1887

Abstract

The aconitase of was purified to homogeneity, albeit in low yield (0·6%). It was shown to be a monomeric protein of 95000 or 97500 by gel filtration and SDS-PAGE analysis, respectively. The N-terminal amino acid sequence resembled that of the enzyme ( product), but the similarity at the DNA level was insufficient to allow detection of the gene using a 456 bp probe. Phages containing the gene were isolated from a - gene bank by immunoscreening with an antiserum raised against purified bacterial enzyme. The gene was located at 28 min (1350 kb) in the physical map of the chromosome by probing Southern blots with a fragment of the gene. Attempts to locate the gene using the same procedure with oligonucleotide probes encoding segments of the N-terminal amino acid sequence were complicated by the lack of probe specificity and an inaccuracy in the physical map of Kohara . ( 50, 495–508, 1987). Aconitase specific activity was amplified some 20-200-fold in cultures transformed with pGS447, a derivative of pUC119 containing the gene, and an apparent four-fold activation—deactivation of the phagemid-encoded enzyme was observed in late exponential phase. The aconitase antiserum cross-reacted with both the porcine and ( 120000) enzymes.

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1991-11-01
2021-08-06
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