Purification of the native form of tyrosine aminotransferase from rat liver

Hargrove, James L.; Granner, Daryl K.

doi:10.1016/0003-2697(80)90303-6

Cited by 17 publications

(1 citation statement)

References 20 publications

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“…(97.4 units -mg of protein-') and by Hargrove and Granner [14] for the enzyme from rat liver (616 units -mg of protein-'). The considerably greater mass of TAT in T. cruzi epimastigotes, which can be calculated as about 3 % of the total soluble protein, in comparison with TATs from other sources, may be a consequence of this low specific activity.…”

Section: Discussionmentioning

confidence: 98%

Purification and partial structural and kinetic characterization of tyrosine aminotransferase from epimastigotes of Trypanosoma cruzi

Montemartini

Santomé

Cazzulo

et al. 1993

Biochemical Journal

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Tyrosine aminotransferase was purified to homogeneity from epimastigotes of Trypanosoma cruzi by a method involving chromatography on DEAE-cellulose, gel filtration on Sephacryl S-200 and chromatography on Mono Q in an f.p.l.c. system. The purified enzyme showed a single band in SDS/PAGE, with an apparent molecular mass of 45 kDa. Since the apparent molecular mass of the native enzyme, determined by gel filtration, is 91 kDa, the native enzyme is a dimer of similar subunits. The amino-acid composition was determined, as well as the sequences of three internal peptides obtained by CNBr cleavage at Met residues. Both criteria suggest considerable similarity with the tyrosine aminotransferases from rat and from human liver. The enzyme contains nine 1/2 Cys residues, three free and the others forming three disulphide bridges. The enzyme is not N-glycosylated. The isoelectric point is 4.6-4.8. The optimal pH for the reaction of the enzyme with tyrosine as a substrate is 7.0. The apparent Km values for tyrosine, phenylalanine and tryptophan, with pyruvate as a co-substrate, were 6.8, 17.9 and 21.4 mM, respectively, whereas those for pyruvate, alpha-oxoglutarate and oxaloacetate, with tyrosine as a substrate, were 0.5, 38 and 16 mM respectively. The purified tyrosine aminotransferase acts as an alanine aminotransferase as well and the activity seems to reside in the same enzyme molecule. The results suggest that the enzyme is a general aromatic-amino-acid transaminase, with high sequence similarity to tyrosine aminotransferases from rat and human liver.

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Section: Discussionmentioning

confidence: 98%

Purification and partial structural and kinetic characterization of tyrosine aminotransferase from epimastigotes of Trypanosoma cruzi

Montemartini

Santomé

Cazzulo

et al. 1993

Biochemical Journal

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Expression and amplification of cloned rat liver tyrosine aminotransferase in nonhepatic cells

Kohli

Stellwagen

1990

Journal Cellular Physiology

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A full-length cDNA for the rat liver enzyme tyrosine aminotransferase has been used to construct mammalian expression vectors by recombinant DNA techniques. These vectors, which have employed either a simian virus 40 or a Rous sarcoma virus promoter, were transfected into a variety of nonhepatic mammalian cell lines in culture. Transient expression of tyrosine aminotransferase was readily observed after transfection into monkey COS cells and mouse L cells. Stable clones that express cloned tyrosine aminotransferase have been isolated from mouse L cells, hamster Wg1a fibroblasts, and Chinese hamster ovary (CHO) cells. A vector capable of expressing both tyrosine aminotransferase and dihydrofolate reductase was stimulated to undergo amplification by treatment with methotrexate in a CHO cell line deficient in the latter enzyme. Levels of tyrosine aminotransferase as much as 50-fold higher than typically seen in glucocorticoid-induced hepatoma cells were achieved in some CHO clones by this technique. The tyrosine aminotransferase produced at these highly amplified levels appeared structurally normal and had no major harmful effects on the cells.

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Regulation of the synthesis of tyrosine aminotransferase: the relationship to mRNATAT

Granner¹,

Hargrove²

1983

Enzyme Induction and Modulation

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Purification of the native form of tyrosine aminotransferase from rat liver

Cited by 17 publications

References 20 publications

Purification and partial structural and kinetic characterization of tyrosine aminotransferase from epimastigotes of Trypanosoma cruzi

Purification and partial structural and kinetic characterization of tyrosine aminotransferase from epimastigotes of Trypanosoma cruzi

Expression and amplification of cloned rat liver tyrosine aminotransferase in nonhepatic cells

Regulation of the synthesis of tyrosine aminotransferase: the relationship to mRNATAT

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