Biomedical subjects
Publications and source records attributed to Y Kobatake.
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The plasmodium of the true slime mold Physarum polycephalum was treated with EDTA or EGTA and the effect of the treatment on the chemotactic response was examined by measuring the chemotactic motive force with the double-chamber method. The results obtained were as follows: (1) The treatment of the plasmodium with 5 mM EDTA (pH 7.0, 20 min) did not give any significant effect on the protoplasmic streaming or motility. (2) The plasmodium treated with EDTA exhibited no chemotactic response to non-electrolyte attractants (D-glucose, D-galactose, D-mannose, and maltose) and negative chemotaxis to electrolyte attractants (cyclic AMP and NaH2PO4). (3) The EDTA treatment gave no effect on the chemotactic response to repellents (D-fructose, NaCl, CaCl2, MgCl2). (4) The EDTA-treated plasmodium exhibited changes in the membrane potential in response to both attractants and repellents as similar to the untreated plasmodium. (5) The treatment of the plasmodium with 5 mM EGTA (pH 7.0, 20 min) gave results similar to those obtained with the EDTA treatment. The results obtained suggested that EDTA (or EGTA) treatment did not affect the receptor sites but modified the transduction mechanism from reception into tactic movement.
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The fluorescence of merocyanine 540 (MC) in liposomal and mitochondrial suspensions was measured under various conditions. Under a given condition, both the amount of dye bound to the membrane and the zeta potential were determined simultaneously. It was found that the fluorescence intensity was proportional to the amount of bound dye and correlated with the zeta potential of particles. The fluorescence intensity was represented quantiatively in terms of the Langmuir adsorption isotherm, when the electrostatic interaction acting between MC and membrane surface was properly taken into account. It was concluded that the changes in MC fluorescence in the liposomal and mitochondrial suspensions are mainly attributed to the changes in the surface potential of the membranes.
The membrane potential of mitochondria was estimated from the accumulation of tetraphenyl phosphonium (TPP+), which was determined with the TPP+-selective electrode developed in the present study. The preparation and some operational parameters of the electrode were described. The kinetics for uptake by mitochondria of TPP+ and DDA+ (dibenzyldimethyl ammonium) were analyzed, and it was found that TPP+ permeated the mitochondrial membrane about 15 times faster than DDA+. The final amounts of accumulation of TPP+ and DDA+ by mitochondria were approximately equal. For the state-4 mitochondria, the membrane potential was about 180 mV (interior negative). Simultaneous measurements of TPP+-uptake and oxygen consumption showed that the transition between states 3 and 4 was detectable by use of the TPP+-electrode. After the TPP+-electrode showed that state-4 was reached, the extra-mitochondrial phosphorylation potential was measured. The difference in pH across the membrane was measured from the distribution of permeant anion, acetate, so as to calculate the proton electrochemical potential. The ratio of extra-mitochondrial phosphorylation potential to proton electro-chemical potential, n was close to 3. This value of n was also found to be 3 when ATP was hydrolyzed under the condition that the respiratory chain was arrested. The implication that n = 3 was discussed.
The gustatory receptors of the eel palate were found to be extremely sensitive to amino acids and carboxylic acids. The results obtained are as follows: (a) 11 amino acids which are among naturally occurring amino acids elicited responses in the palatine nerve, but 9 amino acids did not elicit a response even at a high concentration. The effect of D-amino acids was always much less than that of their corresponding L-isomers. There was no appreciable difference in the effectiveness of an alpha-amino acid (alpha-alanine) and beta-amino acid (beta-alanine). (b) The threshold concentrations of the most potent amino acids (arginine, glycine) were between 10(-8) and 10(-9) M. A linear relation between the magnitude of the response and log stimulus concentration held for a wide concentration range for all the amino acids examined. (c) The palatine receptors responded sensitively to various carboxylic acid solutions whose pH was adjusted to neutral. The threshold concentrations varied between 10(-4) and 10(-7) M. The magnitude of the response at 10(-2) M increased with an increase of carbon chain length. (d) The extent of cross-adaptation was examined with various combinations of amino acids. A variety of the response patterns showing complete cross-adaptation, no cross-adaptation, or synergetic interaction was observed. The synergetic interaction was also observed when one amino acid below its threshold concentration was added to the other amino acid below its threshold concentration was added to the other amino acid. No cross-adaptation was observed between amino acids and fatty acids. (e) The treatment of the palate with papain led to loss of the responses to arginine, glycine, and histidine without affecting those to proline and acetic acid. The treatment with pronase E eliminated selectively the response to proline. The possibility that the eel gustatory receptors are responsible for sensing food at a distance was discussed.
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1. The frog gustatory responses to various salt stimuli and distilled water were greatly enhanced after the tongue was treated with an alkaline solution containing salts of low concentration. The incubation of the alkali-treated tongue in solution of pH 6.0 containing Ca2+ restored reversibly the behaviour of the gustatory receptor to that before the treatment, while Mg2+ had no ability to do this. 2. The responses to salt stimuli and distilled water were greatly decreased after the tongue was incubated in solutions of pH 5.3 containing Ca2+. 3. On piece of tongue incubated in a solution of pH 5.3 containing 45Ca released a larger amount of 45Ca by the alkali treatment than another piece incubated in pH 7.0. It was concluded that removal of Ca2+ from the gustatory receptor membrane by the alkali treatment led to enhancement of the responses and binding of extra Ca2+ to the membrane by the incubation in acidic CaCl2 solution led to suppression of the responses. We emphasized that a conformational change of the receptor domains plays an important role in the transduction process of the gustatory response.
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Responses (chemotaxis and changes in membrane potential) of Tetrahymena, Physarum, and Nitella against aqueous solution of homologous series of n-alcohols, n-aldehydes and n-fatty acids were studied for clarifying the hydrophobic character of chemoreceptive membranes. Results were: (1) All organisms studied responded to homologous compounds examined when the concentration of these chemicals exceeded their respective threshold, Cth, and the response, R, were expressed approximately as R=alpha log (C/Cth) for C greater than Cth. (2) Increase of the length of hydrocarbon chain in homologues decreased Cth. Plots of log Cth against the number of carbon atoms, n, in n-alcohols, n-aldehydes and n-fatty acids showed linear relationships as represented by long Cth=-An+B. A and B are positive constants for respective functional end groups of the chemicals and biological membranes used. The above empirical equation was interpreted in terms of the partition equilibrium of methylene groups between bulk solution and membrane phase. Parameter A was shown to be a measure of hydrophobicity of the membrane, and B represented the sensitivity of chemoreception of the membrane. (3) Thresholds, Cth, for various hydrophobic reagents were compared with those of human olfactory reception, T. Plots of log T against log Cth fell on straight lines for respective organisms with different slopes which were proportional to parameter A.
Interaction between salt and sugar receptions in plasmodium of Physarum polycephalum was studied by using double-chamber method. Effect of sugars on salt reception was evaluated by measuring membrane potential and the motive force of tactic movement of the slime mold, where salt concentration in one compartment was increased successively with a fixed sugar concentration. Results are summarized as follows: (1) The presence of D-glucose, D-mannose, D-maltose, or sucrose in medium led to increase of the threshold concentration Cth, for salts (chlorides and nitrates of Li, Na, K), whereas D-ribose decreased the threshold for salt reception. D-galactose showed no appreciable effect on Cth of every salt species examined. No change in Cth for salt reception was observed until concentration of sugars exceeded their respective thresholds. (2) Double logarithmic plots of Cth for salts against sugar concentration followed different straight lines for different cations, whose slopes being closely correlated with the effects of lyotropic number of anions in the absence of sugars. (3) Plots of log Cth against the reciprocal of the absolute temperature, 1/T, gave linear relations, and the slopes of the straight line became small with increase of sugar concentration above their respective thresholds. Experimental results obtained here suggest that the structure of water at the interface of cell membrane plays an indispensable role in the interaction between salt and sugar receptions.
The electrode sensitive to dibenzyl dimethyl ammonium (DDA+), which is considered to be an indicator of the membrane potential, was constructed by using tetraphenyl borone (TPB-) embedded in dichloroethane. Rapid and Nernstian responses were exhibited against DDA+ solutions ranging between 10(-2) and 3 - 10(-6) M in concentration. High selectivity for DDA+ was observed in the presence of various inorganis salts, ADP, ATP, oxidizable substrates and sugars. The electrode developed here was used to measure the DDA+ uptake in Streptococcus faecalis and the results agreed with those reported by Harold, F.M. and Papineau, D. ((1972) J. Membrane Biol. 8, 27-44 and 45-62). While they determined the DDA+ concentration in the medium by measuring the absorbance of the filtrate treated with the ion-exchangers, the electrode can measure directly the DDA+ concentration in the bacterial suspension without any any pretreatment. It was also shown that the electrode can measure the DDA+ uptake in mitochondria during energization.
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Electrophoretic study of microplasmodia of the true slime mold Physarum polycephalum was carried out in the presence of various concentrations of inorganic salts, nucleotides and sugars, and the zeta potential at the surface of the plasmodia was determined from the electrophoretic mobilities. The membrane potential of the plasmodia was also measured under the same external conditions. It was shown that changes in the membrane potential induced by the chemical stimuli agreed approximately with those induced in the zeta potential in all cases examined. These results suggested that the phase boundary potential at the membrane-solution interface is mainly responsible for the membrane potential in the chemoreception of the slime mold.
An electrode sensitive to sulfa drugs was constructed by using the iron(II)-bathophenanthroline chelate embedded in a liquid membrane. Rapid and Nernstian responses were exhibited against solutions of sulfamerazine and sulfisomidine ranging between 10(-3) and 10(-1) M in concentration. High selectivity was observed in the presence of urea, glycine, aminopyrine, or p-amino-benzoic acid. These chemicals are known to interfere in the usual colorimetric analysis of sulfa drugs.