Experimental Studies on the Protoplasmic Streaming in the Myxomycete Plasmodium III

Transcription

1 232 Experimental Studies on the Protoplasmic Streaming in the Myxomycete Plasmodium III The effects of amino acids and some chelating substances on the motive force of the protoplasmic streaming Jiro Ohta1 Botanical Laboratory, Department of Biology, Faculty of Science, Ochanomizu University, Tokyo, Japan Received April 25, 1958 Fujii et al (1955) investigated the effects of various amino acids and some chelating substances on the motility of starfish spermatozoa, which are generally immobile. When histidine, cysteine, glycine, ethylenedia minetetraacetic acide (EDTA) or diethyldithiocarbamate (DDC) were added to the suspensions of these spermatozoa, the active movement of the sperm was observed instantly. They also suggested that the release of zinc from the surface of the spermatozoa is responsible for the initiation of this activity. Similar phenomena have already been reported by Fitting (1933). He has attempted to discover the nature of the chemical stimulus which initiates protoplasmic streaming in the leaf cells of Vallisneria and Elodea, and found that various amino acids, especially histidine, were effective in causing the initiation of cyclosis in previously resting cells. From these reports, the author was led to investigate the effects of these substances on the protoplasmic streaming in the Myxomycete plasmodium. To express the protoplasmic flow of this organism on a quantitative basis, the technique developed by Kamiya (1942, 1953, 1957) was used in this work. By means of it, continuous changes of the motive force responsible for the flow can be represented by the changes of the hydrostatic counter-pressure just sufficient to stop the flow. In the following, the motive forcetime curve was called by the name of "dynamoplasmogram", which is abbreviated as DPG. Materials and methods The material used in the present work was the plasmodium of Physarum polycephalum, which was grown on rolled oats according to the method of Camp (1936). 1 The present work was partly supported by a grant from the Ministry of Education of the Japanese Government.

2 Experimental Studies on the Protoplasmic Streaming III 233 The method of measurements of the motive force responsible for pro toplasmic streaming is the same as Kamiya's (1942, 1953). Therefore, various technical terms and experimental procedures used in the following are based on Kamiya' s paper The method of admitting chemical agents has also been described by Kamiya et al (1957). The experiments were performed in the following manner: the motive force was first measured under the normal state of the material reagent was admitted and finally, the reagent was removed., then the Reagents to be tested were used in aqueous solution adjusted to ph 6.0 by adding 1N or 0.1N NaOH. The following experiments were made at Ž, in course of the experiments the temperature deviation was not more than 0.2 Ž. Results Effects of amino acids All amino acids used in this work were those of alpha-l-type. 1. Histidine Among various amino acids, histidine showed most striking effects on the protoplasmic flow of the plasmodium. When the whole plasmodium was treated with histidine, the motive force increased considerably. This increase was found to occur within the range of concentrations of histidine from 10-2 to 10-3M. Fig. 1. Dynamoplasmogram under the effect of 10-2M histidine. The whole plasmodium was treated evenly during the period between I and II. After the reagent was removed, the motive force was restored to its original state. In some cases, however, the motive force of the recovery process was slightly smaller than that of the original state. Fig. 1 shows an instance in which 10-2M histidine was admitted to both compartments of

3 234 J. Ohta the double-chamber. When only one half of the plasmodium was treated by histidine and the other half was left in its original state, protoplasm showed a tendency to move into the non-treated side, as indicated by the shifting of the wave upwards (Fig. 2). But, in this case, the magnitude of the motive force showed no significant changes. Fig. 2. Dynamoplasmogram under the effect of 10- M histidine admitted to one half of the plasmodium at I. Histidine was removed at the time designated as II. 2. Cysteine The increase of the motive force was also observed when the plas Fig. 3. Dynamoplasmogram under the effect of 10-1M cysteine. The plasmodium was treated evenly during the period between I and II. modium was entirely treated with cysteine (Fig. 3). The effective concent ration was somewhat higher in the case of cysteine ( ~10-3M) than in histidine, and the degree of the increase caused by cysteine was lower than that caused by histidine. When cysteine was admitted to only one compartment of the double

4 Experimental Studies on the Protoplasmic Streaming III 235 chamber, the DPG showed a tendency to shift upwards histidine., as in the case of 3. Glycine Glycine induced the increase of the motive force in its higher concent rations (5 ~ M), as shown in Fig. 4. In some cases, however marked change of the motive force was observed., no Fig. 4. Dynamoplasmogram under the effect of 10-2M glycine. The plasmodium was treated evenly during the period between I and II. 4. Other amino acids Other amino acids such as alanine, leucine and glutamic acid induced no conspicuous effects on the generation of the motive force of protoplasmic flow. Effect of chelating substances 1. Ethylenediaminetetraacetic acid (EDTA) EDTA inhibited the generation of the motive force to a certain extent, at concentrations of M. This inhibition continued even after the elimination of the reagent. As EDTA is a powerful chelating agent for bivalent cations, it seems probable that the release of Magnesium or Calcium from the plasmodium took place by the treatment of EDTA. Therefore, MgCl2- or CaCl2- solu tions were added after the removal of EDTA. Fig. 5 shows an example in which 2 ~10-3M MgCl2 was admitted after the elimination of 2 ~10-3M EDTA. In this case, the motive force not only recovered, but rather increased as compared with its original state. This fact will be discussed later. Addition of CaCl2-solution increased the protoplasmic viscosity considerably and in such a condition measurements of the motive force were not possible. 2. Diethyldithiocarbarnate (DDC) DDC suppressed the production of the motive force at concentrations

5 236 J. Ohta of 10-2M-10-3M. No marked recovery was observed after the elimination of the reagent. In contrast with EDTA, the inhibition of DDC was not eliminated by the addition of MgCl2. Fig. 5. Dynamoplasmogram under the effect of 2 ~10-3M EDTA admitted to the plasmo dium at I. EDTA was removed at II. 2 ~10-3M MgC12 was admitted at III. Discussion The above experiments indicate that the motive force of protoplasmic flow in the plasmodium increased by the application of histidine, cysteine and glycine. Other amino acids have no effect on this movement. These facts are well in comformity with the date reported by Fujii et al (1955) on the initiation of the motility of starfish spermatozoa. It has been sug gested by them that this initiation of motility is due to the metal-chelating action of amino acids. Therefore, it seems probable that the increase of the motive force mentioned above is also caused by the release of metal from the plasmodium. An evidence for this assumption has been obtained from the following results: Histidine solution, in which the plasmodium had been suspended for 30 minutes, always gave a positive dithizone reaction. That is to say, when such a histidine solution was shaken with 5mg per cent dithizone solution in carbon tetrachloride, the green colour of the latter had a tinge of purple. This fact again suggests that the plasmodium was dep rived of a small amount of some metal (perhaps zinc) by histidine. Simi

6 Experimental Studies on the Protoplasmic Streaming III 237 lar reactions took place in the case of cysteine and glycine, but other amino acids showed negative dithizone reaction. As has been mentioned, Fitting (1953) found that the protoplasmic streaming in Vallisneria and Elodea may also be initiated by histidine and other amino acids. He has presented no definite explanation of this pheno mena. From the above discussion, it is very likely that this phenomena is correlated with the release of metal by amino acids, too.i t has been suggested by Kamiya et al (1957) that the production of the motive force of the protoplasmic flow in the plasmodium is based upon the changes of molecular configuration or aggregation of the actomyosin like structural protein, which really exists in this organism, and that the energy required for it is supplied by ATP. Nakajima (1956, 1957) extracted the actomyosine-like protein fraction from the plasmodium and studied its physico-chemical nature precisely. He found that this protein fraction has ATP-ase activity, which is closely associated with concentrations of Mg++ and Ca++. In the previous section of the present work, it was shown that EDTA inhibited the generation of the motive force and its inhibition was removed by the addition of Mg++. This fact indicates that lack of Mg++ caused by EDTA suppressed the production of the motive force. Therefore, this re sult also gives an indirect evidence that the ATP-ase plays an important role in the generation of the motive force of protoplasmic flow. Then the question arises: How does the release of metal initiate or stimulate cellular movements? At the present time, however, we have no explicit answer for the question, except that some metal (perhaps zinc) may participate in actomyosin-atp system. In conclusion, the author desires to express his gratitude to Prof. T. Fujii, University of Tokyo and Prof. N. Kamiya, Osaka University, for their kind guidance and helpful advice. He also wishes to thank to Prof. B. Wada, University of Tokyo, for his constant encouregement and criticism. Summary The effects of various amino acids, ethylenediaminetetraacetic acid (EDTA) and diethyldithiocarbamate (DDC) on the motive force of protoplasmic stream ing in the plasmodium, Physarum polycephalum, were investigated by means of Kamiya's double-chamber method. The following results were obtained. 1. The motive force increased with the treatment with histidine, cysteine and glycine. This increase was greatest in histidine, greater in cysteine than in glycine. These processes were reversible. 2. Other amino acids induced no marked effects on the production of the motive force. 3. EDTA inhibited the generation of the motive force to a certain extent. This inhibition was not removed by the elimination of the reagent.

7 238 J. Ohta But, when Mg++ was admitted after the removal of EDTA, the motive force not only recovered, but rather increased as compared with its original state. 4. DDC suppressed the motive force. No marked recovery was obtained by the addition of Mg++ in this case. 5. It has been suggested that the induced increase of the motive force by amino acids may be due to the release of some metal (perphaps zinc) from the plasmodium. References Camp, W. G A method of cultivating myxomycete plasmodia. Bull. Torrey Bot. Club 63: Fitting, H Untersuchungen uber den Protoplasmastromung auslosenden Reizstoff der Pflanzen. Naturwissenschaften 21: Fujii, T., Utida, S., Mizuno, T. and Nanao, S Effects of amino acids and some chelating substances on the motility and the oxygen uptake of starfish spermatozoa. Jour. Fac. Sci., Univ. of Tokyo 7: Kamiya, N Physical aspects of protoplasmic streaming. A Symposium on "the Structure of Protoplasm" (edited by W. Seifriz) Monograph Amm. Soc. Plant Physiol. Ames-Iowa: The motive force responsible for protoplasmic streaming in the myxomycete plasmodium. Ann. Rep. Sci. Works, Osaka Univ. 1: Nakajima, H. and Abe, S Physiology of the motive force of protoplasmic stream ing. Protoplasma 48: Nakajima, H Some properties of a contractile protein in the slime mould. Seitai no Kagaku (Medical Science) 7: (in Japanese) A contractile proteine in the slime mould. XXII Ann. Meeting Boṭ Soc. Japan. Cytologia vol. 23 no. 2 (pp ) Issued June 5, 1958 Ausgegeben am 5. Juni 1958 Palu le 5 juin 1958