Phosphatase activity in Amoeba proteus at pH 9.0

In the free-living amoeba Amoeba proteus (strain B), after PAAG disk-electrophoresis of the homogenate supernatant, at using 1-naphthyl phosphate as a substrate and pH 9.0, three forms of phosphatase activity were revealed; they were arbitrarily called "fast", "intermediate", and "slow" phosphatases. The fast phosphatase has been established to be a fraction of lysosomal acid phosphatase that preserves some low activity at alkaline pH. The question as to which particular class the intermediate phosphatase belongs to has remained unanswered: it can be both acid phosphatase and protein tyrosine phosphatase (PTP). Based on data of inhibitor analysis, large substrate specificity, results of experiments with reactivation by Zn ions after inactivation with EDTA, other than in the fast and intermediate phosphatases localization in the amoeba cell, it is concluded that only slow phosphatase can be classified as alkaline phosphatase (EC 3.1.3.1).     

Nuclear Synthesis of Cytoplasmic Ribonucleic Acid in Amoeba proteus

The enucleation technique has been applied to Amoeba proteus by several laboratories in attempts to determine whether the cytoplasm is capable of nucleus-independent ribonucleic acid synthesis. This cell is very convenient for micrurgy, but its use requires a thorough starvation period to eliminate the possibility of metabolic influence by food vacuoles and frequent washings and medium renewal to maintain asepsis. In the experiments described here, amoebae were starved for periods of 24 to 96 hours, cut into nucleated and enucleated halves, and exposed to either C-14 uracil, C-14 adenine, C-14 orotic acid, or a mixture of all three. When the starvation period was short (less than 72 hours), organisms (especially yeast cells) contained within amoeba food vacuoles frequently showed RNA synthesis in both nucleated and enucleated amoebae. When the preperiod of starvation was longer than 72 hours, food vacuole influence was apparently negligible, and a more meaningful comparison between enucleated and nucleated amoebae was possible. Nucleated cells incorporated all three precursors into RNA; enucleated cells were incapable of such incorporation. The experiments indicate a complete dependence on the nucleus for RNA synthesis. The conflict with the experimental results of others on this problem could possibly stem from differences in culture conditions, starvation treatment, or experimental conditions. For an unequivocal answer in experiments of this design, ideally the cells should be capable of growth on an entirely synthetic medium under aseptic conditions. The use of a synthetic medium (experiments with A. proteus are done under starvation conditions) would permit, moreover, a more realistic comparison of metabolic capacities of nucleated and enucleated cells.     

Effect of Bacteria and Amoebae on Rhizosphere Phosphatase Activity

The contributions of various components of soil microflora and microfauna to rhizosphere phosphatase activity were determined with hydroponic cultures. Three treatments were employed: (i) plants alone (Bouteloua gracilis (H.B.K.) Lag. ex Steud.) (ii) plants plus bacteria (Pseudomonas sp.), and (iii) plants plus bacteria plus amoebae (Acanthamoeba sp.). No alkaline phosphatase was detected, but an appreciable amount of acid phosphatase activity (120 to 500 nmol of p-nitrophenylphosphate hydrolyzed per h per plant) was found in the root culture solutions. The presence of bacteria or bacteria and amoebae increased the amount of acid phosphatase in solution, and properties of additional activity were identical to properties of plant acid phosphatase. The presence of bacteria or bacteria and amoebae increased both solution and root phosphatase activities at most initial phosphate concentrations.     

Peptide stimulation of phagocytosis in Amoeba proteus

Summary Normal articular cartilages from the weightbearing areas of the femoral condyles of the knee joints of 11 patients (3–20 years old) and of 35 Schwarzkopf sheep (3 months to 2 years old) were studied using the electron microscope. The study has shown that the matrix of normal articular cartilage is not only composed of collagen fibrils and proteoglycans, but also contains two types of elastic system fibres. Small elastic fibres can be identified in the superficial and lower radiate zones of cartilage of man and sheep. Similar to elastic fibres in other tissues, they consist of a central amorphous core and are surrounded by aggregates of 10 nm microfibrils. Another type of elastic system fibres, oxytalan fibres, are found in the intermediate and upper radiate zones of the articular cartilage.     

Dynamics of the cytoskeleton in Amoeba proteus

Fluorescein-labeled muscle actin was microinjected into Amoeba proteus and followed during intracellular redistribution by means of the image-intensification technique. The fully polymerization-competent protein becomes part of the endogenous actomyosin system undergoing dynamic changes over time periods of several hours. Single-frame analysis of long-term sequences enabled the direct demonstration of both the contractile activities and morphological transformations of microfilaments in normally locomoting, immobilized and phagocytozing specimens. In normally locomoting cells the filament layer undergoes continuous changes in spatial distribution depending on the actual pattern of cytoplasmic streaming and cell shape. The highest degree of differentiation is always maintained in the intermediate region between the front and the uroid, thus indicating this segment of the cortex to be the most important site in generating motive force for pseudopodium formation and ameboid movement. In immobilized cells contracted by the application of ruthenium red or relaxed by different anesthetics, the filament layer forms a continuous thick sheath beneath the cell surface or becomes completely disintegrated. In phagocytozing cells the local polymerization of actin at the tip of pseudopodia forming the food-cup and around the nascent phagosome points to a significant participation of the actomyosin system in the process of capturing and constricting prey organisms. Although our results provide clear evidence for the overall importance of motive force generation according to the hydraulic pressure theory, some motile phenomena exist in Amoeba proteus that cannot exclusively be explained by this mechanism.     

Tartrate-sensitive and tartrate-resistant acid phosphatases in Amoeba proteus

In free-living Amoeba proteus (strain B), acid phosphatase (AcP) was examined by disc-electrophoresis in polyacrylamide gel. The tartrate-sensitive amebian AcP was greatly inhibited by dithiothreitol and Cu2+, and only partly inhibited by sodium orthovanadate, ammonium molybdate, EDTA, disodium salt and Mg2+, Ca2+, Zn2+ and Mn2+. On the contrary, it appeared to be resistant to sulfhydryl reagents--4(hydroxymercury) benzoic acid, sodium salt and N-ethylmaleimide. Unlike the tartrate-sensitive enzyme, the tartrate-resistant AcP was greatly inhibited by EDTA and partly inhibited by dithiothreitol, Mg2+ and Cu2+ (Mn2+ > Cu2+), being activated by orthovanadate, molybdate, sulfhydryl reagents, Mg2+, Ca2+ and Zn2+. Both tartrate-sensitive and tartrate-resistant AcPs lack apparently free SH-groups necessary for their catalytic activities. Using 2-naphthyl phosphate as a substrate at pH 4.5, six AcP electromorphs were revealed in cytosol and sediment, four of these being most frequently localized in the former, and two in the latter. Two other AcP electromorphs were confined to the sediment only. Depending on the quantity of sedimented amoebae making a homogenate (0.5 or 2.0 cm3), that was added to Percoll solution, the lysosomal AcP fraction in polyacrylamide gel was represented by one or two tartrate-sensitive electromorphs. Therefore, tartrate-resistant AcP in A. proteus may be a lysosomal enzyme, while tartrate-resistant AcP may correspond to serine/threonine protein phosphatase.     

Effect of Inorganic Phosphate on the Extracellular Acid Phosphatase Activity of Tobacco Cells Cultured in vitro

Acid phosphatase activity in culture medium of tobacco cells growing in suspension increased with the age of the culture from which the medium was obtained. The increase in the activity was accelerated by omitting inorganic phosphate from nutrient medium, and it was depressed by addition of inorganic phosphate or cycloheximide. Amylase and β-galactosidase activities were not induced by the omission of inorganic phosphate. It was concluded that derepression of acid phosphatase synthesis was involved in the increase in the extracellular acid phosphatase activity upon inorganic phosphate depletion.     

Characteristics of trajectory in the migration of Amoeba proteus

Summary. We investigated the behavior of migration of Amoeba proteus in an isotropic environment. We found that the trajectory in the migration of A. proteus is smooth in the observation time of 500-1000 s, but its migration every second (the cell velocity) on the trajectory randomly changes. Stochastic analysis of the cell velocity and the turn angle of the trajectory has shown that the histograms of the both variables well fit to Gaussian curves. Supposing a simple model equation for the cell motion, we have estimated the motive force of the migrating cell, which is of the order of piconewton. Furthermore, we have found that the cell velocity and the turn angle have a negative cross-correlation coefficient, which suggests that the amoeba explores better environment by changing frequently its migrating direction at a low speed and it moves rectilinearly to the best environment at a high speed. On the other hand, the model equation has simulated the negative correlation between the cell velocity and the turn angle. This indicates that the apparently rational behavior comes from intrinsic characteristics in the dynamical system where the motive force is not torquelike.     

Immunodetection and intracellular localization of caldesmon-like proteins in Amoeba proteus

Summary. Caldesmon immunoanalogues were detected in Amoeba proteus cell homogenates by the Western blot technique. Three immunoreactive bands were recognized by polyclonal antibodies against the whole molecule of chicken gizzard caldesmon as well as by a monoclonal antibody against its C-terminal domain: one major and two minor bands corresponding to proteins with apparent molecular masses of 150, 69, and 60 kDa. The presence of caldesmon-like protein(s) in amoebae was revealed as well in single cells after their fixation, staining with the same antibodies, and recording their total fluorescence in a confocal laser scanning microscope. Proteins recognized by the antibodies bind to filamentous actin. This was established by a cosedimentation assay in cell homogenates and by colocalization of the caldesmon-related immunofluorescence with the fluorescence of filamentous actin stained with rhodamine-labelled phalloidin, demonstrated in optical sections of single cells in a confocal microscope. Caldesmon is colocalized with filamentous actin in the withdrawn cell regions where the cortical actomyosin network contracts and actin is depolymerized, in the frontal zone where actin is polymerized again and the cortical cytoskeleton is reconstructed, inside the nucleus and in the perinuclear cytoskeleton, and probably at the cell-to-substratum adhesion sites. The regulatory role of caldesmon in these functionally different regions of locomoting amoebae is discussed.Correspondence and reprints: Department of Cell Biology, Nencki Institute of Experimental Biology, ulica Pasteura 3, 02-093 Warsaw, Poland.Received October 7, 2002; accepted December 2, 2002; published online August 26, 2003     

Activity and thermal stability of acid phosphatase in homogenates of Amoeba proteus, acclimated to various temperatures

Activity and thermoresistance of acid phosphatase were determined in supernatant of Amoeba proteus homogenates using 1-naphthyl phosphate (pH 4.0) and p-nitrophenyl phosphate (pH 5.5). Although tartrate-resistant and tartrate-sensitive acid phosphatases hydrolyse both substrates, the former mainly hydrolyses p-nitrophenyl phosphate and the latter 1-naphthyl phosphate. A decrease in the activity of the total and tartrate-sensitive acid phosphatases, when using 1-naphthyl phosphate, and of the total and tartrate-resistant acid phosphatases, when using p-nitrophenyl phosphate, was found in amoebae acclimated to 10 degrees C (10 degrees-amoebae) compared to those acclimated to 25 degrees C (25 degrees-amoebae). Using 1-naphthyl phosphate, the thermoresistance of the total acid phosphatase was lower in 10 degrees-amoebae than in 25 degrees-amoebae, but the thermostability of tartrate-resistant enzyme was the same in both groups of amoebae. Using p-nitrophenyl phosphate, the thermoresistance of the total and tartrate-resistant acid phosphatases was lower (the latter only slightly) in 10 degrees-amoebae than in 25 degrees-amoebae. It is suggested that at least with the use of 1-naphthyl phosphate a decrease in thermostability of the total acid phosphatase may be due to a decrease in thermoresistance of tartrate-sensitive enzyme. The results obtained confirm the author's previous data on the activity and thermostability of electrophoretic forms of acid phosphatase using 2-naphthyl phosphate in 10- and 25 degrees-amoebae (Sopina, 2001). It is the first case of discovering a correlation between changes in primary cell thermoresistance of amoebae cultured at different temperatures and changes in the activity and thermostability of acid phosphatase in their homogenates, with the number of electrophoretic forms of this enzyme and their mobility being permanent.