Yeast mitochondrial calcium uptake: Regulation by polyamines and magnesium ions

Spermine, spermidine, and magnesium ions modulate the kinetic parameters of the Ca²⁺ transport system ofEndomyces magnusii mitochondria. Mg²⁺ at concentrations up to 5 mM partially inhibits Ca²⁺ transport with a half-maximal inhibiting concentration of 0.5 mM. In the presence of 2 mM MgCl₂, theS _0.5 value of the Ca²⁺ transport system increases from 220 to 490 µM, which indicates decreased affinity for the system. Spermine and spermidine exert an activating effect, having half-maximal concentrations of 12 and 50 µM, respectively. In the case of spermine, theS _0.5 value falls to 50–65 µM, which implies an increase in the transport system affinity for Ca²⁺. Both Mg²⁺ and spermine cause a decrease of the Hill coefficient, giving evidence for a smaller degree of cooperativity. Spermine and spermidine enable yeast mitochondria to remove Ca²⁺ from the media completely. In contrast, Mg²⁺ lowers the mitochondrial buffer capacity. When both Mg²⁺ and spermine are present in the medium, their effects on theS _0.5 value and the free extramitochondrial Ca²⁺ concentration are additive. The ability of spermine and Mg²⁺ to regulate yeast mitochondrial Ca²⁺ transport is discussed.     

Cyclic AMP stimulation of calcium efflux from kidney,liver, and heart mitochondria

Summary The effect of cyclic AMP on subcellular calcium turnover was studied in isolated kidney, liver and heart mitochondria. The calcium concentration of the incubating medium was determined by fluorometric methods after its separation by millipore filtration. Liver and kidney mitochondria take up calcium in exchange for H⁺ and lower the medium calcium to 1 to 40×10^–6 m in less than 2 min. Cyclic AMP produces an instantaneous release of calcium from mitochondria and a rise in the steady-state calcium concentration of the medium. A new medium calcium level of 0.7 to 3×10^–4 m is achieved in less than 3 sec and is proportional to cyclic AMP concentrations between 10^–7 and 3×10^–6 m. Cyclic AMP is inactive above 5×10^–6 m and below 10^–7 m. Cyclic IMP, 5 AMP, dibutyryl cAMP are inactive at any concentration. Cyclic GMP is active at 10^–5 m and competitively inhibits cyclic AMP action. The same staedy-state calcium level is reached from higher or, lower calcium concentrations, i.e. whether cyclic AMP is added before or after the addition of calcium to the mitochondrial suspension. At low calcium or phosphate concentrations, the calcium released by cyclic AMP is immediately reaccumulated by the mitochondria is less than 2 min with a further release of H⁺. This pulse can be repeated by sequential additions of cyclic AMP. The transient or sustained response to cyclic AMP depends on the medium calcium x phosphate product and presumably on the presence or absence of calcium phosphate precipitate inside the mitochondria. These results support the hypothesis that cyclic AMP regulates cytoplasmic calcium by controlling the mitochondrial calcium efflux rate. This mechanism may be involved in the regulation of calcium transport and in some hormonal effects mediated by cyclic AMP.     

Effect of 2,6-Diisopropylphenol and Halogenated Anesthetics on Tetraphenylphosphonium Uptake by Rat Brain Synaptosomes: Determination of Membrane Potential

The effect of 2,6-diisopropylphenol (propofol) in comparison to that of the halogenated anesthetics enflurane, isoflurane, and halothane on tetrapenylphosphonium uptake by rat brain synaptosomes was studied. A direct method to separately measure the synaptosomal and the mitochondrial transmembrane potential by using the tetraphenylphosphonium cation (TPP⁺) was utilized. The latter is a lipophylic charged molecule which distributes between two compartments according to the transmembrane electrical potential in the presence or absence of 60 mM KCl as a synaptosomal membrane depolarizing agent. After previously reporting the damages induced by general anesthetics on isolated mitochondria, the aim of this paper was to study their possible action on the synaptosomal membrane potential and whether or not drugs concentrations damaging isolated mitochondria are also effective on synaptosomal mitochondria. The results indicated that, in the presence of glucose, mitochondria included in synaptosomes were able to maintain a transmembrane potential of 202 ± 8 mV (mean ± SD) while the synaptosomal membrane showed a potential of 78 ± 8 mV (mean ± SD). When anesthetic concentrations (0.6–1 mM propofol, 10–40 M enflurane, 30–50 M isoflurane, 8–15 M halothane) that impair mitochondrial energy metabolism were used, the synaptosomal transmembrane potential was maintained and, in addition, a slight increase of the TPP⁺ taken up was observed as the anesthetic concentration was increased.     

Spermine binding to subsynaptosomal fractions of rat brain cortex

Binding sites for [¹⁴C]spermine have been identified in rat brain cortex subcellular fractions. The binding, characterized by using synaptosomal membranes, is specific for spermine. It was not detected below 20°C and increased about three/four-fold with a temperature rise of 10°C. Binding occurred only in the presence of-SH reducing agents. It was completely suppressed by metal chelating agents, and was stimulated about four-fold by 1–5×10^–5 M Fe²⁺. Smaller increases were observed in the presence of Mn²⁺, Ni²⁺, Ca²⁺, Mg²⁺, and Zn²⁺; in ocntrast, millimolar concentrations of most divalent cations inhibited the binding differently (Mn²⁺=Ni²⁺=Zn²⁺=Co²⁺Mg²⁺>Ca²⁺).Bound radioactive spermine was not displaced by the addition of high concentrations of unlabelled polyamine or chelating agents, nor by precipitation and washing of the membranes with 10 percent trichloroacetic acid, or by boiling of the precipitate in the presence of 1.0 percent SDS and 10 percent -mercaptoethanol. The trichloroacetic acid precipitate showed two radioactive bands, corresponding to low Mr (<8,000) components, after SDS-polyacrylamide gel electrophoresis and fluorography. The Fe²⁺-stimulated [¹⁴C]spermine binding was neither influenced by a previous heating of the membranes at 100°C for 30 minor by trypsin or pronase digestion, whereas the heat-treatment increased the binding occurring in the absence of Fe²⁺ by about two fold. A non-enzymatic formation of a spermine-metal complex tightly bound to some membrane peptide(s) is suggested.     

Conversion of glycerol to 1,3-propanediol by a newly isolated thermophilic strain

Of 60 different thermophilic enrichment cultures, 16 converted glycerol anaerobically to 1,3-propanediol. Two PD-forming strains were further enriched, isolated, and characterised. For the most active strain, AT1, the optimal cultivation parameters for pH and temperature were determined as 5.8 to 6.0 and 58°C, respectively. In batch-fermentations with AT1, 6.4 g propanediol per litre was formed with a productivity of 0.17 g l^–1 h^–1.     

Effects of ethanol and other alkanols on the temperature relations of glucose transport and fermentation inSaccharomyces cerevisiae

Summary Ethanol, isopropanol, propanol and butanol exponentially inhibited the maximum velocity of the glucose transport system ofSaccharomyces cerevisiae, determined by use of the non-metabolizable analogued-xylose. While the exponential inhibition constants increased with the lipid solubility of the alkanols, they were independent of temperature in the range 21°–35°C: the Arrhenius plots (modified according to the theory of absolute reaction rates) of the initial maximum rates of xylose transport were linear and parallel in both the absence and presence of alkanols. Thus, the alkanols did not affect the enthalpy of activation of the glucose transport system (H ^± was 12 190 cal mol^-1) but decreased the entropy of activation. The following entropy coefficients (decrease in activation entropy per unit concentration of alkanol) were obtained: ethanol,-0.84; isopropanol,-1.21; propanol,-1.41 and butanol,-3.18 entropy units per mole per liter. The temperature relations of glucose fermentation with and without ethanol by resting cells over the temperature range studied (15°–35°C) were nearly identical with those of the glucose transport system, suggesting that the latter mediates the rate-limiting step of the former and that this relationship is maintained in the presence of ethanol.     

Cyclic nucleotide- and calcium-independent phosphorylation of proteins in rat brain polyribosome: Effects of ACTH,spermine, and hemin

The incorporation of [-³²P]ATP into proteins of rat brain polyribosomes was studied in vitro. The effects of cyclic nucleotides, calcium, hemin, ACTH, GTP, and spermine were examined. The incorporation of phosphate into proteins increased with time and phosphatase activity was very low; thus, the extent of phosphorylation was predominantly a reflection of protein kinase activity. Phosphorylation of proteins was not sensitive to Ca²⁺ in the presence or absence of either calmodulin or phosphatidylserine. Phosphorylation was also unaffected by cyclic nucleotides in the absence of exogenous enzymes. However, addition of a cMAP-dependent protein kinase together with cAMP resulted in a stimulation of the incorporation of phosphate into 4 phosphoproteins (pp70, pp58, pp43, and pp32); phosphorylation of pp32 was completely dependent on the addition of the kinase. ACTH (1–24), (11–24), and spermine inhibited the endogenous phosphorylation of one protein band (pp30). The phosphorylation of this 30 kD band was also selectively increased by hemin (5 M). Higher concentrations of hemin exerted an inhibitory effect on the majority of the phosphoproteins. Protein phosphatase activity was not influenced by ACTH or spermine. The specific inhibition of pp30 phosphorylation by ACTH or spermine is most probably explained by an interaction with a cyclic nucleotide- and Ca²⁺-independent protein kinase.     

Vacuolar pH oscillations in mesophyll cells accompany oscillations of photosynthesis in leaves: Interdependence of cellular compartments,and regulation of electron flow in photosynthesis

Oscillations of photosynthesis induced in leaves of Vicia faba L. were accompanied by oscillations not only in the pH of the chloroplast stroma, but also by pH oscillations in the cytosol and in the vacuole of leaf mesophyll cells. Cytosolic pH oscillations were in phase with stromal oscillations, but antiparallel to vacuolar pH oscillations. During maxima of photosynthesis, the cytosolic pH exhibited maxima and the vacuolar pH minima. Vacuolar acidification is interpreted to be the result of energized proton transport from the cytosol into the vacuole. Since the ratio of dihydroxyacetone phosphate to phosphoglycerate is maximal during the peaks of photosynthesis (Stitt et al., 1988, J. Plant Physiol. 133, 133–143; Laisk et al., 1991, Planta 185, 554–562), while the activity of NADP-malic dehydrogenase is highest during minima of photosynthesis (Scheibe and Stitt, 1988, Plant Physiol. Biochem. 26, 473–481), the present data indicate in agreement with earlier observations (Yin et al., 1991, Planta 184, 30–34) that light-dependent cytosolic energization is brought about by the oxidation of dihydroxyacetone phosphate rather than of malate. They also indicate that the over-reduction of the electrontransport chain observed during minima of photosynthesis is relieved not predominantly by oxaloacetate reduction and export of the resulting malate from the chloroplasts but by another reaction, presumably oxygen reduction.Abbreviations CDCF 5-(and 6-)carboxy-2,7-dichlorofluorescein     

Role of polyamines on de novo shoot morphogenesis from cotyledons of Brassica campestris ssp. pekinensis (Lour) Olsson in vitro

Summary The promotive effect of ethylene inhibitors (Els), i.e. AgNO₃ and aminoethoxyvinylglycine (AVG) on de novo shoot regeneration from cultured cotyledonary explants of Brassica campestris ssp. pekinensis cv. Shantung in relation to polyamines (PAs) was investigated. The endogenous levels of free putrescine and spermidine in the explant decreased sharply after 1–3 days of culture, whereas endogenous spermine increased, irrespective of the absence or presence of Els. AgNO₃ at 30 M did not affect endogenous PAs during two weeks of culture. In contrast, explants grown on medium containing 5 M AVG produced higher levels of free putrescine and spermine which increased rapidly after three days and reached a peak at 10 days. An exogenous application of 5 mM putrescine also resulted in a similar surge of endogenous free spermine of the explant. More strikingly, shoot regeneration from explants grown in the presence of 1–20 mM putrescine, 0.1–2.5 mM spermidine, or 0.1–1 mM spermine was enhanced after three weeks of culture. However, exogenous PAs generally did not affect ethylene production, and endogenous levels of 1-aminocyclopropane-1-carboxylate (ACC) synthase activity and ACC of the explant. This study shows the PA requirement for shoot regeneration from cotyledons of B. campestris ssp. pekinensis in vitro, and also indicates that the promotive effect of PAs on regeneration may not be due to an inhibition of ethylene biosynthesis.Abbreviations PAs polyamines - AVG aminoethoxyvinylglycine - SAM S-adenosylmethionine - ACC 1-aminocyclopropane-1-carboxylate - Els ethylene inhibitors     

Stoichiometry of proton uptake in isolated pea chloroplasts under different light intensities

The number of protons released inside the chloroplast thylakoids per electron which is transferred through the electron transport chain (H⁺/e^– ratio) was measured in isolated pea chloroplasts at pH 6.0 under continuous illumination and with methyl viologen as an electron acceptor. At saturating light intensity (200 W · m^–2) (strong light) the H⁺/e^– ratio was 3. At low intensity (0.9 W · m^–2) (weak light) the H⁺/e^– ratio was 2 with dark-adapted chloroplasts, but it was close to 3 with chloroplasts that were preilluminated with strong light. It is shown that the presence of azide in the reaction mixture leads to errors in the determination of the H⁺/e^– ratio due to underestimation of the initial rate of H⁺ efflux on switching off the light. To explain the above data, we assume that transformation of the electron transport chain occurs during illumination with strong light, namely, the Q cycle becomes operative.     

Interaction between neurofilaments and mitochondria in cultured cells of the rat hippocampus

Interaction between neurofilaments and mitochondria was studied on the model of cultured hippocampal cells of newborn rats. A treatment by specific toxins -iminodipropionitril or hexanedione resulted in a disintegration and translocation of neurofilaments in the cultured neurons. These effects were accompanied by a considerable decrease in dimensions of mitochondria, an increase in their elongation coefficient, a noticeable increase in spatial density of these organelles, and their translocation within the perinuclear layer of cytoplasm. The role of neurofilaments in the intraneuronal distribution of mitochondria and modifications of their functional state is discussed. The neurofilament system is supposed to considerably influence the processes of division, growth, and translocation of the mitochondria.Neirofiziologiya/Neurophysiology, Vol. 27, No. 1, pp. 3–10, January–February, 1995.     

Arrangement of the electric potential-generating redox chain in the mitochondrial membrane

The intramembrane arrangement of the respiratory chain generating electric potential difference across the mitochondrial membrane has been studied. The accessibility of various respiratory carriers to the non-penetrating electron donors and acceptors, such as ferri-and ferrocyanide, cytochrome c. fumarate and nicotinamide nuclcotides has been used as a test for surface localization of the carrier in the membrane of mitochondria and inside-out (sonicated) submitochondrial particles. Membrane potential formation was detected by measuring the transmembrane flows of the penetrating anion, phenyl dicarbaundecaborne (PCB^–).It is shown that ferricyanide reduction can support PCB^– movement if this electron acceptor interacts with intact mitochondria in the region localized on the oxygen site of the antimycin-sensitive point. The same region is accessible for ferrocyanide whose oxidation by O₂ can be also coupled with PCB^– translocation. Added nicotinamide nuclcotides cannot be utilized by mitochondria for supporting PCB^– movement.PCB^– movement in the inside-out submitochondrial particles can be supported by reduction of ferricyanide or fumarate by NADH, and of NAD⁺ by NADPH, the former process being sensitive to rotenone but not to antimycin. Antimycin-insensitive reduction of feericyanide or of CoQ₆ by succinate is not coupled with PCB^– transport. Neither ferrocyanide nor ferrocytochromec can be used as electron donors in the particles.Penetrating electron donors (TMPDH₂, succinate) and acceptors (menadione) are effective both in mitochondria and particles.It is coucluded that flavin and transhydrogenase regions of the potential-generating redox chain are localized near the inner surface, cytochromec region-near the outers surface of the internal membrane of intact mitochondria. It means that the redox chain includes at least one act of the transmembrane transfer of reducing equivalents between flavins and cytochromec.     

Movement of mitochondria in the ovarian trophic cord of Dysdercus intermedius (Heteroptera) resembles nerve axonal transport

Summary The motile behaviour of mitochondria in the ovarian trophic cord of the red cotton bug, Dysdercus intermedius, was observed optically using video-enhanced differential interference contrast (AVEC-DIC) microscopy. The motion of 258 video-recorded mitochondria was analysed of which 10%–30% were found to move during the observation periods. Of the moving mitochondria 76% travelled towards the oocyte with an average velocity of 3.37 m/ min, and 24% towards the tropharium with 2.84 m/min. The movement was found to be basically of the saltatory type I as known from nerve axons characterized by the absence of directional reversal. In some cases short periods of interrupted motion of type II, i.e. with local oscillations, were observed. Individual mitochondria often showed velocity variations during the excursions. The hemipteran trophic cords are known to contain numerous parallel microtubules. As the observed type of mitochondrial motility resembles axonal transport, a modified transport hypothesis is presented for the microtubule-based motility of organelles in the nurse strands of telotrophic insect ovarioles.     

Biological activities of human interferon and 2′–5′ oligoadenylates in plants

Exogenous human interferon 2 (IFN) and 2–5 oligoadenylates (2–5A) have been shown to cause at least a dual physiological effect in tobacco and wheat: (i) increased cytokinin activity and (ii) induced synthesis of numerous proteins, among which members of two groups of stress proteins have been identified, namely pathogenesis-related (PR) and heat shock (HS) proteins. These effects were observed only by low concentrations of these substances: IFN at 0.1–1 u/ml and 2–5A at 1–10 nM.     

The Possible Role of Glutamate Uptake in Metaphit-Induced Seizures

In a study of the possible mechanism of action of metaphit and phencyclidine in the brain, the uptake of glutamate at the luminal side of the blood-brain barrier (BBB) was studied by means of an in situ brain perfusion technique in normal guinea pigs and in those pretreated with metaphit. Metaphit, an isothiocyanate analog of phencyclidine (PCP), induces time-dependent epileptogenic changes in the electroencephalogram in guinea pig, reaching a maximum 18–24 h after metaphit administration (50 mg/kg IP). In metaphit-pretreated animals a significant reduction of glutamate BBB uptake was found, in comparison with that of controls. Reduction of glutamate transport from blood to brain ranged from 77% to 79% in all brain structures studied. This inhibition was probably due to changes in the properties of saturable components responsible for transport of glutamate across the BBB. Kinetic measurements revealed a saturable amino acid influx into the parietal cortex, caudate nucleus, and hippocampus, with a Km between 3.1 and 5.1 M, and the V_max ranging from 14.3 to 27.8 pmol^–1 g^–1. The nonsaturable component, K_id, was statistically different from zero, ranging from 1.47 to 2.00 M min^–1 g^–1. Influx of glutamate into the brain was not altered in the presence of 1 mM D-aspartate, but it was significantly inhibited in the presence of 1 mM L-aspartate. We conclude that the cerebrovascular permeability of circulating glutamate is due to the presence of a higher-capacity saturable receptor and/or a carrier-mediated transport system (75%) and also a low-capacity diffusion transport system (25%) for the glutamate located at the luminal side of the BBB. The glutamate transport system is probably fully saturated at physiological plasma glutamate concentrations.     

Specific binding of kainic acid to purified subcellular fractions from rat brain

The subcellular distribution of kainic acid (KA) binding sites in rat brain has been studied using a microcentrifugation assay. KA did not bind to myelin or brain cytosol and had few or no binding sites in the nuclear fraction. However, it bound to microsomal components (K _d =128–136 nM; 2.5–4.8 pmol/mg protein), purified synaptic plasma membranes (SPM) (K _d =45–71 nM; 5.8–6.5 pmol/mg), and purified cell-body and intraterminal mitochondria (K _d =11–31 nM; 0.4–1.1 pmol/mg). Bound KA could be totally displaced byl-glutamate orl-aspartate, but several putative antagonists of these amino acids (nuciferin, compound HA-966, 2-amino-4-phosphonobutyrate, and 2-amino-3-phosphonoproprionate) failed to displace KA or did so at very high concentrations (4 mM). Glutamic acid diethyl ester (GDEE) andd,l--aminoadipate (-AA) were more effective (IC₅₀, 0.2–0.8 mM) and showed differential effects in their capacity to displace KA bound to the various subcellular fractions. Thus, GDEE only displaced 40–60% of the KA bound by SPM or mitochondria and did not prevent the binding of KA to microsomes. -AA, on the other hand, was more effective in preventing the binding of KA at high concentrations and displaced between 80 and 100% of the drug. Both compounds showed biphasic curves of KA displacement from synaptic plasma membranes and mitochondria. The overall results indicate the presence of multiple binding sites for KA in brain cells and suggest that KA does not act exclusively at synaptic glutamate receptors. The mechanism of KA action is most likely quite complex, and the drug probably acts at multiple binding sites affecting a number of processes.     

Coupling mechanisms in anionic substrate transport across the inner membrane of rat-liver mitochondria

The translocation of P_i, malate, -oxoglutarate, and citrate across the inner membrane of rat-liver mitochondria has been studied. Investigation on the effect of pH on anionic substrate translocation across the mitochondrial membrane shows that their distribution across the inner membrane can be governed by transmembrane pH difference. However, evidence is presented that the translocation of P_i, but not that of malate, -oxoglutarate, or citrate can bedirectly coupled to an OH^– counterflux (H₂PO ₄ ^– –OH^– exchange-diffusion). and malate-tricarboxylate exchange-diffusion reactions is directly demonstrated. The study of the effect of uncouplers on the efflux from mitochondria of substrate anions, in the absence of counteranion, and on the anion exchange-diffusions shows that uncouplers act in at least two ways: they promote the efflux of P_i from mitochondria and inhibitdirectly the exchange-diffusion reactions. The kinetics of this inhibition are described. These results are discussed in the light of previous work on the effect of uncouplers on the distribution of substrate anions across the inner membrane of isolated mitochondria. Coupling mechanisms in substrate anion translocation and aspects of the energetics of anion translocation are discussed.     

Effect of Halothane in Cortical Cell Cultures Exposed to N-Methyl-D-Aspartate

In vivo studies have shown potent protection by volatile anesthetic agents against cerebral ischemic insults. Volatile agents have also been shown to antagonize glutamatergic neurotransmission at the N-methyl-D-aspartate (NMDA) receptor. This study examined the potential for halothane to reduce neuronal excitotoxic lesions caused by NMDA. Fetal rat cortical cell cultures were allowed to mature 13–16 d. Culture wells (n = 13–16) were treated with 0 mM – 3.96 mM halothane in the presence/absence of 30 M NMDA. Additional cultures were exposed to 30 M NMDA in the presence/absence of 10 M MK-801 or 10 ACEA 1021. Cellular lethality was assessed by measurement of lactate dehydrogenase (LDH) 24 hrs later. A maximal effect of halothane was observed at 0.70 mM (2.1 vol%) wherein a 36% reduction in NMDA-stimulated LDH release occurred relative to untreated controls. Both MK-801 and ACEA 1021 caused complete inhibition of NMDA-stimulated LDH release. These data confirm that halothane has modulatory effects at the NMDA receptor but potency of this drug is less than that of specific antagonists of either glutamate or glycine. These findings suggest that halothane protection in vivo can be partially explained by anti-excitotoxic properties although other mechanisms of action are probably also important.     

1-Anilino-8-naphthalenesulfonate: A fluorescent probe of ion and ionophore transport kinetics and trans-membrane asymmetry

Summary The kinetics of the transport of the 1-anilino-8-naphthalenesulfonate (ANS^–, an anionic fluorescent probe of the membrane surface) across phospholipid vesicle membranes have been studied using a stopped-flow rapid kinetic technique. The method has been used to gain detailed information about the mechanism of transport of this probe and to study ionophore-mediated cation transport across the membrane. The technique has also been exploited to study differences between the inside and outside surfaces of vesicles containing phosphatidyl choline (PC).The following is a summary of the major conclusions of this study. (a) Binding of ANS^– on the outside surface occurs within times shorter than 100 sec while permeation occurs in the time range 5–100 sec. (b) Net transport of ANS^– occurs with cotransport of alkali cations. (c) The transport rate is maximal in the region of the crystalline to liquidcrystalline phase transition, and the increase correlates with changes in the degree of aggregation of the vesicles. (d) Incorporation of phosphatidic acid (PA), phosphatidyl ethanolamine (PE) or cholesterol into PC membranes decreases the rate of ANS^– transport. (e) Neutral ionophores (I) of the valinomycin type increase ANS^– permeability in the presence of alkali cations (M ⁺) by a mechanism involving the transport of a ternaryI–M ⁺-ANS^– complex. The equilibrium constants for formation of these complexes and their rate constants for their permeation are presented. The maximal turnover number for ANS^– transport by valinomycin in dimyristoyl PC vesicles at 35°C was 46 per sec. (f) The partitioning of the ionophore between the aqueous and membrane phases and the rate of transfer of an ionophore from one membrane have been determined in kinetic experiments. (g) A method is described for the detection ofI–M ⁺ complexes on the membrane surface by their enhancement effects on ANS^– fluorescence at temperature below the phase transition temperature on monolayer vesicles. The apparent stability constants for severalI–M ⁺ complexes are given. (h) Analysis of the effect of ionic strength on the ANS^– binding to the inside outside surfaces indicates that the electrostatic surface potential (at fixed ionic strength and surface change) is larger for the inside surface than for the outside surface. (i) Analysis of the dependence of the maximal ANS^– binding for the inside and outside surfaces of vesicles made from PC and a variable mole fraction of PA, PE or cholesterol indicate that the latter three are located preferentially on the inside surface.     

The effect of different algal densities of Scenedesmus acuminatus on the population growth of Moina micrura Kurz (Crustacea: Anomopoda,Moinidae)

Six densities (0.5 × 10⁶, 1.0 × 10⁶, 1.5 × 10⁶, 2.0 × 10⁶, 3.0 × 10⁶, and 4.0 × 10⁶ cells ml^–1) of the micro-alga Scenedesmus acuminatus, were fed to the cladoceran, Moina micrura, in 40-litre glass aquaria. Moina population increased with increasing cell densities of Scenedesmus only up to treatment 3 (i.e. 1.5 × 10⁶ cells ml^–1) where a peak population of 11303 individuals per litre was obtained. Moinapopulation growth was inhibited at higher algal densities. The percentage of egg-bearing females and the number of eggs per egg-bearing females, followed a similar pattern. Comparatively, the peak production density of approximately 11000 Moina per litre, is interesting from a mass production point of view and indicates that S. acuminatus is a satisfactory micro-alga food for M. micrura.