The distribution and properties of branched chain amino acid aminotransferase(EC 2.6.1.42) was investigated in adult Fasciola hepatica. Fasciola hepatica was fractionated by differential centrifugation into nuclear, mitochondrial and cytosolic fractions. The activity of branched chain amino acid aminotransferase was measured by the method of Ichihara and Koyama (1966) . Isozyme patterns of this enzyme was also examined by DEAE-cellulose column chromatography. The results obtained were as follows: The activity in homogenate was found to be 12.69 units/g wet tissue. The activity of this enzyme was relatively high compared with those in rat tissues. The distribution of branched chain amino acid aminotransferase in the subcellular organelles showed that 87.8 percent of the activity was in cytosolic, 10.9 percent in mitochondrial and 1.3 percent was in nuclear fraction. Cytosolic fraction of Fasciola hepatica contained Enzyme I, but not Enzyme II and III, of branched chain amino acid aminotransferase. Enzyme I was eluted by 50 mM phosphate buffer from DEAE-cellulose column and catalyzed the transamination of all three branched chain amino acids. The Enzyme I was purified about 22-folds increase in specific activity after chromatography on DEAE-cellulose. The best substrate among three amino acids (leucine, isoleucine and valine) was L-isoleucine. The optimal temperature of Enzyme I was 45 C and the optimal pH was 8.2. The Km value for leucine of Enzyme I was 4.17 mM. The Km values for alpha-ketoglutarate and pyridoxal phosphate of Enzyme I were 0.41 mM and 4.76 x 10(-3) mM, respectively.
parasitology-helminth-trematoda ; Fasciola hepatica ; biochemistry ; enzyme ; aminotransferase

this report describes the structures of high-mannose-type N-linked oligosaccharides in glycoproteins synthesized by the microfilariae of Dirofioria immitis. Microfilariae of D. immitis were incubated in vitro in media contaning 2-[(3)H] mannose to allow metabolic radiolabeling of the oligosaccharide moieties of newly synthesized glycoproteins. Glycopeptides were prepared from the radiolabeled glycoproteins by digestion with pronase and fractionation by chromatography on concanavalin A-Sepharose. Thirty eight percent of 2-[(3)H] mannose incorporated into the microfilariae of D. immitis glycopeptides was recovered in high mannose-type asparagine-linked oligosaccharides whech were bound to the immobilized lectin. Upon treatment of 2-[(3)H] mannose labeled glycopeptides with endo-beta-N-acetylglucosaminidase H, the high mannose-type chains were released and their structures were determined by high performance liquid chromatography and exoglycosidase digestion. The major species of high mannose-type chains synthesized by microfilariae of D. immitis have the composition Man(5) GlcNAc(2), Man(6) ClcNAc(2), Man(8) GlcNA(2), and Man(8) GlcNAc(2). Structural analyses indicate that these oligosaccharides are similar to high mannose-type chains synthesized by vertebrates.
parasitology-helminth-nematoda ; Dirofilaria immitis ; mannose ; carbohydrates ; oligosaccharieds ; micrlfilariae ; metabolic labeling ; HPLC ; enzyme digestion ; biochemistry

The activity and distribution of aspartate aminotransferase (EC 2.6.1.1) and alanine aminotransferase (EC 2.6.1.2) in adult Fasciola hepatica have been studied. Fasciola hepatica was fractionated by differential centrifugation into nuclear, mitochondrial and cytosolic fractions. The activity of GOT and GPT was measured by the method of Reitman and Frankel. Isozyme patterns of those enzyme were also examined by DEAE-cellulose column chromatography. The results obtained were as follows: The activity of aspartate and alanine aminotransferase was about 0.55 unit and 0.92 unit per 1 g of Fasciola hepatica, respectively. The activity of those enzymes was relatively low compared with those in mammalian tissues. The distribution of aspartate aminotransferase in the subcellular organelles showed that 71 percent of the activity was in cytosolic, 24 percent in mitochondrial and 5 percent was in nuclear fraction. About 22 percent of the total alanine aminotransferase activity was found in the mitochondrial fraction, about 66 percent in the cytosolic fraction. Aspartate aminotransferase from cytosolic fraction was separated into two types of isozymes, whereas alanine aminotransferase from cytosolic fraction gave only one active peak on DEAE-cellulose column chromatography.
parasitology-helminth-trematoda ; Fasciola hepatica ; biochemistry ; enzyme ; aspartate aminotransferase ; alanine ; aminotransferase ; alanine aminotransferase

The malic dehydrogenase activity was determined by the modified method of Ochoa (1955) using tissue homogenates of various parasitic helminths. Worm parasites were mostly collected from local abattoir, and removed from the organ or tissues of the naturally infected animal hosts, and some materials were also obtained from the human hosts. The helminths used in this experiment include 3 kinds of nematodes, 5 kinds of trematodes, and 8 kinds of cestodes. They were throughly washed and homogenized in glass tissue grinder in ice chilled water bath, and then centrifuged. The supernatants were designated as enzyme preparations. The hydrogen concentrations of buffer solution were pH 1.4, 2.7, 3.5, 4.2, 5.2, 7.4, 8.2, 9.3, 10.2, 11.6, and enzymatic reaction of this experiment was performed at incubation temperature of 20, 30, 40, and 50 C. The extinction of Nicotinamide Adenosine Dinucleotide (NAD) was measured by spectrophotometry at the wave length of 340 millimicron. The results of the experiment were as follows: The malic dehydrogenase activity occurred over all kinds of parasitic helminths used in this study. And the activity on sparganum turned out to be highest. All helminths displayed their maximum activity in the range of alkaline pH. A comparison of the effects of temperature and substrate concentration on the enzyme activity was made among these helminths. However, no definite relationship among them has been detected. The significance of the existence of this enzyme in the helminths was briefly discussed.
parasitology-helminth-trematoda-cestoda-nematoda ; Fasciola hepatica ; Eurytrema pancreaticum ; Paramphistomum sp. ; Taenia solium ; Taenia pisiformis ; Dipylidium caninum ; Diphyllobothrium mansoni ; Cysticercus cellulosae ; Cysticercus fasciolaris ; sparganum ; Ascaris lumbricoides ; Ascaridia galli ; Dirofilaria immitis ; Paragonimus westermani ; Clonorchis sinensis ; malic dehydrogenase-biochemistry-enzyme ; malic dehydrogenase ; Nicotinamide Adenosine Dinucleotide

A series of experiments was performed to determine the lactic dehydrogenase activity of various parasitic helminths. The enzyme activity was determined by the modified method of Wroblewshi and LaDue (1955) using tissue homogenate of 16 kinds of worm parasites. The worms were mostly collected alive from local abattoir and removed from the organ or tissues of the naturally infected animal host and some materials were also obtained from the human host. They were thoroughly washed and homogenized in chilled glass tissue grinder, and then centrifuged. The supernatants were designated as enzyme preparations, and their enzyme activity was measured by spectrophotometry at the wave length of 340 millimicron. In order to know the effects of temperature and substrate concentration on the enzyme activity, the extinction of reduced Coenzyme I(NADH) was measured at the various conditions of incubation temperature and substrate concentration. The results of this experiments were as follows: The lactic dehydrogenase activity occurred over all kinds of parasites used in this study. Most worms of nematodes and trematodes displayed their maximum activity in the range of pH 2.7-3.5, and cestodes revealed their maximum activity in the ranges of both pH 2.7-3.5 and pH 7.4. In nematodes and trematodes, the lactic dehydrogenase activity increased slowly as incubation temperature increases except in the case of Eurytrema pancreaticum, while the activity in cestodes decreased inversely. The lactic dehydrogenase activity increased in proportion to the increase of substrate concentration in most of worm parasites.
parasitology-nematode-trematoda-helminth ; lacticdehydrogenase ; nicotinamide dinucloetide ; sodium pyruvate ; Ascaris lumbricoides ; Ascaridia galli ; Dirofilaria immitis ; Fasciola hepatica ; Eurytrema pancreaticum ; Paramphistomum sp. ; Clonorchis sinensis ; Paragonimus westermani ; Taenia saginata ; Taenia solium ; Taenia pisiformis ; Dipylidium caninum ; Diphyllobothrium mansoni ; sparganum, Cysticercus cellulosae ; Cysticercus fasciolaris ; biochemistry- enzyme

Base Sequence ; Binding Sites, Antibody ; Biochemistry ; methods ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; Genetic Vectors ; Immunoglobulin Fc Fragments ; genetics ; metabolism ; Immunoglobulin G ; genetics ; metabolism ; Ligands ; Molecular Sequence Data ; Plasmids ; Protein Binding ; Recombinant Fusion Proteins ; genetics ; isolation & purification ; Staphylococcal Protein A ; genetics ; metabolism