Molecular biology of ATP citrate lyase

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Cui, Liwei
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Biochemistry, Biophysics and Molecular Biology

The Department of Biochemistry, Biophysics, and Molecular Biology was founded to give students an understanding of life principles through the understanding of chemical and physical principles. Among these principles are frontiers of biotechnology such as metabolic networking, the structure of hormones and proteins, genomics, and the like.

The Department of Biochemistry and Biophysics was founded in 1959, and was administered by the College of Sciences and Humanities (later, College of Liberal Arts & Sciences). In 1979 it became co-administered by the Department of Agriculture (later, College of Agriculture and Life Sciences). In 1998 its name changed to the Department of Biochemistry, Biophysics, and Molecular Biology.

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  • Department of Biochemistry and Biophysics (1959–1998)

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The full length Arabidopsis ACL-A1 and ACL-B2 cDNAs have been expressed in E. coli. Western blot analysis of Arabidopsis extracts, probed with antiserum raised against these proteins, indicate that At.ACL-Al and At.ACL-B2 cDNAs code for proteins of 45 kDa and 65 kDa, respectively. To demonstrate that Arabidopsis ACL-A and ACL-B are subunits of ATP citrate lyase, full length ACL-A1 and ACL-B2 cDNAs were co-expressed in S. cerevisiae strain αD273, and a specific ACL activity of 0.775 nmol/min/mg protein was detected from extract from the strain carrying both the transgenes, whereas little activity was detected from extracts of its parental S. cerevisiae strain αD273, and the strain carrying either of the individual transgenes. These results demonstrate, for the first time, that ACL-A and ACL-B constitute enzymatic Arabidopsis ATP citrate lyase. Western blot analysis of Arabidopsis extracts subjected to non-denaturing electrophoresis, probed with anti-At.ACL-A serum, identified a single complex. The anti-At.ACL-B serum appears to detect the same complex. This demonstrates that Arabidopsis ACL-A and ACL-B polypeptides are in a complex. A homozygous Arabidopsis line containing a T-DNA insertional mutation in the ACL-A3 gene had been isolated. Western blot analysis of extracts from Arabidopsis seedlings germinated for 15 days showed little difference, in terms of ACL level, between wild type and T-DNA insertional ACL-A3 Arabidopsis plants. ACL activity also showed little difference between the wild type Arabidopsis plants and T-DNA insertional ACL-A3 mutant plants. These results indicated that ACL-A3 gene does not play a predominant role in determining ACL level at this stage of plant growth. Histochemical GUS assays of ACL-A2:GUS and ACL-A3:GUS transgenic plants showed that ACL-A2 was expressed throughout the seedlings, mature flowering leaves, flowers, siliques; whereas ACL-A3 was expressed only in seedlings. This is also consistent with the lack of difference in ACL activity or ACL accumulation associated with T-DNA insertional ACL-A3 mutation.

Mon Jan 01 00:00:00 UTC 2001