ABSTRACT
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: 1. The activity of aspartate and alanine aminotransferase was about 0.55 unit and 0.92 unit per 1 g of Fasciola hepatica, respectively. 2. The activity of those enzymes was relatively low compared with those in mammalian tissues. 3. The distribution of aspartate aminotransferase in the subcellular organelles showed that 71 % of the activity was in cytosolic, 24 % in mitochondrial and 5 % was in nuclear fraction. 4. About 22 % of the total alanine aminotransferase activity was found in the mitochondrial fraction, about 66 percent in the cytosolic fraction. 5. 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.
ABSTRACT
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: 1. 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. 2. The distribution of branched chain amino acid aminotransferase in the subcellular organelles showed that 87.8 % of the activity was in cytosolic, 10.9 % in mitochondrial and 1.3 % was in nuclear fraction. 3. 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. 4. The Enzyme I was purified about 22-folds increase in specific activity after chromatography on DEAE-cellulose. 5. The best substrate among three amino acids (leucine, isoleucine and valine) was L-isoleucine. 6. The optimal temperature of Enzyme I was 45 degrees C and the optimal pH was 8.2. 7. The Km value for leucine of Enzyme I was 4.17 mM. 8. The Km values for alpha-ketoglutarate and pyridoxal phosphate of Enzyme I were 0.41 mM and 4.76 x 10(-3) mM, respectively.
ABSTRACT
In attempt to investigate histone fractions and non-histones of parasites, nuclei were isolated from Fasciola hepatica by the procedure of Pogo et al. (1966). Histone fractions H1, H2a, H2b, H3 and H4 were prepared from isolated nuclei by the procedure of Johns (1964 and l967). The five histone fractions found in most tissues were also present in the Fasciola hepatica histones. These histone fractions were characterized by amino acid analysis and by polyacrylamide disc gel electrophoresis. Non-histone proteins were extracted from isolated Fasciola hepatica nuclei and separated by SDS-polyacrylamide gel electrophoresis. The results of the experiment were summarized as follows: 1. The yield of whole histone recovered was 2.47 mg per 1 g of Fasciola hepatica. 2. The yield of DNA was 1.02 mg per gm of tissues. Consequently the DNA to histone ratio was 1:2.44. 3. The relative amounts of five fractions, i.e., Hl, H2a, H2b, H3 and H4 were 19.96%, 26.48%, 29.60%, 12.56% and 14.37%, respectively. 4. Amino acid analysis of the individual histone fractions showed that the over-all compositions were similar but not identical to those of corresponding fraction from calf thymus. 5. It was found that histone H2b fraction of Fasciola hepatica contained detectable amounts of epsilon-N-monomethyllysine. No evidence for the presence of methylated lysine or other side-chain derivatives was reported on this histone fraction. 6. In SDS-polyacrylamide disc gel, it showed that 17 protein bands of nuclear acidic protein can be identified visually.
ABSTRACT
The present study was undertaken to observe the quality and quantity of lipid constituents by the developing Ascaris suum eggs. The collected eggs from the uterus of A. suum were classified into 3 groups, i.e., single cell stage, morula stage and embryonated eggs, and were subjected to analyse their lipid fractions. To obtain the morula stage eggs, 10 to 11 incubation days at 30 degree C were needed and for the embryonated eggs, 30 to 31 days were lasted. At the time of experiment, their indices of development by Hoffman were 285(morula stage) and 42 (embryonated stage) respectively. Lipid extraction was done by the methods of Folch et a1. (1957) and Kenny (1952), and then the extracted lipid fractions from the above 3 groups of eggs were separated by thin layer chromatography. Those fractions were also subjected to perform the quantitative analyses of fatty acids, glycerides, cholesterol and phospholipids. The results obtained were summarized as follows. 1. Total amount of fatty acid was decreased from 12.9 mg per gram of eggs (single cell stage) to 6.6 mg/gm (embryonated eggs), whereas the proportion of free fatty acid to total fatty acid was constantly increased from 77.5% to 89.4% during the period of egg development. 2. Total amount of glycerides was also increased from 33.0 mg/gm of single cell stage to 55.9 mg/gm of the embryonated eggs. The most abundant glyceride among 3 glycerides discovered from A. suum eggs was triglyceride, and the least was monoglyceride. 3. The amount of free cholesterol was much larger than that of ester form in general, and it reached maximum in the eggs of morula stage (4.6 mg/gm). The increase of total cholesterol was monitored during the development of A. suum eggs from 3.3 mg/gm to 5.4 mg/gm. 4. The following 8 phospholipids were detected in the embryonated eggs, i.e., lysophosphatidyl choline, phosphatidyl inositol, sphingomyelin, phosphatidyl choline, phosphatidyl glycerol, phosphatidyl serine, phosphatidyl ethanolamine and unknown phospholipid. But in the single cell stage eggs, 4 kinds out of the above 8 phopholipids were not observed, and in the morula stage eggs, 2 kinds were absent among the 8 phospholipids.
