associated with ‘white’ muscle fibres responded faster to metabolic inhibitors than those axon terminals associated with ‘red’ muscle fibres. 3. Correlated ultrastructural and electrophysiological studies inferred that a tentative relationship existed between the miniature potential activities and synaptic vesicle distributions of the nerve-muscle synapses during the phases of metabolic inhibition.

Summary It has been postulated and generally accepted that mitochondria contain two sets of binding sites for calcium, high affinity and low affinity sites. The high affinity binding of calcium is insensitive to low levels of respiratory inhibitors (antimycin plus rotenone) but completely suppressed by uncouplers. We have found that in beef heart mitochondria high affinity binding is completely suppressed by relatively high levels of respiratory inhibitors for the first two coupling sites, and that these levels of inhibitors have no significant effect on oxidative phosphorylation or energy-linked calcium uptake driven by electron flow at the third coupling site. Similar results have also been obtained in rat liver mitochondria. It is concluded that the so-called high affinity binding of calcium observed in the presence of low levels of inhibitors of respiration is actually the result of energy-linked uptake of calcium due to residual electron transfer remaining in the presence of low levels of inhibitors.

The proportions of active (dephospho) and inactive (phospho) forms of pyruvate dehydrogenase, and the corresponding adenine nucleotide contents have been determined in isolated rat liver mitochondria. Uncoupling of oxidative phosphorylation by carbonylcyanide m-chlorophenylhyclrazone leads, in a dose-dependent manner, to conversion of pyruvate dehydrogenase from the inactive to the active form. The effect of uncoupler on enzyme interconversion is counteracted by 2-oxoglutarate which also increases the low ATP/ADP ratios resulting from uncoupling. Similar effects as with carbonylcyanide m-chlorophenylhydrazone are obtained with Ca2+ and rotenone. In the latter case 2-oxoglutarate again restores both the equilibrium of the active and inactive forms of pyruvate dehydrogenase and the mitochondrial ATP/ADP ratio. The elevation of the ATP potential by 2-oxoglutarate is most likely due to substrate level phosphorylation. Different ATP/ADP ratios were established in mitochondria by adding glucose and varying amounts of hexokinase. On plotting the ratios of ATP/ADP against phosphoenzyme/dephosphoenzyme ratios a straight correlation was obtained. From this it can be derived that a drop of ATP/ADP from 1 to values below 0.4 is accompanied by an about eight-fold increase in pyruvate dehydrogenase activity, due to conversion of the phosphoenzyme to the dephospho form. The inactivating effect of a high ATP potential on pyruvate dehydrogenase is missing in the presence of pgruvate as single substrate. It is suggested that, in vivo, an increased (or decreased) supply of free fatty acids leads to an elevation (or lowering) of the mitochondrial ATP potential whereby adenine nucleotide translocase inhibition by long-chain acyl-CoA may be involved. These fluctuations of the mitochondrial energy state may represent a basic mechanism for the regulation of the pyruvate dehydrogenase system.

It has been found that amytal competitively inhibits succinate (+ rotenone) oxidation by intact uncoupled mitochondria. Similar results were obtained in metabolic state 3, the K i value being 0.45 mM. Amytal did not effect succinate oxidation by broken mitochondria and submitochondrial particles (at a concentration which inhibited succinate oxidation by intact mitochondria). Amytal inhibited the swelling of mitochondria suspended in ammonium succinate or ammonium malate but was without effect on the swelling of mitochondria in ammonium phosphate and potassium phosphate in the presence of valinomycin+carbonylcyanide p -trifluoromethoxyphenylhydrazone. Using [ 14 C] succinate and [ 14 C] citrate it has been shown that amytal inhibited the succinate/succinate, succinate/P i , succinate/malate, and citrate/citrate and citrate/malate exchanges. Amytal inhibited P i transport across mitochondrial membrane only if preincubated with mitochondria. Other barbiturates: phenobarbital, dial, veronal were found to inhibit [ 14 C]succinate/anion (P i , succinate, malonate, malate) exchange reactions in a manner similar to amytal. It is concluded that barbiturates non-specifically inhibit the dicarboxylate carrier system, tricarboxylate carrier and P i translocator. It is postulated that the inhibition of succinate oxidation by barbiturates is caused mainly by the inhibition of succinate and P i translocation across the mitochondrial membrane.