Hiromasa Kijima

SEPARATION OF TWO RECEPTOR SITES IN A SINGLE LABELLAR SUGAR RECEPTOR OF THE FLESH-FLY BY TREATMENT WITH p-CHLOROMERCURIBENZOATE

Abstract A 3 min treatment of a single sugar receptor with 0·5 mM p -chloromercuribenzoate (PCMB) did not affect its response to d -fructose, but depressed completely its response to d -glucose. This is the first direct evidence of the presence of two different sites in the sugar receptor of the fly. No specific protection by d -glucose on PCMB treatment suggested that PCMB did not react at a glucose-binding site but did react at a specific site indispensable to simulation by d -glucose. Various sugars were examined and classified into two groups according to the effects of PCMB treatment on the sugar receptor. They correspond to those effective in the furanose and pyranose forms, respectively. The pyranose group was further divided into two subclasses according to the presence or absence of three successive equatorial hydroxyl groups regardless of their positions. The results are discussed in relation to the structures that are common to furanose stimulating sugars.

Impulse frequency and action potential amplitude in labellar chemosensory neurones of Drosophila melanogaster

Abstract The electrophysiological responses of water, salt and sugar receptors in the labellar chemosenory hair of Drosophila were investigated. In contrast to the responses of large flies such as blowfly and fleshfly, spike height changed in parallel with the spike frequencies in all the three kind of receptors, and at the same time, the spike also changed in shape: when the receptor potential was small, the spike was small and biphasic, but when the receptor potential was large, the spike was large and monophasic. These phenomena are consistently explained by assuming that antidromic conduction of spikes in the distal process of the receptor cells is blocked due to inactivation of Na channels by the depolarizing receptor potential.

Statistical analysis of channel current from a membrane patch I. Some stochastic properties of ion channels or molecular systems in equilibrium

The stochastic behavior of single-channel current in a steady-state has been interpreted as the channels state transitions between several open and shut states, and these transitions have been regarded as a homogeneous Markov process. When a channel is in equilibrium, the principle of detailed balance holds for every step in the state transition scheme. Here we show two stochastic properties of a channel, or any molecule obeying a reversible state transition scheme, under the constraint of detailed balance. First, the distribution functions and the probability density functions of shut or open dwell-time are expressed by the sum of exponential terms with positive coefficients. The same holds for the time-dependent open (or shut) frequency after the shut (or open) transition. Second, the time course of state transition from the state SI to SJ (PI,J(t] is proportional to its reverse transition time course (PJ,I(t], even if SI and SJ are widely separated. The same relation holds also for a transition scheme having transition pathways to the absorbing states. If analysis of a channel current record shows it to be incompatible with either of these two properties, the channel is not in equilibrium but in a steady-state with an energy-consuming cyclic flow. These two properties are also useful for the analysis of any molecular process obeying a homogeneous Markov process or a network of first-order chemical reactions.

Effects of sulphhydryl reagents on the labellar sugar receptor of the fleshfly

Abstract A 5 to 7 min treatment with PCMB dissolved in the phosphate buffer deeply depressed the response of the sugar receptor. The depression was easily recovered by subsequent treatment with β-mercaptoethanol and l -cystein in the buffer, but not with the buffer alone. The depression was characterized by a greater decrease in the maximum response (Rm) rather than by a change in the apparent Michaelis constant (Kb). Thus, PCMB was concluded to react with sulphhydryl groups in one or more components, perhaps proteins, of the sugar receptor system, and was suggested to react at a different site from the receptor site and to block a change in the receptor membrane permeability.

Transmitter release at frog end-plate loaded with a Ca2+-chelator, BAPTA: hypertonicity and erythrosin B augment the release independently of internal Ca2+

A Ca2+-chelator, bis-(aminophenoxy)ethane-tetraacetic acid (BAPTA) was loaded into the presynaptic nerve terminal of frog end-plate. The BAPTA-loaded preparation showed little facilitation. However, the facilitation reappeared upon addition of an ionophore, X-537A, supporting the view that the loss of facilitation was due to the Ca2+-buffering action of BAPTA. Both hypertonic conditions and erythrosin B increased both the size of end-plate potentials and frequency of miniature end-plate potentials without any recovery of facilitation at the BAPTA-loaded end-plate. This suggested that transmitter release was increased by both conditions with little change in internal Ca2+ concentration.

α-Glucosidase at the tip of the contact chemosensory seta of the blowfly, Phormia regina

Abstract α-Glucosidase activity was detected at the tip of the labellar contact chemosensory hair of the blowfly, Phormia regina . The enzyme split about 1 pmole of sucrose per hr per hair on average and the Michaelis constant for sucrose was about 50 mM. The activity of the enzyme was not solubilized into the incubation solution, but stuck stably to the tip of the sensory hair. From the cut end of the sensory hair a high activity of α-glucosidase eluted out. But its Michaelis constant was smaller by far than the one at the tip, suggesting that different types of α-glucosidase isozymes exist in the hair. The possibility that the enzyme at the tip of the sensory hair could be the sugar receptor is discussed.

‘Steady/equilibrium approximation’ in relaxation and fluctuation: I. Procedure to simplify first-order reaction

A general procedure to simplify a complex first-order reaction by two approximations, the principle of fast equilibration and the steady-state approximation, is presented. Rate constants are classified into two groups: those of the order of unity and those of the order of epsilon (much less than 1) or less, and are represented in the schemes by thick and thin arrows, respectively. The fast and the slow components are defined: from the fast component at least one thick arrow originates and from the slow component no thick arrow originates. Fast components are divided into several groups. In a group, the fast components are connected by thick arrows in both directions in each reaction step. When at least one thick arrow originates from the components in a group G and terminates on a component not belonging to group G (group G is open), then the steady-state approximation or principle of fast equilibration holds on each component in group G after an induction period To. When no thick arrow originating from group G is directed to components not belonging to group G (group G is closed), the principle of fast equilibration holds on the fast components in group G after To. The induction period To is less than the order of 1/epsilon.

Isozymes of α-glucosidase in the proboscis and legs of flies

Abstract Three isozymes of α-glucosidase were separated and partially purified from the labella of two kinds of flies: the blowfly Phormia and the fleshfly Boettcherisca . Tarsi of the blowfly also contained the three isozymes in almost equal proportions. Each isolated isozyme had similar substrate specificity, but showed different Michaelis constants, pH dependence, and distribution pattern. One of the isozymes showed interesting properties in relation to sugar reception.

Specific S-methylglutathione incorporation into a nematocyst-rich fraction of hydra

1. Specific S-[C]methylglutathione incorporation from Hydra japonica into a nematocyst-rich subcellular fraction was observed. 2. This specific incorporation is not rapid reversible binding, as shown by the lack of saturation of the reaction with time. 3. Saturating kinetics of specific incorporation rate with S-[14C]methylglutathione concentration suggests the existence of intermediate reversible complex between a macromolecular and S-[14C]methylglutathione. 4. Activity of specific incorporation could be solubilized by Triton X-100 treatment of the nematocyst fraction, showing that the incorporation is not due to transport processes. 5. The incorporation was markedly diminished by the addition of cold trichloroacetic acid or urea, or by heat-treatment after the incorporation, showing that the complex is not stabilized by chemical bonding. 6. No chemical changes of free S-[14C]methylglutathione were detected in the reaction mixture, showing that the macromolecule interacting with S-[14C]methylglutathione is not a catalyzing enzyme. 7. These results suggest that this is a new type of glutathione incorporation and could be explained by a type of receptor protein which accumulates glutathione molecules.

α-Glucosidase isozymes and the labellar sugar receptor of the blowfly

Abstract Some properties of four types of the soluble α-glucosidase, especially of P-II α-glucosidase of Amakawa and others, from the labella of the blowfly were examined and compared with those of the sugar receptor of the blowfly. Tris (hydroxymethyl) aminomethane inhibited four types of α-glucosidase and the electrical response of the sugar receptor, all in a competitive manner, but the inhibition constants ( K i ) for these glucosidases were more than 150 times smaller than that for the sugar receptor. l -Serine inhibited α-glucosidases competitively, but the inhibition for the sugar receptor was not clear. The effects of cations and amino acids on α-glucosidases were also examined in relation to those on the sugar receptor.