Purification of maize leaf acetyl CoA carboxylase

--Margaret A. Egli, Burle G. Gengenbach, John W. Gronwald1, David A. Somers, and Donald L. Wyse

1USDA-ARS

Acetyl CoA carboxylase (E.C. 6.4.1.2; ACCase) catalyzes the initial step of fatty acid synthesis, carboxylation of acetyl CoA to form malonyl CoA, the substrate for fatty acid elongation and also for synthesis of flavonoids and other secondary metabolites. ACCase of most monocots, including maize, is inhibited by cyclohexanedione and aryloxyphenoxypropionate herbicides. Herbicide-tolerant maize lines having altered ACCase activity conferred by partially dominant nuclear mutations have been obtained from tissue culture (Parker et al., PNAS 87:7175-7179,1990). We plan to isolate the ACCase gene from wildtype maize to more fully examine its expression and regulation in normal and herbicide-tolerant genotypes. Our immediate objectives are to purify ACCase from wildtype maize, generate ACCase antibodies, and screen expression libraries to obtain ACCase mRNA sequences.

Approximately 60 ug ACCase was obtained from 50 g maize leaves (A619) extracted in buffer containing 0.2 mM PMSF (PNAS 87:7175) and purified as described in Table 1. Similar to other plant and animal ACCases, purified maize leaf ACCase has a native MW of approximately 490 kD (Superose 6 gel filtration), a pI of 6.9, and appears to be a dimer of biotinylated subunits of 220 kD (SDS-PAGE).

High-titer antiserum (rabbit) was obtained by subcutaneous injections of 20 to 100 ug of 220-kD ACCase peptide in SDS gels. Whole immune serum bound to Protein A-agarose immunoprecipitated ACCase activity. On western blots of crude leaf extracts, antibodies bound primarily to one 220-kD polypeptide. We are currently using the ACCase antiserum to screen a maize seedling lambda gt11 cDNA library (S. Gantt, Univ. Minnesota).

ACCase is known to be localized in plastids, the site of fatty acid synthesis, however, mitochondrial and cytosolic forms of similar MW occur in animals (Allred and Roman-Lopez, Biochem. J. 251:881-885). Plants may also contain different ACCase enzymes. We observed two biotinylated polypeptides of approximately 220 kD on western blots of crude leaf extracts probed with avidin, but only one was strongly recognized by ACCase antiserum. The possibility of ACCase isozymes will be further examined by using avidin and antiserum to test for 220-kD polypeptides in different maize organs and tissues, and by using an ACCase mRNA to estimate the number of ACCase genes.

Table 1.  Purification of ACCase from A619 leaves.
 
step Units2 /mg -fold purification activity yield (%) protein yield (%)
crude extract 0.002 1 100 100.00
30-40%
 (NH4)2SO4
0.068 32 475 15.00
S-300 0.377 175 780 4.50
Blue Sepharose 2.23 1030 113 0.11
FPLC Mono-Q 5.84 2704 35 0.013
2incorporation of [14C]HCO3 into acid-stable products.


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