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B6db references: 5922965

type Journal Article
authors Taylor, R. T.; Jenkins, W. T.
title Leucine aminotransferase. II. Purification and characterization
journal J Biol Chem
sel selected
ui 5922965
year (1966)
volume 241
number 19
pages 4396-405
keywords Animal
abstract Leucine transaminase was purified extensively from pig heart muscle. It appeared to be nearly homogeneous by the criteria of both starch gel and free boundary electrophoresis and ultracentrifugation. The molecular weight was found to be 75,000. Purified leucine transaminase is yellow, and our best preparations contained 1 mole of bound pyridoxal phosphate per 75,000 g of protein. The spectrum of the enzyme has absorption maxima at 414 nm and 326 nm in addition to the characteristic 280-rnp peak. Addition of an amino acid substrate shifted the 414-nm peak to 326 nm, whereupon addition of the corresponding or-keto acid partially reversed this shift. Incubation of the enzyme with an amino acid substrate plus a high concentration of phosphate yielded an inactive apoenzyme which could be reconstituted with pyridoxal phosphate. Loss of activity was correlated with a loss of absorption in the visible region. The enzyme activity was inhibited by carbonyl reagents. Reduction with sodium borohydride completely shifted the 414-nm peak to 320 nm and destroyed the transaminase activity. Acid hydrolysis of the reduced enzyme yielded a fluorescent compound which was tentatively identified as t-pyridoxal lysine. Leucine transaminase displayed a bell-shaped pH-activity curve with an apparent optimum at pH 8.3 to 8.5; however, it was most stable at pH 6 to 7. At pH 8.5 and in the presence of 0.1 M 2-mercaptoethanol, this single enzyme catalyzed transamination between L-glutamate, L-leucine, L-valine, or L-isoleucine and their alpha-keto forms. Under arbitrary conditions, the relative rates of transamination were of the same order of magnitude with a maximum difference of 5-fold.
last changed 2009/04/15 12:01

B6db references