Purification and properties of branched chain amino acid aminotransferase from <i>Fasciola hepatica</i>

, Lee, Jung Ho; , Lee, Dong Wook; , Lee, Hi Sung; , Song, Chul Yong

doi:1983.21.1.49

Korean J Parasito > Volume 21(1):1983 > Article

Original Article


Korean J Parasitol. 1983 Jun;21(1):49-57. English. Published online Mar 20, 1994. http://dx.doi.org/10.3347/kjp.1983.21.1.49
Copyright © 1983 by The Korean Society for Parasitology


Purification and properties of branched chain amino acid aminotransferase from Fasciola hepatica
Jung Ho Lee,Dong Wook Lee,Hi Sung Lee and Chul Yong Song
Department of Biochemistry, College of Medicine, Chung-Ang University, Korea.
Department of Biology, College of Liberal Arts and Sciences, Chung-Ang University, Korea.


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℃ 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 × 10^-3 mM, respectively.

Figures


	Fig. 1 Chromatography of BCAT on DEAE-cellulose.(●-●, protein; ○-○, enzyme activity; …… concentration of buffer)


	Fig. 2 Effect of temperature on activity of branched chain amino acid aminotransferase.


	Fig. 3 Effect of pH on activity of branched chain amino acid aminotransferase.


	Fig. 4 Effect of substrate (leucine) concentration on activity of branched chain amino acid aminotransferase.


	Fig. 5 Effect of α-ketoglutarate concentration on activity of branched chain amino acid aminotransferase.


	Fig. 6 Effect of pyridoxal phosphate concentration on activity of branched chain amino acid aminotransferase.

Tables


	Table 1 Distribution of branched chain amino acid aminotransferase in Fasciola hepatica


	Table 2 Purification of branched chain amino acid aminotransferase from Fasciola hepatica


	Table 3 Isozyme patterns of branched chain amino acid aminotransferase from Fasciola hepatica


	Table 4 Substrate specificities of branched chain amino acid aminotransferase from Fasciola hepatica


	Table 5 Km values of leucine for Enzyme I of Fasciola hepatica


	Table 6 Km values of α-ketoglutarate and pyridoxal phosphate for Enzyme I of Fasciola hepatica

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