Effect of Monovalent Thallium Ions on Differentiation and Multiplication of Acanthamoeba castellanii

The vegetatively multiplying Acanthamoeba castellanii cells are transformed into cysts under unfavourable feeding conditions. The cyst formation may also be induced by treatment of the cells with DNA-synthesis inhibitors or by placing the cells into special ionic medium containing magnesium and calcium at pH 9, with aeration. During Acanthamoeba encystment the morphology of the cells changes significantly, namely a cellulose-protein cyst wall appears which is easily seen under the light and electron microscope. The process of encystment in Acanthamoeba castellanii is considered as a useful simple model of cytodifferentiation of eukaryotic cells.
This communication describes the effects of monovalent thallium ions on the differentiation and multiplication of Acanthamoeba cells growing in optimal feeding conditions. Thallium ions being potassium analogues are readily accumulated by cells. On the other hand, thallium ions, unlike potassium ions, are able to form complexes with some anions, which results in disturbances of some cellular functions.
Thallium ions, added to the growth medium of 2–3-days old Acanthamoeba culture at a concentration of 0.05–1.0 mM inhibit the population growth inducing the differentiation of cells into cysts. The increase of the thallium ion concentration up to 5 or 10 mM in the growth medium causes the very fast multiplication of Acanthamoeba cells. However, at these thallium ion concentrations no cysts can be observed.
Thus, on the basis of the experimental data it seems likely that thallium ions play some role in increasing the rate of multiplication and in switching on the differentiation process (encystment) in Acanthamoeba cells.     

Effects of Taurine on Calcium Ion Uptake and Protein Phosphorylation in Rat Retinal Membrane Preparations

The effects of taurine on ATP-dependent calcium ion uptake and protein phosphorylation of rat retinal membrane preparations were investigated. Taurine (20 mM) stimulates ATP-dependent calcium ion uptake by twofold in crude retinal homogenates. In contrast, it inhibits the phosphorylation of specific membrane proteins as shown by acrylamide gel electrophoresis and autoradiography. The close structural analogue of taurine, 2-aminoethylhydrogen sulfate, demonstrates similar effects in both systems, i.e., stimulation of ATP-dependent calcium ion uptake and inhibition of protein phosphorylation, whereas isethionic acid and guanidinoethanesulfonate have no effect on either system. A P1 subcellular fraction of the retinal membrane preparation that contains photoreceptor cell synaptosomes has a higher specific activity for the uptake of calcium ions. Phosphorylation of specific proteins in the P1 fraction is also inhibited by the addition of 20 mM taurine. Taurine has no effect on retinal ATPase activities or on phosphatase activity, thus suggesting that it directly affects a kinase system.     

Effect of Chlorophenol Analogues on the Oxidative Phosphorylation in Rat Liver Mitochondria

The inhibition of chlorophenol analogues on oxidative phosphorylation in rat liver mitochondria was studied using polarographic technique and some new findings that not only pentachlorophenol (PCP) but also other analogues inhibited the oxidative phoshorylation in a similar manner were made. The inhibitory activity was found to be roughly correlated with its dissociation constant of the inhibitor, PCP being the strongest, varying with the number and position of chlorine atoms in the molecule. The mode of the inhibition was classified into three types and discussed in detail.     

Effect of Sodium Ions on Calcium-Loaded Rat Heart Mitochondria and Frog Myocardium

Journal of Evolutionary Biochemistry and Physiology - The effect of calcium load on rat heart mitochondria [RHM(K+)], in which K+ ions in the matrix were partially replaced by Na+ ions [RHM(Na+)],...     

The Effect of ATP on Uptake of Monovalent Cations and Anions by Red Beet Slices

Externally added ATP inhibits sodium and potassium uptake byred beet tissue; this inhibition seems to be competitive. Theeffects of DNP and ATP additions on cation uptake were foundto be additive. ATP did not inhibit chloride and bromide uptake.The results and their implications are discussed.     

The Inhibitory Effect of 3-Nitropropionate on the Oxidative Phosphorylation in Rat Mitochondria

The effect of 3-nitropropionate (3-NPA)on oxidative phosphorylation by using mitochondria prepared from both rat liver and brain were investigated in connection with the toxicity of this material. It was found that 3-NPA inhibited oxidative phosphorylation. In this inhibition, the uptake of inorganic phosphate was blocked but the oxygen uptake was not influenced at all. Furthermore, increase in ATPase activity of intact mitochondria was shown by the addition of 3-NPA. Results showed that 3-NPA disturbed oxidative phosphorylation as an uncoupler. However, the degree of inhibition by 3-NPA was not so high in comparison with other well-known uncouplers.

Thus the toxicity of 3-NPA is not due to the inhibition of oxidative phosphorylation. 3-NPA also does not affect on cytochrome oxidase activity.     

Electron Transport and Oxidative Phosphorylation in Arum Spadix Mitochondria

Arum spadix mitochondria exhibited a rapid cyanide-resistantoxygen uptake when oxidizing malate, NADH₂ or succinate, anda slower, cyanide-sensitive oxygen uptake when oxidizing ascorbate+tetramethylphenylenediamine(TMPD). Cytochrome oxidase does not therefore appear to functionas the terminal oxidase in the presence of cyanide, and therather low cytochrome c oxidase activity obtained using ascorbate+TMPDmay exclude it from possessing a major role even in the absenceof cyanide. ATP synthesis has been shown to accompany substrateoxidation. In the presence of antimycin A the P: O ratio accompanyingmalate oxidation was reduced by half, while phosphorylationaccompanying NADH₂ or succinate oxidation was almost completelyabolished. It is proposed that electrons from exogenous NADH₂enter the electron transport chain at a site after that whereendogenous NADH2 donates electrons and that electrons from exogenousNADH₂ are not coupled to ATP synthesis at site 1. The cyanide-resistant,non-phosphorylating electron-transport pathway may functionin the absence of cyanide and account for the low efficiencyof energy conservation observed in this tissue.     

The Effect of Temperature on Oxidative Phosphorylation with Insect Flight Muscle Mitochondria

The effect of different temperatures on the biochemical activity and morphology of insect flight muscle mitochondria was examined. It was found that respiration and phosphorylation have the same thermal response at temperatures of 25°C. and below. The energy of activation for both systems is approximately 12,300 calories. Oxidation and phosphorylation can be uncoupled effectively by temperature, for at temperatures above 25°C. there is more rapid heat inactivation of phosphorylation. This is evident from reduced P/O values as well as from morphological deterioration in the mitochondrial population. The thermal response of both this sarcosomal enzyme system and the respiration in the living fly are quantitatively similar.     

Phosphorylation of Ion channels

The introduction of highly specific reagents such as enzymes and inhibitors directly into living cells has proven to be a powerful tool in studying the modulation of cellular activity by protein phosphorylation. The use of exogenous kinases can be thought of as a pharmacological approach: this demonstrates that phosphorylation can produce modulation, but does not address the question of whether the cell actually uses this mechanism under normal physiological conditions. The complementary approach, the introduction of highly specific inhibitors such as R subunit or PKI, does ask whether endogenous kinase activity is necessary for a given physiological response. Together these two approaches have provided rather compelling evidence that cAMP-dependent and calcium/phospholipid-dependent protein phosphorylations can regulate membrane excitability. In several cases single-channel analysis has allowed the demonstration that an ion channel itself or something very close to the channel is the phosphorylation target, and it seems reasonable to assume that this will also be the case for many if not all of the other systems described above. Have any general principles emerged from the results to date? Certainly it seems clear that protein phosphorylation regulates not one but many classes of ion channels. As summarized in the Table, different channels can be modulated in different cells, some channels are activated while others are inhibited, and in some cells more than one channel is subject to modulation by phosphorylation. The list in the Table is probably not yet complete, and indeed it is not inconceivable that all ion channels can under appropriate conditions be regulated by phosphorylation. What aspect of channel function is altered by phosphorylation? The total membrane current, I, carried by a particular species of ion channel is given by Npi, where N is the number of active channels in the membrane, p is the probability that an individual channel will be open, and i is the single-channel current. In principle a change in I, the quantity measured in whole cell experiments, could be caused by a change in any one (or more) of the parameters, N, p or i (see Fig. 1). In the two cases in which single-channel measurements have allowed this question to be investigated, changes in N (Shuster et al., 1985) and p (Ewald et al., 1985) have been observed. Here again it seems unlikely that any one mechanism operates in all cases, and it would not be surprising to find that phosphorylation of some other channel results in a change in i.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of Pyrrolnitrin on Electron Transport and Oxidative Phosphorylation in Mitochondria Isolated from Neurospora crassa

Pyrrolnitrin, at low concentrations, uncouples oxidative phosphorylation in Neurospora mitochondria. At higher concentrations, pyrrolnitrin inhibits electron transport both in the flavine region and through cytochrome oxidase.     

The Effect of γ-Aminobutyric Acid on Substrate-Level Phosphorylation in Brain Mitochondria

Abstract: A consequence of the metabolism of γ-aminobutyric acid (GABA) via the "GABA shunt" should be a decreased rate of substrate-level phos- phorylation of GDP to GTP. ³²P₁ labeling of nucleotides was, therefore, studied in uncoupled brain mitochondria with α-ketoglutarate or a-ketoglutarate + GABA as substrates. The addition of an equimolar amount of GABA resulted in an approximately 50% reduction of the final specific activity of all mitochondrial nucleotides. This effect was completely reversed by aminooxyacetic acid. GABA did not affect the time course of nucleotide labeling. Although delineation of the mechanism involved requires further study, these preliminary results suggest an important modulatory role of GABA in the intermediary metabolism of brain mitochondria.     

Intermediate Reactions of Oxidative Phosphorylation in Mitochondria From Cabbage

Respiratory control ratios between 2.0 and 9.0 were obtained by comparison of the respiratory rates of cabbage mitochondria in the presence and in the absence of individual components of the system used to provide ADP and by comparing the rates before and after exhaustion of added ADP. These results indicate that respiration in cabbage mitochondria is controlled by the availability of ADP, which serves as the phosphate acceptor.Pentachlorophenol (PCP), 2,4-dinitrophenol (DNP), gramicidin and oleic acid inhibited phosphorylation to a greater extent than respiration in the cabbage mitochondria, but these reagents did not stimulate respiration in the absence of a phosphate acceptor. Respiration was stimulated by DNP only in the presence of added ATP.2,4-Dinitrophenol, pentachlorophenol, dicumarol and gramicidin did not stimulate ATPase activity either in the presence or absence of added Mg(2+). Oleic acid stimulated ATPase activity in the presence of added Mg(2+), but did not stimulate respiration even in the presence of added ATP.The ATP-(32)Pi exchange rate was increased many fold in the presence of added Mg(2+). Oleic acid and 2,4-dinitrophenol inhibited the exchange almost completely.     

Spermidine Uptake by Mitochondria of Helianthus tuberosus

In the present work evidence is provided that spermidine, a polyamine largely present in plant tissues, may be transported, at physiological concentrations, into the matrix space of mitochondria isolated from tubers of Helianthus tuberosus L. cv OB1 (Jerusalem artichoke). It is concluded that the movement of spermidine strictly depends on membrane potential, since it is drastically blocked by valinomycin and only slightly sensitive to nigericin. Mg²⁺ and K⁺ inhibit the transport of spermidine in line with the general concept that these cations compete for the same binding sites on the mitochondrial membrane. In contrast to previous data on mammalian mitochondria, spermidine uptake by plant mitochondria does not depend on the presence of inorganic phosphate. This latter result, along with evidence that Ca²⁺ does not affect accumulation of spermidine, indicates that the control of the polyamine uptake mechanism in plant mitochondria is distinct from that of mammalian systems.     

Feedback Regulation and Time Hierarchy of Oxidative Phosphorylation in Cardiac Mitochondria

To determine how oxidative ATP synthesis is regulated in the heart, the responses of cardiac mitochondria oxidizing pyruvate to alterations in [ATP], [ADP], and inorganic phosphate ([P_i]) were characterized over a range of steady-state levels of extramitochondrial [ATP], [ADP], and [P_i]. Evolution of the steady states of the measured variables with the flux of respiration shows that: (1) a higher phosphorylation potential is achieved by mitochondria at higher [P_i] for a given flux of respiration; (2) the time hierarchy of oxidative phosphorylation is given by phosphorylation subsystem, electron transport chain, and substrate dehydrogenation subsystems listed in increasing order of their response times; (3) the matrix ATP hydrolysis mass action ratio [ADP] × [P_i]/[ATP] provides feedback to the substrate dehydrogenation flux over the entire range of respiratory flux examined in this study; and finally, (4) contrary to previous models of regulation of oxidative phosphorylation, [P_i] does not modulate the activity of complex III.     

The Respiratory Chain of Plant Mitochondria. II. Oxidative Phosphorylation in Skunk Cabbage Mitochondria

Mitochondria were prepared from the spadices of skunk cabbage (Symplocarpus foetidus) whose respiratory rate with succinate and malate showed 15% to 30% sensitivity to cyanide inhibition, and which showed respiratory control by added ADP. The observed respiratory control ratios ranged from 1.1 to 1.4. The change in pH of the mitochondrial suspension was recorded simultaneously with oxygen uptake: alkalinization of the medium, expected for phosphorylation of ADP, coincided with the period of acceleration in oxygen uptake caused by addition of an ADP aliquot. The ADP/O ratios obtained were 1.3 for succinate and 1.9 for malate. In the presence of 0.3 mm cyanide, the ADP/O ratio for succinate was zero, while that for malate was 0.7. These results are consistent with the existence of an alternate oxidase which interacts with the flavoprotein and pyridine nucleotide components of the respiratory chain and which, in the presence of cyanide, allows the first phosphorylation site to function with an efficiency of about 70%. In the absence of respiratory inhibitors, the efficiency of each phosphorylation site is also about 70%. This result implies that diversion of reducing equivalents through the alternate oxidase, thereby bypassing the 2 phosphorylation sites associated with the cytochrome components of these mitochondria, occurs to a negligible extent during the oxidative phosphorylation of ADP or State 3.Addition of ADP or uncoupler to skunk cabbage mitochondria respiring in the controlled state or State 4, results in reduction of cytochrome c and the oxidation of the cytochromes b, ubiquinone and pyridine nucleotide. A site of interaction of ADP with the respiratory chain between cytochromes b and cytochrome c is thereby identified by means of the crossover theorem. Flavoprotein measured by fluorescence is also oxidized upon addition of ADP or uncoupler, but flavoprotein measured by optical absorbance changes becomes more reduced under these conditions. Depletion of the mitochondria by pretreatment with ADP and uncoupler prevents reduction of most of the fluorescent flavoprotein by succinate. These results indicate that skunk cabbage mitochondria contain both high and low potential flavo-proteins characterized by different fluorescence/absorbance ratios similar to those demonstrated to be part of the respiratory chain in mitochondria from animal tissues.