Purification and properties of cytosolic alanine aminotransferase from the liver of two freshwater fish, Clarias batrachus and Labeo rohita.
Cytosolic alanine aminotransferase (c-AAT) was purified up to 203- and 120-fold, from the liver of two freshwater teleosts Clarias batrachus (air-breathing, carnivorous) and Labeo rohita (water-breathing, herbivorous), respectively. The enzyme from both fish showed similar elution profiles on a DEAE-Sephacel ion exchange column. SDS-PAGE of purified enzymes revealed two subunits of 54 and 56 kDa, in both fish. The apparent Km values for l-alanine were 18.5+/-0.48 and 23.55+/-0.60 mM, whereas for 2-oxoglutarate the Km values were observed to be 0.29+/-0.023 and 0.33+/-0.028 mM for the enzyme from C. batrachus and L. rohita, respectively. With l-alanine as substrate, aminooxyacetic acid was found to act as a competitive inhibitor with KI values of 6.4 x 10(-4) and 3.4 x 10(-4) mM with c-AAT of C. batrachus and L. rohita, respectively. However, when 2-oxoglutarate was used as substrate, aminooxyacetic acid showed uncompetitive inhibition with similar KI values for purified c-AAT from both fish. Temperature and pH profiles of the enzyme did not show any marked differences between the two fish examined. These results suggest that liver c-AAT, isolated from these two fish species adapted to different modes of life, remain unaltered structurally. However, at the kinetic level, liver c-AAT from C. batrachus exhibits significantly higher affinity for the substrate l-alanine and decreased affinity for its metabolic inhibitor, in comparison to that of the enzyme purified from L. rohita. Such functional changes seem to be of physiological significance and also provide preliminary evidence for subtle changes in the enzyme as a mark of metabolic adaptation in the fish to different physiological demands.
School of Medicine, University of California San Diego, La Jolla, CA 92093, USA. assrivastava@ucsd.edu
Anand S
AS
Srivastava
Ichiro
I
Oohara
Tohru
T
Suzuki
Steve
S
Shenouda
Surender N
SN
Singh
Dharam P
DP
Chauhan
Ewa
E
Carrier
D000410:Alanine Transaminase / Q000737:chemistry / Q000302:isolation & purification; D000818:Animals; D002384:Catalysis; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000737:chemistry* / Q000201:enzymology*; D004795:Enzyme Stability; D005260:Female; D005399:Fishes / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000737:chemistry* / Q000201:enzymology*
D000410:Alanine Transaminase