Purification and characterization of aspartate aminotransferase isoenzymes from carrot suspension cultures.

Abstract:

:Three aspartate aminotransferase isoenzymes were identified from extracts of carrot (Daucus carota L.) cell suspension cultures. These isoenzymes were separated by DEAE chromatography and were analyzed on native gradient polyacrylamide gels. The relative molecular weights of the isoenzymes were 111,000 +/- 5000, 105,000 +/- 5000, and 94,000 +/- 4000 daltons; they were designated forms I, II, and III, respectively. Form I, the predominant form, has been purified to apparent homogeneity (>300-fold) using immunoaffinity chromatography with rabbit anti-pig AAT antibodies. Form I has a subunit size of 43,000 M(r), as determined on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Isoelectric focusing (IEF)-PAGE has resolved three bands at a pl of approximately 5.2. Form I may be composed of subunits of similar molecular weight and different charges, and the three bands with AAT activity on the IEF-PAGE gel are a combination of hetero- and homodimers. Form I has a broad pH optimum of 7.5 to 10.0. K(m) values of 23.6, 2.8, 0.05, and 0.22 millimolar were obtained for glutamate, aspartate, oxaloacetate, and alpha-ketoglutarate, respectively. The mode of action is a ping-pong-bi-bi mechanism.

journal_name

Plant Physiol

journal_title

Plant physiology

authors

Turano FJ,Wilson BJ,Matthews BF

doi

10.1104/pp.92.3.587

subject

Has Abstract

pub_date

1990-03-01 00:00:00

pages

587-94

issue

3

eissn

0032-0889

issn

1532-2548

journal_volume

92

pub_type

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