Branched chain aminotransferase isoenzymes. Purification and characterization of the rat brain isoenzyme.
This paper presents the first complete purification of the branched chain aminotransferase (EC 2.6.1.42) from rat brain cytosol (BCATc). On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the enzyme appeared as a single band with a molecular mass of 47 kDa; however, gel exclusion chromatography suggested that BCATc is a dimer. Comparison of tryptic peptide maps of BCATc and the mitochondrial form of the enzyme (BCATm) indicated that they are different proteins. Experiments with protein labeling reagents, in particular sulfhydryl reagents, also suggested that there may be some distinct structural differences in BCATc and BCATm. Nevertheless, BCATc and BCATm showed similar specificities for amino acid and alpha-keto acid substrates. Both enzymes transaminated branched chain amino acids, their straight chain analogs, L-alloisoleucine and glutamate. A broader range of alpha-keto acids than amino acids was accepted as substrate including alpha-ketobutyrate and the alpha-keto acid of methionine. Both enzymes exhibited ping-pong kinetics with apparent Km values for leucine and isoleucine of about 1 and 5 mM for valine, respectively. Km values for alpha-ketoglutarate ranged from about 0.6 to 3 mM depending on the amino acid substrate. Polyclonal antibodies were raised in rabbits against purified BCATc. BCATc antiserum neutralized branched chain aminotransferase activity in rat brain cytosol but did not affect the activity in a heart mitochondrial extract. However, immunoblotting showed that BCATc and BCATm do share common epitopes since BCATm antiserum recognized BCATc on the immunoblots. The tissue distribution of BCATc was examined using BCATc and BCATm antisera. These data showed that BCATc was found in adult and fetal rat brain, cultured cells from fetal rat brain cortex, ovary, and placenta. Brain had the highest activity followed by ovary, fetal brain, and placenta. BCATc was not found in fetal liver, adult rat liver, or a rat hepatoma cell line. These data provide clear evidence that BCATc, unlike BCATm, is restricted to several highly specialized tissues.
Hall
T R
TR
Department of Biochemistry, Wake Forest University, Bowman Gray School of Medicine, Winston-Salem, North Carolina 27157.
Wallin
R
R
Reinhart
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GD
Hutson
S M
SM
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006882:Hydroxyapatites; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008114:Liver Neoplasms, Experimental / Q000201:enzymology; D008297:Male; D008928:Mitochondria / Q000201:enzymology*; D008970:Molecular Weight; D009500:Neutralization Tests; D009928:Organ Specificity; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley; D013379:Substrate Specificity; D000637:Transaminases / Q000302:isolation & purification* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D006882:Hydroxyapatites; D007527:Isoenzymes; D017886:Durapatite; D000637:Transaminases; C019798:branched-chain-amino-acid transaminase