Isolation and characterization of cytosolic alanine aminotransferase isoforms from starved rat liver.
Alanine is the most effective precursor for gluconeogenesis among amino acids and the initial reaction is catalyzed by alanine aminotransferases (AlaATs). It is a less extensively studied enzyme under starvation and known to that the enzyme activity increases in liver under starvation. The present study describes the purification and characterization of two isoforms of alanine aminotransferases from starved male rat liver under starvation. The molecular mass of isoforms was found to be 17.7 and 112.2 kDa with isoelectric points of 4.2 and 5.3 respectively for AlaAT I and AlaAT II. Both the enzymes showed narrow substrate specificity for L-alanine with different Km for alanine and 2-oxoglutarate. Both the enzymes were glycoprotein in nature. Inhibition, modification and spectroscopic studies showed that both PLP and free-SH groups are directly involved in the enzymatic catalysis. PLP activated both the enzymes with a Km 0.057 mM and 0.2 mM for AlaAT I and II respectively.
Vedavathi
M
M
Department of Biochemistry, University of Mysore, Manasagangotri, Mysore, India.
Girish
K S
KS
Kumar
M Karuna
MK
D000410:Alanine Transaminase / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification*; D000625:Aminooxyacetic Acid / Q000494:pharmacology; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006023:Glycoproteins / Q000378:metabolism; D019811:Hydroxylamine / Q000494:pharmacology; D007526:Isoelectric Point; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification; D007700:Kinetics; D008099:Liver / Q000201:enzymology* / Q000502:physiology; D008297:Male; D008970:Molecular Weight; D051381:Rats; D013053:Spectrophotometry; D013194:Staining and Labeling; D013217:Starvation; D013379:Substrate Specificity; D013696:Temperature
D004791:Enzyme Inhibitors; D006023:Glycoproteins; D007527:Isoenzymes; D000625:Aminooxyacetic Acid; D019811:Hydroxylamine; D000410:Alanine Transaminase