Studies on isoenzyme pattern during differentiation (spherulation) of Physarum polycephalum

Plasmodial homogenates of the true slime mold Physarum polycephalum grown on a liquid medium contain carbohydrates which form a complex with protein under conditions of acrylamide electrophoresis and thus make isoenzyme studies from those extracts impossible. A method, using mild homogenization and centrifugation on top of a 30% sucrose solution was developed. This treatment leaves most of the soluble cytoplasmic enzymes in the upper layer above the sucrose, which then can be used for successful isoenzyme or protein studies with polyacrylamide electrophoresis.The activity changes and isoenzyme pattern of 16 different enzymic activities were studied during differentiation (spherulation) of Physarum polycephalum, induced either by starvation or by mannitol. Only one enzyme, esterase, exhibited a conspicuous change in isoenzyme pattern during development.     

Melanin biosynthesis during differentiation of Physarum polycephalum.

Melanin synthesis in the myxomycete Physarum polycephalum occurs during sporulation but not during spherule formation. Melanin-like pigment was extracted from spores. An almost identical substance of polyphenols was extracted from spherules and characterized by its ultraviolet and infrared absorbance spectra. Polyphenol oxidase activity in spherules was very low and showed only one weak isoenzyme band in isoelectric focusing polyacrylamide gels. A much higher activity, and an increasing number of isoenzymes, were detected in sporulating cultures after illumination during the differentiation process. The addition of melanin precursors resulted in the synthesis of brownish-yellow spherules, probably containing dopachrome, whereas the addition of polyphenol oxidase inhibitors resulted in yellow sporangia. The results indicate that melanin synthesis is probably only a stage in maturation but not an essential part of the morphogenetic process itself.     

Isoenzyme pattern and de novo synthesis of phosphodiesterase during differentiation (spherulation) in Physarum polycephalum

Summary Under conditions of CsCl-equilibrium sedimentation, phosphodiesterase in extracts made from growing Physarum microplasmodia forms two bands with buoyant densities of 1.3572 g/ml (Phosphodiesterase I) and 1.2937 g/ml (Phosphodiesterase II). In spherulating cultures induced by starvation, only phosphodiesterase I is present and true de novo synthesis of this enzyme during this differentiation was demonstrated by density labeling with deuterated amino acids. The synthesis is inhibited by cycloheximide, whereas only the total activity but not the density of the enzyme was influenced by actinomycin-C.In spherulating cultures induced by mannitol both isoenzymes are present as in the growing cultures.     

Myosin switching during amoebo-plasmodial differentiation of slime mold, Physarum polycephalum

We reported previously that myosins from amoebal and plasmodial stages in the life cycle of Physarum polycephalum differ in the primary structure of heavy chains and phosphorylatable 18,000 Mr light chains, while Ca-binding 14,000 Mr light chains are common to both myosins (Kohama & Takano-Ohmuro, Proc Jpn acad 60B (1984) 431; Kohama et al., J biol chem 260 (1986) 8022). We have carried out immunofluorescence microscopical studies upon differentiating cultures of amoebic colonies, which show apogamic amoebo-plasmodial differentiation as follows: Typical amoebae differentiate into mono-nucleate intermediate cells with swollen nuclei and then into two or multi-nucleate young plasmodia (Anderson et al., Protoplasma 89 (1976) 29. Antibodies against plasmodial myosin heavy chain (PMHC) and 18,000 Mr plasmodial myosin light chain (PMLC18) stained intermediate cells and young plasmodia, but not typical amoebae. On the other hand, antibody against amoebal myosin heavy chain (AMHC) stained typical amoebae and intermediate cells--but not young plasmodia. Thus staining was detected using antibodies against both PMHC and AMHC in intermediate cells. Intermediate cells were also stained by antibody against another plasmodium-specific cytoskeletal protein, viz., high molecular weight actin-binding protein (HMWP). We propose that synthesis of myosin subunits switches immediately from amoebal to plasmodial type in mono-nucleate cells with swollen nuclei. This myosin switching is associated with the initiation of HMWP synthesis.     

Activity of some enzymes in Physarum polycephalum

Summary The activities of seven enzymes were studied during the first 24 h of spherule formation (differentiation) in cultures of Physarum polycephalum. These enzymes were: isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase, glutamate dehydrogenase, acid phosphatase, phosphodiesterase, -glucosidase, and histidase. Isocitrate dehydrogenase and glucose-6-phosphate dehydrogenase showed a decrease, glutamate dehydrogenase and phosphodiesterase increased about eight-fold, acid phosphatase about two-fold, -glucosidase showed an activity peak at about 15 h after starvation which decreased, after 24 h to its original value, and histidase showed no significant activity change during the period. The inhibition of protein synthesis by cycloheximide resulted in a decrease of enzymic activity at all times during the process, whereas the inhibition of RNA synthesis by actinomycin D had no effect during the early stages of spherulation but resulted in a rather rapid decrease of enzymic activity when added 21 h after the initiation of spherule formation. Attempts to induce higher enzymic activities during spherulation above the levels already observed were unsuccessful.

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Zusammenfassung Der Aktivitätsverlauf, von sieben Enzymen wurde während der ersten 24 Std der Mikrosklerotienbildung verfolgt. Es handelte sich um die Enzyme: Isocitratdehydrogenase, Glucose-6-Phosphat Dehydrogenase, Glutamat-dehydrogenase, saure Phosphatase, Phosphodiesterase, -Glucosidase und Histidase. Isocitratdehydrogenase und Glucose-6-Phosphat Dehydrogenase nahmen während dieser Zeit ab, Glutamatdehydrogenase und Phosphodiesterase stiegen auf das 8fache des Ausgangswertes, saure Phosphatase auf das 2fache. -Glucosidase zeigte ein Aktivitätsmaximum nach ungefähr 15 Std, das aber nach etwa 24 Std wieder bis zu dem Ausgangswert abnahm, während Histidase sich nicht signifikant veränderte. Die Hemmung der Proteinsynthese durch Cycloheximid resultierte zu jedem Zeitpunkt in einer sofortigen Abnahme der Enzymaktivität, während die Inhibition der RNS-Synthese in der ersten Zeit ohne Effekt war, jedoch bei einer Zugabe nach 21 Std nach der Induktion der Spherulation eine ziemlich schnelle Abnahme der Enzymaktivität bewirkte. Versuche, die Enzymaktivitäten zusätzlich zu den schon beobachteten zu induzieren, schlugen fehl.

     

Activity of some enzymes in Physarum polycephalum

Summary The activity levels of seven enzymes were studied in growing plasmodia of Physarum polycephalum. The enzymes were isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase, glutamate dehydrogenase, acid phosphatase, phosphodiesterase, -glucosidase, and histidase. Six of the enzymes showed a continuous increase in activity during the mitotic cycle; glutamate dehydrogenase exhibited a stepwise increase about 5 h after mitosis. Cycloheximide immediately inhibited the activity of all enzymes. Actinomycin D was ineffective in inhibiting enzyme activity until after one mitotic cycle had been completed; this indicates that mRNA was stable for all of these enzymes during the G2 period. Attempts to induce enzyme activity were unsuccessful.

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Zusammenfassung Der Aktivitätsverlauf von sieben Enzymen wurde in wachsenden Plasmodien von Physarum polycephalum untersucht. Es handelte sich um die Enzyme: Isocitrat-Dehydrogenase, Glucose-6-Phosphat-Dehydrogenase, Glutamat-Dehydrogenase, saure Phosphatase, Phosphodiesterase, -Glucosidase und Histidase. Sechs dieser Enzyme wiesen einen kontinuierlichen Aktivitätsanstieg während des Mitosecyclus auf; Glutamat-Dehydrogenase zeigte einen stufenförmigen Anstieg etwa 5 Std nach der Kernteilung. Cycloheximid hemmte sofort die Aktivität der Enzyme, während Actinomycin D erst nach Ablauf eines halben Teilungscyclus inhibierend wirkte. Dies deutet auf eine relativ stabile mRNA hin. Versuche, die Aktivität zu induzieren, schlugen fehl.

     

Effect of ADP-ribosylation on differentiation in Physarum polycephalum

Abstract. We studied ADP-ribosylation in the vegetative life cycle of the myxomycete Physarum polycephalum . Proliferating macroplasmodia are delayed in their progression through the cell cycle by the specific ADP-ribo-syltransferase inhibitor 3-methoxybenzamide. DNA and RNA synthesis is depressed. During the differentiation of microplasmodia into quiescent microsclerotia, ADP-ribosylation strongly increases in an early stage. The same stage is sensitive towards treatment with 3-methoxybenzamide, which delays the termination of the sclerotization process. The increase of ADP-ribosylation is not evenly distributed among all nuclear acceptor proteins. Histones H3 and H4 are modified to a lower extent in relation to H2A and H2B at the time of maximum ADP-ribosylation. Germination of microsclerotia into growing plasmodia is also repressed by 3-methoxybenzamide.