Studies on phosphatase activity in some parasitic helminths

In order to obtain some informations on the nature and relative activity of the phosphatases present in various helminths, biochemical studies have been made in thirteen kinds of worm parasites including the adults and larvae (Fasciola hepatica, Eurytrema pancreaticum, Paramphistomum sp., Taenia solium, Taenia pisiformis, Dipylidium caninum, Diphyllobothrium mansoni, Cysticercus cellulosae, Cysticercus fasciolaris and Sparganum). A comparison based on the analysis of pH-activity curves was made among these helminths. The worm materials were mostly obtained alive from an abattoir and removed from the organs or tissues of the animal hosts naturally infected. Sparganum and Cysticercus cellulosae, however, are collected from the subcutaneous tissue of the patients by surgical removal. The worms thoroughly washed were weighed and transferred with 0.1 M Tris buffer to a chilled glass grinder (Capacity; 15 ml) and homogenized in the cold. The homogenate was centrifuged at 5000 RPM for 30 minutes. The supernatant was pipetted off for determination of the phosphatase activity. Incubation mixtures consisted of 1 ml substrate, 1 ml buffer and 0.5ml extract. The buffers used were Tris (Hydroxymethyl) aminomethane and citric acid monohydrate and the substrate was paranitrophenyl phosphate (1 gm/25 ml). These mixtures were incubated at the temperature of 37 C for 30 minutes in water bath. The absorbance or transferance of mixture was determined colorimetrically by "Spectronic 20 "spectrophotometer at 410 nm against a distilled water blank. The amount of phenol liberated was then calculated from a standard curve using phenol solutions. Controls consisted of unincubated mixtures. The results were deducted from this experiment. The phosphatase activity occurred over all parasitic helminths used in this experiment. In trematodes, pH-activity curves have demonstrated two peaks of phosphatase activity in Fasciola hepatica and Paramphistomum species. However the acid phosphatase activity was predominantly found and the alkaline phosphatase activity was found distinctly to be low in all three species. In Eurytrema pancreaticum, the pH-activity curves displayed two peaks in acid phosphatase activity, one at pH 5.0 and the other pH 9.0. In cestodes, both alkaline and acid phosphatase activity displayed the pH optima 5.0 and 9.0 to 10.0 in the adult tapeworms. However, major activity in the adults is due to the alkaline phosphtases. In contrast to the adults, Cysticercus and sparganum showed the higher activity in acid phosphatases which predominates in the larvae. In all cases of nematodes, the pH optimum for acid phosphatase was 4.0 to 6.0. A preponderance of acid phosphatase activity was shown in the extract of intestine of Ascaris lumbricoides. The aspect that phosphatases are correlated with phosphorylated passage of substances through the cuticle of helminths and may also be involved in carbohydrate metabolism is discussed.

Metabolism of C(14)-glucose by Moniezia expansa and Diphyllobothrium mansoni

The adult worms of cestodes, Moniezia expansa and Diphyllobothrium mansoni employed in this experiment. The worms were divided into three portions, i.e. immature , mature and gravid proglottids, and each proglottids were incubated in a certain incubation period, and the glucose uptake rate, total CO2 production rate, tissue concentration and their radioactivities were employed as previous reports(Rim et al., 1965). The glucose uptake rate by M. expansa was a mean value of 6.46+/-1.23 micromole per hour per gram of wet wt. and the rate by D. mansoni was a mean value of 18.8+/-0.8 micro-mole per hour per gram of wet wt. The higher rates were observed in the mature proglottid of M. expansa and in the immature proglottid of D. mansoni . The total CO(2) production rates by the worms averaged 14.0+/-2.37 micro-mole per hour per gram in M. expansa and 17.51+/-1.54 micro-mole per hour per gram of wet wt. The relative specific activities of respiratory CO(2)(R.S.A CO(2)) averaged 22.2+/-5.15 percent in M. expansa and 54.2+/-2.2 per cent in D. mansoni. In the both worms, the higher values were obtained in the mature proglottids. Therefore, the average value of 8.84+/-2.66 per cent of glucose utilized by M. expansa and 8.23+/-0.50 percent of glucose utilized by D. mansoni from the medium glucose was oxidized into respiratory CO(2). The tissue concentrations of glycogen were a mean of 2.21+/-0.46 percent per gram of wet wt. in M. expansa and 7.56+/-1.24 percent per gram of wet wt. in D. mansoni. The higher concentration of glycogen was observed in the gravid proglottids of M. expansa, however the gravid proglottids of D. mansoni showed lower concentration of glycogen than the other proglottids. The turnover rate of glycogen pool yielded with a mean of 0.04+/-0.01 miligram per hour per gram of wet wt. of M. expansa, whereas a mean of 1.66+/- 0.46 miligram per hour per gram wet wt. of D. mansoni. Therefore, a mean value of 2.58+/-0.93 per cent(R.G.D gly) of glucose utilized by M. expansa and 53.6+/-1.4 percent by D. mansoni was incorproated into the glycogen . These data account for that at least 11.42 per cent of the utilized glucose by M. expansa and 61.83 per cent of the utilized glucose by D. mansoni participated in furnishing the oxidation into respiratory CO2 and the synthetic process into glycogen.

Metabolism of C(14)-acetate by cestodes

The adult worm and plerocercoid larva(sparganum) of Diphyllobothrium mansoni and Moniezia expansa employed in this experiment. The adult worms were divided into three portions, i.e. immature, mature and gravid proglottids, and each proglottids were incubated in 50 cc or 250 cc volume of special incubation flasks with incubation medium consisting of 10 cc of 25 cc of Krebs-Ringer phosphate buffer (pH 7.4). The incubation medium was added C(14)-acetate and non-radioactive carrier Na-acetate so as to contain acetate concentration of 50 mg per cent. The worms were allowed to incubate for 5 hours in the Dubnoff metabolic shaking incubator at 38 C. After incubation period, the lactate and pyruvate appearance rate, total CO(2) production tate, the turnover rates were employed as pervious report(Seo et al., 1965). The quantitative analysis of C(14)-acetate utilized by the adult worm and plerocercoid larva of D. mansoni and M. expansa were compared and discussed in this report. According to these data of the experiment, it is impressed that the fatty acid such as acetate may play a role of major part of their metabolism in the adult worm and plerocercoid larva of D. mansoni , whereas minor part of acetate participated in the metabolism by M. expansa.